Resveratrol Increases Mitochondrial Protein Import in ... · PDF fileii Resveratrol Increases...

Resveratrol Increases Mitochondrial Protein Import in Differentiated

PC12 Cells.

by

Soghra Jougheh Doust

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Physiology University of Toronto

© Copyright by Soghra Jougheh Doust (2010)

ii

Resveratrol Increases the Mitochondrial Protein Import in

Differentiated PC12 Cells.

Master of Science Thesis-2010

Soghra Jougheh Doust

Department of Physiology

University of Toronto

ABSTRACT:

Mitochondrial function is dependent upon mitochondrial protein import (MPI), a complex

process that transports nuclear-encoded proteins into mitochondria. Little is known about

MPI in neurons. We examined the effects of Resveratrol (RSV), a polyphenolic

antioxidant compound from grapes, on MPI in a neuronal cell model, differentiated PC12

cells. RSV (50µM, 24h) increased levels of mtGFP, a nuclear encoded mitochondrially

targeted green fluorescent protein, and mtHsp70, a physiological mitochondrial heat shock

protein, in mitochondria. In addition RSV also increased levels of Tom20, a key

translocase of the outer mitochondrial membrane. The RSV mediated increases in

mitochondrial proteins were independent of increases in mitochondrial mass or changes in

intramitochondrial degradation. RSV also reduced mitochondria membrane potential and

decreased basal levels of reactive oxygen species. Taken together, these findings show

that RSV increases MPI and that this effect may be an important mechanism in the

reported neuroprotective effects of RSV.

iii

ACKNOWLEDGEMENTS

First, I would like to thank Dr. Linda Mills my supervisor. Her guidance, support and

help throughout this journey were unbelievable. I will be always grateful for the

opportunity she gave me to become one of the graduate students in her lab. Thank you for

helping me out to build my confidence and regain my energy in this new country. Your

encouragements and advice made me to work and focus as best as I could to achieve my

goals in my academic life.

Second, I would like to thank everyone in my lab, Jamie, Natalya, Diana, Nam, Chloe,

Neha and Adrian for their contributions and help. Special thanks go to Natalya and Jamie

for their endless support and help. I am grateful for the opportunity to learn and work with

you.

Third, I would like to thank my supervisory committee members Dr. Eubanks, Dr.

Monnier, Dr. Velumian who were patient with me. Special thanks go to Dr. Eubanks and

Dr. Monnier, your advice and encouragements are priceless.

Fourth, I would like to acknowledge my defense committee for taking time out of their

busy schedules, Dr. Eubanks, Dr. Sugita, Dr. Zhen and Dr. Li.

Fifth, I would like to acknowledge Unilever/Lipton and Crother’s family awards

organizations for their financial supports.

I would like to thank my program director Dr. Roberto Mendoza, all of the members of

post graduate medical studies committee as well as my friends in Clinical and Metabolic

Genetics division at the Hospital for Sick Children, for their support, advice and

understanding of this complicated process. Special thanks go to Parvaz for her

contribution in editing my thesis.

I want to thank my parents whose endless love and encouragement made me to be a

stronger person inside and out. Thanks to my dearest siblings in Iran for their love and

support. I dedicate this thesis to my mother and my father’s soul. May rest in peace, my

dear father!

Lastly, most of the thanks go to my loving, supportive, encouraging, and patient husband

Hassan whose faithful support during Master’s degree process is indescribable. Thank

you.

iv

TABLE of CONTENTS

ABSTRACT……………………………………………………..………………… ii

ACKNOWLEDGEMENTS……………………………………………………… iii

LIST of FIGURES………………………………………… ……………………… vii

LIST of TABLES….……………………………………………………………… vi

LIST of ABBREVIATIONS…… ………………………………………………. . viii

INTRODUCTION………...………………………………………………………... 1

1.1. Mitochondria…………………………………………………...…………... 2

1.2. Mitochondrial Functions………………………………………………… … 2

1.3. Neurodegenerative Diseases and Mitochondria ……………….…………... 4

1.4. Mitochondrial Proteins …………………………………..…………………. 5

1.5. Mitochondrial Protein Import Machinery …………………………….……. 6

1.5.1. General Structure …………………………………………...………….. 7

1.5.2. Translocation of Proteins across the Outer and Inner Membrane ……… 7

1.5.3. Regulation of MPI …………………………………...…………………. 11

1.5.4. The Impact of MPI Defects on the Cells …………………….…………. 12

1.6. Mitochondrial Biogenesis …………………………….…………………..… 16

1.7. Resveratrol……………………………………...…………………………... 17

1.7.1 Beneficial Effects of RSV ……………………….……….……………... 18

1.7.2. RSV and Mitochondria Biogenesis……………………………………… 18

1.7.3. RSV and Antioxidation………………………………………………. 19

1.7.4. RSV and Sirtuins……………………………………………………….. 19

1.7.5. Bioavailability and Pharmacokinetics…………………………….…… 21

1.8. PC12 Cells and MPI Studies…………………………………………..…….. 21

1.9. Green Fluorescent Protein……………………………………..…………….. 22

1.10. Rationale…………………………………………………...………………… 26

1.11. HYPOTHESES and SPECIFIC AIMS ……………………………………. 27

MATERIALS AND METHODS…………………………………………..………. 28

2.1. Creation of Stable PC12 Tet-off/MtGFP Cells ……………………………. 29

2.2. Culturing Undifferentiated PC12 Cells…………………………………….. 29

2.3. Culturing Differentiated PC12 Cells ………………………………………. 30

v

2.4. Preparation of 100 mM RSV Stock Solution …………………………...… 30 2.5. Preparation of Stock Solutions (Mitotracker Green, CCCP, Rhodamine 123

and DCF Experiments) …………………………………………………….. 33

2.6. Flow Cytometry ………………………………………………………….. 34

2.6.1. Assessment of MtGFP Import by Flow Cytometry …………...……… 34

2.6.2. Analysis of Intramitochondrial MtGFP Degradation ………………… 34

2.6.3. Analysis of the Mitochondrial Membrane Potential (MMP)

by Flow Cytometry.……………………………………………………… 35

2.6.4. Measurements of the Mitochondria Mass in PC12 Cells ……………… 35

2.6.5. Assessment of ROS Generation in Response to RSV Treatment ……… 36

2.7. Western Blot Analysis of Mitochondrial Protein Import and Synthesis ….... 36

2.7.1. Preparation of the Whole Cell Lysates and Subcellular Fractions …… 36

2.7.2. Determination of Protein Concentrations in WCL, CP and MT ……… 37

2.7.3. Determination of mtGFP protein in WCL and subcellular fraction ….. 38

2.7.4. Western Blot Analysis of Physiological Proteins in WCL and

Subcellular Fractions……………………….………………………………… 39

2.8. Statistical Analysis of Data ………………………………………………… 39

RESULTS………………………………………………………………………..… 41

3.1. RSV Significantly Increases MtGFP Levels in Mitochondria ……………. 42

3.1.1. The Optimal Doses for RSV …………………………………………… 42

3.1.2. Effects of RSV on Cell Survival in Differentiated PC12 Cells ………... 45

3.1.3. 50 µM RSV Increases MtGFP Levels in Mitochondria ………………… 48

3.2. Sustained RSV Slows Intramitochondrial Degradation of MtGFP ………... 59

3.3. RSV Does not Increase Mitochondrial Mass ………………………………. 59

3.4. RSV Has no Effect on Cell Size …………………………………………… 59

3.5. 50µM RSV Increases the Import of Physiological Mitochondrial Proteins... 65

3.6. RSV and its Effects on Mitochondrial Function …………………………… 74

3.6.1. RSV Decreases ROS Generation……………………………….………. 74

3.6.2. 50 µM RSV Decreases Mitochondria Membrane Potential (MMP) 24h Post

Treatment…………………………………………….………………….. 74

DISCUSSION………………….…………………………………………………… 75

4.1. MPI in Neurons ……………………………………………………………. 78

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4.2. Flow Cytometry and Western Blot Show That RSV Increases

MtGFP in Mitochondria ……………………………………………………. 78

4.3. RSV Does Not Initially Alter Intramitochondrial MtGFP Degradation…… 80

4.4. RSV Increases Physiological Mitochondrial Proteins in Mitochondria …... 80

4.5. RSV Does Not Alter Mitochondria Mass ……………………………...….. 82

4.6. RSV Increases the Expression of Mitochondrial Proteins Selectively …… 82

4.7. RSV Reduces Mitochondrial Membrane Potential ……………………..… 83

4.8. RSV Decreases ROS Generation …………………………………………. 84

4.9. MtGFP as a Model of MPI………………………………………………... 85

4.10. A Model for Effects of RSV on Mitochondrial Protein Import …………. 86

4.11. Future Studies on RSV, MPI, ROS Generation and Ca2+ ………………. 89

4.12. Conclusion and Significance ……………………………………..……… 89

REFERENCES…………………………………………………………….……… 91

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LIST of FIGURES

Figure 1 Simplified schematic of mammalian mitochondrial import machinery 9

Figure 2 Clinical features of mitochondrial diseases. ………………………… 15

Figure 3 MtGFP in Mitochondria in differentiated PC12 cell………………… 25

Figure 4 A model in studying mtGFP import in live PC12 cells……………… 31

Figure 5 Effects of the exposure of different doses of RSV after 24h on the mtGFP signal and cell death……………………………………………… 43

Figure 6 Effects of the exposure of different doses of RSV after 24h on the mtGFP signal and cell death…………………………………………………. 46

Figure 7 50µM RSV increases the mtGFP signal (import) for up to 3 days .. 49

Figure 8 50µM RSV increases mtGFP import as early as 12 h post exposure and

continues to increase the signal for up to 2 days…………………… 51

Figure 9 50µM RSV increases mtGFP levels in mitochondria ………….….. 53

Figure 10 50µM RSV does not alter mtGFP expression……………………… 55

Figure 11 There is no detectable mtGFP in cytoplasmic fractions…………… 57

Figure 12 There is no detectable amount of Tom20 and mtHsp70 in CP fractions 58

Figure 13 50µM RSV does not alter the intramitochondrial mtGFP degradation in the first 24h …………………………………….………………………. 61

Figure 14 Mitochondria mass was not affected by 50µM RSV ………………. 63

Figure 15 RSV does not affect cell size ……………………………………… . 64

Figure 16 50µM RSV increases Tom20 levels in mitochondria ……………… 66

Figure 17 50µM RSV increases Tom20 expression …………………………… 68

Figure 18 50µM RSV increases mtHsp70 levels in mitochondria …………….. 70

Figure 19 50µM RSV does not change mtHsp70 expression ………………….. 72

Figure 20 50µM RSV decreases mitochondrial ROS generation ……………… 75

Figure 21 RSV reduces mitochondrial membrane potential (MMP) 24h post treatment ……………………………………………………………………… 76

Figure 22 A model illustrating the underlying mechanisms involved in RSV induced changes in MPI …………..………………………………………….. 87

viii

LIST of TABLES

Table 1 Mitochondrial disorders……………………………………….…. 14 Table 2 EtOH concentrations……………………………………………… 32

ix

ABBREVIATIONS

AB antibody AD Alzheimer’s Disease AMP adenosine mono phosphate AMPK AMP kinase ADP adenosine diphosphate ANOVA analysis of variance ATP adenosine triphosphate A� peptide amyloid beta peptide Ca2+ Calcium ion Cai

2+ intracellular calcium cAMP 3'-5'-cyclic adenosine monophosphate CCCP carbonyl cyanide m-chlorophenylhydrazone COX cytochrome C oxidase Cpn10 chaperonin 10 CytC cytochrome C ddH2O double distilled H2O DDP1 deafness-dystonia peptide 1 DMSO dimethyl sulfoxide DNA deoxyribonucleic acid EDTA ethylenediaminetetraacetic acid ER endoplasmic reticulum Erv1 essential for respiration and vegetative growth 1 protein FADH2 reduced flavin adenine dinucleotide FL1 first fluorescent detector FL3 third fluorescent detector FSC forward scatter FOXOs fork head box transcription factors O G(g) gram GAPDH glyceraldehyde-3-phosphate dehydrogenase GFP green fluorescent protein GIP general import pore h(hrs) hours HBSS Hank’s buffered salt solution Hcl hydrochloric acid Hsp heat shock protein HD Huntington’s Disease IM inner membrane IMS intermembrane space IP immunoprecipitation IV intravenous K+ potassium ion KCl potassium chloride LDL low density lipoprotein

x

MELAS mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke

MDH malate dehydrogenase Mg Magnesium mg milligram Mia40 mitochondrial intermembrane space assembly protein 40 ml milliliter mM millimolar MMP mitochondrial membrane potential Mn Manganese MnTBAP Mn(III)tetrakis (4-benzoic acid) porphyrin MPP mitochondrial processing protease mRNA messenger ribonucleic acid mt mitochondrial mtDNA mitochondrial deoxyribonucleic acid mtGFP mitochondrially-targeted GFP fusion protein MT Green mitotracker green mtHsp70 mitochondrial heat shock 70 protein MTJ Mohr-Tranebjaerg syndrome MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide mtTFA mitochondrial transcription factor A Na+ sodium ion NaCl sodium chloride NADH reduced nicotinamide adenine dinucleotide NGF nerve growth factor ng nanogram NF nuclear factor nm nanometer nM nanomolar OD optical density OM outer membrane Oxa1 oxidase assembly 1 PAM presequence translocase-associated motor PBS phosphate buffer saline PC12 pheochromocytoma 12 cells PCR polymerase chain reaction PD Parkinson Disease

PGC-1α peroxisome proliferator-activated receptor gamma coactivator 1 alpha PI propidium iodide PKA cyclic AMP-dependent protein kinase PMT photomultiplier tube PP protein phosphatase PO per os QR1 quinone reductase 1 Rh123 rhodamine 123 RNA ribonucleic acid

xi

ROS reactive oxygen species RPMI 1640 Roswell Park Memorial Institute medium 1640 RT room temperature SAM sorting and assembly machinery SD standard deviation SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM standard error of the mean SIR2 silence information regulators 2 Sirt sirtuins SSC side scatter TBS-T tris-buffered saline tween-20 Tet tetracycline tetO tetracycline operon TF transcription factors TIM translocase of the inner mitochondrial membrane TOM translocase of the outer mitochondrial membrane TRE tetracycline response element tTA tetracycline-controlled transactivator µg microgram µl microliter µM micromolar WCL whole cell lysate

1

Introduction

2

1.1. Mitochondria

. Mitochondria are dynamic organelles that integrate environmental signals to regulate

energy production, apoptosis and calcium (Ca2+) homeostasis (Mihara 2000). As such they

are critical for the development and survival of most prokaryotic and eukaryotic cells. Other

mitochondrial functions include but are not limited to, beta-oxidation, ketone body synthesis,

urea cycle and amino acid metabolism (Yudkoff et al., 2001). Mitochondria also have a role

in immune response in viral infections (Hiscott et al., 2006). Although the following sections

focus mainly on mitochondrial function in neurons, most of this information may be

generalized to other cell types.

Mitochondria consist of four main compartments: the outer mitochondrial membrane

(OM), the inner mitochondrial membrane (IM), the inter-membrane space (IMS), and the

matrix (Schon and Manfredi, 2003). The OM is smooth and permeable, while the IM is

relatively impermeable and folded to form invaginations, or cristae. The granular

mitochondrial matrix is of variable density and contains large number of enzymes and

inclusions such as calcium salts, organic crystals, ribosomes and nucleic acids (Calabrese et

al., 2001). The respiratory chain which produces adenosine triphosphate (ATP) consists of

five complexes that are located on the IM: complex I (reduced nicotinamide adenin

dinucleotide (NADH) ubiquinone oxidoreductase), II (succinate ubiquinone oxidoreductase),

III (ubiquinone-cytochrome C reductase), IV (cytochrome C oxidase), and V (ATP synthase).

1.2. Mitochondrial Functions

The primary function of mitochondria is oxidative phosphorylation (OXPHOS) which

provides a highly efficient route for eukaryotic cells to generate adenosine 5'-triphosphate

(ATP) from energy-rich molecules (Chan 2006; Kakkar and Singh 2007; Devin and Rigoulet

2007; Ryan and Hoogenraad 2007). Electrons from oxidative substrates such as NADH and

reduced flavin adenine dinucleotide (FADH2) produced during glycolysis, and the citric acid

cycle respectively, is transferred to oxygen, via a series of redox (oxidation-reduction)

reactions, to generate water. In the process, protons are pumped from the matrix across the

mitochondrial inner membrane through respiratory complexes I, III, and IV. When protons

3

return to the mitochondrial matrix down their electrochemical gradient, ATP is synthesized

via complex V (the F0F1-ATP synthase complex) (Chan 2006; Devin and Rigoulet 2007).

