Understanding the Role of Sirtuin 3

in a Cell Model of Parkinson’s Disease

by

Kristin Elisa Lizal

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Cell and Systems Biology

University of Toronto

© Copyright by Kristin Elisa Lizal 2013

ii

Understanding the Role of SIRT3 in a Cell Model of Parkinson’s Disease

Kristin Elisa Lizal

Master of Science

Graduate Department of Cell and Systems Biology

University of Toronto

2013

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the degeneration of

dopaminergic neurons in the substantia nigra pars compacta. It is now believed that the

mitochondrion is central to the pathologies of PD. Sirtuin 3 (SIRT3) is a mitochondrial

nicotinamide adenine dinucleotide dependent protein deacetylase. Within the mitochondria,

SIRT3 deacetylates substrates of oxidative phosphorylation and antioxidant pathways to promote

cellular functioning. Given that mitochondria impairments are central to PD, and that SIRT3

promotes mitochondrial health, the mechanisms underlying the protective effects of SIRT3 in a

catecholaminergic SH-SY5Y cell model of PD were assessed. The results of this current study

demonstrated that SIRT3 overexpression is cytoprotective in SH-SY5Y cells since it stabilizes

mitochondrial membrane potential and ATP levels and reduces reactive oxygen species after

exposure to toxins that mimic cell death mechanisms in PD. Taken together, SIRT3’s beneficial

effects presents itself as an excellent candidate for Parkinson’s disease medicine.

iii

Acknowledgments

First of all, I would like to thank my supervisor Dr. Joanne Nash who always has pushed me to

be a better and more independent scientist. I appreciate your constructive criticisms and the time

you have dedicated to guide me throughout my master’s degree. I also want to thank my

wonderful supervisory committee Dr. James Eubanks and Dr. Philippe Monnier who have given

me their inputs and directions to better my project. Dr. Patrick McGowan, I also want to thank

you for agreeing to be my external reviewer.

To my loving parents, thank you so much for your countless support, guidance, motivation and

care. Both of you are my biggest role models and have always pushed me to pursue a higher

education. Thank you so much for picking me up during my late night experiments and also

driving me to the lab during the weekends and holidays.

Graduate school would not be the same without the special people who I have established

wonderful friendships with. I want to specifically mention Dr. Chris Yong-Kee for the numerous

hours you have spent training me in the lab and also for being a supportive mentor. I want to

thank Sherri Thiele for her easy-going personality, advice and insights. Special thanks to Mary

Lee (my rock since day 1), Sam Khalouie (always can count on you to have a relaxing chat

during a hectic day), Betty Chen (my sister from another mother), Kewei Xu (my go-to person),

Melanie Ratnam (thank you for being my confidant), Sahara Khadem (thank you for your

optimism even though we have only known each other for a short period of time), Ervis Kola

(my master’s degree buddy). Cindy Leong, thank you for being a great volunteer and helping me

out during my busy days. Elena Sidorova, thank you so much for all your help, especially for

your inputs and insights on SIRT3. Michelle Duong and Adam Pham-Hung - thank you for your

support throughout this journey, you both are such great friends. A huge thanks to Raymond

Nagar for his countless help, support, motivation and advice - even though you are in a different

time zone!

Last but not least, I want to thank God for his guidance and care in every step of my journey and

for making the last two years the greatest learning experience yet.

iv

Dedicated to My Dad and My Mom

v

Table of Contents

Acknowledgments ..................................................................................................................... iii

Table of Contents ....................................................................................................................... v

List of Figures ......................................................................................................................... viii

List of Abbreviations ................................................................................................................. ix

CHAPTER 1 ............................................................................................................................. 1

1 INTRODUCTION ............................................................................................................... 1

1.1 PARKINSON’S DISEASE ............................................................................................. 1

1.2 CAUSES OF CELL DEATH IN PARKINSON’S DISEASE .......................................... 1

1.2.1 Oxidative Phosphorylation Impairment in PD ...................................................... 1

1.2.2 Genetic links between Electron Transport Chain Impairment and PD .................. 2

1.2.3 Mitochondrial Toxins Mimicking Cell Death Mechanisms in PD ........................ 5

1.2.4 Oxidative Stress in PD......................................................................................... 6

1.2.5 Proteolytic System Impairment in PD .................................................................. 8

1.2.6 Proteolytic system inhibitors that mimic cell death mechanisms in PD ................ 9

1.2.7 Involvement of Ca2+

in PD ................................................................................ 10

1.3 OTHER IMPORTANT FUNCTIONS OF THE MITOCHONDRIA ............................. 11

1.3.1 Apoptotic signaling ........................................................................................... 11

1.3.2 Mitochondrial dynamics .................................................................................... 12

1.4 THE MAMMALIAN SIRTUINS.................................................................................. 14

1.4.1 Nuclear Sirtuins ................................................................................................. 14

1.4.2 Cytoplasmic Sirtuins ......................................................................................... 15

1.4.3 Mitochondrial Sirtuins ....................................................................................... 15

1.5 SIRT3 ........................................................................................................................... 19

1.6 MECHANISMS OF SIRT3 ........................................................................................... 19

vi

1.6.1 SIRT3 and Metabolism ...................................................................................... 19

1.6.2 SIRT3 and Protein Synthesis ............................................................................. 20

1.6.3 SIRT3 and Oxidative Stress ............................................................................... 21

1.6.4 SIRT3 and Electron Transport Chain ................................................................. 22

1.6.5 Benefits of SIRT3 ............................................................................................. 26

1.7 RATIONALE ............................................................................................................... 27

1.8 HYPOTHESIS AND AIMS .......................................................................................... 28

CHAPTER 2 ........................................................................................................................... 29

2 MATERIALS AND METHODS ....................................................................................... 29

2.1 Cell Culture .................................................................................................................. 29

2.2 Toxins........................................................................................................................... 29

2.2.1 Dose Response Curves ...................................................................................... 29

2.3 Construction of Stably Transfected Cells ...................................................................... 30

2.4 Western Blot Analysis of SIRT3 Expression ................................................................. 30

2.4.1 Sample Preparation ........................................................................................... 30

2.4.2 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) ...... 30

2.5 Assessment of Human SIRT3 Ectopic Expression in SH-SY5Y cells ............................ 31

2.6 Measurement of Reactive Oxygen Species .................................................................... 32

2.7 Quantification of Mitochondrial Membrane Potential .................................................... 32

2.8 Assessment of ATP Levels ........................................................................................... 33

2.9 Cell Viability Assay ...................................................................................................... 33

2.10 Cell Death Quantification.............................................................................................. 34

2.11 Statistical Analysis ........................................................................................................ 34

CHAPTER 3 ........................................................................................................................... 35

3 RESULTS

3.1 Human SIRT3 expression in stably transfected SH-SY5Y cells..................................... 35

vii

3.2 Dose response curves of toxins that mimic Parkinson’s disease cell death mechanisms

in SH-SY5Y cells ......................................................................................................... 40

3.3 Effects of SIRT3 on cell viability in SH-SY5Y cells ..................................................... 43

3.4 Effect of SIRT3 on cell death in SH-SY5Y cells ........................................................... 46

3.5 Effect of SIRT3 on reactive oxygen species (ROS) levels in SH-SY5Y cells ................ 49

3.6 Effect of SIRT3 on mitochondria membrane potential (∆Ψm) in SH-SY5Y cells .......... 53

3.7 Effect of SIRT3 on ATP levels in SH-SY5Y cells ......................................................... 58

4 DISCUSSION ..................................................................................................................... 61

viii

List of Figures

Figure 1. Overview of mitochondrial SIRT3 substrates ………………………………………...18

Figure 2. Mechanistic overview of SIRT3’s interaction with mitochondrial metabolic pathways,

antioxidant pathways and urea cycle showing sources of NAD+ ……………………………….25

Figure 3. Ectopic overexpression of Human SIRT3 in SH-SY5Y cells…………………………37

Figure 4. Localization of Human SIRT3 transgene expression in SH-SY5Y

cells………………………………………………………………………………………………39

Figure 5.Dose response curves of toxins that mimic PD cell death mechanism in SH-SY5Y

cells………………………………………………………………………………………………42

Figure 6. Effect of SIRT3 on cell viability in SH-SY5Y cells….................................................45

Figure 7. Effect of SIRT3 on cell death in SH-SY5Y cells ……………………………………..48

Figure 8. Effect of SIRT3 on reactive oxygen species in SH-SY5Y cells ………………………52

Figure 9. Effect of SIRT3 on mitochondrial membrane potential in SH-SY5Y cells …………..57

Figure 10. Effect of SIRT3 on ATP levels in SH-SY5Y cells……………………………..…….60

ix

List of Abbreviations

∆Ψm

Mitochondrial membrane potential

•O2-

Superoxides

•OH

Hydroxyl radicals

A.U.

Arbitrary units

AB

Alamar blue

ACS2

AcetylCoA synthase 2

ADP

Adenosine diphosphate

ADP

Adenosine diphosphate

AIF

Apoptosis inducing factor

ALP

Autophagy-lysosome pathway

AMR

ATP monitoring reagent

ANOVA

Analysis of variance

ANT

Adenine nucleotide translocator

ATP

Adenosine triphosphate

BAT

Brown adipose tissue

Ca2+

Calcium

Cat D

Cathepsin D

CMA

Chaperone-mediated autophagy

CM-H2DCFDA

2’,7’-dichlorodihydrofluorescein diacetate

CO2

Carbon dioxide

CPS1

Carbamoyl phosphatase synthase

CR

Caloric restriction

CypD

Cyclophilin D

DA

Dopamine

x

DAQ

Dopamine quinone

DAT

Dopamine transporter

DMEM

Dulbecco’s modified eagle medium

DMN

Dorsal motor nucleus of the vagus

DMSO

Dimethyl sulfoxide

DNA

Deoxyribonucleic acid

DOPAC

3,4-dihydroxyphenylacetic acid

Drp1

Dynamin-related protein 1

E1

Ubiquitin-activating enzyme

E2

Ubiquitin conjugating enzyme

E3

Ubiquitin protein ligase

ETC

Electron transport chain

Fe

Iron

Fe2+

Reduced iron

Fis1

Fission protein 1

FOXO

Forkhead transcription factor O

FSC

Forward scatter

GAP-43

Growth-associated protein

GDH

Glutamate dehydrogenase

GPX

Glutathione peroxidase

GSH

Reduced glutathione

GSSG

Glutathione disulfide

GTPases

Guanosine triphosphatases

H+

Proton

H2O2

Hydrogen peroxide

xi

HIF-1α

Hypoxia-inducible factor 1-alpha

HMGCS2

3-hydroxy-3-methylglutaryl CoA synthase 2

HSC

Heat shock cognate protein

hSIRT3

Human SIRT3

HSP

Heat shock protein

IC50

Inhibitory concentration 50

IDH2

Isocitrate dehydrogenase 2

LAMP-2A

Lysosome-associated membrane protein

LB

Lewy body

LC

Locus coeruleus

LCAD

Long-chain acyl-CoA dehydrogenase

MAP

Mitogen activated protein

Mfn1

Mitofusion 1

Mfn2

Mitofusion 2

MMP

Mitochondria matrix peptidase

MOM

Mitochondrial outer membrane

MPP

Mitochondrial permeability pore

MPP+

1-methyl-4-phenylpyridinium ion

MPTP

1-methyl-4-phenylpyridinium

MRP

Mitochondrial ribosomal protein

MRPL10

Mitochondrial ribosomal protein L10

mtDNA

Mitochondrial DNA

NaCl

Sodium chloride

NAD+

Nicotinamide adenine dinucleotide

NADPH

Nicotinamide adenine dinucleotide phosphate

xii

NAP

Naphthazarin

NBM

Nucleus basilis of Meynert

NeuN

Neuronal nuclei

NMDA

N-methyl-D-aspartate

NO•

Nitric oxide

NRR

Nucleotide releasing reagent

OTC

Ornithine transcarbamoylase

OXPHOS

Oxidative phosphorylation

p53

Protein 53

PARP-1

Poly (ADP-ribose) polymerase-1

PBS

Phosphate buffer saline

PD

Parkinson’s disease

PGC-1α

Peroxisome proliferator-activated receptor γ coactivator 1-

alpha

Pi

Inorganic phosphate

PI

Propidium iodide

PINK1

PTEM induced putative kinase 1

Pol I

Polymerase I

PSI

N-carbobenzyloxy-Ile-Glu(O-t-butyl)-Ala-leucinal

P-value

Probability value

RA

Retinoic acid

rDNA

Ribosomal DNA

RNA

Ribonucleic acid

ROI

Region of interest

ROS

Reactive oxygen species

ROT

Rotenone

xiii

SDHA

Succinate dehydrogenase flavoprotein

SDHB

Succinate dehydrogenase iron sulfur

SEM

Standard error of mean

SH-SY5Y

Human cathelominergic neuroblastoma cell line

SIRT

Sirtuin

SNc

Substantia nigra pars compacta

SOD2

Superoxide dismutase 2

SSC

Side scatter

TBST

Tris-buffered saline and Tween 20

TFAM

Mitochondrial transcription factor A

TH

Tyrosine hydroxylase

TMRE

Tetramethylrhodamine, Ethyl Ester, Perchlorate

TRAP1

TNF receptor-associated protein 1

UCH-L1

Ubiquitin C-terminal hydrolase L1

UCP

Uncoupling protein

UPS

Ubiquitin-proteasome system

VMAT

Vesicular monoamine transporter

1

CHAPTER 1

1 INTRODUCTION

1.1 PARKINSON’S DISEASE

Parkinson’s disease (PD) is a neurodegenerative disease with a pervasiveness of 1-2% in the

population over the age of 50 (Xie et al., 2010). It is characterized by the progressive

degeneration of dopamine (DA) neurons projecting from the substantia nigra pars compacta

(SNc) to the striatum. Commonly observed motoric symptoms are muscle rigidity, gait

imbalance and resting tremors whereas non-motor symptoms include depression, dementia,

sensory loss and sleep disturbances (Novikova et al., 2006). Furthermore, abnormal aggregations

of α-synuclein protein known as Lewy Bodies are seen in diagnosed brains. Most cases of PD are

sporadic (cause unknown) whilst approximately 10-20% of PD cases have been linked to genetic

mutations (Trancikova et al., 2011).

