APOPTOSIS SIGNALING ASSOCIATED WITH AMYLOID … · The studies presented in this thesis were...

UNIVERSIDADE DE LISBOA

FACULDADE DE FARMÁCIA

APOPTOSIS SIGNALING ASSOCIATED WITH

AMYLOID -INDUCED CELLULAR

DYSFUNCTION AND DEGENERATION

RELEVANCE FOR ALZHEIMER'S DISEASE

Ricardo Jorge Soares Viana

DOUTORAMENTO EM FARMÁCIA

BIOQUÍMICA

2010

UNIVERSIDADE DE LISBOA

FACULDADE DE FARMÁCIA

APOPTOSIS SIGNALING ASSOCIATED WITH

AMYLOID -INDUCED CELLULAR

DYSFUNCTION AND DEGENERATION

RELEVANCE FOR ALZHEIMER'S DISEASE

Ricardo Jorge Soares Viana

Tese de Doutoramento em Farmácia (Bioquímica), apresentada à

Universidade de Lisboa através da Faculdade de Farmácia

Research advisor:

Professor Cecília M. P. Rodrigues

Lisboa

2010

The studies presented in this thesis were performed at the Research Institute for

Medicines and Phamaceutical Sciences (iMed.UL), Faculty of Phamacy, University of

Lisbon under the supervision of Professor Cecília M. P. Rodrigues. Part of these studies

was also performed at the Department of Pathology, New York University School of

Medicine, New York, NY, USA, in collaboration with Professors Agueda Rostagno and

Jorge Ghiso.

Ricardo Jorge Soares Viana was the recipient of a Ph.D. fellowship

(SFRH/BD/30467/2006) from Fundação para a Ciência e Tecnologia (FCT), Lisbon,

Portugal. This work was supported by grants PTDC/BIA-BCM/67922/2006 and

PTDC/SAU-FCF/67912/2006 from FCT and FEDER.

De acordo com o disposto no ponto 1 do artigo nº 41 do Regulamento de Estudos

Pós-Graduados da Universidade de Lisboa, deliberação nº 93/2006, publicada em Diário

da República – II Série nº 153 – 5 de Julho de 2003, o Autor desta dissertação declara que

participou na concepção e execução do trabalho experimental, interpretação dos

resultados obtidos e redacção dos manuscritos.

A um sonho de criança

Resumo

Resumo

vii

A doença de Alzheimer (AD) é uma doença neurodegenerativa devastadora, de

causa ainda desconhecida e para a qual não existe cura. Afecta principalmente pessoas

idosas e, com o aumento gradual da esperança média de vida, tornou-se um dos mais

graves problemas de saúde pública. O período médio de sobrevivência do indivíduo

doente, após o diagnóstico, é de apenas 8 anos. Caracteriza-se por originar uma perda

progressiva da memória e da capacidade de raciocínio, mudanças de humor, mudanças de

personalidade e perda de independência. As mutações responsáveis por AD e pelas

formas familiares da doença representam apenas 5% do total de casos. Embora raras,

essas mutações estão muito bem estudadas e definidas. As primeiras mutações que foram

identificadas situam-se no gene da proteína precursora do péptido β amilóide (Aβ), o qual

se localiza no cromossoma 21. As formas mutantes do Aβ, ao contrário da sua forma

nativa, têm a particularidade de serem bastante agressivas para as células endoteliais

vasculares do cérebro, originando a chamada angiopatia amilóide cerebral. Em termos

fisiopatológicos, a AD caracteriza-se, ainda, pela perda massiva de neurónios, o que

origina perturbações na função sináptica. Esta perda de neurónios começa no hipocampo,

numa área que desempenha uma função primordial na formação de novas memórias, e

rapidamente atinge outras zonas do cérebro. As placas amilóides e as tranças

neurofibrilhares, resultantes da hiperfosforilação da proteína tau, são os únicos elementos

discriminadores da doença, utilizados para realizar o seu diagnóstico definitivo aquando

da autópsia.

Estudar e compreender o fenómeno da morte celular programada é de todo

imperativo. A apoptose é um processo altamente regulado, que envolve vários organitos

e, como tal, pode ser iniciado em diversos locais da célula. Na realidade, cada organito

possui sensores que lhes permitem aferir a homeostasia dos processos bioquímicos que

regula. Sempre que a homeostasia esteja comprometida, de uma forma que coloque em

risco a sobrevivência da célula, os organitos comunicam sinais de morte celular entre si,

para que a célula seja removida, sem comprometer as células adjacentes. Assim, a

descodificação da complexa rede de sinalização intracelular, responsável pela morte

celular programada do tipo apoptótico, pode representar a chave para retardar, ou até

prevenir, a neurodegenerescência associada à AD. O conceito de que a morte celular não

é meramente aleatória e caótica, mas antes pode seguir um processo programado e

regulado, representou um estímulo para desenvolver o trabalho apresentado nesta tese.

Resumo

viii

Sabendo que a aplicação aguda de formas solúveis do péptido Aβ mimetiza a

toxicidade observada na AD, estudaram-se os efeitos do Aβ na morte celular.

Inicialmente, procurámos entender o papel da agregação do Aβ na morte celular e, para

isso, tirámos partido do facto de os péptidos mutantes de Aβ terem características

particulares de agregação, para além de uma afinidade selectiva para as células

vasculares. Assim, usando células primárias de endotélio vascular humano,

determinámos que o péptido mutante AβE22Q apresenta um estado conformacional

intermédio, entre o observado para a formas nativas de Aβ40 e Aβ42. Além disso,

mostrámos que, ao contrário das formas nativas, o péptido mutante AβE22Q activa a

apoptose nestas células através da translocação da Bax para a mitocôndria, com a

consequente libertação do citocromo c para o citosol. Este efeito conseguiu ser revertido

com a pré-incubação das células com um agente anti-apoptótico, o ácido tauro-

ursodesoxicólico (TUDCA). Contudo, apesar do seu potente efeito anti-apoptótico, o

TUDCA não interferiu directamente com as propriedades de agregação características do

Aβ in vitro. Estes resultados demonstram que a agregação, por si só, não é suficiente para

induzir toxicidade. Sugerem, antes, que os efeitos tóxicos são devidos a conformações

específicas que os péptidos adquirem e que estas, sim, conseguem danificar a célula e

despoletar a via mitocondrial de apoptose.

Tendo em conta que durante o processo de agregação do péptido Aβ, este forma

espécies desnaturadas e que o retículo endoplasmático (ER) é o organito responsável pela

conformação espacial das proteínas, estudámos em que medida o Aβ solúvel afecta o ER.

A exposição de células PC12 do tipo neuronal ao Aβ provocou uma resposta imediata de

sinalização molecular, que envolveu a caspase-12, independentemente da activação da via

do stresse do ER e que culminou na morte celular. Assim, observaram-se níveis proteicos

diminuídos dos marcadores de stresse do ER, incluindo o GRP94, ATF-6α, CHOP e

eIF2α. Este efeito mostrou-se independente de mecanismos de degradação proteica,

mediados pelo protessoma ou pela autofagia, tendo sido parcialmente contrariado pelo

TUDCA. Por sua vez, com a inibição das calpaínas conseguiu-se bloquear a activação da

caspase-12 e a diminuição do ATF-6α. Muito interessante foi, ainda, o facto de ser

possível bloquear a diminuição dos níveis da proteína GRP94 através da inibição da via

secretória de proteínas, pela utilização da geldanamicina e brefeldina. Este mecanismo de

sinalização pelo péptido Aβ coloca o GRP94 no meio extracelular, abrindo uma

oportunidade única para o desenvolvimento de novas estratégias terapêuticas.

Resumo

ix

Para finalizar, tendo em conta que a cinase do terminal amínico da proteína c-Jun

(JNK) é um sensor primordial do stresse celular, activado na presença do Aβ, bem como

o facto de a JNK activar a caspase-12 e poder ser activada por vias despoletadas no ER,

realizámos estudos para compreender o envolvimento desta cinase no contexto de

exposição de células PC12 do tipo neuronal ao péptido Aβ solúvel. Os nossos resultados

mostraram que a JNK é indispensável à apoptose induzida pelo Aβ e que está localizada

no núcleo, no seu pico de actividade. Além disso, através do silenciamento da JNK,

confirmámos que a caspase-2 é um alvo específico da JNK, quando activada pelo Aβ.

Demonstrámos, ainda, que a caspase-2 activa está localizada no complexo de Golgi,

através da clivagem do seu substrato específico, a golgina 160, que é uma proteína

estrutural do complexo de Golgi. Através da pré-incubação com TUDCA, conseguiu-se,

também aqui, reverter a activação da JNK e a sua translocação para o núcleo, tendo sido

ainda possível reverter a activação da caspase-2. Estes resultados sugerem que o

complexo de Golgi possa desempenhar um papel fundamental na descodificação da

sinalização de morte induzida pelo péptido Aβ.

Os estudos incluídos nesta tese enfatizam a noção de que a AD é uma patologia

complexa, que envolve uma rede de sinalização entre vários organitos celulares, para

além de contribuirem para uma melhor compreensão da comunicação subcelular, no que

respeita aos efeitos tóxicos desencadeados pelo Aβ. A caracterização destas vias de

sinalização e de formas de as modular, abrem novas perspectivas para a regulação da

disfunção celular e degenerescência induzidas pelo Aβ.

Palavras chave: Ácido tauro-ursodesoxicólico; Apoptose; GRP94; JNK; Organitos

celulares; Péptidos β amilóide

Abstract

Abstract

xiii

Alzheimer‟s disease (AD) is a devastating neurological disorder characterized by

massive loss of neurons and disruption of synaptic function. Cell death appears to

involve several organelles, and decoding this complex signaling network of inter-

organellar cross-talk might represent the key for delaying or preventing AD.

We first compared aggregation/fibrillization properties, secondary structure, and

cell death pathways of wild-type amyloid β (Aβ) peptides and AβE22Q Dutch variant,

both in the presence and absence of anti-apoptotic tauroursodeoxycholic acid (TUDCA).

Our data dissociate the apoptotic properties of Aβ peptides in human cerebral endothelial

cells from their distinct mechanisms of aggregation in vitro, while confirming the

perspectives for modulation of amyloid-induced mitochondrial apoptosis by TUDCA.

Next, we investigated the ability of the endoplasmic reticulum (ER) to sense

stress directly caused by the Aβ toxic core. Aβ exposure triggered an early signaling

response by the ER, and caspase-12-mediated apoptosis in PC12 neuronal-like cells,

independently of the ER-stress pathway. Indeed, ER stress markers, including GRP94,

ATF-6α, CHOP and eIF2α were strongly donwregulated by Aβ, in a protein degradation-

independent manner, and partially restored by TUDCA. Calpain inhibition prevented

caspase-12 activation and ATF-6α downregulation. However, GRP94 downregulation

was associated with protein secretion, which in turn was partially rescued by inhibition of

the secretory pathway. Thus, Aβ triggers an ER response beyond the usual ER stress.

Finally, we further investigated the role of the c-Jun N-terminal kinase (JNK) as

early stress sensor of Aβ toxicity that is activated by ER pathways. We reported that JNK

acted as the proximal stress sensor, which translocated to the nucleus and activated

caspase-2 in PC12 neuronal-like cells. Caspase-2 then cleaved golgin-160, suggesting a

unique signaling through the Golgi complex. Importantly, TUDCA modulated the

JNK/caspase-2 apoptotic pathway triggered by Aβ.

In conclusion, these studies contribute to the understanding of the complex sub-

cellular communication involved in Aβ toxicity. Further characterization of these

signaling pathways and exact targets are likely to provide new perspectives for

modulation of amyloid-induced cellular dysfunction and degeneration.

Keywords: Amyloid-β peptides; Apoptosis; Cell organelles; GRP94; JNK;

Tauroursodeoxycholic acid

xv

Acknowledgements

Não poderia deixar de começar por agradecer à pessoa que possibilitou que este

trabalho fosse desenvolvido, a Professora Cecília Rodrigues. Será de todo impossível

detalhar uma relação profissional muito frutuosa que durou quatro anos. Contudo, sou

impelido a enfatizar uma característica impressionante do seu carácter: o seu sentido de

missão! A sua entrega incondicional, aliada à enorme capacidade de trabalho, são traços

marcantes da sua pessoa. Posso por isso testemunhar, o quão confortável é para quem está

a fazer um doutoramento, saber que se tem sempre a quem recorrer, independentemente

dos dias e horas! Assim, cara Professora, como o seu trabalho é feito de partilha, na

investigação que faz, e dádiva, nos conhecimentos que transmite como professora,

gostava antes de partir de lhe deixar o meu bem-haja e pensar que também lhe deixo a si,

alguma coisa de mim.

The choice to do a PhD is much more than a simple job for some years. It is a

journey that changes the rest of our lives. In many years from now, I am sure I will

remember the particular moment of my journey in the New York University, at Professors

Agueda Rostagno and Jorge Ghiso laboratory. I was received in the very best warm way

and treated as one of the laboratory members. Thank you for letting me be part of the

excellence of your laboratory, and also for your humane characteristics that made you so

special. I also want to thank Silvia in name of all the laboratory members for making my

staying in NY a great time. I could not finish without expressing the feelings I nurture for

Milton and André. You were my support, my friends, my "homy" place. You are

responsible for the "New Yorker" feeling that I still have today, and I have no words to

express how thankful I am.

Para os meus queridos companheiros de laboratório, deixo mais do que um

agradecimento. Partilho convosco o sentimento que me percorre quando fecho os olhos e

penso em cada um de vós: Felicidade! "Aquele sorriso maroto" invade-me o rosto e leva-

Acknowledgements

xvi

me a viajar pelas nossas memórias. Foram anos intensos que vivemos juntos e nos quais

fomos actores principais de momentos memoráveis, dos quais não me poderei esquecer: o

apoio incondicional que a Rita e a Filipa me deram em todas as situações no decorrer

desta maratona; a minha companheira de percurso, a Márcia; a preocupação carinhosa e

subtil da Susana; as brincadeiras com o Rui; as gargalhadas com a Joana; as conversas

com a Daniela; a "família" que ganhei com o Pedro; e o ar fresco que a "geração"

Benedita, Duarte e Joana Xavier trouxeram, tendo a certeza que irão dar continuidade a

este laboratório duma forma ainda melhor do que aquela que já foi conseguida. Quero

ainda agradecer à Professora Isabel Moreira da Silva pela forma atenciosa e afável com

que sempre me tratou. Quero também agradecer às Professoras Elsa Rodrigues, Maria

João Gama e Margarida Castro Caldas, bem como aos membros do laboratório Inês,

Maria, Andreia e Miguel pela sempre boa disposição que vos caracteriza e por todas as

discussões científicas partilhadas.

Agradeço à minha família Coimbrã. À Universidade de Coimbra na pessoa dos

professores da Faculdade de Farmácia, o meu bem-haja! Aos meus amigos Hugo,

"Salgados", Ruivo, Rodri, Batista e André, … quero simplesmente abraçar-vos para sentir

o calor da vossa amizade que aqui não consigo descrever. Como sabeis, o que criamos em

Coimbra não se escreve, vive-se!

Agradeço ao meu irmão Miguel, ao Guilherme e Susana por serem um pilar

fundamental da minha vida. Mi, a fé e a confiança que depositas em mim, empurram-me

para lugares que não existem e que ainda não foram tocados! Obrigado por me fazeres

sentir um ser especial.

Aos meus pais, estou eternamente grato por terem semeado dentro de mim os

valores, princípios e moral do meu carácter. Hoje, em tudo o que faço, colho o fruto do

vosso trabalho e penso sempre na bênção que tive e tenho de vos ter como meus pais.

Por último, agradeço a quem reconheço o trabalho mais árduo, à minha mulher

Charlene. Agradeço todos os dias que passamos juntos; o sofrimento que suportaste para

eu prosseguir com o meu sonho; a harmonia que me proporcionas; a felicidade que

despertas em mim; a minha personalidade intensa que aguentas com um sorriso; a alegria

que me dás por chegar a casa; … todo o equilíbrio que me ofereces. Por tudo isto e muito

mais, não consigo expressar em palavras a gratidão que tenho para contigo. Só sentindo a

força do brilho dos meus olhos quando te vejo!

xvii

Abbreviations

AD Alzheimer‟s disease

AICD amyloid precursor protein intracellular domain

APOE-4 4 allele of the apolipoprotein E

APP amyloid precursor protein

ASK1 apoptosis signal-regulating kinase 1

ATF-6 activating transcription factor 6

Aβ amyloid β

AβE22Q mutation in which Gln replaces Glu at position 22 of amyloid β

Bak Bcl-2 antagonist/killer

BAR bifunctional apoptosis regulator

BAX Bcl-2-associated X protein

Bcl-2 B-cell leukemia/lymphoma 2

Bcl-xL B-cell leukemia/lymphoma extra long

BI1 BAX inhibitor 1

C99 β-cleaved C-terminal fragment of amyloid precursor protein

Ca2+

calcium

CHOP CCAAT/enhancer binding protein homologous protein

CTF amyloid precursor protein C-terminal fragment

eIF2α eukaryotic translation initiation factor 2α

ER endoplasmatic reticulum

ERAD endoplasmic reticulum-assisted degradation

FADD Fas associated death domain

GC Golgi complex

GDV granulovacuolar degeneration bodies

GRP78/Bip 78-kDa glucose-regulated protein

GRP94 94-kDa glucose-regulated protein

GSK-3β glycogen synthase kinase 3β

HCHWA- D hereditary cerebral hemorrhage with amyloidosis- Dutch type

Abbreviations

xviii

hsp heat shock protein 90

IRE1 inositol-requiring kinase 1

JIP c-Jun N-terminal kinase-interacting protein

JNK c-Jun N-terminal kinase

MAPK mitogen-activated protein kinase

MVBs multivesicular bodies

NFTs neurofibrillary tangles

NSR nuclear steroid receptor

PCD programmed cell death

PERK double stranded ribonucleic acid-activated protein kinase-

like endoplasmic reticulum kinase

PI3K phosphatidyl inositol 3-kinase

PS1 presenilin-1

PS2 presenilin-2

ROS reactive oxygen species

sAPP soluble amyloid precursor protein

TRAF2 tumor necrosis factor receptor-associated factor 2

TRAIL-R1 tumor necrosis factor-related apoptosis inducing ligand receptor 1

TUDCA tauroursodeoxycholic acid

TUNEL terminal transferase dUTP nick-end labeling

UDCA ursodeoxycholic acid

UPR unfolded protein response

Publications

The present thesis is based on work that has been published, or submitted for

publication, in international peer-reviewed journals:

Viana RJ, Nunes AF, Castro RE, Ramalho RM, Meyerson J, Fossati S, Ghiso J,

Rostagno A, Rodrigues CM. Tauroursodeoxycholic acid prevents E22Q Alzheimer's Aβ

toxicity in human cerebral endothelial cells. Cellular and Molecular Life Sciences 2009;

66: 1094-1104.

Viana RJ, Ramalho RM, Nunes AF, Steer CJ, Rodrigues CM. Modulation of amyloid-β

peptide-induced toxicity through inhibition of JNK nuclear localization and caspase-2

activation. Journal of Alzheimer's Disease 2010; 22: 557-568.

Viana RJ, Steer CJ, Rodrigues CMP. Amyloid-β peptide-induced secretion of

endoplasmic reticulum chaperone glycoprotein GRP94 in PC12 neuronal-like cells. 2010

(submitted).

The following manuscripts have also been published during the Ph.D. studies:

Ramalho RM, Viana RJ, Low WC, Steer CJ, Rodrigues CM. Bile acids and apoptosis

modulation: an emerging role in experimental Alzheimer's disease. Trends in Molecular

Medicine 2008; 14: 54-62.

Ramalho RM, Viana RJ, Castro RE, Steer CJ, Low WC, Rodrigues CM. Apoptosis in

transgenic mice expressing the P301L mutated form of human tau. Molecular Medicine

2008; 14: 309-317.

http://www.ncbi.nlm.nih.gov/pubmed/19189048









Publications

xx

Santos DM, Santos MM, Viana RJ, Castro RE, Moreira R, Rodrigues CM. Naphtho[2,3-

d]isoxazole-4,9-dione-3-carboxylates: potent, non-cytotoxic, antiapoptotic agents.

Chemico-Biological Interactions 2009; 180: 175-182.

Amaral JD, Viana RJ, Ramalho RM, Steer CJ, Rodrigues CM. Bile acids: regulation of

apoptosis by ursodeoxycholic acid. Journal of Lipid Research 2009; 50: 1721-1734.

Viana RJ, Fonseca MB, Ramalho RM, Nunes AF, Rodrigues CM. Organelle stress

sensors and cell death mechanisms in neurodegenerative diseases. CNS & Neurological

Disorders Drug Targets 2010; 9: 679-692.







Table of Contents

Resumo ....................................................................................................................... v

Abstract ..................................................................................................................... xi

Acknowledgements .................................................................................................. xv

Abbreviations ......................................................................................................... xvii

Publications ............................................................................................................. xix

1 General Introduction ............................................................................................... 1

1.1 Alzheimer's disease ................................................................................. 3

1.1.1 Epidemiology .......................................................................................... 3

1.1.2 Molecular neuropathology ...................................................................... 4

1.1.3 Risk factors ............................................................................................. 7

1.1.4 Amyloid precursor protein .................................................................... 13

1.2 Cell death mechanisms ......................................................................... 26

1.2.1 Death receptor and mitochondrial pathways of apoptosis .................... 28

1.2.2 Role of apoptosis in Alzheimer‟s disease ............................................. 30

1.2.3 Role of organelle dysfunction in Alzheimer‟s disease ......................... 35

1.2.4 Inhibition of neuronal apoptosis by bile acids ...................................... 40

Objectives ............................................................................................................ 45

2 Tauroursodeoxycholic acid prevents E22Q Alzheimer‟s amyloid β toxicity in

human cerebral endothelial cells .......................................................................... 47

2.1 Abstract ................................................................................................. 49

2.2 Introduction ........................................................................................... 50

2.3 Materials and methods .......................................................................... 52

Table of Contents

xxii

2.3.1 Peptide synthesis ................................................................................... 52

2.3.2 Peptide aggregation .............................................................................. 52

2.3.3 Peptide structural analysis .................................................................... 53

2.3.4 Culture of human brain microvascular endothelial cells ...................... 54

2.3.5 Induction of apoptosis ........................................................................... 54

2.3.6 Measurement of cell death .................................................................... 54

2.3.7 Immunocytochemistry .......................................................................... 55

2.3.8 Bax translocation and cytochrome c release ......................................... 56

2.3.9 Statistical analysis ................................................................................. 57

2.4 Results ................................................................................................... 57

2.4.1 TUDCA does not affect Aβ peptide aggregation and secondary

structure ................................................................................................ 57

2.4.2 TUDCA inhibits AβE22Q-induced apoptosis in brain microvascular

endothelial cells .................................................................................... 59

2.4.3 TUDCA prevents AβE22Q-induced mitochondrial Bax translocation

and cytochrome c release ...................................................................... 61

2.5 Discussion ............................................................................................. 63

3 Amyloid β peptide-induced secretion of endoplasmic reticulum chaperone

glycoprotein GRP94 in PC12 neuronal-like cells ................................................ 69

3.1 Abstract ................................................................................................. 71

3.2 Introduction ........................................................................................... 72


3.3.1 Cell culture and culture conditions ....................................................... 74


3.3.3 Immunoblotting .................................................................................... 75

3.3.4 RNA isolation and RT-PCR ................................................................. 76

3.3.5 Heat shock protein GRP94 ELISA assay ............................................. 76


Table of Contents

xxiii

3.4 Results ................................................................................................... 77

3.4.1 Aβ-induced apoptosis is mediated by caspase-12 activation and

modulated by TUDCA in PC12 neuronal-like cells ............................. 77

3.4.2 Aβ-induced, caspase-12-mediated cell death is independent of ER

stress ..................................................................................................... 79

3.4.3 ATF-6α and GRP94 downregulation is independent of protein

degradation ........................................................................................... 80

3.4.4 GRP94 downregulation is independent of Ca2+

deregulation ............... 82

3.4.5 Protein secretion inhibitors block GRP94 downregulation .................. 83

3.5 Discussion ............................................................................................. 85

4 Modulation of amyloid β peptide-induced toxicity through inhibition of JNK

nuclear localization and caspase-2 activation....................................................... 89

4.1 Abstract ................................................................................................. 91

4.2 Introduction ........................................................................................... 92


4.3.1 Cell culture and induction of apoptosis ................................................ 93


4.3.3 Inhibition of JNK pathway ................................................................... 94

4.3.4 Immunoblotting .................................................................................... 95

4.3.5 Immunocytochemistry .......................................................................... 95

4.3.6 Short interference-mediated silencing of the jnk gene ......................... 96

4.3.7 Expression of Golgin-160 cleavage products ....................................... 96


4.4 Results ................................................................................................... 97

4.4.1 Aβ-induced apoptosis is mediated by JNK activation and modulated

by TUDCA in PC12 neuronal-like cells ............................................... 97

4.4.2 p-JNK localizes to the nucleus and mediates Aβ-induced caspase-2

activation ............................................................................................. 100

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xxiv

4.4.3 Aβ-induced caspase-2 activation mediates cell death via the Golgi

complex ............................................................................................... 102

4.5 Discussion ........................................................................................... 104

5 Concluding Remarks .......................................................................................... 109

References .............................................................................................................. 117

Table of Contents

xxv

Figures

Figure 1.1. Processing of APP ................................................................................ 6

Figure 1.2. Tau structure and function ................................................................... 8

Figure 1.3. Structure of APP ................................................................................ 11

Figure 1.4. Intracellular trafficking of APP.......................................................... 15

Figure 1.5. Oxidative stress and mitochondrial failure ........................................ 17

Figure 1.6. Synaptic dysfunction in AD ............................................................... 19

Figure 1.7. The regulation of intracellular Ca2+

compartmentalization ............... 21

Figure 1.8. UPR events and connection to the cell death machinery. .................. 24

Figure 1.9. Pathways of apoptosis ........................................................................ 29

Figure 1.10. Schematic overview of organelle dysfunction and cell death

mechanisms ........................................................................................ 35

Figure 1.11. Proposed mechanisms of UDCA and TUDCA inhibition of

apoptosis ............................................................................................ 42

Figure 2.1. Structural studies of wild-type and Dutch-variant Aβ peptides ......... 58

Figure 2.2. AβE22Q-induced apoptosis in HCEC is inhibited by TUDCA. ........ 60

Figure 2.3. AβE22Q-induced Bax translocation and cytochrome c release in

HCEC ................................................................................................ 61

Figure 2.4. TUDCA inhibits AβE22Q-induced cytochrome c release in

HCEC. ............................................................................................... 62

Figure 2.5. TUDCA inhibits AβE22Q-induced cytochrome c release in

HCEC ................................................................................................ 63

Figure 3.1. TUDCA inhibits cell death induced by Aβ, brefeldin and

thapsigargin in PC12 neuronal cells. ................................................. 78

Figure 3.2. TUDCA modulates Aβ-induced caspase-12 processing in PC12

neuronal cells. .................................................................................... 79

Figure 3.3. Aβ-induced apoptosis in PC12 neuronal cells is independent of

ER stress.. .......................................................................................... 80

Figure 3.4. Aβ-induced downregulation of ATF-6α and GRP94 in PC12

neuronal cells is independent of degradation mechanisms. ................ 81

Figure 3.5. Aβ-induced downregulation of GRP94 in PC12 neuronal cells

is independent of Ca2+

deregulation ................................................... 83

Table of Contents

xxvi

Figure 3.6. Aβ-induced downregulation of GRP94 in PC12 neuronal cells

is blocked by protein secretion inhibitors. ......................................... 84

Figure 4.1. TUDCA inhibits apoptosis induced by Aβ in PC12 neuronal cells. .. 97

Figure 4.2. TUDCA modulates Aβ-induced p-JNK and p-c-Jun levels in

PC12 neuronal cells. .......................................................................... 98

Figure 4.3. Aβ induces p-JNK translocation to the nucleus in PC12 neuronal

cells. ................................................................................................. 100

Figure 4.4. TUDCA modulates Aβ-induced active caspase-2 levels in PC12

neuronal cells. .................................................................................. 101

Figure 4.5. Silencing of JNK inhibits Aβ-induced caspase-2 activation in

PC12 neuronal cells. ........................................................................ 102

Figure 4.6. Caspase-2 localizes to the Golgi complex and cleaves golgin-160

in Aβ-induced cell death in PC12 neuronal cells. .......................... 103

1 General Introduction

General Introduction

3

1.1 Alzheimer's disease

Alzheimer‟s disease (AD) is a fatal progressive neurodegenerative illness, and

the most common form of dementia. It is, therefore, a fundamental disorder of cognitive

awareness, integrating reasoning, abstraction, language, and memory, one of the defining

components of human consciousness (Nussbaum and Ellis, 2003). This condition was

first described by Alois Alzheimer in 1906. He presented a clinical/pathological report of

a 51-year old woman, Auguste Deter, who had died of what came to be termed “dementia

praecox.” At autopsy, it was noted that there were extracellular deposits, termed

“plaques”, and intracellular deposits or “tangles”, in the neocortex of the brain. Tangles

are a common hallmark of brain injury and are seen after a variety of other insults, such

as stroke and head trauma. Hence, their presence does not give any clue as to the etiology

of the cognitive decline seen in AD. On the other hand, the plaque deposits, although

also seen with other injuries, do appear to bear a relationship to the underlying cause of

the dementia (Holtzman, 2010).

1.1.1 Epidemiology

The onset of AD is inevitably followed by increasing mental and physical

incapacitation, including loss of ability to look after one‟s own daily needs, followed by

institutionalization and death. AD is the most common cause (60-80%) of dementia

worldwide. The prevalence of AD is less than 1% in individuals aged 60-64 years, but

the probability of developing the disorder increases with age. The estimated rates of

occurrence in various age groups are as follows: 65-74 years, 2.5%; 75-79 years, 4%; 80-

84 years, 11%; and 85-93 years, 24%. It has been estimated that 4.6 million new cases

will appear every year, at a rate of one new case every 7 seconds. With life expectancy

increasing, the numbers are predicted to double every 20 years to 81.1 million by 2040

(Ferri et al., 2005). In Portugal, there are ~ 60,000 patients with AD, although this may

represent an under estimation, given the difficulty of diagnosis.