Mitochondria respond to short-term and long-term changes in cellular energy

requirements. They may either increase or decrease the oxidative phosphorylation rate in

response to short term changes (Das 2003; Devin and Rigoulet 2007; Peuchen et al., 1996).

However, a response to long-term changes in cellular energy demand usually involves the

expression of genes encoding mitochondrial enzymes, and, in some cases, changes in

mitochondrial biogenesis (Devin and Rigoulet 2007). Studies suggest that there are two main

mechanisms involved in a long-term adaptation: modulation of the mitochondrial enzyme

content as a response to energy demand, and kinetic regulation by modifications

(phosphorylations) of some respiratory chain complex subunits. Here, the cyclic adenosine

monophospate (cAMP) signaling pathway plays a major role in molecular signaling, leading

to the mitochondrial response (Devin and Rigoulet 2007). The subcellular distribution varies

in response to different stimuli, especially in neurons due to their complex morphology and

region-specific energy demands (e.g., at the synapse and at the growth cone) (Kann and

Kovacs 2007).

One of the key features of mitochondria is reactive oxygen species (ROS) generation

(Balaban et al., 2005; Finley and Haigis 2009). ROS are generated as a consequence of ATP

production in mitochondria, particularly at complex I and III of the electron transport chain

(ETC) (Genova et al., 2004). Mitochondria consume an excessive amount of O2 in ATP

synthesis and OXPHOS process and consequently generate ROS. As such, mitochondria are

not only the primary source of ROS, but they are also susceptible to ROS induced damage,

such as oxidative stress. Oxidative stress is caused by an imbalance between the production

of reactive oxygen and the eradication of these free radicals by antioxidants. Disturbances in

this normal redox state can cause toxic effects through the production of peroxides and free

radicals that damage all components of the cell, including proteins, lipids, and DNA (Devin

and Rigoulet 2007). Oxidative stress in mitochondria damages mitochondrial membranes and

mtDNA, this damage ultimately leads to mitochondrial dysfunction (Balaban, et al., 2005;

Goldenthal et al., 2004). Recent studies suggest that excessive accumulation of ROS is a

common mechanism in the initiation and/or progression of multiple neurodegenerative

4

diseases (Acevedo-Torres et al., 2009; Deocaris et al., 2008; Kann and Kovacs 2007) and

brain aging (Balaban et al., 2005).

Low molecular mass antioxidants in the brain and ROS scavenging enzymes can help the

brain prevent the adverse effects of oxidative stress. Superoxide dismutase (SOD), is an

example of an enzyme which reduces superoxide to H2O2, and is present in different

compartments of mitochondria in the forms of metalloproteins consisting of Manganese

(SOD2) or Copper and Zinc (SOD1). The other enzyme is glutathione peroxidase which

eliminates H2O2 by oxidation of glutathione to produce water.

Mitochondria also contribute to intracellular calcium (Cai2+) homeostasis through the

sequestration and release of Ca2+ (Mihara 2000). ROS regulate the activity of redox sensitive

enzymes and ion channels within the cell, including Ca2+ channels (Feissner et al., 2009). In

turn, Ca2+, a key regulator of mitochondrial functions, acts at multiple signaling cascades

within the mitochondria to stimulate ATP synthesis. Previous studies showed that

dysregulation of mitochondrial Ca2+ (for example in the case of mitochondrial matrix Ca2+

overload), can lead to enhanced generation of ROS, triggering the permeability of transition

pore, release of cytochrome C and leading to apoptosis (Brookes et al., 2004; Feissner et al.,

2009; Kakkar and Singh 2007). Therefore, the balance between mitochondrial and

cytoplasmic Ca2+ levels is important in neuronal viability.

1.3. Neurodegenerative Diseases and Mitochondria

Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD),

Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), hereditary spastic

paraplegia, and cerebellar degeneration, are progressive diseases of the central nervous

system. These diseases are thought to be caused by neuronal death, or changes in axonal

myelination, that gradually leads to neuronal dysfunction.

Aging is also a determinant risk factor in neurodegenerative disorders. Recent studies

suggest that mitochondrial dysfunction plays a central role in both aging and in

neurodegenerative diseases. On the other hand, neuronal dysfunction and death could in itself

also contribute to mitochondrial dysfunction (Balaban et al., 2005; Beckman and Ames 1998;

5

Calabrese et al., 2001; Chan 2006; Jacobs 2003; Kowald 2001; Reddy 2008).

Mechanistically, oxidative stress, mtDNA pathological mutations and deletions, alterations in

mitochondrial dynamics (fusion and fission), defects in nucleus- mitochondria cross talk, and

subsequent changes in protein synthesis, import and assembly in mitochondria are all

suggested causes of mitochondrial dysfunction, and, in turn neuronal dysfunction and death

(Acevedo-Torres et al., 2009; Balaban et al., 2005; Beal 2005; Beal 2007; Biesalski 2002;

Hood et al., 2003; Hood and Joseph 2004; Rehling et al., 2001; Sirk et al., 2007;

Stojanovski, et al., 2007; Truscott, et al., 2001; Truscott et al., 2003; Wiedemann et al.,

2004; Yang et al., 2008).

For example, in AD direct interaction of mitochondria with beta-amyloid (�-amyloid) and

the amyloid precursor protein leads to increased ROS generation (Beal 2007; Kann and

Kovacs 2007). In recent years, down regulation of one of the mitochondria biogenesis

modulators, the peroxisome proliferator-activated receptor gamma coactivator 1 alpha

(PGC1-�), has been shown in HD (Cui et al., 2006; see also mitochondria biogenesis

section). Furthermore, autosomal recessive PD caused by parkin, DJ1, and PINK1 genes is

also linked to oxidative stress and mitochondrial dysfunction (Beal 2004). Discovery of

disease specific proteins that interact with mitochondria has introduced a new era in

treatment of neurodegenerative diseases (Beal 2007; Reddy 2008).

1.4. Mitochondrial Proteins

Mitochondria are compartmented organelles composed of the matrix, the inner membrane

and the outer membrane. The endosymbiotic hypothesis proposes that mitochondria

originated from a species of alpha Proteobacteria that survived endocytosis by another cell,

and became incorporated into the cytoplasm (symbiotes). Over a gradual evolutionary

process, these symbiotes transferred their genetic information into the nuclear chromosomes

of their hosts and consequently lost some of their functions, thus becoming dependent on

their hosts (Dolezal et al., 2006; Lister et al., 2005; Margulies and Parenti 1968).

Mitochondria, despite having their own genetic material, depend on nuclear genes. The

mitochondrial genome is a 16 kilobase circular double stranded DNA (mtDNA). Each cell

has thousands of copies of mtDNA that encodes 37 genes. These genes are essential in

6

maintaining respiratory functions of the mitochondria by encoding protein subunits of

respiratory complexes (Wallace 2005). Of these genes, thirteen are responsible for encoding

the proteins of complexes I, III, IV and V. Complex II proteins are encoded only by nuclear

genes. In addition, circular mtDNA encodes twenty-two mitochondrial tRNAs and two

rRNAs that are involved in translation of mtDNA transcriptions (Chan 2006).

Although mitochondria possess their own mtDNA, more than 99% of mitochondrial

proteins are encoded in the nucleus, and after their synthesis in cytosol must be targeted and

imported into mitochondria (Sirk et al., 2003; Truscott et al., 2001). Most of the

mitochondrial proteins are synthesized by free cytosolic ribosomes as preproteins and are

imported post translation. These mitochondrial preproteins contain a presequence; an amino

terminal extension containing 10-80 amino acids. The sequence is cleavable, positively

charged, and contains a high number of basic hydrophobic and hydroxylated amino acids

(Rehling et al., 2001; Stojanovski et al., 2007; Truscott et al., 2003; Wiedemann, et al.,

2004).

1.5. Mitochondrial Protein Import Machinery

About ninety nine percent (99%) of mitochondrial proteins are must be imported from the

cytosol into mitochondria (Neupert 1994). The import of mitochondrial proteins is a complex

process involving the orchestrated actions of multiple mitochondrial translocases and

chaperons in a system called the mitochondrial protein import (MPI) machinery.

Our knowledge of the mammalian MPI machinery is an integration of in vitro studies in

isolated mammalian mitochondria, and proteomic studies in budding yeast (Saccharomyces

cerevisiea), red bread mold (Nurospora crrasa), thale cress (Arabidopsis thaliana),

nematodes (Caenorhabditis elegans) (Curran et al., 2004; Elstner et al., 2009; Paschen et al.,

2000; Prokisch et al., 2002). In recent years, many of the essential components of import

machinery such as preproteins, translocases and chaperones has been identified by proteins -,

metabolic labeling and autoradiography of specific proteins –in mitochondrial fractions, but

our understanding of MPI particularly in neurons, is very limited.

7

1.5.1. General Structure

Mitochondrial function is dependent on the MPI machinery, that directs nuclear-encoded

mitochondrial proteins these to one of the four compartments of mitochondria: the outer

membrane (OM), the inter membrane space (IMS), the inner membrane (IM) and the matrix

(see figure 1).

The translocases of the outer membrane (TOM complex) and the sorting and assembly

machinery (SAM) are the two major components of the MPI located on the OM. The SAM

complex assembles the proteins with complex configuration destined for the OM, but the

proteins destined to IMS, IM and matrix, will be transported via a translocase of the inner

membrane TIM23, and the presequence translocase-associated motor (PAM) complex, or

through another translocase of the inner membrane TIM22. The TIM23 complex is

responsible for transport of matrix targeted and IMS preproteins, and the TIM22 complex for

transports of proteins to the IM.

1.5.2. Translocation of Proteins across the Outer and Inner Membrane

The translocases of the outer, (TOMs) and the translocases of the inner membrane (TIMs)

have a critical role in MPI. All mitochondrial proteins encoded by nuclear genes initially

enter mitochondria via the TOM complex. From there the proteins are directed to the proper

mitochondrial sub-compartment. For example, precursor proteins possessing a cleavable

amino-terminal targeting signal (N-MTS) are destined to the IMS, the inner membrane, or

the matrix. Proteins which have uncleavable targeting sequences are destined to either the

OM or IM. Proteins which are destined to the OM either have specific targeting sequence in

the amino- or carboxy-terminus (single pass proteins), or they are �-barrel proteins which

have structural elements carrying their targeting information (Rapaport 2002). The TOM

complex consists of at least 7 proteins (e.g. Tom70, Tom37, Tom22, and Tom20) of which

Tom20, Tom22 and Tom70 are the primary receptor units (Hood 2003). Tom20 mainly

recognizes and binds to the hydrophobic surfaces of preproteins with cleavable presequences

(Truscott et al., 2003). Tom70 mainly binds to preproteins possessing internal targeting

sequences to forms dimers in the OM. General Import Pore (GIP) in mammals contains

Tom22 which is a pore forming protein. Tom22 preferentially delivers presequence

8

containing preproteins from the primary receptors to the GIP. Tom22 works as a central

import receptor in GIP because it can bind to preproteins from both Tom20 and Tom70

(Bohnert et al., 2007). In addition, Tom40, Tom5, Tom6 and Tom7 may have some roles in

stabilizing the TOM complex (Truscott et al., 2003). The interaction of newly synthesized

proteins with cytoplasmic chaperones prevents them from aggregation and keeps them

unfolded. The chaperone- preprotein complex and the targeting sequence directs these

proteins to the proper TOM receptor. Of the cytosolic chaperones, Heat shock protein70

(Hsp70) transfers precursor proteins containing presequences to the Tom20/22 receptor

complex. Hsp70 in conjunction with Hsp90 (which delivers proteins with internal targeting

sequences) transfers proteins to Tom70. Proteins delivered to Tom70 are first passed to

Tom20/22, and then subsequently transferred to GIP (MacKenzie and Payne 2007).

Preproteins that carry their targeting information (β barrel proteins) require the sorting and

assembly machinery (SAM) to complete their translocation to the OM. These proteins are

initially delivered to the IMS via TOM complex. It is suggested that the import and oxidative

folding of small IM space proteins is mediated by mitochondrial intermembrane space

assembly (Mia40) and a protein essential for respiration and vegetative growth 1 (Erv1) The

transport of proteins further into the IM and matrix is mediated by the TIM complex.

Mitochondrial proteins destined for the IM bind to small TIM complexes (Tim8/13,

Tim9/10) found in the IMS that direct them to the TIM22 complex (Bohnert et al., 2007).

9

Figure 1: Simplified schematic of mammalian mitochondrial protein import

machinery. Precursors with a cleavable N-terminal presequence are directed from the cytosol to the outer membrane by cytosolic Hsp70. Precursors with internal targeting signals are chaperoned by mitochondrial import stimulation factor (MSF). At the outer membrane proteins interact with TOM receptors (Tom70, 20 and 22) and then proteins are directed to GIP formed by Tom40. The components of the translocases of the inner membrane (TIM complex) , Tim50, and the smaller Tim isoforms subsequently direct the precursors either to the TIM22 channel to be inserted into the IM, or to the TIM23 channel to be pulled into the matrix via an ATP-dependent action of mitochondrial Hsp70 (mtHsp70) and the mitochondrial membrane potential (MMP). Inside the matrix, the presequences are cleaved by mitochondrial processing peptidase (MPP), and refolded by Hsp60 and chaperonin 10 (Cpn10) into mature proteins (Modified from Hoogenraad et al. 2002; Hood et al 2003).

10

40

20 702222

OM

IMS

TOM Complex

preprotein with presequence

(e.g. mtGFP)

preprotein with internal

targeting sequence

44

22

12

TIM22 Complex

��

MPP

Cytosol

IM

mtHsp70

TIM23 Complex

Small TIMs

Folded protein

Matrix

54

mtGFP

Figure 1: MPI Machinery

TOM: translocases of outer membrane, TIM: tranlocases of inner membrane, OM: mitochondrial

outer membrane, IMS: intermembrane space, IM: inner membrane, mitochondrial proteins

studied in this thesis (Modified from Hoogenraad et al. 2002; Hood et al 2003).

10

11

Tim22 is the pore-forming unit of the TIM22 complex that also includes Tim18,

Tim54, and the adaptor protein Tim12. Presequence containing preproteins are directed

from the TOM complex to the TIM23 complex in the inner membrane. Tim23, the pore-

forming protein, forms TIM23 complex along with Tim17, and Tim50.

Insertion of preproteins in the IM is dependent on the inner mitochondrial membrane

potential (MMP). MMP is a source of energy for translocation and insertion of

preproteins to TIM22. The PAM complex is essential for completion of preprotein

transport into the matrix via Tim23 pore, in an ATP dependent manner. MtHsp70 has 2

different roles in the MPI machinery. It is a central component of PAM and stabilizes

unfolded proteins en route to the matrix. MtHsp70 operates with other chaperones such

as the J-protein Pam18, the J-like component Pam16, the adaptors Tim44 and Pam17, and

the nucleotide exchange factor Mge1. MtHsp70 leads translocations of the proteins by

cycling between ATP- and ADP-bound states (and its affinity for preproteins alters in

these states) which is regulated by the nucleotide exchange factor Mge1. To complete the

transport process, the mitochondrial processing peptidase (MPP) cleaves the targeting

preprotein sequence to allow refolding into their mature and active conformations with

the cooperation of Hsp60 and Hsp10 (Hood et al., 2003).

The group of mediator proteins that deliver proteins in between the mitochondrial sub

compartments are also a part of MPI. For example, oxidase assembly 1 (Oxa1) is an

inner membrane protein which delivers some proteins from the matrix into the inner

membrane (MacKenzie and Payne 2007; Wiedemann et al.,2004).

1.5.3. Regulation of MPI

Much of the work on MPI has focused on the specific proteins of MPI machinery.

Converging evidence suggest that protein import into mitochondria changes in response

to mitochondrial demand, but how this happens is not clear. Studies on isolated

cardiomyocytes and skeletal muscle cells showed enhanced protein import into the

mitochondria. This is consistent with an increase in the expression of some components

of the import machinery (Tom20, mtHsp70) after contractile activity, thyroid hormone

treatment and in senescent cells (Craig and Hood 1997; Grey et al., 2000; Hood et al.,

12

2003; Mattson et al., 2000). Neurons are particularly vulnerable to mitochondrial

dysfunction and recent studies suggest that MPI is a key factor in neuronal survival, (see

below), although there is little direct data on MPI in neurons. Studies by Wright et al.

indicate that oxidative stress inhibits import and the processing of the mitochondrial

matrix proteins in vitro (Wright et al., 2001). Recent studies showed that Mge1 which is a

mitochondrial matrix co-chaperone, alters mtHsp70 affinity to unfolded proteins and

consequently prevents the mitochondrial matrix from becoming overloaded from

unfolded and misfolded protein aggregates (Hood et al., 2003).