1.2 CAUSES OF CELL DEATH IN PARKINSON’S DISEASE

Accumulating evidence suggests that mitochondrial dysfunction may be central to cellular death

in PD. Despite other factors linked to PD such as genetic mutations, oxidative stress, ubiquitin

proteasome system impairment and lysosomal damage, the mitochondria appear to be the most

affected, triggering the mitochondrion-induced cell death.

1.2.1 Oxidative Phosphorylation Impairment in PD

The mitochondria generate over 90% of energy utilized for cellular functioning via oxidative

phosphorylation (Yang et al., 2009). Oxidative phosphorylation involves the activities of five

complexes (Complex I-V), which are located in the inner mitochondrial membrane. Electrons get

transported between each complex in order to create proton (H+

ions) movement from the matrix

to the intermembrane space. This, in turn, produces the proton concentration gradient or also

known as mitochondrial membrane potential (∆Ψm), which is utilized for ATP-synthase to

convert ADP to ATP. However, electrons can leak during their transfer from one complex to

another. Specifically, electrons are more susceptible to escape from Complex I and III to the

matrix during oxidative phosphorylation. In the matrix, these leaked electrons will react with

2

oxygen to form reactive oxygen species (ROS) such as superoxides (•O2-), hydroxyl radicals

(•OH) and nitric oxide (NO•). Accumulation of ROS can have detrimental effects on cellular

functioning since the free radicals can escape from the mitochondria and affect other cellular

activities. ROS can exert its negative effects by inducing oxidative DNA damage, lipid

peroxidation, protein oxidation and nitration. In particular, hydroxyl radicals (•OH) have been

demonstrated to react with the double bonds of DNA bases, resulting in damaged DNA content

(Cooke et al., 2003). Moreover, the mitochondrion is unique in a way that it is capable of

producing its own mitochondrial DNA (mtDNA). mtDNA encodes for 37 mitochondrial genes.

Thirteen of these genes are responsible for the expression of proteins involved in oxidative

phosphorylation while the remaining genes regulate transfer and ribosomal RNA for

mitochondrial protein generation (Keane et al., 2011). Thus, the increased in ROS can also

damage mtDNA which subsequently interfere with mitochondrial functions.

Under basal conditions, ROS generated from oxidative phosphorylation is relatively low and is

usually broken down by the mitochondrial antioxidant enzymes (Lass et al., 1997). However,

deficiency of the respiratory chain complexes can lead to increased electrons leakage, resulting

in increased ROS-induced cellular damage. Postmortem analysis of PD brains have shown that

Complex I and III dysfunctions are present and most likely result in ROS overload and cause

neuronal death in PD (Dickson et al., 2009; Mizuno et al., 1989; Haas et al., 1995; Shinde &

Pasupathy, 2006).

1.2.2 Genetic links between Electron Transport Chain Impairment and PD

The involvement of respiratory chain dysfunction in PD was further confirmed by genetic

mutation cases.

1.2.2.1 SNCA (α-synuclein)

One of the hallmarks of PD is the abnormal α-synuclein protein aggregation that is referred to

Lewy body (LB) inclusions, suggesting a possible mutation in α-synuclein protein expression in

PD. α-synuclein, encoded by the SNCA gene, is primarily cytoplasmic and localized in

presynaptic terminals. It functions as a synaptic transmission regulator and has been suggested to

play a role in neuronal plasticity. In its native state, α-synuclein is soluble, monomeric and

unfolded. Upon binding with phospholipids, its structure conforms to the α-helical form.

Furthermore, α-synuclein has the tendency to self-aggregate into oligomers, protofibrils and

3

fibrillar β-pleated sheet structures, which are observed in LB (Shulman et al., 2011). Despite its

primary localization in the cytoplasm, α-synuclein has also been found in the mitochondria of

post-mortem PD brains, suggesting that this protein has a mitochondrial targeting sequence

(Devi et al., 2008). In the mitochondria, α-synuclein accumulation has been shown to impair

complex I and IV function, induce mtDNA damage, loss of mitochondria membrane potential,

increased levels of mitochondrial ROS and mitochondrial-dependent apoptosis (Stichel et al.,

2007; Tanaka et al., 2001). Taken together, this suggests that an alteration of α-synuclein

expression by the SNCA gene can lead to abnormal accumulation of toxic aggregates that can

interfere with mitochondrial function resulting in cellular death.

1.2.2.2 PTEN induced putative kinase 1 (PINK1)

PTEN induced putative kinase 1 (PINK1) gene mutation is an autosomal recessive form of

Parkinsonism (Valente et al., 2004). PINK1 gene encodes for the 581 amino-acid protein with an

N-terminal mitochondrial targeting sequence, a transmembrane domain and a highly conserved

serine/threonine kinase domain with homology to the Ca2+

/calmodulin family (Exner et al.,

2012). Suggested substrates of PINK1 include the mitochondrial serine protease HtrA2/Omi,

TNF receptor-associated protein 1 (TRAP1) and complex I of the electron transport chain.

HtrA2/Omi is a mitochondrial protein that translocates to the cytosol during apoptosis and

interacts with apoptosis inhibitor proteins. PINK1 mutation in PD results in hypophosphorylated

HtrA2/Omi and more susceptibility to apoptotic signaling (Plun-Favreau et al., 2007). TRAP1 is

a mitochondrial chaperone of the HSP90 family (Pridgeon et al., 2007). Phosphorylation of

TRAP1 by PINK1 suppresses mitochondrial cytochrome c release, mitochondrial pore opening

thus abnormal PINK1 expression can lead to increased pro-apoptotic signaling. PINK1 also

regulates the enzymatic activities of complex I of the electron transport chain and the loss of

PINK1 reduces ATP production for synaptic activities in the brain (Morais et al., 2009). Taken

together, PINK1 appears to serve an important role in stabilizing ATP production and preventing

mitochondrion-induced apoptosis.

1.2.2.3 Parkin

Parkin gene mutation is an autosomal recessive form of Parkinsonism (Kitada et al., 1998). The

parkin gene encodes the cytosolic 465 amino-acid protein with an ubiquitin domain at the N-

terminus and a Ring-between-Ring domain at the C-terminus (Valente et al., 2004). It functions

4

as an E3 ubiquitin protein ligase that mediates the assembly of ubiquitin monomers, targeting

proteins for degradation. Parkin mutation causes impaired ubiquitination and proteasomal

degradation thus leading to accumulation of unwanted proteins such as α-synuclein, which can

lead to mitochondrion-induced cell death discussed in section 1.2.2.1. Aside from its role in the

proteolytic system, Parkin also targets various mitochondrial substrates including PARIS,

mitochondria transcription factor A (TFAM) and complex I of the ETC. PARIS functions as a

transcriptional repressor and its overexpression leads to the inhibition of peroxisome proliferator-

activated receptor γ coactivator 1-α (PGC-1α) which is a master regulator of mitochondrial

biogenesis. Parkin is able to suppress PARIS so PGC-1α can be upregulated. Thus mutation in

Parkin expression can lead to the inhibition of PGC-1α expression and mitochondrial biogenesis

(Kuroda et al., 2006). TFAM functions as a regulator of mitochondrial transcription activities

and mitochondrial DNA (mtDNA). Parkin interacts with TFAM to ensure gene expression in the

mitochondria is optimal. Downregulation of TFAM leads to cell death, suggesting Parkin’s vital

role in the mitochondria (Ekstrand et al., 2007). Other evidence linking Parkin gene to

mitochondrial function is the post mortem analysis of PD brains showing that there is reduced

complex I activity in the mutant Parkin group (Müftüoglu et al., 2004). The link between Parkin

and the respiratory chain was further supported by Palacino et al. (2004). These researchers

demonstrated that suppression of Parkin gene in null mice resulted in reduced enzymatic

activities of Complex I and IV, leading to impaired electron transport chain and increased ROS

in brain tissue. Taken together, Parkin mutation has been implied to cause impaired ubiquitin

degradation pathway, decreasing mitochondrial biogenesis and impairing mitochondrial

functions.

1.2.2.4 DJ-1

Mutations in the DJ-1 gene area rare form of autosomal recessive Parkinsonism (Bonifati et al.,

2003). DJ-1 encodes for the 189-amino acids belonging to the ThiJ/PfpI protein family. It is

primarily localized in the cytoplasm but has also been observed to reside in the mitochondria

(Zhang et al., 2005). Its function has been linked to developing more resistance against oxidative

stress since the loss of DJ-1 results in increased vulnerability to hydrogen peroxide and MPTP

(Keane et al., 2011). Furthermore, the loss of DJ-1 has been shown to increase mitochondrial

fragmentation, suggesting its role in mitochondrial morphology maintenance (Irrcher et al.,

5

2010). Taken together, DJ-1 appears to play a protective role against oxidative stress and

mitochondrial fragmentation.

1.2.3 Mitochondrial Toxins Mimicking Cell Death Mechanisms in PD

In addition to genetic cases and post mortem studies linking PD to mitochondria dysfunctions,

there is also an extensive evidence to suggest that PD like pathologies can be induced utilizing

toxins that impair mitochondrial functions such as rotenone, MPTP and paraquat.

1.2.3.1 Rotenone

Rotenone, the active ingredient found in many pesticides, is another complex I inhibitor that can

induce specific nigrostriatal neuronal death and α-synuclein aggregates. Rotenone is lipophilic

and readily crosses the blood brain barrier to enter neurons. Once inside the neuron, it enters the

mitochondria without the aid of transporters (Keane et al., 2011). In rodents, chronic rotenone

exposure can produce Lewy body like inclusions and can also trigger PD like behaviors such as

hypokinesia and rigidity (Betarbet et al., 2000). In dopaminergic SH-SY5Y cell line, rotenone

resulted in the loss of mitochondrial membrane potential, release of cytochrome c and increased

cellular death (Imamura et al., 2006). It is thought that rotenone can induce ROS generation by

damaging complex I at 4Fe-4S clusters, which increases ubisemiquinone formation (a primary

electron donor in superoxide generation) (Li et al., 2003; Panov et al., 2005).

1.2.3.2 1-methyl-4-phenylpyridinium (MPTP)

1-methyl-4-phenylpyridinium (MPTP) is a meperidine analogue with heroin-like properties

(Ziering et al., 1947). In primates, MPTP administration resulted in α-synuclein positive Lewy-

body like inclusions (Kowall et al., 2000). MPTP readily enters glial cells and gets converted to

the active metabolite 1-methyl-4-phenylpyridinium ion (MPP+) by monoamine oxidase B. MPP

+

has an affinity for the neuronal dopamine transporter (DAT); thus this toxin specifically

accumulates in the dopaminergic neurons. Once MPP+ enters a neuron, it gets taken up by the

mitochondria via the mitochondrial transmembrane gradient, inhibits complex I activity and

increases electron leakage leading to the genesis of ROS (Nicklas et al., 1985). MPP+

specifically prevents electron movement from the iron sulfur cluster of complex I to Q10

complex, resulting in impaired electron transport chain and ATP levels depletion (Ramsay et al.,

1987).

6

1.2.3.3 Paraquat

1,1-dimethyl-4,4-bipyridinium dichloride (Paraquat) is an herbicide that has a similar structure as

MPP+, suggesting a similar toxin mechanism. Exposure to paraquat has been demonstrated to

increase α-synuclein levels and aggregation (Manning-Bog et al., 2002). Paraquat exhibits a

weak complex I inhibitory activities in dopaminergic neurons but most of its harmful effects are

due to its ability to undergo redox cycle in the mitochondria, resulting in lipid peroxidation

(Richardson et al., 2005).

1.2.4 Oxidative Stress in PD

Another important factor contributing to PD pathology is the accumulation of oxidative stress

that can trigger various cellular damage as discussed in section 1.2.1. Dopaminergic nigral

neurons in the substantia nigra pars compacta appear to be more susceptible to oxidative stress

damage for several reasons: 1) the metabolism of dopamine. 2) low levels of antioxidant

enzymes. 3) high levels of iron. These factors contribute to the increase in mitochondrial ROS

generation and also impair the electron transport chain, leading to mitochondrion-induced cell

death.

1.2.4.1 Dopamine metabolism toxicity in PD

The dopamine molecule is structurally unstable and can auto-oxidate to form free radicals such

as DA quinones (DAQ) and superoxide radicals. This reaction is accelerated in the presence of

enzymes such as tyrosine hydroxylase (TH), which is a limiting enzyme in dopamine synthesis.

Furthermore, 3,4-dihydroxyphenylacetic acid (DOPAC), another dopamine metabolite, can

undergo further oxidation to generate ROS and DOPAC-quinones. Under normal conditions, DA

is stored in synaptic vesicles and is ready to be released during a synaptic activity. However,

problems arise when vesicular storage of DA gets disrupted and DA leaks to the cytoplasm. This

results in an increased DA cytosolic content thus increased auto-oxidation of DA. In fact,

inhibition of vesicular monoamine transporter (VMAT2), a protein responsible for transporting

DA into its vesicles, resulted in increased amount of DA oxidation and DA cell death (Caudle et

al., 2007). Likewise, increasing cytosolic levels of dopamine via overexpression of dopamine

transporter (DAT) has been shown to increase the amount of DA cell death (Chen et al., 2008). It

has also been suggested that ROS or reactive quinones may get into the mitochondria and

7

interfere with the electron transport chain, specifically Complex I and IV to inhibit mitochondrial

respiration (Brenner-Lavie et al., 2009).

1.2.4.2 High levels of iron in PD brains

The substantia nigra pars compacta is also known to contain higher levels of iron (Fe) compared

to other brain regions (Synder & Connor, 2009). Iron can exist in two forms: the normal Fe3+

state or the reduced Fe2+

state. In PD, it has been demonstrated that Fe3+

to Fe2+

ratio is altered

from 2:1 to 1:2 for unknown reasons. Fe2+

is an integral component in Fenton reaction where

hydrogen peroxide (H2O2) is converted to the highly reactive hydroxyl radical (•OH). Thus,

increasing the reduced form of iron (Fe2+

) will upregulate free radical generation. Moreover,

research has shown that iron readily binds to α-synuclein, which accelerates its aggregation

process which contributes to Lewy Body formations (Peng et al., 2010). Alterations in iron

import and export are also thought to contribute to the accumulation of iron in a cell (Friedman

et al., 2011). In fact, there is a decrease in the iron storage protein ferritin levels in the brains of

PD patients, hence lowering the iron storage capacity, which results in more iron released into

the cytosol to partake in Fenton reaction. Iron can also be taken up by the mitochondria via the

transferrin receptor 2. Increased levels of iron have been observed in the mitochondria of

substantia parts compacta neurons of PD patients (Mastroberardino et al., 2009). Since the

mitochondria produce high levels of ROS through its oxidative phosphorylation, high iron levels

in the mitochondria are more likely to participate in the Fenton reaction to create more ROS and

cause ROS induced cellular damage.

