Neuronal synapse formation regulated by intercellular ...

Neuronal Synapse Formation Regulated by Intercellular Adhesion Molecule-5 (ICAM-5)

DIVISION OF BIOCHEMISTRY AND BIOTECHNOLOGYDEPARTMENT OF BIOSCIENCESFACULTY OF BIOLOGICAL AND ENVIRONMENTAL SCIENCESUNIVERSITY OF HELSINKI

LIN NING

dissertationes biocentri viikki universitatis helsingiensis 112013

112013L

IN N

ING

Ne

uro

na

l Syn

ap

se F

orm

atio

n R

eg

ula

ted

by In

terce

llula

r Ad

he

sion

Mo

lecu

le-5

(ICA

M-5

)


LIN NING

Division of Biochemistry and Biotechnology Department of Biosciences

Faculty of Biological and Environmental Sciences And

Helsinki Graduate Program in Biotechnology and Molecular Biology University of Helsinki

Academic Dissertation

To be presented for public examination with the permission of the Faculty of Biological and Environmental Sciences University of Helsinki

In the Auditorium 1041 at Viikki Biocenter 2 Viikinkaari 5 Helsinki On September 13th 2013 at 12 orsquoclock noon

2

Supervisors Professor Carl G Gahmberg Department of Biosciences Faculty of Biological and Environmental Sciences University of Helsinki Finland

Docent Li Tian Neuroscience Center University of Helsinki Finland

Pre-examiners Docent Sari Lauri Neuroscience Center Department of Biosciences Faculty of Biological and Environmental Sciences University of Helsinki Finland

Professor Jorma Keski-Oja Department of Pathology Haartman Institute University of Helsinki Finland

Opponent Professor Melitta Schachner Institut fuumlr Biosynthese Neuraler Strukturen Zentrum fuumlr Molekulare Neurobiologie Universitaumltsklinikum Hamburg-Eppendorf Germany

ISSN 1799-7372ISBN 978-952-10-9041-7 (paperback) ISBN 978-952-10-9042-4 (PDF)

Unigrafia Helsinki 2013

3

ldquoUnfortunately nature seems unaware of our intellectual need for

convenience and unity and very often takes delight in complication and

diversityhellip Besides we believe that we have no reason for scepticism While

awaiting the work of the future let us be calm and confident in the future of

our workrdquo

Santiago Ramʊn y Cajal

1906

4

To the memory of my Grandmother

5

TABLE OF CONTENTS

ORIGINAL PUBLICATIONS 6

ABBREVIATIONS 7

ABSTRACT 9

REVIEW OF THE LITERATURE 10

1 Synapses and spines 10

11 Ultrastructure of synapses 1012 Synaptogenesis 1213 Dendritic spines and spine maturation 14

2 Actin binding proteins in synapse formation 16

21 Actin polymerization depolymerization 1622 Actin nucleation 1723 Actin cross-linking proteins 17

3 NMDA receptors 21

31 Structure 2132 Functions 2233 NMDAR induced signaling pathway in regulation of spine morphology 23

4 Cell adhesion molecules in synapse formation and plasticity 24

41 Integrins 2542 Immunoglobulin superfamily (IgSF) adhesion molecules 2743 Neuroligins and neurexins 2944 Cadherin superfamily 3045 Eph receptors and ephrins 31

5 ICAM-5 34

51 History 3552 Molecular feature 3553 Expression 3654 Binding partners 3655 Functions 38

AIMS OF THE STUDY 40

MATERIALS AND METHODS 41

RESULTS 42

DISCUSSION 47

CONCLUDING REMARKS 53

ACKNOWLEDGEMENTS 54

REFERENCES 55

6

ORIGINAL PUBLICATIONS

This thesis is based on following publications referred to by their Roman numerals in the

text

I Nyman-Huttunen H Tian L Ning L and Gahmberg C G (2006) Alpha-actinin-

dependent cytoskeletal anchorage is important for ICAM-5-mediated neuritic outgrowth JCell Sci 199 3055-3066

II Tian L Stefanidakis M Ning L Van Lint P Nyman-Huttunen H Libert C

Itohora S Mishina M Rauvala H and Gahmberg C G (2007) Activation of NMDA

receptors promotes dendritic spine development through MMP-mediated ICAM-5

cleavage J Cell Biol 178 (4) 687-700

III Ning L Tian L Smirnov S Vihinen H Llano O Vick K Davis R L Rivera

C and Gahmberg C G (2013) Interactions between Intercellular Adhesion Molecule-5

(ICAM-5) and E1 integrins regulate neuronal synapse formation J Cell Sci 126 77-89

IV Ning L Nyman-Huttunen H Paetau S Tian L Gahmberg C G (2013) ICAM-5

regulates spine maturation by competing with NMDA receptors for Į-actinin binding

Submitted

These authors contributed equally to the work

These articles are reproduced with the permission of their copyright holders

7

ABBREVIATIONS

ABD actin binding domain

ABP actin-binding protein

AD Alzheimerrsquos disease

ADF actin depolymerization factor

APP amyloid precursor protein

AMPA Į-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

AMPAR AMPA receptor

AZ active zone

CAM cell adhesion molecule

CaM calmodulin

CaMK II calmodulin-dependent protein kinase II

CASK calmodulin-dependent serine protein kinase

CH calponin homology

CNS central nervous system

DIV day in vitro

ECM extracellular matrix

ELISA enzyme-linked immunosorbent assay

EGF epidermal growth factor

EM electron microscope

EPSC excitatory postsynaptic current

ERM ezrin radixin moesin

FAK focal adhesion kinase

GABA Ȗ-aminobutyric acid

GAP GTPase-activating protein

GST glutathione S-transferase

ICAM intercellular adhesion molecule

Ig immunoglobulin

IgSF immunoglobulin superfamily

IPSC inhibitory postsynaptic current

kDa kilodalton

KO knockout

LFA-1 lymphocyte function associated antigen-1

LTP long-term potentiation

LTD long-term depression

mAb monoclonal antibody

mEPSC miniature excitatory post-synaptic current

mIPSC miniature inhibitory post-synaptic current

MMP matrix metalloproteinase

NCAM neuronal cell adhesion molecule

NMDA N-methyl-D-aspartate

NMDAR NMDA receptor

8

NT neurotransmitter

PIP2 phosphatidylinositol (45)-bisphosphate

PIP3 phosphatidylinositol (345)-trisphosphate

PI3-kinase phosphatidylinositide 3-kinase

PS presenilin

PSA polysialic acid

PSD postsynaptic density

RNAi RNA interference

shRNA small hairpin RNA

sICAM-5 soluble ICAM-5

SPR surface plasmon resonance

SR spectrin repeat

SV synaptic vesicle

SynCAM synaptic cell adhesion molecule

TLN telencephalin

VN vitronectin

WT wild type

9

ABSTRACT

In the central nervous system (CNS) synapses form the connections between neurons enabling

unidirectional signal transmission in the neuronal network Synapse formation is a process during

which the initial contacts between axons and dendrites undergo changes in morphology and protein

composition and differentiate into fully functional neurotransmitting units Synapse formation is

regulated by a plethora of factors working in orchestration In particular cell adhesion molecules

(CAMs) a subset of membrane receptors with their extracellular domains extending into synaptic

clefts initiate cellular signals that regulate synapse formation upon binding to their ligands

Intercellular adhesion molecule-5 (ICAM-5 telencephalin) is a neuron-specific

member of the ICAM family As a two-faceted molecule ICAM-5 controls immune responses in

the brain by regulating neuron-lymphocyte communication through binding to lymphocyte

function-associated antigen-1 (LFA-1) by modulating T-cell activation through ICAM-5

ectodomain cleavage or by mediating neuron-microglia interaction On the other hand ICAM-5

plays an important role in neuronal development including synapse formation To date ICAM-5 is

the only identified CAM that negatively regulates synaptic development Deletion of ICAM-5 from

mice leads to accelerated synapse formation enhanced capacity of synaptic transmission and

improved memory and learning Clinically changes in ICAM-5 levels have been detected in

various diseases such as acute encephalitis epilepsy and Alzheimerrsquos disease (AD)

The major goal of my thesis is to address the molecular mechanisms by which ICAM-

5 regulates synapse formation as well as maturation of dendritic spines the post-synaptic

components of excitatory synapses

In my study ICAM-5 was observed as a substrate for the matrix metalloproteinase

(MMP)-2 and -9 Activation of N-methyl-D-aspartate (NMDA) receptors in neurons elevated the

level of MMP activity and subsequently induced ICAM-5 ectodomain cleavage which in turn

promoted spine maturation In addition I identified ȕ1-integrins expressed at the pre-synaptic

membranes as counter-receptors for ICAM-5 The binding site was located to the first two

immunoglobulin (Ig) domains of ICAM-5 This trans-synaptic interaction occurred at the early

stage of synapse formation which inhibited the MMP-induced ICAM-5 cleavage and thereby

served as a protective mechanism that prevented spine maturation Moreover Į-actinin an actin

cross-linking protein was found to be a binding partner for the cytoplasmic tail of ICAM-5 This

binding linked ICAM-5 to the actin cytoskeleton and was important for the membrane distribution

of ICAM-5 as well as ICAM-5-mediated neurite outgrowth NMDA receptor (NMDAR) activity is

known to be one of the major regulators of actin-based spine morphogenesis The GluN1 subunit of

the NMDAR has been reported to bind to Į-actinin We found here that GluN1 and ICAM-5

competed for the same binding region in Į-actinin Activation of NMDAR changed Į-actinin

binding property of ICAM-5 resulting in Į-actinin accumulation and actin reorganization in

developing spines

In conclusion my thesis defines a novel ICAM-5-dependent mechanism which

regulates synapse formation spine maturation and remodeling

10

REVIEW OF THE LITERATURE

1 Synapses and spines Complex cognitive and motor functions are encoded and processed in a network of neurons in the brain About 150 years ago scientists thought that the neuronal network was a continuous syncytial reticulum with exchanging cytoplasm (Gerlach 1872 Golgi 1873) This theory was challenged by Santiago Ramoacuten y Cajal who argued that this network was composed of anatomically discontinuous separated cells (Ramoacuten y Cajal 1937) named neurons (Waldeyer-Hartz 1891) The segmented neurons are connected with each other via specialized cell-cell contacts called synapses(from Greek words ıȣȞ and ȣʌĲİȚȞ meaning to clasp together) In the human brain one neuron is innervated by thousands of other neurons forming a complex network via 1 000 trillion synapses

Rather than just being the physical contact sites synapses also create an important machinery of transmission of information between neurons The information transduced in the form of an electrical potential in neurons is first converted into chemical signals and then transformed back to electrical signals to be relayed in the target cells

Our brain undergoes continuous changes subject to the genetically programmed development as well as the experience that the brain gains through interaction with the environment Synapses are highly dynamic structures throughout their lifetime As basic unit of neuronal information storage dynamics of synapses is intimately associated with brain development and plasticity (Holtmaat and Svoboda 2009 Kasai et al 2010) Therefore understanding how synapses develop and work is of central importance in decoding brain functions and developing new therapies for brain dysfunctions

11 Ultrastructure of synapses In the CNS a chemical synapse is composed of a pre-synaptic structure (usually axonal terminals of the afferent neurons) a post-synaptic structure (dendrites of the efferent neurons) and a narrow synaptic cleft separating them The pre- and post-synaptic structures are made up of highly organized multiprotein complexes with distinctive anatomical properties that facilitate neuronal signal conversion and transmission

Fast chemical synapses can be divided into two categories based on their responsiveness to action potentials 1) Excitatory synapses through which a pre-synaptic action potential increases the probability of action potential occurring in the post-synaptic cell 2) Inhibitory synapses through which the pre-synaptic action potential changes the membrane potential and makes it more difficult to fire action potentials in the post-synaptic cells Glutamate is the primary neurotransmitter (NT) used in excitatory synapses in the mammalian brain Below the structure and formation of synapses refer to the excitatory glutamatergic synapses in the CNS

Pre-synaptic structures

The pre-synaptic structure is the machinery for NT secretion Under electron microscopy (EM) they are button-like structures (aka pre-synaptic bouton) swelling at the terminals or along the course of axons The pre-synaptic bouton is characterized by a synaptic vesicle (SV)-rich domain at the proximal part of the plasma membrane and an active zone (AZ) lying underneath of the plasma membrane (Fig 1)

SVs are lipid bilayer-enveloped spheres filled with NTs The size of SVs varies from 20 to 70 nm depending on the type of neurons and NT content NTs are bioactive peptides and amino acids and so far more than 50 of them have been identified On the surface of SVs several proteins are anchored into the lipid bilayer which are important for SV trafficking docking and fusion as well as for NT release

The AZ seen as the ldquoelectron-denserdquo area in EM is the site for SV docking and NT release More than one AZ is often found in a pre-synaptic terminal The AZ is composed of a dense collection of scaffold proteins such as Munc 13 Rab3-interacting molecule (Rim) family Bassoon Piccolo and calmodulin (CaM)-dependent serine protein kinase (CASK) (Sudhof 2012)

11

These proteins are the core organizers of the AZ which serve as a platform for anchorage of membrane proteins SV proteins and cytoskeletal proteins Gene mutagenesis studies provided lines of evidence that they are crucial for SV exocytosis and NT release Deletion of scaffold protein genes leads to impaired NT release (Augustin et al 1999 Schoch et al 2002 Altrock et al 2003) Additionally they are also responsible for recruitment of Ca2+ channels to the AZ (Sudhof 2012)

Along the membrane of the pre-synaptic terminal there are numerous surface proteins including CAMs and ion channels Pre-synaptic CAMs specifically bind to their ligandsreceptors located at the post-synaptic membrane or extracellular matrix (ECM) proteins in the synaptic cleft

Synaptic clefts

A synaptic cleft is a gap about 30 nm in width between the pre- and post-synaptic membranes It is filled with proteins and carbohydrate-containing molecules such as ECM proteins and extracellular domains of membrane proteins

Post-synaptic structures

The post-synaptic structure opposed to the pre-synaptic bouton across the synaptic cleft is the site of receiving the pre-synaptic input and transforming it into a post-synaptic signal In excitatory synapses the post-synaptic sites are made up of dendritic spines the folded plasma membranes protruding from the dendritic shafts Immediately underneath the post-synaptic membrane lie the

Figure 1 The structure of excitatory synapses (A) A schematic structure of an excitatory synapse (B) An excitatory synapse seen under EM A astrocytes S spines AX axons asterisks PSDs black arrows AZs red arrows SVs

12

post-synaptic densities (PSDs) electron-dense areas precisely aligned with the pre-synaptic AZs The PSD is a disk-like structure of 200-500 nm in diameter and 30-60 nm in thickness (Harris et al 1992) It contains a high concentration of NT receptors signaling proteins scaffold proteins and cytoskeletal elements

NT receptors usually multi-transmembrane protein complex line up along the post-synaptic membrane in close apposition to AZs and bind to NTs released from the pre-synaptic terminals Some NT receptors named ionotropic receptors or ligand-gated ion channels are also ion channels Upon binding to NTs these receptors open the ion pores and change the electrical potential in the post-synaptic neurons In glutamatergic synapses NMDARs and Į-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) are the typical ionotropic receptors Another type of NT receptors metabotropic receptors do not contain ion pores They send secondary signals to voltage-gated ion channels and regulate channel gating Usually they evoke a slower but more endurable post-synaptic response in comparison to ionotropic receptors

Scaffold proteins form the framework of the post-synaptic architecture They contain multiple binding motifs for membrane proteins and thereby recruit them to the PSD A large number of scaffold proteins have been identified in the PSDs In excitatory synapses the most important ones include PSD-95SAP 90 family ProSAPShank family proteins and Homer (Okabe 2007)

CAMs bind to scaffold proteins via their cytoplasmic domains and to their ligands via the extracellular domains Interactions between CAMs and their ligands contribute to the specificity of synaptic contacts and initiate signaling pathways that regulate morphological and functional maturation of synapses (Dalva et al 2007 Missler et al 2012)

12 Synaptogenesis Synaptogenesis is a process that occurs continuously in the brain during which specific contact sites between an axon and its targeting dendrite are established and developed into fully functional synapses Synaptogenesis is predominant in developing brain paralleling the differentiation of neurons and assembly of neuronal circuitry The rudimentary synapses are detectable on the postnatal day 1 in rat hippocampus (Fiala et al 1998) and the number of synapses multiples during the second and third postnatal weeks (Harris and Stevens 1989 Harris et al 1992) After reaching the peak level at the end of the third postnatal week the number of synapses stabilizes with a slight decrease in adulthood (Papa et al 1995 Boyer et al 1998) In the adult brain synapse formation also exists due to learning and memory formation and during recovery after brain injury (Raisman and Field 1990 Kelsch et al 2010)

Even though synapse formation is an uninterrupted process gene transcription protein composition and the structure of synapses are fundamentally different at individual stages of synaptogenesis (Fig 2)

Figure 2 Schematic model of the three stages of synaptic formation (1) Formation of initial synaptic contacts between the axon and the dendrite (2) Assembly of protein complexes at the synaptic contacts (3) Maturation of synaptic contacts

13

Initial synaptic contact formation

In the developing brain initial contacts usually form between axonal growth cones and dendritic filopodia both of which are highly flexible and actively search for their binding partners CAMs owing to their adhesiveness mechanically hinge together the membranes of axon growth cones and filopodia and form the initial contacts These contacts are transient unstable and require further signaling for their maintenance and differentiation Specific signaling proteins including secreted factors ECM proteins and CAMs induce the development of appropriate synaptic contacts whereas mismatched synaptic contacts are gradually lost due to the lack of stabilizing signals Neuronal activity has minimal effect on the initial contact formation as pharmacological and genetic inhibition of NT release does not affect synapse morphology (Craig 1998 Augustin et al 1999)

Assembly of pre- and post-synaptic molecular complexes

Subsequently molecular assembly of synaptic structures and delivery of synaptic components occur at the initial synapses Time-lapse studies revealed that many components of synaptic junctions are assembled rapidly within tens of minutes after the formation of the initial contacts (Friedman et al 2000 Ziv and Garner 2004)

In hippocampal glutamatergic synapses recruitment of presynaptic proteins likely occurs ahead of post-synaptic protein assembly (Friedman et al 2000) Instead of local recruitment of individual proteins most synaptic proteins are packed into clusters and delivered as ldquopacketsrdquo AZ scaffold proteins such as Piccolo Bassoon and Rim3 are packed in 80-nm dense core vesicles Accumulation of these scaffold proteins occurs shortly after the initial contacts have formed which enables them to serve as a platform for the subsequently recruited synaptic components (Ziv and Garner 2004) SV proteins are delivered in small clear-centered vesicles and their delivery to nascent synapses comes later than that of scaffold proteins (Ahmari et al 2000)

The mechanism of post-synaptic component assembly is becoming better understood PSD-95 the major scaffold protein of PSD is likely one of the first components recruited to the differentiating post-synaptic structure In fact PSD-95 clustering is detectable 20 min after axon-dendrite contacts form (Friedman et al 2000 Okabe et al 2001) It is not completely clear whether PSD-95 is delivered to synaptic sites in prefabricated non-synaptic clusters or by local insertion from a diffusive cytoplasmic pool Nevertheless accumulating evidence favors the latter idea (Bresler et al 2001 Marrs et al 2001) NMDAR trafficking to synaptic sites seems independent from that of PSD-95 as transporting NMDAR clusters are almost all PSD-95 negative (Rao et al 1998 Friedman et al 2000 Washbourne et al 2002) An elegant study conducted by Washbourne et al (2004) using time-lapse imaging showed that NMDAR targeting to nascent synaptic sites relies on a combination of two pathways (1) non-synaptic NMDAR trafficking to synaptic sites through lateral diffusion and (2) cytoplasmic NMDAR directly inserting into the synaptic membrane AMPAR trafficking is not associated with NMDARs as they use different sets of PDZ-domain proteins for the cytoplasmic anchorage (Scannevin and Huganir 2000 Barry and Ziff 2002) Similar to NMDARs AMPARs are also delivered by both local insertion and lateral membrane diffusion mechanisms (Passafaro et al 2001 Borgdorff and Choquet 2002)

In addition other post-synaptic proteins such as calmodulin-dependent protein kinase II (CaMKII) Shank and Homer are also recruited to synaptic sites during synaptogenesis Therefore synaptic assembly is the organization of highly heterogeneous complexes of signaling scaffolding and structural proteins

Maturation of synapses

Following the assembly of synaptic proteins synapses undergo morphological changes and expand in size Pre-synaptically axonal boutons are enlarged and the number of SVs increases Post-synaptically filopodia become more stable and develop gradually into mushroom spines (Fig 3) (Okabe et al 2001) The contact area between the pre- and the post-synaptic membranes is widened During this phase the composition of synaptic proteins is no longer under dramatic change Instead accumulated synaptic components on both synaptic sites are being reorganized which lead to

14

functional maturation of synaptic architecture Synaptic transmission properties are altered during this stage because functional

AMPARs are recruited to postsynaptic sites and the composition of NMDAR subunits is altered AMPAR insertion usually lags behind that of NMDARs and requires NMDAR-dependent neuronal activity In addition alterations in pre-synaptic release probability and number and organization of SVs also contribute to synaptic transmission changes (Hall and Ghosh 2008)

13 Dendritic spines and spine maturation Morphology and function of dendritic spines

In excitatory synapses dendritic spines represent more than 90 of the post-synaptic structures Spines are small bulbous protrusions extending from the dendritic shafts typically characterized by an enlarged head connected to the dendritic shaft through a narrow neck The geometry of spines makes them relatively independent units from dendritic shafts which creates boundaries for diffusible synaptic molecules and compartmentalizes post-synaptic signaling

Spines vary from 001 m3 to 08 m3 in volume and range between 1-10 spinesm of dendritic shaft in density (Harris and Kater 1994 Harris 1999 Sorra and Harris 2000) Despite their minute size the morphology of spines is surprisingly variable throughout the lifetime Based on observations on fixed cells and tissues spines were classified into four categories thin stubby mushroom-shaped and irregular spines (Fig 3) (Harris et al 1992 Harris and Kater 1994) Additionally filopodia the thin pointy headless dendritic protrusions are widely accepted to be the precursors of spines (Fig 3) (Ziv and Smith 1996 Marrs et al 2001 Yoshihara et al 2009) During development the flexible filopodia and thin spines are gradually replaced by enlarged stable spines (Papa et al 1995)

This classification was later criticized of being too naʀve to pinpoint the heterogeneity of spine morphology in living neurons With the employment of time-lapse imaging in studies of

Figure 3 Structure of spines (A) Schematic representation of the morphology of a filopodium and three common types of spines (B) Fine structure of a fragment of dendrite from cultured hippocampal neurons is visualized by EGFP Big arrowheads mushroom spines big arrows thin spines small arrowhead stubby spines small arrows filopodia Scale bar = 10 m

15

spine morphogenesis it is found that spine morphology is under constant changes In developing neurons spines are highly mobile switching between morphological categories within minutes to hours (Parnass et al 2000) whereas they are more stable and less inter-categorically switching in mature neurons (Dunaevsky et al 1999) The diversity of spine morphology may reflect a dynamic status of an individual spine during its lifetime or the synaptic efficiency the spine obtained according to its experience Thin spines are more plastic to synaptic activity while mature spines with larger spine heads that accommodate higher number of AMPARs have higher synaptic transmission capacity (Matsuzaki et al 2001)