1.5.4. The Impact of MPI Defects

Many of the proteins of mitochondrial import machinery e.g. translocases, are nuclear

encoded. They are targeted and imported into mitochondria by the import machinery as

described, however some of these proteins do not obey the general rules of standard

import (e.g.Tom20).

Proper mitochondrial functioning is dependant upon interactions of nuclear encoded

proteins with mtDNA encoded proteins (Beal 2007; Cannino et al., 2007; Ryan and

Hoogenraad 2007). Converging evidences from previous studies show synthesis,

targeting, and import of nuclear encoded proteins contribute to human diseases (figure 2

and table 1). The consequence of defects in the MPI machinery, as an essential

component of mitochondrial function, is not yet clearly understood.

Given the fact that mitochondrial functions (e.g. ATP synthesis, Ca2+ homeostasis,

redox activities and protein import) are dependent upon their proteins, any error or

sustained defect in protein synthesis, mitochondrial targeting signals, in MPI, and in

protein assembly will lead to mitochondrial and subsequent cellular dysfunction. MPI is a

dynamic process which can be affected by various conditions. Mackenzie and Payne in

2007 review some of the human disorders attributed to errors or defects in protein

targeting signaling as well as import and assembling pathways (MacKenzie and Payne

2007).

13

Studies have shown that at least one progressive neurodegenerative disease is directly

caused by a defect in MPI. Human Deafness Dystonia Syndrome (HDD) or Mohr-

Tranebjaerg (Jin et al., 1996; Paschen et al., 2000) is an X-linked neurodegenerative

disorder characterized by post-lingual progressive sensorineural deafness, dystonia,

spasticity, dysphagia, mental deterioration and cortical blindness. HDD is caused by

mutations in the IMS protein deafness dystonia peptide 1 (DDP1/Tim8), which is

homologous to the fungal protein Tim8. In mammalian mitochondria, DDP1/Tim8 is

found in complex with Tim13 (table 1). It is thought that a decrease in Tim8 - which

helps in import of Tim23 from the outer membrane to Tim23 complex of IM-, causes a

decrease in Tim23 levels which in turn causes defects in mitochondrial respiration

complexes (figure 2 and table 1).

14

Mitochondrial genetic disorders :

Rearrangements (partial deletions and

duplications) and point mutations

Nuclear genome

Rearrangements:

Chronic progressive external ophthalmoplegia

(CPEO)

Kearns-Sayre syndrome

Diabetes and deafness

Pearson marrow-pancreas syndrome

Sporadic tubulopathy

Point mutations:

Protein encoding genes

LHON (G11778A, T14484C, G3460A)

NARP/Leigh syndrome (T8993G/C)

tRNA genes

MELAS (A3243G, T3271C, A3251G)

MERRF (A8344G, T8356C)

CPEO (A3243G, T4274C)

Myopathy (T14709C, A12320G)

Cardiomyopathy (A3243G, A4269G, A4300G)

Diabetes and deafness (A3243G, C12258A)

Encephalomyopathy (G1606A, T10010C)

rRNA genes

Non-syndromic sensorineural deafness (A7445G)

Aminoglycoside induced non-syndromic deafness

(A1555G)

Autosomal dominant progressive external ophthalmoplegia

(with 2� multiple mtDNA deletions)

•Mutations in adenine nucleotide translocator (ANT1)

•Mutations in DNA polymerase (POLG)

•Mutations in Twinkle helicase (C10orf2)

Mitochondrial neuro-gastrointestinal

encephalomyopathy (with 2� multiple mtDNA deletions)

•Mutations in thymidine phosphorylase (TP)

Myopathy with mtDNA depletion

•Mutations in thymidine kinase (TK2)

Encephalopathy with liver failure

•Mutations in deoxyguanosine kinase (DGK)

Primary disorders of the respiratory chain

Leigh syndrome

•Complex I deficiency: mutations in complex I subunits

(NDUFS2, 4, 7, 8, and NDUFV1)

•Complex II deficiency: mutations in complex II flavoprotein

subunit (SDH)

Leukodystrophy and myoclonic epilepsy

•Complex I deficiency: mutations in complex I subunit

(NDUFV1)

Cardioencephalomyopathy

•Complex I deficiency: mutations in complex I subunit

(NDUFS2)

Optic atrophy and ataxia

•Complex II deficiency: mutations in complex II flavoprotein

subunit (SDH)

Disorders of mitochondrial protein import

Dystonia-deafness

•Mutations in deafness-dystonia protein DDP1 (TIMM8A)

Disorders of assembly of the respiratory chain

Leigh syndrome

•Complex IV deficiency: mutations in COX assembly protein

(SURFI)


(COX10)

Cardioencephalomyopathy


(SCO2)

Hepatic failure and encephalopathy


(SCO1)

•Complex IV deficiency: mutations in protein affecting COX

mRNA stability (LRPPRC)

Tubulopathy, encephalopathy, and liver failure

•Complex III deficiency: mutations in complex III assembly

(BCS1L)

Table 1: Mitochondrial Disorders Modified from Chinnery and Schon, biology online website

15

Figure 2: Clinical features of mitochondrial diseases. Mitochondrial disease may present

with single organ involvement (sensorineural deafness, diabetes, visual failure, myopathy,

or cardiomyopathy), or multisystemic involvement. There are several mitochondrial

syndromes (see also table 1). The combination of neurological disease and extra-

neurological involvement should raise the suspicion of a mitochondrial disorder (from

Chinnery and Schon, biology online website).

16

1.6. Mitochondrial Biogenesis

Mitochondrial biogenesis requires the co-ordinated expression of mitochondrial and

nuclear genes. Mitochondria also modulate protein import when proteins are mutated,

and/or damaged proteins (by oxidative stress) are produced in the cells (Ryan and

Hoogenraad 2007). Communication between mitochondria and the nucleus are essential

in these processes. Mitochondrial signaling may be mediated by changes in metabolites

and ion flow (e.g., Ca2+) or by structural changes of the organelle itself (Ryan and

Hoogenraad 2007).

Biogenesis and proliferation of mammalian mitochondria are influenced by exercise,

nutrients, and hormones. Given the fact that energy demands can require differences in

mitochondrial abundance in different parts of a cell (e.g. axons in neurons) or in specific

organs (muscle cells or hepatic system) the regulatory mechanisms to control

mitochondrial biogenesis are critical. Recent studies have identified transcriptional

factors and coactivators that control the co-ordinate expression of mitochondrial and

nuclear genes. Recent studies have identified mitochondrial transcription factor A

(mtTFA) which stimulates the transcription of mtDNA and the nuclear respiratory factor

1 (NRF1) a transcription factor which regulates mtTFA. The peroxisome proliferator

activated receptor coactivator-1 (PGC-1) is considered a universal system that integrates

mitochondrial biogenesis in vertebrates. PGC-1 coactivators are PGC-1�, PGC-1�, and

the PGC-related coactivator (PRC). Transcriptional activation of PGC-1 is induced by

external stimuli in a tissue-specific manner leading to the transcriptional activation of a

very large number of genes that encode mitochondrial proteins, including elements of the

MPI machinery (Canto and Auwerx 2009; Diaz and Moraes 2008; Onyango et al., 2009;

Ryan and Hoogenraad 2007).

17

1.7. Resveratrol

Background: Resveratrol (RSV) (trans-3, 5, 4’- trihydroxystilbene) is one the

phytoalexins - compounds produced by plants in response to environmental stress (such

as insect, animal or pathogenic attacks, or ultraviolet radiation and ozone). RSV has been

identified in more than 70 species of plants, including mulberries and peanuts. Skin and

the roots of purple grapes are a good source of RSV. Interest in RSV spiked in 1992,

when RSV in red wine was proposed to have cardioprotective properties (Baur and

Sinclair 2006). The skin of grapes contains about 50 to 100 micrograms of RSV per

gram, however its concentration in red wine is much smaller (1.5 to 3 mg/L). There are

considerable amounts of RSV in white wine and other types of wine, as well as in grape

juice and the concentration of RSV appears to depend both on the type of grape and

environmental factors (de la Lastra and Villegas 2005). Studies show that beneficial

effects of red wine and the skin and seeds of purple grapes are related to numerous

polyphenols, e.g., flavonoids (quercetin, catechins, procyanidin) and RSV. RSV was first

isolated from the roots of White Hellebore in 1940 by Takaoka, and later, in 1963, by

Nanomora (Baur and Sinclair 2006) and from the roots of Polygonum cuspidatum or

Japanese Knotweed (a plant of South Asian herbal medicine) (Anekonda 2006; Baur and

Sinclair 2006; Dasgupta and Milbrandt 2007).

Chemical properties: RSV exists as two isomeric forms: the biologically inactive cis-

form, and the active trans- form. UV exposure converts the trans-form to cis- form.

Trans- form of RSV is synthesized for in vivo, in vitro and ex vivo experiments. The

purified form of RSV extracted from Knotweed is also available as dietary supplements.

RSV is a stilbene and its estrogenic effects mimic diethylstilbestrol, a synthetic estrogen.

Physiologic effects of RSV: RSV is thought to have a cholesterol lowering effect

which hypothetically is responsible for the reduced risk of heart disease in people having

a Mediterranean diet. The fact that there is an epidemiologic link between moderate red

wine consumption (in spite of high fat diet), and decreased incidence of cardiovascular

disease in the French population (the French Paradox) suggests that some red wine

ingredients like RSV do have beneficial effects) (Anekonda 2006; Nanji and French

1986).

18

1.7.1. Beneficial Effects of RSV

Recent reviews (Anekonda 2006; Baur et al., 2006; de la Lastra and Villegas 2005; de

la Lastra and Villegas 2007; Opie and Lecour 2007) have reviewed the effects of RSV.

Since 1992 a number of studies suggested that RSV can prevent or delay multiple

diseases including cancer, cardiovascular diseases and ischemic injuries (neuroprotective

effects in response to brain ischemic injuries) (Gao et al., 2006; Zhang et al., 2003).

Other studies have shown that RSV increases resistance to stress and extends the lifespan

of yeast and vertebrates. The mechanism by which RSV confers these beneficial effects is

not clear. A series of studies have demonstrated that RSV mimics the effect of calorie

restriction in lower organisms by interacting with specific genetic pathways (Baur et al.,

2006; Calabrese et al., 2008; Civitarese et al., 2007; Dali-Youcef et al., 2007; Ghosh

2008; Guarente 2007; Smith et al., 2009). Calorie restriction, defined as consumption of

around 60% of normal diet, has anti-aging effects in organisms ranging from yeasts to

mammals. Calorie restriction has some obvious limitations in humans but the new

category of drugs called CR mimetics, e.g., RSV, are promising in prevention and

treatment of human diseases (Baur and Sinclair 2006; Roth, Lane, Ingram 2005).

1.7.2. RSV and Mitochondria Biogenesis

In 2006 Lagouge et al (Lagouge et al., 2006) reported that RSV induces OXPHOS

genes and mitochondrial biogenesis by increasing mitochondrial size and mtDNA content

in obese mice. Similarly, Baur et al in 2006 showed that RSV improves health and

survival of mice on a high calorie diet and has beneficial effects on metabolism (Baur et

al., 2006). These effects are associated with an increase in insulin sensitivity, a decrease

in insulin-like growth factor, an increase in AMP kinase activated protein (AMPK), an

increase in PGC-1α activity, and an increase in mitochondria number. RSV’s effect on

mitochondria biogenesis is considered to be tissue dependant. RSV induces

mitochondrial biogenesis in endothelial cells (Csiszar et al., 2009b), liver, and neurons

(Dasgupta and Milbrandt 2007; Onyango et al., 2009) but not in heart (Lagouge et al.,

2006). Importantly, at least some of these effects could be mediated by sirtuins (silence

information regulators) or SIRTs, from the histone deacteylase family (Baur et al., 2006).

19

1.7.3. RSV and Antioxidation

The close relationship between RSV and mitochondrial function became evident in

several ischemic brain injury studies performed by Zini et al. (Zini et al., 1999; Zini et

al., 2002). Zini and colleagues suggested that the mechanisms by which RSV might

conserve mitochondrial function in response to ischemic insult is by reducing ROS

generation (antioxidant properties) and by stabilizing the mitochondrial membrane. RSV

has long been considered to be an antioxidant; however the mechanisms by which RSV

responds to oxidative stress are not clear. Recent studies suggested that RSV suppresses

peroxidation of lipids and other macromolecules. Floreani et al in 2002 (Floreani et al.,

2002) showed that RSV induces catalase and quinone reductase 1(QR1) activities in

cardiac tissue and decreases the amount of ROS generated by Menadione. Thus, RSV

might act directly as a ROS scavenger and/ or might induce cellular natural antioxidants.

RSV’s cardioprotective effects are also thought by to be mediated its antioxidative

properties. RSV inhibits oxidation of low-density lipoprotein (LDL) particles (a known

risk factor for coronary heart disease and myocardial infarction), by increasing expression

of paraoxonase 1 gene (Holvoet 2004). It also changes the qualities of pro-oxidants in

alcohol. However the results of other studies failed to support an antioxidant role for

RSV (Turrens et al., 1997).

1.7.4. RSV and Sirtuins

RSV activates Sirt1 and increases the life span of yeast, nematode Caenorhabditis

elegans and fruit fly Drosophila Melanogaster. In recent years agents that target PGC1-�

and sirtuins have been proposed as new therapies in neurodegenerative as well as other

diseases (Chaturvedi and Beal 2008). In November 2008, researchers reported that

dietary supplementation with RSV significantly reduced plaque formation in the brains of

Alzheimer’s transgenic mice (Karuppagounder et al., 2009).

Sirtuins are a family of NAD+ dependent deacetylases that first were found in

Saccharomyces cerevisiae and named silent information regulator 2 (Sir2) proteins. It has

been shown that overexpression of Sir2 genes in yeast, worms and flies extends lifespan.

There are seven mammalian SIRT proteins (Sirt 1-7) (Dali-Youcef et al., 2007; Pfister et

20

al., 2008; Trapp and Jung 2006), and Sirt1 is the closest homologue to Sir2. It has been

postulated that the main function of sirtuin proteins might be to promote survival and

stress resistance in times of adversity. An in vitro study identified RSV as the most potent

Sirt1 activators (among 18 deacylators) (Anekonda 2006).

Little is known about the role of sirtuins in neuroprotection. Recent studies show that

RSV treatment attenuated polyQ toxicity in Huntington’s models (HdhQ111 knock-in

mice and polyQ mutant transgenic C. elegans) by inducing induced Sirt1 in mice and

Sir2 genes in worms (Parker et al., 2005). Other studies strongly implicated sirtuins in

neuroprotection and showed that CR mimetics can increase sirtuins in the brain (Araki et

al., 2004; Bedalov and Simon 2004). Araki et al suggested that the delayed axonal

degeneration in Wallerian degeneration mouse models could be due to the increased

synthesis of nicotinamide adenine dinucleotide (NAD) and increased expression of Sirt1,

causing activation of other genes responsible for neuronal protection (Araki et al.,

2004). In addition, Bedalov and Simon (2004) showed that RSV treatment prior to

axotomy decreases axonal degeneration.

RSV presumably modulates and rescues neurological functions in other neurological

disorders, such as brain ischemia, stroke, seizure, and epilepsy. In rat hippocampal

neurons, RSV inhibited voltage-activated K+ currents, suggesting that it may be useful

for treating ischemic brain injury (Gao et al., 2006).

It has been shown that RSV and its Sirt1 modulation effect may play a significant role

in protecting the neurons from brain insults in AD (Anekonda 2006). It was proposed that

Sirt1 activation by RSV suppresses the apoptotic proteins p53 and FOXOs (fork head box

transcription factors O) (Kim et al., 2007; Kobayashi et al., 2005) thus conferring

neuronal protection in AD brains. Further, RSV and subsequent Sirt1 expression in

Sprague–Dawley rat microglia and astrocytes induced nitric oxide synthase (iNOS) and

cathepsin B, protected neurons against A�-induced toxicity, and inhibited nuclear factor

NF-�B signaling, suppressing two apoptotic factor (Tsai et al., 2007). The molecular

effects of RSV on sirtuins other than Sirt1 are still unknown. Interestingly, RSV did not

show significant enzyme activation of human Sirt2 (Bouras et al., 2005). Taken together,

21

the emerging evidence from recent studies demonstrates that RSV activates Sirt1 and

other biological factors in yet undefined downstream pathways.