AD is fourth in the list of leading causes of death in industrialized societies,

preceded only by heart disease, cancer, and stroke. It affects individuals of all races and

ethnic groups, occurring slightly more commonly in women than in men. Although AD

is emerging as the most prevalent and socially disruptive illness of aging populations, its

causes and cure remain enigmas (Aguzzi and O'Connor, 2010).

Chapter 1

4

1.1.2 Molecular neuropathology

AD is characterized by two neuropathological hallmarks, including extracellular

deposits, known as amyloid or “senile” plaques, and intracellular neurofibrillary tangles

(NFTs) (Spillantini and Goedert, 1998). In addition to these deposits, the AD brain is

characterized by a dramatic loss of neurons and synapses in many areas of the central

nervous system, particularly in regions involving higher-order cognitive functions, such

as learning and memory. Because memory loss may occur by other causes, definitive

diagnosis of AD requires postmortem examination of the brain, which must contain

sufficient numbers of plaques and tangles to qualify as affected by AD (Braak and Braak,

1998; Dickson, 1997).

Brain regions, such as the basal forebrain and hippocampus that exhibit plaques,

typically possess a reduced number of synapses. Neurites found associated with the

plaques are also often damaged, suggesting a toxic effect for amyloid β (Aβ) peptide, the

major constituent of amyloid plaques (Mattson, 2004). In addition, the levels of many

neurotransmitters are greatly reduced, including serotonin, noradrenaline, dopamine,

glutamate, and especially acetylcholine. Reduced neurotransmitter levels are thought to

be responsible for the broad and profound clinical symptoms of AD (Nussbaum and Ellis,

2003).

1.1.2.1 Amyloid plaques

Amyloid plaques are primarily composed of a 4.3-kDa amyloid peptide, first

isolated from the brains of patients with AD in 1984. The Aβ peptide is a 39-43 amino

acid sequence that forms extraneuronal aggregates with a fibrillar, β-pleated structure.

Moreover, Aβ peptide accumulation can be observed both in brain and blood vessels.

Aβ is cleaved from the amyloid precursor protein (APP) (Fig. 1.1), a 695-770

amino acid transmembrane protein found in virtually all peripheral and brain cells (Selkoe

and Schenk, 2003). Although there is no conclusive evidence of the function of APP, an

increasing body of data suggests its involvement in regulating neurite outgrowth, synaptic

plasticity, and cell adhesion (Mattson, 2004).

APP is normally cleaved within the Aβ domain by α-secretase, liberating a

soluble N-terminal fragment (sAPPα) and a membrane-bound C-terminal fragment (C83).

Alternatively, APP can be cleaved by β-secretase at the N-terminus of the Aβ domain,


5

yielding s-APPβ and C99. The latter membrane-bound fragment, then undergoes

intramembrane cleavage by γ-secretase at the C-terminus of Aβ, resulting in the liberation

of Aβ into the cell (Yamazaki and Ihara, 1998). The secretion of Aβ follows, allowing

the peptide to participate in extracellular aggregation and to become incorporated into

growing plaques.

The β-site of APP cleaving enzyme (BACE1) is responsible for the β-secretase

activity; γ-secretase is composed of four essential subunits, including presenilin-1 (PS1)

or presenilin-2 (PS2), together with nicastrin, anterior pharynx-defective 1 (APH-1), and

presenilin enhancer 2 (PEN-2) (Thinakaran and Koo, 2008). The γ-secretase complex

cleaves at multiple sites within the transmembrane domain of APP, generating Aβ

peptides ranging in length from 38 to 42 residues (Selkoe and Wolfe, 2007). Nearly 90%

of secreted Aβ ends at residue 40, giving Aβ40, whereas Aβ42 accounts for only less than

10%, and peptides ending at residues 38 are minor components (Thinakaran and Koo,

2008). Notably, the pattern and distribution of these species varies among the different

topographical lesions. Parenchymal deposits consist of Aβ42 as the major component,

whereas vascular Aβ, particularly in the large leptomeningeal vessels, is primarily

composed of Aβ40 species, organized in large concentric sheets and replacing the smooth

muscle cell layer (Roher et al., 1993). Amyloid associated with arterioles and small

cortical arteries contains a mixture of Aβ40 and Aβ42, while deposits affecting the

capillary network are mainly composed of Aβ42 (Kokjohn and Roher, 2009). The

reasons for this selectivity as well as its importance for the pathogenesis of the disease

remain largely unclear (Rostagno et al., 2010).

The term “soluble Aβ” is generally applied either to newly generated, cell-

secreted Aβ or to that fraction of tissue or synthetic Aβ that is taken into the aqueous

phase of a non-detergent containing extraction buffer. “Misfolded” and “aggregated” Aβ

are terms used to describe very early, nonspecific changes in Aβ folding states or

solubility states, respectively (e.g., aggregated Aβ solutions usually scatter light to a

greater extent than do solutions of soluble Aβ). “Oligomeric” Aβ refers to peptide

assemblies with limited stoichiometry (e.g., dimers, trimers, etc.), while protofibrils are

structures of intermediate order between aggregates and fibrils (Fig. 1.1) (Gandy, 2005).

Chapter 1

6

Querfurth and LaFerla, 2010

Figure 1.1. Processing of APP. (A) Non-amyloidogenic processing of APP, with sequential

cleavage by membrane-bound α- and γ-secretase. α-secretase cleaves within the Aβ sequence,

thus preventing the generation of intact Aβ peptide. sAPPα ectodomain is secreted to

extracellular milieu and C83 is released. C83 is then processed by γ-secretase, releasing

extracellular p3 (3 kDa) fragment. Alternatively, amyloidogenic processing starts by β-secretase

cleavage at the N-terminus of Aβ, releasing a shorter sAPPβ and C99 fragments. The C99

fragment is subsequently cleaved within the transmembrane domain by γ-secretase to originate

Aβ and AICD. AICD is targeted to the nucleus, signaling transcription activation. Soluble Aβ is

a hydophobic peptide that is prone to aggregation. (B) Protofibrils (upper left) and annular or

porelike profiles (lower left) are transient structures observed during in vitro formation of mature

amyloid fibrils. In the right panel, self-association of 2 to 14 Aβ monomers into oligomers is

dependent on concentration (right, left immunoblot). In the right immunoblot, oligomerization is

promoted by oxidizing conditions (lane 2) and divalent metal conditions (lane 3). AICD, APP

intracellular domain; GPI, glycophosphatidylinositol.


7

Numerous mutations have been identified in the gene encoding APP, which alter

its expression or processing (Rocchi et al., 2003). These mutations are associated with

early onset AD and are implicated in the autosomal dominant form of the disease, which

constitutes ~ 5% of all cases. Patients with Down‟s syndrome, who have three copies of

the APP gene, show diffuse Aβ deposits as early as in the second decade of life. These

eventually develop into mature neuritic plaques that are indistinguishable from those

found in the brains of patients with AD.

1.1.2.2 Neurofibrillary tangles

The chief component of intracellular NFTs is tau, a microtubule-associated

protein abundant in six different isoforms in the adult brain. In AD, highly

phosphorylated and glycosylated tau protein forms paired, helically wound fragments

(paired helical filaments), ~ 10 nm in diameter, which associate to give insoluble tangles

in nerve cell bodies and dendrites (Fig. 1.2) (Spillantini and Goedert, 1998). Tangles are

a hallmark of many dementia-related diseases, including corticobasal ganglionic

degeneration, frontotemporal dementia, myotonic dystrophy, Niemann-Pick disease, and

progressive supranuclear palsy. The wide variety of neurological disorders in which

NFTs occur suggests that paired helical filament formation is a nonspecific marker of

certain types of neuronal injury.

1.1.3 Risk factors

Besides ageing, which is the most obvious risk factor for AD, no specific

environmental toxin has been found to be consistently associated with the disease, and

there have been no randomized clinical trials as yet to support any specific dietary

intervention. Some evidence suggests that dietary intake of homocysteine-related

vitamins, vitamin B12 and folate; antioxidants, such as vitamin C and E; unsaturated fatty

acids; and also moderate alcohol intake, especially wine, could reduce the risk of AD.

Epidemiological studies point to depression, traumatic head injury and cardiovascular and

cerebrovascular factors, including cigarette smoking, midlife high blood pressure,

obesity, hypercholesterolaemia, atherosclerosis, coronary heart disease and diabetes as

increasing disease risk, while anti-inflammatory medications seem to reduce risk. Some

studies even suggest a beneficial role of psychosocial factors as though, higher education,

physical exercise and mental activity (reviewed in Selkoe and Schenk, 2003). Such

Chapter 1

8

reports may point to a role of previously unconsidered pathways in the etiology of the

disease, but the mechanistic interpretation of retrospective epidemiological studies is

challenging (Citron, 2010).


Figure 1.2. Tau structure and function. Tau is a highly soluble microtubule-binding protein

that contributes to microtubule assembly and cohesion. Four repeat sequences (R1-R4) make up

the microtubule-binding domain (MBD) of tau. Tau is abnormally hyperphosphorylated in AD by

numerous kinases, leading to the formation of NFTs. Normal phosphorylation of tau occurs on

serine (S; inset) and threonine (T; inset) residues, numbered according to their position in the full

tau sequence. Between the N‑terminal portion of the protein (projection domain) and the MBD

lies a basic proline-rich region (P); these amino acids are phosphorylated by GSK-3β, CDK5 and

its activator subunit p25, or MAPK. Non proline-directed kinases, such as Akt, Fyn, PKA,


9

CaMKII, and MARK are also shown. By activating these kinases, certain inflammatory cytokines

can also trigger tau hyperphosphorylation. Hyperphosphorylated sites specific to paired helical

filament tau in AD tend to flank the MBD. Excessive kinase, reduced phosphatase activities, or

both cause hyperphosphorylated tau. Hyperphosphorylation of tau, particularly that mediated by

MARK, CDK5 and GSK-3β destabilizes microtubules, causing impairments in axonal transport

and neuronal dysfunction. CDK5, cyclin-dependent kinase 5; MAPK, mitogen-activated protein

kinase; PKA, protein kinase A; CaMKII, calcium-calmodulin protein kinase 2; MARK,

microtubule affinity-regulating kinase. From Querfurth and LaFerla, 2010.

1.1.3.1 Genetics

Family history is the second-greatest risk factor for AD after age, and the

increasing understanding of its genetics has been vital to the knowledge of the pathogenic

mechanisms that cause the disease. Genetically, AD is complex and heterogeneous and

appears to follow an age-related dichotomy: rare and highly penetrant early-onset familial

AD mutations in different genes are transmitted in an autosomal dominant fashion, and

late-onset AD that represents the vast majority of the cases and is absent of obvious

familial segregation (Bertram and Tanzi, 2005).

1.1.3.1.1 Sporadic disease

The designation of late-onset AD is used when the onset of the disease occurs

at 65 years or older, and represents ≥ 95% of all AD cases. Although genetic analysis of

segregation and twin studies have suggested a major role of genetic factors in this form of

AD (Mayeux et al., 1991), until now, only one factor has been effectively established, the

4 allele of the apolipoprotein E (APOE-4) gene on chromosome 19q13 (Schmechel et

al., 1993; Strittmatter et al., 1993). In contrast to all other association-based observations

in AD, the risk outcome of APOE-4 has been consistently replicated by different

laboratories that developed several studies transversal to numerous ethnic groups (for

meta-analysis, see Farrer et al., 1997). The APOE-4 genotype, which occurs in 16% of

the population (Roses, 1997) is the major susceptibility gene identified for both rare

early-onset and sporadic late-onset AD (Coon et al., 2007). In addition to the increased

risk by 4-allele, it has also been reported in several studies a weak, albeit significant,

protective effect for the minor allele, 2. Contrasting to mutations in the known early-

onset familial AD genes, APOE-4 is neither necessary nor sufficient to cause AD, since

Chapter 1

10

many 4-positive individuals do not develop AD, and many AD patients do not carry

these genes. Instead, APOE-4 operates as a genetic risk modifier by decreasing the age

of onset in a dose-dependent manner. Despite its long-known and well-established

genetic association, the molecular effects of APOE-4 in AD pathogenesis are not yet

fully understood, but are expected to include Aβ-aggregation/clearance and/or cholesterol

homeostasis (Bertram and Tanzi, 2005).

1.1.3.1.2 Familial disease

Early-onset familial AD (FAD) represents only a small fraction of ≤ 5% of all

AD cases. This designation is used when onset ages are younger than 65 years, showing

autosomal dominant transmission within affected families. To date, more than 160

mutations in 3 genes have been reported to cause early-onset FAD. These genes include

the APP on chromosome 21 (Goate et al., 1991), PS1 on chromosome 14 (Sherrington et

al., 1995), and PS2 on chromosome 1 (Levy-Lahad et al., 1995; Rogaev et al., 1995).

PS1 represents the most frequently mutated gene and accounts for the majority of AD

cases with onset prior to age of 50 (Bertram and Tanzi, 2005). Of the genes directly

related to the development of familial AD, PS1 and PS2 mutations affect the levels of

Aβ42 production, whereas nucleotide changes within the APP molecule have a

differential effect depending on the location of the mutated residue (Fig. 1.3).

Alterations in Aβ domain that result from amino acid replacement in regions

adjacent to β-secretase cleavage site change the kinetics of APP enzymatic processing.

As a consequence, increased production of Aβ42 and Aβ40 is observed, maintaining the

ratio Aβ42/Aβ40 unaffected, which manifests in a classic early-onset AD clinical

phenotype (Revesz et al., 2009). Two examples are Swedish double mutation

(K670N/M671L) (Mullan et al., 1992) that increases the production of Aβ40 and Aβ42 by

> 5-fold, and Italian mutation (A673V) (Di Fede et al., 2009) that shows enhanced

production of both Aβ species by ~ 2-fold, maintaining the ratio Aβ42/Aβ40 unaltered.

However, different processing is observed for mutations in regions adjacent to the γ-

secretase cleavage sites. These mutations are typically associated with increased

production of Aβ42 and lower levels of Aβ40, analogous to what is observed for

mutations in the presenilin genes (Suzuki et al., 1994). As a result, the ratio of

Aβ42/Aβ40 is several fold elevated, reaching in some cases ~ 18-fold values. Amino acid


11

substitutions within residues 21-23 and 34 of the Aβ peptide are, with few exceptions

(Wakutani et al., 2004), associated with prominent vascular compromise.

Gandy, 2005

Figure 1.3. Structure of APP. (A) Schematic structure and topography of APP. (B) Area

surrounding the Aβ domain, α-, β- and γ-secretase cleavage sites, and amino acid location in

selected FAD missense mutations.

1.1.3.2 Cerebral amyloid angiopathy

Cerebral amyloid angiopathy (CAA) due to Aβ deposition is a key pathological

feature of FAD. It is an age-associated condition, characterized by deposition of Aβ in

the medial layer of cerebral blood vessels (Coria and Rubio, 1996). It is also a significant

cause of intracerebral hemorrhage in patients with AD and certain related disorders (Coria

and Rubio, 1996; Melchor et al., 2000). Specific missense mutations in the APP gene

occurring inside the Aβ region comprising residues 21-23 is considered a „„hot spot‟‟ for

mutations due to the high number of genetic variants reported in this area of the molecule.

Mutations within this amino acid cluster typically show strong vascular compromise and

primarily associate with CAA, hemorrhagic strokes and dementia. Mutations in the Aβ

sequence are concentrated at positions 21-23 and are termed Dutch (E22Q) (Miravalle et

al., 2000), Italian (E22K) (Miravalle et al., 2000), Iowa (D23N) (Van Nostrand et al.,

Chapter 1

12

2001) , Arctic (E22G) (Nilsberth et al., 2001) , Flemish (A21G) (Demeester et al., 2001)

and New Italian (L34V) (Obici et al., 2005) mutations. In general, all mutants are

thought to have stronger neurotoxicity than wild-type Aβ, which may correlate with their

ability to form protofibrils. However, not all mutants have the same capacity to induce

neurotoxic effects. This suggests that the substitution of different amino acids may confer

distinct structural properties, influencing the onset and aggressiveness of the disease.

The first mutation inside Aβ domain was described in a condition known as

hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D), an

autosomal dominant disorder clinically defined by recurrent strokes, vascular dementia,

and fatal cerebral bleeding in the fifth to sixth decades of life. In this missense mutation,

a Gln replaces a Glu residue at position 22 (AβE22Q) resulting from a single nucleotide

transversion (G for C) at codon 693 (Levy et al., 1990). The clinical manifestations for

the carriers of this mutation include periodic episodes of cerebral hemorrhages that are

associated with massive amyloid deposition in the walls of leptomeningeal and cortical

arteries and arterioles, as well as in vessels in the brainstem and cerebellum (Bornebroek

et al., 1999). Besides the vascular involvement, Dutch familial cases also develop

parenchymal amyloid deposits resembling the diffuse preamyloid lesions seen in AD.

However, dense-core plaques and NFTs are rare or even not present at all (Levy et al.,

1990). The non-fibrillar diffuse plaques show differences in the extent of fibrillary

material, ranging from fine diffuse plaques in young patients to more fibrillar, dense

content in old patients (Maat-Schieman et al., 2000). Thus, the non-fibrillar diffuse

plaques, variably associated with reactive astrocytes and activated microglia (Maat-

Schieman et al., 2004) evolve into more fibrillar, dense lesions (Maat-Schieman et al.,

2000). Nevertheless, the degree of dementia appears to be independent of plaque

involvement and neurofibrillary degeneration, which is absent or limited, and

contrastingly correlates with the severity of CAA (Natte et al., 2001).

Aβ40 is the predominant Aβ species of HCHWA-D vascular amyloid, with wild-

type and Dutch-type mutated sequences present at a 1:1 molar ratio. In contrast, Aβ42 is

only a minor component of the vascular deposits (Herzig et al., 2004) and is uniquely

associated with species bearing the mutation, and not with those originated from the

normal allele (Prelli et al., 1990). So far, it has not been possible to identify Aβ42 wild-

type or carrying the E22Q substitution in the cortex by Western blot, and ELISA results

show an Aβ42/Aβ40 considerably lower than in AD cases (Herzig et al., 2004).


13

In vitro cell culture studies show that E22Q has potent toxic effects on

cerebrovascular endothelial and smooth muscle cells (Davis and Van Nostrand, 1996;

Miravalle et al., 2000), likely reflecting the in vivo hemorrhage-associated phenotype.

The E22Q mutant is also a potent angiogenesis inhibitor, disturbing the fibroblast growth

factor 2 (FGF-2) signaling pathway, downregulating phosphorylation of the FGF-2

receptor, and the Akt survival signaling (Solito et al., 2009). Altogheter, these effects

lead to endothelial dysfunction and probably contribute, solely or partially, to the

disturbances in membrane permeability and hemorrhagic manifestations in the family

members (Rostagno et al., 2010).

1.1.4 Amyloid precursor protein

APP in mammals belongs to a family of conserved type I membrane proteins

that include APP (Goldgaber et al., 1987), APP like protein 1 (APLP1) (Wasco et al.,

1992) and 2 (APLP2) (Slunt et al., 1994). Importantly, the Aβ peptide domain is not

conserved and is only present in APP and not in any of the other APP-related genes.

Although APP family is abundantly expressed in the brain, and APLP1 expression is

restricted to neurons (Lorent et al., 1995), APP and APLP2 can also be found in the

majority of other tissues as well. The human APP gene is located on the long arm of

chromosome 21 (Lamb et al., 1993). The expression results in alternative splicing that

generates APP mRNAs encoding isoforms that go from 365 to 770 amino acid residues.

The most important Aβ peptide encoding proteins have 695, 751 and 770 amino acids,

and are denominate as APP695, APP751 and APP770 (Zheng and Koo, 2006).

The exact physiological role of APP is not clearly understood. However, several

functions have been recognized based on the variety of APP extracellular subdomains,

such as cell surface receptor (Lorenzo et al., 2000), cell adhesion (Soba et al., 2005),

neurite outgrowth and synaptogenesis (Herard et al., 2006). Furthermore, the APP

intracellular domain presents a high degree of sequence conservation between the

intracellular domain of APP, APLP1, and APLP2, predicting that it is a critical domain

for regulation of APP function (Zheng and Koo, 2006).

In fact, APP can be phosphorylated at multiple sites in both extracellular and

intracellular domains (Hung and Selkoe, 1994). Among these, the phosphorylation at the

threonine residue (Thr668) in APP intracellular domain has received most attention.

Several kinases have been implicated in this phosphorylation event, including the cyclin-

Chapter 1

14

dependent kinase 5 (CDK5), c-Jun N-terminal kinase 3 (JNK3), and glycogen synthase

kinase 3β (GSK-3β) (Iijima et al., 2000; Kimberly et al., 2005; Muresan and Muresan,

2005a). APP Thr668 phosphorylation has been described to control APP localization to

the growth cones and neurites (Muresan and Muresan, 2005a). Notably, Thr668

phosphorylated APP is preferentially transported to the nerve terminals (Muresan and

Muresan, 2005b), and Thr668 phosphorylated APP fragments are increased in AD, but

not in control subjects (Lee et al., 2003). This evidence supports the theory that APP

Thr668 phosphorylation participate in AD pathogenesis by controling Aβ production.

The preceding facts relate to the positive or beneficial functions of APP. Interestingly,

there is also extensive research about the cytotoxic apoptotic properties of APP,

particularly when APP or the β-cleaved C-terminal fragment of APP ("C99" or "C100")

are upregulated (Yankner et al., 1989). Overexpression of the C100 APP C-terminal

fragment (CTF) is related with neuronal degeneration (Oster-Granite et al., 1996),

possibly through alteration of APP signal transduction. Furthermore, APP CTF may be

cytotoxic through amyloid precursor protein intracellular domain (AICD), and appears to

require an intact caspase site within the cytosolic tail (Lu et al., 2003).

Finally, APP has also been strongly involved in axonal transport regulation, at

least in part through adaptor protein JIP-1 (Sisodia, 2002), a member of the c-Jun N-

terminal kinase-interacting protein (JIP) family. This fact has particular significance,

since APP is transported in axons via the fast anterograde transport machinery, and

protein processing or modifications are known to take place during transit in axons

(Zheng and Koo, 2006).

1.1.4.1 Subcellular trafficking, maturation and processing

APP processing takes place in various organelles, during its normal secretory

pathway, and also at the cell surface. In both neuronal and non-neuronal cells, APP is

recognized to be transported through the secretory pathway, a continuum transport in

separate membrane-enclosed organelles that ultimately reach the cell surface (Fig. 1.4).

Throughout this secretory transport, post-translational modifications of the newly

synthesized APP proteins are prone to occur, which may influence APP cleavage and Aβ

production.

During its trafficking through the different subcellular organelles, APP can be

processed originating different APP fragments. Although most cell types seem to


15

metabolize APP using the same basic molecular mechanisms, the relative influence of the

different pathways is considerably different, particularly between neurons and non-

neuronal cell lines (Hartmann, 1999).

Thinakaran and Koo, 2008

Figure 1.4. Intracellular trafficking of APP. After synthesis, APP enters the endoplasmatic

reticulum followed by the Golgi complex, and is transported via the constitutive secretory

pathway to the cell membrane (black bars) in the basolateral surface of the cell (route 1). Once in

the plasma membrane, APP is rapidly internalized (route 2), and then shuttled through endocytic

and recycling compartments back to the cell surface (route 3), or degraded in the lysosome.

Amyloidogenic processing involves transit through the endocytic organelles, where APP

encounters β- and γ-secretases. Non degradable lysosomal Aβ42 is recycled back to the cell

surface. In AD, this process is enhanced by the overproduction of Aβ42 in the endossomes, or the

protection of APOE4 over Aβ42 against lysosomal degradation, resulting in accumulation of

excessive Aβ42 (toxic forms) at the cell membrane. Non-amyloidogenic processing is thought to

be located near the plasma membrane, where α-secretase is present.

Most of the APP processing occurs after complete maturation of the protein,

even though some immature APP may also be cleaved by secretases at a low rate in the

endoplasmatic reticulum (ER) or the cis-Golgi complex subcellular compartments.

Mature APP is processed rapidly, with turnover of ~ 30-45 min, as it is transported to or

from the cell surface via the secretory or endocytic pathways, respectively (Koo et al.,

1996; Kuentzel et al., 1993; Yamazaki et al., 1996). Moreover, only small amounts of

APP were detected at the cell surface when compared to the total cellular pool (Kuentzel

et al., 1993). This is consequence of rapid removal of APP at the cell surface. APP half-

life was reported to be shorter than 10 min (Lai et al., 1995), either by APP proteolytic

Chapter 1

16

cleavage or endocytosis. Around 30% of cell surface APP is processed to sAPP and

secreted (Koo et al., 1996), while the remaining cell surface CTFs may be cleaved by γ-

secretase locally, or in the endocytic pathway in endosomes, or further degraded in

lysosomes (Kaether et al., 2006). Both cell surface CTF cleavage product and

unprocessed full-length APP are re-internalized through coated pits and vesicles by

receptor-mediated endocytosis (Yamazaki et al., 1996). If endocytosed, internalized APP

half life is ~ 30 min (Koo et al., 1996), with a pool of endosomal APP being delivered to

lysosomes.

1.1.4.2 Pathological role of intracellular Aβ

There is substantial evidence from transgenic mouse models that intracellular Aβ

initiates cellular dysfunction, before it accumulates in extracellular plaques (Hsia et al.,

1999). Moreover, a recent study characterizing intracellular accumulation of Aβ in

humans, including patients with AD, concluded that intracellular Aβ was abundantly

present, but did not correlate with plaque load or NFT formation (Wegiel et al., 2007). In

addition, the disease-associated isoform Aβ42 seems to be more prone to intracellular

accumulation than Aβ40. Also, intracellular Aβ occurs most frequently in the

hippocampus and entorhinal cortex, which are the brain regions to be affected first in AD

(Gouras et al., 2000). Expression of APOE-4 also increases intracellular Aβ (Zerbinatti

et al., 2006). Within neurons, Aβ42 seems to localize in multivesicular bodies (MVBs),

which are considered late endosomes and are generated from the early endosome system.

Immunogold electron microscopy in brains of patients with AD demonstrated that Aβ42

is localized to the external membrane of MVBs (Takahashi et al., 2002). The

accumulation of non-fibrillar Aβ within neuronal MVBs has since been shown in

APPxPS1 transgenic mice (Langui et al., 2004), and Aβ-containing MVBs were

frequently in the perinuclear region. Neurons from APPxPS1 transgenic mice also

exhibited Aβ-positive granules within the peri-nuclear region of the cell body, which was

double labeled largely with lamp 2, a lysosomal membrane protein, cathepsin D, another

lysosomal hydrolase, and MG160, a Golgi complex marker (Langui et al., 2004).

Recent studies have demonstrated that Aβ accumulation within MVBs is

pathological, leading to disrupted MVB organization through impairment of the

ubiquitin-proteasome system (Almeida et al., 2006). Inhibition of the proteasome by Aβ

has been demonstrated in animal models and cell lines (Almeida et al., 2006; Oh et al.,


17

2005). Proteasome inhibition in the 3xTg-AD mice showed oligomeric Aβ accumulation

within neuronal cell bodies (Oddo et al., 2006; Tseng et al., 2008). Taken together, these

findings suggest that oligomeric Aβ accumulation within neuronal cell bodies has

pathological consequences, such as proteasome impairment that leads to the increase of

tau protein. In addition, proteasome inhibition, both in vivo and in vitro, result inelevated

Aβ levels, suggestive that the proteasome degrades Aβ, and that Aβ must be within the

cytosolic compartment for this degradation to occur (LaFerla et al., 2007).

In Tg2576 mice, accumulation of Aβ has also been reported to take place in

mitochondria (Manczak et al., 2006), where all subunits of the γ-secretase have been

located (Hansson et al., 2004). Progressive accumulation of intracellular Aβ in

mitochondria is associated with reduced enzymatic activity of respiratory chain

complexes III and IV, and decreased rate of oxygen consumption (Fig. 1.5) (Caspersen et

al., 2005). These observations facilitate the understanding of the large number of

mitochondrial alterations described in AD (Keil et al., 2006).


Figure 1.5. Oxidative stress and mitochondrial failure. Mitochondrial dysfunction has been

implicated in the etiology and development of AD. APP may be targeted to mitochondria and

Chapter 1

18

interfere with protein import. Mitochondria have been reported to contain active γ-secretase

complexes, which are involved in cleaving APP to form Aβ, and contain also PS1. ROS and

reactive nitrogen species (RNS) are generated from respiratory complexes I, II, and III. The

resulting free radicals attack the cell and organelle membrane lipids, increasing the levels of toxic

aldehydes hydroxynonenal (HNE) and malondialdehyde, and inhibit the mitochondrial aldehyde

dehydrogenase. Oxidative damage to membrane-bound, ion-specific ATPases and stimulation of

calcium (Ca2+) entry mechanisms, such as glutamate N-methyl-D-aspartate (NMDA) receptors,

membrane-attack complex of complement, and ion-selective amyloid pore formation, cause

cytosolic and mitochondrial Ca2+ overload. Cellular Aβ binds to alcohol dehydrogenase and

directly targets the respiratory chain complex IV (cytochrome c oxidase), and key Krebs-cycle

enzymes, leading to increased ROS levels and ATP depletion. Aβ also promotes damage in

mitochondrial DNA (mtDNA), which results in augmented dysfunction in respiratory chain

complexes and further increase in ROS. Lipid peroxidation products also promote tau

phosphorylation and aggregation, which in turn inhibit complex I. As the mitochondrial

membrane potential collapses and the mitochondrial permeability-transition pore (MPP) opens,

caspases are activated and ROS are released to the cytosol. Cytosolic ROS/RNS in turn were

demonstrated to activate KATP channels, which causes alterations in mitochondrial membrane

potential (Ψm) and further augments ROS levels. Aβ also induces the stress activated protein

kinases p38 and JNK, as well as p53, which are further linked with apoptosis. Substrate

deficiencies, notably NADH and glucose, combine with electron transport uncoupling to further

diminish ATP production. GLUT1, 4 , glucose transporter 1, 4.