Previous studies have shown that information in the brain is stored as a strengthening or weakening of synaptic connectivity and correspondingly an expansion or shrinkage of spine size (Yuste and Bonhoeffer 2001 Kasai et al 2003 Portera-Cailliau et al 2003 Holtmaat et al 2006) Therefore spines are thought to be crucial components of the machinery of learning and memory formation at the cellular level

Spine morphogenesis

Despite substantial progress in our understanding of the biology of spines and synapses the mechanisms by which spines originate still remain unclear One widely accepted model proposed that filopodia constitute precursors of spines and their contacts with axonal boutons initiate and facilitate the transformation of themselves to spines This model is supported by several key experiments Using live-cell imaging in cultured neurons Ziv and Smith observed a filopodia-to-spine transformation when filopodia made contacts with axonal terminals which was accompanied by a decrease of filopodia motility and length and an enlargement of the distal region of filopodia (Ziv and Smith 1996) This transformation was also found by other scientists using hippocampal tissue slices and in the neocortex of living animals (Dailey and Smith 1996 Maletic-Savatic et al 1999 Marrs et al 2001 Okabe et al 2001)

Apparently not all spines originate from filopodia For example in young pyramidal neurons the majority of synapses are formed between axonal terminals and dendritic shafts instead of filopodia As the neurons mature shaft synapses are replaced by spine synapses (Harris et al 1992) suggesting that these spines emerge from dendritic shafts directly

Due to the heterogeneity of neurons and synapses different mechanisms may be employed for spine morphogenesis depending on the microenvironment or the dynamic status of an individual spine

Actin in dendritic spines

Dendritic spines are actin-rich structures (Caceres et al 1983) An actin network composed of interconnected actin bundles and branched actin filaments serves as the backbone of spines The neck of spines is filled with loosely tangled longitudinal actin filaments and the head contains a dense network of cross-linked branched actin filaments (Hotulainen and Hoogenraad 2010)

In developing spines the actin network is highly mobile undergoing constant turnover (Star et al 2002) Reorganization of the actin cytoskeleton is a major driving force for spine morphogenesis Pharmacological manipulation of actin dynamics results in morphological changes of spines Inhibition of actin polymerization by latrunculin A or cytochalasins induces transformation from spines into filopodia (Zhang and Benson 2001) and prohibits extension and retraction of small filopodia from the surface of spine heads (Allison et al 1998 Fischer et al 1998) In contrast actin stabilizing drugs prevent neuronal excitotoxicity-induced spine loss in cultured hippocampal neurons (Halpain et al 1998)

Accumulating lines of evidence have shown an interplay among actin organization synaptic activity and higher brain functions (Cingolani and Goda 2008) A variety of signaling pathways that regulate spine morphology and synaptic functions ultimately converge at the assembly disassembly and stabilization of actin filaments (Okamoto et al 2004 Ethell and Pasquale 2005 Tada and Sheng 2006 Cingolani and Goda 2008) Activation and inhibition of glutamate receptors result in altered actin dynamics and turnover time (Fischer et al 2000 Star et al 2002) Disruption of actin structure or inhibition of actin polymerization leads to memory loss

16

(Krucker et al 2000 Honkura et al 2008) In addition regulation on actin binding proteins (ABPs) directly affects memory and learning (Pontrello et al 2012 Huang et al 2013)

A plethora of molecules or protein complexes control spine morphology Ion channels and NT receptors regulate ion fluxes changing the membrane potential of spines and increasing the concentration of intracellular Ca2+ By binding to their ligands CAMs are activated and initiate signaling through their cytoplasmic domains Ca2+ and small GTPases regulate the activity of a variety of ABPs which directly control actin assembly and disassembly Among the large group of spine regulators ABPs NT receptors and CAMs are reviewed below

2 Actin binding proteins in synapse formation Dynamics of the actin network is controlled by continuous changes in the length of actin filaments and in the complexity of interconnection between actin filaments There are two forms of actin in spines monomeric or globular-actin (G-actin) and filamentous-actin (F-actin) which is composed of polymerized G-actin connected through non-covalent interaction Formation of a new actin filament starts from a nucleation seed a trimeric G-protein complex to which G-protein monomers can be added to both ends and eventually form filamentous structures A fast growing end (the barbed end) of an actin filament exhibits net polymerization and in the opposite direction a slow growing end (the pointed end) depolymerizes (Pantaloni et al 2001 Hotulainen and Hoogenraad 2010) Processes contributing to actin dynamics include actin polymerization depolymerization and nucleation ABPs play multiple roles during these processes and therefore are implicated in spine formation plasticity and synaptic function (Fig 4)

21 Actin polymerization depolymerization ABPs that regulate actin polymerization and depolymerization directly control the length of actin filaments

Profilin facilitates actin polymerization by increasing the binding efficiency of G-actin to the barbed end of F-actin (Pollard et al 2000) and enhances actin nucleation by activating the Cdc42-Arp23 signaling pathway (Yang et al 2000) The accumulation of profilin II in spine heads

Figure 4 Schematic diagram of ABPs in dendritic spines From Open Neurosci J Lin and Webb 2010

17

stabilizes spine structures by reducing actin dynamics (Pollard et al 2000) Targeting of profilin II the brain-specific splicing variant to spines is regulated at the molecular level by NMDAR activation synaptic activity and at the behavioral level by lateral amygdala-related contextual memory (Ackermann and Matus 2003 Lamprecht et al 2006) Cofilin and its related actin-depolymerization factor (ADF) promote disassembly of actin filaments by depolymerizing or by severing the existing actin filaments (Carlier et al 1997 Bamburg 1999) ADF and cofilin are required for proper actin turnover and spine morphogenesis Knockdown of cofilin by RNA intereference (RNAi) reduces actin turnover which lead to increased spine length and irregular spine heads (Hotulainen et al 2009) Ablation of Lin11-Isl-1-Mec-3 (LIM) -kinase-1 (LIMK-1) a negative regulator of ADFcofilin results in abnormal spine morphology enhanced long-term potentiation (LTP) and impaired fear responses and spatial memory (Arber et al 1998 Meng et al 2002)

Gelsolin is an actin severing protein It inserts into two associated actin subunits and breaks the actin filaments into small fragments Ca2+ activates gelsolin (McGough et al 2003) whereas phosphatidylinositol (45)-bisphosphate (PIP2) dissociates gelsolin from actin filaments and therefore blocks its severing activity (Sun et al 1999) Axon growth cones from gelsolin-- mice exhibit increased length of filopodia due to impaired filopodia retraction (Lu et al 1997) In addition in neurons from gelsolin-- mice NMDAR activity-induced actin stabilization is impaired suggesting that gelsolin is important in synaptic consolidation (Star et al 2002)

22 Actin nucleation Some ABPs mediate the branching of actin filaments and contribute to the complexity of the actin network

Arp23 is a complex of Arp2 Arp3 and five other actin-related proteins It directly binds to actin filaments and mediates the formation of new actin filaments at the side of the existing ones (Mullins et al 1998 Pollard 2007) Arp23 is an effector of Rho GTPases Rac1 and Cdc42 and can be activated by both of them Two other ABPs WASP and cortactin form a complex with Arp23 which activate Arp23 and promote actin nucleation Rac1 and Cdc42-WASP-Arp23 pathways regulate dendritic spine morphogenesis and Arp23 plays an important role in promoting the enlargement of spine heads (Soderling et al 2007 Wegner et al 2008)

Formins are another group of ABPs regulating actin nucleation In contrast to Arp23 formin family proteins induce elongation of unbranched actin filaments Rho GTPase activity is required for the activation of most of the formin proteins (Pruyne et al 2002 Zigmond 2004) For instance by removing its autoinhibition the Rho GTPase Rif activates mammalian diaphanous-related formin2 (mDia2) and promotes the formation of filopodial protrusion (Hotulainen et al 2009)

23 Actin cross-linking proteins Some ABPs do not control the length of filaments directly instead they crosslink neighboring actin filaments and hinge them together to form actin bundles and networks

Neurabin I and neurabin II (spinophilin) are two related ABPs which form homo- or hetero-dimers and bundle actin filaments Both of them interact with protein phosphatase 1 (PP1) in dendritic spines and mediate phosphorylation of spinal proteins such as NMDAR AMPAR and myosin regulatory light chain (Fernandez et al 1990 Yan et al 1999) Phosphorylation of neurabin II by kinases for instance protein kinase A and CaMKII reduces its binding to actin leading to altered actin organization (Hsieh-Wilson et al 2003 Grossman et al 2004 Futter et al 2005) Overexpression of neurabin II in cultured hippocampal neurons increases the length of dendritic protrusions whereas neurabin II gene deletion leads to reduced long-term depression (LTD) and impaired learning function in mice (Feng et al 2000 Stafstrom-Davis et al 2001)

Drebrin A is an actin side-binding protein that crosslinks actin filaments and forms thick and curving bundles (Shirao et al 1994) Drebrin A has multiple functions in spines Firstly it increases the length of actin filaments by blocking the binding between myosin to actin which prevents actin contraction Secondly it induces actin polymerization by recruiting profilin to the

18

barbed end of F-actin Overexpression of drebrin A triggers an increase in the length of filopodia and spines (Hayashi and Shirao 1999 Mizui et al 2005) In addition drebrin A serves as an upstream regulator of many other actin modifying proteins such as Į-actinin tropomyosin and gelsolin Drebrin blocks the actin binding activity of these proteins by competing for actin binding Moreover drebrin A binds to PSD-95 Clustering of drebrin A in spines induces PSD-95 synaptic recruitment and actin assembly (Takahashi et al 2003)

Additionally myosin II is an ATP-driven actin motor protein which promotes actin contractility therefore regulating spine motility (Cheng et al 2000) The activity of myosin II is regulated by CaMK II through phosphorylation (Means and George 1988)

231Ƚ-ActininĮ-Actinin which belongs to the spectrin superfamily is a highly conserved actin crosslinking protein There are four isoforms of the Į-actinin identified named Į-actinin-1 -2 -3 and -4 The four isoforms share highly homologous primary sequences but have their distinctive tissue- and cell type-specific expression profiles Į-Actinin-1 and -4 the non-muscle isoforms are ubiquitously expressed all over the body and often seen in focal contacts and stress fibers (Blanchard et al 1989 Otey and Carpen 2004) Į-Actinin-2 and -3 the muscle isoforms are localized to the Z-disk in striated muscle fibers which anchor actin filaments and connect adjoining sarcomeres (Beggs et al 1992) Į-Actinin-2 is widely expressed in skeletal cardiac and extraocular muscles Į-Actinin-3 is only expressed in a subset of muscle fibers (type II fast) (North and Beggs 1996 Mills et al 2001) Interestingly Į-actinin isoforms are all present in the brain despite of the low expression level of Į-actinin-3 (Mills et al 2001 Kremerskothen et al 2002 Kos et al 2003)

Genetic studies have revealed different functions of Į-actinin family members A mutation in ACTN3 (encoding Į-actinin-3) (R577X) has been found in a significant proportion of the population particularly in Asians Mutation of this gene is related to athletic performance especially the endurance-related performance (Yang et al 2003 Roth et al 2008) Deficiency of Į-actinin-4 leads to glomerular disease due to abnormal morphology of the podocytes (Kos et al 2003) In human patients carrying a point mutation in ACTN4 suffer from the familial kidney disease focal and segmental glomerulosclerosis (Kaplan et al 2000)

Figure 5 Į-actinin structure and binding partners (Top) An D-actinin monomer consists of an N-terminal ABD domain a central rod domain with four SRs and a C-terminal CaM domain D-Actinin binding partners recognize different domains in the protein (Bottom) Two D-actinin monomers form an anti-parallel dimer

19

Structure

Under physiological conditions Į-actinin exists as homodimers composed of two rod-shape monomers Each Į-actinin molecule has an N-terminal actin-binding domain (ABD) followed by a rod domain with multiple spectrin repeats (SR) and a C-terminal CaM-like domain (Fig 5) There is usually a flexible neck between the ABD and the rod domain contributing to the conformational change of Į-actinin (Sjoblom et al 2008)

The ABD contains two tandem calponin homology (CH) domains each of which is composed of four principal helices (Djinovic Carugo et al 1997) There are three major actin binding sites in ABD located in the N-terminal helix of CH1 the C-terminal helix of CH1 and the interdomain linker between CH1 and CH2 domain (Sjoblom et al 2008) The ABD is highly conserved during evolution suggesting an essential role of Į-actinin-actin binding (Sheterline et al 1995)

Two Į-actinin polypeptides bind to each other side-by-side through the rod domains in an anti-parallel fashion and thereby assemble actin filaments into bundles (Ylanne et al 2001) The rod domain is composed of multiple SRs and in vertebrate Į-actinin has four SRs The number of SRs varies among species and determines the length and flexibility of the rod domain which results in different actin cross-linking capacities (Virel and Backman 2004) Adjacent SRs are connected through short and rigid linkers which make the rod domain a strong and elastic platform for the docking of proteins A crystallography study revealed that the rod domain is both curved axially and twisted (Tang et al 2001 Ylanne et al 2001)

The C-terminal CaM-like domain composed of four EF hand motifs regulates the conformational change of Į-actinin In the muscle isoforms Į-actinin-2 and -3 the CaM-like domain is closed by the interaction with the neck region and its binding to other proteins is inhibited (Young and Gautel 2000) Binding of PIP2 to Į-actinin triggers the conformational change of the CaM-like domain which releases this domain from the neck region and enables its binding to other partners (Young et al 1998 Edlund et al 2001) The non-muscle isoforms Į-actinin-1 and -4 bind to Ca2+ through the EF hands and regulate the actin-binding efficiency of Į-actinin (Burridge and Feramisco 1981 Tang et al 2001)

Binding partners and regulation

As an ABP Į-actinin does not directly add to or sever the length of actin filaments instead it binds to the side of actin filaments and hinges them into bundles or networks The rigid yet flexible nature of Į-actinin makes it an ideal actin crosslinking protein which confers the stability and dynamics of the actin cytoskeleton In vitro data shows that Į-actinin regulates the transformation of actin from an isotropic network to bundles of parallel filaments depending on the concentration and affinity of Į-actinin to actin (Wachsstock et al 1993)

Į-Actinin binds a large group of transmembrane proteins including CAMs cell surface receptors ion channels signaling proteins and metabolic proteins Į-Actinin provides mechanical anchorage for these proteins and links them to the actin cytoskeleton Moreover Į-actinin engages signaling proteins into an interconnected complex and regulates their activities therefore facilitating signaling transduction (Otey and Carpen 2004 Sjoblom et al 2008)

The integrin family (see page 24-27) is one of the first studied binding partners for Į-actinin at the adhesion sites The cytoplasmic tails of integrin subunits ȕ1 ȕ2 and ȕ3 bind directly to Į-actinin (Otey et al 1990 Otey et al 1993 Heiska et al 1996) Interference with the link between integrins and Į-actinin attenuates integrin activation and perturbs integrin-dependent cell motility migration proliferation and focal adhesion assembly (Gluck and Ben-Zeev 1994 Duncan et al 1996 Yamaji et al 2004 Stanley et al 2008 Tadokoro et al 2011 Roca-Cusachs et al 2013) Reciprocally the activity of Į-actinin is regulated by integrin-mediated signaling pathways For example focal adhesion kinase (FAK) a tyrosine kinase downstream of integrins induces tyrosine phosphorylation of Į-actinin and reduces its binding to actin upon activation by integrin signaling (Izaguirre et al 2001)

Another type of well-studied binding partners for Į-actinin at adhesion sites are the ICAMs By affinity chromatography several ICAM family members have been found to bind to Į-

20

actinin (Carpen et al 1992b Heiska et al 1996 Nyman-Huttunen et al 2006) Unlike integrins ICAMs do not undergo signaling-induced activation Therefore the change of avidity of ICAMs by clustering seems a major way to regulate their adhesiveness Į-Actinin binds to the ICAM-1 cytoplasmic amino acid sequence 478-505 The interaction of ICAM-1 to Į-actinin is important in maintaining its membrane distribution in B cells and in regulating leukocyte extravasation (Carpen et al 1992b Celli et al 2006) Binding between the cytoplasmic tail of ICAM-2 and Į-actinin is implicated in neuroblastoma cell motility and metastasis (Heiska et al 1996 Yoon et al 2008) Other CAMs binding to Į-actinin include syndecan-4 (Greene et al 2003) L-selectin (Pavalko et al 1995) and the platelet glycoprotein Ib-IX (Feng et al 2002)

Signaling proteins bind to Į-actinin and regulate its activity For instance phosphatidylinositol (345)-triphosphate (PIP3) binds directly to Į-actinin (Shibasaki et al 1994) The binding of PIP2 and PIP3 to Į-actinin attenuates its actin-binding capacity (Fraley et al 2003 Corgan et al 2004) Production of PIP3 induced by activation of phosphatidylinositide 3-kinases (PI3-kinase) reduces the affinity of Į-actinin to ȕ integrins and disables Į-actinin in bundling actin filaments which further releases the link between integrins and stress fibers (Greenwood et al 2000 Fraley et al 2003)

At stress fiber dense cores many Į-actinin-binding proteins are characterized by LIM or PDZ domains (te Velthuis et al 2007) such as Zyxin and Cysteine-rich protein (CRP) (Beckerle 1997 Li and Trueb 2001) They both bind to and form a tripartite complex with Į-actinin which serves as a scaffold to regulate the binding capacity and the cellular distribution of the two former proteins (Beckerle 1997)

Importantly in neuronal synapses several cell surface receptors and ion channels have been found to bind to Į-actinin the GluN1 and GluN2B subunits of NMDAR (Wyszynski et al 1997) GluA4 subunit of AMPAR (Nuriya et al 2005) adenosine A2A receptors (Burgueno et al 2003) metabotropic glutamate receptor type 5b (Cabello et al 2007) and L-type Ca2+ channel (Sadeghi et al 2002) to name a few It is likely that the interaction between these receptors and Į-actinin regulates their expression activity and function

Functions of Į-actinin in neuronal synapses

In neurons the isoform Į-actinin-2 is specifically concentrated to the PSD of glutamatergic synapses (Wyszynski et al 1998) Expression of Į-actinin-2 is temporarily and spatially regulated during the postnatal development Į-Actinin-2 is detectable on postnatal day 1 in the neonatal cerebral cortex of the rat brain The expression increases reaches a platform within the first two postnatal weeks and persists into adulthood In the adult rat brain Į-actinin-2 immunoreactivity is prominent in the forebrain especially in the striatum hippocampus and cortex (Wyszynski et al 1998) In rat striatum Į-actinin-2 exhibits a neuron type-specific expression pattern - highly expressed in substance-P containing neurons which project to the substantia nigra pars reticulata but not in the population of neurons expressing nNOS and somatostatin (Dunah et al 2000)

In cultured hippocampal neurons overexpression of Į-actinin-2 promotes filopodia elongation and thinning and impairs synaptic protein recruitment (Nakagawa et al 2004) Co-expression of Į-actinin-2 and SPAR (Spine-Associated Rap GTPase-activating protein) in cultured neurons induces the enlargement of spine heads and thinning of filopodia at the same time indicating a combinatorial role of different actin adaptor proteins in spine morphogenesis (Hoe et al 2009) Knockdown of Į-actinin-2 in cultured hippocampal neurons has little effect on spine morphology likely due to the compensatory effects from other Į-actinin isoforms (Nakagawa et al 2004) These data suggest an important role of Į-actinin in regulating spine morphology

The molecular mechanisms by which Į-actinin mediates spine formation and synaptic plasticity are still under investigation The interaction of NMDAR with Į-actinin may contribute to these regulatory mechanisms By in vitro binding assays two subunits GluN1 and GluN2B were found to bind to Į-actinin that colocalizes with NMDAR in spines (Wyszynski et al 1997 Wyszynski et al 1998) Interestingly co-immunoprecipitation of GluN1 and Į-actinin demonstrates that only a fraction of GluN1 subunit interacts with Į-actinin (Dunah et al 2000) suggesting that a subset of cell type- or composition-specific NMDAR binds to Į-actinin or that the interaction between NMDAR and Į-actinin is transient and highly dynamic

21

CaM binds to GluN1 and inhibits the binding of Į-actinin-2 to GluN1 (Wyszynski et al 1997) Upon Ca2+ influx CaM binds to GluN1 and displaces the binding of Į-actinin which triggers inactivation of NMDAR and reduces the probability of channel opening (Zhang et al 1998 Krupp et al 1999) By this means the negative feedback loop among CaM Į-actinin and NMDAR serves as a regulator for NMDAR-dependent synaptic plasticity

3 NMDA receptors In the current view the structural plasticity of synapses reflects synaptic strength which is regulated by neuronal activity (Ehrlich et al 2007) In excitatory glutamatergic synapses NMDAR and AMPAR are the major types of ionotropic glutamate receptors They receive the NT inputs from the pre-synaptic terminals change the electrical properties of the post-synaptic compartments and thereafter initiate signals underlying actin cytoskeleton reorganization NMDARs are most abundant in nascent synapses and are the major contributors for glutamatergic synaptic transmission in developing neurons (Wu et al 1996) The composition of NMDAR and the AMPARNMDAR ratio changes over time during synapse maturation (Hall et al 2007)

31 Structure NMDAR is composed of two heterogenic subunits organized into a tetramer in a ldquodimer-to-dimerrdquo manner There are three types of NMDAR subunits named GluN1 GluN2 and GluN3 (the subunits were previously denoted as NR1 NR2 and NR3) They are synthesized in the endoplasmic reticulum and transported to the plasma membrane

GluN1 and GluN2 subunits are highly homologous in their sequences and share similar domain

Figure 6 Schematic model of NMDAR structure The NMDA receptor is a tetramer formed by two GluN1 subunits and two GluN2 subunits (bottom) (Top) For a clear illustration only one GluN1 and one GluN2 subunit are shown Both GluN1 and GluN2 subunits contain a large extracellular domain four transmembrane domains (M1-M4) and a cytoplasmic tail In the extracellular domains GluN1 subunit has a glycine binding site and GluN2 has a glutamate binding site Adapted from Neurology Benarroch 2011

22

structures The primary amino acid sequence of NR subunits can be divided into eight segments From the N-terminus it starts from a signal peptide the first ligand-binding domain followed by three hydrophobic domains the second ligand-binding domain the fourth hydrophobic domain and the C-terminal domain (Fig 6) Crystallography studies suggest that the GluN subunit is a four-pass transmembrane protein The N-terminal signal peptide and the two ligand-binding domains are located at the extracellular part Of the four hydrophobic domains M1 M3 and M4 form Į-helix structures and span across the phospholipids bilayer M2 does not cross the membrane and is folded as a hairpin loop linking M1 and M3 at the cytoplasmic part S1 and S2 domains interact with each other via hydrogen bonds salt bridges and hydrophobic interactions to form hetero- or homodimers between GluN subunits Separation of GluN subunits leads to dysfunction of NMDAR

GluN1 is an obligatory subunit of NMDAR In the rodent brain there are eight splicing variants of GluN1 subunits named GluN1-1 -2 -3 and -4 each of which has two isoforms the one with an N-terminal N1 exon (GluN1-a) and the one without (GluN1-b) (Dingledine et al 1999) Expression of GluN1 appears as early as the embryonic day 14 and peaks at the third postnatal week followed by a slight decline at the adult stage (Akazawa et al 1994 Laurie and Seeburg 1994 Monyer et al 1994) The temporal expression pattern is similar among all splicing variants Among all splicing variants GluN1-2 is the most abundant one and its expression is almost homogenous throughout the brain GluN1-1 and GluN1-4 show moderate expression levels and their expression patterns compensate for each other GluN1-1 is restricted to the rostral parts and GluN1-4 is more enriched in the caudal parts GluN1-3 has very low expression levels only weakly detected in the cortex and hippocampus of the postnatal brain (Paoletti 2011)