1.7.5. Bioavailability and Pharmacokinetics:

Like other oral medications and dietary supplements, the efficacy of oral (PO) RSV

administration depends on its absorption, metabolism distribution, and clearance. Our

knowledge about RSV’s pharmacokinetics and bioavailability in human is limited. Rat

studies show that after intravenous (IV) administration of purified RSV, plasma

concentration of aglycone drop rapidly with a half life of about 0.13 h, and then increased

4-8h after drug administration. In this study, the bioavailability of oral RSV was

estimated to be about 38% (de la Lastra and Villegas 2005). Wallace et al in 2005

showed that after IV and PO administration of RSV in human volunteers, a very small

amount of RSV found in the plasma and most of the oral dose was found in urine.

1.8. PC12 Cells and MPI Studies

While primary cultures are the optimal model in neuronal studies, clonal cell lines

provide significant experimental convenience in terms of cost and availability. There are

both rat and human (neuroblastoma) cell lines available. In our lab we use rat

pheochromocytoma (PC12) cells as a neuronal model in the MPI and mitochondrial

morphology studies.

Pheochromocytoma is a neuro-endocrine tumor originated from chromaffin cells of

medullary adrenal. This cell line was established by Greene and Tischler in 1976 and has

been extensively used ever since as a neuronal model in neurochemical and

neurobiological studies (Greene and Tischler 1976). PC12 cells differentiate in the

presence of nerve growth factor (NGF) into neuronal cells that have properties of

sympathetic neurons. Differentiated PC12 cells have axons and dendrites which are

useful for outgrowth studies. Greene and Tischler also showed that differentiated cells

can synthesize and store catecholamine neurotransmitters. In addition, PC12 cells grow

very rapidly and can be produced in large amounts making them useful for the studies

like protein import studies that require extraction and purification of proteins. In addition

the PC12 cells could be easily transfected with various clones.

22

1.9. Green Fluorescent Protein

Green Fluorescent Protein (GFP) transfected cells have been used in gene expression

and protein localizations studies. Wild- type GFP (wtGFP) consists of 238 amino acids

and exhibits bright green fluorescence when exposed to blue light. GFP traditionally

refers to the protein first isolated from the jellyfish Aequorea victoria, however, there are

other species which have GFP. GFP has a half life of about 26 hours. GFP major

excitation peak is at 395nm and minor peak is at 470nm, its emission peak is at 509nm

(Chalfie et al., 1994; Shimomura 2005; Tsien 1998). Modifications of wtGFP have

increased its stability. The cycling and oxidation of the 3 amino acids Ser65-Tyr66-Gly67

is responsible for GFP to emit a green light. GFP does not need cofactors for its emission

and formation of dimers does not affect the emission of a monomer. Numerous studies

have assessed protein import to mitochondria using isolated mitochondria and/or yeast

systems. Other studies have used muscle cells, fibroblasts, COS-7 and HeLa cells (Craig

and Hood 1997; Kanazawa et al., 1997; Terada et al., 1997; Wright et al., 2001; Yano et

al., 1998). However, comparable studies on neurons have not been done. The only studies

that measured MPI in vivo in a neuronal model were conducted on PC12 cells in our lab

(Sirk et al., 2007; Sirk et al., 2003). In our lab, studies on different aspects of

mitochondrial functions were performed on differentiated PC12 cells stably transfected

with nuclear encoded mitochondrially targeted GFP fusion protein (mtGFP) (Sirk et al.,

2003).

To make a mtGFP construct the N-terminal mitochondrial targeting sequence of

cytochrome c oxidase (COX) VIII was fused to the 5’ end of GFP. Then this construct

was incorporated in a vector which is responsive to Tetracycline (Tet). MtGFP synthesis

has been regulated negatively in the presence of Tet (Clontech, 2008). This is an

important feature of PC12 cells in our lab. Expression of mtGFP is under the control of

the tetracycline response element (TRE). The TRE is located upstream of the CMV

promoter and contains Tet operator (tetO) sequences. In the absence of tetracycline, a

Tet-controlled transactivator (tTA) binds to the tetO sequences and drives the expression

of mtGFP. Conversely, in the presence of tetracycline, tTA binding to tetO is reversibly

inhibited, and, consequently, mtGFP transcription is inhibited (Sirk et al., 2003).

23

Tetracycline allows our cells to be turned “On and Off”. This means that in the presence

of Tet mtGFP signal is declined and mtGFP in the mitochondria degrades until no signal

is detectable (mtGFP-off cells). In contrast, after removing Tet, mtGFP signal is induced

and increases over time as mtGFP is synthesized and subsequently imported into the

mitochondria (mtGFP-on cells). Since mtGFP fluorescence reflects GFP imported into

the mitochondrial matrix, turning on mtGFP expression and then measuring mtGFP

fluorescence allows measuring the MPI in vivo (Sirk et al., 2007; Sirk et al., 2003). In our

lab we have developed a rapid and sensitive technique for the MPI quantification by

measuring mtGFP expression using flow cytometry. MtGFP is not fluorescent until it has

been imported into mitochondria. This is because GFP fluorescence is strongly dependent

upon proper folding of the protein and mtGFP like most mitochondrial proteins must be

in a linear conformation to be import-competent (Sirk et al., 2003, Rizzuto et al., 1995

and 1996). The matrix targeted mitochondrial protein mtGFP contains the targeting

presequence which prevents intracytoplasmic folding of mtGFP. Subsequently, the

cleavage of the targeting presequence within mitochondrial matrix allows the proper

folding of mtGFP. Consequently mtGFP fluorescence in PC12 cells directly reflects the

mtGFP signal in the mitochondria. Previous studies by Sirk et al. (in 2003) confirmed

that mtGFP signal is minimal or undetectable in cytoplasm of PC12 cells (Figure 3A). In

addition cytoplasmic levels of mtGFP protein (pre-import) are low to undetectable under

normal conditions (Sirke te al 2003; 2007; (Yano et al., 1998). Recent studies

(Shulyakova Master’s thesis 2008) indicate that, as a general rule, levels of most nuclear

encoded mitochondrial proteins are low in the cytoplasm since they are either rapidly

imported into mitochondria after synthesis, or rapidly degraded by proteasomes (Figure

3B) .

The import of mitochondrially targeted proteins is frequently quantified by

immunoprecipitation and autoradiography of newly synthesized proteins in the

mitochondria, or by Western blot. Quantifying protein levels in whole cell lysates

(WCLs) by Western blot provides a measure of total protein levels, while quantification

of protein levels in cytoplasmic and mitochondrial fractions provides a measure of levels

of pre-imported and imported nuclear-encoded mitochondrial proteins respectively.

Previous studies in our lab demonstrated that flow cytometry could be used to monitor

24

the import and intramitochondrial turnover of mtGFP in live cells (Sirk et al., 2007; Sirk

et al., 2003). Continuing studies on MPI in our lab have also shown that glucose

deprivation decreases Tom20 and mtGFP import and Tom20 overexpression restores

Tom20 levels and rescues mtGFP import (Phan, Master’s thesis 2006). In addition,

exposure to sublethal amyloid beta peptide (A�1−42) inhibited the import of mtGFP and

of physiological mitochondrial proteins, resulting in the accumulation of ‘ectopic’

mitochondrial proteins in the cytoplasm, mitochondrial deficits, increased vulnerability to

oxygen-glucose deprivation and changes in mitochondrial morphology (Sirk et al., 2007).

A recent study indicates that Tom20 overexpression and/or antioxidants can provide

protection against oxidative stress in vitro (Phan et al., in preparation). These studies

suggest that even a minimal sustained decline in MPI has a negative impact on

mitochondrial activities.

25

Figure 3: MtGFP in mitochondria in differentiated PC12 cells. A: A live,

differentiated PC12 cell was imaged by confocal microscopy. The green fluorescent

elongated particles are mtGFP labeled mitochondria distributed in the cell body. Since

mtGFP levels in cytoplasmic fractions are low to undetectable under normal

conditions (by Western blot) and mtGFP is not fluorescent prior to being imported

into mitochondria, the fluorescent signal reflects only mtGFP imported into

mitochondria (Sirk et al., 2003). B: Almost immediately after synthesis, mtGFP either

imported into mitochondria or degraded in the cytoplasmic proteasomes (Shulyakova

Master’s Thesis, 2008).

Cytoplasm

Nucleus

mtGFP synthesis

Proteasomes

(Degradation)

Mitochondria

MPI

26

1.10. Rationale

My study focuses on MPI in differentiated PC12 cells, a well-documented model of

mammalian neurons. The rationale for my work is based upon the following. First,

neuronal survival depends on mitochondria and mitochondrial function depends on

mitochondrial protein import (MPI). Second, changes in MPI have implications for

mitochondrial activities. Literature shows that at least one of the progressive

neurodegenerative diseases (MTJ) is caused by defective protein import and many other

neurodegenerative diseases are related to defects in mitochondrial proteins. Third, MPI

enhancement improves the outcome of Alzheimer’s disease (AD), Parkinson’s disease

(PD), Huntington’s disease (HD), stroke, diabetes and neuronal regeneration and may

also slow down aging. Fourth, studies have shown that RSV treatment increases

mitochondrial biogenesis which in turn requires increase in MPI. However, it is not

known if RSV affects MPI, and if it does affect MPI what are the underlying

mechanisms.

I hypothesize that RSV treatment increases MPI in neurons independently of

increased mitochondria biogenesis.

27

1.11. HYPOTHESES and SPECIFIC AIMS

A. RSV regulates MPI in neurons. Specifically, in differentiated PC12 cells RSV

alters the import of mitochondrially targeted GFP (mtGFP) and other

physiological mitochondrial proteins.

B. RSV can increase MPI independently of mitochondrial biogenesis.

Specific Aims

1. To establish dose-response for RSV toxicity in differentiated PC12 cells.

2. To determine if RSV alters Tom20, a major translocase protein that is not

imported in classical sense, and/or the import of mtGFP, mtHsp70, a chaperone

protein, and mtTFA, a protein involved in mitochondria biogenesis.

3. To identify the signaling pathways triggered by RSV, specifically the role of ROS

and mitochondrial membrane potential.

28

Materials and Methods

29

2.1. Creation of Stable PC12 Tet-off/MtGFP Cells

A rat pheochromocytoma cell line (PC12) stably expressing GFP in mitochondria

under regulation of tetracycline was previously developed in our lab (Sirk et al., 2003).

Mitochondrially targeted GFP was created by cloning the COX VIII N-terminal

targeting sequence into the 5’ end of wt-GFP. PC12 cells were co-transfected with a

pTRE/mtGFP in which mtGFP expression is regulated by pTRE/Tet-off system. In this

system when tetracycline is present, mtGFP is not expressed in the cells, and upon

removal of tetracycline mtGFP expression is rapidly (within 15 minutes) resumes.

2.2. Culturing Undifferentiated PC12 Cells

Undifferentiated Tet-off/ mtGFP PC12 cells were cultured in T75 flasks flask

(Falcon, #353136)or on 10 cm tissue culture plastic dishes (Falcon, # 35003) in a 5%

CO2 and 95% air humidified incubator. The cultures were grown in the presence of RPMI

1640 media (Gibco, #11875-093) containing10% of horse serum (Gibco, #16050-122)

and 5% fetal bovine serum (Gibco, #16000-044), and 0.5% Penicillin-Streptomycin

(10.000 Unit per milliliter(ml)). The cells were fed at least 3 times a week (every other

day) by complete media exchange and passaged when the cultures were 80% confluent.

The optimal dose of Tet to effectively inhibit mtGFP synthesis is 50 nanogram (ng)/ml

(Sirk et al., 2003). To suppress mtGFP expression and create mtGFP negative (mtGFP-

off) cells, PC12 cells were grown in the presence of Tet for at least 3 days.

To induce and obtain maximal mtGFP expression levels Tet removed from media and

the cells were cultured in the absence of Tet for at least 7days (mtGFP-on cells, see figure

3A).

30

2.3. Culturing Differentiated PC12 Cells

PC12 cells were differentiated by using15% serum media supplemented with 2.5S

NGF (Harlan Bioproducts, Indianapolis, IN, #005017) for a final NGF concentration of

25ng/ml.

Cells were differentiated for 4-5 days prior to each experiment in T75 flasks or 10 cm

tissue culture dish. After 4-5 days of differentiation cells were harvested by gentle

trituration with Ca2+- and Mg2+- free Hank’s Balanced Salt Solution (HBSS) (Gibco,

#14170), centrifuged at 1500 rpm for 4 minutes to pellet the cells and then cells were re-

plated at a density of 4x106/dish or ml in 1% serum (0.66% horse serum, 0.33% fetal

bovine serum) plus NGF (with or without Tet) on plastic dishes coated with 5% rat tail

collagen . Cells were left overnight in 1% serum plus NGF media to promote neurites

outgrowth.

The expression of mtGFP was induced 2-3 h prior to drug addition, by washing out

Tet from the media. To wash out Tet the plating media was replaced with 1% serum plus

NGF media (without Tet) and the cells were incubated for 10 minutes. This procedure

was repeated 3 times to completely remove Tet from the media (Figure 4).

2.4. Preparation of 100mM RSV Stock Solution

RSV (Tocris Bioscience, Ellisville, Missouri, USA, #1418) was dissolved in 95%

Ethanol (EtOH, Commercial EtOH, #9386) to make 100 mM (millimolar) stock

solutions. To keep the EtOH concentration at lowest, intermediate (10- 25 and 40mM)

stock solutions were prepared with appropriate amounts of 100mM stock solutions

diluted in 1% serum media plus 25ng/ml NGF. Control media contained the equal

amount of EtOH diluted in 1% serum plus 25ng/ml NGF. For final concentrations of

EtOH at different doses of RSV in the RSV dose response experiments see table 2.

31

mtGFP M

GFP-offN

Tet

M

GFP-on

mtGFP

N

Figure 4: A model for studying mtGFP import in live PC12 cells. Differentiated

PC12 cells are stably transfected with an inducible mitochondrially targeted GFP

fusion protein (mtGFP). After expression, the targeting sequence of mtGFP facilitates

its import into mitochondria. Addition of Tet to the media inhibits mtGFP synthesis

and import (Tet-off system); these GFP-off cells do not fluoresce. Upon Tet removal

(Tet washout) the synthesis of the mtGFP is induced rapidly and mtGFP is imported

into mitochondria (GFP-on). MtGFP is not fluorescent until it is cleaved and folded in

mitochondrial matrix (N: nucleus, M: mitochondria, Tet: tetracycline).

32

Table 2: EtOH concentrations (%) in intermediate stock solutions used in RSV dose response experiments. µM=micromolar; mM=millimolar; sltn=solution.

Final concentration of RSV(µM) % EtOH in 10 ml % EtOH in 1 ml

5µM (intermediate stock sltn=10mM) 0.00475 0.000475





200µM(intermediate stock sltn=10mM) 0.18 0.018

33

2.5. Preparation of Stock Solutions (Mitotracker Green, CCCP, Rhodamine 123 and

DCF Experiments)

To prepare stock solutions of 1mM Mitotracker Green FM (MT Green) (Invitrogen-

Molecular Probes, #M7514), 10mM carbonyl cyanide m-chlorophenylhydrazone (CCCP)

(Sigma, #C2759), 4mM 5’6’CMH2DCF-DA (Molecular probe, #C-6827) each drug was

dissolved in dimethyl sulfoxide (DMSO) to the appropriate concentration. All stock

solutions were stored as aliquots at -20˚C until immediately prior to use. A 2.63mM stock

solution of Rhodamine 123 (Rh123) (Molecular Probes, #R302) was prepared in ddH2o

and was stored at 4˚C until use.

Flow cytometry measures the fluorescence signal and analyzes cell size. Flow

cytometry measures signal in one cell at a time but can measure several hundred cells per

second (Invitrogen website, Flow cytometry tutorial).

Flow cytometry experiments were performed on the basis of protocols previously

described by Sirk et al. (Sirk et al., 2003). Generally, 4-5 day differentiated PC12 cells

were plated on a 5% collagen coated 12-well dishes (Falcon, #3043) at a density of 2.5 x

105 cells per well in 1% serum plus NGF (plus 50ng/ml Tet for GFP-off cells) media and

incubated overnight. Following incubation, media was replaced with RSV contained

media and after specific time points cells were harvested in ice cold Ca2+- and Mg2+-free

Phosphate Buffer Saline (PBS) (1mM KH2PO4, 10mM Na2HPO4, 137mM NaCl, and

2.7mM KCl, pH 7.4) and analyzed by flow cytometry.

In general, to measure the signal from the live GFP-on cells, dead cells were gated

out by the addition propidium iodide (PI). One microliter per milliliter (µl/ml) of a one

milligram per milliliter (mg/ml) PI (Molecular Probes, #P-1304) stock solution was

added to the cells, and incubated for approximately 5 minutes prior to cell harvesting.