Intraneuronal Aβ is associated with synaptic dysfunction (Fig. 1.6), which could

underlie cognitive deficits. The 3xTg-AD mouse model of AD exhibits intraneuronal

accumulation of Aβ at 4 months of age, when cognitive deficits are first detected (Billings

et al., 2005). Accordingly, these mice show no plaque formation, little somatodendritic

tau and no hyperphosphorylated tau species. Finally, the depletion of intraneuronal Aβ

with immunotherapy restores cognition to control non-transgenic levels (Billings et al.,

2005).


19


Figure 1.6. Synaptic dysfunction in AD. Aβ is thought to have a physiological role in

modulating synaptic activity, the disruption of which probably underlies cognitive dysfunction.

Excess build-up of Aβ and synaptotoxic Aβ oligomers induce neurotransmitter receptor

internalization and inhibition. Aβligomers, impair synaptic plasticity by altering the balance

between long-term potentiation (LTP) and long-term depression (LTD) and reducing the numbers

of dendritic spines. At high concentrations, oligomers may suppress basal synaptic transmission.

Because Aβ oligomers can also enhance LTD through activation of glutamate receptors,

stimulation of glutamate receptors results in excitotoxicity that, under conditions of reduced

energy availability or increased oxidative stress, leads to Ca2+ influx into postsynaptic regions of

dendrites. This, in turn, can trigger apoptosis. Aβ facilitates endocytosis of NMDA receptors and

AMPA. Aβ also binds to the receptors of p75 neurotrophin (p75NTr) and brain-derived

neurotrophic factor (BDNF) also known as tyrosine kinase B receptor (trkBr), exacerbating a

situation in which levels of BDNF and nerve growth factor (NGF) are already suppressed. Aβ

impairs nicotinic acetylcholine receptor (nAChr) signaling and acetylcholine (Ach) release from

the presynaptic terminal. Thus, ACh levels are markedly reduced in AD. Levels of nAChR are

diminished in the AD brain, in part by Aβ-induced internalization. pCaMKII, phosphorylated

Ca2+ calmodulin-dependent protein kinase 2; pCREB phosphorylated cyclic AMP response-

element-binding protein; VGCC voltage-gated Ca2+ channel.

Chapter 1

20

A large body of evidence indicates that the accumulation of intracellular Aβ

induces important expression of ER stress markers. Immunohistochemical studies

showed that neurons in postmortem brain samples of AD patients present staining of

phosphorylated (activated) unfolded protein response (UPR) kinases, such as double

stranded ribonucleic acid-activated protein kinase-like ER kinase (PERK, also known as

EIF2AK3) and inositol-requiring kinase 1 (IRE1, also known as ERN1) (Hoozemans et

al., 2009). These proteins were clearly upregulated in hippocampal neurons in AD,

particularly in neurons containing granulovacuolar degeneration. Interestingly, pPERK-

positive neurons also presented abundant staining for GSK-3β. This is a relevant

observation since it points out that ER stress may trigger the expression of GSK-3β, a

well-known tau kinase, and in that way enhance NFT formation (Resende et al., 2008).

1.1.4.2.1 Endoplasmic reticulum stress

The ER fulfills multiple cellular functions (reviewed in Xu et al., 2005). The

lumen of the ER is an exceptional compartment, holding the highest concentration of Ca2+

within the cell, due to active transport of Ca2+

ions by Ca2+

ATPases (Fig. 1.7). The

lumen is an oxidative environment, important for generation of disulfide bonds and

proper folding of proteins destined for secretion or display on the cell surface. Because of

its role in protein folding and transport, the ER is also rich in Ca2+

-dependent molecular

chaperones, such as 78-kDa glucose-regulated protein (GRP78), also known as Bip

(GRP78/Bip), 94-kDa glucose-regulated protein (GRP94), and calreticulin, which

stabilize protein folding intermediates (reviewed in Orrenius et al., 2003). Importantly,

Ca2+

is a vital second messenger associated with the most fundamental molecular

pathways within the cell. Thus, its intracellular free levels are tightly regulated by the ER

to avoid cell death induced by intracellular Ca2+

dysregulation.

GRP94 has been extensively linked to cellular Ca2+

homoeostasis (Macer and

Koch, 1988). One of the major characteristics that GRP94 shares with other ER stress

proteins is that its expression is induced trough a transcriptional feedback loop (Little et

al., 1994), when cells are challenged with Ca2+

ionophores (Drummond et al., 1987).

GRP94 binds Ca2+

and is one of approximately six luminal proteins that serve as the

major Ca2+

buffers of the ER (Cala and Jones, 1994; Macer and Koch, 1988; Nigam et al.,

1994). Purified GRP94 is predicted to bind 28 mol of Ca2+

/mol of protein by one

estimate (Macer and Koch, 1988) and 16-20 mol/mol by another, with a few high-affinity


21

(Kd 1-5 μM) binding sites and other lower-affinity sites (Kd ∼ 600 μM). Furthermore,

GRP94 affects the biosynthesis of several secreted and membrane-bound proteins, such as

immunoglobulins (Melnick et al., 1994) or Toll-like receptors (Randow and Seed, 2001),

and acts as a peptide-binding protein (Blachere et al., 1997; Srivastava and Udono, 1994).

Its peopensity to bind several different peptides has been used to enhance immune

responses. When injected into mice, GRP94-bound peptides are transferred on to major

histocompatability complex (MHC) class I proteins that then activate peptide-specific T-

cells (Berwin et al., 2002; Li et al., 2005; Suto and Srivastava, 1995).

Orrenius, 2003

Figure 1.7. The regulation of intracellular Ca2+

compartmentalization. Cellular Ca2+ influx

into the cytosol through the plasma membrane is a tightly control process, both temporally and

spatially, that occurs largely by receptor-operated (for example, glutamate receptors), voltage-

sensitive and store-operated channels. Once inside the cell, Ca2+ can either interact with Ca2+-

binding proteins or become sequestered into the ER or mitochondria. At rest, cytosolic Ca2+

levels are maintained at relatively low levels, relative to the extracellular space and the

intracellular stores by the presence of Ca2+-binding buffering proteins, via the extrusion of

cytosolic Ca2+ across the plasma membrane through Ca2+ ATPase (PMCA) pumps and

exchangers, and also due to sequestration into intracellular stores. Then, after activation

(electrical, synaptic or receptor (R) mediated), they rise rapidly to the low micromolar range. The

ER is the largest Ca2+ intracellular store via the pumping of cytosolic calcium of

Chapter 1

22

sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps. Inside the ER Ca2+ is maintained

elevated by Ca2+-binding buffering proteins (calreticulin, calsequestrin, GRP94). Calcium release

from the ER occurs through inositol triphosphate receptors (InsP3Rs or IP3Rs) and ryanodine

receptors (RyRs). Release through InsP3Rs requires the activation of G proteins (G) on the cell

surface that induce phospholipase C (PLCγ) to cleave phosphatidylinositol-4,5-bisphosphate

(PtdIns(4,5)P2) into diacylglycerol (DAG) and Ins(1,4,5)P3, although it might be possible to

activate release without Ins(1,4,5)P3. Alternatively, release can also occur through tyrosine-

kinase-coupled receptors. Mitochondria pump cytosolic Ca2+ electrophoretically via a uniport

transporter and can release it again through three different pathways: reversal of the uniporter,

Na+/H+-dependent Ca2+ exchange, or as a result of permeability transition pore (mPTP) opening.

The mPTP can also flicker to release small amounts of Ca2+. Ca2+ efflux from cells is regulated

primarily by the PMCA, which binds calmodulin and has a high affinity for Ca2+. Ca2+ efflux

might also be mediated by the Na+/ Ca2+-exchanger (NCX).

Disturbances in ER function or loss of integrity lead to ER stress, which may be

caused by accumulation of unfolded proteins or changes in Ca2+

homeostasis within the

ER (Lindholm et al., 2006). Most neurodegenerative disorders, including AD are

associated with aggregation of misfolded proteins (Rao et al., 2004). When viable

proteins acquire pathological conformations, physiological functions in the cell are

affected, which often culminates in cell death. Pathogenic pathways involve membrane

permeabilization, through either a channel mechanism or by hydrophobic interaction of

prefibrillary oligomers with cellular targets (Jellinger, 2009).

To eliminate misfolded proteins, cells can activate a large number of intracellular

proteases and chaperones, which integrate the cellular protein quality-control system of

the ER. The two principal routes of intracellular protein catabolism are the ubiquitin-

proteasome system (UPS), and the autophagy-lysosome pathway or autophagy. Both

pathways were described as having a dual role in nervous system homeostasis, including

both protection and degeneration (Jellinger, 2009).

When protein misfolding occurs, the ER triggers a signaling pathway known as

UPR (Fig. 1.8). Initially cytoprotective, the UPR will trigger a typical apoptotic cascade

if the cellular insult is not efficiently removed, representing a last resort of multicellular

organisms to dispense of dysfunctional cells. The UPR is essential for the non-lysosomal

degradation and clearance of altered proteins that have the potential to induce cellular

damage (Kim et al., 2008). It is characterized by the coordinated activation of multiple


23

ER-resident sensors, including PERK, IRE1, and activating transcription factor 6 (ATF-

6). Once activated, these proteins trigger signaling events, such as increased expression

of ER chaperones, inhibition of protein entry into the ER, blockage of mRNA translation,

and acceleration of altered protein export from the ER to the cytosol for ubiquitination

and proteasome-mediated degradation (reviewed in Schroder and Kaufman, 2005).

Normally, the N-termini of these transmembrane ER proteins are held by ER chaperone

GRP78/Bip, preventing their aggregation. But when misfolded proteins accumulate,

GRP78/Bip is released, allowing aggregation of these transmembrane signaling proteins,

and launching the UPR (Xu et al., 2005).

Disturbances in Ca2+

regulation can also induce the UPR and perturb cellular

events that control cell fate (Fig. 1.8). B-cell leukemia/lymphoma 2 (Bcl-2) family

proteins represent the strongest link between ER Ca2+

regulation and the cell death

machinery, as several Bcl-2 proteins reside in the ER membrane and modulate ER Ca2+

homeostasis. In fact, Bcl-2 and B-cell leukemia/lymphoma extra long (Bcl-xL) decrease

basal Ca2+

concentrations in the ER (Foyouzi-Youssefi et al., 2000), whereas Bcl-2-

associated X protein (Bax) has an opposite effect (Foyouzi-Youssefi et al., 2000).

Several studies have demonstrated an association of ER stress and disturbed Ca2+

homeostasis in AD pathogenesis (LaFerla, 2002). The involvement of ER stress in AD

has been confirmed by detection of hyperactivated PERK-eukaryotic translation initiation

factor 2α (eIF2α) pathway (Unterberger et al., 2006). In addition, PS1 and PS2 proteins,

members of the multiprotein γ-secretase complex that mediates the intramembranous

cleavage of APP, are abundantly expressed in neurons and localized in the ER membrane,

further suggesting a link between AD and ER stress (Lindholm et al., 2006). Mutant PS1-

expressing cells show altered Ca2+

homeostasis, increased Aβ production (Levitan et al.,

2001), and enhanced sensitivity to ER stress-induced apoptosis (Lindholm et al., 2006).

Similarly, PS1 mutant transgenic mice display abnormalities in ER Ca2+

regulation and

increased neuronal vulnerability toward cell death and excitotoxic injury. It has also been

demonstrated that mutant PS1 binds to and inhibits the UPR protein IRE1, suppressing

the activation of the UPR (Katayama et al., 1999). IRE1 activates downstream signals,

including the transcription of the ER chaperone GRP78/Bip (Katayama et al., 2004).

Recent studies revealed increased levels of GRP78/Bip and PERK in AD brains, further

confirming the occurrence of ER stress in AD (Hoozemans et al., 2009; Hoozemans et al.,

2005). Finally, ER stress can directly activate caspase-12. Primary cortical neurons from

caspase-12 knockout mice showed reduced susceptibility toward ER stress. Neurons

Chapter 1

24

were resistant to Aβ-induced cell death (Nakagawa et al., 2000), suggesting that ER stress

and activation of caspase-12 may contribute to neuronal death in AD.

Kim et al., 2008

Figure 1.8. UPR events and connection to the cell death machinery. UPR signaling pathways

induced by ER stress are represented, and some connections to the cell death machinery are also

shown. In non-stressed cells (not shown), the ER chaperone GRP78/Bip binds to the luminal

domains of the ER-stress sensors IRE1α, PERK and ATF-6, maintaining these proteins in an

inactive state. Upon accumulation of misfolded proteins in the ER, GRP78/Bip preferentially

binds to unfolded or misfolded proteins, thus driving the equilibrium of GRP78/Bip binding away

from IRE1α, PERK and ATF6, triggering the ER stress (shown). These three proteins are the

initiators of the three main signaling cascades of the UPR. The release of GRP78/Bip allows

IRE1α to dimerize, activating its protein kinase activity (through autophosphorylation) and

intrinsic ribonuclease activity. A IRE1α dimer binds tumor necrosis factor receptor-associated

factor 2 (TRAF2), activating apoptosis signal-regulating kinase 1 (ASK1) and downstream

kinases that activate p38 MAPK and JNK. IRE1α signaling is positively regulated by binding of

the multidomain pro-apoptotic Bax and Bak. The interaction of Bax/Bak with IRE1α is required

for the recruitment of TRAF2 and ASK1 leading to the activation of the mitogen-activated protein

kinase (MAPKs) JNK and p38, through specific mitogen activated protein kinase kinase (MKK).

The intrinsic ribonuclease activity of IRE1α then removes a 26-base intron from X-box-binding

protein 1 (XBP1) mRNA. The spliced XBP1 mRNA encodes a potent transcription factor that

translocates to the nucleus, activating the expression of genes involved in restoring protein folding

or degrading unfolded proteins. The release of GRP78/Bip also results in the activation of PERK,

through PERK homodimerization and trans-autophosphorylation. Activated PERK then


25

phosphorylates the eIF2α, reducing global mRNA translation. At the same time, favoring the

translation of selected mRNAs, such as ATF-4 mRNA, that are preferentially translated in the

presence of phosphorylated eIF2α. ATF-4 activates the transcription of UPR target genes

encoding factors involved in restoring ER homeostasis such as, amino-acid biosynthesis, the

antioxidative-stress response and apoptosis, as well as autophagy genes. In contrast to PERK and

IRE1, release of BiP from ATF-6 induces its translocation to the Golgi complex where it is

processed by the site-1 (S1P) and site-2 (S2P) generates an ATF-6α. This fragment migrates to

the nucleus, activating the transcription of genes mainly involved in ER-assisted degradation

(ERAD) and ER homeostasis. Upon severe ER stress, ATF-4, XBP1, and ATF-6 can upregulate

the expression of the proapoptotic transcription factor C/EBP homologous protein (CHOP), which

mediates apoptosis by the upregulation of proapoptotic BH3-only protein Bim, and by

suppressing Bcl-2 expression. CHOP activity is enhanced through phosphorylation by

p38MAPK. Phosphorylation by JNK in turn activates Bim, while inhibiting Bcl-2 functions. The

autophagy protein Beclin is inhibited by direct binding to Bcl-2.

1.1.4.2.2 JNK stress sensor

The JNK signaling pathway has been identified as a key event in AD-

associated apoptosis. The IRE1α/tumor necrosis factor receptor-associated factor 2

(TRAF2)/apoptosis signal-regulating kinase 1 (ASK1) pathway activates stress kinases

(Kim et al., 2008; Salminen et al., 2009; Urano et al., 2000), which have deep functional

effects on neuronal homeostasis (Bogoyevitch and Kobe, 2006; Dhanasekaran and Reddy,

2008; Sekine et al., 2006). ER stress activates ASK1, which subsequently triggers JNK

and p38 mitogen-activated protein kinase (MAPK) signaling (Fig. 1.8). However, it is

not fully understood whether ER stress is the only inducer of ASK1. For example, Aβ

induces neuronal apoptosis through reactive oxygen species (ROS)-mediated ASK1

activation rather than via ER stress (Kadowaki et al., 2005). However, ER stress can

activate ROS production via Ca2+

mitochondrial signaling. The ASK1-mediated JNK

pathway plays a key part in AD pathogenesis (Okazawa and Estus, 2002). This stress

signaling kinase can regulate APP processing (Colombo et al., 2009) and control the

phosphorylation state of APP at the Thr668 site, which is important for both APP

cleavage and degradation in physiological conditions. Inhibition of JNK-mediated

phosphorylation of APP causes it to follow the proteasome degradation pathway

(Colombo et al., 2007). It may also induce accumulation of intracellular Aβ (Colombo et

al., 2009; Shoji et al., 2000), phosphorylate tau protein and trigger aggregation of NFTs

Chapter 1

26

(Reynolds et al., 1997; Vogel et al., 2009). Furthermore, JNK mediates the activation of

several molecules, including caspase-2 (Troy et al., 2000), p53 (She et al., 2002), and

Bcl-2 family members (Sorenson, 2004) and potentiate inflammatory responses via AP-1

activation (Manning and Davis, 2003).

A histopathological analysis of AD brains showed accumulation of activated

pJNK in granules within hippocampal pyramidal cells (Lagalwar et al., 2007). These

granules normally co-localize with granulovacuolar degeneration bodies (GVD)

(Lagalwar et al., 2007), which also contain GSK-3β (Leroy et al., 2002), a recognized

PERK target kinase. GVD are large cytoplasmic vacuoles (Lagalwar et al., 2007) that

result from autophagic vacuoles usually seen in AD (Nixon, 2007). Interestingly, it has

been shown that excessive ER stress can induce autophagic uptake of accumulated

material from the overloaded ER (Yorimitsu and Klionsky, 2007). Both activated pJNK

and pGSK-3β are present in pre-tangle accumulations of tau protein (Ishizawa et al.,

2003; Zhu et al., 2001b), which are different structures from GVDs. Furthermore, recent

data demonstrated that JNK can induce caspase cleavage of tau protein, but also that

GSK-3β activation is required for tau aggregation (Sahara et al., 2008). In addition, c-

Jun, an immediate-early proapoptotic protein of the JNK pathway was found to be

colocalized with fragmented DNA in neurons (Anderson et al., 1996). Enhanced

expression of transcriptional factors c-Jun and c-Fos, increased levels of c-Jun mRNA,

and phosphorylation of c-Jun on its N-terminal transactivation domain have all been

observed in neuronal apoptosis (Sastry and Rao, 2000). Finally, Aβ itself may induce

activation of JNK and c-Jun (Morishima et al., 2001). Consistently, Aβ-induced cell

death is attenuated in cortical neurons from JNK3-null mice, while JNK3 mediates cell

death through the activation of c-Jun and enhanced expression of apoptosis antigen-1

ligand (FasL) (Morishima et al., 2001).

1.2 Cell death mechanisms

Programmed cell death (PCD) plays a key role in the development of the

nervous system. However, deregulation of cell death programs are involved in

developmental and neoplastic disorders of the nervous system, and increasing evidence

suggests that such deregulation may also occur in neurodegenerative diseases. In fact,

many neurological disturbances are characterized by gradual loss of specific groups of


27

neurons, resulting in disorders of movement and cognitive function, such as AD (Vila and

Przedborski, 2003).

Cell death represents a highly heterogeneous process with distinct morphological

features, which results from activation of diverse, although partially overlapping,

biochemical cascades. Dying cells are engaged in a cascade of reversible molecular

events, until a first irreversible process takes place. Often known as the “point of no-

return”, this limiting molecular event is still an undeciphered enigma. It has been

proposed that a cell should be considered dead when any of the following molecular or

morphological criteria is met: (1) loss of cell plasma membrane integrity, as defined by

the incorporation of vital dyes in vitro (e.g., propidium iodide); (2) cell fragmentation into

discrete bodies; and/or (3) engulfment of cellular corpse, or its fragments by adjacent

cells in vivo (Kroemer et al., 2009). The precise characterization of the molecular

machinery of cell death is expected to have repercussions on the development of

innovative therapeutic strategies.

Although PCD has often been equated with apoptosis, nonapoptotic forms of cell

death also exist. In fact, at least three different forms of PCD are distinguishable,

including type I, also known as nuclear or apoptotic, type II or autophagic, and type III or

cytoplasmic. These are activated at particular times of nervous system development, but

may also occur after various insults, such as DNA damage or accumulation of damaged

proteins. Apoptosis is the best characterized type of PCD, where cells typically round up,

form blebs, and undergo chromatin condensation, nuclear fragmentation, and formation

of apoptotic bodies (Galluzzi et al., 2009). These morphological changes are largely the

result of the activation of cysteine proteases, termed caspases.

In comparison with apoptosis, little is known about other nonapoptotic forms of

PCD. Autophagy is the process by which intact organelles and/or large portions of the

cytoplasm are engulfed within double-membraned autophagic vacuoles for degradation.

The massive accumulation of autophagic vacuoles may represent either an alternative

pathway of cell death, or an ultimate attempt of cells to survive by adapting to stress. In

fact, autophagic cell death is crucial during development in Drosophila melanogaster

(Mohseni et al., 2009), and knockout of essential autophagy genes has been shown to

protect cultured mammalian cells from cell death (Maiuri et al., 2007). Nevertheless,

both pharmacologic and genetic inhibition of autophagy does not prevent cell death

(Galluzzi et al., 2008). Type III PCD, or cytoplasmic cell death, is a necrosis-like form of

Chapter 1

28

PCD that may involve swelling of the ER and mitochondria, but lacks typical apoptotic

features, such as apoptotic bodies and nuclear fragmentation (Bredesen et al., 2006).

1.2.1 Death receptor and mitochondrial pathways of apoptosis

Caspase activation by the death receptor of extrinsic pathway of apoptosis

involves the binding of extracellular death ligands, such as FasL or tumour necrosis

factor-α (TNFα) to transmembrane death receptors (Fig. 1.9). Engagement of death

receptors with their cognate ligands provokes the recruitment of adaptor proteins, such as

the Fas-associated death domain protein (FADD), which in turn recruit and aggregate

several molecules of caspase-8, thereby promoting its autoprocessing and activation.

Active caspase-8 then proteolytically processes and activates caspase-3 and -7, provoking

further caspase activation events that culminate in substrate proteolysis and cell death. In

some situations, extrinsic death signals can cross-talk with the intrinsic pathway through

caspase-8-mediated proteolysis of the Bcl-2 homology domain 3 (BH3)-only protein Bid

(BH3-interacting domain death agonist). Truncated Bid (tBid) can promote

mitochondrial cytochrome c release and assembly of the apoptosome, comprising seven

molecules of apoptotic protease-activating factor-1 (APAF1) and the same number of

caspase-9 homodimers (reviewed in Kroemer et al., 2007).

In the mitochondrial or intrinsic pathway of apoptosis, diverse stimuli that

provoke cell stress or damage typically activate one or more members of the BH3-only

protein family (Fig. 1.9). BH3-only proteins act as pathway-specific sensors for various

stimuli and are regulated in distinct ways. BH3-only protein activation above a crucial

threshold overcomes the inhibitory effect of the anti-apoptotic Bcl-2 family members and

promotes the assembly of Bcl-2 agonist killer 1 (Bak)-Bax oligomers within

mitochondrial outer membranes. These oligomers permit the efflux of intermembrane

space proteins, such as cytochrome c, into the cytosol. On release from mitochondria,

cytochrome c can seed apoptosome assembly. Active caspase-9 then propagates a

proteolytic cascade of further caspase activation events (reviewed in Kroemer et al.,

2007).


29

Benn and Woolf, 2004

Figure 1.9. Pathways of apoptosis. The extrinsic apoptotic pathway involves specific death

ligands, TNFα or FasL, that are recognized by death receptors TNFR1 (TNF receptor 1) and Fas,

located in the cell surface. TNFR1 stimulation recruits proteins to form a complex, which signals

to survival pathways (blue arrows) or the JNK- and caspase-2-induced death pathways (black

arrows). Fas stimulation induces FADD-mediated activation of procaspase-8 or DAXX (death-

associated protein)-mediated activation of JNK. JNK transforms Bid in jBid, promoting its

translocation to the mitochondria and release of SMAC/DIABLO (second-mitochondrial derived

activator of caspases). The intrinsic apoptotic pathway is triggered at the mitochondria by several

stimulus. (a), Membrane permeabilization occurs through BH3-only proteins that sequester and

inhibit the repression of Bax by Bcl-2, promoting Bax translocation to mitochondria and the

release of cytochrome c, SMAC/DIABLO, AIF (apoptosis-inducing factor) and HTRA2/OMI

Chapter 1

30

(high temperature requirement serine protease 2). (b), Cytochrome c binds to APAF1 (apoptosis

protease activating factor-1) to recruit and activate caspase-9, that together form the apoptosome,

which activates the downstream executioner caspases-3 and -7. SMAC/DIABLO sequesters and

inhibits IAP (inhibitor of apoptosis) proteins. AIF causes DNA degradation through PARP1

(poly(ADP-ribose) polymerase-1). ER stress mediated apoptosis occurs through caspase-12 and

caspase-3 activation. AIP1, ASK1-interacting protein 1; AKT/PKB, protein kinase B; AP1,

activator protein-1; BAD, Bcl-2-associated death protein; Bak, Bcl-2 agonist killer 1; Bid (BH3-

interacting domain death agonist) tBid (truncated Bid); Bim, Bcl-2-interacting mediator of cell

death; BMF, Bcl-2 modifying factor; Bok, Bcl-2-related ovarian killer protein; FADD,FAS-

associated death domain; FLICE, FADD-like interleukin-1β; HRK, 5/harakiri; IκB, inhibitory

subunit of NF-κB; IKK, IκB kinase; NF-κB, nuclear factor-κB; NIK, NF-κB-inducing kinase;

NOXA, phorbol-12-myristate-13 acetate-induced protein (PMAIP1); PDK1/2, 3- phospho-inoside

dependent kinase 1/2; PI3K, phosphatidyl inositol 3-kinase; PUMA, p53-upregulated modulator

of apoptosis; RAIDD, RIP-associated ICH-1 homologous protein with a death domain; UV,

ultraviolet light; XIAP, X-chromosome linked inhibitor of apoptosis protein; TRADD, TNF-

receptor death domain.

1.2.2 Role of apoptosis in Alzheimer’s disease

The study of apoptosis in neurodegenerative diseases remains a challenging task.

For years, the question on whether apoptosis plays a key role as the major form of cell

death in neurodegenerative diseases has been largely discussed, especially because

synaptic loss and electrophysiological abnormalities typically precede cell loss. In

addition, given the slow progression of most neurodegenerative diseases, in contrast with

the rapid progression of a cell through apoptosis, only a few cells exhibiting apoptotic

features are found in postmortem human brain tissue. Therefore, the synchronous

detection of a substantial number of apoptotic neurons at any given time point is, indeed,

unlikely. In turn, apoptotic processes may be terminated or not yet begun by the time the

tissue is available for examination. In fact, the collected tissue is usually representative of

the end-stage disease, being removed much later than it would be ideal to treat patients

and prevent neuronal cell death. Finally, the criteria used to classify the type of cell death

as apoptosis is often based solely on morphological assessment and biochemical assays,

which may also account for other forms of PCD. For example, the terminal transferase

dUTP nick-end labeling (TUNEL) assay is usually used as a marker of apoptosis in

human brain tissue. However, TUNEL can also label necrotic cells, which is why more


31

than one assay should be considered when determining the type of cell death

experimentally (Galluzzi et al., 2009). Despite these limitations, there is now a consensus

for a major role of apoptosis in human neuronal cell death and neurodegenerative

processes. Studies on the pathological mechanisms underlying neuronal apoptosis in

hereditary forms of neurodegenerative diseases have provided important clues, by linking

neuronal apoptosis with oxidative stress and mitochondrial dysfunction. Nevertheless,

the most prominent facts underlying the role of apoptosis in neurodegenerative diseases

came from studies using either in vitro cell culture systems or in vivo models of disease.

Although with inherent limitations, as these models tend to examine the effect of only one

toxin or genetic defect, usually involving acute exposure to neurological insults, they

have greatly increased our fundamental knowledge. This knowledge may now be used

for the development of new drugs that interfere with the apoptotic cascade in human

neurodegenerative diseases (Vila and Przedborski, 2003).

Regardless of the various genetic and environmental factors that may lead to the

manifestation of AD, several features of the neurodegenerative process are common to

different experimental models, including increased levels of oxidative stress, metabolic

compromise, perturbed Ca2+

regulation, ER stress, mitochondrial dysfunction, DNA

damage, activation of apoptotic biochemical cascades, autophagy and abnormalities in

protein processing and degradation (Culmsee and Landshamer, 2006). Interestingly, both

synaptic loss and neuritic dystrophy are affected early in AD, with no apparent cell loss.

Although these events precede extensive neuronal loss, it is unknown whether the

corresponding neuronal cell bodies are truly healthy. Conversely, it is also unclear

whether early dendritic and axonal defects kill the corresponding neurons, or whether

dendritic, axonal and neuronal deaths are each direct consequences of extracellular A.

Nevertheless, the pathogenic cascade of AD finally results in neuronal cell and synapse

loss.

Discussion of the nature of neuronal loss in AD remains controversial. Although

autophagy has been suggested to play a central role in AD neurodegeneration (Ling and

Salvaterra, 2009), it is still not clear whether it does play a causative role or, inversely, a

protective role, or if it is a consequence of the disease process itself (Hung et al., 2009).

In contrast, the detection of cleaved caspases and the accumulation of their cleaved

substrates in post-mortem AD brain tissue, represent a small part of the whole frame

supporting the hypothesis that apoptosis plays a key role in AD (Ribe et al., 2008). In

Chapter 1

32

addition, biochemical and morphological hallmarks of apoptosis have been associated

with neuropathological features found in AD brain tissue, including NFTs and amyloid

plaques. This further underlies the role of neuronal apoptosis in the progression of the

disease (Culmsee and Landshamer, 2006).