1.2.4.3 Low levels of antioxidant enzymes in PD brains

Lowered levels of mitochondrial antioxidant molecules have also been observed in PD brains. In

the mitochondria, the glutathione molecule plays an important role in the antioxidant pathway.

Glutathione can exist in two different forms: the oxidized form or known as glutathione disulfide

(GSSG) and the reduced glutathione form (GSH). With the presence of NADPH, GSSG is

readily converted to GSH. GSH is then utilized by gluthathione peroxidase (GPX) to scavenge

and break down ROS. In PD, an abnormal decrease in antioxidant molecule GSH has been

observed. A theory proposes that the lowered level of GSH is linked to a decrease in synthesis

and recycling from GSSG to GSH thus less GSH availability to be used by GPX (Martin &

Teismann, 2009). Since the SNc in PD brains is under constant stress from ROS accumulation in

8

the mitochondria from the respiratory chain impairment, ROS-induced damaged is more likely to

occur. Moreover, the fact that there are low levels of antioxidant enzymes available in the SNc of

PD brains can exacerbate ROS-induced damage, resulting in dopaminergic neuronal death.

1.2.5 Proteolytic System Impairment in PD

Proteolytic stress is another factor implicated in the pathology of PD. Proteolytic stress refers to

when levels of unwanted proteins accumulate and exceed the degradation capacity of the cell.

This can result in the formation of protein aggregates that interfere with cellular function. There

are two main cellular degradation systems: the ubiquitin-proteasome system (UPS) and the

autophagy-lysosome pathway (ALP). The UPS is responsible for smaller sized molecule

degradation whereas ALP can digest larger molecules and also dysfunctional organelles. In PD,

post mortem analysis and genetic mutations have suggested that proteolytic system impairment

may contribute to cell death pathology of the disease. The abnormal aggregations of α-synuclein

to form Lewy Body inclusions imply that these proteins were not degraded in effectively.

Accumulation of α-synuclein has also been demonstrated to impair mitochondrial function,

resulting in mitochondrion-induced apoptosis. Moreover, a mutation in ubiquitin C-terminal

hydrolase L1 (UCH-L1) gene has been linked to neurodegeneration observed in PD pathology.

1.2.5.1 Ubiquitin Proteasome System (UPS)

Activation of ubiquitin monomers is an ATP-dependent reaction. Typically, ubiquitin-activating

enzyme (E1) gets activated and interacts with ubiquitin conjugating enzyme (E2). Together, both

E1 and E2 complex then bind to ‘target’ proteins with the assistance of ubiquitin protein ligase

(E3). Finally, these ubiquitinated proteins are degraded by the 26S/20S proteasome subunit. In

PD, it has been hypothesized that there is a loss of the α-subunit function of the 26S/20S

proteasome complex. Impairment to the α-subunit can interfere with the overall 26/20S

proteasome subunit stability and reduced PA700, integral component in initiating protein

degradation, binding to the proteasome subunit (Voges et al., 1999). As a result, unwanted

proteins accumulate and aggregate which can trigger the JNK and apoptotic pathways (Sherman

& Goldberg, 2001).

9

1.2.5.2 Autophagy-lysosome pathway (ALP)

Another main important proteolytic pathway is the autophagy-lysosome pathway (ALP). There

are three major types of ALP which include macroauthophagy, microautophagy and chaperone-

mediated autophagy (CMA). During UPS failure, ALP gets recruited as a compensation to

degrade accumulation of unwanted proteins. Impairment of CMA has been implicated in PD

(Cuervo et al., 2004). CMA complex or known as lysosome-associated membrane protein

(LAMP-2A) complex consists of heat shock cognate protein 70 (HSC70), heat shock protein 70

(HSP70) chaperone and Hsp70 cochaperones (Xilouri & Stefanis, 2011). HSP70 and HSP90

function to stabilize the assembly and disassembly of LAMP-2A complexes. Once assembled,

LAMP-2A complex is responsible in the translocation of ‘target’ protein for degradation by the

lysosome. In PD brains, a decrease in both LAMP-2A and HSP70 levels in the SNc were

observed (Alvarez-Erviti et al., 2011). Furthermore, another study has shown that suppression of

the LAMP-2A gene in dopaminergic cells result in an increased amount of α-synuclein (Xillouri

& Stefanis, 2011). Thus, proper UPS and ALP functioning is essential in order to prevent

accumulation of protein aggregates that can impair cellular functioning.

1.2.6 Proteolytic system inhibitors that mimic cell death mechanisms in PD

The involvement of the proteolytic system impairment in PD pathology was further

demonstrated utilizing toxins that impair either the UPS or the lyososomal system. By inhibiting

the proteolytic system, PD like pathologies can manifest and trigger mitochondrion-induced cell

death. This toxicity is most likely mediated through the accumulation of undegraded α-synuclein

proteins, which can interfere with mitochondrial function as discussed in section 1.2.2.1.

1.2.6.1 5,8-dihydroxy-1,4-naphthoquinone (Naphthazarin)

Naphthazarin exert its damaging effects by producing free radical species within the cell, leading

to the collapse of the lyososomal membrane. First, it is metabolized to its semiquinone form by

intracellular reductases and then undergoes redox cycling to create superoxide radicals (Roberg,

Johansson & Ollinger, 1999). It is believed that the destabilization of lyososomal membrane is an

early event of pro-apoptotic signaling cascade (Ollinger and Brunk, 1995; Brunk et al., 1995). In

particular, elevated levels of free radicals have been strongly linked to lysosomal disintegration.

By exposing cells to naphthazarin, lysosomal membranes are disrupted thereby releasing

cathepsin D (cat D) from the lysosome to the cytosol to trigger mitochondrial cytochrome c

10

release. Furthermore, there has been evidence suggesting a potential link between lysosomal

dysfunction and mitochondrial function. In vitro, naphthazarin exposure results in the loss of

mitochondrial membrane potential and ATP depletion (Roberg, Johannson & Ollinger, 1999).

Since the lyososomal system plays a role in degradation of α-synuclein, its loss of function can

lead to α-synuclein accumulation in the mitochondria and interfere with the respiratory chain

observed in PD.

1.2.6.2 Proteasome inhibitor N-carbobenzyloxy-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI)

Proteasome inhibitor N-carbobenzyloxy-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI) is a derivative of

the naturally occurring Epoximicin peptide aldehyde inhibitors of the proteasome. It interferes

with the proteasome subunit by binding to the primary site for peptide bond cleavage of the 20S

subunit. By covalently binding the subunit, PSI blocks the proteolytic function of the proteasome

subunit (Salama and Arias-Carrion, 2011). Systemic administration of PSI in rats exhibited PD

like pathologies. Neurodegeneration was observed in nigrostriatal system, locus coeruleus (LC),

dorsal motor nucleus of the vagus (DMN), nucleus basalis of Meynert (NBM), raphe nuclei – all

areas involved in PD (McNaught et al., 2004; Zeng et al., 2006). Furthermore, it resulted in the

formation of Lewy body (LB) like inclusions in neurons of the substantia nigra compacta (SNc),

LC and DMN (McNaught et al., 2004; Zeng et al., 2006). Thus, it is evident that the proteasome

system is actively involved in the degradation of α-synuclein proteins, since its inhibition

resulted in LB like inclusions. As discussed in section 1.2.2.1, α-synuclein build up can interfere

with mitochondrial functions and result in cell death observed in PD. Moreover the proteasome

system requires ATP for its activities thus depletion in ATP levels caused by mitochondrial

dysfunctions will exacerbate the toxicity.

1.2.7 Involvement of Ca2+

in PD

The mitochondrion plays a vital role in calcium (Ca2+

) signaling which is an integral factor in

regulating oxidative phosphorylation (OXPHOS) and apoptosis. The rate of OXPHOS is

regulated via a positive feedback of ADP and inorganic phosphate (Pi). This means that a higher

ADP and Pi will stimulate OXPHOS while low ADP and Pi will decrease OXPHOS. However, it

has been observed that OXPHOS can take place independent of changes in ADP and Pi levels

(Duchen, 2000). Intramitochondrial Ca2+

can regulate OXPHOS by interacting with pyruvate

dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and F0F1ATPase thus

11

stimulating ATP production. In fact, increasing intramitochondrial Ca2+

content alone is enough

to stimulate oxidative phosphorylation, suggesting that Ca2+

is a key component for ATP

production (Territo et al., 2001). However, high levels of Ca2+

can be detrimental to the cell.

Studies have suggested that 20-80nmol Ca2+

/mg is a safe and tolerable concentration whereas

inducing 300nmol Ca2+

/mg usually can trigger the opening of mitochondrial pore opening within

minutes (also known as excitoxicity) (Gunter et al., 2004). Excitoxicity usually occurs due to the

overactivation of N-methyl-D-aspartate (NMDA) receptors, resulting in the recruitment of

glutamate and ultimately high amount of Ca2+

cellular influx. Due to the mitochondria’s ability

to uptake Ca2+

, intracellular increased of Ca2+

can cause impaired mitochondrial function. By

accumulating in the mitochondria, Ca2+

has been demonstrated to alter mitochondrial membrane

potential, impaired ATP synthesis and increase ROS levels (Nicholls & Budd, 1998). As

discussed in section 1.2.1, there is a plethora amount of evidence linking between complex I

impairment and PD pathologies. There is also evidence suggesting that complex I inhibition

increases mitochondria’s susceptibility to Ca2+

toxicity (Sherer et al., 2001). In mice, an increase

in glutamate levels in the SNc has been observed after exposure to complex I inhibitors,

suggesting the possible role of Ca2+

toxicity in PD pathologies (Meredith et al., 2009).

1.3 OTHER IMPORTANT FUNCTIONS OF THE MITOCHONDRIA

Aside from participating in oxidative phosphorylation, Ca2+

storage, antioxidant defenses, the

mitochondrion is responsible for other crucial cellular functions including mitochondrion-

induced apoptotic signaling and mitochondria morphology.

1.3.1 Apoptotic signaling

The mitochondria play an important role in mediating apoptosis. Apoptotic events include the

opening of mitochondrial permeability pore (MPP), the release of pro-apoptosis factors such as

cytochrome c, apoptosis inducing factor (AIF) from the intermembrane space to the cytosol

cytoskeletal breakdown and DNA fragmentation. Once released, these pro-apoptosis factors can

initiate apoptosis via caspase pathway. The opening of MPP will result in dispersion of ions

across the membrane, the loss of mitochondria membrane potential and ATP depletion. Opening

of MPP is closely mediated by the pro-apoptotic proteins such as Bax and Bak and also by the

anti-apoptotic proteins of the Bcl-2 family.

12

Several stimuli can trigger mitochondrion-induced apoptosis. Stressors such as cytokines,

hypoxia, toxins, radiation and reactive oxygen species (ROS) can induce pro-apoptotic signaling

(Elmore, 2007). Apoptosis is closely regulated by Bcl2 protein families. Bcl2 proteins can be

divided into two categories: pro-apoptotic and anti-apoptotic. Pro-apoptotic proteins include

Bcl10, Bax, Bak, Bid, Bad, Bim, Bik and Blk whereas anti-apoptotic proteins consist of Bcl2,

Bclx, BclXL, BclXS, Bclw and Bag. Pro-apoptotic proteins are capable of interacting directly on

the outer mitochondrial membrane and can trigger the opening of MMP. These proteins are also

hypothesized to interact with cytochrome c, Smac/DIABLO and HtrA2/Omi and control its

release from the mitochondria intermembrane space to the cytosol (Elmore, 2007). Once

released, cytochrome c will bind and activate Apaf-1 and procaspase-9 to form apoptosome

whereas Smac/DIABLO and HtrA2/Omi function by inhibiting IAP (inhibitors of apoptosis

proteins) to induce apoptosis. Once the apoptotic mechanism is underway, more apoptotic

proteins such as AIF, endonuclease G and CAD are released from the mitochondria. These

proteins are translocated to the nucleus to initiate DNA chromatin condensation and DNA

fragmentation (Joza et al., 2001). Moreover, Caspase-3 gets recruited to induce cytoskeleton

breakdown and cell disintegration into apoptotic bodies.

Apoptosis can be prevented with interaction between pro-apoptotic and anti-apoptotic proteins.

Pro-apoptotic protein Bad can inhibit anti-apoptotic proteins BclXl or Bcl2 to promote cellular

death (Yang et al., 1995). Without inhibition by Bad, both Bcl2 and BclXl prevent the release of

pro-apoptotic factor cytochrome c from the mitochondria. Thus healthy and functional

mitochondria are essential in order to have a controlled balance between the pro-apoptotic and

anti-apoptotic pathways.

1.3.2 Mitochondrial dynamics

The mitochondria undergo continuous fission and fusion processes in order to alter their

morphology. In mammals, proteins such as dynamin-related guanosine triphosphatases

(GTPases), mitofusion 1 (Mfn1), mitofusion 2 (Mfn2) are involved in mitochondrial fusion. The

C-terminal membrane binding domains of Mfn1 and Mfn2 are attached to the mitochondrial

outer membrane (MOM) whereas their N-terminal GTPase domain faces the cytoplasm. Both

Mfn 1 and 2 mediate fusion through their active N-terminal GTPase domains by binding

mitochondrial membranes together. Once opposing MOMs are tethered together, mitochondria

13

inner membranes (MIM) are fused and mitochondrial components will be mixed together

(Ishihara et al., 2004). Both Mfn1 and Mfn2 are thought to affect MOM fusion while Opa1 is

suggested to form cristae junctions and MIM fusion. On the other hand, mitochondrial fission

involves proteins such as dynamin-related protein 1 (Drp1) and fission protein 1 (Fis1). Drp1

self-oligomerizes on the mitochondrial surfaces and interacts with Fis1 to assemble into foci that

act as potential scission sites for fission events (Smirnova et al., 2001).