The interaction between PI and double stranded nucleic acids of dead cells yields a

fluorescent signal that is exclusively nuclear. Using the FACScan Flow cytometer

(Becton Dickinson, San Jose, CA) 10000 cells from each sample were analyzed. In all

cases, debris and cells that clumped together were gated out based on their FSC (Forward

scattering) and SSC (Side Scattering) profiles. The mtGFP signal was measured with the

34

first fluorescent detector (FL1) photomultiplier tube (PMT) channel, which measures in

the green range of the spectrum; 510-545 nanometer(nm). PI fluorescence was measured

using the FL3 PMT channel, which measures in the red range of spectrum (above 650

nm). MtGFP-on and mtGFP-off cell populations provided baseline mtGFP signal

intensity used in the data analysis.

2.6. Flow Cytometry

2.6.1. Assessment of MtGFP Import by Flow Cytometry

In each experiment there were two groups of differentiated PC12 cells: experimental

cells treated with RSV, and a control group treated with equal amount of EtOH (vehicle

controls). Tet was washed out from the cells prior to addition of RSV or EtOH as

previously described. Two to three hours after Tet wash out, the media was replaced with

RSV and EtOH containing media for experimental and control groups respectively. Cells

were incubated for up to 3 days and at different time points (12, 24, 48 and 72h), were

collected in ice-cold PBS and the mtGFP signal was analyzed by flow cytometry. In

all the experiments, mtGFP and PI signal of each cell was determined. Cells from three

different wells were analyzed per each experimental condition; measurements were done

in 10000 cells from each well. Based on the signal from mtGFP positive and negative

control cells the 10000 cells from each sample were divided into 4 populations; (a)

mtGFP-on cells, PI positive (dead) cells, (b) mtGFP-on, PI negative (live) cells, (c)

mtGFP-off cells, PI positive cells, and (d) mtGFP-off, PI negative cells. Typically,

analysis was performed on the GFP-on, PI negative population.

2.6.2. Analysis of Intramitochondrial MtGFP Degradation

To monitor mtGFP turnover/degradation rates, GFP-on cells were differentiated for 4

days (Tet washed out on the first day of differentiation) in 15% serum plus NGF media.

At the time of 50µM RSV or 0.5µl/ml EtOH treatment, 50ng/ml Tet was added to both

RSV treated and control cells media to inhibit mtGFP synthesis. Cells were harvested

with ice-cold PBS, and the mtGFP signal was quantified by flow cytometry at T=0, 24,

48, 72 and 96h post addition of Tet (at T=0). MtGFP fluorescence was calculated as

35

geometric mean and expressed as a percentage of mtGFP always ON cells (maximum

attainable GFP signal).

2.6.3. Analysis of the Mitochondrial Membrane Potential (MMP) by Flow Cytometry

Differentiated GFP-off cells were plated on 5% collagen coated 12-well dishes at a

density of 2.5 x 105 cells per well in 1% serum plus NGF. To prevent mtGFP expression

50ng/ml Tet added to the media. Rhodamine123 (Rh123, Molecular Probes, #R-302) was

used to assess mitochondrial membrane potential. Rh123 is a lipophilic dye for which

uptake by mitochondria is dependent upon inner membrane potential. A 125�M stock

solution was prepared by dissolving Rh123 in ddH2O. PC12 cells were loaded with

Rh123 to achieve 0.5�M final concentration and incubated for 30 min. To ensure that

Rh123 was used in non-quenched mode, a set of PC12 cells was incubated with 20�M

Rh123 to create a positive control. Negative controls, where mitochondrial potential was

lost, were prepared by incubating PC12 cells with 20�M CCCP. CCCP is a protonophore

mitochondrial membrane uncoupler that blocks MMP and consequently decreases Rh123

uptake by mitochondria. After over night incubation with CCCP the cells were labeled

with 0.5�M Rh123. Rh123 fluorescence is in the green spectrum (511nm), and Rh123

signal was assayed in FL1 channel and calculated as geometric means.

2.6.4. Measurements of the Mitochondria Mass in PC12 Cells

To assess mitochondria mass in cells treated with 50µM RSV and 0.5µl/ml EtOH

mitochondria were stained with MitoTracker Green FM (MT Green) (Invitrogen,

#M7514). Differentiated GFP-off cells were plated on a 5% collagen coated 12-well

dishes (Falcon, #3043) at a density of 2.5 x 105 cells per well in 15% serum plus NGF

and 50ng/ml Tet media. Differentiated PC12 cells were kept in tetracycline containing

medium and prepared in the same manner as for measurements of the mitochondrial

membrane potential. Cells were loaded with MT Green to obtain 20nM final

concentration. After 30 minutes incubation the medium was replaced with ice-cold PBS

to wash out the MT Green. Cells were then harvested with ice-cold PBS. The MT signal

was measured using the FL1 channel; MT Green is excited at 490nm and emits light at

516nm.

36

2.6.5. Assessment of ROS Generation in Response to RSV Treatment

ROS generation has been shown to interfere with mitochondrial protein import. To

determine whether the changes in ROS generation could be implicated in the observed

increase in mitochondrial protein import, ROS generation was measured by use of the

dye 5’6’CMH2DCF-DA in differentiated PC12 cells treated with 50µM RSV and controls

(0.5µl/ml EtOH). Cells prepared and treated with RSV and EtOH as in MMP experiment

except that the cells were incubated with 10µM DCF (Molecular probe, #C-6827) for 30

minutes. PI was also added to the cells for about 5 minutes and the cells were harvested

in cold PBS for flow cytometry. DCF enters the cell and has its acetate group cleaved by

esterases trapping the non-fluorescent DCFH internally. Cellular hydrogen peroxide,

hydroxyl groups and other free radicals oxidize DCFH to DCY, the fluorescent form.

Positive controls were incubated with 10mM H2O2 (30% w/v) (Sigma,#H3410). The

DCF signal was measured using the FL1 channel.

2.7. Western Blot Analysis of Mitochondrial Protein Import and Synthesis

2.7.1. Preparation of the Whole Cell Lysates and Subcellular Fractions

Western blot analysis was used to determine the effects of 50µM RSV treatment on

levels of mtGFP in WCLs and mitochondrial (MT) fractions. Differentiated GFP-off cells

were plated on 5% collagen coated 100mm dishes (Falcon, #3003) at a cell density of 2 x

106 cells per dish in 15% serum plus NGF and 50ng/ml Tet media and were incubated

overnight. Following Tet washout, media was replaced with 50µM RSV or 0.5µl/ml

EtOH in 1% serum plus NGF media and cells were incubated for 12h, 24h, or 48 h. Cells

were harvested with ice-cold PBS, collected in cooled centrifuge tubes, and spun down at

4000g for 4 minutes. The supernatant was removed and the pellet was resuspended in

1ml PBS and transferred to an Eppendorf tube. Cells were spun down at 10000g for 30 s.

This washing procedure was repeated 3 times. After washing was complete, WCLs or

MT and cytoplamic (CP) fractions were prepared from the cell pellets.

37

To prepare WCLs, cells were resuspended in 100-250µl of cell lysis buffer [50mM

Tris-HCl (pH 7.5), 150mM NaCl, 1% nonidet P-40, 0.5% sodium deoxycholate, 1 tablet/

25ml protease inhibitor (Roche), ddH20] depending on the pellet size.

To prepare MT and CP fractions cells were resuspended in 250 - 500µl (based on

pellet size) of digitonin release buffer [(250mM sucrose, 17mM MOPS (pH 7.4), 2.5mM

ethylene diamine tetraacetic acid (EDTA), protease inhibitors and 0.8 mg/ml of freshly

added digitonin] and allowed to stand at room temperature (RT) for 1 min. After that the

cell suspension was transferred into a pre-cooled 2ml Dounce glass homogenizer. Cell

plasma membranes were disrupted using 20-40 strokes with the low clearance pestle. The

lysate was spun at 1,000g for 5 minutes in order to pellet down unbroken cells and nuclei.

The supernatant was removed and spun at 21,000g for 30 minutes at 4°C yielding both

the mitochondrial fraction (pellet) and the cytoplasmic fraction (supernatant). The

mitochondrial fraction was resuspended in 250 – 500µl of cell lysis buffer (50mM Tris-

hydrochloric acid (HCl) (pH 7.5), 150mM NaCl, 1% Nonidet P40, 0.5% sodium

deoxycholate, and protease inhibitors) and triturated to break the mitochondrial

membrane. If necessary, samples were frozen at –80°C until use.

2.7.2. Determination of Protein Concentrations in WCLs, CP and MT

To determine concentration of proteins in WCLs and subcellular fractions a

colorimetric DC Protein Assay (BioRad, #500-0116) was used according to

manufacturer’s manual. The BioRad assay is an improved version of the Lowry assay and

is based on the reaction of proteins with an alkaline copper tartrate solution and Folin

reagent. The assay is performed in two steps leading to color development. Color

development is primarily due to presence of the amino acids tyrosine and tryptophan,

and, to a lesser extent, cystine, cysteine, and histidine. A differential change in color

occurs in response to various protein concentrations. Developed color is blue with

maximum absorbance at 750nm and minimum absorbance at 405nm.

38

The assay was used in the micro-plate format. Absorbance was read at 630nm, single

wavelength using an ELISA plate reader. The protein concentration was determined by

comparing absorbance of unknown protein concentrations to absorbance of known

concentrations of protein standards (BioRad, #500-0005). Measurements for each

unknown were performed in triplicates.

2.7.3. Determination of MtGFP Protein in WCLs and Subcellular Fractions

To determine levels of mtGFP in WCLs, MT and CP fraction the equivalents of

25µg of WCLs and CP, and 15µg of MT were mixed with the equivalvolume of 2x SDS

reducing electrophoresis loading buffer (4% SDS, 10% B-mercaptoethanol, 20%

glycerol, 0.02% bromophenol blue, 12.5mM Tris-HCl, pH 6.8). Samples were heated at

100˚C for 5 minutes to denature the proteins and subsequently resolved on a 12%

Tris/Glycine SDS-PAGE gel (pH 8.8) with a 4% stacker (pH 6.8) (30 Volts through

stacker followed by 70 - 80 Volts through separating gel for approximately 2h). The

proteins were then transferred onto a nitrocellulose membrane (Bio-Rad) (30 Volts,

overnight at 4˚C). Following rinsing steps in tris-buffered saline tween-20 (TBS-T)

buffer (20mM Tris-base, 137mM NaCl, 1M HCl, 0.1% Tween-20, ddH20, pH 7.6) the

membrane was blocked with 5% blotting grade nonfat dry milk (Bio-Rad, # 170-6404)

for 1 h and then probed overnight at 4˚C with primary antibody for mtGFP

(1:3000, rabbit anti-GFP from Molecular Probes, Eugene, OR, # A-6455). Following 3

rinsing steps in TBS-T, membrane was incubated for 1 hour in an anti-rabbit horseradish

peroxidase (HPR)-linked secondary antibody (Amersham, #LNA934VI/93/01) at a

1:15000 dilution. To visualize the bands, membrane was exposed to reagents for enzyme-

linked chemiluminescence (Life Science Products, #NEl105) for 1 min and the bands

were recorded on Kodak MR-1 X-ray film (Rochester, NY).

39

2.7.4. Western Blot Analysis of Physiological Proteins in WCLs and Subcellular

Fractions

To measure physiological protein levels (mtHsp70, mtTFA, Tom20, glyceraldehyde-

3-phosphate dehydrogenase (GAPDH) Western blots of WCLs and subcellular fractions

were carried out as previously described in section 2.7.3 for mtGFP protein. Using

primary and secondary antibodies the proteins of interest were probed. The

concentrations for primary antibodies were as following: Anti-Tom 20 rabbit polyclonal

antibody (Santa Cruz Biotechnology Inc., #SC-11415), 1: 2000 dilution; anti-Mortalin

(mtHsp70) mouse monoclonal antibody (Affinity Bioreagents, #MA-3-028), 1:500-

1:1000 dilution; anti-mtHsp70 mouse monoclonal antibody (Stressgen, #SPS-825),

1:500- 1:1000 dilution; anti-mtTFA goat polyclonal antibody (Santa Cruz Biotechnology

Inc., #SC-23588), 1:500 dilution; anti-GAPDH mouse monoclonal antibody

(Calbiochem, #CB-1001), 1:15000 dilution. Secondary antibodies were as follows: anti-

mouse HPR-linked secondary antibody for mtHsp70 were used at 1:2000-1:5000, and for

GAPDH were used at of 1:5000 and for PGC1-� at 1:2000 dilutions; anti-rabbit HPR-

linked secondary antibody (Amersham, #LNA934VI/93/01) for Tom20 was used at

1:5000 dilution, and anti-goat HPR-linked secondary antibody for mtTFA was used at

1:5000 dilution .

2.8. Statistical Analysis of Data

Statistical analyses were performed using Excel 2003. Protein concentrations

obtained from the Bio-Rad assay were calculated using Excel 2000. P values were

calculated by using single ANOVA. To address the problem of multiple comparisons in

RSV dose-response experiments, a Bonferroni correction was used. Briefly, if you

perform n tests, each of them with a given probability (�), the probability that at least one

of them will be significant is � n*�. In ANOVA we want this probability to equal �. By

solving for �, we get � = � / n.

In other words, If an experimenter is testing n dependent or independent hypotheses

on a set of data, the family error rate which is the probability of making one or more false

discoveries among all the hypotheses when performing multiple pairwise tests is

40

generated by testing each individual hypothesis at a statistical significance level of 1/n

times. Thus if we want the significance level for the whole family of tests to be (at most)

�, then the Bonferroni correction would test each of the individual tests at a significance

level of (�/n). Statistically significant simply means that a given result is unlikely to have

occurred by chance assuming the null hypothesis is correct.

Unless otherwise stated all experimental data is from 3 or more independent

experiments (n=3). To obtain flow cytometry data, each experimental parameter was

measured in three wells. The mean fluorescence for each well was calculated from 7000

to 10000 cells/ well and data from the triplicates wells pooled. Mean values for an

individual experiment were the average value of these three wells and were therefore

based on 21000-30000 cells. Data were normalized by expressing it as percentage of

control values.

Data collected from Western blot experiments were obtained from 3 or more separate

experiments. Band intensities were measured using the Fluo-S Multi-Imager program

(Quantity One, Bio-Rad). Briefly, a box was drawn around the largest band, excluding as

much background as possible. This box then was copied and pasted around every band in

the blot. A similar box was placed in an area closest to each band to get background

density readings. The density was determined for each band (background subtracted) and

expressed as OD/mm2. For WCLs differences in protein loading were controlled for by

expressing each sample relatively to GAPDH (loading control). For mitochondrial

fractions samples were loaded in triplicates, by use of Bradford quantification equal

amount of protein were loaded in each well then an average was obtained for each

sample.

41

Results

42

3.1. RSV Significantly Increases MtGFP Levels in Mitochondria

3.1.1. The Optimal Dose for RSV

Mitochondrial protein import was assessed by using a differentiated PC12 cell line

stably transfected with a Tet negatively regulated inducible mitochondrially targeted

GFP (mtGFP). As shown by previous studies in our lab (Sirk et al., 2003 and 2007)

mtGFP is not fluorescent until its import process is completed by cleavage and folding

in mitochondrial matrix, thus the mtGFP signal reflects mtGFP imported into

mitochondria. Optical sectioning by confocal microscopy also confirmed that there is

no mtGFP signal in cytoplasm and parallel western blot studies showed no mtGFP in

cytoplasmic fractions under normal conditions (Sirk et al., 2003; 2007; Shulyakova

Master’s thesis 2008). RSV treatment resulted in a dose-dependent increase in the

mtGFP signal (see figure 5A).

43

Figure 5: Effects of the exposure of different doses of RSV after 24h on mtGFP signal

and cell death. A: Graph represents the flow cytometry data of the effects of different doses of RSV on mtGFP signal in one experiment. Differentiated PC12 cells treated with 5, 10, 25, 50, 75, 100, and 200 µM of RSV for 24h. The mtGFP signal with 50µM of RSV was 656 ± 31 SD arbitrary units (AU), with 75µM was 591± 25 SD and with 100µM was 571 ± 15 SD (AU) which were much higher than the mtGFP signal in controls (480 ±19 AU). Each sample done in triplicate, 10000 cells per each sample counted. The mean GFP fluorescent signal from GFP-on-PI- cells (70-80% of 10000 cells counted) used for graphs. B: Graph shows the results of the effects of different doses of RSV on cell death under basal conditions in one experiment. The average percent cell death ± SD after 24h of RSV treatment with 5, 10, 25, 50,75, 100 and 200 µM doses and controls were 15.8% ± 0.9% SD, 14.3% ±1.6% SD, 14.9% ± 1.4% SD, 11.8% ± 1.2% SD, 10.4% ± 2.4% SD, 14.8% ± 1.7% SD, 16.7% ± 2.1% SD and 18.1 ±1.1 SD respectively. Each experiment done in triplicate samples; 10000 cells counted per each sample. The %PI stained cells used for graphs.