GluN2 is the glutamate-binding subunit which determines the functional properties of NMDARs (Traynelis et al 2010) GluN2 has four splicing variants named from GluN2A-GluN2D which exhibit distinct expression patterns throughout the brain (Akazawa et al 1994 Monyer et al 1994) GluN2B and GluN2D are the two first expressed variants and their expression starts from embryonic day 14 In the prenatal brain GluN2B is the predominant subunit in telencephalon and spinal cord while GluN2D is abundant in diencephalon mesencephalon and spinal cord After birth the expression of these two variants persists throughout the early postnatal development In contrast GluN2A and GluN2C appear only after birth They are first detected in hippocampus and cerebellum respectively and reach their peak levels between the second and third postnatal week After that GluN2A declines to the adult level while GluN2C expression remains high and becomes the predominant subunit in the adult brain (Monyer et al 1994 Zhong et al 1995) During the early postnatal development there is a switch of GluN2B- to GluN2A-containing NMDAR and the delivery of GluN2A subunit to synapses is dependent on synaptic activity (Barria and Malinow 2002 Yashiro and Philpot 2008)

GluN3 consisting of two splicing variants GluN3A and GluN3B is expressed as a triheteromer with GluN1 and GluN2 subunits (Ciabarra et al 1995) GluN3 is an inhibitory subunit of NMDAR When binding to GluN1 the receptor becomes Mg2+-insensitive Ca2+-impermeable and does not respond to glutamate and NMDA (Sucher et al 1995) Temporarily in rodents GluN3A expression is detectable in the prenatal CNS reaches its peak level at postnatal day 8 and then declines to adult levels by postnatal day 20 GluN3B expression is low before birth increases during early postnatal development and persists at a high level in adult brains Spatially GluN3A is expressed in the spinal cord the brain stem hypothalamus thalamus hippocampus amygdala and part of cortical cortex (Ciabarra et al 1995 Sucher et al 1995) GluN3B was thought to be exclusive in the brain stem and the spinal cord (Sucher et al 1995 Wong et al 2002) however a recent study suggested that they are ubiquitously expressed in the CNS (Wee et al 2008)

32 Functions As ionotropic NT receptors NMDARs and AMPARs both regulate excitatory synaptic transmission by gating the ion flux across the plasma membrane In comparison to AMPARs NMDARs are permeable to Ca2+ ions and mediate slow and prolonged synaptic responses in a voltage-dependent manner due to blockade of the ion channels by Mg2+ at the resting state

23

Activities of both pre- (glutamate release) and post-synaptic (depolarization) neurons are required to remove the Mg2+ blockade and open up the ion pore Once the ion channels are open Na+ and Ca2+ flux into the post-synaptic neurons which elicit electrical signals and initiate a multitude of signaling pathways leading to the long-lasting modifications in synaptic efficacy that persist for hours and even days Two generic terms LTP and LTD are used to describe such activity-dependent changes of synaptic function LTP is the long-lasting increase while LTD is the long-lasting decrease in synaptic strength They are considered to be the cellular mechanism underlying enduring changes in brain function (Bliss and Collingridge 1993 Malenka and Bear 2004) Usually LTP corresponds to an increase of spines and insertion of glutamate receptors in the PSD LTD is accompanied by shrinkage or loss of spines and glutamate receptor removal from spines (Bosch and Hayashi 2012) NMDARs are able to trigger both LTP and LTD depending on the context of synapses and the level of stimulation (Luscher and Malenka 2012)

A variety of alterations in synaptic properties occur upon NMDAR activation among which modification on spine morphology is one of the major events constituting synaptic plasticity In acute hippocampal slices from the postnatal day 2-5 mice inhibition of NMDAR results in a ~35 decrease in the density and turnover of dendritic filopodia (Portera-Cailliau et al 2003) In agreement downregulation of NMDAR by RNAi in isolated hippocampal neurons leads to increased spine motility (Alvarez et al 2007) Furthermore initiation of NMDAR-dependent LTP promotes de novo spine formation and filopodia elongation (Engert and Bonhoeffer 1999 Maletic-Savatic et al 1999 Toni et al 1999) In addition mice lacking the GluN3A subunit of NMDAR exhibit an increased density and size of spines likely due to its inhibition in NMDAR Ca2+

permeability when binding to GluN1 (Das et al 1998) Given their promotional role in spine maturation it is extraordinary that NMDAR activation is also required for experience-dependent spine elimination and maintenance of the plasticity of pruning spines (Bock and Braun 1999) Intense activation of NMDAR results in spine shrinkage and collapse associated with prolonged increase of intracellular Ca2+ concentration (Halpain et al 1998) These data suggest that NMDARs regulate bi-directionally spine maturation which can promote both spine expansion and elimination depending on the overall synaptic input in a neural network

As the structural basis of spines the actin cytoskeleton is essential in regulating NMDAR activity-dependent spine morphogenesis Activation of NMDAR blocks actin motility in spines which is accompanied by rounding up of spine heads with an increased stable actin pool (Fischer et al 2000 Star et al 2002 Brunig et al 2004) NMDAR-dependent LTP and LTD alter the amount of F-actin in spine heads (Colicos et al 2001 Ackermann and Matus 2003 Fukazawa et al 2003) Moreover disruption of actin cytoskeleton also reduces the number of NMDAR in spines which pinpoints a bi-directional interplay between glutamate receptor-mediated synaptic activity and actin dynamics (Matsuzaki et al 2004) Actin regulatory proteins are also subject to NMDAR activity For example activation of NMDAR induces redistribution of cortactin from the spines to the dendritic shafts and of profilin II and Abp 1 in an opposite way (Ackermann and Matus 2003 Hering and Sheng 2003 Qualmann et al 2004 Haeckel et al 2008)

The incorporation of AMPAR in synaptic transmission is a key step for the maturation of synapses (Wu et al 1996 Aizenman and Cline 2007) The volume of spine heads is tightly associated with its content of AMPAR NMDAR also modulates spine composition by regulating AMPAR trafficking toward synapses Previous studies have demonstrated that AMPAR insertion induced by NMDAR activity is highly dependent on the type of synaptic stimulation High-frequency synaptic stimulation activates NMDAR leading to LTP which promotes the insertion of GluR1-containing AMPAR into synapses (Shi et al 1999 Hayashi et al 2000) In contrast low-frequency stimuli which causes NMDAR-dependent LTD in spines results in the removal of AMPAR from synapses (Carroll et al 1999 Beattie et al 2000)

33 NMDAR induced signaling pathway in regulation of spine morphology When NMDAR is activated Ca2+ enters spines and the increased intracellular Ca2+ concentration triggers a variety of signaling pathways contributing to actin-regulated spine plasticity

Firstly Ca2+ influx recruits actin regulators to PSD which facilitate the formation of a signaling protein complex For example in cultured hippocampal neurons increased intracellular

24

concentration of Ca2+ following activation of NMDAR induces a translocation of RhoA Rho-kinase (ROCK) and Rho guanine-nucleotide-exchange factor (GEF) Lcf from dendritic shaft to spines where they form complexes with profilin or spinophilin and therefore affects actin filament polymerization (Grossman et al 2002 Hsieh-Wilson et al 2003 Grossman et al 2004 Ryan et al 2005 Schubert et al 2006)

Alternatively Ca2+ regulates the enzyme activity of kinases and phosphatases that directly modulate ABPs CaMKII a prominent kinase that has a number of substrates in spines can be activated by Ca2+ influx CaMKII directly binds to NMDAR and many other PSD proteins The binding is Ca2+-sensitive and depends on the phosphorylation status of CaMKII (Omkumar et al 1996 Shen and Meyer 1999 Shen et al 2000) Specific inhibition of CaMKII blocks LTP as well as filopodia extension and spine formation (Jourdain et al 2003) SynGAP a Ras GTPase activating protein maintains the dynamic state of filopodia during spine maturation (Vazquez et al 2004) Downregulation of synGAP activity by CaMKII phosphorylation (Chen et al 1998) at least partially confers the mechanism by which NMDAR-dependent Ca2+ influx promotes spine enlargement

The mitogen-activated protein kinase (MAPK) is another important regulator in neuronal activity-dependent spine formation Activation of MAPK requires CaMKII signaling and subsequently initiates signaling that induces protein synthesis and prolonged changes in spines such as LTP and LTD CaMKII promotes activation of the small GTPase Ras via Ras guanine nucleotide exchange factor (RasGEF) resulting in upregulation of two MAPKs Erk1 and Erk2 (Sweatt 2004) Stimulation of this CaMKII-Ras-MAPK pathway triggers AMPAR insertion into the PSD followed by LTP (Zhu et al 2002) In contrast LTD employs a different small GTPase Rap1 even though Ca2+ is also required to initiate the signaling pathway Two Rap1 GTPase activating proteins SPAR and SPAL positively regulate Rap1 activity and activate the downstream protein p38MAPK which leads to removal of AMPAR from spine heads (Pak et al 2001 Roy et al 2002 Zhu et al 2002)

4 Cell adhesion molecules in synapse formation and plasticity

Figure 7 Schematic representation of the structures of CAMs at neuronal synapses

In the CNS CAMs are the ldquostickyrdquo proteins located on the plasma membrane of synapses and integrate the pre- and post-synaptic components as a whole unit CAMs are mostly transmembrane proteins (Fig 7) Their extracellular domains bind to the ligands across the synaptic cleft which

25

provide a mechanical force keeping synapses in place The cytoplasmic domains bind to adaptor proteins that organize the cytoplasmic structure or initiate signals underlying alterations of synaptic structure and function Accumulating data indicate that CAMs play a pivotal role throughout the lifetime of a synapse The most important CAMs at synapses include integrins immunoglobulin superfamily (IgSF) proteins neuroligin and neurexins cadherin superfamily proteins as well as ephrins and their receptors

41 Integrins Integrins are a large group of heterodimeric proteins consisting of two single transmembrane subunits Through interactions with their binding partners on both sides of the plasma membrane integrins connect the extracellular environment and the intracellular signaling

Structure

Each integrin molecule is composed of non-covalently linked Į and ȕ subunits both of which are type I (with an intracellular C-terminal and an extracellular N-terminal) transmembrane polypeptides To date 18 Į-subunits and 8 ȕ-subunits have been found forming 24 heterodimeric integrins in mammals (Hynes 2002) The large ectodomains of integrins bind to their ligands and the cytoplasmic tails are linked to the actin cytoskeleton via a spectrum of adaptor proteins Through integrins extracellular stimuli are translated into intracellular signaling cascades (Legate and Fassler 2009)

Integrins exist in different activity forms which correspond to different ligand binding capacities At the resting state integrins stay at a low-affinity conformation characterized by a bent ectodomain toward the plasma membrane and associated cytoplasmic tails of Į and ȕ subunits Upon activation integrins increase their ligand binding affinity or avidity In the former case the enhanced affinity is a result of a conformational change from the bent closed conformation to the extended open conformation In the latter case increased avidity as an increase of local integrin concentration results from integrin clustering (Gahmberg 1997 Gahmberg et al 2009) Activation of integrins can be triggered by extracellular factors through binding to their ligands (outside-in activation) or by a cytoplasmic chain-of-events that terminates in the binding of adaptor proteins to the integrin cytoplasmic tails (inside-out activation) (Campbell and Humphries 2011 Margadant et al 2011)

Distribution

In mammals integrins are expressed in a variety of tissues but ȕ2 integrins are confined only to leukocytes

In the adult mouse brain the mRNAs of 14 integrin subunits including Į1 Į2 Į3 Į4Į6 Į7 ĮV ȕ1 ȕ3 ȕ4 ȕ5 ȕ6 and ȕ7 were detected by RT-PCR (Murase and Hayashi 1996 Pinkstaff et al 1998 Chan et al 2003) At the protein level at least 14 out of the 24 integrin heterodimers are expressed (Einheber et al 1996 Nishimura et al 1998 Pinkstaff et al 1999 Schuster et al 2001) Some examined integrins exhibit cell type-specific and region-specific distribution (Table 1) Their specific expression patterns suggest that different integrin heterodimers have their distinctive functions and are implicated in the adhesive events for a subset of cells

Functions

Integrins are versatile CAMs in the developing and adult brains have been implicated in neural migration neurite outgrowth spine maturation and synapse plasticity (Milner and Campbell 2002 Clegg et al 2003 Schmid and Anton 2003)

An emerging body of evidences has revealed the roles of integrins in synaptogenesis and spine maturation Among the 24 integrin heterodimers ȕ1 ȕ3 and ȕ5 subtypes are enriched in dendritic spines (Shi and Ethell 2006) Overexpression of constitutively active Į5 integrin in cultured hippocampal neurons increases the length of dendritic protrusions but decreases the number of them Downregulation of Į5 integrin by RNAi leads to smooth spine-less dendritic

26

shaft (Webb et al 2007) Blocking the ȕ1 and ȕ3 integrin function with antibodies or RGD-containing peptide an established integrin ligand (Pierschbacher et al 1985) results in filopodia elongation and formation (Shi and Ethell 2006) When treating hippocampal organotypic slices with the same method a decreased number of synapses in the CA1 region was observed (Nikonenko et al 2003) Recently it was reported that a conditional knockout (KO) of ȕ1 integrin in excitatory neurons results in loss of synapses and less elaborated dendritic arbors in the hippocampal CA1 region by an inactivating Arg kinase-p190RhoGAP involved signaling pathway (Warren et al 2012) Moreover indirect regulation of integrin activity also affects spine morphology For instance overexpressing autoactivating MMP-9 promotes the ȕ1 integrin activation which contributes to thinning and elongation of spines (Michaluk et al 2009) Ephrin A3-induced EphA4 activation inhibits the ȕ1 integrin signaling which alters spine morphology (Bourgin et al 2007) These results emphasize the importance of integrins especially the ȕ1 and ȕ3subtypes in filopodia extension from dendritic shafts However they do not seem to be obligatory for the enlargement of spines and formation of synapses

Integrins Regional localization Cellular localization

Į1-7ȕ1 CC HC OB HT BS CB Pyramidal neurons granule cells glial cells

Į8ȕ1 CC HC OB SN VT SOC Pyramidal neurons granule cells glial cells

ĮVȕ(135) CC HC OB HT CB BS Pyramidal neurons granule cells glial cells

ĮVȕ6 CC HC OB HT CB Pyramidal neurons granule cells

ĮVȕ8 CC HC CB Pyramidal neurons granule cells glial cells

Į4ȕ7 CC HC OB HT CB Pyramidal neurons

ĮLȕ2 ĮMȕ2 HC Glial cells leukocytes (ĮLȕ2) neurons (ĮLȕ2)

In mature synapses integrins are required for synaptic plasticity Using RGD-containing peptides Xiao et al (1991) showed that blockade of integrin function in hippocampal slices induces a reversible dose-dependent decay of late-LTP in CA1 region However the effect of RGD-containing peptides is much less prominent for early-LTP (Staubli et al 1998) In line with these

results functional blocking antibodies against the D3 D5 and Dv integrin subunits also attenuate the stabilization of LTP (Kramar et al 2002 Kramar et al 2003) Recently it was shown that

induction of LTP by theta burst stimulation causes a rapid activation of E1 integrins lasting up to 45

min during which E1 integrins have no response to a second theta burst stimulation Trafficking of

the E1 integrins toward the plasma membrane probably contributes to the delayed response to stimulation (Babayan et al 2012) These data indicate that integrins are important for the maintenance and consolidation of existing LTP instead of LTP induction

To analyze the specific roles of individual integrin subunits genetic approaches that

downregulate integrin expression have been employed Mice with an D3 integrin null mutation

show less LTP maintenance compared with the wild type (WT) mice Mice with the D3 and D5double heterozygous mutations exhibit a deficit in the probability of presynaptic release (Chan et al

Table 1 Distribution of integrins in the brain CC cortical cortex HC hippocampus OB olfactory bulb HT hypothalamus BS brainstem CB cerebellum SN substantia nigra VT ventral tegmentum SOC superior olivary complex Data were originally from (Einheber et al 1996 Murase et al 1996 Pinkstaff et al 1998 Pinkstaff et al 1999 Nishimura et al 1998 Einheber et al 2001 Schuster et al 2001 Chan et al 2003 Clegg et al 2003 Wakabayashi et al 2008 Wu et al 2012)

27

2003) Deletion of the E1 integrin gene from the mouse forebrain excitatory neurons at the embryonic stage results in impaired LTP and at the later postnatal stage leads to abolished AMPAR-dependent synaptic transmission and abrogated NMDAR-induced LTP (Chan et al 2006

Huang et al 2006) Likewise deletion of D3 and D8 integrins from the forebrain excitatory neurons during the postnatal development also causes deficits in LTP (Chan et al 2007 Chan et al 2010)

However genetic deletion of E3 integrins or pharmacological disruption of their ligand interactions fails to alter LTP LTD and short-term synaptic plasticity (McGeachie et al 2012) Like mice Drosophila melanogaster also requires integrins for synaptic plasticity By random mutagenesis

the gene Volado encoding the integrin DPS3 subunit was found to be essential for synaptogenesis and synaptic transmission in Drosophila mushroom body Deletion of Volado causes abnormally evoked synaptic current and reduced Ca2+-dependent transmission (Rohrbough et al 2000)

There are at least two mechanisms by which integrins could contribute to LTP Firstly integrin activation initiates a kinase cascade which induces multiple changes in spines RGD-containing peptide-induced integrin activation leads to increased phosphorylation in FAK proline-rich tyrosine kinase 2 and Src family kinases (Bernard-Trifilo et al 2005) Blocking Src kinase and CaMKII inhibits integrin-dependent synaptic plasticity (Kramar and Lynch 2003 Lin et al 2003) Many integrin-activated signaling pathways converge at the regulation of actin cytoskeleton assembly which is directly linked to consolidation of LTP (Kramar et al 2006 Shi and Ethell 2006) Secondly integrin activation regulates the expression composition and trafficking of glutamate receptors underlying synaptic function For example RGD-containing peptide treatment induces the phosphorylation of NMDAR subunits GluN2A and GluN2B Changes in NMDAR

subunit composition regulated by E3 integrins contribute to altered NMDAR synaptic responses (Chavis and Westbrook 2001 Lin et al 2003 Bernard-Trifilo et al 2005) In cultured neurons

pharmacological perturbation of E3 integrins results in an increased endocytosis of GluA2-containing AMPAR (Cingolani et al 2008) Recently it was found that GluA2 directly binds to the

E3 integrin subunit (Pozo et al 2012) Reciprocally activation of glutamate receptors also regulates

integrin signaling Stimulation of AMPARs increases the membrane insertion of D5 and E1 integrin subunits through a protein kinase C-involved exocytotic pathway which further stabilizes the membrane integrins (Lin et al 2005)

Due to the impact of integrins on synaptic plasticity they are the important candidates contributing to memory formation and learning Mice with reduced expression of integrins Į3 Į5and Į8 are defective in spatial memory (Chan et al 2003) KO of the Į3 or ȕ1 integrin genes from mouse forebrain excitatory neurons impairs hippocampal-dependent working memory (Chan et al 2006 Chan et al 2007 Warren et al 2012) In addition ȕ1 integrin-conditional KO mice exhibit higher sensitivity to cocaine exposure (Warren et al 2012) In Drosophila removing of the integrin gene Volado compromises short-term olfactory memory which can be rescued by re-expression of Volado (Rohrbough et al 2000)

42 Immunoglobulin superfamily (IgSF) adhesion molecules A large group of CAMs characterized by extracellular Ig domains belong to the IgSF Ig domains loop-like structures formed by disulfide bonds from two discrete cysteins constitute the adhesion sites mediating homophilic or heterophilic binding to their ligands In the CNS examples of extensively studied IgSF proteins include the synaptic cell adhesion molecule (SynCAM) and the neural cell adhesion molecule (NCAM) ICAM-5 one of the key molecules in my doctoral project belongs also to this superfamily

421SynCAMIn vertebrate the SynCAM protein family consists of at least four members with many splicing variants The extracellular domain of SynCAM composed of three extracellular Ig domains mediates the in trans binding to their ligands such as SynCAMs and nectins (Biederer et al 2002 Biederer 2006) The cytoplasmic domain contains Four1 protein- Ezrin-Radixin-Moesin (FERM)- and PDZ- binding motifs and interacts with multiple scaffold proteins such as CASK syntenin-1 synaptophysin protein 41 and Farp1 (Biederer et al 2002 Hoy et al 2009 Cheadle and Biederer

28

2012)SynCAMs are widely expressed among a variety of regions in the developing and

adult brains (Fujita et al 2005 Thomas et al 2008) In neurons SynCAMs are detectable in both pre- and post-synaptic structures in excitatory and inhibitory synapses (Biederer et al 2002 Fogel et al 2007)

SynCAM 1 expressed at the axons which maintains the shape and mobility of axonal growth cones is needed for appropriate axo-dendritic contact formation (Stagi et al 2010) Once the synaptic contacts are made SynCAM 1 rapidly clusters at the nascent synaptic contacts followed by recruitment of synaptic components including PSD-95 (Stagi et al 2010) the NMDARs along with the adaptor protein 41 B and the AMPARs with the protein 41 N (Hoy et al 2009) Post-synaptic SynCAM 1 contributes also to the differentiation of the pre-synaptic machinery In a co-culture system isolated neurons were mixed with non-neuronal cells transfected with SynCAM 1 On the axons making contacts with non-neuronal cells pre-synaptic proteins syntaxin-1 and synaptophysin-1 are clustered and colocalized with SynCAM 1 indicating an assembly of pre-synaptic components in the contacting areas The induced pre-synaptic structures are functionally active since they exhibit spontaneous and evoked neurotransmitter release similar to endogenous synapses (Biederer 2006) Homophilic binding of SynCAM 1 is involved since axons lacking SynCAM 1 fail to form such clusters Using similar system heterophilic binding between SynCAM 1 and SynCAM 2 was found to promote synapse formation with increased pre-synaptic terminals and excitatory transmission (Fogel et al 2007) In addition lateral assembly of SynCAM 1 by cis interaction between the 2nd and 3rd Ig domains affects synapse formation as well Interference with this interaction at the early stage of synaptogenesis results in impaired synaptic induction and at the late stage decreases the size of synapses (Fogel et al 2011)

Loss-of-function and gain-of-function studies using transgenic mice support the results obtained with cultured neurons Overexpression of SynCAM 1 induces excitatory synapse formation and transmission in mice without altering the ultrastructure of synapses In contrast ablation of the SynCAM 1 gene leads to a decreased number of excitatory synapses (Robbins et al 2010)

SynCAM 1 not only regulates synapse formation but also affects the plasticity of synapses SynCAM 1 -- mice exhibit abrogated LTD and improved spatial learning (Robbins et al 2010)

422NCAMThe NCAM protein family is one of the first identified CAMs (Jorgensen and Bock 1974) There are a large number of NCAM splicing isoforms Some of them are transmembrane proteins and others lacking the cytoplasmic tails are anchored to the plasma membrane through a glycophosphatidylinositol linker The extracellular domains of NCAM family proteins contain five N-terminal Ig domains followed by two fibronectin type III modules They exhibit homophilic binding to NCAM molecules in both cis and trans manners and heterophilic binding to many other ligands including ECM proteins CAMs and growth factors The cytoplasmic domains of NCAM have also multiple binding partners such as ABPs phosphatases and kinases ion channels NT receptors and can even directly bind to actin