Mutations in mitochondrial DNA (mtDNA) can accumulate overtime and cause detrimental

effects to mitochondria health. Wallace and Fan (2009) suggested that if there are approximately

70-90% mtDNA mutations, pathological diseases are more likely to develop. Indeed, a study by

Chen et al. (2010) showed that loss of mitochondrial fusion in skeletal muscle resulted in

mtDNA point and deletion mutations, which propose the idea that exchanging mitochondrial

content via fusion can increase tolerance against mtDNA mutations. Conversely, knocking out

Drp1 and Fis1 genes in cells result in high levels of mutant mtDNA compared to wild-type

mtDNA (Malena et al., 2009). Hence, modifications in expression levels of fusion and fission

proteins can affect the separation of mutant and wild-type mtDNA which play an important

factor in regulating mitochondrial function. Moreover, degradation of damaged or energy-

deficient mitochondria is necessary to prevent apoptosis (Wohlgemuth et al., 2010). Fission is

closely related to degradation of dysfunctional mitochondria. There have been reports where

accumulation of enlarged or highly interconnected mitochondria has low ATP production, loss of

cristae structure and a swollen morphology (Terman and Brunch, 2005). Likewise, suppression

of fission protein Fis1 can cause prolonged mitochondrial fusion, increased ROS levels and

reduced mitochondrial membrane potential (Lee et al., 2007; Yoon et al., 2006). Therefore, a

balance in fusion and fission machinery is critical in order to reduce mtDNA mutations, degrade

unhealthy mitochondria and maintain ATP production.

Taken together, mitochondrial dysfunctions appear to be the most significant contributor to

cellular death in Parkinson’s disease. Post mortem analysis and genetic cases of PD suggest that

mitochondria dysfunction may be the trigger of cell death. Furthermore, dopaminergic nigral

neurons have been shown to be more susceptible to mitochondrial oxidative stress overload.

Proteolytic system dysfunctions can also result in the accumulation of α-synuclein and interfere

with the respiratory chain of the mitochondria. Moreover, toxins utilized to recapitulate PD like

pathologies appear to impair mitochondrial functions. The mitochondria also play an important

14

role in mediating apoptosis, excitoxicity and mitochondrial morphology. Thus, converging

evidence suggests that mitochondrial health is essential to prevent cell death. Hence, to find a

treatment to prevent further cell death in PD, the mitochondrion is an excellent organelle to

target.

1.4 THE MAMMALIAN SIRTUINS

The sirtuins are a group of nicotinamine adenine dinucleotide (NAD+) dependent deacetylases

and/or ADP-ribosylases (Michan & Sinclair, 2007). Acetylation/deacetylation is an important

posttranslational modification since removal of an acetyl group can affect various pathways

involved in metabolism and ultimately overall functioning of the cell (Glozak et al., 2005). On

the other hand, ADP ribosylation is involved in various signaling pathways within the cell and is

involved in DNA repair (Hassa et al., 2006). In mammals, seven sirtuins have been identified:

Sirtuin 1(SIRT1) - Sirtuin 7(SIRT7). Each of these sirtuin is localized in various locations in the

cell – the nucleus, the cytoplasm and the mitochondria.

1.4.1 Nuclear Sirtuins

SIRT1 is NAD+ dependent deacetylase predominantly localized in the nucleus and serves an

important role in anti-apoptotic regulation, insulin secretion, mitochondrial biogenesis and anti-

apoptotic effects. SIRT1 has been linked with substrates such as: p53 which leads to reduction of

its pro-apoptotic effect, peroxisome proliferator-activated receptor gamma coactivatior-1α (PGC-

1α) and forkhead box O1 (FOXO1) which promotes glucogenesis, insulin sensitivity and

mitochondrial biogenesis (Puigserver et al., 2003; Puroshotham et al., 2009). SIRT1 also

deacetylases the transcriptional repressor of glucogenesis STAT3 and inhibits its activity thus

enhancing glucogenesis (Nie et al., 2009).

SIRT6 is another NAD+ dependent deacetylase localized in the nucleus and serves to regulate

genome stability, metabolism and inflammatory response. In addition to NAD+ dependent

deacetylase, SIRT6 was also shown to carry out its function as ADP-ribosylase in mouse (Liszt

et al., 2005). From previous findings, SIRT6 is thought to interact with substrates such as acetyl-

H3K9 and acetyl-H3K56, which are important in regulating DNA repair, telomere function,

genomic stability and cellular senescence (Michishita et al., 2008; Yang et al., 2009). SIRT6

also suppresses nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)

(Kawahara et al., 2009) resulting in decreased proinflammatory cytokines signaling. In order to

15

maintain metabolic balance, SIRT6 suppresses the transcription factor hypoxia-inducible factor

1-alpha (HIF-1-ɑ) to downregulate glucose uptake and glycolysis (Zhong et al., 2010).

SIRT7 is the only Sirtuin localized in the nucleolus. It is a NAD+ deacetylase and appears to

regulate RNA polymerase I (Pol I) transcription machinery. By interacting with RNA Pol I,

SIRT7 is presumed to regulate the transcription of rDNA during transcriptional elongation

(Grummt and Pikaard, 2003). Through this interaction, SIRT7 ensures cell proliferation and

prevents apoptosis.

1.4.2 Cytoplasmic Sirtuins

SIRT2 is a NAD+ dependent deacetylase primarily found in the cytoplasm and has been

implicated in regulating cytoskeletal functioning, cell cycle and functioning. In the cytoplasm,

SIRT2 deacetylates microtubules (North et al., 2003) and it is able to migrate to the nucleus

during G2/M transition to deacetylase histone H4 and stabilize chromatin condensation during

metaphase (Vaquero et al., 2006). Moreover, SIRT2 has been linked to activate transcription

factors class O, FOXO1 and FOXO3 to regulate cell cycle, apoptosis, metabolism and DNA

repair (Li et al., 2007; Wang and Tong, 2009; Zhu et al., 2012).

1.4.3 Mitochondrial Sirtuins

SIRT3 is NAD+ dependent deacetylase which localizes in the mitochondria and targets

important substrates involved in metabolic pathways, antioxidant defenses and cellular death

signaling. Details of SIRT3 and its functions will be discussed later in this thesis.

SIRT4 is another mitochondria sirtuin and has been suggested to serve a role in metabolism. The

only known substrate of SIRT4 is glutamate dehydrogenase (GDH). SIRT4 interacts with GDH

and suppresses GDH activities via ADP-ribosylation in order to suppress insulin secretion

(Ahuja et al., 2007; Haigis et al., 2006). Moreover, SIRT4 has been implicated to regulate fatty

acid oxidation but the mechanisms of this interaction are unclear (Nasrin et al., 2010).

SIRT5 is the last NAD+ dependent deacetylase localized in the mitochondria and has been

implicated to modulate the urea cycle. It has been reported to deacetylates carbamoyl

phosphatase synthase (CPS1), the rate limiting enzyme of the urea cycle in the mitochondria.

16

Deacetylating CPS1 prevents ammonia build up after prolonged fasting thus reducing cellular

toxicity (Nakagawa et al., 2009).

Since it is believed that mitochondria dysfunction is central to Parkinson’s disease, enhancing

mitochondrial function may be therapeutic. Therefore, the focus of this thesis will be on the

mitochondrial sirtuins.

Lombard et al. (2007) has shown that 20% of mitochondrial proteins are acetylated, suggesting

that the acetylation/deacetylation process is integral to the organelle’s functioning. Glozak et al.

(2005) suggested the removal of acetyl group is a key regulator in cell metabolism and survival.

SIRT3, SIRT4 and SIRT5 have been suggested to be localized in the mitochondria and each has

been implicated to serve various functions. Interestingly, amongst the mitochondria sirtuins,

SIRT3 appears to be the main deacetylase. Lombard et al. (2007) demonstrated that knocking out

SIRT3 in mice resulted in a high amount of hyperacetylated proteins within the mitochondria.

However, suppressing SIRT4 or SIRT5 in mice did not significantly affect levels of acetylated

proteins. This suggests that SIRT3 is the major sirtuin responsible for deacetylating proteins in

the mitochondria. SIRT3 targets various targets such as catabolic pathways, electron transport

chain complexes, antioxidant defenses, urea cycle and mitochondrial pore opening (Figure 1).

17

Figure 1.

18

Figure 1. Overview of SIRT3 mitochondrial substrates

SIRT3 deacetylates various mitochondrial substrates in oxidative phosphorylation, antioxidant

defenses, urea cycle, mitochondrial protein synthesis pathway and cellular death signaling. In

oxidative phosphorylation pathway, SIRT3 interacts with glutamate dehydrogenase (GDH),

isocitrate dehydrogenase 2 (IDH2), 3-hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS2),

long-chain acyl-CoA dehydrogenase (LCAD), acetylCoA synthase 2 (ACS2) and Complex I-V.

In anti-oxidant pathway, SIRT3 interacts with superoxide dismutase 2 (SOD2). In urea cycle,

SIRT3 interacts with ornithine transcarbamoylase (OTC). In cellular death pathway, SIRT3

interacts with Cyclophilin D (CypD). In mitochondrial protein synthesis pathway, SIRT3

interacts with mitochondrial ribosomal protein L10 (MRPL10).

19

1.5 SIRT3

There has been a debate as to where SIRT3 is truly localized. One study has suggested that

human SIRT3 (hSIRT3) is a nuclear 44kDa protein and has an N-terminal mitochondrial

localization sequence. It gets translocated to the mitochondria where it is cleaved by

mitochondria matrix peptidase (MMP) to its active 28kDa form (Schwer et al., 2002). On the

other hand, mouse SIRT3 (mSIRT3) has been shown to lack the N-terminal mitochondrial

localization sequence and exists in its 28kDa form already (Yang et al., 2000). These two

contradicting findings raise the question as to whether mouse and human SIRT3 function via

different mechanisms. Furthermore, Scher et al. (2007) suggested that hSIRT3 is nuclear and

only transported to the mitochondria during conditions of stress where it gets cleaved to its active

28kDa form. To challenge the findings by Scher et al. (2007), another study demonstrated that

hSIRT3 is exclusively mitochondrial (Cooper and Spelbrink, 2008). Despite the controversies,

much evidence supports the notion that SIRT3 carries out its true deacetylating functions in the

mitochondria.

1.6 MECHANISMS OF SIRT3

1.6.1 SIRT3 and Metabolism

In the absence of glucose, the mitochondrion has the ability to utilize fatty acids, amino acids and

acetates as an alternative source of energy. In the fatty acid catabolism (or β-oxidation), long-

chain acetyl coenzyme A dehydrogenase (LCAD) is the key enzyme that breaks down fatty acids

and produces acetyl-coA, which readily enters the Krebs cycle (Figure 2A). In SIRT3 knockout

mice, LCAD is hyperacetylated in the liver leading to a decrease in enzymatic activities, β-

oxidation and ATP level (Hirschey et al., 2010). This suggests that SIRT3 deacetylates LCAD

since SIRT3 wild type mice had more LCAD activity compared to knockout SIRT3.

In amino acid catabolism, glutamate dehydrogenase (GDH) is the key enzyme that converts

glutamate to α-ketoglutarate and produces NADPH as a byproduct (Figure 2A). SIRT3

deacetylates GDH in mice and has been shown to increase GDH activity compared to knockout

SIRT3 mice (Schlicker et al., 2008). Furthermore, NADPH generated from glutamate to GDH

conversion is a key component in stimulating the anti-oxidant pathway.

20

In acetate catabolism, the initial step for the conversion from acetate to acetyl-CoA is catalyzed

by acetyl-CoA synthase 2 (ACS2). Upregulation of ACS2 will result in increased acetyl-CoA

which is an important component to stimulate the Krebs cycle. Overexpression of SIRT3 has

been demonstrated to decrease acetylation levels of ACS2, suggesting that it functions as a

deacetylase on ACS2 (Figure 2A) (Schwer et al., 2006).

Ketone body production is closely regulated by 3-hydroxy-3-methylglutaryl CoA (HMGCS2).

Ketogenesis is triggered during fasting and results in the upregulation of HMGCS2 expression,

increased glucagon and cyclic-AMP (Madsen et al., 1999). SIRT3 has been demonstrated to

deacetylate HMGCS2 (Figure 2A), the rate-limiting enzyme for the synthesis of the ketone β-

hydroxybuterate from aceteoacetyl-CoA thus enhancing ketogenesis (Shimazu et al., 2010).

Lastly, SIRT3 deacetylases ornithine transcarbamoylase (OTC), which is one of the urea cycle

enzymes (Figure 2B). Acetylation of OTC was previously shown to impair proper hydrogen

formation and decreases its enzymatic activity (Hallows, Yu & Denu, 2011). Thus SIRT3’s

ability to deacetylate and upregulate OTC can work in favor of the urea cycle to prevent toxic

ammonia built-up.

Taken together, SIRT3 appears to play an important role in regulating alternative metabolic

pathways in the absence of the glucose which can be beneficial during caloric restriction.

1.6.2 SIRT3 and Protein Synthesis

SIRT3 has been linked to the regulation of mitochondrial protein synthesis. The mitochondrion is

unique in such a way that this organelle has its own DNA (mtDNA). Previously, it has been

shown that mitochondrial ribosomal proteins (MRP) play a crucial role in regulating

mitochondrial protein synthesis, cell growth and apoptosis (Miller, Koc & Koc, 2008). Recently,

it has been shown that mitochondria ribosomal protein L10 (MRPL10) is another substrate that

SIRT3 can deacetylate (Yang et al., 2010). By SIRT3 deacetylating activity on MRPL10, there

was a downregulation of mitochondrial protein synthesis observed. The opposite effect was

observed when MRPL10 was acetylated, suggesting that SIRT3 plays a role in regulating

mitochondrial protein synthesis. Even though the mitochondria produces 90% of ATP in

mammalian cells, caloric deprivation studies have suggested that reduction in metabolic

activities is able to reduce cell death and promote longevity (Yang et al., 2010). With caloric

21

restriction, SIRT3 expression was increased, suggesting that high amount of ATP is not always

necessarily better. Taken together, SIRT3 appears modulate mitochondrial protein synthesis via

its deacetylating activity on MRPL10.