44

A

Figure 5

B

ControlRSV(µM)

ControlRSV(µM)

45

The effects of RSV on the mtGFP signal and cell death were measured by flow

cytometry after 24h of treatment.

Since flow cytometry measurements of any fluorescent signal vary between

different experiments due to changes in laser intensity (data not shown), for

cumulative results, data from multiple experiments were normalized to controls and

expressed as percent control.

RSV at 50, 75 and 100µM concentrations increased the mtGFP signal compared to

time and dose matched EtOH controls 24h post treatment (see figure 6A).

The increase in the mtGFP signal did not significantly differ among 50µM (25.1%

± 5.0% SEM), 75µM (21.5% ± 1.2% SEM) and 100µM (20.5% ± 4.5% SEM) thus I

used 50µM RSV in all of the subsequent experiments because at this dose we had the

desirable effects with less amount of the drug (cost effective) and lower concentration

of EtOH (avoiding EtOH intoxication). In addition this concentration was safely used

in previous studies by other researchers (Aggarwal B.B, Shishodia S., review 2006).

3.1.2. Effects of RSV on Cell Survival in Differentiated PC12 Cells

To determine whether RSV has any toxic effects on differentiated PC12 cells, cell

death was evaluated by flow cytometry using PI. PI labels the nuclei of dying cells by

diffusing into the cells and intercalating in between double stranded DNA. The

fluorescent signal from highly stained nucleus is quantified by flow cytometry.

RSV treatment at 50, 75 and 100µM for up to 24h showed no neurotoxicity but

significantly reduced the normally low basal rates of cell death compared to time

matched controls (figure 6B). Further experiments using 5, 10 and 25µM RSV did not

reduce cell death. As shown in figure 5B (which shows the values from one

experiment), cell death in cells treated with 50 or 75 µM RSV was reduced by 35% ±

6% SD and 42%±13% respectively, compared to time matched controls. Cumulative

results from 4 independent experiments (figure 6B)

46

Figure 6: Effects of the exposure of different doses of RSV after 24h on the

mtGFP signal and cell death. A: Graph shows the mtGFP signal in cells treated with 25, 50, 75, 100 and 200µM RSV in 5 independent experiments. RSV significantly increased mtGFP signal at 50µM (25.1%±5.0% SEM, n=5, p<0.001), 75 µM (21.5%±1.2% SEM, n=5, p<0.001) and 100µM (20.5%±4.5% SEM, n=4, p<0.001). Bars represent mean of mtGFP signal in independent experiments, n=5 for 50and75 µM, n=4 for 25 and100 µM, and n=3 for 200 µM RSV. Each experiment done in triplicate samples, 10000 cells per sample counted. The mean mtGFP fluorescent signal from mtGFP-on + PI- cells (70-80% of 10000 cells) used for graphs; *p<0.01 (ANOVA and Bonferroni Post-test) versus dose matched vehicle (EtOH) controls. B: Graph shows cumulative results of independent experiments for cell death in response to RSV at 24h. The cell death determined with PI staining. Bars represent the means of percent dead cells (PI stained cells) in n=5 experiments for 50 and 75µM, n=4 for 25 and100µM and n=3 for 200µM, each experiment done in triplicate samples; each sample represents the PI stained gated cells from 10000 cells;*p<0.01(ANOVA and Bonferroni Post-test) versus controls.

47

Figure 6

***

A

B

* * *

ControlRSV(µM)

ControlRSV(µM)

48

(n=5 for 50 and 75 µM; n=4 for 100 µM; p<0.001) show that RSV at 100µM also

decreases basal cell death. Overall my results show that RSV at 24h promotes cell

survival.

3.1.3. 50 µM RSV Increases MtGFP Levels in Mitochondria

To monitor the effects of 50µM RSV on the mtGFP signal over time mtGFP was

measured by flow cytometry for up to 72h post induction of mtGFP synthesis.

MtGFP synthesis was induced by removing Tet from the media 2-4h prior to RSV

addition. As shown in figure 7 the mtGFP signal increased in both RSV treated cells

and in controls at all of the time points examined (12h, 24h, 48h and 72h). Figure 8

shows that starting at 12h the mtGFP import in RSV treated cells was significantly

higher compared to controls. The mtGFP import was higher in RSV treated cells

versus controls at 24h and 48h (there was no significant increase in mtGFP import at

48h compared to 24h).

49

Figure 7: 50µM RSV increases the mtGFP signal (import) for up to 3 days. A: The panel shows a representative dot plot of the relative mtGFP signal in a population of mtGFP-off cells (top) and RSV treated cells (bottom) separated into 4 quadrants; top right: PI positive (dead) mtGFP-on cells, bottom right: PI negative (live) mtGFP-on cells, Top left: PI positive mtGFP-off cells, and bottom left: PI negative mtGFP-off cells. Data for mtGFP import studies are generally taken from live cells expressing mtGFP, i.e. lower right quadrant (mtGFP-on + PI- ; ~ 70-80% of the total population). B: Representative flow cytometry histogram traces taken at 24h post-addition of RSV and EtOH (blue trace= RSV, yellow trace= EtOH (control), purple= mtGFP-off, red= mtGFP-on cells) for one experiment. Each histogram represents the relative mtGFP signal from 10000 cells. C: Graph represents the mtGFP signal in 4 different populations of the cells: mtGFP-off (purple line), mtGFP-on (red line), RSV treated (blue line) and dose matched EtOH controls (Ctrl=yellow line). Experiments done at 12h, 24h, 48h and 72h by flow cytometry. I: The purple line (bottom) shows that the relative mtGFP signal in the population of mtGFP-off cells at 12h, 24h, 48h and 72h were 19.8, 25.8, 20.53, 21.66 (AU) respectively. These cells were used as mtGFP negative controls. II: The red line is showing mtGFP signal in mtGFP-on cells. MtGFP signal (AU) at 12h, 24h, 48h and 72h were 269.0±2.3SD, 397.8±4.2SD, 466.7±6.69SD, 676.0±7.1SD respectively. These cells were used as mtGFP positive controls. III and IV: MtGFP signal (AU) in RSV(50µM) treated cells -blue line- at 12h, 24h,48h and 72h were 301.3±2.6SD, 406.3 ± 4.7SD, 833.6 ± 10.0SD, 930.2± 32.2SD and the signal (AU) for their time matched controls (yellow line) were 262.7±6.7SD, 320.0±1.0SD, 602.8±23.1SD,731.1±32.1SD respectively. The mtGFP signal in flow cytometry experiments are generally taken from live cells. Each experiment done in triplicate samples and 10000 cells per sample counted; mean GFP fluorescent signal from mtGFP-on + PI- cells (70-80% of 10000 counted cells) used for graphs .

50

0

200

400

600

800

1000

12h 24h 48h 72h

mtG

FP

sig

nal

(A

U)

Time(h)

mtGFP signal over timeGFP on

GFP Off

Control

RSV

A

Figure 7

B

C

IV

III

II

I

mtGFP-off---- RSV----

mtGFP-on---- Control----

51

*

**

*

Figure 8: 50µM RSV increases mtGFP import as early as 12h post exposure and

continues to increase the signal for up to 2 days. The graph shows the cumulative

results of the effects of RSV on mtGFP import over time. At 12h the mean mtGFP

signal in RSV treated cells was 11.0%�1.9% SEM higher than time matched controls

(n=4). MtGFP signal continue to increase significantly for up to two days. MtGFP

signal at 24h was 25.0%�4.6% (n=8) and at 48h was 38.6%�4.3% (n=5) higher than

time matched controls . Bars represent the means, expressed as percent control �

SEM, each experiment done in triplicate and each sample represents the mean GFP

signal from 70-80% of 10000 cells counted ; *p<0.05, **p<0.01 (ANOVA) versus

time matched controls.

52

Western blots of mitochondrial fractions confirmed that 50µM RSV increased

mtGFP levels in mitochondria 12h, 24h and 48h post treatment compared to time

matched controls (figures 9C). RSV altered mtGFP levels in WCLs (which reflect

expression and import into mitochondria) by 24h (but not at 12h and/or 48h) compared

to time matched controls (figure 10). The representative mitochondrial fractions and

WCLs Western blots at 12h, 24h and 48h are shown in figures 9B and 10A.

Altogether, my results from flow cytometry and Western blot indicate that mtGFP

levels in mitochondria are increased with 50µM RSV treatment in differentiated PC12

cells by 48h.

Western blotting did not detect any mtGFP in cytoplasmic fractions (figures 11,

12). Loading 50µg proteins did in some cases result in a faintly detectable band in the

CP fractions consistent with previous studies using immunoprecipitation and

autoradiography (Sirk et al., 2003). The fact that the CP fractions are also negative

for Tom20 confirms that there is no mitochondrial contamination.

53

Figure 9: 50µM RSV increases mtGFP levels in mitochondria. To determine the effects of 50µM RSV on mtGFP levels in mitochondria and total mtGFP levels Western blot performed on RSV and control treated cells for up to 48h. A: Graph shows increased intramitochondrial mtGFP levels by 24h in one Western blot experiment. MtGFP levels in mitochondria of RSV treated cells were 123.5 ± 9.4 SD (AU) versus 92.6 ± 10.8 SD (AU) in time matched controls. Each experiment was done in triplicates. Bars represent the mean mtGFP levels in arbitrary units (AU) ± SD in one experiment. B: Representative Western blot of the mtGFP in mitochondrial fractions (15µg of protein loaded) from control cells and RSV treated cells at 12h, 24h and 48h. Mitochondrial fractions were loaded in triplicates. C: Western analysis showed that mtGFP levels at 12h (n=4), 24h (n=9) and 48h (n=5) post RSV treatment were 21.0% ± 4.6% SEM, 33.3% ± 3.4% SEM and 25.8% ± 11.7% SEM higher compared to time matched controls, respectively. Bars represent the mean of GFP levels in mitochondria, expressed as percent control ± SEM; each experiment done in triplicate; *p<0.05 (ANOVA) versus time matched controls.

54

50µM RSVControl

28kDa

28kDa12h

24h

48h

28kDa

mtGFP

Mitochondria

Figure 9A

B

C

mtG

FP

in

mit

och

on

dri

a

(% o

f co

ntr

ol)

ControlRSV(µM)

** *

55

Figure 10: 50µM RSV alters mtGFP expression by 24h. A: Representative Western blot of the mtGFP in WCLs (25µg of protein loaded) from control cells and RSV treated cells at 12h, 24h and 48h. Each sample has been probed for both anti-mtGFP (top panels) and anti-GAPDH (bottom panels) antibodies. B: Total mtGFP level in WCLs was increased in response to 50µM RSV treatment by 24h but not at 12 and 48h. Bars represent the means, expressed as percent control, normalized to GAPDH levels of n=3 for 12h, n=6 for 24h and n=4 for 48h experiments.

56

B

12h 24h 48h

35kDa

28kDa

GAPDH

28kDa 28kDa

35kDa35kDa

mtGFP

Control RSV Control RSV Control RSV

Whole Cell

Figure 10

A

0

20

40

60

80

100

120

140

12 24 48

mtG

FP

no

rmal

ized

to

GA

PD

H(%

of

con

tro

l)

Time(h)

Total mtGFP (WCL)

*

57

WCL CP

28kDa

GAPDH35kDa

mtGFP

WCL CP

Control RSV

Figure 11: There is no detectable mtGFP in cytoplasmic fractions. Western blots

of Whole Cell Lysates (WCL) and cytoplasmic (CP) fractions extracted from

differentiated PC12 cells were probed with anti-mtGFP antibody and anti-GAPDH

antibody. MtGFP was detected in WCL but not in CP fractions confirming the purity

of mitochondrial fractions. Mitochondrial fractions used for detection of the target

mitochondrial proteins in the Western blot experiments. Protein load was 10µg of

WCL and 50µg of CP fractions per each well. The WCL and CP fractions are loaded

from one experiment.

58

CP

Control RSV

28kDa

Tom20

mtGFP

72kDa

17kDa

mtHsp70

GAPDH

34kDa

Figure 12: There is no detectable amount of mtGFP, Tom20 and mtHsp70 in CP

fractions. Western blots using 50µg protein loads of CP fractions showed no detectable

amount of mtGFP, Tom20 and mtHsp70 in CP fractions. The GAPDH used as positive

control for these blots. The 2 wells loaded with cytoplasmic fractions of control and

RSV treated cells.

59

3.2. Sustained RSV Slows Intramitochondrial Degradation of MtGFP

To assess whether intramitochondrial degradation of mtGFP alters in response to

50µM RSV, I monitored mtGFP degradation for up to 4 days (96h). To inhibit the

mtGFP synthesis 50ng/ml Tet added to mtGFP-on cells at the time of RSV addition

(Time zero or T=0) and then the mtGFP signal measured in RSV treated and control

cells. As expected the mtGFP signal declined rapidly after Tet addition in both RSV

and control cell populations. Sirk et al in 2003 using autoradiography showed that

mtGFP synthesis ceases very rapidly by Tet addition. This study showed that by 120

minutes after the addition of Tet, only a small residual amount of newly synthesized

mtGFP could be detected by autoradiography in the whole cell lysate (Sirk et al.,

2003). Cumulative results showed that the mtGFP signals in control and RSV treated

cells did not significantly change by 24h (figure 13). However as shown in figure 13

by 48h mtGFP signal degradation appeared to be significantly slower in RSV treated

cells.

3.3. RSV Does not Increase Mitochondrial Mass

To determine if the increase in mtGFP in mitochondria (shown by flow cytometry

and Western blot data) reflected an increase in mitochondria mass, we used Mito

Tracker (MT) Green FM to measure mitochondrial mass by flow cytometry (figure

14). The MT Green signal was not significantly different between control and RSV

treated cells; (p>0.05, n=6).

3.4. RSV Has no Effect on Cell Size

To determine if RSV treatment altered cell size (which could potentially alter the

flow cytometry signal), the forward scattering values from flow cytometry data

analyzed and the results shown in figure 15 indicate that the cell size did not differ

between RSV treated and controls at 24h (n=5; p>0.05) and 48h (n=5; p>0.05).

Taken together, the increase in mtGFP levels in mitochondria after 24h of RSV

treatment, coupled with unchanged levels in WCLs, unchanged intramitochondrial

degradation, unchanged mitochondria mass, and unaltered cell size suggest that at 24 h

60

RSV increases mtGFP import. Subsequently (by 48h) degradation of mtGFP within

mitochondria is also slowed.

61

Figure 13: 50µM RSV does not alter the intramitochondrial mtGFP degradation

in the first 24h. To determine the effects of 50 µM RSV on intramitochondrial mtGFP degradation, 50ng/ml Tet was added to cultures at the time of 50µM RSV or EtOH (control) addition to inhibit mtGFP synthesis. The mtGFP signal (remaining in the mitochondria) was monitored by flow cytometry for up to 4 days. A: Main graph shows mtGFP decline as percent of GFP signal in GFP- on cells at time zero (T0). Trend lines (orange for controls and green for RSV treated cells) were fitted to the scatter plots using exponential regression. The graph shows that the mtGFP decay over time is exponential [f=a*exp (-b*x)] with R2 values of 0.83 and 0.75 respectively. There was no significant decline in mtGFP signal in RSV treated compared to time matched controls after 24h of Tet exposure. By 48h, 72h and 96h, mtGFP remained significantly higher in RSV treated cells compared to control cells. At 24h the mtGFP signal in controls showed a 46.5% ± 4.7% SEM decline (of the signal of the mtGFP-on cells at T=0) and in RSV treated cells showed 39.0 ± 8.3%SEM. At 48h mtGFP signal had a 63.3% ± 2.7% SEM decline in control cells but a 49.6% ± 5.6% SEM in RSV treated cells which indicates that after 48h the signal in RSV treated cells was significantly higher than the signal in control cell. The mtGFP signal at 72h in control cells was reduced by 68.5% ± 1.2% SEM compared to 55.0% ± 2.8% SEM in RSV treated cells. By 96h the mtGFP signal in controls and RSV treated cells were reduced by 72.6% ± 1.2% SEM and 57.8% ± 2.4% SEM in control and RSV treated cells respectively. The mtGFP half-life was calculated to be 42h for control and 59h for RSV treated cells; n=7 for 24h; n=6 for 48h; n=3 for 72h and 96h; each experiment done in triplicates; *p<0.05; **p<0.01 (ANOVA) versus time matched controls. To clarify the mtGFP signal reduction at 48h an inset is included in the figure. B: Graph in inset shows the mtGFP signal in controls and RSV treated cells at 24h and 48h post Tet exposure. There was no difference in the mtGFP signal in controls compared to RSV treated cells at 24h post exposure to Tet. Bars represent the means, expressed as percent of mtGFP signal of mtGFP-on cells at T=0; n=7 for 24h; n=6 for 48h.