In mammals the extracellular domains of NCAM contain multiple glycosylation sites which generates three isoforms NCAM-120 -140 and -180 named according to their different molecular weights (120 140 and 180 kilodaltons (kDa) respectively) The three isoforms have their distinct expression patterns and functions (Persohn et al 1989 Schuster et al 2001 Polo-Parada et al 2004)

In the CNS both in vivo and in vitro data pinpoints the role of NCAM in synaptogenesis synaptic plasticity and memory formation In rat hippocampal slices blockade of NCAM adhesion activity using antibodies results in attenuation of NMDAR-dependent LTP in the CA1 neurons (Luthl et al 1994) and amnesia in a passive avoidance task in living animals (Doyle et al 1992) Mice lacking NCAM exhibit reduced LTP and impaired spatial and contextual memory (Cremer et al 1994 Senkov et al 2006)

Polysialylation is a special type of glycosylation which adds a heavy negatively

29

charged carbohydrate polysialic acid (PSA) to NCAM Polysialylation changes the surface charge and binding property of NCAM (Rutishauser 2008) and therefore makes up a major regulatory mechanism for NCAM functions Proper adhesiveness of NCAM is essential for NCAM-mediated cell adhesion therefore the level of polysialylation on NCAM needs to be strictly monitored and regulated In the mouse brain PSA expression immediately follows the appearance of NCAM from the embryonic day 8-85 peaks at perinatal days and diminishes after two postnatal weeks (Probstmeier et al 1994) Two enzymes 2 8-polysialytransferases type II (ST8SiaIISTX) and type IV (ST8SiaIVPST) catalyze the synthesis of PSA and are responsible for its attachment to NCAM Simultaneous deletion of both enzymes in mice generates lethal phenotypes in mice including specific brain wiring defects progressive hydrocephalus postnatal growth retardation and precocious death Interestingly triple KO of ST8SiaIIST8SiaIVNCAM1 partially rescues these phenotypes suggesting that abnormally enhanced NCAM functions when lacking PSA contributes to these phenotypes (Weinhold et al 2005) Application endoneuraminidase-N an enzyme specific removing PSA from NCAM results in delayed induction of LTP and LTD in mouse hippocampal organotypic slices (Muller et al 1996) In cultured neurons treatment of endoneuraminidase-N abrogates NCAM-induced synapse formation and prohibits the induction of LTP (Dityatev et al 2004) In an agreement addition of PSA-NCAM but not NCAM alone restores contextual memory in NCAM -- mice (Senkov et al 2006) These data collectively suggest that polysialylated NCAM promotes the formation and plasticity of synapses and is important for selective memory formation Moreover PSA is also important for appropriate axonal targeting and synaptic contact formation (Seki and Rutishauser 1998 El Maarouf and Rutishauser 2003)

Other IgSF proteins such as nectins L1 and SALM to name but a few play also distinctive roles in synapse formation and spine maturation (for reviews see (Dalva et al 2007 Missler et al 2012))

43 Neuroligins and neurexins Structure

Neuroligins form a family of type I transmembrane proteins expressed at the post-synaptic terminals There are four neuroligin isoforms in rodents (Ichtchenko et al 1996) named neuroligin-1 to -4 Among them neuroligin-1 is exclusively expressed at excitatory synapses neuroligin-2 and -4 at inhibitory synapses and neuroligin-3 is detectable from both types of synapses (Missler et al 2012) The extracellular domains of neuroligins are highly glycosylated which mediate the in cisbinding between two neuroligin molecules and form constitutive dimers (Comoletti et al 2003) The dimerization of neuroligins is important for the clustering of their ligands neurexins and the recruitment of pre-synaptic complexes The cytoplasmic tails contain a PDZ-protein recognizing sequence which mediate the interaction with synaptic scaffolding proteins (Irie et al 1997 Poulopoulos et al 2009)

Neurexins are the most well-known ligands for neuroligins at pre-synaptic terminals In mammals there are three neurexins encoded by individual genes (namely neurexin-1 to -3) and each of them produces two isoforms (Į- and ȕ-neurexins) Į-Neurexin has a large extracellular domain with six Laminin A-neurexin-and-sex hormone-binding protein (LNS) domains whereas ȕ-neurexin only contains a single LNS-domain (Ushkaryov et al 1992 Ushkaryov and Sudhof 1993) Similar to neuroligins the cytoplasmic domain of neurexins contains a PDZ-protein binding sequence which links neurexins to scaffold proteins like CASK Munc 18 interacting protein (MINT) and protein 41 (Hata et al 1996 Biederer and Sudhof 2000 Biederer and Sudhof 2001)

The five conserved splicing sites in neurexins and two in neuroligins generate a large number of splicing variants The interactions between neurexins and neuroligins are highly specific and splicing variant-sensitive These splicing isoforms are expressed differentially among different brain regions (Ushkaryov and Sudhof 1993 Ullrich et al 1995) suggesting that each neurexin splicing variant serves as a synaptic address code and the neurexinneuroligin interaction contributes to the specificity of synaptic targeting

30

Functions

In cultured neurons overexpression of neuroligins results in an increased number of synapses and spines whereas knockdown of these genes causes a loss of synapses (Prange et al 2004 Levinson et al 2005 Chih et al 2006) In the co-culture system neuroglin-1 and -2 expressed in non-neuronal cells trigger de novo formation of pre-synaptic specializations in the contacting axons (Scheiffele et al 2000) Likewise clustering of neuronal neurexin induced by antibody cross-linking promotes pre-synaptic assembly (Dean et al 2003) indicating that neuroligin-1-mediated pre-synaptic differentiation is dependent on neurexin clustering The neurexin-neuroligin signaling is bidirectional Transfection of neurexin-1ȕ-SS4 in non-neuronal cells induces aggregation of PSD95 and NMDARs at excitatory synapses whereas another splicing isoform neurexin-1ȕ +SS4 transfection leads to aggregation of gephyrin and Ȗ-aminobutyric acid (GABA)-A receptor at inhibitory synapses (Graf et al 2004 Boucard et al 2005 Chih et al 2006) These results provide further evidence that synaptic differentiation regulated by neuroligins is splicing-dependent

The role of neurexins and neuroligins in synapse formation and plasticity were further analyzed in vivo using genetic approaches that regulate their expression levels Elevated expression neuroligin-1 selectively increases the density of glutamergic synapses Neuroligin-1 binds directly to PSD-95 and another scaffold molecule S-SCAM which form a complex with NMDARs at synapses Neuroligin-1 overexpression induces NMDAR clustering and in turn enhances NMDAR-mediated excitatory synaptic transmission (Prange et al 2004 Chubykin et al 2005) In line with the gain-of-function studies neuroligin-1 -- mice exhibit decreased NMDAR-mediated neurotransmission Downregulation of neuroligin-1 by infecting mouse lateral amygdala with lentivirus carrying small hairpin RNA (shRNA) leads to attenuated NMDAR-mediated excitatory post-synaptic currents (EPSCs) and decreased NMDARAMPAR ratio (Chubykin et al 2005 Kim et al 2008) The suppression of neuroligin-1 also prevents induction of NMDAR-dependent LTP and causes memory impairment (Kim et al 2008) Neuroligin-2 is restricted to the inhibitory synapses Overexpression of neuroligin-2 results in increased density of GABAergic synapses elevated frequency of miniature inhibitory postsynaptic currents (mIPSCs) and a decreased ratio of excitatory to inhibitory synaptic transmission (EI) (Hines et al 2008) Mice harboring triple KO of neuroligin -1-2-3 die at birth due to respiratory failure because of impaired neurotransmission in neurons of the brainstem respiratory center however the morphology and density of synapses are not altered (Varoqueaux et al 2006) These in vivo data suggest that neurexin and neuroligins are more important in plasticity of mature synapses but not essential for formation of nascent synapses

Importantly mutations in genes encoding neuroligin-3 and -4 were found in patients with familial autism mental retardation and Asperger syndrome (Jamain et al 2003 Zoghbi 2003 Laumonnier et al 2004) Thus understanding the function of neuroligins is of central importance for developing therapies for these psychiatric diseases In addition to neuroligins there are other post-synaptic ligands for neurexins LRRTMs bind to neurexins in a Ca2+ -dependent manner in competition with neuroligins LRRTM induces pre-synaptic differentiation in cultured hippocampal neurons (Siddiqui et al 2010 Ko et al 2011) Loss-of-function of LRRTM2 and LRRTM3 together with neuroligin-1 and -3 leads to decreased excitatory synapse density and overexpression of LRRTMs increases synapse density (Ko et al 2011)

44 Cadherin superfamily Structure

The cadherin protein superfamily is a group of Ca2+-dependent CAMs composed of more than 100 members which can be subdivided into classical cadherins procadherins and desmosomal cadherins (Takeichi 2007) All cadherin superfamily members are characterized by varying number of cadherin repeats in their extracellular domains which mediate the binding of cadherins to their ligands Classical cadherins have five cadherin repeats exhibiting both homophilic and heterophilic binding properties The intracellular domains of cadherins bind to the cytoplasmic adaptor proteins

31

catenins (Shimoyama et al 2000 Kwiatkowski et al 2007) Procadherins consist of more than 70 members containing a different number of cadherin repeats and their cytoplasmic domains do not bind to catenins (Morishita and Yagi 2007)

Functions

Neuronal-cadherin (N-cadherin) is localized to nascent synapses and forms clusters soon after the axon-dendrite contacts form (Togashi et al 2002) As synapses mature N-cadherin gradually becomes concentrated in excitatory synapses and absent from GABAergic synapses (Benson and Tanaka 1998) In mature neurons N-cadherin is restricted to the areas surrounding the active zones at pre-synaptic terminals (Elste and Benson 2006)

During early development N-cadherin contributes to the accuracy of neural circuitry Blockade of N-cadherin function in the chick optic tectum generates deficits in retinal axon targeting and ganglion cell dendritic arborization (Inoue and Sanes 1997 Marrs et al 2006) Genetic deletion of N-cadherin in mouse and zebrafish do not prevent synapse formation (Erdmann et al 2003 Kadowaki et al 2007) Cultured hippocampal neurons transfected with a dominant-negative N-cadherin exhibit normal synapse formation (Bozdagi et al 2004) However in these neurons clustering of pre- and post-synaptic proteins is perturbed recycling of synaptic vesicles is reduced and the size and number of spines decreases (Togashi et al 2002 Bozdagi et al 2004) These results suggest that N-cadherin is more important in stabilizing and expanding existing synapses rather than inducing filopodia extension from dendritic shafts

As N-cadherin binding proteins catenins are also implicated in synapse formation For instance p120 catenin null mice exhibit a significant loss of synapse density (Elia et al 2006)

Likewise targeted mutation of the neuronal form of D-catenin results in increased length and

motility of spines (Togashi et al 2002) On the contrary overexpression of D-catenin leads to an increase in the density and a decrease in the motility of dendritic spines (Abe et al 2004)

In addition to N-cadherin other classical cadherin members as well as the procadherin family also contribute to synapse formation For example knockdown of Cad11 or Cad13 leads to reduction of synaptic density and excitatory synapse transmission (Paradis et al 2007)

In mature synapses cadherins contribute to synaptic plasticity Inhibition of cadherin function by pharmacological blockade RNAi or gene deletion results in attenuated synaptic transmission impaired LTP and reduction of synaptic vesicle cycling (Jungling et al 2006 Okuda et al 2007) Reciprocally cadherin trafficking clustering and interaction with catenin is affected by NMDAR-dependent neuronal activity suggesting interplay between neuronal activity and cadherin-mediated synaptic function (Bozdagi et al 2004)

45 Eph receptors and ephrins Structure

Eph receptors (Ephs) consist of 13 members which are subdivided into two classes according to their ligand binding specificity EphAs bind to ephrin-As which are glycophosphatidylinositol-anchored proteins whereas EphBs bind to ephrin-Bs the transmembrane form Given the specific binding property crosstalk between EphAs with ephrin-Bs and EphBs with ephrin-As gives rise to heterogeneity of Ephs and ephrin interaction

Ephs are all transmembrane proteins The extracellular domain of them contains an N-terminal ephrin-binding globular domain a cystein-rich domain and two fibronectin type III repeats (Yamaguchi and Pasquale 2004) The cytoplasmic domain contains a tyrosine kinase domain

proximal to the plasma membrane a sterile-D-motif (SAM) followed by a PDZ protein-binding domain The tyrosine kinase domain mediates phosphorylation of a set of downstream signaling proteins and the PDZ protein-binding domain interacts with cytoplasmic adaptor proteins or glutamate receptors The structure of ephrins is less understood It is known that ephrin-Bs contain a PDZ protein-binding domain which is missing in ephrin-As

Functions

32

Ephs and ephrins are widely expressed in both the peripheral nervous system (PNS) and the CNS In hippocampal synapses EphB2 is primarily localized to the post-synaptic membrane while ephrins are present in both pre- and post-synaptic terminals (Torres et al 1998 Dalva et al 2000 Grunwald et al 2001) Moreover ephrin-A3 has been found in astrocytes mediating the interaction between glial cells and neuronal synapses (Murai et al 2003)

The striking phenomenon that EphB1-3 triple -- mice exhibit almost complete absence of dendritic spines and postsynaptic specialization shed light on the importance of EphBs in spine formation (Henkemeyer et al 2003) This phenotype can be rescued by EphB2 expression (Kayser et al 2006) EphB2 -- mice display a less severe phenotype in terms of synapse morphology but the number of NMDARs at synapses is significantly decreased indicating that the effect of EphB on postsynaptic differentiation may rely on its modulation on NMDARs (Henderson et al 2001) In fact EphB2 lacking the cytoplasmic domain is not able to rescue the KO phenotype Therefore the interaction between the cytoplasmic tail of EphB and NMDARs possibly contributes to EphB-dependent post-synaptic differentiation EphA4 null mice show normal synapse morphology and differentiation indicating that EphAs might not be as important as EphBs in regulating synapse formation (Grunwald et al 2004)

Ephs and their ligands ephrins are engaged in regulating the formation of excitatory synapses Like other synaptic CAMs Ephephrin interaction initiates bi-directional signals which recruit synaptic proteins to nascent synaptic contacts Addition of the recombinant ephrin-B-Fc fusion protein into cultured neurons and brain slices induces clustering of NMDARs and AMPARs accompanied by increased number of synapses and altered spine morphology (Dalva et al 2000 Henkemeyer et al 2003 Kayser et al 2006 Lim et al 2008) Co-culture of HEK-293 cells overexpressing EphB2 with cortical neurons triggers differentiation of pre-synaptic specialization in contacting axons (Kayser et al 2006) Surprisingly there is no difference in synapse number and morphology between WT mice and the ones lacking ephrin-B2 or -B3 suggesting that these two ephrins are dispensable for synapse formation (Grunwald et al 2004) Crosstalk between ephrin-A and EphBs may compensate for the effect from loss of ephrin-B2 and -B3

EphB receptors are associated with various regulatory proteins which directly drive actin cytoskeleton assembly and spine morphological changes For instance kalirin-7 and Tiam-1 are two downstream signaling proteins of EphB receptors Activation of EphB receptors evokes tyrosine phosphorylation of kalirin-7 and Tiam-1 (Penzes et al 2003 Tolias et al 2007) both of which in turn activate the Rho GTPase Rac1 and subsequently promote actin polymerization and spine expansion In contrast EphA receptors activate Rho GTPase Cdk5 that inhibits actin polymerization resulting in spine retraction (Murai et al 2003)

In mature neurons Eph receptors and ephrins regulate synaptic plasticity Deletion of either ephrin-B2 or -B3 from the postnatal forebrain significantly reduces LTP in response to tetanic stimulation without affecting the basal synaptic transmission (Grunwald et al 2004 Rodenas-Ruano et al 2006) Likewise ablation of EphA4 a high-affinity receptor for ephrin-B2 and -B3 impairs early phase LTP (Grunwald et al 2001) suggesting that the interactions between EphA4 and ephrin-B2 and -B3 contribute to the formation of LTP

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CAMs Description Synapse formation Synaptic plasticity Learning and memory

Į3 integrins Conditional KO in forebrain

ND Impaired LTP in CA1 normal basal transmission and pre-pulse facilitation 1

Deficits in hippocampal memory 1

Null ND Impaired LTP maintenance 2

ND


ND Deficits in LTP in CA13 Normalhippocampal memory 3

Į3 Į5 Į8integrins

Null ND Deficits in LTP in CA1 2 Impaired spatial memory normal fear conditioning 2

ȕ1 integrins Conditional KO in forebrain

Loss of synapses less elaborated dendritic arbors 4

Impaired LTP in CA1 and AMPAR-mediated synaptic transmission 56

Impaired hippocampal working memory 6

ȕ3 integrins KO ND Normal LTP LTD and short-term synaptic plasticity 7

Impaired anxiety-like behavior 7

SynCAM-1 KO Decreased excitatory synapses 8

abrogated LTD 8 Improved spatial memory 8

NCAM

NCAM

Null Defective mossy fiber laminations 9

Decreased LTP in CA1 and CA3 10 11

Impaired spatial and contextual memory 12

Conditional KO in hippocampus

Normal mossy fiber laminations 13

Decreased LTP in CA1 normal in CA3 13

Deficits in spatial memory formation 13

ST8SiaIV KO Normal mossy fiber laminations 18

Decreased LTP and LTD in CA1 normal in CA3 18

ND

Neuroligin-1

KO ND Decreased NMDAR-mediated neurotransmission 14

Deficits in memory15

Neurogilin-1-2-3

Triple KO die of respiratory failure

No loss of synapses increased ratio of excitatory to inhibitory synapses 16

ND ND

Neurexin-1Į-2Į-3Į

Triple KO Less inhibitory synapses 17

Decreased pre-synaptic release 17

ND

N-Cadherin KO Normal synapse formation 19

abrogated synaptic transmission impaired LTP and reduced synaptic vesicle cycling 20

ND

Į-catenin Targeted mutation

Increased length and motility of spines 21

ND ND

EphB 1-3 Triple KO Almost complete loss of dendritic spines 22 decreased PSD 23

ND ND

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EphB2 Null Decreased number of NMDAR at synapses 24 slightly increased synapse number in CA1 25

Decreased LTP and LTD in CA1 24 25

Impaired spatial memory 25

Ephrin-B2and -B3

Conditional KO in forebrain

No change decreased LTP and LTD in CA1 26 27

ND

EphA 4 Null No change Decreased LTP and LTD in CA1 25

ND

ICAM-5 KO Increased number and size of spines decreased filopodia 28

Increased LTP in CA1 29enhanced synaptic plasticity in the thalamocortical circuit 30

Improved learning and memory 29

5 ICAM-5

The ICAM protein family belongs to the IgSF It consists of five members named ICAM-1 to ICAM-5 all of which have important roles in mediating cell-cell adhesion or cell-ECM adhesion (Fig 8) Like the other IgSF proteins ICAMs are composed of multiple Ig-like extracellular domains a single transmembrane domain and a short cytoplasmic tail ICAM-1 and ICAM-3

Figure 8 Schematic structure of ICAM family proteins The open loops represent Ig domains and the closed triangles represent glycosylation sites

Table 2 A summary of in vivo evidence for roles of CAMs in synapse formation and plasticity References 1 Chan et al 2007 2Chan et al 2003 3 Chan et al 2010 4 Warren et al 2012 5 Huang et al 2005 6 Chan et al 2006 7 McGeachie et al 2012 8 Robbins et al 2010 9 Cremer et al 1997 10 Muller et al 2000 11 Cremer et al 1998 12 Cremer et al 1994 13 Bukalo et al 2004 14 Chubykin et al 2005 15 Kim et al 2008 16 Varoqueaux et al 2006 17 Missler et al 2003 18 Eckhardt et al 2000 19 Jungling et al 2006 20 Okuda et al 2007 21 Togashi et al 2002 22 Henkemeyer et al 2003 23 Kayser et al 2006 24 Grunwald et al 2001 25 Henderson et al 2001 26 Grunwald et al 2004 27 Rodenas-Ruano et al 2006 28 Matsuno et al 2006 29 Nagamura et al 2000 30 Barkat et al 2011 ND not determined

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contain five extracellular Ig-like domains whereas ICAM-2 and -4 contain two ICAM-5 has a more complex structure and possesses nine Ig-like domains (Gahmberg 1997)

ICAM family members have their distinctive expression profile ICAM-1 is mainly expressed in leukocytes and endothelial cells Its expression level is low at the resting state and can be upregulated upon cytokine activation ICAM-2 is expressed in leukocytes endothelial cells and platelets ICAM-3 is specific for leukocytes and is important in regulating various immune responses ICAM-4 is restricted to red blood cells and erythroid precursors ICAM-5 is the only ICAM expressed in the brain and is neuron-specific (Gahmberg et al 1997)

51 History In 1987 Mori et al produced a series of monoclonal antibodies (mAb) following immunization of mice with the dendrodendritic synaptosomal fractions of the rabbit olfactory bulb They found that one of these mAbs 271A6 specifically recognized an antigen in the gray matter of the neocortex piridorm cortex hippocampus striatum septum amygdalaloid nucleus and olfactory bulb These regions are all derived from the telencephalon segment of the prenatal brain and constitute the major portion of the mammalian brain during development However other segments of the prenatal brain including diencephalon mesencephalon metencephalon or myelencephalon are devoid of immunoreactivity of this mAb (Mori et al 1987)

Three years later scientists from the same group purified this telencephalon-specific antigen using affinity chromatography with mAb 271A6 This antigen was characterized as a membrane glycoprotein with a total molecular weight of 500 kDa consisting of four subunits of 130 kDa each It was expressed specifically in the telencephalon-derived region in all examined mammalian brains (including mouse rat rabbit guinea pig cat and monkey) and thus was given the name telencephalin (TLN) (Oka et al 1990)

The cDNA encoding rabbit and mouse TLN was cloned in 1994 (Yoshihara et al 1994) and human TLN cDNA was cloned in 1997 (Mizuno et al 1997) The cDNA sequence reveals that TLN is an integral membrane protein which shares high homology with ICAM proteins especially ICAM-1 and -3

The human TLN gene was mapped to chromosome 19p 132 where ICAM-1 -2 and -4 genes are also located (Mizuno et al 1997 Kilgannon et al 1998) The mouse TLN gene was found located to chromosome 9 close to the ICAM-1 gene (Sugino et al 1997) This finding suggests a strong correlation between TLN and ICAMs Subsequently it was recognized as a novel member of the ICAM family and named ICAM-5 (Gahmberg 1997)

52 Molecular feature ICAM-5 cDNA contains 3024 nucleotides among which nucleotide 1-124 is the 5rsquo-untranslated region 125-2860 is the open reading frame and 2861-3024 is the 3rsquo-untranslated region (Yoshihara et al 1994)

Deduced from the cDNA sequence rabbit ICAM-5 contains 912 amino acids Human ICAM-5 contains 924 amino acids and mouse counterpart 917 They are highly homologous to rabbit ICAM-5 with 85 and 82 identity respectively (Yoshihara et al 1994 Mizuno et al 1997) Starting from the N-terminal amino acids 1-29 form the signal peptide which is cleaved in the mature ICAM-5 protein Amino acids 30-821 form a hydrophilic extracellular domain consisting of nine Ig-like domains The 28 cysteine (Cys C) residues in this region form intra-chain disulfide bonds typical of Ig-like loops The amino acids 822-851 constitute the transmembrane domain while amino acids 852-912 compose the short cytoplasmic domain (Fig 9) Based on its amino acid sequence ICAM-5 was predicted to have a molecular weight of 93 kDa Its extracellular domain contains 14 Asparagine (Asn N)-linked glycosylated sites which add 42 kDa to the protein Therefore the full-length ICAM-5 approximates 135 kDa (Yoshihra et al 1994)