Finally, there is increasing evidence for a pivotal role of ER stress in AD-

associated cell death. During ER stress, perturbed Ca2+

homeostasis or altered protein

processing leads to the accumulation of unfolded protein in the ER, which activates the

UPR. In turn, the UPR activates transcription factor C/EBP homologous protein (CHOP),

which may induce apoptosis. In fact, CHOP has been suggested as the link between ER

and mitochondria during apoptotic cell death induced by the A peptide (Costa et al.,

2010).

1.2.2.1 Function of caspases

Substantial evidence demonstrates that multiple caspases are detected in brains

of patients with AD. In fact, caspases-1, -2, -3, -5, -6, -7, -8 and -9 have all been found to

be transcriptionally increased in AD, compared with controls (Pompl et al., 2003).

Caspase-2 was initially described as a neuronally expressed caspase that was

downregulated during brain development (Kumar et al., 1992). Nevertheless, one

paradigm that is supported in both cell culture work as well as primary neurons from

wild-type and caspase-2 null animals is Aβ-induced neuronal death. In this paradigm, the

death pathway is conserved in central and peripheral neurons and in PC12 neuronal cell

line (Troy et al., 2000). Removal of caspase-2 acutely or chronically confers resistance to

Aβ. It is likely that Aβ-mediated death of neurons does not require other caspases

because caspase-2 deficient neurons are resistant to Aβ and, although caspase-3 becomes

activated in response to Aβ, it is neither necessary nor sufficient for death (Troy et al.,

2000). As a therapeutic target, caspase-2 has certain advantages. In the nervous system,

the expression of caspase-2 is neuron-specific; thus, inhibition or ablation of caspase-2 in

the nervous system would only affect neurons. All the evidence suggests that caspase-2

is activated rapidly and is upstream or independent of mitochondria, which makes it an

attractive target for ablation without disturbing mitochondrial function (Troy and Ribe,

2008).

Active caspase-3 has been detected in neurons of AD brains, with a high degree

of colocalization with NFTs and senile plaques (Su et al., 2001). It has recently been


33

shown that caspase-3 is increased and preferentially located in the postsynaptic density

fractions in AD, suggesting an important role for caspase-3 in synapse degeneration

during disease progression (Louneva et al., 2008). In fact, in parallel with its role in

apoptosis, caspase-3 may be also important in AD pathogenesis by cleaving tau in its C-

terminal region; in AD brains, caspase-3-cleaved tau colocalizes with both intracellular

A and activated caspase-3, mainly in tangle bearing neurons. Finally, different caspases,

including caspase-3, have also been shown to cleave APP, generating A-containing

peptides (Gervais et al., 1999; LeBlanc et al., 1999), while amyloid plaques are enriched

in caspase-cleaved APP (Gervais et al., 1999). This suggests that A plaque formation

and toxicity is likely amplified in a caspase-dependent manner. Nevertheless, by cleaving

APP and presenilins, caspase-3 may give rise to C31, which induces cell death through a

caspase-independent process and triggers selective increase of A42 in vitro

(Dumanchin-Njock et al., 2001).

Caspase-9 activity has been specifically implicated in the pathology of AD. It

was demonstrated that neurons and dystrophic neuritis within plaque regions of AD

hippocampal brain sections, display strong activation of caspase-9. Importantly, these

studies suggest that caspase-9 activation precedes NFT formation (Rohn et al., 2002).

Caspase-9 was also shown to directly cleave APP, originating the strong pro-apoptotic

peptide C31 (Lu et al., 2000). Finally, it appears that caspase-9 may induce apoptosis in

AD brains independently of caspase-3 or Apaf-1. This would represent an alternative

circuit to the classical intrinsic pathway of apoptosis in AD (Madden and Cotter, 2008).

Cells from caspase-12 gene deleted mice showed reduced susceptibility towards

ER stress (Nakagawa et al., 2000). Particularly, cultured cortical neurons were resistant

to death caused by the Aβ (Nakagawa et al., 2000). This indicates that ER stress and

activation of caspase-12 may contribute to neuronal death in AD. However, as the

presence of caspase-12 is less likely in human neurons, related molecules with similar

functions may be present. Human caspase-4 shows similar characteristics to mouse

caspase-12 and is localized to the ER and to the mitochondria. Caspase-4 is increased in

AD brains, suggesting that this caspase may play a role in ER stress-induced cell death

(Hitomi et al., 2004).

Altogether, caspases play an active role at distinct levels of A-induced

neurotoxicity and AD. Apart from representing a terminal event in apoptosis associated

with AD, activation of caspases appears to constitute also a proximal event that promotes

Chapter 1

34

the pathology underlying the disease. Nevertheless, caspase-independent pathways of

apoptosis may be relevant to certain neurodegenerative diseases. Such pathways are

mediated through the mitochondrial release of apoptosis-inducing factor (AIF) and/or

EndoG. AIF is normally confined to the mitochondrial intermembrane space, but

translocates to the cytosol and to the nucleus after apoptotic insults. In particular, AIF

has been associated with neuronal death in AD (Adamczyk et al., 2005).

1.2.2.2 Involvement of Bcl-2 family members

Apart from caspases, several other molecules involved in the apoptotic cascade

have also been linked to AD. It has been shown, for instance, that Bcl-2 family members

Bax and Bcl-2 co-localize in the frontal cortex of AD patients (Lu et al., 2005). In

hippocampal tissue, apoptotic proteins c-Fos, c-Jun, and Bak were shown to be increased

(Sajan et al., 2007). In vitro, A induces expression of pro-apoptotic Bcl-2 proteins,

including Bax, and down regulation of anti-apoptotic members such as Bcl-2, Bcl-xL and

Bcl-w (Yao et al., 2005). Increased Bax expression has also been associated with senile

plaques in AD hippocampal brain tissue and neurons with tau-positive NFTs (MacGibbon

et al., 1997). Consistently, A fails to induce apoptosis in Bax-deficient neurons

(Selznick et al., 2000), whereas overexpression of Bcl-xL and Bcl-2 attenuates A-

induced apoptosis (Song et al., 2004; Tan et al., 1999). A also induces Bcl-2-interacting

mediator of cell death (Bim) in cultured hippocampal and cortical neurons. Moreover,

Bim was found to be up-regulated in entorhinal cortical neurons of postmortem AD

human brains (Biswas et al., 2007b).

A successful therapeutic intervention would seem to entail an anti-apoptotic gain

of function and/or a pro-apoptotic loss of function to promote mitochondrial function

stabilization (Letai, 2005). Therefore Bax and BH3-only proteins inhibitors can represent

potential targets for therapeutic intervention in AD and other neurodegenerative

disorders. However, while several small molecule inhibitors of anti-apoptotic members

of the Bcl-2 family have been reported (Jana and Paliwal, 2007), only a few inhibitors of

pro-apoptotic members are described. Bombrun et al described molecules that inhibit

cytochrome c release from mitochondria and suggested that this action was mediated by

inhibition of Bax channel-forming activity (Bax channel blockers) (Bombrun et al.,

2003). Hetz et al later described and evaluated two novel Bax channel inhibitors Bci1

and Bci2 (Hetz et al., 2005). It was demonstrated that these compounds prevent


35

activation of mitochondria-induced apoptosis, augmenting protection against ischemia-

induced tissue damage. Becattini et al (Becattini et al., 2004), using NMR-based

strategies, designed a series of compounds that bind and suppress the BH3-only protein

Bid.

1.2.3 Role of organelle dysfunction in Alzheimer’s disease

When apoptosis was first characterized, cytoplasmic organelles were described

as morphologically intact (Kerr et al., 1972). However, it is now well established that

most organelles of the dying cell manifest subtle biochemical alterations, in particular,

partial proteolysis and membrane permeabilization, which may contribute to the apoptotic

phenotype. Importantly, each organelle can sense stressful and pathogenic alterations that

initiate local or global responses, leading to either adaptation, or cell death if a critical

damage threshold is reached (Fig. 1.10).

Figure 1.10. Schematic overview of organelle dysfunction and cell death mechanisms. The

death ligand interacts with the respective death receptor at the plasma membrane, recruiting

adaptor proteins such as FADD, and activating caspases-8. The initiator caspase then cleaves

effector caspases-3, which activates key downstream targets and executes apoptosis. This process

Chapter 1

36

is mitochondrial independent and can be inhibited through diverse mechanisms, including the

activation of inhibitors of apoptosis (IAPs). Excessive glutamate and other death signals activate

intracellular cascades involving increased levels of ROS, Ca2+, and p53. For example, DNA

damage insults engage the p53 apoptotic pathway and this increases DNA fragmentation. Death

stimuli target mitochondria either directly or through transduction by translocation of Bax and

Bad to the mitochondrial membrane. Mitochondria then release cytochrome c and other

apoptogenic factors into the cytosol. Cytochrome c induces oligomerization of Apaf-1, recruiting

and activating procaspase-9. Activated caspase-9 then cleaves and activates effector caspase-3.

The opening of the permeability transition pore (PTP) occurs in the apoptotic cascade during

Ca2+-dependent cell death, disrupting the mitochondrial structure and releasing apoptogenic

factors. In addition, the ER senses local stress through chaperones, Ca2+-binding proteins and Ca2+

release channels, which might transmit Ca2+ responses to mitochondria. ER stress-induced cell

death is also mediated by the UPR via the proteasome. Similarly, cell death may result from

compromised chaperone-mediated autophagy, a mechanism in equilibrium with the ER. Further,

the GC constitutes a privileged site for the generation of the pro-apoptotic mediator ganglioside

GD3 and facilitates local caspase-2 activation. Finally, axonal transport is critical in sensing and

enhancing cell damage. In this regard, hyperphosphorylated tau impairs axonal transport and leads

to cell death. GC, Golgi complex. From Viana et al., 2010.

1.2.3.1 Mitochondria

Mitochondrial function is compromised in brain cells of AD patients.

Impairment of tricarboxylic acid (TCA) cycle enzymes of mitochondria was found to

correlate with the clinical state of AD (Bubber et al., 2005). Recently, it was

demonstrated that A induces apoptosis in primary hippocampal neurons which derive, at

least in part, from mitochondria dysfunction, as assessed by depolarized membrane

potential, decreased cytochrome c oxidase activity and ATP levels, and cytochrome c

release (Yang et al., 2009). These results indicate that A induces neuronal death by

activating an apoptotic pathway involving impaired mitochondria function and cellular

homeostasis. A is also involved in the disruption of Ca2+

homeostasis, through ER-

mediated stress, thereby inducing mitochondrial dysfunction (Pereira et al., 2004).

Furthermore, A-mediated mitochondrial dysfunction also enhances generation of ROS.

In addition to membrane lipid or protein damage, ROS can mediate DNA damage, which

activates fatal apoptotic signaling through the tumor suppressor p53 and poly(ADP-

ribose) polymerase (PARP) (Culmsee and Landshamer, 2006).


37

Based on the above, it is clear that therapeutic strategies that modulate

mitochondrial-dependent cell death pathways may provide a key tool to protect neurons.

An increasing number of growth factors activate cell survival in neuronal cells. By

activating counter receptors, growth factors stimulate cellular intrinsic pathways, which

lead to the upregulation of cytoprotective proteins (Bogaerts et al., 2008). In addition,

mitochondrial membrane permeabilization can be inhibited by the knockout of

cyclophilin D, a mitochondrial matrix protein, or by the use of cyclosporin A or other

pharmacological inhibitors of cyclophilin D (Nakagawa et al., 2005). Overexpression of

anti-apoptotic members of the Bcl-2 family has a major neuroprotective effect (Mehta et

al., 2007), and the use of peptidomimetics designed to act like Bcl-2 antiapoptotic

proteins should also be evaluated. Antioxidants and small compounds, such as lipoic

acid, cystamine, vitamin E, and carnitine have shown neuroprotective effects in

neurodegenerative conditions (Lee et al., 2009). FK 506, a calcineurin inhibitor, and

Dimebon, an antihistamine drug, are neuroprotective mainly by inducing mitochondria

stabilization (Lee et al., 2009). Nicotinamide, resveratrol, and sirtuins modulate the

tricarboxylic acid cycle in mitochondria and provide neuroprotective effects in

neurodegenerative conditions (Lee et al., 2009). Rosiglitazone, known as a peroxisome

proliferator activated receptor-gamma agonist and an anti-diabetic drug in the

thiazolidinedione class of drugs, was neuroprotective in a subset of patients with AD

(Watson et al., 2005).

These studies suggest that targeting mitochondria, especially the mitochondrial

membrane permeabilization, to block the release of pro-apoptotic proteins and stabilize

energy metabolism may provide optimal neuroprotection.

1.2.3.2 Golgi complex

The involvement of the Golgi complex (GC) in PCD and neurodegenerative

diseases has recently been the focus of intense research (Fan et al., 2008). The GC

undergoes irreversible fragmentation during apoptosis, in part as a result of caspase-

mediated cleavage of several GC-associated proteins. In fact, it has been demonstrated

that GC cisterns are smaller in apoptotic PC12 cells (Sesso et al., 1999). In addition, Fas

receptor mediated and staurosporine-induced apoptosis triggered caspase mediated

cleavage of several GC proteins, including p115 and golgin-160 (Mukherjee et al., 2007;

Mancini et al., 2000). Moreover, GM130, an integral membrane protein, was rapidly

Chapter 1

38

diminished during Fas-mediated apoptosis associated with GC fragmentation (Walker et

al., 2004).

Worthy of note is the demonstration that GC fragmentation occurs in an early,

preclinical stage of neurodegeneration (Karecla and Kreis, 1992; Mourelatos et al., 1996).

As a consequence, neuronal GC integrity has been suggested as a reliable index of initial

degeneration (Stieber et al., 1996).

Although some evidence suggests that GC fragmentation is involved in the

apoptotic process, other results argue that GC fragmentation is not a consequence of

apoptosis (Diaz-Corrales et al., 2004; Gomes et al., 2008). Nevertheless, given its central

location and essential role in membrane trafficking, the GC is ideally positioned to sense

and integrate information regarding the status of the cell. For example, the structure of

the GC might be particularly conducive to sense small changes in lipid composition of the

bilayer (Mancini et al., 2000). In fact, the GC may represent a novel apoptotic signaling

organelle. Interestingly, several signaling proteins implicated in apoptotic pathways are

enriched at GC membranes, including TNF-R1, Fas, phosphatidyl inositol 3-kinase

(PI3K), beclin, GD3 synthase (α-2,8- sialytransferase), and BRUCE, a novel ubiquitin-

conjugating enzyme that contains a baculovirus inhibitor of apoptosis (BIR) motif. GC

membranes are also likely to be the site of ceramide signaling, which has been implicated

in the induction of apoptosis by conversion of ceramide to ganglioside GD3 (De Maria et

al., 1997; Rippo et al., 2000). Finally, substantial evidence indicates that the GC might

participate as a functional Ca2+

storage as it contains all Ca2+

/manganese-ATPases

(SPCAs), and is regarded as a inositol 1,4,5- trisphosphate (IP3)-sensitive intracellular

Ca2+

store. The GC accommodates IP3Rs and neuronal Ca2+

sensor, and possesses Ca2+

binding proteins Cab45 and CALNUC in its lumen. This organelle takes up Ca2+

using

both sarco(endo)-plasmic-reticulum Ca2+

-ATPases (SERCA) and secretory pathway

SPCAs. Thus, the integrity of the GC is required for normal Ca2+

homeostasis and

signaling (reviewed in Fan et al., 2008). It is expected that continuous attention to the GC

function should provide a more detailed and accurate understanding of disease

mechanisms and potential drug targets during neurodegeneration.

1.2.3.3 Endoplasmic reticulum

Several studies demonstrated ER stress and disturbed Ca2+

homeostasis in AD

pathogenesis (LaFerla, 2002). The involvement of ER stress in AD has been confirmed


39

by detection of hyperactivated PERK-eIF2α pathway (Unterberger et al., 2006). In

addition, PS1 and PS2 proteins, members of the multiprotein α-secretase complex that

mediates the intramembranous cleavage of APP, are abundantly expressed in neurons and

localized in the ER membrane, further suggesting a link between AD and ER stress

(Lindholm et al., 2006). Mutant PS1-expressing cells show altered Ca2+

homeostasis

(Leissring et al., 2000), increased Aβ production (Levitan et al., 2001), and enhanced

sensitivity to ER stress-induced apoptosis (Lindholm et al., 2006), (Terro et al., 2002).

Similarly, PS1 mutant transgenic mice display abnormalities in ER Ca2+

regulation and

increased neuronal vulnerability toward cell death and excitotoxic injury. It has also been

demonstrated that mutant PS1 binds to and inhibits the UPR protein IRE1, suppressing

the activation of the UPR (Katayama et al., 1999). IRE1 activates downstream signals,

including the transcription of the GRP78/Bip (Katayama et al., 2004). Recent studies

revealed increased levels of GRP78/Bip and PERK in AD brains, further confirming the

occurrence of ER stress in AD (Hoozemans et al., 2005), (Hoozemans et al., 2009).

Finally, ER stress can directly activate caspase-12. Furthermore, primary cortical neurons

from caspase-12 knockout mice showed reduced susceptibility towards ER stress. On the

other hand, neurons were resistant to Aβ-induced cell death (Nakagawa et al., 2000),

suggesting that ER stress and activation of caspase-12 may contribute to neuronal death

in AD.

Targets for drug discovery in the context of ER stress in neurodegeneration are

still under validation. A screen for compounds that block tunicamycin-induced death of a

neuronal cell line resulted in the discovery of salubrinal, a compound that prevents

dephosphorylation of eIF2α. This results in increased eIF2α phosphorylation and

inactivation, thereby generating stronger PERK responses (Boyce et al., 2005).

Salubrinal was reported to protect neuronal cells from death triggered by various ER

stress inducers (Reijonen et al., 2008; Smith et al., 2005). However, this inhibitor of

eIF2α activity also impairs long-term memory in a mouse model (Costa-Mattioli et al.,

2007), suggesting that the approach would not be acceptable as a direction for chronic

therapies.

It has been described that chemical inhibitors of ASK1 might provide

cytoprotection in neurodegenerative disorders (Kawaguchi et al., 2008). As JNK is

activated downstream of ASK1, and JNK is known to activate Bid while inhibiting Bcl-2,

it would be interesting to explore whether chemical inhibitors of JNK may have

cytoprotective activity in such context (Salh, 2007).

Chapter 1

40

An additional potential strategy for ameliorating ER stress induced by inclusion

bodies is to stimulate autophagy, efficiently clearing insoluble protein aggregates from

cells. Chemical screens for enhancers of autophagy have been reported, which have

identified compounds that improve clearance of protein inclusions from cultured cells ,

(Zhang et al., 2007), (Sarkar et al., 2007) . Among the compounds purported to increase

autophagy, without signs of cellular toxicity, are several drugs already approved by the

US Food and Drug Administration. These include antipsychotics (such as fluspirilene,

trifluoperazine, pimozide) and Ca2+

-channel modulators (such as nicardipine, niguldipine,

amiodarone), acting through mechanisms that are distinct from that of rapamycin (a

mammalian target of rapamycin (mToR) inhibitor) (Zhang et al., 2007). A different

strategy proposed for removing insoluble protein inclusions is to increase chaperone

activity in cells, especially cytosolic hsp70. It would be interesting to explore the efficacy

of chemical chaperones that serve as ligands to stabilize protein structure and promote

protein folding, analogous to what has been described for compounds such as

SR121463A (1-[4-(Ntertbutyl carbamoyl)-2-methoxybenzene sulphonyl]-5-ethoxy-3-

spiro-[4-(2-morpholinoethoxy)cyclohexane] indol-2-one, fumarate) (Serradeil-Le Gal et

al., 1996), and others (Burrows et al., 2000; Tamarappoo and Verkman, 1998). Thus,

previous studies are encouraging and suggest that targeting the cellular protein quality

control system of the ER is an attractive strategy that warrants further exploitation for the

treatment of neurodegenerative conditions associated with accumulation of damaged

molecules within the cell.

1.2.4 Inhibition of neuronal apoptosis by bile acids

The therapeutic role of the endogenous bile acid ursodeoxycholic acid (UDCA)

has been firmily established in the treatment of liver diseases (Lazaridis et al., 2001;

Paumgartner and Beuers, 2002). Interestingly, after conjugation with taurine,

tauroursodeoxycholic acid (TUDCA) administrated in high doses can be delivered to

other tissues, including the brain (Rodrigues et al., 2003a). In vitro, TUDCA inhibits

apoptosis induced by several stimuli in diverse cell types, such as in neuronal cells (Fig.

1.11) (Rodrigues et al., 2000b; Solá et al., 2003). Furthermore, the protective role of

TUDCA has been extended to several mouse models of neurological disorders, including

Hungtington‟s disease (Keene et al., 2002; Keene et al., 2001; Rodrigues et al., 2000b),

Parkinson‟s disease (Duan et al., 2002; Ved et al., 2005), and acute ischemic (Rodrigues


41

et al., 2002) and hemorrhagic stroke (Rodrigues et al., 2003a). Interestingly, pretreatment

with TUDCA also significantly reduced glutamate-induced apoptosis of rat cortical

neurons (Castro et al., 2004).

Prompted by these results, the anti-apoptotic role of TUDCA has been

investigated in experimental models of AD. Initial studies using primary rat neurons and

astrocytes incubated with Aβ showed increased levels of apoptosis, which was prevented

by TUDCA and UDCA (Rodrigues et al., 2000a). The anti-apoptotic effects of TUDCA

were further confirmed in PC12 neuronal cells incubated with Aβ (Ramalho et al., 2004).

Similar results were seen in in vitro models of familial AD that consist of mouse

neuroblastoma cells expressing APP with the Swedish mutation or double-mutated human

APP and PS1 (Ramalho et al., 2006), as well as in primary cortical neurons incubated

with fibrillar Aβ42. Of note, in vitro cellular models of AD have limitations to their use

and the results should be interpreted with caution.

Because of their potential role in Aβ-induced toxicity, mitochondria were

isolated and co-incubated with TUDCA to assess its protective effect against apoptosis.

In fact, UDCA and, more efficiently, TUDCA inhibited Aβ-induced mitochondrial

membrane permeabilization and consequent cytochrome c release in isolated neuronal

mitochondria (Rodrigues et al., 2000a). Moreover, using electron paramagnetic

resonance spectroscopy analysis, it was demonstrated that TUDCA prevented Aβ-driven

modifications in mitochondrial membrane redox status, lipid polarity and protein order

(Rodrigues et al., 2001). The effects of TUDCA on the Aβ-affected mitochondria were

further confirmed by the observation of decreased Bax translocation in the presence of

this bile acid (Solá et al., 2003).

Consistent with results obtained in non-neurological models, it was also shown

that TUDCA might interfere with upstream targets of mitochondria, including cell cycle

related proteins (Fig. 1.11). In fact, TUDCA incubations resulted in inhibition of E2F-1

induction, p53 stabilization and Bax expression in PC12 neuronal cells (Ramalho et al.,

2004). Further, TUDCA protected PC12 cells against p53- and Bax-dependent apoptosis

induced by E2F-1 and p53 overexpression, respectively. The role of p53 in mediating the

effects of TUDCA was further confirmed using an in vitro model of familial AD

(Ramalho et al., 2006). In neuroblastoma cells, TUDCA modulated p53 activity and Bcl-

2 family changes. Moreover, overexpression of p53 was sufficient to induce apoptosis,

which was in turn reduced by TUDCA. It is still not clear, however, how TUDCA

interferes with p53 expression and activation. Importantly, TUDCA interacts with a

Chapter 1

42

specific region of the mineralocorticoid receptor (MR) ligand-binding domain and

dissociates it from hsp90 in neuronal cells incubated with Aβ (Solá et al., 2006). As a

consequence, TUDCA translocates into the nucleus with MR, modulating its

transactivation and inhibiting Aβ-induced apoptosis. The elucidation of the molecular

targets affected by the TUDCA-mediated regulation of MR, which might include p53,

would be important to fully understand the anti-apoptotic action of the bile acid in

neuronal acids. In fact, it was demonstrated recently that UDCA reduces p53

transcriptional activity and its ability to bind DNA in hepatocytes (Amaral et al., 2007).

Thus, it is tempting to speculate that a similar effect would occur in neurons exposed to

Aβ.

Figure 1.11. Proposed mechanisms of UDCA and TUDCA inhibition of apoptosis.UDCA

negatively modulates the mitochondrial pathway by inhibiting Bax translocation, ROS formation,

cytochrome c release, and caspase-3 activation. UDCA can also interfere with the death receptor

pathway, inhibiting caspase-3 activation. Moreover, TUDCA inhibits apoptosis associated with

ER stress, by modulating intracellular Ca2+ levels, and inhibiting calpain and caspase-12

activation. Importantly, UDCA interacts with NSR, leading to NSR/hsp90 dissociation and

nuclear translocation of the UDCA/NSR complex. Once in the nucleus, UDCA modulates the


43

E2F-1/p53/Bax pathway, thus preventing apoptosis. Finally, UDCA downregulates cyclin D1 and

Apaf-1, further inhibiting the mitochondrial apoptotic cascade. Cyt c, cytochrome c; hsp90, heat

shock protein 90; NSR, nuclear steroid receptor. From Amaral et al., 2009.

Notably, TUDCA prevented caspase-2 activation in neuroblastoma cells. A

recent study suggests that Aβ-induced cell death also required the activation of caspase-2

(Troy et al., 2000). Caspase-2 triggers apoptosis through activation of the mitochondrial

pathway and it has also been described as a target of the JNK pathway. Further

investigations are warranted to elucidate the mechanism(s) by which TUDCA interferes

with this signaling pathway. Finally, recent data also showed that TUDCA modulates

Aβ-induced apoptosis, in part, by activating a PI3K signaling pathway, underscoring the

pleiotropic effect of the bile acid (Solá et al., 2003). In fact, incubation with wortmannin,

an inhibitor of the PI3K pathway, significantly reduced the ability of TUDCA to

modulate p53-induced apoptosis (Ramalho et al., 2006).

TUDCA also mitigates the toxic downstream effects of Aβ. In primary rat

cortical neurons incubated with fibrillar Aβ42, TUDCA inhibited the levels of apoptosis

and caspase-3 activation and abrogated the caspase-3-cleavage of tau into a toxic species

(Ramalho et al., 2008). Thus, by interfering with apoptotic pathways, both at the

mitochondrial and transcriptional levels, TUDCA not only increased the survival of

neurons but also prevented downstream abnormal conformations of tau. This might have

beneficial consequences in slowing cognitive decline.

In the last decade, we assisted to a major advance in understanding the role of

anti-apoptotic bile acids as neuroprotective agents. The association of the ubiquitous

anti-apoptotic properties of certain hydrophilic bile acids with their safety profile, renders

this group of molecules as a potential therapeutic alternative in neurodegenerative

disorders. However, the exact cellular mechanism(s) of anti-apoptotic bile acid effects

are not clearly understood. Thus, further exploitation of molecular pathways by which

these molecules modulate cell death is warranted to consolidate our understanding

therapeutic potential of specific bile acids.

Chapter 1

44

45

Objectives

The studies presented in this thesis were driven by the ambition to further

understand cell death mechanisms in AD, with the hopes to contribute to rational

therapeutic strategies. Supported by the knowledge that apoptosis plays a role in

neurodegeneration associated with AD, we designed three major objectives. First, our

goal was to determine the physical properties of Aβ peptide toxic assemblies and

elucidate the cellular mechanism(s) of toxicity. Second, we aimed at investigating stress

signaling events triggered by soluble Aβ at the ER level and specifically characterizing

the JNK stress pathway. Finally, we intended to further explore alternative pathways of

neuroprotection by TUDCA.

The specific questions addressed in this thesis are:

1. Are pro-apoptotic properties of Aβ peptides associated with their distinct

mechanisms of aggregation/fibrillization?

2. Does soluble Aβ trigger early stress signaling cascades involving the ER?

3. What is the role of JNK as early stress sensor of Aβ toxicity that is activated

by the ER?

Ultimately, the overarching goal of the research presented in this thesis is to

expand the understanding of the pathological mechanisms of AD. The conjugation of this

information with existing anti-apoptotic tools, such as the use of TUDCA, will likely

provide valuable insights to develop novel, more effective therapeutic strategies for AD.

2 Tauroursodeoxycholic acid prevents E22Q

Alzheimer’s amyloid β toxicity in human

cerebral endothelial cells

Ricardo J. S. Viana1, Ana F. Nunes

1, Rui E. Castro

1, Rita M. Ramalho

1, Jordana

Meyerson2, Silvia Fossati

2, Jorge Ghiso

2,3, Agueda Rostagno

2, Cecília M. P. Rodrigues

1

1Research Institute for Medicines and Pharmaceutical Sciences, Faculty of Pharmacy,

University of Lisbon, Lisbon, Portugal; and Departments of 2Pathology and

3Psychiatry,

New York University School of Medicine, New York, NY, USA

Cellular and Molecular Life Sciences 2009; 66: 1094-1104

Chapter 2

48

Reprinted from Cellular and Molecular Life Sciences, vol 66, Issue 6, R. J. S. Viana, A.

F. Nunes, R. E. Castro, R. M. Ramalho, J. Meyerson, S. Fossati, J. Ghiso, A. Rostagno

and C. M. P. Rodrigues. Tauroursodeoxycholic acid prevents E22Q Alzheimer‟s Aβ

toxicity in human cerebral endothelial cells, pages 1094-1104, Copyright 2009, with

permission from ©Birkh_user Verlag, Basel, 2009. All rights reserved.

TUDCA prevents A Dutch mutant toxicity

49

2.1 Abstract

The vasculotropic E22Q mutant of the amyloid β (Aβ) peptide is associated with

hereditary cerebral hemorrhage with amyloidosis Dutch type. The cellular mechanism(s)

of toxicity and nature of AβE22Q toxic assemblies are not completely understood.