1.6.3 SIRT3 and Oxidative Stress

SIRT3 has been implicated in the deacetylation of substrates that are involved in the anti-oxidant

pathway, apoptotic pathway, and can interact with complex I and III of ETC to reduce ROS

generation. By deacetylation, SIRT3 interacts with the Krebs cycle enzyme glutamate

dehydrogenase (GDH) and isocitrate dehydrogenase 2 (IDH2) (cite), which produces NADPH in

the mitochondria. NADPH is necessary to convert oxidized gluthathione (also known as

gluthathione disulfide (GSSG)) into reduced gluthathione (GSH). GSH then activates the

mitochondria gluthathione peroxidase (GPX) to detoxify ROS. Furthermore, SIRT3 has been

demonstrated to deacetylate another important antioxidant enzyme known as manganese

superoxide dismutase (SOD2) (Figure 2C). By activating the SOD2 enzyme, it can assist in the

breaking down of ROS to hydrogen peroxide (H2O2) which then gets broken down to water by

GPX. Specifically, SIRT3 deacetylates three lysine residues of SOD2 which are Lys 53, Lys89

and Lys 122 (Tao et al., 2010). Moreover, SIRT3 is believed to interact with complex I and

complex III of the ETC. Evidence suggests that electron leakage from complex I and III

comprise 90% of ROS production within the mitochondria (Jing et al., 2011). Hence by

deacetylating complex I and III, SIRT3 may decrease the electron leakage that is central to ROS

generation.

Additionally, SIRT3 has been shown to modulate the apoptotic pathway. A high level of

unmanaged oxidative stress can trigger the pro-apoptotic pathway which is mediated by the

mitochondria. In cardio myocytes, SIRT3 has been shown to interact with Lys 166 residue of

cyclophilin D (CypD), a protein involved in regulation of mitochondria pore opening (Hafner et

al., 2010). Apoptotic events involve the opening of the mitochondria permeability pore which

leads to the collapse of mitochondria membrane potential, depletion of ATP and release of pro-

apoptotic factors to the cytosol. By deacetylating CypD, SIRT3 inhibits its activity and triggers

its detachment from the adenine nucleotide translocator (ANT). As a result, opening of the pore

is halted and apoptosis is decreased. Collectively, this evidence suggests that SIRT3 is protective

22

against oxidative stress because it can enhance the anti-oxidant pathway and inhibit mitochondria

pore opening.

1.6.4 SIRT3 and Electron Transport Chain

SIRT3 deacetylation activities affect the mitochondrial electron transport chain (ETC). Evidence

has shown that SIRT3 plays an important role in deacetylating complex I to V. When SIRT3 is

suppressed, an increase in acetylation is observed in each of these complexes. In particular,

SIRT3 deacetylates the mitochondrial complex I component NDUFA9 (Anh et al., 2008). In

complex II, SIRT3 interacts with succinate dehydrogenase flavoprotein (SDHA) and succinate

dehydrogenase iron sulfur (SDHB) subunits and has been observed to deacetylate the F1α

subunit of complex V (Finley et al., 2011). Furthermore, suppression of SIRT3 leads to 74% and

60% reduction in complex III and IV activities respectively, suggesting that deacetylation by

SIRT3 is necessary for optimal functioning in these complexes (Kendrick et al., 2011). The role

of SIRT3 in enhancing the ETC was further supported in SIRT3 knockout mice exhibiting a

reduction in global ATP levels (Anh et al., 2008). Taken together, SIRT3 appears to play a vital

role in regulating proper mechanisms of the electron transport chain.

23

Figure 2.

A.)

B.)

24

Figure 2.

C.)

25

Figure 2. Mechanistic overview of SIRT3’s interaction with metabolic pathways, anti-

oxidant pathway and urea cycle showing sources of NAD+

A.) SIRT3 and various mitochondrial metabolic pathways. SIRT3 deacetylates long-chain

acyl-CoA dehydrogenase (LCAD) for B-oxidation or fatty acid catabolism, HMGSA for

ketogenesis, isocitrate dehydrogenase 2 (IDH2) for Krebs cycle, acetylCoA synthase 2

(ACS2) for acetate catabolism and glutamate dehydrogenase (GDH) for amino acid

catabolism

B.) SIRT3 and Urea cycle. SIRT3 deacetylates ornithine transcarbamoylase (OTC)

C.) SIRT3 and Anti-oxidant pathway. SIRT3 deacetylates glutamate dehydrogenase GDH

and isocitrate dehydrogenase 2 (IDH2) to produce NADPH as a byproduct, which is

necessary to stimulate the anti-oxidant pathway. SIRT3 also deacetylates SOD2 which

breaks down ROS to hydrogen peroxide (H2O2).

26

1.6.5 Benefits of SIRT3

SIRT3 has been closely linked with caloric restriction (CR) studies. Previous findings have

shown that SIRT3 expression was elevated in adipose tissue, skeletal muscle and liver during

CR, and this correlated with longevity extension compared to non-restricted diet treatments

(Lombard et al., 2011). This evidence suggests that SIRT3 has a role in CR and provides

protective effects. Also, SIRT3 has been implicated to function as a tumor suppressor. Kim et al.

(2010) demonstrated that mice lacking SIRT3 formed a mammary tumor after 24 months whilst

this was not the case in wild type SIRT3 group. It is thought that SIRT3 mediates its protective

effects via its interaction with the transcription factor hypoxia-inducible factor, H1F-1α. HIF-1α

is known to promote tumor growth via activating the aerobic glycolysis pathway. Indeed,

silencing SIRT3 resulted in increased ROS and H1F-1α activation and large tumor formation

whereas SIRT3 overexpression caused the smallest tumor (Bell et al., 2011).

More recently, SIRT3’s protective effects have been more evident in the brain. A study by

Someya et al. (2010) demonstrated that caloric restriction (CR) in wild type SIRT3 aged mice

protected against hair cells and spiral ganglia neurons loss in the inner ear cochlea. However, in

SIRT3 knockout mice, CR did not prevent hear loss as observed in wild type SIRT3. This

suggests that the protective effects of CR require the presence of SIRT3 in order to prevent age

related oxidative stress and promote longevity. Next, Kim et al. (2011) demonstrated that SIRT3

overexpression can prevent against NMDA induced damage in primary mouse cortical neurons.

SIRT3 expression was increased when neurons are depleted in NAD+ under NMDA toxicity via

poly (ADP-ribose) polymerase-1 (PARP-1) activation. Furthermore, SIRT3 has also been

implicated to be neuroprotective in Huntington Disease. Fu et al. (2012) showed that Viniferin

was shown to decrease ROS and prevented the loss of mitochondrial membrane potential.

Interestingly, the mechanisms of Viniferin were shown to be mediated by SIRT3, since SIRT3

knockout resulted in inhibited Viniferin-mediated AMP-activated kinase activation thus lowering

Viniferin’s protective effects. Furthermore, Weir et al. (2012) showed that SIRT3 expression is

upregulated in β-amyloid (a hallmark of Alzheimer’s disease pathology) overexpressing mice

and promoted neuronal longevity. They also observed that SIRT3 expression was enhanced in

Alzheimer’s brain tissue samples.

27

1.7 RATIONALE

Thus, there is a large amount of evidence suggesting SIRT3 has a protective role in regulating

oxidative stress damage via its deacetylating activities. The ability of SIRT3 to enhance

oxidative phosphorylation and antioxidant pathways in the mitochondria make it an interesting

target for a protective treatment in mitochondria-dysfunction related diseases. In the brain,

SIRT3 has shown protective potential against hearing loss, NMDA induced toxicity, Alzheimer’s

disease and Huntington’s disease (Someya et al., 2010; Kim, Lu & Alano, 2011; Fu et al., 2012;

Weir et al., 2012).

Since it is believed that mitochondrial dysfunction is central to Parkinson’s disease pathology, I

decided to evaluate SIRT3’s protective potential in a cell model of PD. So far, no study has been

performed to assess effects of SIRT3 overexpression in Parkinson’s disease thus this will be the

first study to do so. Here, stably transfected catecholaminergic undifferentiated SH-SY5Y cell

line overexpressing SIRT3 were utilized. SH-SY5Y cells have the ability to synthesize dopamine

and noradrenaline because the cells express tyrosine and dopamine-β-hydroxylases (Oyarce and

Fleming, 1991). Moreover, they also can express dopamine transporter (DAT), a protein found

only in dopaminergic neurons within the central nervous system (Takashi et al., 1994). In order

to recapitulate some PD pathologies, cells were be exposed to rotenone (mitochondrial complex I

inhibitor), dopamine (oxidative stressor), 5,8-Dihydroxy-1,4-napthoquinone or naphthazarin

(lysosome system inhibitor) and proteasome inhibitor N-carbobenzyloxy-Ile-Glu(O-t-butyl)-Ala-

leucinal (PSI). After incubation with the toxins, reactive oxygen species (ROS), mitochondrial

membrane potential (∆Ψm), ATP levels, cell viability and cell death were quantified.

28

1.8 HYPOTHESIS AND AIMS

Hypothesis:

Ectopic overexpression of SIRT3 protects against cellular damage induced by mitochondrial

dysfunction, oxidative stress, lysosome and proteasome system dysfunction.

Utilizing the stably transfected SH-SY5Y cells, the following aims were assessed:

1. To assess whether SIRT3 overexpression can be cytoprotective against toxins that mimic

cell death mechanisms in PD

2. If SIRT3 is cytoprotective against toxins mimicking cell death mechanism in PD, how

does it exert its cytoprotective functions?

a. Assess the effect of SIRT3 overexpression on ROS levels

b. Assess the effect of SIRT3 overexpression on mitochondrial membrane potential

c. Assess the effect of SIRT3 overexpression on ATP levels

29

CHAPTER 2

2 MATERIALS AND METHODS

2.1 Cell Culture

Human neuroblastoma catecholaminergic undifferentiated SH-SY5Y cells (CRL-2266, ATCC)

were cultured in 15 cm plates in Dulbecco’s Modified Eagle Medium (DMEM) (4.5g/L glucose,

L-Glutamine and sodium pyruvate) (319-005-CL, Wisent) with 10% bovine calf serum, heat

inactivated (074-250, Wisent) in an incubator chamber at 37oC in 5% CO2 (MCO-20AIC,

Sanyo).

2.2 Toxins

Toxins selected for the experiments were: Rotenone (R8875, Sigma-Aldrich), dopamine

hydrochloride (H8502, Sigma-Aldrich), N-carbobenzyloxy-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI)

(ENZ260089M005, Biolynx) and 5,8-Dihydroxy-1,4-napthoquinone (Naphthazarin) (388513,

Sigma-Aldrich). Rotenone, PSI, Naphthazarin and dopamine were dissolved in dimethyl

sulfoxide (DMSO) (DMS666, BioShop).

2.2.1 Dose Response Curves

Prior to the experiments, dose response curve was performed to establish the appropriate

concentration to use. Amount of cell death was measured using the fluorescence probe

Propidium Iodide (P1304MP, Invitrogen) (Excitation: 535nm, Emission: 617nm). This dye

works by passive diffusion into non-living cells and binds to nucleic acids, which then results in

fluorescent signal. Thus higher intensity reading will signify more cell death.

SH-SY5Y cells were plated at the density of 1 x 105 in a 96-well plate. Twenty-four hours later,

toxins (rotenone, dopamine, naphthazarin and PSI) were added at concentrations in the range of

0-100µM. Cells were then incubated with the toxins for 24 hours and washed once with DMEM

without phenol red (319-050-CL, Wisent). Propidium Iodide (2µM) was added to the cells and

incubated for 1 hour at 37oC in 5% CO2. Cells were then washed 1 more time with DMEM

without phenol red before fluorescent intensity readings were taken using a spectrophotometer.

The concentration that exhibited 40-50% of cell death was utilized for subsequent experiments.

30

Concentrations selected were: 30nM (Rotenone), 65µM (Dopamine), 30nM (PSI) and 300 nM

(Naphthazarin).

2.3 Construction of Stably Transfected Cells

SH-SY5Y cells were plated at the density of 5 x 105

in a 6-well plate. Twenty four hours later,

the transfecting reagent Lipofectamine 2000 (10µL) (11668-027, Invitrogen) was incubated with

DMEM without bovine calf serum (236µL) and pLKO-MYC (4µg) or pLKO-SIRT3MYC (4µg)

plasmids for 20 minutes before being added to the cells. Since both pLKO vectors have

resistance against Blasticidin S (380-089-M100, Alexis Biochemicals), a concentration of 5µM

was added to the SH-SY5Y cells to select for SIRT3-MYC and MYC transfected cells. Once

colonies were formed, individual colonies were picked, and plated in a 96 well plate and cultured

in DMEM with 5µM of selective media. Twenty days later, the colonies were transferred to a 24

well plate and treated with 400µL DMEM with 5µM of Blasticidin. Protein expression of SIRT3

was then confirmed by Western blot.

2.4 Western Blot Analysis of SIRT3 Expression

2.4.1 Sample Preparation

Stably transfected MYC-SIRT3, MYC and Naïve SH-SY5Y cells were plated at the density of

5.0x105 in 10 cm plates. Forty-eight hours later, the cells were scraped and harvested using

Laemelli Buffer (950µL) + β-mercaptoethanol (50µL) solution. Sample was then boiled for 10

minutes and protein assay was performed using Protein Assay Dye Reagent (500-0006, Bio-Rad)

as per manufacturer’s protocol. Absorbance reading was taken using spectrophotometer (595nm)

to quantify protein concentration of the sample.

2.4.2 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)

Once protein concentration had been determined via protein assay method (section 2.4.1), 38µg

of protein was loaded into each lane of 12% resolving, 4% stacking acrylamide gel and was ran

at 150 V for 1.5 hour in electrophoresis buffer (25mM Tris, 192 mM glycine and 0.1% SDS).