ICAM-5 is heavily glycosylated The extracellular region of human ICAM-5 contains 15 N-glycosylation sites and all oligosaccharides can be released by N-glycosidase F-digestion (Mizuno et al 1997 Ohgomori et al 2009) Mutation analysis of the 15 N-glycan sites individually revealed that Asn54 is important for ICAM-5 targeting to filopodia in neurons (Ohgomori et al

36

2012)

53 Expression ICAM-5 expression is spatially highly specific It is confined to telencephalon-derived regions in the brain (Mori et al 1987 Oka et al 1990) A conserved sequence in the 5rsquo-flanking region of ICAM-5 cDNA is responsible for the telencephalon-specific expression of ICAM-5 (Mitsui et al 2007)

At the cellular level ICAM-5 is exclusively expressed in neurons and not in glial cells In the rabbit olfactory bulb ICAM-5 is only present in granule neurons but not in principal neurons mitral and tufted cells (Murakami et al 1991) In cultured hippocampal neurons ICAM-5 is detected in pyramidal neurons but not in GABAergic inhibitory neurons (Benson et al 1998)

At the subcellular level ICAM-5 is confined to the soma-dendritic membrane but not to the axonal membrane (Yoshihara et al 1994 Benson et al 1998) The cytoplasmic sequence of ICAM-5 especially Phe905 is important for the dendritic targeting of ICAM-5 (Mitsui et al 2005)

Temporarily in rodents ICAM-5 is absent from the embryonic brain appears abruptly at birth increases during the early postnatal weeks and reaches stable levels in adulthood (Yoshihara et al 1994) In other examined species including rabbit cat and human the temporal expression of ICAM-5 follows similar pattern as that in rodents paralleling the critical period of dendritic development and synaptogenesis (Mori et al 1987 Imamura et al 1990 Arii et al 1999)

54 Binding partners The most interesting binding partners for ICAM-5 include two integrins vitronectin and presenilin (Fig 9) The extracellular Ig domains transmembrane domains as well as the cytoplasmic tail of ICAM-5 all contribute to the interactions

Figure 9 Schematic representation of the structure of human ICAM-5 and the interaction sites to its binding partners

37

LFA-1 (CD11aCD18)

The first identified counter-receptor for ICAM-5 is integrin LFA-1 (CD11aCD18) (Mizuno et al 1997 Tian et al 1997) LFA-1 belongs to the ȕ2 integrin family and is a leukocyte-specific integrin In the brain microglia and infiltrated T-lymphocytes are the possible source of LFA-1 Interestingly all other ICAM family members bind to integrin LFA-1 (Gahmberg 1997)

ICAM-5 binds to LFA-1 through its first Ig-like domain (Tian et al 2000) The binding site in LFA-1 resides in the I-domain of the integrin alpha chain LFA-1 binds to ICAM-5 with low affinity at resting state upon integrin activation an axial movement of the Į7 helix increases the binding affinity of LFA-1 to ICAM-5 (Zhang et al 2008)

ȕ1 integrins

ICAM-5 binds to ȕ1 integrins particularly the Į5ȕ1 integrin in trans The binding site is located in the first two Ig-like domains (See below Page 45-46)

ICAM-5 homophilic binding

The homophilic association of ICAM-5 is formed between the 1st and the 4th-5th Ig-like domains from the counter cells The 2nd Ig-like domain is not directly involved in the homophilic binding but plays a regulatory role since the Ig domains 1-2 display better homophilic binding than the single Ig domain 1 (Tian et al 2000)

Presenilin (PS)

PS-1 and -2 are implicated in familial AD They are responsible for the cleavage of amyloid protein precursors (APP) into Aȕ42 peptide a major constituent of the plagues in the brains of AD patients The full-length PS consists of eight transmembrane domains which are usually endoproteolyzed into an N-terminal and a C-terminal fragment The two fragments interact with each other forming stable complexes and the complete structure is required for its biological function PS1 binds to both ICAM-5 and APP The first transmembrane domain and the C-terminus of PS1 form a circular structure which embraces the transmembrane domains of both ICAM-5 and APP Five amino acids (Val829-Trp833) in the N-terminal part of ICAM-5 transmembrane region and 11 amino acids (Thr639-Lys649) at the C-terminus of the APP transmembrane domain are required for the interaction with PS1 (Annaert et al 2001)

ERM proteins

ERM family proteins belonging to the Protein 41 superfamily are ABP consisting of three closely related members ezrin radixin and moesin The N-terminal FERM domains bind to transmembrane receptors and their C-termini bind to F-actin thus linking the plasma membrane to actin filaments (Tsukita and Yonemura 1999) ICAM-5 is colocalized with ERM proteins within dendritic filopodia In yeast-two hybrid analysis ICAM-5 binds directly to ezrin and radixin through its juxtamembrane cytoplasmic region (Furutani et al 2007) Four amino acids within this region Lys864 Lys865 Tyr868 and Val870 are crucial for this binding The C-terminus of ERM contains a phosphorylation site at Thr567 which regulates the activity of ERM protein (Furutani et al 2007)

Vitronectin (VN)

VN is an ECM protein which is present abundantly on blood vessel basement membranes and transported to a variety of tissues through the blood flow In the brain VN is produced and secreted by pericytes surrounding small blood vessels leptomeninges reactive astrocytes and olfactory ensheathing glia (Niquet et al 1996)

ICAM-5 binds to VN through the second Ig-like domain Upon binding to VN ICAM-5 induces the phosphorylation of ERM proteins and their recruitment to filopodia along with other filopodia components F-actin and PIP2 (Furutani et al 2012)

Į-Actinin

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The cytoplasmic domain of ICAM-5 binds to D-actinin and the binding site partially overlaps with that of ERM proteins (See below page 47)

55 Functions The more complex structure and specific regional expression make ICAM-5 a special member of the ICAM family Interestingly ICAM-5 plays a role in both immunity and neuronal development in the brain

The immune functions of ICAM-5

In the CNS neurons are sensitive to the immune stimuli in their microenvironment The substancesgenerated from an immune response can challenge the survival and proper functioning of neurons and therefore controlling this response is critical for CNS homeostasis It has been established that the CNS neurons actively interact with immune cells and participate in the regulation of immune response through a variety of mechanisms (Tian et al 2009 Jurgens and Johnson 2012) In a contact-independent mechanism neurons release soluble factors including cytokines neuropeptides neurotrophins and neurotransmitters to inhibit the activation of microglia and T cells In a contact-dependent mechanism neurons directly bind to microglia or T cells suppressing their activation or inducing their apoptosis

As a ligand for leukocyte integrin LFA-1 ICAM-5 can bind to T-lymphocytes (Tian et al 1997 Tian et al 2000) By binding to T cells ICAM-5 may present them to neighboring glial cells which may lead to inactivationapoptosis of the former cells Moreover soluble ICAM-5 (sICAM-5) cleaved from the membrane-bound ICAM-5 directly interacts with T-cells Different from its close relative ICAM-1 sICAM-5 suppresses T cell activation by reducing the expression of the activation markers CD69 CD40 and CD25 Therefore sICAM-5 serves as an anti-inflammatory factor and is involved in maintaining immune privilege in the CNS (Tian et al 2008)

sICAM-5 is generated by MMP-2 and -9 in activated neurons or T-cells Elevated levels of MMP have been found in many CNS immune disorders and have been shown to attenuate synaptic plasticity Abnormal sICAM-5 levels in the cerebrospinal fluid and serum have been reported in many CNS immune diseases such as acute encephalitis temporal-lobe epilepsy and ischemia (Rieckmann et al 1998 Guo et al 2000 Lindsberg et al 2002 Borusiak et al 2005) Thus serum sICAM-5 level has been used as an indicator to diagnose some forms of brain dysfunction (Jansen et al 2008)

Microglia are the residential macrophages in the brain Morphologically they vary from the ramified resting form to the round activated form In the healthy brain most of microglia stay at the resting state Upon brain injury or infection microglia are activated and become neurotoxic or neuroprotective ie phagocyting dying neurons and promoting regeneration of damaged neurons The interaction between T cells and microglia is dynamically regulated This regulation could provide apoptotic signals to activated T cells and suppress brain damage caused by T cell activation ICAM-5 induces LFA-1-mediated morphological change of microglia which is accompanied by LFA-1 redistribution on microglia (Mizuno et al 1999) However the interaction between LFA-1 and ICAM-5 seems more important for the regulation of microglia function rather than the formation of adhesive contacts between neurons and microglia as evidenced by ICAM-5 KO mouse brain that displays normal formation of neuron-microglia contacts (Hasegawa et al 2007) Through binding to microglia ICAM-5 may indirectly attenuate T-cell activation by down-regulating antigen presenting properties of microglia Interestingly a growing body of evidence suggests that resting microglia participate in the formation and plasticity of synapses under physiological condition indicating that ICAM-5 may be implicated in the regulation of microglia on neuronal synapse formation and plasticity

The neuronal functions of ICAM-5

The distinctive temporal expression of ICAM-5 suggests its importance in neurite outgrowth and synapse formation In patients with holoprosencephaly a developmental disorder in which the two

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cortical hemispheres are not divided normally ICAM-5 expression is abnormal (Arii et al 2000) suggesting a role of ICAM-5 in brain segmental organization

A carpet of chimeric ICAM-5-Fc protein induces neurite outgrowth and arborization of dendrites in cultured hippocampal neurons through homophilic binding (Tamada et al 1998 Tian et al 2000) Antibodies against ICAM-5 or chimeric ICAM-5-Fc proteins block this homophilic binding and therefore attenuate dendritic outgrowth (Tian et al 2000) ICAM-5 appears to play a central role in synaptic development and plasticity ICAM-5-deficient mice exhibit increased hippocampal LTP and enhanced learning and memory capacity (Nakamura et al 2001) Moreover during a critical period of mouse auditory cortex connectivity formation ICAM-5-deficient mice display higher plasticity of the thalamocortical synapses in response to acoustic stimuli suggesting that loss of ICAM-5 results in an improved experience-dependent change (Barkat et al 2011) As a post-synaptic protein ICAM-5 is described as a negative regulator of the morphological maturation of dendritic spines Overexpression of ICAM-5 significantly increases the density and length of filopodia whereas ablation of ICAM-5 promotes the enlargement of spine heads (Matsuno et al 2006)

Therefore to dissect the cellular and molecular mechanisms by which ICAM-5 regulates spine maturation and synapse formation is instrumental in understanding ICAM-5-mediated brain plasticity and function

In addition ICAM-5 immunoreactivity is reduced in the brain of AD patients (Hino et al 1997) PS-1 which is often mutated in AD patients is important for the turnover of ICAM-5 In PS-1-deficient mouse neurons ICAM-5 accumulates in autophagic vacuole-like structures due to failure to fuse into endosomelysosome (Esselens et al 2004) Accumulation of ICAM-5 is likely associated with AD suggesting that ICAM-5 may be implicated in neurodegeneration

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AIMS OF THE STUDY

The neuron-specific adhesion molecule ICAM-5 serves as a negative regulator of spine maturation The current studies were carried out to understand the mechanisms by which ICAM-5 is engaged in spine morphogenesis and synapse formation To this end we will pursue the following aims

1 To understand the molecular mechanism of ICAM-5 ectodomain cleavage and its contribution to NMDAR-dependent spine morphogenesis

2 To examine whether ICAM-5 is a ligand for E1 integrins and to characterize the interaction in neurons

3 To elucidate the functional relevance of the ICAM-5E1 integrin interaction in synapse formation

4 To study the cytoplasmic interactions of ICAM-5 specifically the interaction complex of

ICAM-5D-actininNMDAR

5 To uncover the functional role of the ICAM-5D-actininNMDAR complex for synapse formation

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MATERIALS AND METHODS

Material and methods used in this study are described in detail in the original publications

Materials or Methods Original Publication

Antibodies I-IV

Animals I-IV

Primary neuron culture I-IV

Cell lines I-IV

Human placental Į5ȕ1 integrin purification III

Recombinant protein constructs and purification I-IV

In vitro protein binding assay I-IV

ELISA I III IV

Coimmunoprecipitation I-IV

SDS-PAGE I-IV

Western blotting I-IV

Gelatinase zymography II

Immunohistological staining II

Immunofluorescent staining I-IV

Confocal microscopy I-IV

Time-lapse imaging II IV

Electron microscopy III

Mass spectrometry II IV

Flow cytometry II

RNAi II III

Mouse brain homogenization and fractionation II III

Synaptosome fractionation III

Patch clamp recording III

Cell adhesion assay III

Beads recruitment assay III

Cell stimulation II-IV

Statistical analysis I-IV

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RESULTS

1 ICAM-5 is susceptible to the MMP-2- and -9-mediated cleavage upon activation of the glutamate receptors (II) MMPs are a group of zinc-dependent endopeptidases which are able to degrade extracellularly CAMs and ECM proteins Many members of MMPs have been implicated in synapse formation and plasticity (Huntley 2012) In this study we first examined whether MMP-2 and -9 are responsible for ICAM-5 cleavage

Recombinant mouse ICAM-5 ectodomain D1-9-Fc protein was incubated with active MMP-2 and -9 and the resulting fragments were detected by Western blotting (II Fig 2 D-F) Both MMPs cleaved ICAM-5 D1-9-Fc (170 kDa) and produced four different fragments which were identified as a 40 kDa N-terminal fragment (NTF40) a 130 kDa C-terminal fragment (CTF130) a NTF80 and a CTF55 by matrix-assisted laser desorptionionization-time of flight (MALTI-TOF) mass mapping (II Suppl Fig 2) The different lengths of fragments resulted from two individual cleavage sites MMP-9 preferably cleaved ICAM-5 at D2 domain generating a long C-terminal fragment the CTF130 In contrast MMP-2 mainly targeted the cleavage site between the D8 and D9 domain preferably generating the NTF80 (II Fig 2 D-F)

It is known that NMDAR activation leads to the elevation of MMP expression and activity (Meighan et al 2006 Nagy et al 2006) Therefore NMDA in place of the cytotoxic purified MMPs was applied to cultured neurons as a more physiological method to study MMP-mediated ICAM-5 cleavage Fourteen day in vitro (DIV) cultured hippocampal neurons were

treated with 5 PM NMDA without or with MMP inhibitors for 16 hr Cell culture media was then collected to detect the amount of released N-terminal ICAM-5 fragment- sICAM-5 which was cleaved from the membrane-bound form NMDA treated neurons generated significantly more sICAM-5 (II Fig 1 and Fig 2 A sICAM-5110) This increase was inhibited by MMP inhibitors (GM6001 CTT MMP2amp9 inhibitor II and MMP9 inhibitor I) but not by the negative MMP inhibitor controls (II Fig 2 A and B) MMP-mediated ICAM-5 cleavage was also confirmed by knocking down of MMPs by RNAi in cultured neurons which resulted in a reduced amount of sICAM-5 in the cell culture media (II Fig 2 C) Moreover in the brain of MMP-2 or -9 KO mice the amount of membrane-remaining full length ICAM-5 was increased compared to that in the WT mouse brain in the first postnatal week providing a piece of evidence for the existence of MMP-mediated ICAM-5 cleavage in vivo (II Fig 4 A-C)

2 MMP-mediated ICAM-5 cleavage promotes spine maturation (II) To clarify the expression of ICAM-5 in spines at different maturation stages we transfected cultured hippocampal neurons with EGFP and immunostained for ICAM-5 We found that ICAM-5 was more abundantly expressed in filopodia and thin spines but less in mature mushroom spines (II Fig 5)

Since MMPs are involved in synapse formation and plasticity (Huntley 2012) the results above led us to assume that removal of ICAM-5 by MMPs may be important for spine maturation Indeed after treatment with NMDA hippocampal neurons exhibited a significant increase in spine density especially mushroom spines (II Fig 6 and 7) By time-lapse imaging

enlargement of spine heads was observed within 30 min in WT neurons in response to the 20 PMNMDA treatment However this enlargement was not observed in ICAM-5 -- neurons which exhibited bigger spines compared with WT before the treatment (II Fig 8 B) The effect of NMDA was reversed by the NMDAR antagonist MK801 and at least one of the MMP inhibitors (II Fig 7 and 8)

Spine morphogenesis is highly dependent on actin dynamics NMDAR activity can shape spine morphology through its modulation of the organization of the cytoskeleton Therefore it was interesting to study whether NMDA-induced ICAM-5 cleavage and the related spine maturation are associated with actin reassembly We found that when cultured neurons were treated with cytochalasin D and latrunculin A the actin polymerization inhibitors that bind to the barbed ends of actin filaments and actin monomers respectively the levels of sICAM-5 were significantly

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increased (II Fig 3 A and B) Additionally after NMDA treatment cultured hippocampal neurons were lysed and fractionated into plasma membrane and cytoskeletal fractions by ultracentrifugation The amount of ICAM-5 attached to the actin cytoskeleton was significantly decreased upon the NMDA treatment indicating that NMDA stimulation broke the anchorage of ICAM-5 to the actin cytoskeleton (II Fig 3 C and D) Moreover using a neural crest-derived cell line Paju we observed a considerable increase of ICAM-5 cleavage in cells transfected with the cytoplasmic-

tail-deleted ICAM-5 (Paju-ICAM-5-cp) in comparison with those with the full-length ICAM-5 (Paju-ICAM-5-fl) (II Fig 3 E)

To study the potential role of sICAM-5 in spine maturation we treated cultured

neurons with 10 Pgml the recombinant mouse ICAM-5 D1-4-Fc protein during 9-12 DIV We found that sICAM-5 induced a significant increase of filopodia length and density in WT neurons compared to ICAM-5 -- neurons However the effect of sICAM-5 on spine density was minimal (II Fig 9 B-D)

3 ICAM-5 is a ligand for the pre-synaptic ȕ1 integrins (III) ICAM-5 -- neurons have a higher level of overlap between spine heads and synaptophysin-labeled pre-synaptic terminals in comparison to WT neurons (Matsuno et al 2006) suggesting the existence of ICAM-5 binding partner(s) from axons mediating the formation of synaptic contacts All ICAM family members act as ligands for the integrin LFA-1 In the CNS ȕ1 integrins constitute the integrin subtypes widely expressed across the brain and known to regulate axonal guidance neurite outgrowth synapse formation and plasticity Therefore they make up a group of possible candidates for ICAM-5 counter-receptors from axons

Interaction between ICAM-5 and ȕ1 integrins was first detected by co-immunoprecipitation When ICAM-5 was immunoprecipitated from mouse forebrain homogenates integrin subunit ȕ1 and Į5 were co-precipitated with ICAM-5 and so was vice versa (III Fig 3 A and B) In contrast the Į3 and Į8 integrin subunits were not co-precipitated with ICAM-5 (data not shown) Therefore Į5ȕ1 integrin is likely the most prominent binding partner for ICAM-5 in the brain

To characterize the interaction we took again the advantage of Paju cells transfected with a full-length ICAM-5 (III Fig 3 C Paju-ICAM-5-fl) or a cytoplasmic-tail-deleted ICAM-5 (III Fig 3 C Paju-ICAM-5 ǻcp) When the cytoplasmic domain of ICAM-5 was missing the binding between ICAM-5 and ȕ1 integrins was not altered indicating that the binding site is primarily located at the extracellular domain of ICAM-5 (III Fig 3 C)

The direct interaction between ICAM-5 and ȕ1 integrins was confirmed by enzyme-linked immunosorbent assays (ELISA) Recombinant human ICAM-5 D1-9-Fc was found to bind to the coated purified Į5ȕ1 integrin (III Fig 3 D)

To map the binding sites in ICAM-5 we employed cell adhesion assays The culture dishes were coated with purified recombinant proteins ICAM-5 D1-2-Fc D1-9-Fc and two negative controls (human IgG and ICAM-2-Fc) and 106ml Paju cells expressing ȕ1 integrins were allowed to bind Compared with controls both D1-2-Fc and D1-9-Fc increased cell adhesion D1-2-Fc showed a better binding (III Fig 4 A) To further confirm that this increased cell adhesion was mediated by ICAM-5ȕ1 integrin interaction antibodies and inhibitory peptides against ICAM-5 or ȕ1 integrins were applied Antibodies recognizing the first two Ig-like domains of ICAM-5 (179D 179K and 246 H) effectively blocked the cell adhesion (III Fig 4 B and Table 1) Similarly the ȕ1-integrin-blocking antibody 2253 also prevented the cell adhesion In contrast RGD-containing peptide and its negative control had negligible effects Notably the ȕ1-integrin-activating antibody TS216 significantly promoted the cell adhesion (III Fig 4 B and Table 1)

To examine the distribution pattern of ȕ1 integrins cultured hippocampal neurons were fixed at 15 DIV and immunostained for ȕ1 integrins (III Fig 5 A red) synapsin I (a presynaptic marker) and PSD-95 (a postsynaptic marker) ȕ1 integrins had a considerable higher colocalization with synapsin I than PSD-95 (III Fig 5 A and C) In comparison ICAM-5 was prominently colocalized with PSD-95 (III Fig 5 B and D) as reported earlier (Yoshihara et al 1994 Benson et al 1998) Studies by immuno-EM confirmed the pre-synaptic distribution of ȕ1integrins even though we did not exclude the presence of ȕ1 integrins in the post-synaptic sites and

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dendritic shafts (III Suppl Fig 3) The biochemical approach of synaptosomal fractionation provided an additional piece of evidence for the pre-synaptic distribution of ȕ1 integrins (III Fig 5 E)

The above results support our hypothesis that ȕ1 integrins act as the pre-synaptic counter receptors for ICAM-5 and that the interaction may occur at the early stage of synaptogenesis

4 Trans-synaptic interaction between ICAM-5 and ȕ1 integrins mediates synapse formation (III) The first question we encountered when studying the role of ICAM-5ȕ1 integrin interaction in synapse formation was the time frame in which the two proteins contact We therefore examined the expression of ȕ1 integrins and their colocalization with ICAM-5 in individual morphological types of spines Cultured hippocampal neurons were transfected with EGFP and fixed at 15 and 22 DIV followed by immunostaining for ȕ1 integrins In filopodia ȕ1 integrins were weakly expressed at the tips In thin spines ȕ1 integrins were localized juxtaposed to the spine heads In mature spines ȕ1 integrins overlapped with the spine heads (III Fig 6 A and B) The colocalization of ICAM-5 and ȕ1 integrins was then analyzed using similar techniques Immunoreactivity of ȕ1 integrins and ICAM-5 overlapped at the tip of filopodia and thin spines (III Fig 6 C arrows) but was devoid from each other in the heads of mushroom spines in which ȕ1 integrin still remained but ICAM-5 was excluded (III Fig 6 C arrowheads)