62

0

20

40

60

80

100

120

-24 0 24 48 72 96Time (h)

**

**

Figure 13

Intramitochondrial GFP degradation

B

A

*

mtG

FP

sig

nal

(%G

FP

-on a

t T

ime

0)

mtG

FP

sig

nal

(%G

FP

-on a

t T

ime

0)

ControlRSV(µM)

ControlRSV(µM)

63

Figure 14: Mitochondria mass was not affected by 50µM RSV. Flow cytometry

determined the effects of a 24h RSV(50µM ) treatment on mitochondria mass by use

of MT Green staining. The RSV and vehicle controls (EtOH) were loaded with 20nM

of MT Green. In each experiment a high dose MT Green (50 nM) sample was also

prepared to ensure that the MT Green signal is not quenched. The signal from mtGFP-

off cells without MT Green used as background signal. Graph shows the cumulative

results of 6 independent MT Green experiments on RSV treated versus dose matched

EtOH controls. Bars represent the mean, expressed as percent control � SEM, of n=6

experiments; each experiment done in triplicates; p>0.05 (unpaired Student’s t-test).

ControlRSV(µM)

64

0

20

40

60

80

100

120

24h 48h

Cel

l si

ze (

% o

f co

ntr

ol)

Time (h)

RSV and cell size

Figure 15: RSV does not affect cell size. In flow cytometry, forward scattering signal

correlates with cell volume. Since the fluorescent signal could be potentially affected

by alterations in cell size, to determine the effects of RSV on cell size, the flow

cytometry analyzed on the basis of forward scattering data. Bars represent the

mean, expressed as percent control � SEM, of n=5 experiments; each experiment done

in triplicates, p>0.05 (ANOVA).

ControlRSV(µM)

65

3.5. 50µM RSV Increases the Import of Physiological Mitochondrial Proteins

To determine the effects of 50µM RSV on physiologic mitochondrial proteins I

assessed levels of Tom20 (which is not imported in the classical sense), (Becker et

al., 2008) and mtHsp70 (a nuclear-encoded mitochondrial protein targeted to the

matrix), by Western blot of WCLs and mitochondrial fractions.. Tom20 is a

translocase of the outer mitochondrial membrane involved in the import of the

majority of preproteins and of all matrix targeted proteins (Grey et al., 2000),

mtHsp70 is a mitochondrial matrix chaperone critical for protein import into the

matrix and protein folding in the matrix (Deocaris et al., 2008; Geissler et al., 2001).

As shown in figures 16 and 17 levels of Tom20 in mitochondria increased in RSV

treated cells by 38% at 24h and by 37% at 48h compared to time matched controls.

Total Tom20 levels in WCLs increased about 40% in response to 50µM RSV

treatment at 48h compared to time matched controls. However Tom20 expression was

unchanged at 24h.

Figures 18 A and B show that mtHsp70 levels in mitochondria significantly

increased by 20% in RSV treated cells compared to time matched controls by 24h and

40% by 48h. Levels of mtHsp70 in WCLs (which reflect expression, import, and

turnover) did not change at 24h and 48h (figures 19 A and B).

66

Figure 16: 50µM RSV increases Tom20 levels in mitochondria. To determine the effects of 50µM RSV on Tom20 levels in mitochondria and Tom20 expression, Western blot performed on RSV treated and control cells at 24h and 48h. A: Representative Western blot of the Tom20 in mitochondrial fractions (15µg of protein loaded) from control cells and RSV treated cells at 24 and 48 h. Mitochondrial fractions were loaded in triplicates. The right sided triplicates are showing RSV treated and the left sided triplicates showing controls. B: At 24h Tom20 levels in mitochondria increased by 38% ± 11% SEM compared to time matched controls (n=4; p<0.05) and after 48h increased by 37% ± 4% SEM (n=5; p<0.05). Bars represent the means, expressed as percent control ±SEM; n=5 for 24h; n=6 for 48h experiments; each experiment done in triplicates; *p<0.05 (ANOVA) versus time matched controls.

67

Figure 16

A

B

50µM RSVControl

17kDa

Tom20

17kDa

24h

48h

Mitochondria

* *

68

Figure 17: 50µM RSV increases Tom20 expression. A: Representative Western blot of Tom20 in WCLs (25µg of protein loaded) from control cells and RSV treated cells at 24h and 48h. Each sample has been probed for both anti-Tom20 (upper panels) and anti-GAPDH (lower panels) antibodies. B: Total Tom20 levels in WCLs increased in response to 50µM RSV treatment at 48h by 40% ± 12% SEM compared to time matched controls (n=5; p<0.05). However Tom20 expression was unchanged after 24h (n=5; p>0.05). Bars represent the means, expressed as normalized values to GAPDH levels.

69

Figure 17

A

*

B

Whole Cell

GAPDH 35kDa35kDa

17kDa17kDa

24h 48h

Tom20

Control RSV Control RSV

70

Figure 18: 50µM RSV increases mtHsp70 level in mitochondria. To determine the effects of 50µM RSV on mtHsp70 levels in mitochondria and total amount of mtHsp70, Western blot using anti-mtHsp70 antibodies performed on RSV and control cells at 24h and 48h. A: Representative Western blot of the mtHsp70 in mitochondrial fractions (15µg of protein loaded) from control cells and RSV treated cells at 24 and 48h. Mitochondrial fractions were loaded in triplicates. The right sided triplicates are showing RSV treated and the left sided triplicates showing controls. B: The mean mtHsp70 in mitochondria detected by Western blot at 24h was 20% ± 3% SEM and after 48h was 40% ± 10% SEM. Bars represent the means, expressed as percent control ± SEM, n=3 for 24h and n=4 for 48h experiments; each experiment done in triplicates; *p<0.05(ANOVA).

71

Figure 18

Mitochondria

mtHsp7050µM RSVControl

72 kDa

72 kDa

24h

48h

B

A

*

*

72

Figure 19: 50µM RSV does not change mtHsp70 expression. A: Representative Western blot of the mtHsp70 in WCLs (25mg of protein loaded) from control and RSV treated cells at 24h and 48h. Each sample has been probed for both anti-mtHsp70 (upper panels) and anti-GAPDH (lower panels) antibodies. B: Total mtHsp70 levels in WCLs do not show any significant changes in response to 50µM RSV treatment at 24h and 48h. Total mtHsp70 levels expressed as percent control normalized to GAPDH. Bars represent the means, expressed as normalized values to GAPDH levels, n=3 for both 24h and 48h experiments (p>0.05; ANOVA).

73

GAPDH 35kDa35kDa

72kDa72kDa

24h 48h

mtHsp70

Control RSV Control RSV

Figure 19

A

B

Whole Cell

mtH

sp70 n

orm

aliz

ed t

o G

AP

DH

(% o

f co

ntr

ol)

74

3.6. RSV and its Effects on Mitochondrial Function

3.6.1. RSV Decreases ROS Generation

To investigate the effects of RSV on ROS formation, we used the fluorescent

indicator 5’6’CMH2DCF-DA and measured the DCF signal by flow cytometry. A

20µM dose of hydrogen peroxide was used as a positive control. As shown in figure

20, 50µM RSV significantly reduced basal ROS generation;, by 24h. DCF signal

declined by 16% compared to levels in control cells.

3.6.2. 50µMRSV Decreases Mitochondria Membrane Potential (MMP)

The cumulative results of 4 experiments shown in figure 21 indicate that 50µM

RSV (for 24h) reduces MMP compared to time matched controls. Note: Since the

uptake of Rhodamine123 (Rh123) is dependant upon MMP, a decrease in the Rh123

signal (in non-quench mode), indicates a decline in MMP as the mitochondria are now

more positive (less hyperpolarized) relative to the cytoplasm. Conversely an increase

in Rh123 occurs when MMP increases (mitochondria become more hyperpolarized).

Exposure to 75µM RSV resulted in a more severe decline in MMP; the mean Rh123

signal was 26% ± 1.2 SEM lower than in time matched controls (n=3; p<0.001; data

not shown).

75

Figure 20: 50µM RSV decreases mitochondrial ROS generation. To determine the

effects of 50µM RSV on basal ROS generation, the RSV treated cells and controls

were labeled with DCF and the signal measured by flow cytometry. To evaluate DCF

function, each experiment consists of a 20µM H2O2 sample as a positive control. In

one experiment the DCF signal in arbitrary units (AU) for H2O2, RSV treated and time

matched controls were 1843 � 58 SD, 522 � 69 SD and 422 � 15 SD respectively

(data not shown). Graph shows a cumulative result of 4 independent experiments.

50µM RSV for 24h decreased basal ROS in mitochondria by 16 � 4% SEM compared

to time matched controls. Each experiment done in triplicates and each sample

indicates the mean DCF signal of 70-80% of 10000 cells counted (n=4; *p<0.05

(unpaired Student’s t-test) compared to time matched controls).

*

ControlRSV(µM)

76

*ControlRSV(µM)

Figure 21: RSV reduces mitochondrial membrane potential (MMP) 24h post

treatment. To measure the changes in mitochondrial membrane potential

(MMP), differentiated PC12 cells were treated with RSV and stained with

Rhodamine123 (Rh123). Addition of CCCP (20µM) to the media blocks the MMP

and consequently decreases Rh123 signal: this population considered as negative

control in Rh123 experiments. To ensure that Rh123 (0.5µM) is not quenched in

RSV treated and control cells, a high concentration of Rh123 (20µM) added to the

cells and used as positive control. In one experiment, the mean values of Rh123

signal in arbitrary unites (AU) were as following: CCCP group: 54 � 3 SD; 20µM

Rh123 group: 544 � 8 SD; Rh123 labeled (0.5µM Rh123) RSV treated cells

group was 69 � 4 SD (AU) and Rh123 labeled (0.5µM Rh123) time matched

controls 98�2 SD (data not shown). Graph shows the cumulative results of 4

independent experiments. 50µM RSV decreased MMP significantly after 24h.

Bars represent the means, expressed as percent control � SEM, The mean Rh123

signal after 24h was 16% � 4%SEM lower than time matched controls; n=4;

*p<0.05 (unpaired Student’s t-test) ; each experiment done in triplicates and

10000 cells per each sample counted. The mean Rhodamine signal (from 70-80%

of 10000 counted cells) used for graphs. Experiments using 75µM RSV also

showed a significant and further decrease (26% � 2% SEM) in MMP in RSV

treated cells compared to time matched controls (n=3; p<0.001; data not shown).

77

Discussion

78

RSV and Mitochondria

4.1. MPI in Neurons

It is clear that mitochondrial integrity and function is critically dependant upon

mitochondrial protein import (MPI) a sophisticated and poorly understood process by

which nuclear encoded proteins are imported into mitochondria. To date many of the MPI

machinery components have been identified but still little is known about the regulation

of MPI. Converging evidence suggest that protein import to mitochondria changes in

response to mitochondrial demand but how this happens is not clear. Studies on isolated

cardiomyocytes and skeletal muscle cells showed enhanced protein import into the

mitochondria coincident with an increase in the expression of some components of the

import machinery (Tom20, mtHsp70) after contractile activity, thyroid hormone

treatment and in senescent cells (Craig and Hood 1997; Grey et al., 2000; Hood et al.,

2003; Mattson et al., 2000). Studies by Wright et al have also shown that oxidative stress

inhibits import and the processing of the mitochondrial matrix proteins in vitro (Wright et

al., 2001).

Neurons are particularly vulnerable to mitochondrial dysfunction and recent studies

suggest that MPI is a key factor in neuronal survival but to date there is very little data on

MPI in neurons. My study, for the first time, shows that RSV increases the import of

nuclear encoded proteins into mitochondria in a neuronal model.

4.2. Flow Cytometry and Western Blot Show That RSV Increases MtGFP in

Mitochondria

RSV significantly increased levels of mtGFP in mitochondria. Flow cytometry

experiments showed that mtGFP increased in response to RSV treatment as early as 12h

compared to time matched controls and this increase continued for the duration of the

RSV exposure up to 72h. Previous studies have shown that while mtGFP labels virtually

all mitochondria within the cell (confocal imaging), mtGFP fluorescence is not detectable

in the cytoplasm (Sirk et al., 2003 and 2007; Shulyakova Master’s thesis 2008), thus the

mtGFP signal directly reflects mitochondrial mtGFP. Western blots of cytoplasmic

79

fractions also reveal that very little mtGFP is present in the cytoplasm under normal

conditions, and trace amounts are only detected with higher protein loads. This is also the

case for physiological mitochondrial proteins which, like mtGFP, are rarely detected in

Western blots of CP fractions unless high (>50 µg) of protein are loaded. After

proteasome inhibition (Shulyakova, Master’s thesis, 2008) mtGFP and other

physiological proteins increase markedly in CP fractions indicating that normally

substantial amounts of many mitochondrial proteins are rapidly degraded in the

cytoplasm prior to their import. The fact that mtGFP and other mitochondrial proteins do

increase in the cytoplasm after inhibiting MPI (Sirk et al., 2003 and 2007; Wright et al.,

2001) may reflect proteasome overload. In the case of mtGFP, the lack of any

fluorescence in the cytoplasm under normal conditions is not surprising given the very

low levels of mtGFP present. However, why is no cytoplasmic fluorescence observed

under conditions (MPI or proteasome inhibition) where Western blots reveal substantial

amounts of mtGFP protein in the cytoplasm? The answer lies in the fact that mtGFP

fluorescence is dependant upon protein folding and this folding occurs only after import

in the mitochondrial matrix (see introduction for mtGFP import). Many mitochondrial

proteins are prevented from folding by cytoplasmic chaperones in the cytoplasm (Neupert

and Herrmann 2007). In addition although mtGFP, like most mitochondrial proteins, is

thought to be imported into mitochondria post-translation (Neupert and Herrmann 2007;

Truscott et al., 2003) it remains possible that some portion of it may be imported co-

translationally as has been reported for other proteins (Ahmed et al., 2006; Ahmed and

Fisher 2009). In this case import starts prior to completion of synthesis, which by

definition prevents folding. Western blot of mitochondrial fractions confirmed that RSV

increased mtGFP levels in mitochondria at 12h, 24h and 48h by 11%, 25% and 24%

respectively compared to time matched controls confirming the increase in mtGFP signal

detected by flow cytometry. Western blot of cytoplasmic fractions did not detect any

mtGFP, but increasing the protein load 2-3 folds (50µg) did in some cases result in a faint

signal in the CP fractions (see figures 11 and 12). This is consistent with previous studies

using immunoprecipitation and autoradiography which show that under normal

conditions mtGFP levels in the cytoplasm are low to undetectable (see also discussion

above).

80

4.3. RSV Does Not Initially Alter Intramitochondrial MtGFP Degradation

Little is known about the mechanisms or signaling pathways involved in the effects of

RSV on mitochondria. The increase I observed in mtGFP levels in mitochondria in RSV

treated cells could be due to increased import, decreased intramitochondrial degradation,

or increased mitochondria biogenesis (mitochondria mass). RSV did not alter mtGFP

degradation in the first 24h, but mtGFP degradation by 48h showed a sustained decline.

These results indicate that the increase in mitochondrial mtGFP at 24h is not due to

delayed intramitochondrial turnover/degradation. However since mtGFP degradation did

slow by 48h, degradation may have contributed to the increased levels of mtGFP at later

time points. How RSV slows down the mtGFP degradation is not clear but mitochondrial

proteases are likely involved.

4.4. RSV Increases Physiological Mitochondrial Proteins in Mitochondria

I examined two mitochondrial proteins that play essential roles in mitochondrial

protein import machinery; mtHsp70 and Tom20. Tom20 is a translocase of the outer

mitochondrial membrane involved in the import of the majority of preproteins and of all

matrix-targeted proteins (Grey et al., 2000), mtHsp70 is a mitochondrial matrix

chaperone critical for protein import into the matrix as well as protein folding in the

matrix. Tom20 is not imported via the common import pathway (Popov-Celeketic et al.,

2008) but is integrated into outer membrane by an N-terminal alpha-helix which is

necessary for its targeting and membrane anchoring. Studies also show that Tom20

preproteins require Mim1 to be inserted into the outer membrane (Hulett et al., 2008;

Popov-Celeketic et al., 2008) and that this process does not require Tom receptors, or

the Tom40 pore and SAM complex (Becker et al., 2008). Increased mitochondrial

Tom20 in the absence of an increase in total Tom20 suggests that RSV increases

insertion of Tom20 into the OM. RSV may also reduce cytoplasmic degradation of

Tom20 prior to its integration which would maximize the available Tom20. RSV also

decreased basal ROS which suggests that this might be one of the mechanisms by which

insertion of Tom20 into the outer membrane is up regulated. Consistent with this

interpretation oxidative stress has been shown to reduce levels of Tom20 in cardiac

81

tissue, in isolated mitochondria of COS cells and in PC12 cells (Boengler et al., 2006;

Wright et al., 2001; Phan et al., in preparation).