The gels were then transferred to nitrocellulose membrane (Bio-Rad) for 16 hours at 25 V in

transfer buffer (48mM Tris, 39mM glycine, 0.04% SDS and 20% methanol) at 4ºC. After the

transfer, the membrane was blocked for 1 hour in 1% non-fat powdered milk TBST solution

(20mM Tris, 500mM NaCl, 0.05% Tween 20) and was incubated overnight with SIRT3 rabbit

31

primary antibody (2627, Cell Signaling) (1:500 dilution in 0.1% non-fat powdered milk TBST

solution) at 4ºC. The membrane was then washed 3 times with TBST with 5 minutes incubation

between each wash and probed with HRP-conjugated rabbit secondary antibody for 1 hour

(7074, Sigma) (1:1000 dilution in 0.1% non-fat powdered milk TBST solution) 4ºC. Once the

secondary antibody incubation was over, the membrane was washed another 3 times with TBST

with 5 minutes incubation between each wash. The membrane was then treated with

chemiluminesence reagent (RPN2232, GE Healthcare Life Sciences) for 5 minutes and imaged

using Bio-Rad Gel Doc imager machine.

To check for equal loading, the membrane was stripped using a restore Western blot stripping

buffer (21059, ThermoScientific) for 2 hours at room temperature and re-probed overnight at 4ºC

for β-actin (mouse) (A5441, Sigma) (1:1000 dilution) followed by 1 hour incubation with HRP-

conjugated mouse secondary antibody (7076, Cell Signalling) (1:2000 dilution). Densitometry

technique was later utilized to quantify SIRT3 protein levels normalized to β-actin across

samples using ImageJ software.

2.5 Assessment of Human SIRT3 Ectopic Expression in SH-SY5Y cells

Stably transfected SIRT3MYC SH-SY5Y cells were plated at the density of 5.0x105 in a 6 well

plate. Twenty four hours later, cells were transiently transfected with the mitochondria marker

pOCTmito-DSRed2 plasmid (5µg) using Lipofectamine 2000 (10µl) with transfection protocol

outlined in section 2.3. Forty-eight hours post transfection, cells were washed 3 times with

PBSX1 and then fixed with 4% paraformaldehyde for 20 minutes before being washed 3 times

again with PBSX1. Cells were then permeabilized with 0.1% Triton X-100 + 100mM glycine

solution for 20 minutes. Once permeabilized, cells were washed 3 times with PBSX1 and then

blocked with 5% bovine calf serum in PBSX1 for 1 hour at room temperature. Cells were then

incubated overnight at 4ºC with MYC-Tag mouse monoclonal primary antibody (2276, Cell

Signaling) (1:500 dilution in 1% bovine calf serum PBSX1 solution).

The next day, cells were washed 3 times with PBSX1 with 5 minutes incubation between each

wash. Cells were then incubated in the dark at room temperature for 1 hour with AlexaFluro488

mouse secondary antibody (016-540-084, Jackson Immunoresearch) (1:1000 dilution in 1%

bovine calf serum PBSX1 solution). Once the incubation period was over, cells were then

washed 3 times with PBSX1 with 5 minutes incubation between each wash. Coverslips were

32

then mounted using fluorescence mounting medium (S3023, Dako) on microslide glass (48311-

703, VWR). Cells were imaged using confocal microscopy (LSM 510 Meta, Zeiss) with 100x

objective.

2.6 Measurement of Reactive Oxygen Species

Reactive oxygen species (ROS) was measured using the fluorescence dye 2',7'-

dichlorodihydrofluorescein diacetate (CM-H2DCFDA) (Excitation: 492nm, Emission: 517nm)

(C6827, Invitrogen). This dye works by the cleavage process of its acetate groups by intracellular

esterases and the interaction of its thiol chloromethyl group with intracellular glutathione and

thiols. Hence its fluorescence intensity will indirectly signify ROS within the cells.

Stably transfected SH-SY5Y MYC and SIRT3MYC cells were plated at the density of 2.5x105 in

a 24 well plate. Twenty four hours later, toxins (rotenone, dopamine, naphthazarin and PSI) were

added to the cells and incubated for 24 hours at 37oC, 5% CO2. Cells were then washed twice

with DMEM without phenol red and treated with CM-H2DCFDA (10µM) for 30 minutes in the

dark at 37oC in 5% CO2. After the incubation period, cells were washed twice with DMEM

without phenol red to remove excess dye before being measured for ROS using flow cytometry

(BD LSRFortessa).

2.7 Quantification of Mitochondrial Membrane Potential

Mitochondrial membrane potential was measured utilizing the fluorescence probe

Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE) (T-669, Invitrogen) (Excitation:

549nm, Emission: 574nm) that was dissolved in DMSO. Since TMRE is a voltage-dependent

dye, it readily enters the active mitochondrion due to the mitochondrion’s more negative charge.

Once inside the organelle, it undergoes redox reaction and fluoresces. Thus higher fluorescence

intensity will depict a higher membrane potential of the mitochondria.

Stably transfected MYC and SIRT3MYC SH-SY5Y cells were plated at the density of 5.0x105

on a 6 well plate. Twenty four hours later, toxins (rotenone, dopamine, naphthazarin and PSI)

were added and incubated for 24 hours at 37ºC in 5% CO2. Following the incubation period,

cells were treated with TMRE (20nm) for 20 minutes in the dark at 37ºC in 5% CO2 and then

washed once with DMEM without phenol red. Coverslips were transferred to a live imaging

chamber and then 1mL of DMEM without phenol red was added to the imaging chamber. Cells

33

were imaged live using fluorescence microscopy (Apotome, Zeiss) with the magnification of 40x

at 37ºC in 5% CO2.

One hundred cells were analyzed per experimental group using Zen2009 Software (Zeiss).

Region of interest (ROI) was traced around each cell and mean fluorescence reading of each

ROI/cell was recorded.

2.8 Assessment of ATP Levels

ATP was quantified using bioluminescent technique (ViaLightTM HS kit, Lonza). ATP was

measured utilizing bioluminescence which works by the enzyme luciferase catalyzing ATP and

luciferin reaction. Light becomes the byproduct of this reaction therefore the higher

luminescence intensity signifies higher level of ATP present within the cells.

Stably transfected SIRT3MYC and MYC SH-SY5Y cells were plated at the density of 1.0x105 in

a white luminescence 96 well plate. Twenty-four hours later, cells were exposed to toxins

(rotenone, dopamine, naphthazarin and PSI) and incubated overnight at 37ºC in CO2. 100µL of

nucleotide releasing reagent (NRR) was added into each well and then incubated for 5 minutes.

Following the incubation period, the cells were then treated with 20µL of ATP monitoring

reagent (AMR) and luminescence readings were taken using a spectrophotometer.

2.9 Cell Viability Assay

Cell viability was assessed utilizing the redox sensitive dye Alamar Blue (DAL1025, Invitrogen).

This dye readily enters a cell and is reduced from the inactive non fluorescence resazurin to the

active red fluorescence resorufin form that fluoresces (Excitation: 530nm, Emission: 590nm).

Thus, higher fluorescence intensity signifies higher cell viability in the experimental group.

Stable cell lines of SH-SY5Y expressing MYC and SIRT3MYC were plated at the density of

1.0x105 in a 96 well plate. Twenty four hours later, cells were treated with toxins (rotenone,

dopamine, naphthazarin and PSI) and Alamar Blue (10% of the final volume). Cells were left

overnight to incubate for 24 hours at 37ºC in 5% CO2. Fluorescence reading was then taken

using a spectrophotometer (BMG Lab Tech).

34

2.10 Cell Death Quantification

Cell death was measured using the fluorescence probe Propidium Iodide (2μM) with a protocol

described in section 2.2. Stably transfected MYC and SIRT3MYC SH-SY5Y cells were plated at

the density of 1.0x105 on a 96 well plate. Twenty four hours later, cells were exposed to toxins

(rotenone, dopamine, naphthazarin and PSI). Cell death was measured 24 hours after toxin

exposure by incubating the cells with Propidium Iodide (2µM) for 1 hour before quantified using

spectrophotometer (BMG Lab Tech).

2.11 Statistical Analysis

Results were analyzed using either one-way analysis of variance (ANOVA) or two-way ANOVA

using Graphpad Prism software. When one-way ANOVA results were significant, Dunnett’s

multiple comparison test was performed for further statistical analysis. When two-way ANOVA

results were significant, Bonferroni’s post-hoc test was performed for further statistical analysis.

All results were displayed as Mean ± Standard Error of Means (SEM) and a probability value (P-

value) of less than 0.05 was accepted as significant.

35

CHAPTER 3

3 RESULTS

3.1 Human SIRT3 expression in stably transfected SH-SY5Y cells

Since there has been a debate on human SIRT3’s (hSIRT3) expression pattern, ectopic hSIRT3

expression in SH-SY5Y cells was assessed using immunofluorescence and Western blotting. It

has been hypothesized that hSIRT3 is 44kDa in molecular weight is targeted to the mitochondria

by its N terminus mitochondria targeting sequence. At the mitochondria matrix, mitochondrial

peptidase protein (MPP) cleaves hSIRT3 to the active deacetylating 28kDa form (Schwer et al.,

2002). Another theory suggested that hSIRT3 is exclusively mitochondrial (Cooper & Spelbrink,

2008).

In the stably transfected SH-SY5Y cells overexpressing SIRT3, Western blot detected the

endogenous uncleaved SIRT3 form at 44kDa and the cleaved active form at 28kDa for all cell

types (Figure 3A). In SIRT3MYC or SIRT3 overexpressing cells, there was a 29kDa band

detected, which represents the SIRT3MYC ectopic cleaved expression since the MYC-tag

sequence weighs approximately 1kDa. A 45kDa band was also detected in the SIRT3MYC

group which signifies SIRT3MYC ectopic uncleaved SIRT3 expression. Using densitometry

techniques, it was determined that there is a 5-fold increase in ectopic overexpression of the

SIRT3MYC cleaved form compared to MYC SH-SY5Y and NAÏVE SH-SY5Y cells (Figure

3B).

Immunofluorescence images showed that there is co-localization between the SIRT3MYC

(Green) and mitochondria marker MitoDSRed2 (Red) (Figure 4). There was no SIRT3MYC in

the nucleus but a small amount of cytoplasmic SIRT3MYC expression was observed. This

suggests that hSIRR3 transgene expression in SH-SY5Y cells is both mitochondrial and

cytoplasmic when overexpressed in this system.

36

Figure 3.

A)

B)

Naive Myc SIRT3-Myc0

200

400

600

800 Endogenous Mito SIRT3Mito MYC-SIRT3

% E

xp

ress

ion

(n

orm

ali

zed

to

co

ntr

ol)

37

Figure 3. Ectopic overexpression of Human SIRT3 in SH-SY5Y cells.

A) Representative Western blot showing ectopic overexpression and endogenous SIRT3

expression detected within SH-SY5Y cells. At 28kDa, the endogenous form of SIRT3

was present in SH-SY5Y SIRT3MYC, SH-SY5Y MYC and Naïve SH-SY5Y cells.

At approximately 29kDa, SIRT3MYC overexpression was present in SH-SY5Y

SIRT3MYC cells only. To ensure equal loading, samples were also probed for the

housekeeping gene β-actin with a molecular weight of 47kDa. (n=3).

B) Mean of SIRT3 expression in the three cell populations. SH-SY5Y cells stably

transfected with SIRT3MYC showed an approximate 5.2 fold increase in

mitochondrial SIRT3 expression compared to endogenous mitochondrial SIRT3

expression. Endogenous mitochondrial SIRT3 expression was comparable between

the 3 groups. (n=3).

38

Figure 4.

SIRT3-Myc MitoDSRed Merge

39

Figure 4. Localization of human SIRT3 transgene expression in SH-SY5Y cells.

Representative immunofluorescence images of stably transfected SH-SY5Y SIRT3MYC probed

for MYC. Ectopic SIRT3 construct was successfully translocated into the mitochondria of the

SH-SY5Y cells. This was determined by the co-localization signal between the SIRT3MYC

(Green) and mitoDSRed (Red) depicted in the Merge panel. There was no nuclear SIRT3MYC

staining but there was a minimal amount of cytoplasmic SIRT3MYC staining observed. Images

were taken with 100x objective. Scale bar: 10 microns.

40

3.2 Dose response curves of toxins that mimic Parkinson’s disease cell death

mechanisms in SH-SY5Y cells

In order to recapitulate the cell death mechanisms observed in Parkinson’s Disease, the following

toxins were selected: mitochondria complex 1 inhibitor rotenone, oxidative stressor dopamine

hydrochloride, lysosome system inhibitor naphthazarin and the proteasome inhibitor N-

carbobenzyloxy-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI). By the time patients are first diagnosed

with PD, approximately 50% of dopaminergic neurons in the substantia nigra pars compacta

have died. Thus, in order to recapitulate the initial diagnosis, a dose response curve was

performed in order to find the approximate inhibitory concentration 50 (IC50) of the toxins. In

order to measure cell death, the fluorescent dye Propidium Iodide (PI) was utilized. PI readily

enters dead cells and gives a strong fluorescent signal once binded to nucleic acid components of

a cell thus the higher fluorescence intensity indicates a large amount of cell death.

Rotenone caused a significant increase in cell death compared to vehicle at concentrations 0.03-

100µM (*** p < 0.001, n = 4) (Figure 5A). Dopamine resulted in a significant increase in cell

death compared to vehicle at concentration 100µM (*** p < 0.001, n = 4) (Figure 5B).

Naphthazarin resulted in a significant increase in cell death compared to vehicle at

concentrations 0.3-100µM (** p < 0.01, *** p < 0.001, n = 4) (Figure 5C). PSI caused a

significant increase in cell death compared to vehicle at concentrations 0.03-100µM (** p < 0.01,

*** p < 0.001, n = 4) (Figure 5D). Approximate IC50 selected for subsequent experiments were:

0.03µM for rotenone as there was 40.75 ± 3.51% of cell death, 65µM for dopamine as there was

an approximately 40% of cell death, 0.3µM for naphthazarin as there was 39.12 ± 3.2% and

0.03µM for PSI as there was 38.83 ± 3.5% of cell death.

41

Figure 5.

42

Figure 5. Dose response curves of toxins that mimic PD mechanisms in SH-SY5Y cells.