ICAM-5 is known as a negative regulator that slows down the filopodia-to-spine transition (Yoshihara et al 2009) By patch clamp recording we found that ICAM-5 -- neurons had higher frequency of miniature EPSCs (mEPSCs) in comparison with the WT neurons indicating that ICAM-5 inhibits the formation of functional synapses We then explored the implication that ICAM-5ȕ1 integrin interaction delays spine maturation Cultured neurons were treated with antibodies against ICAM-5 or ȕ1 integrins (III Table 1) Interestingly blocking antibodies for ICAM-5 (III Fig 2 A and B 179D 179K) and ȕ1 integrins (III Fig 2 A and B Ha25) both led to an increase in density of mushroom spines and a decrease in that of immature spines In contrast an activating antibody for ȕ1 integrins significantly increased the number of filopodia and reduced that of spines (III Fig 2 A and B TS216) Downregulation of ȕ1 integrins by shRNA in axons displayed also a similar effect as blocking antibodies Dendrites when making contacts with the ȕ1-integrin-knockdown axons exhibited decreased immature and increased mature spines (III Fig 8 C-E)

Our previous study showed that the shedding of ICAM-5 ectodomain promotes spine maturation (II) Therefore it was tempting to analyze whether the ICAM-5ȕ1 integrin interaction affects ICAM-5 shedding Antibodies described earlier (III Table 1) were used to treat cultured neurons The cleaved ICAM-5 in the cell culture medium was detected by Western blotting Notably the blocking antibodies 179D and 179K (against ICAM-5) and Ha25 (against ȕ1integrins) dramatically enhanced the ICAM-5 cleavage whereas ȕ1-integrin-activating antibody TS26 and 9EG7 attenuated the shedding Interestingly added recombinant ICAM-5 D1-2-Fc protein also decreased the cleavage of endogenous ICAM-5 (III Fig 7)

The data above suggest that the ICAM-5ȕ1 integrin interaction prevents spine maturation by inhibiting the extracellular cleavage of ICAM-5

5 Interaction of the ICAM-5 cytoplasmic tail with DD-actinin is implicated in neurite outgrowth (I) Dendritic outgrowth and spine morphogenesis are specific cell adhesion events which are intrinsically driven by the assembly of actin filaments Therefore it became important for us to

investigate the interplay between ICAM-5 and the actin cytoskeleton D-Actinin an actin crosslinking protein is known to bind to ICAM-1 and -2 (Carpen et al 1992a) We therefore tested

whether ICAM-5 also binds to D-actinin The cytoplasmic domain of ICAM-5 was expressed as a glutathione S-transferase

(GST) fusion protein purified by affinity chromatography and immobilized to a matrix in order to

fish for bound proteins from lysates of Paju cells Immunoreactivity of D-actinin (I Fig 1 B a) was

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detected by Western blotting from the bound fraction Direct binding between D-actinin to ICAM-5

was further confirmed by pulling-down purified D-actinin using the GST-ICAM-5 cytoplasmic

domain (I Fig 1 B b) Co-immunoprecipitation of ICAM-5 and D-actinin from mouse brain homogenates further demonstrated that this interaction also exists in vivo (I Fig 2)

To map the binding sites in ICAM-5 a five amino acid peptide KKGEY derived from

the ICAM-5 cytoplasmic sequence 857-861 which resembles the D-actinin binding sites in ICAM-

1 and -2 was used to test the binding to D-actinin Purified KKGEY peptide efficiently bound to D-actinin but substitution of either one or both of the two lysines abolished the binding (I Fig 1 C) Filamin and talin were also pulled down from Paju lysates by the GST-ICAM-5 cytoplasmic domain (data not shown) but purified filamin and talin did not bind to the peptide KKGEY (I Fig 1 D) suggesting that their binding sites are located to sequences other than the KKGEY or they bind to ICAM-5 in an indirect manner

Binding kinetics of D-actinin to the ICAM-5 peptide KKGEY was then determined by surface plasmon resonance (SPR) analysis The binding occurred rapidly and reached a steady state

in seconds with an equilibrium constant Kd = 229 r 205 PM and a maximal binding level Rmax =

235 r 104 RU Based on the data fitting by Sigmaplot the interaction was assumed to be a one-to-one model (I Fig 1 E)

The interaction between the ICAM-5 cytoplasmic domain and D-actinin in vivo was further supported by their colocalization in Paju cells In full-length-ICAM-5 expressing Paju cells

ICAM-5 immunoreactivity exhibited a patchy pattern ICAM-5D-actinin and ICAM-5F-actin were highly colocalized in cell uropod or cell-cell contact sites When the cytoplasmic tail of

ICAM-5 was deleted colocalization of ICAM-5D-actinin and ICAM-5F-actin was abolished and ICAM-5 was distributed evenly throughout the cell body Moreover in cells expressing ICAM-5 with two lysines (Lys K) mutated to alanines (Ala A) the colocalization was also lost (I Fig 4) These results further support our hypothesis that the cytoplasmic tail of ICAM-5 especially Lys857

and Lys858 are essential for binding to D-actinin When activated with a phorbol ester (PDBu) Paju cells changed morphology and projected

neurite-like structures ICAM-5 was colocalized with D-actinin and F-actin along the growth cone membranes when its cytoplasmic tail was intact suggesting the cytoplasmic anchorage of ICAM-5 may regulate neurite outgrowth By quantification Paju cells with the full-length ICAM-5 possessed significantly longer neurite length as compared to the ones with the cytoplasmic-tail-truncated ICAM-5 (I Fig 5) Furthermore infusion of a KKGEY-containing peptide into Paju-

ICAM-5 cells which blocked the binding between ICAM-5 and D-actinin compromised PDBu-

induced neurite outgrowth (I Fig 6) indicating that the ICAM-5D-actinin interaction mediates neurite outgrowth

6 GluN1 competes with ICAM-5 for DD-actinin binding (IV)

The rod domain of D-actinin which contains four SRs constitute the binding region for many

membrane proteins due to the negative charge of this region To figure out which SR of D-actinin binds to ICAM-5 we performed ELISAs The four SRs were subcloned and expressed as GST-fusion proteins followed by affinity chromatography purification The ICAM-5 cytoplasmic protein was coated on a surface and the SR GST-fusion proteins were allowed to bind The amount of bound SR GST-fusion proteins was detected by a GST antibody The whole rod domain with the four SRs was an efficient binder to the ICAM-5 cytoplasmic domain and the second SR (R2) showed an equally strong binding (IV Fig 2 D)

NMDARs are known to bind to D-actinin through the subunits GluN1 and GluN2B

GluN1 is an obligatory subunit in all NMDARs and is colocalized with D-actinin in spine heads In

this study we also investigated the binding between GluN1 and D-actinin in detail The cytoplasmic tail of GluN1 was expressed as a His6-tagged fusion protein and purified by affinity

chromatography The binding between the His6-GluN1 cytoplasmic protein to D-actinin was examined by ELISAs As reported earlier GluN1 bound to the whole rod domain Like ICAM-5

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GluN1 also bound efficiently to the R2 domain In addition the R4 domain exhibited relatively weaker binding to GluN1 as well (IV Fig 2 E)

We then analyzed whether the cytoplasmic domains of ICAM-5 and GluN1 compete in binding to the R2 domain We immobilized the R2 domain GST-fusion protein to glutathione matrix When increasing the concentration of ICAM-5 the amount of GluN1 bound to R2 was reduced Reciprocally increasing GluN1 concentration also decreased the ICAM-5 binding to R2

However ICAM-5 appeared to have a higher affinity to D-actinin compared to GluN1 When ICAM-5 was at 100 nM the GluN1 binding to R2 domain was significantly decreased at 500 nM the GluN1R2 binding was completely blocked In contrast at the GluN1 concentration as high as 1000 nM ICAM-5 was still seen binding to the R2 domain The required concentration for a half-maximum-inhibition was 55 nM for the ICAM-5 cytoplasmic protein and 340 nM for the GluN1 (IV Fig 3)

The interaction of ICAM-5D-actininGluN1 was then characterized in vivo In line

with its in vitro binding property D-actinin preferably bound to ICAM-5 rather than GluN1 in mouse brain as well as in Paju-ICAM-5 cells When ICAM-5 was genetically deleted or its

cytoplasmic tail ablated the interaction between GluN1 and D-actinin was increased (IV Fig 4 A and B) We have previously observed that activation of NMDAR promoted spine maturation accompanied by ICAM-5 detachment from the actin cytoskeleton (II) Herein we found that in

cultured neurons NMDAR activation led to the dissociation of ICAM-5 from D-actinin and a

concomitant increased attachment of GluN1 to D-actinin (IV Fig4 C)

7 Regulation of ICAM-5DD-actinin interaction is important for spine maturation (IV)

Since ICAM-5 D-actinin and NMDARs are known regulators of spine morphogenesis we next tested whether their interplay is important for spine maturation Cultured hippocampal neurons were studied by immunofluorescent staining between 14 and 21 DIV a time frame in which the majority of spines mature and ICAM-5 is excluded from spine heads During this period the

ICAM-5D-actinin colocalization was decreased and the GluN1D-actinin colocalization was

increased D-Actinin exhibited a punctuate distribution pattern and with development the D-actinin puncta expanded in size (IV Fig 1)

NMDAR activation which resulted in a reciprocal change in bindings of ICAM-5 and

GluN1 to D-actinin respectively also led to an alteration in their colocalization the ICAM-5D-

actinin colocalization was decreased and that of GluN1D-actinin was increased in response to the

NMDA treatment Furthermore D-actinin formed bigger puncta which essentially overlapped with GluN1 but were devoid of ICAM-5 immunoreactivity (IV Fig 5)

By time-lapse imaging we found that D-actinin was highly dynamic in filopodia of

the WT neurons Upon the NMDA treatment randomly moving D-actinin rapidly shifted to either the distal or to the proximal part in filopodia longitudally Within 20 min of the NMDA application

D-actinin clusters were formed in the roots or the tips of filopodia resulting in D-actinin

punctuation In ICAM-5 -- neurons before the NMDA treatment D-actinin already exhibited a punctuated pattern in filopodiaimmature spines and appeared to be more stable in comparison to

that in the WT neurons The NMDA treatment failed to induce D-actinin clustering in ICAM-5 --

neurons indicating that NMDA-induced D-actinin clustering is dependent on ICAM-5 (IV Fig 6) Moreover as the major cytoskeletal component in spines F-actin was also analyzed in

parallel After the NMDA treatment F-actin became enriched in pearl-like puncta along dendritic

shafts and the colocalization of F-actin to D-actinin was dramatically increased ICAM-5 --

neurons exhibited bigger F-actin puncta and better colocalization between F-actin and D-actinin In contrast to the WT neurons ICAM-5 -- neurons showed little response to the NMDA treatment in

terms of F-actin distribution and colocalization with D-actinin (IV Fig 7)

These results suggest that regulation of ICAM-5D-actinin GluN1 binding plays an

important role in the D-actinin distribution in spines and thereby mediates spine morphogenesis through the F-actin reorganization

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DISCUSSION

ICAM-5 is a telencephalic neuron-specific molecule sharing high homology with the previously described ICAM-molecules (Yoshihara and Mori 1994 Gahmberg 1997) Like the other ICAMs ICAM-5 serves as a ligand for the ȕ2 integrin LFA-1 expressed on peripheral blood leukocytes and microglia and regulates immune responses in the brain (Mizuno et al 1997 Tian et al 1997 Mizuno et al 1999 Tian et al 2000 Tian et al 2008) The expression of ICAM-5 temporally parallels the period of dendritic outgrowth and synaptogenesis and ICAM-5 has been functionally implicated in these processes (Imamura et al 1990 Yoshihara et al 1994 Tian et al 2000 Matsuno et al 2006) ICAM-5 is the only CAM shown to act as a negative regulator of spine maturation Genetic ablation of ICAM-5 results in increased LTP improved memory and learning function and enhanced neural circuit plasticity (Nakamura et al 2001 Barkat et al 2011) Therefore it is of paramount importance to understand the underlying mechanisms of ICAM-5 functions

1 ȕ1 integrins the pre-synaptic partners of ICAM-5 In this study ȕ1 integrins a major integrin subfamily expressed in neurons in multiple brain regions were identified as the pre-synaptic receptors for ICAM-5 Interaction between ȕ1 integrins

and ICAM-5 was initially observed by co-immunoprecipitation (III) D3 D5 and D8 integrin subunits are located in synapses and have their distinctive functions in synapse formation and

plasticity Herein we found that D5ȕ1 but not D3 and D8 integrins was the predominant binding partner for ICAM-5 Using purified proteins we confirmed that ICAM-5 and ȕ1 integrins bind directly to each other through the extracellular domains

ICAM-5 promoted the ȕ1-integrin-mediated cell adhesion suggesting that the interaction occurs in trans ICAM-5 extracellular portion D1-2 was efficient for ȕ1-integrin-mediated cell adhesion suggesting that the binding sites are located in the first two Ig domains of ICAM-5 (III) Unexpectedly the whole ectodomain of ICAM-5 was not as an efficient binder as D1-2 The lower binding capacity is likely due to the intra-molecular interactions that mask the binding sites for ȕ1 integrins A similar phenomenon was seen for ICAM-5 binding to LFA-1 (Tian et al 2000)

Many studies have revealed that ȕ1 integrins are expressed in spines but the pre-synaptic distribution of them has previously not been well documented Employing different methods including immunofluorescence in combination with confocal microscopy immuno-EM and synaptosome fractionation we confirmed that ȕ1 integrins are also present at pre-synaptic terminals (III) In line with our results Hellwig et al (2011) also reported the existence of pre-synaptic ȕ1 integrins by EM It is possible that ȕ1 integrins have different roles in pre- and post-synaptic sites during synaptogenesis In nascent synapses the pre-synaptic ȕ1 integrins are likely more dominant and serve as counter-receptors for ICAM-5 The interaction between ICAM-5 and ȕ1 integrins is apparently more important at the early stage of synaptogenesis since they are tightly opposed to each other in the tips of filopodia or the heads of thin spines

The role of the ICAM-5 ȕ1 integrin interaction is to fine-tune the ectodomain cleavage of ICAM-5 Inhibition of this interaction by blocking antibodies against ICAM-5 or ȕ1integrins promoted the cleavage of membrane-bound ICAM-5 and the release of sICAM-5 By contrast activating antibodies against ȕ1 integrins attenuated the generation of sICAM-5 (III) ȕ1integrins bound to ICAM-5 within the first two Ig domains but the preferred MMP cleavage site in ICAM-5 was located close to the 9th Ig domain (II III) Possibly instead of masking the cleavage site of MMPs by physical binding ȕ1 integrins could regulate the conformation of ICAM-5 Binding to ȕ1 integrins ICAM-5 may adopt a tightly packed extracellular structure which makes the cleavage sites less accessible to MMPs Interestingly addition of the ICAM-5 D1-2 protein also decreased ICAM-5 cleavage This is likely due to (1) the homophilic binding between D1-2 and membrane-bound ICAM-5 which also protects ICAM-5 from MMPs or (2) increased the avidity of pre-synaptic ȕ1 integrins induced by ICAM-5 D1-2 binding which further enhances the trans-synaptic ICAM-5ȕ1 integrin interaction ICAM-5 ectodomain cleavage is accompanied by

48

morphological changes of spines Inhibition of the interaction by antibodies or axonal ȕ1 integrin shRNA shifted the filopodia-to-spine axis toward the more mature spiny structures whereas enhancing the interaction by activating antibodies reversed the morphological change toward the more immature status Therefore binding of pre-synaptic ȕ1 integrins to ICAM-5 provides a protective mechanism against ICAM-5 cleavage Dissociation of ICAM-5 from ȕ1 integrins facilitates ICAM-5 cleavage which impacts spine maturation

Maintaining the flexibility of spines is of particular importance for developing synapses The initial synaptic contacts may be inaccurate The mismatched synaptic contacts need to be discarded and only the correct ones are stabilized by further signaling It is widely accepted that dynamic spines are more plastic and prone to be re-shaped While many other molecules act as inducers for synapse formation the ICAM-5ȕ1 integrin interaction buffers the maturation process providing sufficient time for neurons to distinguish the ldquorightrdquo synapses from the ldquowrongrdquo ones and preventing over-stabilization of mismatched synapses In mature spines ȕ1 integrins remain bound to the post-synaptic membrane and cover the whole spines while ICAM-5 is excluded At this stage other post-synaptic molecules may bind to ȕ1integrins after ICAM-5 is cleaved Previous studies have shown that ȕ1 integrins interact with many CAMs or ECM proteins such as NCAM L1 Eph RTKsephrins cadherin-related neuronal receptor 1 (CNR1) and reelin and are involved in signaling pathways related to these proteins (Dulabon et al 2000 Mutoh et al 2004 Diestel et al 2005 Bourgin et al 2007) Different CAMs have their distinctive functions at specific stages of synapse formation When one has accomplished its role it will be withdrawn and other CAMs will replace it

2 Removal of ICAM-5 by MMPs promotes spine maturation Detailed studies on ICAM-5 distribution at spines revealed that ICAM-5 is abundantly expressed in filopodia and immature spines but weakly expressed in mature mushroom spine heads (Matsuno et al 2006) This phenomenon is consistent with the discovery that ICAM-5 immunoreactivity labels post-synaptic membranes but avoids post-synaptic density (Sakurai et al 1998) implying that there is a mechanism by which ICAM-5 is gradually removed from the synapses during the maturation of spines

In this study we have shown that ICAM-5 is a substrate of MMP-2 and -9 In vitrorecombinant mouse ICAM-5 was cleaved both by active MMP-2 and -9 (II) It is known that the expression of MMPs is up-regulated by activation of glutamate receptors in a transcription factor-dependent pathway In vivo application of NMDA and AMPA increased the levels of cleaved ICAM-5 at steady state We found that the production of cleaved ICAM-5 induced by the NMDA treatment was blocked by NMDAR antagonists MMP inhibitors and MMP RNAi Moreover in MMP-2 and -9 -- mice levels of ICAM-5 are abnormally high in neonates supporting the idea that ICAM-5 is subject to MMP-mediated proteolytic processing (II)

Accompanied by ICAM-5 cleavage the elevation of MMPs induced a reduction in the immunoreactivity of ICAM-5 in spine heads and dendrites This MMP-dependent ICAM-5 cleavage promoted spine head enlargement and MMP inhibitors blocked the cleavage of ICAM-5 and spine enlargement (II) Later Conant et al (2010) found that tetanic stimulation of hippocampal slices a classic protocol to induce LTP also promotes the MMP-dependent ICAM-5 cleavage with increased synaptic strength Therefore MMP-induced ICAM-5 cleavage not only promotes morphological changes of spines but also their functional maturation

An increasing amount of evidences support that MMPs are also implicated in spine maturation and synaptic plasticity under physiological conditions Researchers have started to draw their attention to the relationship between MMP function and neuronal activity Expression secretion and activation of MMPs can be induced by neuronal activity and during memory and learning processes (Meighan et al 2006 Nagy et al 2006) Reciprocally MMPs in turn regulate spine morphology and synaptic plasticity by controlling glutamate receptor trafficking and functions (Michaluk et al 2009) The interplay between ȕ1 integrins MMP and NMDARs may jointly contribute to the fine-tuning of ICAM-5 cleavage at synapses MMPs described as molecular scissors sheer ectodomains of adhesion molecules remove unnecessary adhesions and keep cell-cell and cell-matrix adhesion at the desired level Interaction of ICAM-5 to its counter-

49

receptor ȕ1 integrins is important to prevent its cleavage by MMP and the transition of filopodia into mature spines Once neuronal activity or other signals activate MMPs overriding the ȕ1integrin-mediated protection ICAM-5 is cleaved and the inhibition is abolished Thereafter filopodia are free for further development

Under pathological conditions such as brain trauma inflammation and infection MMP activities are excessively elevated which likely contribute to neuronal and synaptic injury (Agrawal et al 2008) Interruption of MMP functions leads to abnormal cleavage of cell surface proteins which contribute to a variety of neurological diseases For instance in patients with acute encephalitis MS or epilepsy the levels of sICAM-5 are elevated and may be caused by dysfunction of MMPs A recent discovery shows that a highly addictive psychostimulant methamphetamine also induces ICAM-5 cleavage through activation of MMP-3 and -9 (Conant et al 2011) providing a new perspective on the function of ICAM-5 in the brain

The subsequent signaling events of ICAM-5 cleavage leading to spine morphogenesis are not completely understood Nevertheless release of sICAM-5 seems play multiple roles in the development of neurons Our previous studies indicated that ICAM-5 promotes dendritic elongation through homophilic binding with counter dendrites in the early life of neurons (Tian et al 2000) Herein we found that homophilic binding between released sICAM-5 and membrane-bound ICAM-5 promotes filopodial formation by increasing the number and length of spines (II) This data expands our knowledge of sICAM-5 in the context of NMDAR-dependent spine maturation In agreement blockade of ionotropic glutamate receptors leads to a decrease in the density and turnover of dendritic filopodia whereas activation of them results in an increase in the length of filopodia (Portera-Cailliau et al 2003) Alternatively sICAM-5 was found by others to bind to post-synaptic ȕ1 integrins which consequently induce cofilin phosphorylation (Conant et al 2011) Cofilin promotes spine growth by facilitating actin polymerization (Hotulainen et al 2009) Due to heterogeneity of spine morphogenesis during maturation it is possible that sICAM-5 promotes filopodial elongation and spine enlargement at the same time through different binding partners in different subsets of spines However the mechanisms by which sICAM-5 distinguishes the post-synaptic ICAM-5 from ȕ1 integrins remain to be investigated Another putative pathway may lie in the dynamic interaction between ICAM-5 and the actin cytoskeletal adaptor proteins We found that NMDA treatment leads to ICAM-5 dissociation from the actin cytoskeleton When actin polymerization was blocked the NMDA-induced ICAM-5

cleavage was enhanced Moreover Paju-ICAM-5-cp cells lacking the cytoplasmic domain exhibited a higher level of ICAM-5 cleavage in comparison to Paju-ICAM-5-full-length (II) These data imply that lacking the cytoplasmic anchorage to the actin cytoskeleton renders ICAM-5 susceptible to cleavage Reciprocally shedding of the ectodomain may change the cytoplasmic interaction with the actin cytoskeleton In agreement earlier studies have shown that both the extracellular and intracellular domains of ICAM-5 are required for the maintenance of filopodia (Matsuno et al 2006) In sum in depth studies focusing on the cytoplasmic region of ICAM-5 in actin dynamics are required to further understand the role of ICAM-5

3 From extracellular to intracellular signaling

D-Actinin is an actin crosslinking protein that binds to the cytoplasmic regions of ICAM-1 and -2

(Carpen et al 1992b Heiska et al 1996) In neurons D-actinin is concentrated in spines and regulates spine morphogenesis (Wyszynski et al 1998 Nakagawa et al 2004) These properties

make D-actinin an ideal candidate for ICAM-5 cytoplasmic binding

The binding site to D-actinin is located to the sequence between amino acids 857-861 (ICAM-5857-861 KKGEY) at the membrane proximal part of the ICAM-5 cytoplasmic tail (I) The

two Lys are crucial for binding to D-actinin When replaced by Ala binding of the peptide to D-actinin was abolished Interestingly when replaced by arginine (Arg R) the mutated peptides ICAM-5-K857R or ICAM-5-K858R exhibited weak binding suggesting that the positive charge of Arg somehow favors the binding even though not enough for good binding (I) The crystal

structure of D-actinin reveals the highly acidic property of the rod domain which constitutes the major binding sites for many transmembrane proteins In parallel positively charged amino acids

50

were found in the D-actinin binding sites of many transmembrane proteins such as ICAM-1

(RKIKK) ICAM-2 (VRAAWRRL) E1-integrin (FAKFEKEKMN) and E2-integrin (RRFEKEKLKSQ) (Carpen et al 1992a Tang et al 2001 Ylanne et al 2001 Otey and Carpen