Comparative assessment of structural parameters and cell death mechanisms elicited in

primary human cerebral endothelial cells by AβE22Q, and wild-type Aβ revealed that

only AβE22Q triggered the Bax mitochondrial pathway of apoptosis. AβE22Q neither

matched the fast oligomerization kinetics of Aβ42 nor reached its predominant β-sheet

structure, achieving a modest degree of oligomerization with a secondary structure that

remained a mixture of β- and random-conformations. The endogenous molecule

tauroursodeoxycholic acid (TUDCA) was a strong modulator of AβE22Q-triggered

apoptosis but did not significantly change secondary structures and fibrillogenic

propensities of Aβ peptides. These data dissociate the pro-apoptotic properties of Aβ

peptides from their distinct mechanisms of aggregation/fibrillization in vitro, providing

new perspectives for modulation of amyloid toxicity.

Keywords: Apoptosis; cerebral amyloid angiopathy; E22Q mutant Aβ peptide;

mitochondria; TUDCA

Chapter 2

50

2.2 Introduction

Cerebral amyloid angiopathy (CAA), a common feature of Alzheimer‟s disease

(AD), is an age-associated condition characterized by the deposition of amyloid peptides

in cortical and leptomeningeal vessels causing capillary disruption and endothelium

dysfunction, and playing a significant role in intracerebral hemorrhage (Coria and Rubio,

1996; Rensink et al., 2003; Vinters, 1987). Different proteins are associated with CAA

(reviewed in Revesz et al., 2003; Yamada, 2000). The most common is amyloid β (Aβ),

which is the main component of the deposits seen in normal aging and sporadic

Alzheimer‟s disease (AD) that originates by processing of the amyloid precursor protein

(APP) (reviewed in Ghiso and Frangione, 2002).

Of the several mutations identified in the APP gene, substitutions at residues

flanking the Aβ region affect the rate of enzymatic APP processing and the production of

Aβ42, resulting in an early-onset AD phenotype (Selkoe, 1999). On the contrary,

mutations within the Aβ sequence, particularly at residues 21-23, generate Aβ variants

Dutch, Iowa, Flemish, Artic, and Italian (Grabowski et al., 2001; Hendriks et al., 1992;

Levy et al., 1990; Miravalle et al., 2000) that preferentially associate with CAA,

hemorrhagic strokes and/or dementia. One of the most aggressive clinical phenotypes

described in familial AD is associated with a glutamine to glutamic acid substitution at

residue 22 (E22Q), found in a disorder known as hereditary cerebral hemorrhage with

amyloidosis Dutch type (HCHWA-D) (Levy et al., 1990). The disease is characterized by

recurrent strokes and vascular dementia in the absence of neurofibrillar pathology, with

fatal cerebral bleeding resulting from the massive amyloid deposition in leptomeningeal

vessels and cortical arteries and arterioles (reviewed in Zhang-Nunes et al., 2006).

Parenchymal mature plaques characteristic of AD are rare in this kindred, while diffuse

preamyloid deposits are relatively frequent and show a remarkable relationship with age.

They are more abundant in younger patients, suggesting that these lesions may develop

into the less profuse mature fibrillar counterparts (Maat-Schieman et al., 2005; Maat-

Schieman et al., 2000; Natte et al., 2001).

In general, Aβ mutants have been demonstrated to exert stronger toxicity than

the wild-type counterparts in both neuronal (Murakami et al., 2003) and cerebrovascular

cells (Davis and Van Nostrand, 1996; Eisenhauer et al., 2000; Miravalle et al., 2000; Van

Nostrand et al., 2001), albeit striking differences exist among the variants themselves.

Differences in toxicity likely correlate with distinct structural properties conferred by the


51

amino acid substitutions, which typically translate into enhanced fibrillogenic properties

(Nilsberth et al., 2001; Wisniewski et al., 1991). These, in turn, influence the onset and

aggressiveness of the respective clinical phenotypes (reviewed in Ghiso and Frangione,

2002).

One of the most studied mutants is the aggressive intra-Aβ genetic variant E22Q.

Following intraventricular injection in rats, this variant dramatically disrupts synaptic

plasticity. Together with the Arctic (E22G) mutant, it is 100-fold more potent than wild-

type Aβ40 at inhibiting long-term potentiation (Klyubin et al., 2004). The Dutch Aβ

peptide has also been shown to exert potent effects on vessel wall cells, exhibiting anti-

angiogenic properties (Paris et al., 2005) and inducing apoptosis in cerebral endothelial

and smooth muscle cells, under conditions in which wild-type Aβ had no effects (Davis

and Van Nostrand, 1996; Eisenhauer et al., 2000; Miravalle et al., 2000; Van Nostrand et

al., 2001). Nevertheless, the cellular mechanism(s) by which Aβ mutants induce toxicity

remains poorly understood and requires further investigation.

Tauroursodeoxycholic acid (TUDCA) is an endogenous bile acid known to play

a key role in modulating the apoptotic threshold in various cells types by interfering with

classical mitochondrial pathways of cell death (Rodrigues et al., 1998). Its antiapoptotic

effects have been demonstrated in several pathological conditions, including different

neurological disorders (Colak et al., 2008; Duan et al., 2002; Keene et al., 2002; Macedo

et al., 2008; Rodrigues et al., 2003a). TUDCA modulates Aβ-induced cell death by

inhibiting the mitochondrial pathway of apoptosis and enhancing survival signaling in rat

cortical neurons (Solá et al., 2003). Moreover, it can interfere with targets upstream of

mitochondria, including cell cycle-related proteins E2F-1 and p53 (Ramalho et al., 2004),

through functional modulation of nuclear steroid receptors (Solá et al., 2006). These data

strongly suggest that TUDCA may also be beneficial in reducing other manifestations of

brain amyloid toxicity, such as CAA.

Many studies have been devoted to elucidate the mechanisms of Aβ

neurotoxicity. However, there is limited information available regarding the effect(s) of

amyloid peptides on endothelial vessel wall cells that are in immediate contact with the

vascular amyloid deposits in CAA. In this study, we compared aggregation/fibrillization

properties, secondary structure, and cell death pathways of the Dutch variant and wild-

type Aβ40 and Aβ42, both in the presence and absence of TUDCA. Our data dissociate

the apoptotic properties of Aβ peptides in human cerebral endothelial cells and their

Chapter 2

52

distinct mechanisms of aggregation in vitro, while providing new perspectives for

modulation of amyloid-induced apoptosis.

2.3 Materials and methods

2.3.1 Peptide synthesis

Synthetic wild-type Aβ40 and Aβ42 peptides as well as Aβ40 peptide containing

the E22Q substitution were synthesized by James I. Elliott at Yale University using N-

tert-butyloxycarbonyl chemistry and then purified by reverse phase-high performance

liquid chromatography on a Vydac C4 column (Western Analytical, Murrieta, CA)

(Ghiso et al., 2004; Miravalle et al., 2000). Molecular masses were corroborated by

matrix-assisted

laser desorption ionization time-of-flight (MALDI-TOF) mass

spectrometry, and concentration assessed by amino acid analysis. All peptides were

supplied as single components eluted from the reverse phase columns with experimental

molecular masses of 4329.41 Da for Aβ40 (theoretical mass, 4329.86 Da), 4514.24 Da

for Aβ42 (theoretical mass, 4514.10 Da), and 4328.80 Da for Aβ40E22Q (theoretical

mass, 4328.87 Da).

2.3.2 Peptide aggregation

Synthetic Aβ homologues were dissolved to 1 mM in hexafluoro-isopropanol

(HFIP; Sigma Chemical Co., St. Louis, MO), a pre-treatment that breaks down -sheet

structures and disrupts hydrophobic forces leading to monodisperse A preparations

(Dahlgren et al., 2002). Following lyophilization to remove HFIP, peptides were

subsequently solubilized in deionized water and added of an equal volume of a 2x-

concentrated phosphate buffered saline (PBS), pH 7.4, to a final concentration of 1 mg/ml

in 1 x PBS. Peptides, in the presence or absence of 100 µM TUDCA (Sigma), were

either incubated at 37oC for up to 48 h for the aggregation studies or diluted into culture

media at the required concentration for the toxicity experiments. For the aggregation

studies, structural properties were assessed by Western blot analysis, circular dichroism

(CD) spectroscopy, and Thioflavin T binding.


53

2.3.3 Peptide structural analysis

For the Western blot analysis, 200 ng aliquots of each peptide either freshly

solubilized or after 24 and 48 h of agregation, in the presence or absence of TUDCA,

were separated on 16.5% Tris-Tricine SDS-PAGE. After electrophoresis, proteins were

electrotransferred to nitrocellulose membranes (Hybond-ECL; GE Healthcare Life

Sciences, Piscataway, NJ) using 10 mM 3-cyclohexylamino-1-propanesulfonic acid

(Sigma) buffer, pH 11.0, containing 10% (v/v) methanol, at 400 mA for 2 h. After

blocking in 5% nonfat milk in PBS containing 0.1% Tween 20, the membranes were

immunoreacted with a combination of mouse monoclonal anti-Aβ antibodies 4G8

(epitope: residues Aβ18-22) and 6E10 (epitope: residues Aβ3-8), both from Covance

(Princeton, NJ), at a 1/3000 dilution each, followed by incubation with horseradish

peroxidase (HRP)-labeled F(ab‟)2 anti-mouse IgG (1/5000; GE Healthcare Life

Sciences). Fluorograms were developed with SuperSignal West Pico Chemiluminescent

Substrate (Thermo Scientific, Rockford, IL) and exposed to Hyperfilm ECL (GE

Healthcare Life Sciences).

The secondary structure of Aβ peptides was estimated by CD spectroscopy as

previously described (Ghiso et al., 2004; Miravalle et al., 2000). Spectra in the far-UV

light (190–260 nm; band width: 1 nm; intervals: 1 nm; scan rate: 60 nm/min) yielded by

the different peptides at each time point of aggregation were recorded at 24°C with a

Jasco J-720 spectropolarimeter (Jasco Corp., Tokyo, Japan), using a 0.2 mm-path quartz

cell and a peptide concentration of 1 mg/ml. For each sample, 15 consecutive spectra

were obtained, averaged and baseline-subtracted. Results were expressed in terms of

mean residue ellipticity (degree-cm2 dmol

_1) (Kelly et al., 2005).

Thioflavin T binding was assessed essentially as previously described (LeVine,

1999; Walsh et al., 1999). Six µl aliquots of each of the peptide aggregation time-point

samples were added of 10 µl of 0.1 mM Thioflavin T (Sigma) and 50 mM Tris-HCl

buffer, pH 8.5 to a final volume of 200 µl. Fluorescence was recorded after 300 s in a

LS-50B luminescence spectrometer (Perkin Elmer, Waltham, MA) with excitation and

emission wavelengths of 435 nm (slit width = 10 nm) and 490 nm (slit width = 10 nm),

respectively. Each sample was analyzed in duplicate.

Chapter 2

54

2.3.4 Culture of human brain microvascular endothelial cells

Primary human cerebral endothelial cells (HCEC; Cell Systems, Kirkland, WA)

were grown in CS-C Complete Medium on dishes coated with Attachment Factor (Cell

Systems), at 37ºC in a 5% CO2 atmosphere. For cell death assays and

immunocytochemistry, cells were plated at a density of 4x104

cells/ml. For

immunoblotting, cells were plated at a density of 1x105

cells/ml. In all cases, cells were

allowed to rest for 1 day prior to treatment with the Aβ peptides.

2.3.5 Induction of apoptosis

HFIP pre-treated Aβ peptides were dissolved to 500 µM in PBS, as indicated

above, and subsequently added of warm F12K Nutrient Mixture (Invitrogen/Gibco,

Carlsbad, CA) supplemented with 1% fetal bovine serum (FBS; Invitrogen/Gibco) to a 50

µM final concentration. These freshly prepared peptide solutions were incubated with

HCEC, cultured as described above, for 12 or 24 h. In co-incubation experiments, 100

µM TUDCA was added to the cell cultures 12 h prior to the addition of Aβ peptides. As

negative controls, cells were separately incubated either in the absence of amyloid

peptides, or with TUDCA alone. In all cases, attached cells were either fixed for Hoechst

staining and immunocytochemistry analysis, or processed for viability assays. For

Western blot analysis, attached and floating cells were combined prior to the preparation

of cytosolic and mitochondrial protein fractions and subsequent protein extraction.

2.3.6 Measurement of cell death

Cell viability was measured by trypan blue exclusion and by the lactate

dehydrogenase (LDH) viability

assay (Sigma) according to the manufacturer's

instructions. Apoptotic nuclei were detected by Hoechst labeling after cell fixation with

4% formaldehyde in PBS, for 10 min at room temperature. Following incubation with

Hoechst dye 33258 (Sigma) at 5 g/mL in PBS for 5 min, and PBS washes, slides were

mounted with PBS:glycerol (3:1, v/v) and fluorescence visualized with an Axioskop

fluorescence microscope (Carl Zeiss GmbH, Hamburg, Germany). Fluorescent nuclei

were scored and categorized according to the condensation and staining characteristics of

chromatin. Normal nuclei showed non-condensed chromatin dispersed over the entire

nucleus. Apoptotic nuclei were identified by condensed and fragmented chromatin

contiguous to the nuclear membrane, as well as nuclear fragmentation and presence of


55

apoptotic bodies. Three random microscopic fields per sample of approximately 250

nuclei were counted and mean values expressed as the percentage of apoptotic nuclei.

2.3.7 Immunocytochemistry

After 24 h of exposure to the respective amyloid peptides, both in the presence

and absence of TUDCA, HCEC were either directly processed for mitochondria labeling

in live cells or fixed in 4% formaldehyde in PBS as above for the immunocytochemical

evaluation of cytochrome c and Bax. Staining of mitochondria in Aβ-challenged cells

was performed by incubation for 30 min with 100 nM Red-Fluorescent MitoTracker

Probe (Invitrogen/Molecular Probes, Eugene, OR) to allow for the selective incorporation

of the dye and subsequent staining of the organelles. Immunofluorescence signals were

evaluated using fluorescence microscopy as above. Bax mitochondrial immuno-

colocalization was performed with the Tyramide Signal Amplification Kit

(Invitrogen/Molecular Probes), according to the manufacturer´s instructions. Briefly,

cells were fixed with 4% formaldehyde as above, washed with PBS, and incubated with

1% blocking reagent, 0.1% Triton X-100 for 1 h, at room temperature. Incubation with

mouse monoclonal anti-Bax antibody (Santa Cruz Biotechnology, Santa Cruz, CA) (1:50

in blocking solution; overnight, 4ºC) was followed by HRP-conjugated goat anti-mouse

IgG (Bio-Rad Laboratories, Hercules, CA) (1:200; 1 h, room temperature). Cells were

subsequently incubated with Alexa Fluor 350-labeled tyramide (1:100 in amplification

buffer/0.0015% H2O2; 10 min, room temperature), washed with PBS, and mounted in

PBS: glycerol (3:1, v/v). Fluorescence was visualized as above.

For cytochrome c immunostaining, after Aβ challenge and fixation, HCEC were

incubated for 1 h with PBS containing 0.3% Triton X-100 (PBST) and 20 mg/ml bovine

serum albumin (BSA). This was followed by 2 h incubation with monoclonal antibody

anti-cytochrome c (BD Biosciences, Franklin Lakes, NJ; 1:200 in PBST containing 5

mg/ml BSA) and 1 h reaction with Alexa Fluor 488 conjugated anti-mouse secondary

antibody (Invitrogen; 1:200 in PBST with 5 mg/ml BSA). Slides were mounted with

Dapi-containing Mounting Medium (Vectashield, Burlingame, CA) and images acquired

in a Nikon Eclipse E-800 Deconvolution Microscope, using Image-Pro Plus software

(Media Cybernetics, Silver Springs, MD) and Autodeblur (AutoQuant Imaging, Inc.,

Watervliet, NY) for deconvolution. Specificity of immune detection was assessed by

omission of the primary antibody.

Chapter 2

56

2.3.8 Bax translocation and cytochrome c release

Sub-cellular distribution of Bax and cytochrome c in amyloid-challenged HCEC

was determined using mitochondrial and cytosolic protein extracts. Briefly, cells were

collected by centrifugation at 600g for 5 min at 4°C. The pellets were washed once in

ice-cold PBS and resuspended with 3 volumes of isolation buffer (20 mM HEPES/KOH,

pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT)

supplemented with Complete protease inhibitor cocktail tablets (Roche Diagnostics

GmbH, Mannheim, Germany), in 250 mM sucrose. After chilling on ice for 15 min, cells

were disrupted by 40 strokes of a glass homogenizer, and homogenates were centrifuged

twice at 2500g for 10 min at 4°C to remove unbroken cells and nuclei. The supernatants

were further centrifuged at 12000g for 30 min at 4°C, and the pellets containing the

mitochondrial fraction resuspended in isolation buffer and frozen at -80°C. For cytosolic

proteins, the 12000g supernatants were collected, filtered sequentially through 0.2 μm and

0.1 μm Ultrafree MC filters (Millipore, Bedford, MA) to remove other cellular organelles,

and frozen at -80°C. Protein concentrations of the subcellular fractions were determined

using the Bio-Rad protein assay kit according to the manufacturer‟s specifications. For

Western blot detection of cytochrome c and Bax, typically 10 μg of mitochondrial and 50

μg of cytosolic proteins were separated on 15% SDS-PAGE and transferred onto

nitrocellulose membranes. After sequentially blocking with 15% H2O2 and 5% non-fat

milk (1 h, room temperature), membranes were separately incubated overnight at 4°C

with mouse monoclonal anti-Bax (Santa Cruz Biotechnology) and anti-cytochrome c

(PharMingen, San Diego, CA) diluted 1:500, and 1:2000, respectively. Finally,

immunoblots were incubated with HRP-conjugated secondary antibodies (Bio-Rad

Laboratories; 1:5000, 3 h, room temperature) and processed for chemiluminescence

detection using the SuperSignal Chemiluminescent Substrate (Thermo Scientific).

Mitochondrial contamination of the cytosolic protein extracts was assessed with

cytochrome c oxidase II (Cox II; Santa Cruz Biotechnology; 1:200). Cox II and -actin

(Sigma; 1:25000) were used as loading controls for mitochondrial and cytosolic fractions,

respectively.


57

2.3.9 Statistical analysis

Statistical analysis was performed using GraphPad InStat version 3.00 (Graph-

Pad Software) for the analysis of variance and Bonferroni‟s multiple comparison tests.

Values of p < 0.05 were considered significant.

2.4 Results

2.4.1 TUDCA does not affect Aβ peptide aggregation and secondary structure

It has been postulated that Aβ toxicity correlates with peptide aggregation

propensity. Based on our previous findings demonstrating an inhibitory role of TUDCA

on Aβ neurotoxicity, we studied its effect on the aggregation/fibrillization properties of

the Dutch variant comparing with wild-type Aβ40 and Aβ42. As indicated in Figure

2.1A, wild-type Aβ40 remained mostly monomeric up to 48 h of incubation, with the

appearance of only faint dimeric bands on Western blot analysis at this time point. In

contrast, as expected, Aβ42 showed high tendency to aggregate. Dimers and even faint

tetrameric species were observed immediately after solubilization, whereas high

molecular mass aggregates were readily visible at 24 h and accentuated at 48 h. AβE22Q

showed an intermediate behavior with the formation of SDS-resistant oligomeric

assemblies clearly noticeable at 48 h incubation but of lower molecular masses than those

of Aβ42. Notably, in all cases, co-incubation with TUDCA resulted in no significant

changes in the aggregation properties of the different peptides.

The effect of TUDCA on the fibrillogenic propensity of the peptides was

assessed by Thioflavin T binding. Co-incubation with TUDCA did not modify the

inherent properties of each peptide (Fig. 2.1B). In fact, Aβ42 continued to express the

highest fluorescence values, while Aβ40 levels remained negligible even at 48 h.

AβE22Q showed an intermediate behavior, in agreement with the aggregation pattern

shown in Fig. 2.1A. Secondary structure analysis by CD spectroscopy illustrated both the

different structural characteristics of the peptides and the lack of effect of TUDCA (Fig.

2.1C). The initial Aβ40 structure showed a minimum at 198 nm, characteristic of

unordered conformations. Within 48 h of incubation, the peptide acquired a β-sheet-rich

structure showing the typical minimum at 218 nm in the CD scan. Aβ42 showed a

predominantly β-sheet conformation even immediately after solubilization. AβE22Q

exhibited an intermediate structural configuration, in agreement with the data illustrated

in Fig. 2.1A and B, with a mixture of random and β-sheet components when freshly

Chapter 2

58

solubilized and an increase in the β-sheet component at 48 h. However, AβE22Q did not

achieve the predominant β-sheet conformation of the Aβ42 in the time frame of the

experiments. Corroborating the Western blot and Thioflavin T data, co-incubation with

TUDCA did not induce conformational changes in the secondary structures of any of the

Aβ peptides studied.

Figure 2.1. Structural studies of wild-type and Dutch-variant Aβ peptides. HFIP-treated

peptides at 1 mg/ml in PBS were aggregated at 37oC for up to 48 h. Peptide

aggregation/fibrillization propensity as well as secondary structure were analyzed both in the

presence or absence of 100 µM TUDCA. (A) Oligomerization of wild-type and Dutch-variant

peptides assessed by Western blot probed with a combination of monoclonal anti-Aβ antibodies

4G8 and 6E10 after separation on 16.5% Tris-Tricine SDS-PAGE. (B) Fibrillization of wild-type

and Dutch-variant peptides estimated by fluorescence evaluation (Excitation/Emission

wavelengths 435 nm/490 nm, respectively) after Thioflavin T binding. Solid lines, Aβ peptides

alone; broken lines, Aβ peptides in the presence of TUDCA. (C) Secondary structure of wild-

type and Dutch-variant peptides assessed through the respective spectra in the far-UV light (190-

260 nm) in a Jasco J-720 spectropolarimeter via a 0.2 mm-path quartz cell, at 24 (top) and 48 h


59

(bottom) of aggregation. Blue, Aβ peptides alone; red, Aβ peptides in the presence of TUDCA;

green, TUDCA alone.

The effect of TUDCA on the fibrillogenic propensity of the peptides was

assessed by Thioflavin T binding. Co-incubation with TUDCA did not modify the

inherent properties of each peptide (Fig. 2.1B). In fact, Aβ42 continued to express the

highest fluorescence values, while Aβ40 levels remained negligible even at 48 h.

AβE22Q showed an intermediate behavior, in agreement with the aggregation pattern

shown in Fig. 2.1A. Secondary structure analysis by CD spectroscopy illustrated both the

different structural characteristics of the peptides and the lack of effect of TUDCA (Fig.

2.1C). The initial Aβ40 structure showed a minimum at 198 nm, characteristic of

unordered conformations. Within 48 h of incubation, the peptide acquired a β-sheet-rich

structure showing the typical minimum at 218 nm in the CD scan. Aβ42 showed a

predominantly β-sheet conformation even immediately after solubilization. AβE22Q

exhibited an intermediate structural configuration, in agreement with the data illustrated

in Fig. 2.1A and B, with a mixture of random and β-sheet components when freshly

solubilized and an increase in the β-sheet component at 48 h. However, AβE22Q did not

achieve the predominant β-sheet conformation of the Aβ42 in the time frame of the

experiments. Corroborating the Western blot and Thioflavin T data, co-incubation with

TUDCA did not induce conformational changes in the secondary structures of any of the

Aβ peptides studied.

2.4.2 TUDCA inhibits AβE22Q-induced apoptosis in brain microvascular

endothelial cells

Morphologic evaluation of apoptosis induced by Aβ peptides in HCEC was

performed by fluorescence microscopy following Hoechst staining (Fig. 2.2A). The

results showed that nuclear fragmentation was differentially triggered by Aβ peptides,

starting at 12 h of treatment, with a maximum at 24 h, under the conditions tested.

Interestingly, the A22Q variant was very aggressive in HCEC, increasing apoptosis by

almost 3-fold compared to controls (p < 0.001), in contrast to wild-type Aβ40 and Aβ42

which had almost no effect (Fig. 2.2B). Consistent with its vasculotropic properties,

A22Q did not significantly increase cell death in primary rat cortical neurons

(unpublished observations). TUDCA was highly effective at modulating A22Q-

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60

induced toxicity, significantly inhibiting peptide-induced nuclear fragmentation to levels

comparable to controls (p < 0.05). Trypan blue dye exclusion and LDH levels (data not

shown) were in accordance with the morphological results. Detailed characterization of

the structural assemblies of A22Q indicated that the induction of apoptosis correlated

with the presence of peptide oligomers of low molecular mass and intermediate

Thioflavin-binding capacity (Fig. 2.1). Notably, pre-treatment with TUDCA, did not

induce significant changes either in the peptide structure or in the

aggregation/fibrillization propensity.

Figure 2.2. AβE22Q-induced apoptosis in HCEC is inhibited by TUDCA. Cells were

incubated either with vehicle (control), 50 μM of wild-type or Dutch-variant Aβ peptides, ± 100

μM TUDCA for 24 h. In co-incubation experiments, cells were pre-treated with TUDCA for 12 h

and the bile acid was left in the culture medium with Aβ peptides. (A) Fluorescence microscopy

of Hoechst staining shows condensed or fragmented nuclei indicative of apoptosis in control cells

(a), and in cells exposed to TUDCA (b), Aβ40 (c), and Aβ42 (d). Nuclear condensation or

fragmentation was markedly increased in cells incubated with AβE22Q (e), but less evident in

cells exposed to AβE22Q plus TUDCA (f). (B) Histogram shows the percentage of apoptotic


61

cells (mean ± SEM) identified by condensed and fragmented chromatin contiguous to the nuclear

membrane, as well as nuclear fragmentation and presence of apoptotic bodies, following Hoechst

staining and fluorescence microscopy. †p < 0.001 from control; *p < 0.05 from AβE22Q.

2.4.3 TUDCA prevents AβE22Q-induced mitochondrial Bax translocation and

cytochrome c release

To further investigate the cell death pathways differentially triggered by Aβ

peptides in HCEC, we sought to determine the specific involvement of the mitochondrial

pathway of apoptosis. Our results showed that the AE22Q mutant was very effective at

triggering cytochrome c translocation from mitochondria to the cytosol when compared to

Aβ40 and Aβ42 (Fig. 2.3), which exhibited almost negligible effects. This is consistent

with the lack of ability of the wild-type peptides to induce major apoptotic changes in

HCEC. Data on Bax translocation from the cytosol to mitochondria, assessed by Western

blot, were in agreement with cytochrome c changes (Fig. 2.3).

Figure 2.3. AβE22Q-induced Bax translocation and cytochrome c release in HCEC. Cells

were incubated either with vehicle (control), or 50 μM wild-type or Dutch-variant Aβ peptides for

24 h. Mitochondrial and cytosolic protein fractions were extracted for Western blot analysis.

Representative immunoblots of Bax and cytochrome c cellular distribution are shown.

Cytochrome c oxidase II (Cox II) and -actin were used as loading controls for mitochondrial and

cytosolic fractions, respectively. Cyt c, cytochrome c.

Chapter 2

62

The role of TUDCA in preventing AE22Q-induced, mitochondria-dependent

cell death was assessed by immunofluorescence microscopy. We confirmed that

incubation of HCEC with the AE22Q mutant resulted in cytochrome c release from

mitochondria as illustrated by the strong cytoplasmic staining, which was mostly

abolished by TUDCA (Fig. 2.4).

Figure 2.4. TUDCA inhibits AβE22Q-induced cytochrome c release in HCEC. Cells were



and the bile acid was left in the culture medium with Aβ peptides. Fluorescence microscopy

shows mitochondrial localization of cytochrome c in control cells (a) and in cells exposed to

TUDCA (b), Aβ40 (c), and Aβ42 (d). Strong cytoplasmic staining was evident in cells incubated

with AβE22Q (e). Notably, co-incubation with TUDCA mostly abolished the effect induced by

the Dutch-variant peptide (f).

In addition, our results demonstrated that TUDCA significantly reduced co-

localization of Bax with mitochondrial markers after incubation of HCEC with AE22Q

(Fig. 2.5A). In fact, TUDCA reduced mitochondrial co-localization of Bax to control

values (Fig. 2.5B; p < 0.05).


63

Figure 2.5. TUDCA inhibits AβE22Q-induced cytochrome c release in HCEC. Cells were



and the bile acid was left in the culture medium with Aβ peptides. Fluorescence microscopy

shows mitochondrial localization of cytochrome c in control cells (a) and in cells exposed to

TUDCA (b), Aβ40 (c), and Aβ42 (d). Strong cytoplasmic staining was evident in cells incubated

with AβE22Q (e). Notably, co-incubation with TUDCA mostly abolished the effect induced by

the Dutch-variant peptide (f).

2.5 Discussion

Extensive CAA is the neuropathological hallmark of familial AD cases linked to

intra-Aβ mutations. These Aβ genetic variants show potent vasculotropic properties and

result in a predominant deposition of Aβ peptides ending at position 40 (Castano et al.,

1996; Kumar-Singh et al., 2002; Shin et al., 2003). These are also the species that target

Chapter 2

64

the vasculature in sporadic AD, in contrast with the primary association of Aβ42 with the

parenchymal deposits (Miller et al., 1993; Mori et al., 1992).

Aggregation/fibrillization of Aβ plays a critical role in neurodegeneration. It is

now considered that the transition from soluble monomeric species circulating under

normal conditions to the oligomeric, protofibrillar and end-point fibrillar assemblies

contribute significantly to disease pathogenesis. In particular, intermediate oligomeric

and protofibrillar forms seem to display the most potent effects in neuronal cells inducing

synaptic disruption and neurotoxicity (reviewed in Caughey and Lansbury, 2003; Walsh

and Selkoe, 2007). In contrast, the abundance of mature amyloid plaques correlates

poorly with AD severity (Lue et al., 1999; McLean et al., 1999). Mutants with

accelerated formation of intermediate species and increased stability of these structures,

such as the Arctic (E22G) variant, elicit an earlier and stronger effect on neuronal cells

compared with wild-type peptides (Nilsberth et al., 2001; Whalen et al., 2005). In

contrast, the effect of Aβ genetic variants on cerebral vessel wall cells, specifically on

endothelial cells has not been so well characterized. The E22Q peptide has been shown

to selectively exert deleterious effects on these cells, significantly increasing apoptotic

responses in comparison to the wild-type Aβ40 (Eisenhauer et al., 2000; Miravalle et al.,

2000). Nevertheless, the nature of the amyloid assemblies inducing toxicity remains

uncertain. In the present study, we characterized in detail the conformational properties

of the E22Q peptide triggering apoptotic responses in HCEC and demonstrated its

intermediate fibrillogenic/aggregation properties and content of -sheet conformation

compared with wild-type A40 and A42. In one extreme, Aβ40 exhibited virtual lack of

oligomerization; in the other, Aβ42 showed high molecular mass assemblies with strong

fibrillar components. The absence of apoptosis resulting from endothelial challenge with

both wild-type peptides suggests that the intermediate conformation of the E22Q

oligomeric assemblies accounts for the potent, specific apoptotic effect of this genetic

variant.