A) – D) Line graphs depicting dose response curves of rotenone, dopamine, naphthazarin

and PSI in SH-SY5Y cells. SH-SY5Y cells were exposed to toxins for 24 hours and then

cell death was measured using the fluorescent probe Propidium Iodide. There was

significant increase in cell death at concentrations: 0.03-100µM for rotenone, 100µM for

dopamine, 0.3-100µM for naphthazarin and 0.03-100µM for PSI. Approximate IC50

selected for the toxins were: 30nm for rotenone, 65µM for dopamine, 300nm for

naphthazarin and 30nm PSI. (n = 4, * p < 0.05, ** p < 0.01, *** p < 0.001, One-way

ANOVA, Dunnett’s multiple comparison test, error bars indicate ± SEM)

43

3.3 Effects of SIRT3 on cell viability in SH-SY5Y cells

To determine whether SIRT3 was cytoprotective, the effect of SIRT3 overexpression on cell

viability was assessed. In order to measure this, the Alamar Blue (AB) dye was incubated with

the cells for the duration of 24 hours. AB is a redox-sensitive dye which gets reduced from the

inactive blue resazurin form to the active red fluorescence resorufin in the mitochondria and

cytoplasm. Given that only healthy cells are able to carry out redox reaction to convert this dye

to the active fluorescence form, fluorescence intensity will be proportionate to cell viability.

High fluorescence intensity will depict cell high viability whereas low fluorescence intensity will

depict low cell viability.

Interestingly, there was no significant difference in AB fluorescence intensity between MYC

control and SIRT3-MYC overexpressing cells under basal conditions. However, there was a

significant decrease in cell viability in MYC control cells after treatment with PSI (30 nm) (83.1

± 4.8%, * p < 0.05, n = 4), mitochondria complex 1 inhibitor rotenone (30 nm) (69.2 ± 2.5%, ***

p < 0.001, n = 4), lysosome system inhibitor naphthazarin (300 nm) (75.0 ± 4.3%, *** p < 0.001,

n = 4) and oxidative stressor dopamine (65 µM) (66.6 ± 3.6%, *** p < 0.001, n = 4) compared to

vehicle treatment (Figure 6). In SIRT3-MYC overexpressing cells, cell viability did not decrease

after exposure to toxins.

44

Figure 6.

Vehicle ROT DA NAP PSI0

20000

40000

60000

80000

**********

MYC Cells

AB

Flu

ore

scen

ce In

ten

sit

y (

A.U

.)

Vehicle ROT DA NAP PSI0

20000

40000

60000

80000SIRT3-MYC Cells

AB

Flu

ore

scen

ce In

ten

sit

y (

A.U

.)

45

Figure 6: Effect of SIRT3 on cell viability in SH-SY5Y cells.

Graph to show mean Alamar Blue (AB) fluorescence intensity (A.U.) of MYC Control and

SIRT3-MYC overexpressing SH-SY5Y cells after treatment with rotenone, dopamine,

naphthazarin and PSI for 24 hours. In MYC Control SH-SY5Y cells, cell viability was decreased

after exposure to toxins compared to vehicle treatment. However, in SIRT3-MYC cells,

fluorescence intensity was maintained after exposure to toxins, suggesting SIRT3’s ability to

preserve cell viability. (n = 4, * p<0.05, *** p<0.001, One-way ANOVA, Dunnett’s multiple

comparison test, error bars indicate ± SEM)

46

3.4 Effect of SIRT3 on cell death in SH-SY5Y cells

Since SIRT3 appears to prevent toxin-induced reduction in cell viability (Figure 6), the effect of

SIRT3 on cell death was also assessed using the fluorescence probe Propidium Iodide. Cell death

did not increase in SIRT3-MYC overexpressing cells after exposure to rotenone (30nm),

dopamine (65µM), naphthazarin (300nm) and proteasome system inhibitor (PSI) (30nm)

compared to SIRT3-MYC vehicle treatment (Figure 7). However, cell death was significantly

increased in MYC (control) cells when compared to MYC vehicle treatment after exposure to

rotenone (51.7 ± 6.6%), dopamine (59.0 ± 6.1%), naphthazarin (34.9 ± 4.5%) and PSI (38.4 ±

5.0%) (* p < 0.05, ** p < 0.01, *** p < 0.001, n = 4). Thus, this suggests that SIRT3-MYC

overexpression protects against toxin-induced cell death in SH-SY5Y cells. There was no

significant difference in cell death between SIRT3 and control cells under normal conditions,

which is consistent to findings from Figure 6.

47

Figure 7.

Vehicle ROT DA NAP PSI0

20

40

60

80

******

***

MYC Cells

Cell D

eath

(%

to

Co

ntr

ol)

Vehicle ROT DA NAP PSI0

20

40

60

80SIRT3-MYC Cells

Cell D

eath

(%

to

Co

ntr

ol)

48

Figure 7: Effect of SIRT3 on cell death in SH-SY5Y cells

Graphs showing mean cell death following toxin exposure. SH-SY5Y overexpressing SIRT3-

MYC cells did not display an increase in toxin-induced cell death after treatment with rotenone,

dopamine, naphthazarin and PSI compared to SIRT3 vehicle treatment. In control MYC SH-

SY5Y cells, there was a significant increase in cell death after exposure to toxins compared to

control vehicle treatment. (n = 4, * p < 0.05, ** p < 0.01, *** p < 0.001, One-way ANOVA,

Dunnett’s multiple comparison test, error bars indicate ± SEM)

49

3.5 Effect of SIRT3 on reactive oxygen species (ROS) levels in SH-SY5Y cells

Given that SIRT3 overexpression is cytoprotective in SH-SY5Y cells against toxins that mimic

cell death mechanisms of PD (Figure 6; Figure 7); next, the underlying mechanisms of SIRT3

overexpression in SH-SY5Y cells were assessed. One of the most popular theories attempting to

explain the root cause of neurodegenerative diseases such as Parkinson’s disease is the free

radical theory. This theory believes that the accumulation of reactive oxygen species (ROS)

results in irreversible cell damage and an overall functional decline (Harman, 1956). The main

source of ROS generation within a cell is the mitochondria, thus attenuating ROS in this

organelle might improve the overall health of the cell. Previous findings have shown that SIRT3

has the ability to enhance anti-oxidant pathway enzymes manganese superoxide dimutase

(SOD2) and glutamate dehydrogenase (GDH) (Schlicker et al., 2008; Tao et al., 2010). Thus, the

effect of SIRT3 overexpression in SH-SY5Y cells on ROS levels was assessed. In order to

measure ROS in SH-SY5Y cells, the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate

(CM-H2DCFDA) was utilized. This dye works by the cleavage of its acetate groups by

intracellular esterases and the interaction of its thiol chloromethyl group with intracellular

glutathione and thiols. Hence the fluorescence intensity is proportionate to ROS levels within the

cells. To ensure proper workings of the ROS probe CM-H2DCFDA, two controls were utilized;

unlabeled cells to ensure there was no auto fluorescence of untreated cells (Dark blue line) and

5μM of Rotenone (a complex 1 mitochondrial inhibitor known to induce ROS) (Pink line)

(Figure 6B; 6C) (Li et al., 2003; Panov et al., 2005).

In normal conditions, overexpression of SIRT3 in SH-SY5Y cells decreased ROS significantly

by 47.46 ± 1.94% compared to control MYC cells (### p < 0.001, n = 4) (Figure 8). After

exposure to rotenone (30nm), dopamine (65µM), naphthazarin (300nm) and PSI (30nm), control

MYC cells had a significant increase in ROS by 36.01 ± 4.72%, 43.97 ± 16.73%, 23.61 ± 2.81%

and 25.99 ± 1.35% respectively compared to vehicle (*** p < 0.001 for both naphthazarin and

PSI and * p < 0.05 for both rotenone and dopamine, n = 4). However, there was no significant

increase in ROS in SIRT3 overexpressing SH-SY5Y cells after exposure to toxins.

50

Figure 8.

51

Figure 8.

D)

Vehicle ROT DA NAP PSI0

20000

40000

60000

80000

100000

120000

**** ***

*

MYC Cells

CM

-H2D

CF

A F

luo

rescen

ce In

ten

sit

y (

A.U

.)

Vehicle ROT DA NAP PSI0

20000

40000

60000

80000

100000

120000 SIRT3-MYC Cells

###

CM

-H2D

CF

A F

luo

rescen

ce In

ten

sit

y (

A.U

.)

52

Figure 8: Effect of SIRT3 on ROS levels in SH-SY5Y cells

A) Representative flow cytometry scatter plot depicting three SH-SY5Y cell populations

gated according to size (forward scatter or FSC) and granularity (side scatter of SSC).

Cells low in FSC represent the dead cell population (Pink dots), cells high in FSC

represent the clumped cell population (Green dots) and excluded from analysis. The

remaining cells (Blue dots) were included in data analysis.

B) Representative flow cytometry histogram showing CM-H2DCFA fluorescence intensity

(A.U.) of the MYC (control) SH-SY5Y cell population of interest (after gating). There

was a right shift in histogram peaks after cells were treated with toxins (Black line, Red

line, Green line, Orange line) compared to the Control treatment (Light blue line). This

suggests that exposure to toxins resulted in higher levels of ROS. Dark blue line denotes

negative control (cells treated without any dye) and Pink line represents positive control

(cells treated with 5µM Rotenone).

C) Representative flow cytometry histogram depicting CM-H2DCFA fluorescence intensity

(A.U.) of SIRT3 overexpressing SH-SY5Y cell population of interest (after gating).

Histogram peaks of SIRT3 cells treated with toxins (Black line, Red line, Green line,

Orange line) were comparable to SIRT3 control treatment (Light blue line), suggesting

that SIRT3 overexpression was able to maintain ROS.

D) Bar graph depicting mean CM-H2DCFA fluorescence intensity (A.U.) of SH-SY5Y

MYC and SIRT3MYC overexpressing cells in various conditions. In normal conditions,

SIRT3MYC demonstrated lower fluorescence intensity compared to MYC (n = 4, ### p

< 0.001, Two-way ANOVA, Bonferonni post-hoc test, error bars indicate ± SEM). After

treatment with rotenone, dopamine, naphthazarin and PSI for 24 hours, MYC cells had

significantly higher fluorescence intensity. In SIRT3-MYC cells, fluorescence intensity

was maintained after toxins exposure, suggesting that SIRT3 was able to protect against

ROS induced toxicity while control MYC cells could not. (n = 4, * p < 0.05, *** p <

0.001, One-way ANOVA, Dunnett’s multiple comparison test, error bars indicate ±

SEM)

53

3.6 Effect of SIRT3 on mitochondria membrane potential (∆Ψm) in SH-SY5Y cells

So far, the present study has shown that overexpression of SIRT3 in SH-SY5Y cells can reduce

ROS level under basal conditions and prevents ROS from increasing after toxic insults (Figure

8). Several physiological factors have been found to regulate mitochondria ROS generation. One

of these factors is the mitochondrial membrane potential (∆Ψm) where high ∆Ψm has been

linked to favoring ROS production (Zhang and Gutterman, 2007). Since ROS and ∆Ψm are

closely related to each other, ∆Ψm is the next important measurement to further assess the

underlying mechanisms of SIRT3 overexpression in SH-SY5Y cells.

The voltage-dependent fluorescent dye Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE)

was utilized in order to measure the differences in ∆Ψm between SIRT3-MYC overexpressing

cells and MYC control cells under normal conditions. In addition to measuring ∆Ψm under basal

conditions, the protective role of SIRT3 on ∆Ψm was assessed after exposure to toxins that

mimic pathologies of PD: mitochondria complex I dysfunction (rotenone) (30nm), oxidative

stressor (dopamine) (65µM), lysosome system dysfunction (naphthazarin) (300nm) and

proteasome system malfunction (PSI) (30nm). Since TMRE is readily consumed by the

mitochondria’s more negative charge, fluorescence intensity will be proportionate to ∆Ψm. As

controls, unlabeled cells were utilized to ensure no auto-fluorescence and 5μM Rotenone, which

has been shown to collapse ∆Ψm in dopaminergic cells (Moon et al., 2005).

The results showed that stable overexpression of SIRT3-MYC in SH-SY5Y cells significantly

decreased ∆Ψm compared to MYC control cells by 42.95 ± 6.07% under basal conditions

(Figure 9C) ( # p < 0.05, n = 4). Exposure to rotenone, dopamine, naphthazarin and proteasome

system inhibitor for 24 hours resulted in a significant ∆Ψm reduction in MYC control cells by

19.91 ± 1.35 %, 12.63 ± 2.85 %, 11.51 ± 0.92 % and 16.03 ± 2.70 % respectively ( * p < 0.05, **

p < 0.01, n = 4). In contrast, SIRT3-MYC cells maintained ∆Ψm in the presence of the toxins

(Figure 9C).

54

Figure 9.

A)

55

Figure 9.

B)

56

Figure 9.

C)

VEHICLE ROT DA NAP PSI0

10

20

30

40

* * ** *

MYC Cells

TM

RE

Flu

ore

scen

ce In

ten

sit

y (

A.U

.)

VEHICLE ROT DA NAP PSI0

10

20

30

40SIRT3-MYC Cells

#

TM

RE

Flu

ore

scen

ce In

ten

sit

y (

A.U

.)

57

Figure 9: Effect of SIRT3 on mitochondrial membrane potential in SH-SY5Y cells.

A) Representative live fluorescent microscopy images of MYC control SH-SY5Y cells

stained with voltage dependent dye TMRE (Red). Control cells that were exposed to

rotenone (ROT), dopamine (DA), naphthazarin (NAP) and PSI for 24 hours exhibited

lower fluorescence intensity compared to control group. Images were taken at 40X. Scale

bar: 10µM.

B) Representative live fluorescent microscopy images of SH-SY5Y cells overexpressing

SIRT3-MYC stained with TMRE (Red). In SIRT3 cells, exposure to ROT, DA, NAP and

PSI for 24 hours did not alter in fluorescence intensity compared to SIRT3 control group.

Images were taken at 40X. Scale bar: 10µM.