2004) The opposite charges found on the D-actinin rod domain and its binding partners suggest

that electrostatic forces-mediated interactions Through interaction with D-actinin ICAM-5 becomes linked to actin filaments

NMDARs interact with D-actinin through the subunits GluN1 and GluN2B (Wyszynski et al 1997) Taken together based on the regulation of NMDAR activity on ICAM-5 cleavage and its dissociation from actin filaments it is plausible that NMDARs may directly

compete with ICAM-5 for interaction with D-actinin thereby affecting ICAM-5 cytoplasmic linkage

We have characterized here the binding sites in D-actinin for ICAM-5 and GluN1

respectively We found that ICAM-5 and GluN1 competed for binding to D-actinin in the R2 region of the rod domain ICAM-5 had a somewhat higher affinity for R2 at resting state When

NMDARs were activated binding between GluN1 and D-actinin was increased and that between

ICAM-5 and D-actinin was decreased (IV)As a dendrite-specific CAM ICAM-5 mediates dendrite-dendrite contact through

homophilic binding which promotes dendritic elongation and arborization (Tian et al 2000) We

have observed here that ICAM-5 colocalizes with D-actinin and F-actin in Paju cells at the cell-cell contact sites and the growth cones of neurites When the cytoplasmic domain of ICAM-5 was lacking or outcompeted by a peptide KKGEY the colocalization was abolished and the neurite outgrowth was inhibited (I) Dendritic outgrowth is a specific process constituted of a series of cell adhesionmigration events and actin filaments are central in controlling the morphology and movement of migrating cells Therefore the cytoplasmic interaction of ICAM-5 with the actin

cytoskeleton through D-actinin may contribute to ICAM-5-mediated neurite outgrowth during early neuronal differentiation NMDAR is likely not a major regulator during this process since its expression in young neurons is weak

A possible role of ICAM-5 in filopodia and immature spines is to maintain the dynamics of Į-actinin and prevent Į-actinin clustering This interpretation is supported by 1) time-lapse imaging showing that ICAM-5 -- neurons contained relatively stable clustered Į-actinin whereas in WT neurons Į-actinin was highly mobile and the filopodia showed increased flexibility 2) Į-actinin tended to form puncta in neuronal areas devoid of ICAM-5 immunoreactivity including mature spines or spines of ICAM-5 -- neurons 3) activation of NMDARs which decreased ICAM-5 Į-actinin binding led to Į-actinin clustering and stabilization of filopodia and immature spines

Using live-cell imaging we observed that upon NMDAR activation Į-actinin rapidly moved toward the heads and roots of filopodia and became concentrated in these areas The spines then became less mobile and enlarged in Į-actinin clustered areas Hotulainen et al (2009) earlier reported that the head and root of filopodia are the major sites for actin polymerization During spine maturation actin-binding proteins such as Arp23 facilitate actin branching and therefore promote spine head enlargement Certainly a stable actin filament network requires bundling activity from actin cross-linking proteins It is possible that an increased local concentration of Į-actinin facilitates actin filament bundling and contributes to fixation of actin filaments resulting in more stable mature spines In agreement with this we found that F-actin co-clustered with Į-actinin in response to NMDAR activation The assembly of the actin cytoskeleton is intimately associated with synaptic efficacy and memory functions (De Roo et al 2008 Bosch and Hayashi 2012) Spine maturation includes stabilization of the actin cytoskeleton and increased growth and complexity of the spinal actin network resulting in increased spine volume better AMPAR accommodation and enhanced synaptic transmission capacity Therefore the altered Į-actinin dynamics and stabilization of the actin cytoskeleton in ICAM-5 -- neurons may contribute to the improved memory and higher LTP in ICAM-5 -- mice

The direct trigger of Į-actinin clustering is still unknown but neuronal activity seems to play a pivotal role We here showed that ICAM-5 and GluN1 both bound to Į-actinin and

51

competed for the same binding region ICAM-5 had a slightly higher binding affinity NMDAR activation led to the dissociation of Į-actinin from ICAM-5 and its association with GluN1 (IV) Once ICAM-5 is dissociated Į-actinin is free and starts to cluster under the control of other regulatory molecules Į-Actinin clustering may directly be due to the physical interaction with GluN1 as a result of NMDAR trafficking to the PSD and enrichment in spine heads during spine maturation Another possibility is that NMDAR activation induces signaling pathways that regulate Į-actinin dynamics Several studies have shown that activation of glutamate receptors altered the activity or distribution of actin binding proteins which in turn regulate the actin dynamics underlying spine morphology and synaptic function (Star et al 2002 Sekino et al 2006 Takahashi et al 2009 Pontrello et al 2012)

Another ABP the ERM family protein ezrin was also shown to interact with ICAM-5

and the binding sequence partially overlaps with that of D-actinin (Furutani et al 2007) Activation of ERM is regulated by post-translational modification The active C-terminal Thr phosphorylated ERM colocalizes with ICAM-5 in filopodia which promotes filopodia elongation Recently it was found that VN binds to the second Ig domain of ICAM-5 (Furutani et al 2012) Interestingly VN-deficient mice exhibit reduced dendritic spine density (Bell et al 2010) which resembles the phenotype of ICAM-5 -- mice Binding to VN induces ICAM-5 binding to ERM and phosphorylation of ezrin and promotes filopodial outgrowth (Furutani et al 2012) Thus it is plausible that the ICAM-5 interaction with the cytoskeleton in filopodia is complex and involves several components the regulatory elements yet to be identified

Even though ICAM-5 is gradually excluded during spine maturation what happens to the ICAM-5 fragment after the ectodomain shedding remains unclear Interestingly a recent study shows that ADP-ribosylation factor 6 (ARF6)-dependent endocytosis is responsible for ICAM-5 removal during spine maturation ARF6 activation triggered by EFA6A a guanine nucleotide exchange factor leads to ERM protein dephosphorylation and dissociation from ICAM-5 which further internalizes ICAM-5 Once endocytosed ICAM-5-containing vesicles are either recycled back to the dendritic surface or are targeted for lysosomal degradation (Raemaekers et al 2012) It is not surprising that transmembrane proteins are prone to endocytosis or degradation when they lose anchorage to the actin cytoskeleton Therefore cleaved ICAM-5 may be removed in this way

4 ICAM-5 a multi-faceted player in the brain Since the discovery of ICAM-5 numerous binding partners for ICAM-5 have been identified and ICAM-5-regulated signaling pathways underlying distinctive functions of ICAM-5 start to emerge As neuron-specific CAM ICAM-5 regulates neuronal development and immune response in the brain Excitingly ICAM-5 may be a double facet player that links the CNS to the immune system and modulates the interplay between them (Gahmberg et al 2008)

In During dendritic outgrowth ICAM-5 exists as monomers exhibits homophilic binding through the extracellular domain and mediates dendritic branching and elongation The anchorage of ICAM-5 cytoplasmic tail to Į-actinin is likely a downstream signaling event of the homophilic binding which is implicated in dendritic outgrowth During synaptic formation ICAM-5 from the filopodial membrane binds to the pre-synaptic ȕ1 integrins since the initial synaptic contact formed This interaction prevents spine maturation by restricting ICAM-5 ectodomain cleavage NMDAR activation induces ICAM-5 cleavage accompanied by Į-actininbinding to GluN1 Į-actinin clustering and actin cytoskeleton reorganization which further promote spine maturation

On the other hand ICAM-5 binds to integrin LFA-1 enabling the direct binding between neurons and infiltrating leukocytes Neuron-derived sICAM-5 primarily due to MMP cleavage was shown to regulate T-cell activation which in turn promotes further production of sICAM-5 through elevated MMP activity Moreover the recombinant ICAM-5 ectodomain protein induces a morphological change in microglia (Mizuno et al 1999) Our preliminary data indicate that ICAM-5 directly binds to microglia even though the counter receptor has not been identified

The CNS and the immune system were used to be recognized as relatively independent systems and the immune responses in the brain are neurotoxic which clear up invaded antigens at the price of neuronal injury To date accumulating studies have robustly shown

52

that the immune system can actively regulate neurogenesis neural circuit formation and plasticity and higher brain functions (Yirmiya and Goshen 2011) Synapse formation thereby is no longer solely modulated by neuron-neuron contacts but also requires the reciprocal interactions between neurons and the immune cells Microglia in addition to their canonical role of surveying the microenvironment in the brain and taking up cell debris are now thought to be active regulators in synaptic pruning and plasticity although the underlying mechanisms remain to be uncovered (Tremblay 2011 Bechade et al 2013 Kettenmann et al 2013) The binding capacity of ICAM-5 to heterogeneous counter receptors makes it an ideal mediator between the two systems We can assume that under different physiological environments ICAM-5 may have alternative binding partners either from neurons or from immune cells By binding to ȕ1 integrins from the axonal terminals of neurons ICAM-5 is implicated in synaptogenesis and negatively regulates spine maturation By binding to microglia which can engulf and destruct synapses ICAM-5 may mediate synaptic plasticity and elimination of synapses In the future it would be interesting to explore how ICAM-5 distinguishes between different binding partners from heterogeneous cells and whether there is a competition between these binding partners for ICAM-5 under different physiological conditions

Figure 10 A schematic model depicting the molecular mechanisms by which ICAM-5 regulates neuronal synapse formation (Left) In immature neurons ICAM-5 from filopodia binds to pre-synaptic E1 integrins through the extracellular domain and its cytoplasmic tail binds to D-actinin At this stage filopodia are flexible and D-actinin remains highly motile E1 integrins are also present at the post-synaptic sites For a clear illustration E1 integrins are only shown in one of three filopodia (Right) Induced by NMDAR activation MMPs initiate ICAM-5 cleavage generating a large sICAM-5 and a small membrane-remaining ICAM-5 fragment The remaining ICAM-5 loses the binding capacity to D-actinin In replace of ICAM-5 NMDAR binds to D-actinin resulting in D-actinin clustering and actin cytoskeleton reorganization which further promotes spine enlargement The sICAM-5 binds to the full-length membrane-bound ICAM-5 from neighboring filopodia and facilitates filopodia formation through homophilic binding On the other hand the sICAM-5 binds to post-synaptic E1 integrins initiating cofilin phosphorylation and promoting spine enlargement In mature spines pre-synaptic E1 integrins still bind to the post-synaptic structures likely through the interaction with other CAMs

53

CONCLUDING REMARKS

As a negative regulator of synaptogenesis ICAM-5 maintains filopodial formation and prevents spine maturation Therefore the removal of ICAM-5 is a pivotal event that initiates the filopodia-to-spine transition ICAM-5 elimination may be achieved by ARF6-mediated endocytosis from spine heads which would promote spine maturation In this study we have defined a novel alternative mechanism of ICAM-5 removal by cleaving the ectodomain of ICAM-5 Stimulation of MMP-2 and -9 activities by NMDAR activation induces ICAM-5 cleavage resulting in (1) loss of ICAM-5 extracellular interaction with

the pre-synaptic E1 integrins and (2) ablation of ICAM-5 cytoplasmic linkage to D-actinin

which further triggers actin cytoskeleton reorganization through D-actinin clustering Therefore ICAM-5 regulates spine maturation at multiple levels This study has broadened our view of the mechanism by which ICAM-5 regulates spine maturation and helps to understand the ICAM-5-dependent higher brain functions at the cellular and molecular level

54

ACKNOWLEDGEMENTS

This study was carried out in Division of Biochemistry and Biotechnology Faculty of Biological and Environmental Sciences University of Helsinki during year 2007-2013 under the supervision of Professor Carl G Gahmberg and Docent Li Tian The work has been financially supported by the Helsinki Graduate Program in Biotechnology and Molecular Biology the Magnus Ehrnrooth foundation the Finnish Society of Science and Letters and the Academy of Finland

I want to express my deepest gratitude to my professor Carl who not only provided me with the excellent facilities but also gave me the patience support trust and freedom throughout my study His wealth of knowledge and his enthusiasm for science remind me what a real scientist is

I am heartfeltly thankful to Tian Without her I wouldnrsquot have come across the magic world of neuroscience Since guiding me doing the first experiment here she has given me the wholehearted help and brought me inspirations and ideas in every fruitful discussion Thank you for everything Tian my tutor my friend and my role model

I thank Docents Pia Siljander and Sari Lauri for their tuition and following up my work especially Pia who always challenged and encouraged me Sari again and Professor Jorma keski-Oja are acknowledged for their careful reviewing of my thesis and the constructive comments

I am grateful to the research groups led by Drs Claudio Rivera Eija Jokitalo Heikki Rauvala and Ronald Davis for their cooperation in my work especially to Claudio for his consistent guidance and intelligent input to my work and to Olaya for her help and friendship Besides Mika and Kimmo from the Light Microscope Unit have been very helpful for my imaging work

I have been lucky enough to work in a friendly and open environment I want to thank all the present and previous members in Carlrsquos lab and in the Division in particular Liisa Hetti Maria A Emiliano Suneeta Maria P Sarah Tuomas Pirjo Yvonne and Katarina for the help and friendship over these years I want to thank Esa who generously shared with me his knowledge on cloning protein purification and quality coffee and inspired me with his precision attitude Irsquove learnt a lot from you my friend I wouldnrsquot forget to thank my girls Farhana Sonja Tahira and Daniela the ldquohi hi-ha hardquo we had in and outside of the lab is an important part of memory for me I owe many thanks to Miku for all the stimulating discussions and her invaluable feedback on my thesis Importantly this dissertation is impossible without the excellent and persistent technical assistance from Leena Seija and Outi Thank you very much

My warmest gratitude goes to my beautiful friends Yurui Madonna and Elina I am blessed to have you always being there for me at the good moments and the bad moments

I wish to devote a special ldquothank yourdquo to Minh Dang who is greatly influential to me Without his encouragement and help I would never have become who I am

Last but not least I couldnrsquot say enough about my lovely parents It was their endless unconditional love that carried me all the way here 㹋㹋ᖗᛳ䇶៥ⱘཛྷཛྷǄ

Helsinki 8th August 2013

55

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56

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57

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Seki T amp Rutishauser U 1998 Removal of polysialic acid-neural cell adhesion molecule induces aberrant mossy fiber innervation and ectopic synaptogenesis in the hippocampus The Journal of Neuroscience The Official Journal of the Society for Neuroscience 18 3757-3766 Sekino Y Tanaka S Hanamura K Yamazaki H Sasagawa Y Xue Y Hayashi K amp Shirao T 2006 Activation of N-methyl-D-aspartate receptor induces a shift of drebrin distribution disappearance from dendritic spines and appearance in dendritic shafts Molecular and Cellular Neurosciences 31 493-504 Senkov O Sun M Weinhold B Gerardy-Schahn R Schachner M amp Dityatev A 2006 Polysialylated neural cell adhesion molecule is involved in induction of long-term potentiation and memory acquisition and consolidation in a fear-conditioning paradigm The Journal of Neuroscience The Official Journal of the Society for Neuroscience 26 10888-109898 Shen K amp Meyer T 1999 Dynamic control of CaMKII translocation and localization in hippocampal neurons by NMDA receptor stimulation Science (New York NY) 284 162-166 Shen K Teruel MN Connor JH Shenolikar S amp Meyer T 2000 Molecular memory by reversible translocation of calciumcalmodulin-dependent protein kinase II Nature Neuroscience 3 881-886Sheterline P Clayton J amp Sparrow J 1995 Actin Protein Profile 2 1-103 Shi SH Hayashi Y Petralia RS Zaman SH Wenthold RJ Svoboda K amp Malinow R 1999 Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation Science (New York NY) 284 1811-1816 Shi Y amp Ethell IM 2006 Integrins control dendritic spine plasticity in hippocampal neurons through NMDA receptor and Ca2+calmodulin-dependent protein kinase II-mediated actin reorganization The Journal of Neuroscience The Official Journal of the Society for Neuroscience 26 1813-1822 Shibasaki F Fukami K Fukui Y amp Takenawa T 1994 Phosphatidylinositol 3-kinase binds to alpha-actinin through the p85 subunit The Biochemical Journal 302 ( Pt 2) 551-557 Shimoyama Y Tsujimoto G Kitajima M amp Natori M 2000 Identification of three human type-II classic cadherins and frequent heterophilic interactions between different subclasses of type-II classic cadherins The Biochemical Journal 349 159-167 Shirao T Hayashi K Ishikawa R Isa K Asada H Ikeda K amp Uyemura K 1994 Formation of thick curving bundles of actin by drebrin A expressed in fibroblasts Experimental Cell Research 215 145-153 Siddiqui TJ Pancaroglu R Kang Y Rooyakkers A amp Craig AM 2010 LRRTMs and neuroligins bind neurexins with a differential code to cooperate in glutamate synapse development The Journal of Neuroscience The Official Journal of the Society for Neuroscience 30 7495-7506 Sjoblom B Salmazo A amp Djinovic-Carugo K 2008 Alpha-actinin structure and regulation Cellular and Molecular Life Sciences CMLS 65 2688-2701 Soderling SH Guire ES Kaech S White J Zhang F Schutz K Langeberg LK Banker G Raber J amp Scott JD 2007 A WAVE-1 and WRP signaling complex regulates spine density synaptic plasticity and memory The Journal of Neuroscience The Official Journal of the Society for Neuroscience 27 355-365 Sorra KE amp Harris KM 2000 Overview on the structure composition function development and plasticity of hippocampal dendritic spines Hippocampus 10 501-511 Stafstrom-Davis CA Ouimet CC Feng J Allen PB Greengard P amp Houpt TA 2001 Impaired conditioned taste aversion learning in spinophilin knockout mice Learning amp Memory (Cold Spring Harbor NY) 8 272-278 Stagi M Fogel AI amp Biederer T 2010 SynCAM 1 participates in axo-dendritic contact assembly and shapes neuronal growth cones Proceedings of the National Academy of Sciences of the United States of America 107 7568-7573 Stanley P Smith A McDowall A Nicol A Zicha D amp Hogg N 2008 Intermediate-affinity LFA-1 binds alpha-actinin-1 to control migration at the leading edge of the T cell The EMBO Journal 27 62-75 Star EN Kwiatkowski DJ amp Murthy VN 2002 Rapid turnover of actin in dendritic spines and its regulation by activity Nature Neuroscience 5 239-246

71

Staubli U Chun D amp Lynch G 1998 Time-dependent reversal of long-term potentiation by an integrin antagonist The Journal of Neuroscience The Official Journal of the Society for Neuroscience 18 3460-3469 Sucher NJ Akbarian S Chi CL Leclerc CL Awobuluyi M Deitcher DL Wu MK Yuan JP Jones EG amp Lipton SA 1995 Developmental and regional expression pattern of a novel NMDA receptor-like subunit (NMDAR-L) in the rodent brain The Journal of Neuroscience The Official Journal of the Society for Neuroscience 15 6509-6520 Sudhof TC 2012 The presynaptic active zone Neuron 75 11-25 Sugino H Yoshihara Y Copeland NG Gilbert DJ Jenkins NA amp Mori K 1997 Genomic organization and chromosomal localization of the mouse telencephalin gene a neuronal member of the ICAM family Genomics 43 209-215 Sun HQ Yamamoto M Mejillano M amp Yin HL 1999 Gelsolin a multifunctional actin regulatory protein The Journal of Biological Chemistry 274 33179-33182 Sweatt JD 2004 Mitogen-activated protein kinases in synaptic plasticity and memory Current Opinion in Neurobiology 14 311-317 Tada T amp Sheng M 2006 Molecular mechanisms of dendritic spine morphogenesis Current Opinion in Neurobiology 16 95-101 Tadokoro S Nakazawa T Kamae T Kiyomizu K Kashiwagi H Honda S Kanakura Y amp Tomiyama Y 2011 A potential role for alpha-actinin in inside-out alphaIIbbeta3 signaling Blood117 250-258 Takahashi H Sekino Y Tanaka S Mizui T Kishi S amp Shirao T 2003 Drebrin-dependent actin clustering in dendritic filopodia governs synaptic targeting of postsynaptic density-95 and dendritic spine morphogenesis The Journal of Neuroscience The Official Journal of the Society for Neuroscience 23 6586-6595 Takahashi H Yamazaki H Hanamura K Sekino Y amp Shirao T 2009 Activity of the AMPA receptor regulates drebrin stabilization in dendritic spine morphogenesis Journal of Cell Science 122 1211-1219 Takeichi M 2007 The cadherin superfamily in neuronal connections and interactions Nature ReviewsNeuroscience 8 11-20 Tamada A Yoshihara Y amp Mori K 1998 Dendrite-associated cell adhesion molecule telencephalin promotes neurite outgrowth in mouse embryo Neuroscience Letters 240 163-166 Tang J Taylor DW amp Taylor KA 2001 The three-dimensional structure of alpha-actinin obtained by cryoelectron microscopy suggests a model for Ca(2+)-dependent actin binding Journal of Molecular Biology 310 845-858 te Velthuis AJ Ott EB Marques IJ amp Bagowski CP 2007 Gene expression patterns of the ALP family during zebrafish development Gene Expression Patterns GEP 7 297-305 Thomas LA Akins MR amp Biederer T 2008 Expression and adhesion profiles of SynCAM molecules indicate distinct neuronal functions The Journal of Comparative Neurology 510 47-67 Tian L Kilgannon P Yoshihara Y Mori K Gallatin WM Carpen O amp Gahmberg CG 2000 Binding of T lymphocytes to hippocampal neurons through ICAM-5 (telencephalin) and characterization of its interaction with the leukocyte integrin CD11aCD18 European Journal of Immunology 30 810-818 Tian L Lappalainen J Autero M Hanninen S Rauvala H amp Gahmberg CG 2008 Shedded neuronal ICAM-5 suppresses T-cell activation Blood 111 3615-3625 Tian L Nyman H Kilgannon P Yoshihara Y Mori K Andersson LC Kaukinen S Rauvala H Gallatin WM amp Gahmberg CG 2000 Intercellular adhesion molecule-5 induces dendritic outgrowth by homophilic adhesion The Journal of Cell Biology 150 243-252 Tian L Rauvala H amp Gahmberg CG 2009 Neuronal regulation of immune responses in the central nervous system Trends in Immunology 30 91-99 Tian L Yoshihara Y Mizuno T Mori K amp Gahmberg CG 1997 The neuronal glycoprotein telencephalin is a cellular ligand for the CD11aCD18 leukocyte integrin Journal of Immunology (Baltimore Md 1950) 158 928-936 Togashi H Abe K Mizoguchi A Takaoka K Chisaka O amp Takeichi M 2002 Cadherin regulates dendritic spine morphogenesis Neuron 35 77-89