The limited high-order oligomerization/fibrillization observed in AβE22Q

correlates well with the findings from nuclear magnetic resonance spectroscopy and

molecular dynamics simulation studies, which highlighted the effect of the mutation in

fibril stabilization (Ma and Nussinov, 2006; Zheng et al., 2007). These studies indicated

that the wild-type Aβ peptide, although mostly unstructured in solution, bears some

regions exhibiting structural order and protease resistance. A fragment stretching from


65

amino acids 21 through 30 adopts a stable bend structure which appears to nucleate

monomer folding (Lazo et al., 2005). The turn is stabilized by hydrophobic interactions

between Val24 and Lys28 and by long-range electrostatic interactions between Lys28 and

either Glu22 or Asp23. The computational studies predicted that the E22Q mutation

would translate into destabilization of the turn and enhanced oligomerization propensity

(Grant et al., 2007; Krone et al., 2008), consistent with our findings. Also, a contributing

factor to the nucleation propensity of the Dutch variant may be constituted by the

depletion of the Glu22-Lys28 salt-bridge as a consequence of the Glu for Gln substitution

(Krone et al., 2008).

The major form of apoptosis in mammalian cells proceeds through the

mitochondrial pathway, which involves mitochondrial outer membrane permeabilization

and modulation by the Bcl-2 family of proteins. While some members of this family,

typically located in the mitochondrial membrane, are potent inhibitors of apoptosis,

others, including Bax, are pro-apoptotic and predominantly cytosolic (reviewed in Youle

and Strasser, 2008). Upon commitment to apoptosis, and after mitochondrial

translocation, Bax undergoes conformational changes, oligomerizes and forms pores in

the outer mitochondrial membrane, allowing the release of proteins, including

apoptogenic cytochrome c, to the cytoplasm. These events facilitate downstream stages

of the cell death cascades, leading to DNA fragmentation and formation of apoptotic

bodies. Our data clearly demonstrates that AβE22Q is a strong inducer of the Bax pro-

apoptotic mitochondrial pathway in HCEC and highlights its role in various downstream

events. In fact, E22Q increased mitochondrial Bax and, consequently, cytosolic

cytochrome c. As expected, both events were accompanied by downstream nuclear

fragmentation. Although apoptosis indicators have not been investigated in HCHWA-D

cases, it is noteworthy that studies in AD patients have demonstrated alterations in the

expression of apoptosis-related genes, such as Bax (Mattson, 2000; Sajan et al., 2007),

suggesting that this process also takes place in vivo.

TUDCA is an endogenous bile acid that modulates the apoptotic threshold by

interfering with classical mitochondrial pathways of cell death (Rodrigues et al., 1998). It

has been previously shown that TUDCA is neuroprotective in pharmacological and

transgenic mouse models of Huntington‟s disease (Keene et al., 2002; Keene et al., 2001),

reduces lesion volumes in rat models of ischemic and hemorrhagic stroke (Rodrigues et

al., 2003a; Rodrigues et al., 2002), improves survival and function of nigral transplants in

a rat model of Parkinson‟s disease (Duan et al., 2002), and partially rescues from

Chapter 2

66

mitochondrial dysfunction a Parkinson‟s disease model in C. elegans (Ved et al., 2005).

Additionally, TUDCA modulates Aβ-induced neuronal death by inhibiting the

mitochondrial machinery and enhancing survival signaling (Solá et al., 2003), most likely

through functional modulation of nuclear steroid receptors (Solá et al., 2006). In

particular, TUDCA was shown to mitigate primary neurons mitochondrial insufficiency

and toxicity by inhibiting Bax translocation from cytosol to mitochondria (Rodrigues et

al., 2000b) and subsequent downstream events ending in caspase activation, substrate

cleavage and ultimately cell death. In this study, we show that TUDCA additionally

protects brain microvascular endothelial cells from the apoptotic insult triggered by the

potent vasculotropic E22Q peptide through suppression of Bax translocation, cytochrome

c release, and subsequent cell death. Notably, despite its potent anti-apoptotic role,

TUDCA pre-treatment did not significantly alter the aggregation properties, fibrillogenic

capacity, or secondary structure of the Aβ peptides in our experimental in vitro paradigm.

Future experiments should clarify if TUDCA has any differential effect in the peptide

fibrillization occurring in cell culture conditions.

The present data highlight a dichotomy between aggregation/fibrillization

patterns and apoptotic properties of Aβ peptides in HCEC. Whether these observations

downplay the role of high molecular mass structural assemblies in the induction of

apoptosis, or simply indicate that TUDCA directly affects upstream pathways remains to

be elucidated. It is conceivable that TUDCA may bind to the monomers/small oligomers

of the Aβ peptides affecting their capability to insert into lipid bilayers and form channels

(Jang et al., 2008; Jang et al., 2007). Alternatively, as a cholesterol-derived molecule,

TUDCA may insert into cellular membranes (Guldutuna et al., 1993; Rodrigues et al.,

2003b), thus preventing the subsequent insertion of Aβ. Certainly, further studies are

needed to identify the exact targets of TUDCA; nevertheless, this work provides new

perspectives for the modulation of amyloid-induced toxicity in CAA, presenting new

targets for therapeutic interventions.

Acknowledgements

This work was supported by grant PTDC/BIA-BCM/67922/2006 from Fundação

para a Ciência e a Tecnologia (FCT), Lisbon, Portugal, NIH grants NS051715 and

AG10491, and the American Heart Association, USA. RJS Viana is the recipient of a

PhD fellowship from FCT, Portugal (BD/30467/2006); AF Nunes, RE Castro and RM


67

Ramalho are recipients of postdoctoral fellowships from FCT, Portugal

(BPD/34603/2007, BPD/30257/2006, and BPD/40623/2007, respectively).

Chapter 2

68

3 Amyloid β peptide-induced secretion of

endoplasmic reticulum chaperone glycoprotein

GRP94 in PC12 neuronal-like cells

Ricardo J. S. Viana1, Clifford J. Steer

2,3, Cecília M. P. Rodrigues

1


University of Lisbon, Lisbon, Portugal; and Departments of 2Medicine and

3Genetics,

Cell Biology, and Development, University of Minnesota Medical School, Minneapolis,

MN, USA

(Submitted manuscript)

Aβ-induced GRP94 secretion

71

3.1 Abstract

Amyloid β (Aβ) peptide-induced neurotoxicity is typically associated with cell

death through mechanisms not entirely understood. Here, we investigated stress signaling

events triggered by soluble Aβ in differentiated rat neuronal-like PC12 cells.

Morphologic evaluation of apoptosis confirmed that Aβ induced nuclear fragmentation

that was prevented by pre-treatment with the antiapoptotic bile acid tauroursodeoxycholic

acid (TUDCA). In addition, Aβ exposure triggered an early signaling response by the

endoplasmic reticulum (ER) and caspase-12-mediated apoptosis, which was independent

of the ER-stress pathway. Furthermore, ER stress markers, including GRP94, ATF-6α,

CHOP and eIF2α were strongly downregulated by Aβ, independent of protein

degradation, and partially restored by TUDCA. Calpain inhibition prevented caspase-12

activation and reduced levels of ATF-6α. Importantly, Aβ-induced GRP94

downregulation was related to protein secretion and partially rescued through inhibition

of the secretory pathway by geldanamycin and brefeldin. In conclusion, we showed that

the ER is a proximal stress sensor for soluble Aβ-induced toxicity, resulting in caspase-12

activation and cell death in PC12 neuronal cells. Moreover, GRP94 secretion was

associated with Aβ-induced apoptotic signaling. These data provide new information

linking apoptotic properties of Aβ peptide to distinct subcellular mechanisms of toxicity.

Further characterization of this signaling pathway is likely to provide new perspectives

for modulation of amyloid-induced apoptosis.

Keywords: Amyloid β; Apoptosis; Endoplasmic reticulum; Caspase-12; ATF-6α;

GRP94; Tauroursodeoxycholic acid

Chapter 3

72

3.2 Introduction

Alzheimer‟s Alzheimer‟s disease (AD) is associated with the accumulation of

pathogenic amyloid β (Aβ) assemblies in brain resulting in progressive dismantling of

synapses and loss of neurons in selected areas. It is recognized that increased production,

oligomerization and aggregation of βpeptides are crucial factors in the onset of AD.

Toxic Aβ40 and Aβ42 are processed from the amyloid-βprecursor protein (APP) via

cleavage by β- and γ-secretase complexes (LaFerla et al., 2007; Thinakaran and Koo,

2008). APP is a transmembrane protein, which is folded and modified in endoplasmic

reticulum (ER), and then transported through the Golgi complex to the outer membrane.

Currently, it is not clear which cellular compartment processes APP to toxic Aβpeptides

(LaFerla et al., 2007).

ER serves two major functions in the cell, including proper folding of newly

synthesized proteins destined for secretion, cell surface or intracellular organelles, and as

a reservoir of Ca2+

. The ER is highly sensitive to alterations in Ca2+

homeostasis and

perturbations in its environment that result in ER stress. The ER responds by triggering

specific signaling pathways including the unfolded protein response (UPR), the ER-

overload response (EOR), and the ER-associated degradation (ERAD). These pathways

are collectively known as the ER stress response, which is also associated with increased

expression of genes that expand the folding capacity of the ER, preventing new protein

synthesis, and accelerating degradation of misfolded proteins. The UPR is characterized

by the coordinated activation of multiple proteins that can be divided into three groups,

consisting of ER stress-induced cell death sensors, modulators and effectors. The ER

membrane residing proteins inositol-requiring enzyme 1 (IRE1), activating transcription

factor 6 (ATF-6), and double-stranded RNA-activated protein kinase like ER kinase

(PERK) serve as proximal ER stress sensors, which are maintained in an inactive state by

binding to the chaperone 78-kDa glucose-regulated protein, also known as

immunoglobulin heavy chain binding protein or Bip (GRP78/Bip). GRP78/Bip is a distal

sensor and functions as a master regulator of the UPR. During ER stress, the release of

GRP78/Bip results in inhibition of translation (PERK), and upregulation of molecular

chaperones (ATF-6) and ERAD activity (IRE1) (Bernales et al., 2006; Romisch, 2005).

ER stress-induced cell death modulators include members of the Bcl-2 family (Bcl-2,

Bcl-xL, Bak and Bik) (Nutt et al., 2002; Thomenius et al., 2003) and CHOP (Zinszner et

al., 1998), among others.


73

Finally, several molecules that are either present on the ER surface or in the

soluble compartment have been implicated as effectors that cause death in response to

prolonged ER stress. Caspase-12 is an initiator caspase which is specifically involved in

apoptosis (Rao et al., 2001); and its activation may occur through ER stress-induced

calpain or caspase-7 cleavage of procaspase-12 (Nakagawa and Yuan, 2000; Rao et al.,

2001). ER stress-activated IRE1 may also aggregate procaspase-12 at the ER membrane

surface through the cytosolic adaptor tumor necrosis factor associated receptor factor 2

(TRAF2) proteins, resulting in cleavage and activation of caspase-12 (Yoneda et al.,

2001). Most notably, caspase-12, together with caspase-9, serves as a mediator of an

intrinsic apoptosis pathway that is independent of mitochondria (Morishima et al., 2002).

The ER is a repository of antiapoptotic GRP94, a member of the heat shock

protein 90 (hsp90) family (Koch et al., 1986; Lee, 1992; Mazzarella and Green, 1987;

Tanaka et al., 1990). GRP94 is a hydrophobic, ATP-, peptide- (Booth and Koch, 1989;

Tanaka et al., 1990), and calcium-binding protein that contains several helix-loop-helix

structural domains (Csermely et al., 1998). Its antiapoptotic properties (Lee et al., 2008;

Little and Lee, 1995; McCormick et al., 1997) are not fully understood. It has also been

described to act as a vaccine in prophylactic and therapeutic models of cancer

immunotherapy (Basu et al., 2000; Tamura et al., 1997). More recently, GRP94 was

shown to be released from cells undergoing necrotic cell death as a consequence of lethal

viral infection, suggesting a potential physiological role for chaperone proteins in the

regulation of immunological responses to pathological cell death (Basu et al., 2000;

Berwin et al., 2001). Inhibitors of hsp90 and GRP94, such as geldanamycin (GA) have

been investigated in the treatment of cancer. GA inhibits hsp90 and GRP94 by binding

competitively to its N-terminal ATP-binding site, which results in misfolding and

degradation of hsp90 substrates through the ubiquitin-proteasome pathway. While GA-

mediated enhanced degradation of certain hsp90 proteins is now well established,

(reviewed in Pratt, 1998; Sepp-Lorenzino et al., 1995), few studies have reported GA-

mediated inhibition of intracellular trafficking (Barzilay et al., 2004).

In this study, we show that Aβ-induced toxicity activates caspase-12, which is

known to mediate ER-specific apoptosis in PC12 neuronal cells. Unexpectedly, we also

demonstrated that ER stress-induced cell death sensors and modulators are clearly

downregulated. ATF-6α is proteolytically cleaved by calpain I, and GRP94 is secreted to

the extracellular medium, in association with Aβ-induced toxicity.

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74


3.3.1 Cell culture and culture conditions

PC12 cells were grown in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO,

USA) supplemented with 10% heat-inactivated horse serum (Sigma-Aldrich), 5% fetal

bovine serum (Invitrogen Corp., Carlsband, CA, USA) and 1% penicillin/streptomycin

(Invitrogen), and maintained at 37°C in a humidified atmosphere of 5% CO2. Cells were

plated and differentiated in the presence of nerve growth factor (NGF) (Promega Corp.,

Madison, WI), as previously described (Park et al., 1998). Cell density was maintained at

1 x 105 cells/cm

2 for morphological assessment of apoptosis and viability assays, or 4 x

105 cells/cm

2 for transfection assays and protein extraction.

To induce apoptosis, cells were exposed to 25 µM of Aβ (25-35) peptide active

fragment (Bachem AG, Bubendorf, Switzerland) for 1, 2, 8 and 24 h. This concentration

was found to be ideal in maximizing the cellular response of Aβ with minimum

exacerbated toxic effects in neuronal-like PC12 cells (data not shown). Brefeldin and

thapsigargin (Sigma-Aldrich) were used at 18 and 3 µM, respectively, as positive controls

for ER-specific apoptosis. To test the effect of antiapoptotic TUDCA, cells were

incubated in medium supplemented with 100 µM TUDCA (Sigma-Aldrich), or no

addition (control), for 12 h, and then exposed to 25 µM of Aβ (25-35) for 1, 2, 8 and 24 h.

Cells were fixed for microscopic assessment of apoptosis or processed for cell viability

assays. In addition, total proteins were extracted for immunoblotting.

In parallel experiments, cells were pretreated for 30 min before Aβ incubation

with SP600125, the c-Jun N-terminal kinase (JNK) inhibitor (Calbiochem, Darmstadt,

Germany), at either 10 µM for 24 h, or 50 µM SP600125 for 3 h. Similarly, cells were

pretreated with 10 µM calpain inhibitor I (Roche Applied Science, Mannheim, Germany)

for 24 h or 50 µM calpain inhibitor I for 3 h. Proteasome and caspase inhibition during

apoptosis was achieved by pretreating cells with either 10 µM MG-132 and z-VAD.fmk

(Calbiochem) for 24 h, or 50 µM MG-132 and z-VAD.fmk for 3 h. To determine the

effect of Ca2+

deregulation, cells were pretreated with 50 µM ZnCl2, 100 nM

xestospongin C, 20 µM nifedipine, 10 µM dantrolene and 0.05 to 5 µM cyclosporin A

(Sigma-Aldrich) for 3 h. Finally, cells were pretreated with 0.05 to 5 µM GA and 9 µM

brefeldin (Sigma-Aldrich) for 3 h to inhibit protein secretion.


75


Cell viability was measured by trypan blue exclusion. Apoptotic nuclei were

detected by Hoechst labeling after cell fixation with 4% formaldehyde in phosphate

buffer saline (PBS), for 10 min at room temperature. Following incubation with Hoechst

dye 33258 (Sigma-Aldrich) at 5 g/mL in PBS for 5 min, and PBS washes, slides were

mounted with PBS:glycerol (3:1, v/v) and fluorescence visualized with an Axioskop

fluorescence microscope (Carl Zeiss GmbH, Hamburg, Germany). Fluorescent nuclei

were scored and categorized according to the condensation and staining characteristics of

chromatin. Normal nuclei showed non-condensed chromatin dispersed over the entire

nucleus. Apoptotic nuclei were independently identified by condensed and fragmented

chromatin contiguous to the nuclear membrane, as well as nuclear fragmentation and

presence of apoptotic bodies. Three random microscopic fields per sample of ~ 250

nuclei were counted and mean values expressed as the percentage of apoptotic nuclei.

3.3.3 Immunoblotting

Steady-state levels of caspase-12, ATF-6α, KDEL, CHOP and eIF2α were

determined by Western blot analysis. Briefly, 80 µg of total and nuclear proteins were

separated by 12% sodium dodecyl-polyacrylamide gel electrophoresis. After

electrophoretic transfer onto nitrocellulose membranes, immunoblots were incubated with

15% H2O2 for 15 min at room temperature. Following a block with 5% milk solution,

membranes were incubated overnight at 4°C with primary mouse monoclonal antibody

reactive to KDEL (10C3) (Abcam, Cambridge Science Park, Cambridge, UK), primary

rabbit polyclonal antibodies reactive to ATF-6α (H-280), GADD153 (R-20) (Santa Cruz

Biotechnology, Santa Cruz, CA, USA), Anti-eIF2α [pS52

] (Invitrogen). Finally,

secondary goat anti-mouse or anti-rabbit IgG antibody conjugated with horseradish

peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) was added for 3 h at room

temperature. The membranes were processed for protein detection using the SuperSignal

substrate (Pierce Biotechnology, Rockford, IL, USA). β-Actin (AC-15) (Sigma-Aldrich)

was used as loading control for total proteins. Protein concentrations were determined

using the Bio-Rad protein assay kit (Bio-Rad) according to the manufacturer‟s

specifications.

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76

3.3.4 RNA isolation and RT-PCR

Transcript expression of GRP94 was determined by RT-PCR. Total RNA was

extracted from rat PC12 cells using the TRIZOL reagent (Invitrogen). For RT-PCR, 5 g

of total RNA was reverse transcribed using oligo(dT) (Integrated DNA Technologies Inc.,

Coralville, IA) and SuperScript II reverse transcriptase (Invitrogen). Specific

oligonucleotide primer pairs were incubated with cDNA template for PCR amplification

using the Expand High FidelityPLUS

PCR System (Roche Applied Science, Mannheim,

Germany). The following sequences were used as primers: GRP94 sense 50-

TACTATGCCAGTCAGAAGAAAACG-30; GRP94 antisense 50-CATCCTTT

CTATCCTGTCTCCATA-30; β-actin sense 50-CGTCTTCCCCTCCATCGTG-30; and β

-actin antisense 50-CCAGTTGGTTACAATGCCGTG-30. The product of the β-actin

RNA was used as control.

3.3.5 Heat shock protein GRP94 ELISA assay

The rat hsp90 glycoprotein 96 (hsp90 gp96) ELISA Kit (Cusabio Biotech

Co.,Newark, USA) was used to measure GRP94 levels, according to the manufacturer‟s

protocol. Supernatant samples were applied to plates containing biotinylated antibody for

GRP94 (1:100). After 1 h incubation at 37oC, plates were washed and incubated with

HRP-avidin (1:100) for an additional 1 h, after which the chemiluminescence substrate

solution was added and incubated for 10-30 min. The reaction was stopped and the

optical density was measured after 30 min using a microplate reader set to 450 nm.


The relative intensities of protein bands were analyzed using the Quantity One

Version 4.6 densitometric analysis program (Bio-Rad) and normalized to the respective

loading controls. Statistical analysis was performed using GraphPad InStat Version 3.00

(GraphPad Software, San Diego, CA) for the analysis of variance and Bonferroni‟s

multiple comparison tests. Values of p < 0.05 were considered significant.


77

3.4 Results

3.4.1 Aβ-induced apoptosis is mediated by caspase-12 activation and modulated

by TUDCA in PC12 neuronal-like cells

Soluble Aβ (25-35) fragment induces apoptosis in PC12 neuronal-like cells,

which is abrogated by TUDCA pre-treatment (Ramalho et al., 2004; Viana et al.,2010).

ER is a potential intracellular target of Aβ protein (Nakagawa et al., 2000), and TUDCA

exerts protection from apoptosis, in part, by preventing caspase-12 activation (Xie et al.,

2002). In this study, neuronal PC12 cells pre-exposed to NGF for 8 days were treated

with either Aβ (25-35), or ER-specific apoptotic stimuli brefeldin and thapsigargin, with

or without TUDCA, and assessed for cell death and caspase-12 fragmentation. We

confirmed that Aβ (25-35)-induced apoptosis after 24 h was reduced to almost control

levels in cells pre-treated with TUDCA (p < 0.05) (Fig. 3.1A). General cell death as

assessed by trypan blue dye exclusion was in accordance with the morphological data

(Fig. 3.1B). Similar results were obtained in PC12 cells exposed to 10 M Aβ40 (data

not shown). TUDCA was also effective at modulating brefeldin- and thapsigargin-

induced apoptosis (Fig. 3.1A).

After Aβ (25-35) exposure, caspase-12 processing peaked at 24 h, and pre-

treatment with TUDCA markedly reduced Aβ-induced effects by ~ 70% (p < 0.05) (Fig.

3.2). The activation of caspase-12 by calpains in apoptosis has been previously

documented (Nakagawa and Yuan, 2000). Moreover, the JNK pathway is activated in

response to ER stress by IRE1 (Nishitoh et al., 1998; Urano et al., 2000), which may

activate caspase-12 through TRAF2, the same cytosolic adaptor of JNK at the ER

membrane surface (Yoneda et al., 2001). To test if calpains or JNK plays a role in

mediating neuronal cell death induced by Aβ, we incubated PC12 cells in the presence or

absence of calpain inhibitor I or SP600125 JNK inhibitor for 24 h. After 24 h of

incubation with Aβ, calpain inhibition, but not JNK inhibition, prevented caspase-12

fragmentation by almost 50% (p < 0.05), suggesting that calpains are involved in Aβ-

induced caspase-12 activation.

Chapter 3

78

Figure 3.1. TUDCA inhibits cell death induced by Aβ, brefeldin and thapsigargin in PC12

neuronal cells. Cells were incubated with either 25 µM soluble Aβ (25-35), 9 and 18 µM

brefeldin, 1.5 and 3 µM thapsigargin, or no addition (control), ± 100 µM TUDCA for 24 h. In co-

incubation experiments, TUDCA was added 12 h prior to incubation with Aβ, brefeldin or

thapsigargin. (A) Total and trypan blue stained cells were counted and cell viability calculated as

the number of surviving cells per total number of cells. (B) Cells were fixed and stained with

Hoechst for microscopic assessment of apoptosis, and the percentage of apoptotic cells was

determined. Cells exposed to Aβ, brefeldin or thapsigargin alone showed more nuclear

fragmentation compared with cells pre-treated with TUDCA. The results are expressed as mean ±

SEM for at least three different experiments. *p < 0.01 from control; †p < 0.05 from Aβ,

brefeldin or thapsigargin alone.


79

Figure 3.2. TUDCA modulates Aβ-induced caspase-12 processing in PC12 neuronal cells.

Cells were incubated with 25 µM Aβ, or no addition (control), ± 100 µM TUDCA, 10 µM

SP600125 and 10 µM calpain inhibitor I for 24 h. In co-incubation experiments, TUDCA was

added 12 h prior to incubation with Aβ. SP600125 and calpain inhibitor I were added 30 min prior

to incubation with Aβ. Total proteins were extracted for western blot analysis as described in

Materials and Methods. Representative immunoblot of procaspase-12 and cleaved caspase-12 in

cells exposed to Aβ, ± TUDCA, SP600125 or calpain inhibitor I (top), and active caspase-12

levels (bottom). The results are normalized to endogenous β-actin protein levels and expressed as

mean ± SEM arbitrary units of at least four different experiments. *p < 0.05 from control; †p <

0.05 from Aβ alone.

3.4.2 Aβ-induced, caspase-12-mediated cell death is independent of ER stress

Caspase-12 Caspase-12 can be activated under specific ER stress stimuli

(Nakagawa et al., 2000). In addition, Aβ causes ER stress (Lee do et al., 2010) and

proteosomal dysfunction (Shringarpure et al., 2000). To determine whether ER stress

plays a role in Aβ-induced apoptosis mediated by caspase-12 in PC12 neuronal cells, we

evaluated several ER stress markers by immunoblotting (Fig. 3.3). ER stress makers

were upregulated or activated after either brefeldin or thapsigargin incubation, but not

after Aβ exposure for 24 h. Interestingly, ATF-6α, eIF2α, CHOP and GRP94 were

markedly downregulated after Aβ incubation. This effect was partially inhibited by

TUDCA (Fig. 3.3). Thus, ER is strongly involved in Aβ-induced apoptosis, although

with no evidence of ER stress activation.

Chapter 3

80

Figure 3.3. Aβ-induced apoptosis in PC12 neuronal cells is independent of ER stress. Cells

were incubated with 25 µM Aβ, or no addition (control), ± 100 µM TUDCA for 2 h. Positive

controls for ER stress were brefeldin 18 µM and thapsigargin 3 µM for 2 h. In co-incubation

experiments, TUDCA was added 12 h prior to incubation with Aβ, brefeldin or thapsigargin.

Total proteins were extracted for western blot analysis as described in Materials and Methods.

Representative immunoblots of ER stress markers ATF-6α, eIF2α, CHOP, GRP94 and

GRP78/Bip. β-Actin was used as loading control.

3.4.3 ATF-6α and GRP94 downregulation is independent of protein degradation

Cells containing a mutated presenilin-1 (PS1) gene showed significantly

inhibited induction of GRP78 mRNA expression when stimulated for ER stress

(Katayama et al., 1999). More importantly, GRP78 and GRP94 are decreased in brains of

sporadic AD patients (Katayama et al., 1999). The reduction of GRP94 protein levels

may be explained by selective proteolytic cleavage by calpains activated during

etoposide-induced apoptosis (Reddy et al., 1999). In this study, basal levels of GRP94

and ATF-6α mRNA expression were not altered in PC12 cells when exposed to Aβ for 2

and 24 h (data not shown). We tested if GRP94 and ATF-6α cleavage was mediated by

proteases, such as caspases and calpains, or by the proteasome, since calpain inhibitors

might also interfere with the proteasome activity. Our results showed that general

caspase inhibitor z-VAD-fmk and JNK inhibitor SP600125 did not prevent cleavage of

GRP94 and ATF-6α, after 24 h of Aβ incubation (Fig. 3.4A). Nevertheless, both calpain

and proteasome inhibition were partially protective (Fig. 3.4A). Proteasome activity

remained unchanged and autophagy was not activated by Aβ incubation under the


81

conditions tested (data not shown). Thus, typical mechanisms for protein degradation,

such as the proteasome and autophagy were not triggered by Aβ incubation in PC12

neuronal-like cells.

Figure 3.4. Aβ-induced downregulation of ATF-6α and GRP94 in PC12 neuronal cells is

independent of degradation mechanisms. Cells were incubated with 25 µM Aβ, or no addition

(control), ± 10 µM of SP600125, calpain inhibitor I, MG132, and z-VAD.fmk for 24 h. For

shorter incubations with Aβ, 50 µM of each inhibitor was used for 2 h. In co-incubation

experiments, the inhibitors were added 30 minutes prior to incubation with Aβ. Total proteins

were extracted for western blot analysis as described in Materials and Methods. (A)

Representative immunoblot of ATF-6α and GRP94 in cells exposed to Aβ, ± 10 µM of SP600125,

calpain inhibitor I, MG132, and z-VAD.fmk for 24 h. (B) Representative immunoblot of ATF-6α

and GRP94 in cells exposed to Aβ, ± 50 µM of SP600125, calpain inhibitor I, MG132, and z-

VAD.fmk for 2 h. β-Actin was used as loading control.

Since caspase-12 activation coincided with the early reduction of GRP94 at 2 h

of Aβ incubation, we tested if GRP94 and ATF-6α cleavage was selectively mediated by

Chapter 3

82

calpains or the proteasome, and not a consequence of general protein degradation. Cells

were pretreated with 50 µM of each inhibitor fro 30 min (Reddy, et al. 1999), followed by

2 h of Aβ incubation. In contrast to MG-132, pretreatment with calpain inhibitor I

prevented ATF-6α cleavage (Fig. 3.4B). However, GRP94 downregulation was not

blocked by any of the inhibitors. These results suggested that ATF-6α is selectively

cleaved by calpains during Aβ-induced apoptosis, whereas GRP94 is not cleaved by

proteases during apoptosis.

3.4.4 GRP94 downregulation is independent of Ca2+

deregulation

GRP94 has been linked directly to cellular Ca2+

homeostasis (Biswas et al.,

2007a). GRP94 binds Ca2+

and is one of approximately six luminal proteins that serve as

the major buffers of the ER (Cala and Jones, 1994; Macer and Koch, 1988; Nigam et al.,

1994). A key mechanism by which Aβ is believed to alter cellular Ca2+

homeostasis

involves disruption of plasma membrane Ca2+

permeability. At the extracellular level, Aβ

exerts actions on endogenous plasmalemmal ion channels, such as L-type channels (Fox

et al., 1987), and pore formation (Arispe et al., 1993). Furthermore, Aβ oligomers

induce massive calcium entry into mitochondria, which opens the mitochondrial

permeability transition pore (mPTP) (Sanz-Blasco et al., 2008), and Aβ targets the mPTP

resulting in damage amplification (Du et al., 2008). We used Ca2+

blockers to determine

whether GRP94 reduction was due to unregulated flux of extracellular Ca2+

induced by

Aβ exposure. ZnCl2, which blocks pore formation was unable to prevent GRP94

downregulation (Fig. 3.5A). Combinations of ZnCl2 with calpain inhibitor I and TUDCA

were also ineffective, as was pretreatment with nifedipine that blocks L-type channels.