Tom20 increased by 24 and 48h in response to RSV treatment. Since protein import

is dependent upon Tom20 (Boengler et al., 2006; Wright et al., 2001; Phan et al., in

preparation) it seems likely that the RSV mediated increase in Tom20 facilitates the

import of mtGFP and mtHsp70, both of which are matrix targeted mitochondrial proteins.

These findings are consistent with studies on C2C12 muscle cells showing that

increasing or decreasing Tom20 increased or decreased expression and import of another

matrix targeted protein, malate dehydrogenase (MDH) (Grey et al., 2000). Previous

studies in our lab show sublethal glucose deprivation and reperfusion (GD/RP) decreased

the Tom20 levels and this decline was associated with a decline in the import of matrix

targeted proteins e.g. mtHsp70 and mtGFP. Furthermore over-expression of Tom20 or

treatment with antioxidants (e.g. Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP)

or RSV) prevented the increase in ROS, decline in Tom20, and the decline in MPI (Phan

et al.,in preparation).

Levels of mtHsp70 in mitochondria increased after 24h and 48h of RSV exposure.

MtHsp70 like mtGFP is a nuclear encoded mitochondrial matrix targeted protein and it is

imported into mitochondria using the Tom complex component (Geissler et al., 2001;

Hulett et al., 2008). MtHsp70 can be up regulated by thyroid hormones in isolated

cardiac mitochondria (Schneider and Hood 2000) and by over expression of Tom20 or by

use of antioxidants (Phan et al., under revision) which suggests that mtHsp70 can be

regulated by multiple factors. Studies by Colavecchia show that mtHsp70 over expression

increases the import of MDH (Csiszar et al., 2009a) which suggest mtHsp70 has a

significant role in import of nuclear encoded proteins. This suggests that the effects of

RSV on mtGFP import may be a consequence of the increase in mtHsp70 or Tom20 or

both.

82

4.5. RSV Does Not Alter Mitochondria Mass

RSV improves mitochondrial function and it induces mitochondria biogenesis

(Lagouge et al., 2006) which of necessity requires increased MPI. Mitotracker

experiments showed no increase in mitochondrial mass after 24h of exposure to RSV so

it appears that the observed increase in MPI did not reflect of increased numbers of

mitochondria at least at 24h. Additionally, protein quantification assays “the Bradford”

showed no difference in levels of total mitochondrial protein in mitochondrial fractions

extracted from RSV treated cells compared to controls. As discussed earlier RSV

increases mitochondria biogenesis in muscles (Gerhart-Hines et al., 2007); hepatic tissues

(Lagouge et al., 2006), in endothelial cells (Csiszar et al., 2009b), and in neurons

(Dasgupta and Milbrandt 2007; Onyango et al., 2009). Plausibly the increase in MPI is an

initial response to RSV and increased biogenesis requires a longer exposure to RSV or a

longer time frame.

Mitochondrial biogenesis requires activation of PGC-1α a transcriptional co-

activator that increases the expression of mtTFA, a nuclear-encoded protein imported into

the mitochondrial matrix via Tom20 that is essential for the transcription (Grey et al.,

2000) replication and packaging of mtDNA (Bonawitz et al., 2006), RSV activates

PGC1-α pathway by induction of Sirt1, a protein deacetylase. Studies in vivo (Lagouge

et al., 2006) show that RSV mediated mitochondria biogenesis, involves a decrease in

PGC-1α acetylation and an increase in PGC-1α activity. These studies suggest that

mitochondrial biogenesis is not an immediate response to RSV since an increase in

mitochondria biogenesis was observed in muscle fibers of C57Bl/6J male mice only after

about 15 weeks of RSV treatment.

4.6. RSV Increases the Expression of Mitochondrial Proteins Selectively

RSV increases synthesis of at least two nuclear encoded mitochondrial proteins,

TFAM and NRF1 (Lagouge et al., 2006), but how RSV effects other nuclear encoded

mitochondrial proteins is not clear. In my study, Western blotting of WCLs of RSV

treated cells showed an increase in total mtGFP at 24h but no increase at 12 and 48h.

In addition, mtHsp70 levels in WCLs showed no increase at 24 and 48h, but Tom20

83

levels increased. Although the technical issues (different exposure times) in developing

Western blots (films) should not be underestimated, one possibility is that RSV has

selective effects; that it can induce expression of some proteins like mtGFP (at 24h) and

Tom20 and can increase the import of other mitochondrial proteins without altering their

synthesis. This finding is consistent with studies on isolated mitochondria from cardiac

muscle which show that ischemia has a selective effect on levels of mitochondrial

proteins such that it decreases levels of Tom20 but has no effect on levels of Tom40, and

Tim23 (Boengler et al., 2006).

How could mtHsp70 increase in mitochondria without any alteration in total levels of

these proteins? One possibility is that RSV treatment results in the ‘recruitment” a

reserve supply of proteins (which is normally degraded prior to import, see discussion

above and Shulyakova Master’s thesis 2008). This might occur initially as a direct result

of the RSV mediated decrease in ROS and later as a consequence of increased levels of

Tom20. Since basal ROS generation significantly decreased by 24h of RSV, the decrease

in ROS could also be involved in the increase in Tom20 expression.

This is consistent with Phan et al study (Phan et al., in preparation) in which

antioxidant treatment prevented the decline in ROS, the decline in Tom20, and rescued

MPI. ROS generation plays a key role in ischemia induced decreases in Tom20 in cardiac

muscle (Boengler et al., 2006) and also in the inhibitory effects of paraquat on MPI in

liver cells (Wright et al., 2001).

4.7. RSV Reduces Mitochondrial Membrane Potential

The maintenance of mitochondrial membrane potential (MMP) has an important role

in mitochondrial function and plays a role in the regulation of MPI. Flow cytometry

showed a small but significant reduction in MMP indicating a mild degree of

mitochondrial depolarization at 24h of RSV treatment compared to controls. Since the

translocation of proteins to IMS or matrix is dependant upon maintenance of MMP this

seems at first glanced to be a paradox, RSV increased MPI despite a decrease in MMP.

However previous studies have shown that even a severe loss of MMP does not fully

inhibit MPI. Thus this relatively small decline was likely insufficient to decrease MPI.

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Experiments using 75µM RSV showed a further decrease in MMP (24% decrease in RSV

treated cells compared to controls) suggesting that MPI is relatively resistant to even

moderate changes in MMP.

4.8. RSV Decreases ROS Generation

One of the beneficial effects of RSV is its antioxidant properties. The antioxidant

effects of RSV have been extensively studied in various organs and tissues in vivo and in

vitro.

It is believed that RSV works in both Phases I, II of drugs metabolization process. In

general, Phase I enzymes, consist of cytochrome P450s (CYPs) and flavin

monooxygenases which basically oxidize, reduce or hydrolyze foreign molecules to

prepare them for removal. Phase II enzymes include conjugating and antioxidant

enzymes which detoxify harmful molecules, including toxic products of Phase I enzymes.

cDNA arrays and reverse transcriptase-PCR on hepatic cells showed that RSV generally

down regulates Phase I enzymes and up regulates Phase II enzymes. RSV has been

shown to induce expression of Phase II enzymes in vitro (Cao and Li 2004). RSV also

induces haem oxygenase 1 and quinone reductase 1 (QR1) in vivo, resulting in improved

tolerance of ischemia and increased resistance to Menadione (vitamin K3) toxicity

(Floreani et al., 2003). In addition to induction of QR1, RSV may inhibit QR2. Little is

known about QR2 function but it seems evident that QR2 induces Phase II enzymes. Our

knowledge about the effects of RSV in neurons is very little but RSV reduces the

oxidative stress markers such as glycated albumin in serum of the stroke prone

hypertensive rats (Mizutani et al., 2001). Antioxidant properties of RSV have also been

shown in cancer (Gao et al., 2002; Kimura and Okuda 2001) and cardiovascular diseases

(Wang et al., 2002).

In microarray studies by Motta (Motta et al., 2004), the ribosomal mRNA processing,

striated muscle contraction, electron transport chain, OXPHOS, and ATP synthesis were

amongst the top 30 gene sets which were significantly enriched in RSV-treated mice

which indicate in these systems RSV increases ATP synthesis. Plausibly if RSV enhances

85

ATP in PC12 cells it could enhance mitochondrial import which is dependant upon ATP

and subsequent mitochondria biogenesis.

As briefly summarized (see discussion above and 1.7.3 of introduction), some

previous studies suggest RSV has antioxidant properties. To determine the effects of

RSV on ROS generation I used the fluorescence dye 5’6’CMH2DCF-DA and flow

cytometry. RSV significantly decreased basal ROS generation by 24h (15%, with 50µM

RSV, 24% with 75µM), positive controls (H2O2 treated cells), showed a 5 fold increase in

the DCF signal confirming that the DCF was sensitive to free radicals and H2O2 in

particular.

Mitochondria are a major source of ROS in cells (neurons) thus ROS is a key player

in mitochondria activities. A decrease in ROS generation is expected to be beneficial

generally. Taken in conjunction with previous studies that suggest MPI, and Tom20 in

particular, is sensitive to ROS my data suggest that the effects of RSV on MPI may be

due to the reduction in ROS.

4.9. MtGFP as a Model of MPI

The model I used in my experiments is a PC12 cell line stably transfected with

mitochondrially targeted GFP (mtGFP). PC12 cells are broadly used in neuronal studies

but there are some limitations in using these cells as neurons. First, they are not primary

neurons although they acquire the characteristics of sympathetic neurons in the presence

of NGF e.g., neurite outgrowth. Second, their genetic materials can change over repeated

passage number which can change their phenotype (Das, Freudenrich, Mundy 2004;

Harry et al., 1998).

Using a mtGFP-based assay for mitochondrial protein import offers the following

advantages: first, PC12 cells can be stably or transiently transfected with mtGFP

relatively easily which offers important advantages for studies on protein import. Second,

mtGFP expression in the stable line is negatively regulated by pTRE/Tet-off system that

means when tetracycline is present mtGFP is not expressed in the cells; upon tetracycline

86

wash out mtGFP expression is rapidly induced. This feature of our mtGFP transfected

PC12 cells provides a valuable tool that permits quantification of mtGFP import and turn

over in live cells using flow cytometry and confocal imaging thus reducing the need for

radioisotopes and autoradiography (Shulyakova et al.,2007, Modern research and

educational topics in Microscopy; Sirk et al., 2003 and 2007). Third, examining relatively

minor or even moderate changes in MPI requires a highly sensitive assay and the quality

of many antibodies in such that this can be difficult by Western blot; since mtGFP

synthesis can be “turned on” as desired small changes can be more readily detected.

Further, given the variety of fluorescent dyes now available, this system can be adapted

to assess MPI along with other cellular and mitochondrial activities e.g., changes in

MMP, intracellular Ca2+ and ROS generation in live cells.

4.10. A Model for Effects of RSV on Mitochondrial Protein Import

Mitochondrial protein import is a sophisticated process which is essential for

mitochondrial function in all cells. Previous studies showed that MPI is regulated or

affected by multiple different factors. My results show for the first time that RSV

treatment regulates MPI in neurons although the pathways involved in MPI regulation by

RSV clearly require more investigation. My data also indicate that RSV mediated

decreases in ROS may play a role in MPI regulation. Figure 22, which is my working

model, suggests that this is the case. ROS is a key player in different activities in

mitochondria, thus ROS can activate different signaling pathways which increase

expression and import of mitochondrial proteins. Among them is Tom20, one of the

essential components of MPI machinery that acts as a rate limiting protein in import

process. The other key protein is mtHsp70 which is also important in the import of other

proteins into mitochondria. Previous studies have shown that RSV could induce

mitochondria biogenesis thus the increase in import that I detected in my study may be an

early preparatory stage of mitochondria biogenesis. The model illustrates the underlying

mechanisms involved in RSV induced changes in MPI. RSV mildly reduced the MMP

and that might contribute to of the slower degradation of mtGFP after 24h. The

corresponding outcome on ATP levels is not clear. RSV also increased basal cell survival

but whether the increase in MPI is involved is not clear.

87

Figure 22: A model illustrating underlying mechanisms involved in RSV induced

changes in MPI. Solid black lines are known data from my study, solid blue lines are known data shown by others,dashed lines are possible pathways that might have a role in effects of RSV on MPI. RSV decreases ROS which can activate different signaling pathways such as increase in mitochondrial protein expressions and import. RSV increased the import of Tom20 and mtHsp70 two essential components of MPI machinery which play a key role in import of other mitochondrial proteins. Previous studies suggested that RSV can modulate mitochondria biogenesis through different pathways such as PGC1-�-mtTFA pathway, therefore the increase in MPI detected in my study might be a preparatory stage in mitochondria biogenesis. RSV mildly reduced the MMP and that might contribute to the slower degradation of mtGFP after 24h. It is also possible that the up or down regulation of proteasomes by RSV might play a role in MPI regulation in differentiated PC12 cells.

88

RSV

mtGFP

Tom20

MMP

ROS

mtGFP

mtHsp70

mtGFP

Degradation

Cell

death

Mitochondria

Nucleus

Proteasomes

Mitochondria Biogenesis

(via PGC1-� activation):

TFAM and mtDNA

Working Model

89

4.11. Future Studies on RSV, MPI, ROS Generation and Ca2+

As pointed out earlier, RSV decreased ROS generation and MMP. The former is

generally assumed to be beneficial while the latter is not. Since the interaction between

ROS generation, MMP, ATP levels and Ca2+ play a crucial role in mitochondrial function

and neuronal health, it is important to assess the effects of RSV on Ca2+ homeostasis.

Since Ca2+ can regulate ATP generation it would be imperative to evaluate whether the

effects of RSV on ATP production is via Ca2+ pathways. In addition since ROS

generation is important in Ca2+ regulation defining the interplay between ROS and

intramitochondrial Ca2+ accumulation is also essential. Finally, since recent studies in

our lab indicate that proteasomes are a key factor in MPI regulation (Shulyakova’s

Master’s Thesis, 2008), understanding RSV’s effect proteasomes may identify another

mechanism by which RSV affects MPI. Future studies will be looking at the effects of

RSV on ATP levels, intracellular Ca2+ levels and homeostasis, and the relationships

between ROS, MMP, ATP levels and Ca2+ and on MPI.

4.12. Conclusion and Significance

Mitochondria are a site of numerous biochemical activities from ATP synthesis to

biogenesis. They play a critical role in disease states and developmental abnormalities.

All of these activities could be affected by changes in MPI and supporting data from

recent studies suggest that MPI is a key factor in neuronal survival and health. My study

suggests for the first time that RSV increases MPI in differentiated PC12 cells. RSV

increases the import and synthesis of mtGFP and the import of mtHsp70 and increases

levels of Tom20 indicating that the effects of RSV on mitochondrial proteins may be

protein specific. The increase in MPI initially happens in the absence of any changes in

intramitochondrial degradation or any increases in mitochondrial mass which suggests

that the increase in import is at least initially independent of biogenesis. RSV also

reduced MMP significantly. The increase in MPI could be mediated by different

pathways including the PGC-1� pathway, suppression of proteasomes activity, and

ROS. In my study basal ROS decreased in response to RSV treatment suggesting that the

effects of RSV may be due to RSV acting as an antioxidant.

90

RSV promotes longevity in several organisms and is thought to have considerable

potential for neuroprotection in stroke, nerve injury and some neurodegenerative

disorders e.g., Huntington and Alzheimer’s diseases.. My study shows for the first time

that RSV increases MPI and suggests that MPI might be one of the mechanisms by which

RSV exerts its beneficial effects. There are still numerous questions which require to be

answered in terms of RSV and MPI. For example it is essential to know the effects of

RSV on ATP synthesis and the role of Ca2+ in the up regulation of MPI, induced by RSV.

It will also be of interest to examine the effects of RSV in models of neuronal injury.

Ongoing studies are focused on these issues.

My study broadens our knowledge about MPI and its regulation in neurons. Previous

studies have supported a role for RSV as an antioxidant, a neuroprotective agent and an

anti-aging agent. My studies now suggest that MPI is a new target for RSV and that

modulation of MPI may be a central event in the positive actions of RSV.

91

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