72

Tolias KF Bikoff JB Kane CG Tolias CS Hu L amp Greenberg ME 2007 The Rac1 guanine nucleotide exchange factor Tiam1 mediates EphB receptor-dependent dendritic spine development Proceedings of the National Academy of Sciences of the United States of America 104 7265-7270 Toni N Buchs PA Nikonenko I Bron CR amp Muller D 1999 LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite Nature 402 421-425 Torres R Firestein BL Dong H Staudinger J Olson EN Huganir RL Bredt DS Gale NW amp Yancopoulos GD 1998 PDZ proteins bind cluster and synaptically colocalize with Eph receptors and their ephrin ligands Neuron 21 1453-1463 Traynelis SF Wollmuth LP McBain CJ Menniti FS Vance KM Ogden KK Hansen KB Yuan H Myers SJ amp Dingledine R 2010 Glutamate receptor ion channels structure regulation and function Pharmacological Reviews 62 405-496 Tremblay ME 2011 The role of microglia at synapses in the healthy CNS novel insights from recent imaging studies Neuron Glia Biology 7 67-76 Tsukita S amp Yonemura S 1999 Cortical actin organization lessons from ERM (ezrinradixinmoesin) proteins The Journal of Biological Chemistry 274 34507-34510 Ullrich B Ushkaryov YA amp Sudhof TC 1995 Cartography of neurexins more than 1000 isoforms generated by alternative splicing and expressed in distinct subsets of neurons Neuron 14497-507Ushkaryov YA Petrenko AG Geppert M amp Sudhof TC 1992 Neurexins synaptic cell surface proteins related to the alpha-latrotoxin receptor and laminin Science (New York NY) 25750-56Ushkaryov YA amp Sudhof TC 1993 Neurexin III alpha extensive alternative splicing generates membrane-bound and soluble forms Proceedings of the National Academy of Sciences of the United States of America 90 6410-6414 Varoqueaux F Aramuni G Rawson RL Mohrmann R Missler M Gottmann K Zhang W Sudhof TC amp Brose N 2006 Neuroligins determine synapse maturation and function Neuron51 741-754 Vazquez LE Chen HJ Sokolova I Knuesel I amp Kennedy MB 2004 SynGAP regulates spine formation The Journal of Neuroscience The Official Journal of the Society for Neuroscience 24 8862-8872 Virel A amp Backman L 2004 Molecular evolution and structure of alpha-actinin Molecular Biology and Evolution 21 1024-1031 Wachsstock DH Schwartz WH amp Pollard TD 1993 Affinity of alpha-actinin for actin determines the structure and mechanical properties of actin filament gels Biophysical Journal 65205-214Waldeyer-Hartz W (1891) Ueber einige neuere Forschungen im Gebiete der Anatomie des Centranervensystems Deutsche medicinische Wochenschrift 171213-1218 1244-1216 1287-1219 1331-1212 and 1350-1216 Warren MS Bradley WD Gourley SL Lin YC Simpson MA Reichardt LF Greer CA Taylor JR amp Koleske AJ 2012 Integrin beta1 signals through Arg to regulate postnatal dendritic arborization synapse density and behavior The Journal of Neuroscience The Official Journal of the Society for Neuroscience 32 2824-2834 Washbourne P Bennett JE amp McAllister AK 2002 Rapid recruitment of NMDA receptor transport packets to nascent synapses Nature Neuroscience 5 751-759 Webb DJ Zhang H Majumdar D amp Horwitz AF 2007 Alpha5 Integrin Signaling Regulates the Formation of Spines and Synapses in Hippocampal Neurons The Journal of Biological Chemistry 282 6929-6935 Wee KS Zhang Y Khanna S amp Low CM 2008 Immunolocalization of NMDA receptor subunit NR3B in selected structures in the rat forebrain cerebellum and lumbar spinal cord The Journal of Comparative Neurology 509 118-135 Wegner AM Nebhan CA Hu L Majumdar D Meier KM Weaver AM amp Webb DJ 2008 N-wasp and the arp23 complex are critical regulators of actin in the development of dendritic spines and synapses The Journal of Biological Chemistry 283 15912-15920

73

Weinhold B Seidenfaden R Rockle I Muhlenhoff M Schertzinger F Conzelmann S Marth JD Gerardy-Schahn R amp Hildebrandt H 2005 Genetic ablation of polysialic acid causes severe neurodevelopmental defects rescued by deletion of the neural cell adhesion molecule The Journal of Biological Chemistry 280 42971-42977 Wong HK Liu XB Matos MF Chan SF Perez-Otano I Boysen M Cui J Nakanishi N Trimmer JS Jones EG Lipton SA amp Sucher NJ 2002 Temporal and regional expression of NMDA receptor subunit NR3A in the mammalian brain The Journal of Comparative Neurology 450303-317Wu G Malinow R amp Cline HT 1996 Maturation of a central glutamatergic synapse Science (New York NY) 274 972-976 Wyszynski M Kharazia V Shanghvi R Rao A Beggs AH Craig AM Weinberg R amp Sheng M 1998 Differential regional expression and ultrastructural localization of alpha-actinin-2 a putative NMDA receptor-anchoring protein in rat brain The Journal of Neuroscience The Official Journal of the Society for Neuroscience 18 1383-1392 Wyszynski M Lin J Rao A Nigh E Beggs AH Craig AM amp Sheng M 1997 Competitive binding of alpha-actinin and calmodulin to the NMDA receptor Nature 385 439-442 Yamaguchi Y amp Pasquale EB 2004 Eph receptors in the adult brain Current Opinion in Neurobiology 14 288-296 Yamaji S Suzuki A Kanamori H Mishima W Yoshimi R Takasaki H Takabayashi M Fujimaki K Fujisawa S Ohno S amp Ishigatsubo Y 2004 Affixin interacts with alpha-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction The Journal of Cell Biology 165 539-551 Yan Z Hsieh-Wilson L Feng J Tomizawa K Allen PB Fienberg AA Nairn AC amp Greengard P 1999 Protein phosphatase 1 modulation of neostriatal AMPA channels regulation by DARPP-32 and spinophilin Nature Neuroscience 2 13-17 Yang C Huang M DeBiasio J Pring M Joyce M Miki H Takenawa T amp Zigmond SH 2000 Profilin enhances Cdc42-induced nucleation of actin polymerization The Journal of Cell Biology 150 1001-1012 Yang N MacArthur DG Gulbin JP Hahn AG Beggs AH Easteal S amp North K 2003 ACTN3 genotype is associated with human elite athletic performance American Journal of Human Genetics 73 627-631 Yashiro K amp Philpot BD 2008 Regulation of NMDA receptor subunit expression and its implications for LTD LTP and metaplasticity Neuropharmacology 55 1081-1094 Yirmiya R amp Goshen I 2011 Immune modulation of learning memory neural plasticity and neurogenesis Brain Behavior and Immunity 25 181-213 Ylanne J Scheffzek K Young P amp Saraste M 2001 Crystal structure of the alpha-actinin rod reveals an extensive torsional twist Structure (London England 1993) 9 597-604 Yoon KJ Phelps DA Bush RA Remack JS Billups CA amp Khoury JD 2008 ICAM-2 expression mediates a membrane-actin link confers a nonmetastatic phenotype and reflects favorable tumor stage or histology in neuroblastoma PloS One 3 e3629 Yoshihara Y De Roo M amp Muller D 2009 Dendritic spine formation and stabilization Current Opinion in Neurobiology 19 146-153 Yoshihara Y amp Mori K 1994 Telencephalin a neuronal area code molecule Neuroscience Research 21 119-124 Yoshihara Y Oka S Nemoto Y Watanabe Y Nagata S Kagamiyama H amp Mori K 1994 An ICAM-related neuronal glycoprotein telencephalin with brain segment-specific expression Neuron 12 541-553 Young P Ferguson C Banuelos S amp Gautel M 1998 Molecular structure of the sarcomeric Z-disk two types of titin interactions lead to an asymmetrical sorting of alpha-actinin The EMBO Journal 17 1614-1624 Young P amp Gautel M 2000 The interaction of titin and alpha-actinin is controlled by a phospholipid-regulated intramolecular pseudoligand mechanism The EMBO Journal 19 6331-6340

74

Yuste R amp Bonhoeffer T 2001 Morphological changes in dendritic spines associated with long-term synaptic plasticity Annual Review of Neuroscience 24 1071-1089 Zhang H Casasnovas JM Jin M Liu JH Gahmberg CG Springer TA amp Wang JH 2008 An unusual allosteric mobility of the C-terminal helix of a high-affinity alphaL integrin I domain variant bound to ICAM-5 Molecular Cell 31 432-437 Zhang S Ehlers MD Bernhardt JP Su CT amp Huganir RL 1998 Calmodulin mediates calcium-dependent inactivation of N-methyl-D-aspartate receptors Neuron 21 443-453 Zhang W amp Benson DL 2001 Stages of synapse development defined by dependence on F-actin The Journal of Neuroscience The Official Journal of the Society for Neuroscience 21 5169-5181Zhong J Carrozza DP Williams K Pritchett DB amp Molinoff PB 1995 Expression of mRNAs encoding subunits of the NMDA receptor in developing rat brain Journal of Neurochemistry 64 531-539 Zhu JJ Qin Y Zhao M Van Aelst L amp Malinow R 2002 Ras and Rap control AMPA receptor trafficking during synaptic plasticity Cell 110 443-455 Zigmond SH 2004 Formin-induced nucleation of actin filaments Current Opinion in Cell Biology 16 99-105 Ziv NE amp Garner CC 2004 Cellular and molecular mechanisms of presynaptic assembly Nature ReviewsNeuroscience 5 385-399 Ziv NE amp Smith SJ 1996 Evidence for a role of dendritic filopodia in synaptogenesis and spine formation Neuron 17 91-102 Zoghbi HY 2003 Postnatal neurodevelopmental disorders meeting at the synapse Science (New York NY) 302 826-830

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LIN NING

Division of Biochemistry and Biotechnology Department of Biosciences

Faculty of Biological and Environmental Sciences And

Helsinki Graduate Program in Biotechnology and Molecular Biology University of Helsinki

Academic Dissertation

To be presented for public examination with the permission of the Faculty of Biological and Environmental Sciences University of Helsinki

In the Auditorium 1041 at Viikki Biocenter 2 Viikinkaari 5 Helsinki On September 13th 2013 at 12 orsquoclock noon

2








3





our workrdquo


1906

4


5

TABLE OF CONTENTS


ABBREVIATIONS 7

ABSTRACT 9






3 NMDA receptors 21




5 ICAM-5 34




RESULTS 42

DISCUSSION 47


ACKNOWLEDGEMENTS 54

REFERENCES 55

6



text












Submitted



7

ABBREVIATIONS







AMPAR AMPA receptor

AZ active zone


CaM calmodulin





DIV day in vitro












Ig immunoglobulin



kDa kilodalton

KO knockout










NMDAR NMDA receptor

8

NT neurotransmitter




PS presenilin

PSA polysialic acid






SR spectrin repeat

SV synaptic vesicle


TLN telencephalin

VN vitronectin

WT wild type

9

ABSTRACT




































developing spines



10











11



Synaptic clefts





12








13










14








15



Spine morphogenesis








16







17









18






19

Structure










20









21






22






23








24







25



Structure



Distribution



Functions



26



















27













28










29







30

Functions






31


Functions












Functions

32






33






ND













NCAM

NCAM










ND

Neuroligin-1



Neurogilin-1-2-3



ND ND




ND



ND



ND ND


ND ND

34




Ephrin-B2and -B3



ND


ND




5 ICAM-5




35










36

2012)







37

LFA-1 (CD11aCD18)



ȕ1 integrins




Presenilin (PS)


ERM proteins


Vitronectin (VN)



Į-Actinin

38










39





40

AIMS OF THE STUDY








41




Antibodies I-IV

Animals I-IV


Cell lines I-IV




ELISA I III IV


SDS-PAGE I-IV









Flow cytometry II

RNAi II III








42

RESULTS









43











44












45
























46





























47

DISCUSSION








48







49












50

















51









52



53

CONCLUDING REMARKS




54

ACKNOWLEDGEMENTS











55

REFERENCES


56


57


58


59


60


61


62


63


64


65


66


67


68


69


70


71


72


73


74




















Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

2








3





our workrdquo


1906

4


5

TABLE OF CONTENTS


ABBREVIATIONS 7

ABSTRACT 9






3 NMDA receptors 21




5 ICAM-5 34




RESULTS 42

DISCUSSION 47


ACKNOWLEDGEMENTS 54

REFERENCES 55

6



text












Submitted



7

ABBREVIATIONS







AMPAR AMPA receptor

AZ active zone


CaM calmodulin





DIV day in vitro












Ig immunoglobulin



kDa kilodalton

KO knockout










NMDAR NMDA receptor

8

NT neurotransmitter




PS presenilin

PSA polysialic acid






SR spectrin repeat

SV synaptic vesicle


TLN telencephalin

VN vitronectin

WT wild type

9

ABSTRACT




































developing spines



10











11



Synaptic clefts





12








13










14








15



Spine morphogenesis








16







17









18






19

Structure










20









21






22






23








24







25



Structure



Distribution



Functions



26



















27













28










29







30

Functions






31


Functions












Functions

32






33






ND













NCAM

NCAM










ND

Neuroligin-1



Neurogilin-1-2-3



ND ND




ND



ND



ND ND


ND ND

34




Ephrin-B2and -B3



ND


ND




5 ICAM-5




35










36

2012)







37

LFA-1 (CD11aCD18)



ȕ1 integrins




Presenilin (PS)


ERM proteins


Vitronectin (VN)



Į-Actinin

38










39





40

AIMS OF THE STUDY








41




Antibodies I-IV

Animals I-IV


Cell lines I-IV




ELISA I III IV


SDS-PAGE I-IV









Flow cytometry II

RNAi II III








42

RESULTS









43











44












45
























46





























47

DISCUSSION








48







49












50

















51









52



53

CONCLUDING REMARKS




54

ACKNOWLEDGEMENTS











55

REFERENCES


56


57


58


59


60


61


62


63


64


65


66


67


68


69


70


71


72


73


74




















Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

3





our workrdquo


1906

4


5

TABLE OF CONTENTS


ABBREVIATIONS 7

ABSTRACT 9






3 NMDA receptors 21




5 ICAM-5 34




RESULTS 42

DISCUSSION 47


ACKNOWLEDGEMENTS 54

REFERENCES 55

6



text












Submitted



7

ABBREVIATIONS







AMPAR AMPA receptor

AZ active zone


CaM calmodulin





DIV day in vitro












Ig immunoglobulin



kDa kilodalton

KO knockout










NMDAR NMDA receptor

8

NT neurotransmitter




PS presenilin

PSA polysialic acid






SR spectrin repeat

SV synaptic vesicle


TLN telencephalin

VN vitronectin

WT wild type

9

ABSTRACT




































developing spines



10











11



Synaptic clefts





12








13










14








15



Spine morphogenesis








16







17









18






19

Structure










20









21






22






23








24







25



Structure



Distribution



Functions



26



















27













28










29







30

Functions






31


Functions












Functions

32






33






ND













NCAM

NCAM










ND

Neuroligin-1



Neurogilin-1-2-3



ND ND




ND



ND



ND ND


ND ND

34




Ephrin-B2and -B3



ND


ND




5 ICAM-5




35










36

2012)







37

LFA-1 (CD11aCD18)



ȕ1 integrins

ICAM-5 binds to ȕ1 integrins particularly the Į5ȕ1 integrin in trans The binding site is located in the first two Ig-like domains (See below 5-46)



Presenilin (PS)


ERM proteins


Vitronectin (VN)



Į-Actinin

38










39





40

AIMS OF THE STUDY








41




Antibodies I-IV

Animals I-IV


Cell lines I-IV




ELISA I III IV


SDS-PAGE I-IV









Flow cytometry II

RNAi II III








42

RESULTS









43











44












45
























46





























47

DISCUSSION








48







49












50

















51









52



53

CONCLUDING REMARKS




54

ACKNOWLEDGEMENTS











55

REFERENCES


56


57


58


59


60


61


62


63


64


65


66


67


68


69


70


71


72


73


74




















Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

4


5

TABLE OF CONTENTS


ABBREVIATIONS 7

ABSTRACT 9






3 NMDA receptors 21




5 ICAM-5 34




RESULTS 42

DISCUSSION 47


ACKNOWLEDGEMENTS 54

REFERENCES 55

6



text












Submitted



7

ABBREVIATIONS







AMPAR AMPA receptor

AZ active zone


CaM calmodulin





DIV day in vitro












Ig immunoglobulin



kDa kilodalton

KO knockout










NMDAR NMDA receptor

8

NT neurotransmitter




PS presenilin

PSA polysialic acid






SR spectrin repeat

SV synaptic vesicle


TLN telencephalin

VN vitronectin

WT wild type

9

ABSTRACT




































developing spines



10











11



Synaptic clefts





12








13










14








15



Spine morphogenesis








16







17









18






19

Structure










20









21






22






23








24







25



Structure



Distribution



Functions



26



















27













28










29







30

Functions






31


Functions












Functions

32






33






ND













NCAM

NCAM










ND

Neuroligin-1



Neurogilin-1-2-3



ND ND




ND



ND



ND ND


ND ND

34




Ephrin-B2and -B3



ND


ND




5 ICAM-5




35










36

2012)







37

LFA-1 (CD11aCD18)



ȕ1 integrins




Presenilin (PS)


ERM proteins


Vitronectin (VN)



Į-Actinin

38










39





40

AIMS OF THE STUDY








41




Antibodies I-IV

Animals I-IV


Cell lines I-IV




ELISA I III IV


SDS-PAGE I-IV









Flow cytometry II

RNAi II III








42

RESULTS









43











44












45
























46





























47

DISCUSSION








48







49












50

















51









52



53

CONCLUDING REMARKS




54

ACKNOWLEDGEMENTS











55

REFERENCES


56


57


58


59


60


61


62


63


64


65


66


67


68


69


70


71


72


73


74




















Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

5

TABLE OF CONTENTS


ABBREVIATIONS 7

ABSTRACT 9






3 NMDA receptors 21




5 ICAM-5 34




RESULTS 42

DISCUSSION 47


ACKNOWLEDGEMENTS 54

REFERENCES 55

6



text












Submitted



7

ABBREVIATIONS







AMPAR AMPA receptor

AZ active zone


CaM calmodulin





DIV day in vitro












Ig immunoglobulin



kDa kilodalton

KO knockout










NMDAR NMDA receptor

8

NT neurotransmitter




PS presenilin

PSA polysialic acid






SR spectrin repeat

SV synaptic vesicle


TLN telencephalin

VN vitronectin

WT wild type

9

ABSTRACT




































developing spines



10











11



Synaptic clefts





12








13










14








15



Spine morphogenesis








16







17









18






19

Structure










20









21






22






23








24







25



Structure



Distribution



Functions



26



















27













28










29







30

Functions






31


Functions












Functions

32






33






ND













NCAM

NCAM










ND

Neuroligin-1



Neurogilin-1-2-3



ND ND




ND



ND



ND ND


ND ND

34




Ephrin-B2and -B3



ND


ND




5 ICAM-5




35










36

2012)







37

LFA-1 (CD11aCD18)



ȕ1 integrins




Presenilin (PS)


ERM proteins


Vitronectin (VN)



Į-Actinin

38










39





40

AIMS OF THE STUDY








41




Antibodies I-IV

Animals I-IV


Cell lines I-IV




ELISA I III IV


SDS-PAGE I-IV









Flow cytometry II

RNAi II III








42

RESULTS









43











44












45
























46





























47

DISCUSSION








48







49












50

















51









52



53

CONCLUDING REMARKS




54

ACKNOWLEDGEMENTS











55

REFERENCES


56


57


58


59


60


61


62


63


64


65


66


67


68


69


70


71


72


73


74




















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6



text












Submitted



7

ABBREVIATIONS







AMPAR AMPA receptor

AZ active zone


CaM calmodulin





DIV day in vitro












Ig immunoglobulin



kDa kilodalton

KO knockout










NMDAR NMDA receptor

8

NT neurotransmitter




PS presenilin

PSA polysialic acid






SR spectrin repeat

SV synaptic vesicle


TLN telencephalin

VN vitronectin

WT wild type

9

ABSTRACT




































developing spines



10











11



Synaptic clefts





12








13










14








15



Spine morphogenesis








16







17









18






19

Structure










20









21






22






23








24







25



Structure



Distribution



Functions



26



















27













28










29







30

Functions






31


Functions












Functions

32






33






ND













NCAM

NCAM










ND

Neuroligin-1



Neurogilin-1-2-3



ND ND




ND



ND



ND ND


ND ND

34




Ephrin-B2and -B3



ND


ND




5 ICAM-5




35










36

2012)







37

LFA-1 (CD11aCD18)



ȕ1 integrins




Presenilin (PS)


ERM proteins


Vitronectin (VN)



Į-Actinin

38










39





40

AIMS OF THE STUDY








41




Antibodies I-IV

Animals I-IV


Cell lines I-IV




ELISA I III IV


SDS-PAGE I-IV









Flow cytometry II

RNAi II III








42

RESULTS









43











44












45
























46





























47

DISCUSSION








48







49












50

















51









52



53

CONCLUDING REMARKS




54

ACKNOWLEDGEMENTS











55

REFERENCES


56


57


58


59


60


61


62


63


64


65


66


67


68


69


70


71


72


73


74




















Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

7

ABBREVIATIONS







AMPAR AMPA receptor

AZ active zone


CaM calmodulin





DIV day in vitro












Ig immunoglobulin



kDa kilodalton

KO knockout










NMDAR NMDA receptor

8

NT neurotransmitter




PS presenilin

PSA polysialic acid






SR spectrin repeat

SV synaptic vesicle


TLN telencephalin

VN vitronectin

WT wild type

9

ABSTRACT




































developing spines



10











11



Synaptic clefts





12








13










14








15



Spine morphogenesis








16







17









18






19

Structure










20









21






22






23








24







25



Structure



Distribution



Functions



26



















27













28










29







30

Functions






31


Functions












Functions

32






33






ND













NCAM

NCAM










ND

Neuroligin-1



Neurogilin-1-2-3



ND ND




ND



ND



ND ND


ND ND

34




Ephrin-B2and -B3



ND


ND




5 ICAM-5




35










36

2012)







37

LFA-1 (CD11aCD18)



ȕ1 integrins




Presenilin (PS)


ERM proteins


Vitronectin (VN)



Į-Actinin

38










39





40

AIMS OF THE STUDY








41




Antibodies I-IV

Animals I-IV


Cell lines I-IV




ELISA I III IV


SDS-PAGE I-IV









Flow cytometry II

RNAi II III








42

RESULTS









43











44












45
























46





























47

DISCUSSION








48







49












50

















51









52



53

CONCLUDING REMARKS




54

ACKNOWLEDGEMENTS











55

REFERENCES


56


57


58


59


60


61


62


63


64


65


66


67


68


69


70


71


72


73


74




















Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

Blank Page

8

NT neurotransmitter




PS presenilin

PSA polysialic acid






SR spectrin repeat

SV synaptic vesicle


TLN telencephalin

VN vitronectin

WT wild type

9

ABSTRACT




































developing spines



10











11



Synaptic clefts





12








13










14








15



Spine morphogenesis








16







17









18






19

Structure










20









21






22






23








24







25



Structure



Distribution



Functions



26



















27













28










29







30

Functions






31


Functions












Functions

32






33






ND













NCAM

NCAM










ND

Neuroligin-1



Neurogilin-1-2-3



ND ND




ND



ND



ND ND


ND ND

34




Ephrin-B2and -B3



ND


ND




5 ICAM-5




35










36

2012)







37

LFA-1 (CD11aCD18)



ȕ1 integrins




Presenilin (PS)


ERM proteins


Vitronectin (VN)



Į-Actinin

38










39





40

AIMS OF THE STUDY








41




Antibodies I-IV

Animals I-IV


Cell lines I-IV




ELISA I III IV


SDS-PAGE I-IV









Flow cytometry II

RNAi II III








42

RESULTS









43











44












45
























46





























47

DISCUSSION








48







49












50

















51









52



53

CONCLUDING REMARKS




54

ACKNOWLEDGEMENTS











55

REFERENCES


56


57


58


59


60


61


62


63


64


65


66


67


68


69


70


71


72


73


74




















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Neuronal synapse formation regulated by intercellular ...

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