Next, we inhibited the release of Ca2+

from the ER using xestospongin C and

dantrolene, which block inositol triphosphate receptors (IP3Rs) and ryanodine receptors

(RyRs), respectively. Under the established conditions, only xestospongin C was able to

partially prevent the reduction of GRP94 (Fig. 3.5B). Finally, cyclosporine A, a strong

inhibitor of calcium-induced mPTP opening, did not prevent the reduction of GRP94

protein levels (Fig. 3.5C). Thus, Ca2+

deregulation induced by Aβ incubation is not the

main molecular mechanism of GRP94 downregulation.


83

Figure 3.5. Aβ-induced downregulation of GRP94 in PC12 neuronal cells is independent of

Ca2 deregulation. Cells were incubated with 25 µM Aβ, or no addition (control), ± 50 µM ZnCl2,

100 nM xestospongin C, 20 µM nifedipine, 10 µM dantrolene, and 0.05 or 5 µM cyclosporin A

for 2 h. In co-incubation experiments, the inhibitors were added 30 min prior to incubation with

Aβ. Total proteins were extracted for western blot analysis as described in Materials and Methods.

(A) Representative immunoblot of GRP94 in cells exposed to Aβ, ± 50 µM of ZnCl2.

Combinations of ZnCl2 with 50 µM calpain inhibitor I or 100 µM TUDCA for 2 h are also shown.

Thapsigargin 3 µM was used as positive control. (B) Representative immunoblot of GRP94 in

cells exposed to Aβ, ± 100 nM xestospongin C, 20 µM nifedipine and 10 µM dantrolene. (C)

Representative immunoblot of GRP94 in cells exposed to Aβ, ± 0.5 or 5 µM of cyclosporine A.

Thapsigargin 3 µM and brefeldin 18 µM were used as positive controls; and β-actin as a loading

control.

3.4.5 Protein secretion inhibitors block GRP94 downregulation

GA is an antibiotic that specifically inhibits hsp90 and GRP94 (Chavany et al.,

1996; Pratt, 1998). Inhibition of hsp90 mainly leads to targeted degradation of client

proteins via the ubiquitin proteasome pathway (Smith et al., 1995; Whitesell et al., 1994),

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84

while the best understood response to inhibition of GRP94 is the transcriptional

upregulation of ER chaperones (Lawson et al., 1998). In addition, GA also mediates

inhibition of intracellular trafficking of hsp90 substrates (Barzilay et al., 2004). To

determine the effect of GA on Aβ-induced GRP94 downregulation, we preincubated cells

with different concentrations of GA for 30 min prior to exposure to Aβ for 2 h (Fig. 3.6).

Figure 3.6. Aβ-induced downregulation of GRP94 in PC12 neuronal cells is blocked by

protein secretion inhibitors. Cells were incubated with 25 µM Aβ, or no addition (control), ±

0.05 to 5 µM GA for 2 h. In co-incubation experiments, the inhibitors were added 30 min prior to


85

incubation with Aβ. Total proteins and total RNA were extracted for western blot and RT-PCR

analysis, respectively, as described in Materials and Methods. (A) Representative immunoblot of

GRP94 in cells exposed to Aβ, ± 0.5 and 5 µM GA. Thapsigargin 3 µM and brefeldin 18 µM

were used as positive controls. (B) Representative RT-PCR of GRP94 in cells exposed to Aβ, ±

0.05 to 5 µM of GA. (C) Representative immunoblot of GRP94 in cells exposed to Aβ, ± 9 µM

brefeldin. Combinations of 0.5 µM GA with 100 µg/ml cyclohexemide for 2 h are also

represented. β-Actin was used as loading control. (D) Secreted protein levels of GRP94.

Supernatant anti-GRP94 of cells pretreated with 0.5 µM GA and 9 µM brefeldin, and challenged

with Aβ for 2 h was quantified using an ELISA assay. The results are expressed as mean ± SEM

for at least three different experiments. *p < 0.05 from control; †p < 0.05 from Aβ alone.

We found that GA pretreatment prevented GRP94 downregulation in a

concentration-dependent manner (Fig. 3.6A), without significantly altering GRP94

mRNA expression (Fig. 3.6B). In addition, GA effect was not dependent on de novo

protein synthesis as determined in experiments using cyclohexamide (Fig. 3.6C).

However, a non-apoptotic concentration of brefeldin that inhibits protein secretion was

also able to prevent GRP94 dowregulation (Fig. 3.6C). Finally, an ELISA assay for

GRP94 in the supernatant of cells challenged with Aβ established that GRP94 levels were

increased in the extracellular medium of cells incubated with Aβ, independently of a

necrotic mechanism (Fig. 3.6D). Retention of GRP94 within the cell by GA and

brefeldin was associated with levels of apoptosis of ~ 5%, similar to those seen in control

cells. Thus, Aβ induces early stimulation of GRP94 secretion, an effect that was

mitigated by inhibition of the secretory pathway.

3.5 Discussion

The role of Aβ in the pathogenesis and progression of AD-associated

neurodegeneration has not been firmly established and even remains controversial. The

present study shows that Aβ-induced toxicity activates caspase-12 that mediates ER-

specific apoptosis in PC12 neuronal cells, which in turn is modulated by antiapoptotic

TUDCA. Notably, we demonstrated that ER stress-induced cell death sensors and

modulators are downregulated, and propose that ATF-6α is proteolytically cleaved by

calpains, and GRP94 is secreted to the extracellular milieu during Aβ-induced apoptosis.

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86

There is a growing list of mediators that link ER stress to the apoptotic

machinery. Caspase-12 was proposed to function as the apical caspase responsible for

initiating an apoptotic cascade in response to ER stress and Aβ (Nakagawa et al., 2000).

In the present study, we showed that Aβ-induced apoptosis in PC12 neuronal-like cells

requires caspase-12 activation that peaked after 24 h of Aβ incubation, and which was

inhibited by antiapoptotic TUDCA. Both the JNK pathway (Tan et al., 2006) and

calpains (Nakagawa and Yuan, 2000) have been shown to activate caspase-12. Our

results confirmed that calpain inhibition partially abrogated caspase-12 activation. This

fact suggests a role for intracellular Ca2+

, given that calpains are activated by Ca2+

(Croall

and DeMartino, 1991) and Aβ-incubation results in intracellular Ca2+

overload

(Kuchibhotla et al., 2008).

ER is very sensitive to changes in Ca2+

homeostasis, leading to ER stress and

caspase-12 activation (Nakagawa et al., 2000; Orrenius et al., 2003). In the present study,

we investigated if caspase-12 activation after Aβ-incubation resulted from ER stress. We

found that ER stress markers such as GRP94, ATF-6α, eIF2α and CHOP were

downregulated in PC12 cells exposed to Aβ peptide. This effect was extremely

pronounced for GRP94 levels, which became undetectable after only 2 h of incubation

with Aβ.

ER-resident molecular chaperones, such as GRP78 and GRP94 are

downregulated in brains of AD patients and in PS1 mutant cells (Katayama et al., 1999).

Moreover, it has been demonstrated that in etoposide-induced apoptosis, the reduction of

GRP94 levels is due to calpain cleavage (Reddy et al., 1999). Here, we demonstrated that

under Aβ-induced apoptosis, GRP94 downregulation was calpain-independent. In

contrast, ATF-6α downregulation was prevented by calpain inhibition. This fact,

suggested that ATF-6α is either a substrate for calpain cleavage, or that calpain regulates

proteases that normally cleave and activate ATF-6α. During the ER stress response,

ATF-6α is cleaved with release of its cytoplasmic bZIP domain from the membrane, and

translocation to the nucleus (Haze et al., 1999).

GRP94 is a passive high-capacity reservoir for Ca2+

, whose activity is regulated

by Ca2+

. Furthermore, despite the redundancy of Ca2+

-binding proteins in the ER, the

ability of GRP94 to bind Ca2+

is uniquely important in the cellular context, as cells

become hypersensitive to perturbations in Ca2+

homeostasis in the absence of GRP94.

Here, we demonstrated that deregulation of Ca2+

homeostasis induced by Aβ-incubation

was not related with downregulation of GRP94. In fact, Aβ can induce intracellular Ca2+


87

overload by several mechanisms (reviewed in Demuro et al., 2010). In the present study,

we used nifedipine and ZnCl2 at the plasma membrane level to inhibit L-type Ca2+

channel and zinc pore formation, respectively. Xestospongin C and dantrolene were used

at the ER level to inhibit Insp3Rs and RyRs receptors, respectively. Moreover,

cyclosporine A inhibits Ca2+

regulation between ER-mitochondria through the mPTP pore

formation. Despite the fact that GRP94 binds Ca2+

and serves as the major Ca2+

buffer in

the ER (Cala and Jones, 1994; Macer and Koch, 1988; Nigam et al., 1994), our results

suggested that GRP94 downregulation is not strictly related to Ca2+

deregulation.

Nevertheless, inhibition of Ca2+

deregulation slightly prevented the reduction of GRP94

levels.

GRP94 has been identified in the extracellular space as a consequence of acute

stress in lethal viral infection and necrotic cell death (Basu et al., 2000; Berwin et al.,

2001). Indeed, stress proteins such as heat shock proteins, glucose regulated proteins and

calreticulin are released from cells in a variety of circumstances, thus interacting with

adjacent cells or in some cases entering the bloodstream (reviewed in Calderwood et al.,

2007). Furthermore, GA inhibits GRP94 and hsp90 (Neckers, 2002), and activates a heat

shock response, possibly through increased expression of molecular chaperones (McLean

et al., 2004; Sittler et al., 2001). Nonetheless, only a few reports have suggested that GA

may mediate the inhibition of intracellular protein trafficking (Barzilay et al., 2004). In

the present study, we demonstrated that GRP94 downregulation is blocked in cells

pretreated with GA in a dose-dependent manner. This occurred in a degradation-

independent fashion, since neither the proteasome nor autophagy mechanisms were

activated under the conditions tested. Further, mRNA levels of GRP94 were not

upregulated in the presence of GA, and synthesis of de novo proteins did not appear to be

involved. Next, we tested if the capacity of GA to inhibit intracellular protein trafficking

was the mechanism responsible to block GRP94 downregulation. Brefeldin, a known

inhibitor of the secretion pathway, blocked GRP94 downregulation. Finally, we further

confirmed that GRP94 was secreted to the extracellular medium in the presence of Aβ-

peptide. To our knowledge, this is the first report demonstrating that Aβ exposure in

PC12 neuronal-like cells induces GRP94 secretion. Once secreted, stress proteins may

either increase stress resistance, after binding to stress sensitive recipient cells, such as

neurons, signal tissue destruction and danger to inflammatory cells, or aid in

immunosurveillance, by transporting intracellular peptides to distant immune cells

(Calderwood et al., 2007).

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88

In conclusion, this study provides convincing data that Aβ-induced toxicity

triggered an early signaling response by the ER, independent of the ER-stress pathway.

ER-stress markers were downregulated, including GRP94 that was secreted to the

extracellular milieu in Aβ-induced apoptosis. Additional studies are warranted to

determine the specific role of GRP94 protein on Aβ-induced toxicity. Notably, this

signaling pathway was observed in an apoptotic context, rather than necrotic, which

opens a new window for the development of novel therapeutic strategies in AD.

Acknowledgments


para a Ciência e a Tecnologia (FCT), Lisbon, Portugal. RJS Viana is the recipient of a

PhD fellowship (SFRH/BD/30467/2006) from FCT, Portugal.

4 Modulation of amyloid β peptide-induced

toxicity through inhibition of JNK nuclear

localization and caspase-2 activation

Ricardo J. S. Viana1, Rita M. Ramalho

1, Ana F. Nunes

1, Clifford J. Steer

2,3,

Cecília M. P. Rodrigues1


University of Lisbon, Lisbon, Portugal; and Departments of 2Medicine and

3Genetics,

Cell Biology, and Development, University of Minnesota Medical School, Minneapolis,

MN, USA

Journal of Alzheimer's Disease 2010; 22: 557-568

Reprinted from Journal of Alzheimer's Disease, vol. 22: 557, Ricardo J. S. Viana, Rita M.

Ramalho, Ana F. Nunes, Clifford J. Steer, Cecília M. P. Rodrigues. Modulation of

amyloid-β peptide-induced toxicity through inhibition of JNK nuclear localization and

caspase-2 activation, Copyright 2010, with permission from IOS press. All rights

reserved.

Modulation of JNK/caspase-2 pathway

91

4.1 Abstract

Amyloid β peptide (Aβ)-induced neurotoxicity is typically associated with

apoptosis. In previous studies, we have shown that tauroursodeoxycholic acid (TUDCA),

an endogenous anti-apoptotic bile acid, modulates Aβ-induced apoptosis. Here, we

investigated stress signaling events triggered by soluble Aβ and further explored

alternative pathways of neuroprotection by TUDCA in differentiated rat neuronal-like

PC12 cells. Morphologic evaluation of apoptosis confirmed that Aβ-induced nuclear

fragmentation was prevented by TUDCA. In addition, Aβ exposure resulted in activation

of the early stress c-Jun N-terminal kinase (JNK) pathway, JNK nuclear translocation,

and caspase-2 activation. Knock-down experiments of JNK established caspase-2 as a

specific downstream target of JNK in Aβ-induced apoptosis. Furthermore, active

caspase-2 cleaved golgin-160 and was localized to the Golgi complex. Importantly,

TUDCA abrogated Aβ-induced JNK/caspase-2 signaling. In conclusion, we show that

JNK is the proximal stress sensor for soluble Aβ-induced toxicity, which translocates to

the nucleus, activates caspase-2, and is strongly modulated by TUDCA in PC12 neuronal

cells. Active caspase-2 cleaves golgin-160, suggesting caspase-2-dependent transduction

of Aβ apoptotic signaling through the Golgi complex. These data provide new

information linking apoptotic properties of Aβ peptide to distinct subcellular mechanisms

of toxicity. Further characterization of this signaling pathway and exact targets of

modulation are likely to provide new perspectives for modulation of amyloid-induced

apoptosis by TUDCA.

Keywords: Amyloid β; Apoptosis; c-Jun N-terminal kinase; Caspase-2; Golgi complex;

Tauroursodeoxycholic acid

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92

4.2 Introduction

Alzheimer‟s disease (AD) is the most common human age-related, sporadic

neurodegenerative disorder, and characterized by a global cognitive decline. The

neuropathological hallmarks of AD include synaptic loss and/or dysfunction, diminished

neuronal metabolism, senile plaques, neurofibrillary tangles (NFT), and loss of multiple

neurotransmitter systems (Selkoe, 2001). Brain regions involved in learning and memory

processes are reduced in size in AD patients, resulting from degeneration of synapses and

death of neurons. Although the mechanisms involved in neuronal dysfunction and death

are still not completely understood, there is growing evidence to suggest that apoptosis

may play a key role (Loo et al., 1993; Su et al., 1997) .

The c-Jun N-terminal kinase (JNK) pathway is a central stress signaling pathway

implicated in neuronal plasticity, regeneration and death (Herdegen et al., 1997). Several

lines of evidence suggest a potential involvement of the JNK pathway in AD

pathogenesis. Post-mortem brain sections from AD patients revealed altered subcellular

distribution and activation of JNK (Pei et al., 2001; Zhu et al., 2001b). Expression of c-

Jun, a JNK preferential transcriptional target, was also increased in AD brains (Anderson

et al., 1994; Anderson et al., 1996). In addition, JNK staining was localized in neurons

with intracellular accumulation of amyloid β (Aβ), and those surrounding amyloid

plaques in transgenic mice overexpressing a mutant form of presenilin-1 (Shoji et al.,

2000). Further, JNK phosphorylates tau, the major constituent of NFT, at sites identified

in paired helical filaments (Reynolds et al., 1997). Finally, JNK is activated by Aβ,

mediating both Aβ toxicity and its adverse effect on hippocampal long term potentiation

(Bozyczko-Coyne et al., 2001; Minogue et al., 2003; Morishima et al., 2001; Troy et al.,

2001; Wei et al., 2002). Of note, neurons from c-Jun-null mice are resistant to Aβ

toxicity (Kihiko et al., 1999).

Caspase-2 is the most evolutionarily conserved caspase, suggesting that it might

play a crucial role during programmed cell death. It shares sequence homology with

initiator caspases, but its cleavage specificity is closer to that of effector caspases

(Vakifahmetoglu-Norberg and Zhivotovsky). The limited knowledge that exists

regarding its mechanism of activation and the lack of specific substrates has hampered

further studies of this unique caspase (Bouchier-Hayes et al., 2009). Although caspase-2

can be activated in a JNK-independent mechanism (Marques et al., 2003), the association

between caspase-2 activation and JNK in response to Aβ-induced neuronal death has been

documented in cell culture studies, including those with primary neurons from caspase-2


93

null animals (Troy et al., 2000). In addition, acute or chronic removal of caspase-2 has

been shown to confer resistance to Aβ.

The role of apoptosis in Aβ-induced toxicity suggests that its modulation may

slow the neurodegenerative process. The endogenous hydrophilic bile acid

ursodeoxycholic acid (UDCA) and its taurine-conjugate tauroursodeoxycholic acid

(TUDCA) increase the apoptotic threshold in several cell types (Rodrigues et al., 1998).

In fact, we have demonstrated that TUDCA interrupts classic apoptotic pathways by

inhibiting mitochondrial membrane depolarization and channel formation (Rodrigues et

al., 1999; Rodrigues et al., 2003b; Rodrigues et al., 2000b). TUDCA is also a potent

suppressor of Aβ-induced changes of lipid/protein fluidity and oxidative status in the

membranes of isolated mitochondria (Rodrigues et al., 2001). Finally, we have shown

that TUDCA inhibits Aβ-induced apoptosis in vitro (Ramalho et al., 2004; Solá et al.,

2003), and that TUDCA is neuroprotective in pharmacologic and transgenic mouse model

of Huntington‟s disease (Keene et al., 2002; Keene et al., 2001), and in rat models of

ischemic and hemorrhagic stroke (Rodrigues et al., 2003a; Rodrigues et al., 2002).

UDCA is FDA-approved for the treatment of primary biliary cirrhosis and its therapeutic

effects in liver diseases may result from the ability to modulate cell fate (Paumgartner and

Beuers, 2004). UDCA administration was also safe and tolerable in patients with

amyotrophic lateral sclerosis, and penetrated into the central nervous system in a dose-

dependent manner (Parry et al.).

In this study, we show that JNK is the proximal stress sensor for Aβ-induced

toxicity, and that it translocates to the nucleus and activates caspase-2 in PC12 neuronal

cells. Caspase-2 then induces cleavage of golgin-160, suggesting a unique transduction

mechanism of Aβ apoptotic signaling through the Golgi complex. Importantly, TUDCA

was efficient in modulating the JNK/caspase-2 apoptotic pathway.


4.3.1 Cell culture and induction of apoptosis

PC12 cells were grown in RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO,

USA) supplemented with 10% heat-inactivated horse serum (Sigma-Aldrich), 5% fetal

bovine serum (Invitrogen Corp., Carlsband, CA, USA) and 1% penicillin/streptomycin

(Invitrogen), and maintained at 37°C in a humidified atmosphere of 5% CO2. Cells were

plated and differentiated in the presence of nerve growth factor (NGF) (Promega Corp.,

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94

Madison, WI), as previously described (Park et al., 1998). Cell density was either at 1 x

105 cells/cm

2 for morphological assessment of apoptosis and viability assays, or 4 x 10

5

cells/cm2 for transfection assays and protein extraction. PC12 neuronal cells were then

incubated in medium supplemented with 100 µM TUDCA (Sigma-Aldrich), or no

addition (control), for 12 h, and exposed to 25 µM of peptide active fragment Aβ (25–35)

(Bachem AG, Bubendorf, Switzerland) for 1, 2, 8, and 24 h. Cells were fixed for

microscopic assessment of apoptosis or processed for cell viability assays. In addition,

total proteins were extracted for immunoblotting.


Cell viability was measured by trypan blue exclusion and by the lactate

dehydrogenase (LDH) viability assay (Sigma-Aldrich), according to the manufacturer's

instructions. Apoptotic nuclei were detected by Hoechst labeling after cell fixation with

4% formaldehyde in phosphate buffer saline (PBS), for 10 min at room temperature.

Following incubation with Hoechst dye 33258 (Sigma-Aldrich) at 5 g/mL in PBS for 5

min, and PBS washes, slides were mounted with PBS:glycerol (3:1, v/v) and fluorescence

visualized with an Axioskop fluorescence microscope (Carl Zeiss GmbH, Hamburg,

Germany). Fluorescent nuclei were scored and categorized according to the condensation

and staining characteristics of chromatin. Normal nuclei showed non-condensed

chromatin dispersed over the entire nucleus. Apoptotic nuclei were independently

identified by condensed and fragmented chromatin contiguous to the nuclear membrane,

as well as nuclear fragmentation and presence of apoptotic bodies. Three random

microscopic fields per sample of ~ 250 nuclei were counted and mean values expressed as

the percentage of apoptotic nuclei.

4.3.3 Inhibition of JNK pathway

The involvement of JNK pathway in Aβ-induced toxicity was confirmed by cell

viability assays after inhibition of JNK and c-Jun activation. PC12 cells were transfected

at 80% density with either the binding domain of the JNK interacting protein-1 (JIP-1)

(pCMV-Flag-JBD (JIP-1)) (Dickens et al., 1997), or dominant negative DN-c-Jun

Flag169 plasmids (Ham et al., 1995), using Lipofectamine 2000 (Invitrogen) according

to the manufacturer‟s protocol. At 24 h after transfection, PC12 cells were incubated with


95

25 µM A (25-35) for an additional 24 h. Cell viability was then measured by trypan

blue exclusion and confirmed by LDH viability assays.

4.3.4 Immunoblotting

Steady-state levels of phosphorylated JNK (p-JNK) and c-Jun (p-c-Jun), total

JNK and c-Jun, and caspase-2 proteins were determined by Western blot analysis.

Briefly, 80 µg of total and nuclear proteins were separated by 12% sodium dodecyl-

polyacrylamide gel electrophoresis. After electrophoretic transfer onto nitrocellulose

membranes, immunoblots were incubated with 15% H2O2 for 15 min at room temperature.

Following blocking with a 5% milk solution, membranes were incubated overnight at 4°C

with primary mouse monoclonal antibodies reactive to p-JNK (G-7), primary rabbit

polyclonal antibodies reactive to p-c-Jun (Ser 63/73), caspase-2 (H-19) (Santa Cruz

Biotechnology, Santa Cruz, CA, USA), and primary rabbit monoclonal antibodies

reactive to SAPK/JNK (56G8), c-Jun (60A8) (Cell Signaling Technology, Inc., Danvers,

MA, USA). Finally, secondary goat anti-mouse or anti-rabbit IgG antibody conjugated

with horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) was added for 3

h at room temperature. The membranes were processed for protein detection using the

SuperSignal substrate (Pierce Biotechnology, Rockford, IL, USA). β-Actin (AC-15)

(Sigma-Aldrich), GADPH (6C5) (Santa Cruz) and anti-acetyl-histone H3 (Upstate

Biotechnology, Lake placid, NY, USA) were used as loading controls for total, cytosolic

and nuclear proteins, respectively. Protein concentrations were determined using the Bio-

Rad protein assay kit (Bio-Rad) according to the manufacturer‟s specifications.

4.3.5 Immunocytochemistry

Cellular localization of p-JNK, p-c-Jun and caspase-2 was determined by

immunocytochemistry. Cells were fixed for 30 min in 4% formaldehyde and blocked

with 10% donkey serum, 1% FBS and 0.1% Triton X-100 for 1h, at room temperature.

Cells were then incubated either with mouse monoclonal anti-p-JNK antibody, rabbit

polyclonal anti-p-c-Jun antibody, or rabbit polyclonal anti-caspase-2 (all at 1:50 in

blocking solution; Santa Cruz), overnight at 4ºC. After rising, cells were incubated with

Alexa Fluor 488 conjugated anti-mouse secondary antibody (1:200 in blocking solution;

Invitrogen) or fluorescently labeled anti-rabbit CyTM5 (1:200 in blocking solution;

Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) for 1 h at room

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96

temperature. Finally, cells were stained with 5 g/mL of Hoechst dye 33258 for 5 min, or

co-incubated with mouse monoclonal anti-GM130 (1:200 in blocking solution; BD

Biosciences, Franklin Lakes, NJ, USA), overnight at 4ºC. Adequate controls were

prepared by omitting the primary antibody or using preimmune IgG, which eliminated all

labeling. Subcellular localization of p-JNK, p-c-Jun and caspase-2 were visualized using

an Axioskop fluorescence microscope.

4.3.6 Short interference-mediated silencing of the jnk gene

A pool of 2 specific short interference RNA (siRNA) nucleotides designed to

knock down jnk gene expression in rats (Wang et al., 2007b) was purchased from

Dharmacon (Waltham, MA, USA). A control siRNA containing a scrambled sequence

that does not lead to the specific degradation of any known cellular mRNA was used as

control. PC12 cells were transfected at 80% density with siRNA at the final

concentration of 50 and 75 nm using Lipofectamine 2000. JNK knockdown was

determined at 24 h after transfection. Floating and attached cells were harvested for

preparation of total and nuclear protein extracts, which were then subjected to Western

blot analysis.

4.3.7 Expression of Golgin-160 cleavage products

PC12 cells were transfected with either green fluorescent protein (GFP)-tagged

wild-type golgin-160, or mutant golgin-160 (3DE) using Lipofectamine 2000. Stable cell

lines were selected with 0.4 mg/ml G418 (Invitrogen) and screened as described

previously (Mancini et al., 2000). Cells were plated and treated at ~ 90% confluency with

10 mM sodium butyrate in normal growth medium for 12-15 h to increase expression of

the GFP fusion protein. Cells were exposed to either 25 µM Aβ (25-35), 10 µM

staurosporine (positive control) or left untreated, then lysed and processed for

immunoblotting of GFP (B-2) (Santa Cruz).


The relative intensities of protein bands were analyzed using the Quantity One

Version 4.6 densitometric analysis program (Bio-Rad) and normalized to the respective

loading controls. Statistical analysis was performed using GraphPad InStat Version 3.00


97

(GraphPad Software, San Diego, CA) for the analysis of variance and Bonferroni‟s

multiple comparison tests. Values of p < 0.05 were considered significant.

4.4 Results

4.4.1 Aβ-induced apoptosis is mediated by JNK activation and modulated by

TUDCA in PC12 neuronal-like cells

Soluble Aβ (25-35) fragment induces apoptosis in neuronal PC12 cells and

TUDCA is a strong modulator of cell death (Ramalho et al., 2004). Neuronal PC12 cells

pre-exposed to NGF for 8 days were treated with Aβ, with or without TUDCA and

assessed for cell death, and phosphorylation of JNK and c-Jun. Aβ-induced apoptosis

was reduced to control levels in cells pre-treated with TUDCA (p < 0.05) (Fig. 4.1).

Figure 4.1. TUDCA inhibits apoptosis induced by Aβ in PC12 neuronal cells. Cells were

incubated with 25 µM soluble Aβ (25-35), or no addition (control), ± 100 µM TUDCA for 24 h.

In co-incubation experiments, TUDCA was added 12 h prior to incubation with Aβ. Cells were

fixed and stained for microscopic assessment of apoptosis as described in Materials and Methods.

Fluorescence microscopy of Hoechst staining (top) and percent apoptosis in cells exposed to Aβ ±

TUDCA (bottom). Cells exposed to Aβ alone (a) showed more nuclear fragmentation compared

with cells pre-treated with TUDCA (b). The results are expressed as mean ± SEM for at least

three different experiments. *p < 0.01 from control and †p < 0.05 from Aβ alone. Scale bar, 10

µm.

p-JNK increased over time in the absence of TUDCA, peaking at 2 h, while p-c-

Jun peaked at 24 h. Total JNK and c-Jun remained constant (Fig. 4.2A). Pre-treatment

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98

with TUDCA markedly reduced Aβ-induced JNK and c-Jun phosphorylation (p < 0.05)

(Fig. 4.2B). The protective function of TUDCA was independent of Erk and

phosphatidylinositol 3-kinase/Akt survival activation pathways (data not shown).

Figure 4.2. TUDCA modulates Aβ-induced p-JNK and p-c-Jun levels in PC12 neuronal

cells. Cells were incubated with 25 µM soluble Aβ (25-35), or no addition (control), ± 100 µM


99

TUDCA, for the indicated time points. In co-incubation experiments, TUDCA was added 12 h

prior to incubation with Aβ. Total proteins were extracted for western blot analysis as described

in Materials and Methods. (A) Protein levels of p-JNK and p-c-Jun in cells incubated with Aβ for

1, 2, 8 and 24 h. (B) Protein levels of p-JNK and p-c-Jun in cells exposed to Aβ ± TUDCA for 2

h and 24 h, respectively. The results are normalized to endogenous total JNK and c-Jun

production and expressed as mean ± SEM arbitrary units for at least four different experiments.

(C) Percent cell death measured by trypan blue exclusion after transfection with the binding

domain of JIP-1 and the dominant negative DN-c-Jun Flag169 plasmids. *p < 0.01 and §p <

0.05 from control; ‡p < 0.01 and †p < 0.05 from Aβ alone.

To confirm that JNK activation is required for Aβ-induced cell death in vitro, we

used two dominant interfering forms of the JNK signaling pathway, pCMV-Flag-JBD

(JIP-1) (Dickens et al., 1997) and DN-c-Jun FlagΔ169 (Ham et al., 1995). The binding

domain fragment of scaffolding protein JIP-1 has been shown to bind JNK and reduce its

ability to phosphorylate substrates such as c-Jun, thus inhibiting the JNK pathway. In

addition, DN-c-Jun Flag169 lacks the c-Jun transactivation domain. When

overexpressed, DN-c-Jun Flag169 competes with endogenous c-Jun for binding to DNA

regulatory elements, thus inhibiting c-Jun-dependent transcription. Aβ-dependent general

cell death was essentially abolished in cells transfected with constructs encoding either

pCMV-Flag-JBD (JIP-1) or DN-c-Jun FlagΔ169 (Fig. 4.2C). To assess transfection

efficiency, we have done as described in (Ramalho et al., Journal of Neurochemistry,

2004). Briefly, cells were co-transfected with the luciferase construct, pCMV-Control

vector (Promega Corp., Madison, WI, USA). Based on this reporter, transfection

efficiencies were approximately 70% and did not differ between wild-type and dominant

negative plasmids. After 24 h of incubation with Aβ, cell death was 18% in cells

expressing either pCMV-Flag-JBD (JIP-1) or DN-c-Jun FlagΔ169 constructs, and 30% in

cells expressing the vector only (p < 0.01). Thus, JNK appears to be an early stress

sensor for Aβ-induced toxicity, which in turn is strongly modulated by TUDCA.

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100

4.4.2 p-JNK localizes to the nucleus and mediates Aβ-induced caspase-2

activation

Under specific stress stimuli, JNK is an absolute requirement for neuronal cell

death. Importantly, confinement of JNK inhibition to the cytoplasm failed to protect

neurons, whereas a nuclear-targeted inhibitor provided robust protection (Bjorkblom et

al., 2008). To determine whether p-JNK cellular distribution plays a critical role in Aβ-

induced apoptosis of PC12 neuronal cells, we performed immunofluorescence studies.

After 2 h of Aβ incubation, p-JNK was primarily localized to the nucleus, suggesting a

nuclear signaling mechanism (Fig. 4.3A). Importantly, TUDCA significantly reduced p-

JNK nuclear localization. These results were corroborated by immunoblot analysis of

nuclear protein extracts (Fig. 4.3B).

Figure 4.3. Aβ induces p-JNK translocation to the nucleus in PC12 neuronal cells. Cells

were incubated with 25 µM Aβ, or no addition (control), ± 100 µM TUDCA, for the indicated

time points. In co-incubation experiments, TUDCA was added 12 h prior to incubation with Aβ.


101

(A) Fluorescence microscopy shows immuno-co-localization of p-JNK (in red) with the nucleus

(in blue) at 2 h of Aβ incubation. Scale bar, 10 µm. (B) Representative immunoblots of nuclear

and cytosolic p-JNK. The blots were normalized to nuclear and cytosolic endogenous protein

levels with anti-acetyl-histone H3 and GADPH, respectively.

In addition, p-JNK nuclear localization was associated with increased caspase-2

intermediate active fragment at 30 kDa at 2 h of Aβ incubation, and a maximum at 24 h

(p < 0.05) (Fig. 4.4). Notably, pre-treatment with TUDCA reduced active caspase-2 to

control levels (p < 0.05).

Figure 4.4. TUDCA modulates Aβ-induced active caspase-2 levels in PC12 neuronal cells.

Cells were incubated with 25 µM Aβ, or no addition (control), ± 100 µM TUDCA, for the

indicated time points. In co-incubation experiments, TUDCA was added 12 h prior to incubation

with Aβ. Total proteins were extracted for western blot analysis as described in Materials and

Methods. Representative immunoblot of cleaved caspase-2 (top) and respective protein levels

(bottom) in cells exposed to Aβ ± TUDCA for 1, 2, 8 and 24 h. The results are normalized to

endogenous β-Actin protein levels and expressed as mean ± SEM arbitrary units of at least four

different experiments. §p < 0.05 from control; †p < 0.05 from Aβ alone.

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Next, we used siRNA to knock down JNK isoforms, and investigated whether

caspase-2 is a specific downstream target of JNK. The siRNAs designed against JNK

isoforms were derived from the jnk1 and jnk2 respective conserved motif, and could

recognize different sub-isoforms of the respective gene but not isoforms from the other

gene (Wang et al., 2007b). Caspase-2 activation was markedly reduced after silencing

JNK with siRNA JNK 1/2 pool (Fig. 4.5). Transfection of a scrambled siRNA control did

not disturb the expression of JNK nor the housekeeping gene β-actin, confirming the

specificity of JNK siRNAs. Thus, caspase-2 appears to be a specific downstream target

of the JNK pathway.

Figure 4.5. Silencing of JNK inhibits Aβ-induced caspase-2 activation in PC12 neuronal

cells. Cells were transfected either with control, scrambled siRNA or JNK siRNA pool at 50 and

75 nM. At 24 h after transfection, cells were challenged with 25 µM Aβ, or no addition (control)

for 2 h. Total proteins were extracted for immunoblot analysis as described in Materials and

Methods. Representative immunoblots of p-JNK and caspase-2. The blots were normalized to β-

Actin endogenous protein levels.

4.4.3 Aβ-induced caspase-2 activation mediates cell death via the Golgi complex

Caspase-2 cleavage alone is not an accurate indication of either activity or

activation state (Tu et al., 2006). Interestingly, the only caspase-2 specific substrate

identified thus far is golgin-160, a peripheral Golgi membrane protein (Mancini et al.,

2000). Although golgin-160 may be cleaved by other caspases, namely caspase-3 and

caspase-7, it has been shown to possess a caspase-2 specific cleavage site at aspartate-59,

which is not susceptible to other caspases. To unequivocally demonstrate caspase-2

activation, we generated PC12 cell lines stably expressing GFP-tagged wild-type golgin-

160, or mutant golgin-160 with all three caspase cleavage sites replaced with glutamates.


103

We confirmed that neither caspase-3, nor caspase-7, was activated under the experimental

conditions used (data not shown), suggesting that the GFP-golgin-160 processing pattern

was due to caspase-2 cleavage alone. After induction of apoptosis by staurosporine or

Aβ, the intact wild-type GFP-golgin-160 fusion protein was cleaved, generating a new

fragment of 28 kDa (Fig. 4.6A). However, this caspase-2 cleaved fragment was no longer

detectable in cells expressing mutant GFP-golgin-160 fusion protein. Importantly,

immunocytochemistry analysis revealed that caspase-2 was localized to the Golgi process

after 24 h of Aβ incubation (Fig. 4.6B); and this was reduced by pre-treatment with

TUDCA. In fact, fluorescence intensity of caspase-2 and Golgi co-localization was ~

30% diminished in the presence of TUDCA. Thus, caspase-2 localizes to the Golgi and

acts as an effector element of JNK-dependent Aβ-induced apoptosis, which in turn is

effectively modulated by TUDCA.

Figure 4.6. Caspase-2 localizes to the Golgi complex and cleaves golgin-160 in Aβ-induced

cell death in PC12 neuronal cells. (A) Stable PC12 cells transfected with either wild-type

golgin-160 or mutant golgin-160 were incubated with 25 µM Aβ, 1 µM staurosporine (positive

control), or no addition (control) for 4 h. Total proteins were extracted for western blot analysis

as described in Materials and Methods. A 28-kDa fragment was detected in wild-type golgin-160

expressing cells treated with staurosporine or Aβ, but not in mutant golgin-160 expressing cells.

Representative immunoblots of at least four different experiments are shown. β-Actin protein was

used as loading control. (B) Fluorescence microscopy shows immuno-co-localization of caspase-

2 (in red) with the Golgi complex (in green) at 24 h of Aβ incubation. Scale bar, 10 µm.

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4.5 Discussion

The role of Aβ in the pathogenesis and progression of AD-associated

neurodegeneration has not been firmly established. Aβ-induced toxicity is a multi-

factorial process thought to involve the generation of reactive oxygen species, alteration

of intracellular calcium homeostasis, mitochondrial perturbation, and caspase activation

(Selkoe, 2001). Recent studies corroborated the involvement of apoptosis by showing

that Aβ alters expression of the Bcl-2 family of apoptosis-related genes (Yao et al., 2005),

and that survival signaling pathways are required for protection of Aβ-mediated neuronal

apoptosis (Watson and Fan, 2005). In addition, we have previously reported that anti-

apoptotic TUDCA prevents Aβ-induced apoptosis by inhibiting the mitochondrial

pathway of cell death and regulating cell cycle-related proteins (Ramalho et al., 2006;

Ramalho et al., 2004; Solá et al., 2003). The present study demonstrates that Aβ-induced

apoptosis is, at least partially dependent on JNK/caspase signaling. Further, we show that

JNK translocates to the nucleus and activates caspase-2, which in turn localizes to the

Golgi complex and cleaves golgin-160 during toxic Aβ-induced apoptosis. Importantly,

TUDCA is an effective modulator of Aβ peptide-induced toxicity through inhibition of

JNK nuclear localization and caspase-2 activation. UDCA is FDA-approved for the

treatment of primary biliary cirrhosis in humans. In fact, the therapeutic effects of UDCA

in liver diseases may result from its ability to inhibit apoptosis (Paumgartner and Beuers,

2004).

PC12 cells have been established as an efficient system for cell signaling studies

(Vaudry et al., 2002). In addition, PC12 cells respond to several stress stimuli via the

JNK pathway, including Aβ-induced cell death (Troy et al., 2001), and is a logical choice

for investigating the potential role of JNK in the context of neuronal apoptosis (Waetzig

and Herdegen, 2003). Previous studies have shown that activation of JNK signaling is

closely linked to a variety of apoptotic stimuli; whereas inhibition of JNK provides

protection against neuronal apoptosis in multiple paradigms, including Aβ-induced

neurotoxicity (Bozyczko-Coyne et al., 2001; Morishima et al., 2001; Troy et al., 2001;

Yao et al., 2005, 2007). In addition, the activation of JNK has been reported in

susceptible neurons of AD patients and has been correlated with AD pathogenesis (Zhu et

al., 2001a). JNK promotes apoptosis via both transcriptional and posttranslational

regulation of Bcl-2 family members (Wang et al., 2007a; Yao et al., 2007) c-Jun is also a

major target of the JNK pathway as it contributes to the cellular stress response and is

believed to mediate apoptosis neuronal cell death (Behrens et al., 1999) . In fact, JNK


105

activation is accompanied by an increase in c-Jun Ser-63/73 phosphorylation (Coffey et

al., 2002). Although the constitutively high activity that is characteristic of neuronal JNK

is largely cytoplasmic (Coffey et al., 2000), nuclear localization is critical for cell death

(Bjorkblom et al., 2008). Thus, the suppression of JNK-dependent apoptotic gene

expression may be an effective therapeutic strategy for preventing apoptosis of neuronal

cells.

In the present study, we show that Aβ-induced apoptosis in PC12 neuronal-like

cells requires early JNK activation, confirming this pathway as the proximal stress sensor

for Aβ toxicity. JNK activation peaked after 2 h of Aβ incubation, and was followed by

c-Jun phosphorylation as well as cell death features. Using two dominant interfering

forms of the JNK signaling pathway, we further confirmed that JNK is required for Aβ-

induced apoptosis of PC12 neuronal cells. Moreover, we showed that most early active

JNK is confined to the cell nucleus. Despite the myriad of physiological functions

controlled by JNK in the cytoplasm, our results suggest that Aβ-induced cell death is, in

part dependent on a nuclear site of JNK activity, consistent with previous observations

under different conditions (Bjorkblom et al., 2008). Notably, we demonstrated that

TUDCA inhibited Aβ-induced JNK activation, changes in p-c-Jun levels, and JNK

nuclear translocation. It remains to be determined if JNK-mediated cell death is a strictly

nuclear-specific process, or instead, p-JNK nuclear localization is a pan-cell death

mechanism.

Despite the extensive knowledge regarding JNK pathway activation,

downstream cell signaling components remain unclear in Aβ-induced apoptosis.

Caspase-2 has been described as a downstream target of JNK activation in different cell

models (Murakami et al., 2007; Troy et al., 2001). However, by using chemical

inhibitors, which may lack specificity and block multiple events associated with Aβ-

induced apoptosis, these studies were unable to clarify the association (Bennett et al.,

2001; Bozyczko-Coyne et al., 2001). Here we demonstrate that caspase-2 is a specific

downstream target of Aβ-induced JNK activation. In fact, after depleting cells of JNK by

specific siRNA, Aβ incubations failed to activate both JNK and caspase-2. As expected,

TUDCA was also capable of inhibiting caspase-2 activation.

The role of caspases in the pathogenesis and progression of AD has not been

entirely established. Nevertheless, previous studies have shown that Aβ challenge and

trophic factor withdrawal activate caspase-2 in neurons (Stefanis et al., 1998; Troy et al.,

2000). More importantly, it has been described that protein levels of caspase-2 were

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significantly increased in AD brains (Shimohama et al., 1999). Here, we confirmed

caspase-2 as a mediator of Aβ-induced apoptosis and provided mechanistic evidence for

Aβ-induced caspase-2 activation through the JNK pathway. Caspase-2 is an initiator

caspase, but specific details of how, where, and when it is activated remain unclear.

Furthermore, the mechanism(s) by which caspase-2 induces cell death is unknown.

Unlike other initiator caspases, caspase-2 does not directly activate executioner caspases;

and may act via mitochondrial outer membrane permeabilization to induce apoptosis

(Guo et al., 2002). Moreover, several stimuli of cell death that induce caspase-2-

dependent pathways do not require other caspases (Troy and Ribe, 2008). Interestingly,

caspase-2 deficient neurons are resistant to Aβ and, although caspase-3 becomes activated

in response to Aβ, it is neither necessary nor sufficient for cell death (Troy et al., 2000).

The results suggest that Aβ may require primarily caspase-2 to induce neuronal cell death.

A major contributing factor to the paucity of knowledge on the physiological

activation of caspase-2 is the inability to measure activation of initiator caspases.

Caspase-2 undergoes autocatalytic processing when activated and is also cleaved by

caspase-3 downstream of mitochondrial outer membrane permeabilization (Slee et al.,

1999). However, this does not activate caspase-2 (Baliga et al., 2004), and thus

monitoring cleavage alone is not sufficient to identify caspase-2 as the apical caspase

(Bouchier-Hayes et al., 2009). It is also critical to determine the downstream substrates

of caspase-2 to fully understand its functions. To date, no specific substrates of caspase-

2, other than procaspase-2 itself (Harvey et al., 1996) and golgin-160 (Mancini et al.,

2000) have been identified. Golgin-160 is a peripheral Golgi membrane-associated

autoantigen that is cleaved by caspase-2 in vitro during apoptosis (Mancini et al., 2000).

In the nervous system, the expression of caspase-2 is neuron specific, which makes it an

attractive target for therapeutic intervention. In addition, caspase-2 is activated rapidly

and is upstream or independent of mitochondria, which makes it an attractive target for

inhibition, without disturbing mitochondrial function (Troy and Ribe, 2008).

In the present study, we showed that Aβ-induced activated caspase-2 cleaves its

substrate, the structural Golgi protein golgin-160. After confirming that neither caspase-3

nor -7 were activated, we examined the effects of stable expression of wild-type golgin-

160 and caspase-resistant mutant golgin-160. Cells expressing mutant golgin-160 failed

to present the characteristic caspase-2 cleavage fragment observed in wild-type golgin-

160 expressing cells. Our results suggest that Aβ-induced apoptotic signals can be

transduced at the Golgi complex. In addition, we demonstrated that caspase-2 has a Golgi


107

complex subcellular distribution, which is effectively modulated by TUDCA. The Golgi

complex, with its central location and essential role in membrane traffic, is in an ideal

position to sense and integrate information on the cell status. Interestingly, several

signaling proteins implicated in apoptotic pathways are enriched at Golgi membranes,

including tumor necrosis factor receptor, Fas, an isoform of protein kinase C, and

phophatidyl inositide 3-kinase. Understanding the mechanisms of how proteins such as

golgin-160 regulate Aβ-induced apoptotic signal transduction will be vital in elucidating

the role of the Golgi complex in the apoptosis associated with AD.

In conclusion, this study provides convincing data that Aβ-induced toxicity

triggers the JNK early stress sensor pathway. Our results also demonstrated that early

active JNK is localized primarily to the nucleus, suggesting that this preferential

subcellular distribution is critical for Aβ-induced neuronal death. Moreover, our studies

highlight the importance of caspase-2 and the JNK pathway(s) in the development of Aβ-

induced apoptosis. Caspase-2 is localized in the Golgi complex, where it cleaves a

structural Golgi protein. Notably, TUDCA was able to inhibit JNK early activation and

nuclear translocation, thus protecting cells from death. Caspase-2 apoptotic features were

also considerably inhibited in the presence of TUDCA. These results clarify Aβ-induced

subcellular toxicity mechanism and open new avenues in understanding anti-apoptotic

TUDCA modulation.

Acknowledgments

We are grateful to Dr. Roger Davis, Howard Hughes Medical Institute, and Dr.

Lee Rubin, Harvard University, for providing pCMV-Flag-JBD (JIP-1) and DN-c-Jun

FlagΔ169 plasmids, respectively. We also thank Dr. Carolyn Machamer, Johns Hopkins

University School of Medicine for providing wild-type golgin-160 and mutant golgin-160

(3DE) plasmids.


para a Ciência e a Tecnologia (FCT), Lisbon, Portugal. RJS Viana is the recipient of a

PhD fellowship from FCT, Portugal (BD/30467/2006); RM Ramalho and AF Nunes are

recipients of postdoctoral fellowships from FCT, Portugal (BPD/40623/2007 and

BPD/34603/2007, respectively).

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109

5 Concluding Remarks

Concluding Remarks

111

In modern societies, people live longer than ever and this symbolizes the

hallmark of human kind evolution. Age-related diseases embody the opposite face, with

neurodegenerative diseases representing one of the biggest challenges for modern

medicine. The fact that most people maintain a well-functioning nervous system and

continue being productive through their long lives is truly encouraging.

By mechanisms not fully understood, people who develop neurodegenerative

diseases share common significant neuronal loss. This highlights the importance of

studying and understanding the cell death phenomenon. It is now becoming accepted that

cell death might be initiated through several organelles and specific stress sensors, which

communicate cell death modulating signals. Decoding this complex signaling network of

inter-organellar cross-talk might represent the key for delaying or preventing

neurodegenerative diseases. New and effective therapeutic approaches are expected to

emerge with the integration and understanding of these complex mechanisms.

Detection of apoptosis in post mortem human tissue is complicated by many

conceptual and technical factors. First, the presumed daily rate of neuron loss in

neurodegenerative disorders is low and disappearance of apoptotic cells is rapid (Vila and

Przedborski, 2003). The time period between the condensation of chromatin and the

formation of apoptotic bodies lasts a few minutes (Bursch et al., 1990), and apoptotic

bodies can be detected for only 4-5 hours (Arends et al., 1990). Therefore, early

morphological changes in apoptotic cells are not easily detected, and the chronological

sequence of typical apoptotic morphological changes is controversial. Second, post

mortem specimens typically derive from advanced disease stages, when most of the

neurons affected by the pathological process are already lost. Third, most morphological

post mortem studies rely on the TUNEL technique to document the presence of apoptotic

cells. However, we now know that TUNEL is not specific for apoptosis, especially in

human post mortem tissue, in which factors such as hypoxia can produce TUNEL-

positive, non-apoptotic DNA damage (Kingsbury et al., 1998). Aβ peptide induces

apoptosis in primary neurons in vitro (Loo et al., 1993). Further, pharmacological or

molecular inhibition of particular members of the caspase family, such as caspases-2, -3, -

8 and -12, has been reported to offer partial or complete protection against Aβ-induced

apoptotic cell death in vitro (Giovanni et al., 2000; Ivins et al., 1999; Nakagawa et al.,

2000; Troy et al., 2000). Despite all of the above, scant evidence of caspase activation or

apoptotic cell death has been gathered in animal models of AD and, therefore, in vivo

confirmation for a potential beneficial effect of blocking apoptosis pathways in AD is still

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uncertain. Nevertheless, fragmentation of nuclear DNA has been detected in brains of

patients with AD. More conclusive biochemical evidence that apoptosis might occur in

AD has been provided by the detection of activated caspases-3, -8 and -9 in hippocampus

neurons of brains affected by AD (Rohn et al., 2001; Rohn et al., 2002; Selznick et al.,

1999; Stadelmann et al., 1999).

Aβ is derived from γ-secretase-mediated processing of the APP, which can also

be cleaved by caspases, such as caspase-3, at positions distinct from the classic secretase-

processing sites, in both cultured cells and brains affected by AD (Gervais et al., 1999).

Caspase-mediated cleavage of APP releases not only Aβ, but also a carboxy-terminal

peptide that is a potent inducer of apoptosis (Lu et al., 2000). Similarly, caspase-3-

cleaved fragments of tau, a microtubule-associated protein that is the primary protein

component of the filaments found in AD brains, have also been detected in post mortem

samples (Rohn et al., 2002). Nevertheless, neurons appear to compensate for the global

neuronal loss in AD by expanding their connective interactions and functional capacities

(Cotman and Anderson, 1988). This may explain the fact that neurological deficits are

only clinically detectable when ~ 50 to 70% of neurons are lost in a given population

(Waldmeier and Tatton, 2004).

From a translational viewpoint, research on apoptosis creates exciting

opportunities. The concept that cell death is not merely random and chaotic, but rather

occurs in a programmed and regulated fashion was a stimulus to develop the work

presented in this thesis. In reality, identifying early signaling events in AD pathogenesis

is crucial to unravel novel pharmacological targets, which may be amenable to

therapeutic intervention before significant neuronal degeneration has occurred. Soluble

Aβ accumulation, altered phosphorylation of tau, and decreased synaptic density appear

to occur early in the course of AD-like cytopathology in mouse models and perhaps also

in patients with mild cognitive impairment, a frequent harbinger of AD. In addition,

acute application of soluble forms of human Aβ can rapidly, within minutes and, in some

cases, reversibly alter synaptic transmission and mimetize the toxic effects observed in

AD (Townsend et al., 2006). Aggregation/fibrillization of Aβ plays a critical role in

neurodegeneration. In particular, intermediate oligomeric and protofibrillar forms seem

to display the most potent effects in neuronal cells. Mutant peptides with accelerated

formation of intermediate species and increased stability of these structures affect mainly

vascular cells. The AE22Q variant peptide has been shown to selectively exert

Concluding Remarks

113

deleterious effects on vascular cells, compared to the wild-type Aβ40 (Bossers et al.,

2009; Fadok et al., 1992). Nevertheless, the nature of the amyloid assemblies and the

mechanisms of toxicity remain uncertain. Using human cerebral endothelial cells we

studied the influence of the aggregation/fibrillization cascade on apoptosis-induced

toxicity, and dissected the potential stress pathway. We determined that AβE22Q

presented an intermediate Aβ assembly between wild-type Aβ40 and Aβ42. Aβ40 did not

show any evidence of fibrillar components, which in turn were obvious for Aβ42. In

addition, we determined that neither wild-type Aβ40 nor Aβ42 triggered apoptosis in this

cellular context. On the other hand, AβE22Q strongly activated cell death in a selective

way for human cerebral endothelial cells. Furthermore, we demonstrated that the stress

sensors activated by AβE22Q converged and transduced through the mitochondria.

AβE22Q was a strong inducer of the Bax pro-apoptotic mitochondrial pathway in human

cerebral endothelial cells, activating specific downstream events, such as cytochrome c

release and nuclear fragmentation. Importantly, we showed that TUDCA was effective at

protecting from vasculotropic E22Q peptide, through suppression of Bax translocation to

mitochondria. Notably, despite its potent anti-apoptotic role, TUDCA did not interfere, at

least directly, with the aggregation properties of Aβ peptide in vitro. Future experiments

should clarify whether TUDCA has any differential effect in peptide fibrillization in cell

culture conditions and, ultimately, in vivo. These results demonstrate that the

aggregation/fibrillization cascade per se is not determinant of Aβ-induced apoptotic

toxicity. Rather, it is suggested that specific and selective conformational assemblies for

each Aβ peptide are responsible for the toxic effects. This is in accordance with data

from other groups showing that the low-n oligomers, including dimers, trimers and

tetramers were the most toxic species (Shankar et al., 2008; Townsend et al., 2006).

Nevertheless, insoluble amyloid plaques may also play a pathogenic role, as their

invariant accumulation may represent relatively inert reservoirs of small bioactive

oligomers, which may readily disassemble in the presence of lipids (Martins et al., 2008).

That plaque cores may release locally active Aβ species in vivo is suggested by a

penumbra of synapse loss around cores in APP transgenic mice (Spires et al., 2005).

Despite the fact that mitochondria play a central role in apoptotic cell death,

recent studies have challenged this function and suggested that apoptosis can be induced

by the ER stress pathway, independent of mitochondria (Lamarca and Scorrano, 2009).

In line with this concept, it has been shown that AD does not result from some unusual

toxic phenomenon but, rather, is caused by age-related declines seen in innumerable

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metabolic pathways in all metazoans. Specifically, plaque formation is thought to result

from failure of the ER to catalyze the post-translational processing of Aβ (Erickson et al.,

2005). Furthermore, there is an age-dependent decline in critical chaperones necessary

for catalysis of this process in the ER (Erickson et al., 2006). Corroborating this idea,

ER-resident molecular chaperones such as GRP78 and GRP94 are donwregulated in

brains of AD patients and in PS1 mutant cells (Katayama et al., 1999). These

observations provide an alternative explanation for the cognitive decline seen in the

elderly. The ER catalyzes the post-translational processing of the bulk of intrinsic plasma

membrane proteins, including the newly synthesized synaptic proteins necessary for the

initiation, consolidation, and retrieval of memory (Erickson et al., 2005). If these proteins

are not properly folded in the ER, rather than being inserted into the synaptic membrane,

they are degraded, and the amino acids are recycled.

One obstacle to understanding how Aβ induces toxicity is the ambiguity over

which molecular species are responsible for the biological effect. To overcome this

concern, and minimize the differential effects of Aβ assemblies in the

aggregation/fibrilization cascade, we have used one of the shortest and soluble forms of

Aβ (25-35). We investigated how ER senses the stress directly caused by Aβ toxic core.

Our results showed that Aβ-induced toxicity resulted in activation of caspase-12, which

mediates ER-specific apoptosis in PC12 neuronal cells. Importantly, using the anti-

apoptotic compound TUDCA, or a calpain inhibitor, we were able to revert active

caspase-12 to control levels. Intriguingly, however, we observed that Aβ-induced

toxicity triggered an early signaling response by the ER, independent of the ER-stress

pathway. In fact, ER stress markers such as GRP94, ATF-6α, eIF2α and CHOP/GADD

153 were downregulated as early as 2 h after soluble Aβ incubation. This was

independent of transcription as well as degradation, such as proteasome and autophagy

pathways. Changes in ATF-6α were prevented by calpain inhibitor I, suggesting that the

concentration of intracellular Ca2+

was elevated. However, despite the importance of

GRP94 in Ca2+

regulation, neither calpain inhibitor I nor Ca2+

channels blockers prevented

GRP94 protein changes. Finally, since GRP94 had been identified in the extracellular

space as a consequence of acute stress cell death (Basu et al., 2000; Berwin et al., 2001),

we demonstrated that GRP94 downregulation was blocked in cells pretreated with

inhibitors of the secretory pathway, geldanamycin and brefeldin. Using an ELISA assay

for GRP94 in the supernatant of cells challenged with Aβ, we established that GRP94

levels were increased in the extracellular medium of cells incubated with Aβ. Once in the

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115

extracellular milieu, stress proteins such as GRP94 might increase stress resistance, after

binding to stress-sensitive recipient neurons, signal tissue destruction to inflammatory

cells, and aid in immunosurveillance, by transporting intracellular peptides to distant

immune cells. These findings deserve further exploitation as they might open new

opportunities for alternative therapeutic strategies.

JNK is an early stress pathway that is activated by the ER pathway IRE1α-

TRAF2-ASK1 (Urano et al., 2000). Further, JNK activates caspase-12 (Tan et al., 2006).

We found that in the absence of JNK, through the use of dominant negative forms, Aβ-

induced apoptosis was abrogated in PC12 neuronal-like cells. We have also confirmed

that JNK was the proximal stress sensor with peak activation at 2 h, followed by c-Jun

phosphorylation as well as cell death features. Most early active JNK was confined to the

cell nucleus, the most probable location when signaling for cell death (Bjorkblom et al.,

2008). Notably, we demonstrated that TUDCA inhibited Aβ-induced JNK activation,

changes in p-c-Jun levels, and JNK nuclear translocation. By silencing JNK, we

confirmed that caspase-2 was a specific downstream target of Aβ-induced JNK activation.

Unsurprisingly, TUDCA inhibited caspase-2 activation as determined by cleavage of its

specific substrate, the structural Golgi protein golgin-160. In addition, caspase-2 had a

Golgi complex subcellular distribution, which in turn was effectively modulated by

TUDCA. These results suggest that Aβ-induced apoptotic signals can be transduced at

the Golgi complex and place this organelle in the centre for development of potential

therapeutic strategies.

Our results demonstrating a role for diverse cell organelles in cell death signaling

has advanced our understanding of AD pathogenesis. We have demonstrated stress

sensing and execution of apoptosis by mitochondria of human cerebral endothelial cells,

when faced with toxic mutant Aβ peptides. We have also shown that soluble forms of Aβ

distress the ER, independently of ER stress, and suggested that this response may exist as

a trigger for immunosurveillance protection. Finally, we have described the straight

involvement of the nucleus and Golgi complex during Aβ-induced, JNK-dependent cell

death.

The involvement of several organelles in AD pathogenesis, together with the

large panoply of risk factors suggests that AD may be considered as a "pleiotropic

disease". Therefore, not surprisingly, the usual approach of single therapy, selective to a

target, used to treat AD patients is far from the best solution. A change has recently been

made in a tentative to use cocktails of medicines. This approach is also far from ideal,

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because it does not account totally for safety concerns. Thus, the use of a "pleiotropic

agent", such as TUDCA that modulates several pathways of the disease should the further

addressed in the future, using relevant animal models of AD.

117

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