C) Bar graph depicting mean TMRE fluorescence intensity (A.U.) of SH-SY5Y cells under

various conditions. Under basal conditions, SIRT3MYC overexpressing cells exhibited

lower fluorescence intensity compared to Control MYC cells (n = 4, # p < 0.05, Two-way

ANOVA, Bonferonni post-hoc test, error bars indicate ± SEM). After exposure to ROT,

DA, NAP and PSI, Control MYC SH-SY5Y cells showed significantly lower

fluorescence intensity. However, fluorescence intensity did not change after toxins

exposure in SIRT3-MYC overexpressing cells. (n = 4, * p < 0.05, ** p < 0.01, One-way

ANOVA, Dunnett’s multiple comparison test, error bars indicate ± SEM)

58

3.7 Effect of SIRT3 on ATP levels in SH-SY5Y cells

One of the reasons why maintenance of mitochondria membrane potential (∆Ψm) is crucial for

survival is because it is the driving force of ATP synthesis. So far, SIRT3 overexpression in SH-

SY5Y cells has been demonstrated to preserve ∆Ψm after exposure to toxins such as the

mitochondria complex 1 inhibitor rotenone, oxidative stressor dopamine, lysosome system

inhibitor naphthazarin and proteasome system blocker (PSI) (Figure 9). Given how closely

related ∆Ψm and ATP are, next it was determined if SIRT3 overexpression in SH-SY5Y cells

can also help to protect against ATP depletion caused by toxic insults. In this experiment, ATP

was measured utilizing bioluminescence which works by the enzyme luciferase catalyzing ATP

and luciferin reaction. Light becomes the byproduct of this reaction therefore the higher

luminescence intensity signifies higher level of ATP present within the cells.

In fact, a maintenance in ATP levels in SIRT3-MYC cells was observed after 24 hours exposure

to rotenone (30nm), dopamine (65µM), naphthazarin (300nm) and PSI (30nm) compared to

SIRT3-MYC vehicle treatment (Figure 10). In MYC control cells, ATP luminescence intensity

was significantly depleted after exposure to rotenone, dopamine, naphthazarin and PSI by 43.5 ±

1.9%, 37.8 ± 2.7%, 43.3 ± 8.6% and 32.9 ± 5.3% respectively ( ** p < 0.01, *** p < 0.001, n =

4). SIRT3-MYC cells also showed significantly lower ATP luminescence intensity by 32.5 ±

5.3% compared to MYC control cells under normal condition ( ## p < 0.01, n = 4).

59

Figure 10.

Vehicle ROT DA NAP PSI0

5000

10000

15000

20000

25000 MYC Cells

******

*****

AT

P L

um

inesen

ce In

ten

sit

y (

A.U

.)

Vehicle ROT DA NAP PSI0

5000

10000

15000

20000

25000 SIRT3-MYC Cells

##

AT

P L

um

inesen

ce In

ten

sit

y (

A.U

.)

60

Figure 10: Effect of SIRT3 on ATP levels in SH-SY5Y cells

Graph showing the mean luminescence ATP intensity (A.U.) of SH-SY5Y MYC control and

SIRT3-MYC overexpressing cells under various conditions. MYC control cells showed

significantly higher ATP luminescence intensity compared to SIRT3-MYC overexpressing cells

under normal conditions (n = 4, ## p < 0.01, Two-way ANOVA, Bonferonni post-hoc test, error

bars indicate ± SEM). After exposure to rotenone (ROT), dopamine (DA), naphthazarin (NAP)

and PSI for 24 hours, there was a significant decrease of ATP luminesence intensity in MYC

control cells. However, ATP luminesence intensity was preserved after exposure to toxins in

SIRT3-MYC overexpressing cells (n = 4, ** p < 0.01, *** p < 0.001, One-way ANOVA,

Dunnett’s multiple comparison test, error bars indicate ± SEM).

61

4 DISCUSSION

Given that mitochondrial dysfunction appears to be the primary cause of cell death in

Parkinson’s disease (PD), a molecule that enhances mitochondrial function may be an excellent

candidate for a treatment in halting the neurodegeneration. SIRT3 is a NAD+ dependent

deacetylase residing in the mitochondria and has the capacity to promote mitochondrial health

due to its ability to regulate oxidative phosphorylation, antioxidant defense mechanisms and

mitochondrial pore opening. This study, for the first time, has demonstrated that overexpression

of SIRT3 in SH-SY5Y cells is cytoprotective against toxins that mimic cell death mechanisms of

PD; mitochondria inhibitor (rotenone), oxidative stressor (dopamine), lysosomal system

impairment (naphthazarin) and ubiquitin proteasome system (UPS) dysfunction (PSI). These

toxins have been previously validated by Yong-Kee et al. (2011) as a reliable assay to assess the

protective potential of a molecule in PD medicine. Rotenone, dopamine, naphthazarin and PSI

induce cellular stress via different mechanisms. Despite their differences, these toxins share one

common factor: they cause mitochondrial dysfunctions. Rotenone, which mimics mitochondrial

impairment in PD, inhibits enzymatic activities of complex I resulting in electron leakage and the

generation of reactive oxygen species (ROS). Dopamine, which recapitulates the dopamine

metabolism toxicity in PD, has the ability to autoxidate to produce ROS and inhibit complex I

activities. Both naphthazarin and PSI mimic the protealytic system impairments in PD, leading to

α-synuclein accumulation in the mitochondria and interfere with the respiratory chain. By

inhibiting mitochondrial functions, mitochondrion-induced cell death gets triggered and it is

likely that dopaminergic cell death in PD occur via this cell death pathway.

Despite the substantial amount of evidence indicating SIRT3’s robust deacetylating activities in

the mitochondria, its subcellular localization remains elusive. To date, SIRT3 has been shown to

be inconsistently expressed in the nucleus and mitochondria (Scher et al., 2007; Cooper and

Spelbrink, 2008), leading to numerous debates. In the current study, SIRT3 exists in both the

uncleaved form and cleaved form in SH-SY5Y cells, which supports the original finding by

Schwer et al. (2002). SIRT3 also exhibits a strong localization in the mitochondria and low

amount of expression in the cytoplasm. SIRT3’s expression in the mitochondria is in agreement

with previous findings by Scher et al. (2007) and Cooper and Spelbrink (2008). One possible

explanation for the cytoplasmic localization of SIRT3 in SH-SY5Y cells is perhaps due to the

types of SIRT3 being quantified. In this study, SIRT3 is ectopically overexpressed with Myc

62

epitope tag fused to the SIRT3’s C terminal. Thus, Myc antibody was utilized to identify SIRT3

transgene expression. Previous studies, however, have utilized SIRT3 antibody to identify SIRT3

expression. This means that the SIRT3 antibody detects the endogenous form of SIRT3, not the

transgene form. Hence, two types of SIRT3 were measured; transgene SIRT3 expression (in this

study) and endogenous SIRT3 expression (in previous studies), which provides an explanation to

different localization pattern between the studies. This could imply that the transgene SIRT3

construct utilized in this study may not completely get targeted to the mitochondria. Nonetheless,

it is important to note that only a minimal amount of SIRT3 transgene expression is observed in

the cytoplasm whereas the majority of it is in the mitochondria, confirming that the transgene

SIRT3 is mostly mitochondria targeted and exerts its protective effects in the mitochondria of the

SH-SY5Y cells.

In order to recapitulate the initial diagnosis of PD, an IC50 of rotenone, dopamine, naphthazarin

and PSI were utilized to mimic the 50% cell death observed in the substantia nigra pars compacta

(SNc) during the first diagnosis (Murray et al., 2005). In this study, the IC50 of dopamine in SH-

SY5Y cells is 65µM. The high IC50 of dopamine could be attributed to the sensitivity of SH-

SY5Y cells to certain toxins. In fact, this is not the first study that has demonstrated a high IC50

of dopamine in SH-SY5Y cells. Other studies utilizing SH-SY5Y cells have shown that

dopamine has an IC50 in the range of 40µM- 100µM (Ballesteros et al., 2013; Yong-Kee et al.,

2011), suggesting the SH-SY5Y cells are more resistant to dopamine toxin in general. It is also

possible that the high IC50 of dopamine in SH-SY5Y cells is because these cells are not

differentiated and still undergo proliferation, resulting in decreased susceptibility to toxin

induced cell death. Another interesting finding in this study was the 40% of cell death caused by

rotenone at 30nm and 5µM but exhibiting higher ROS levels and lower mitochondrial membrane

potential (∆Ψm) at 5µM compared to 30nm. A possible explanation to why both concentrations

resulted in similar amount of cell death but not comparable ROS and ∆Ψm may be due to the

time point selected. After 24 hours exposure to 5µM rotenone, SH-SY5Y cells are possibly

undergoing mitochondrial dysfunction due to the rise in ROS and the loss of ∆Ψm. Since

mitochondrial impairment is one of the earliest events of cell death implicated in PD (Yong-Kee

et al., 2012), it is likely that cell death is not occurring yet at this point and will be more apparent

after the 24 hour time point in the 5µM rotenone treatment group. Thus this can be a potential

63

explanation to why 30nm and 5µM resulted in equal amount of cell death but not ROS and ∆Ψm

at the 24 hour time point.

In this study, the overexpression of SIRT3 in SH-SY5Y cells appears to be cytoprotective

against rotenone, dopamine, naphthazarin and PSI by reducing ROS levels and stabilizing ∆Ψm

and ATP levels. Since SIRT3 targets many mitochondrial enzymes, these protective effects were

expected. SIRT3 is capable in attenuating ROS levels by deacetylating superoxide dimutase 2

(SOD2), which is a ROS scavenging enzyme that helps to breaks down superoxides (O2-) into

water (H2O). Furthermore, SIRT3 enhances the production of NADPH by deacetylating

glutamate dehydrogenase (GDH) and isocitrate dehydrogenase (IDH2). High levels of NADPH

are necessary to activate another ROS scavenging enzyme glutathione peroxidase (GPX). SIRT3

overexpression in SH-SY5Y cells also exerts its protective effects by preserving ∆Ψm and ATP

levels. SIRT3 is likely to stabilize ∆Ψm by deacetylating cyclophilin D (CypD) (Hafner et al.,

2010). CypD is an integral component of the mitochondrial pore opening. Mitochondrial pore

opening characterizes cellular death signaling resulting in ion influx, loss of ∆Ψm and the release

of pro-apoptotic factors such as cytochrome c into the cytosol. By deacetylating CypD, SIRT3

inhibits CypD’s activation, resulting in reduced vulnerability to the loss of ∆Ψm during

mitochondrial stress. SIRT3 also enhances various oxidative phosphorylation components hence

this may be why SIRT3 overexpression in SH-SY5Y cells preserves ATP levels even during

mitochondrial impairment induced by rotenone, dopamine, naphthazarin and PSI. SIRT3

deacetylates important metabolic enzymes such as glutamate dehydrogenase (GDH), isocitrate

dehydrogenase 2 (IDH2), long-chain acetyl coenzyme A dehydrogenase (LCAD), acetyl CoA

synthase 2 (ACS2). Moreover, SIRT3 also deacetylates complex I-V of the electron transport

chain thereby regulating the respiratory chain for ATP generation. Therefore, by targeting these

substrates, overexpression of SIRT3 enables the cells to be more resistant against ATP depletion.

Some unexpected findings in this study were; in basal conditions, SIRT3 overexpression in SH-

SY5Y cells resulted in the loss of ∆Ψm and a decrease in ATP levels. The loss of ∆Ψm in SIRT3

overexpressing cells could be attributed to the "uncoupling protein" theory. Uncoupling protein

(UCP) exerts its function by translocating protons from the intermembrane mitochondrial space

back to the matrix during oxidative phosphorylation. By allowing protons to leak into the matrix,

∆Ψm is reduced and heat gets generated. To date, five mammalian UCPs (UCP1-UCP5) have

been identified. The most well characterized UCP is UCP1, which gets upregulated during

64

thermogenesis in brown adipose tissue (BAT). Interestingly, Shi et al. (2005) have demonstrated

a potential link between SIRT3 and UCP1 in BAT, suggesting that SIRT3 may be mediating its

protective effects by stimulating UCP1 expression during thermogenesis. Aside from in BAT,

UCPs have been demonstrated to play beneficial roles in other cell types such as regulating

calcium homeostasis and ROS levels (Andrews, Diano & Tamas, 2005). For example, in SH-

SY5Y cells, overexpression of UCP4 and UCP5 were previously shown to protect against MPTP

and dopamine induced toxicities (Chu et al., 2009; Kwok et al., 2010) thus it is possible that

SIRT3 overexpression in SH-SY5Y cells mediate its protective effects by interacting with UCP4

and UCP5. In neurons, UCP2 gets unregulated during stress, resulting in the loss of ∆Ψm,

reduced mitochondrial Ca2+

uptake and a decrease in ROS (Mattiasson et al., 2003). Hence, if

SIRT3 regulates UCP1 expression in BAT, it is possible that SIRT3 exerts its protective function

by modulating other UCP members such as UCP2, UCP4 and UCP5 in other cell types including

SH-SY5Y cells.

In addition to decreasing ∆Ψm, UCP mediates its effects by reducing ATP levels, an effect

observed in SH-SY5Y cells overexpressing SIRT3. As discussed above, UCP decreases ∆Ψm

and ATP synthesis in order to mediate its protective effects by decreasing ROS generation and

susceptibility to mitochondrial Ca2+

toxicity. Thus, lower ATP levels in basal conditions

observed in SIRT3 overexpressing cells is likely protective rather than toxic if it is mediated

through SIRT3's interaction with UCP. To further support this idea, SIRT3 was recently

demonstrated to deacetylate mitochondrial ribosomal protein L10 (MRPL10), one of the

mitochondrial ribosomal proteins (MRPs) (Yang et al., 2010). The MRPs are responsible for

synthesizing proteins that comprise the electron transport chain (ETC). By deacetylating

MRPL10, SIRT3 inhibits its activity, causing a downregulation in ETC protein synthesis (Yang

et al., 2010). As a result, both ETC and ATP synthesis are attenuated. Therefore, in SH-SY5Y

cells overexpressing SIRT3, SIRT3 appears to exert its protective functions by reducing ATP

synthesis to ensure the mitochondrion is not overworked and promote mitochondrial health. This

effect is likely mediated through SIRT3's interaction with UCP and MRPL10.

It is also important to note that this study has utilized the undifferentiated SH-SY5Y cell model.

Since PD targets mature neurons, differentiated SH-SY5Y cells would have been a more

appropriate model for this study. Differentiated SH-SY5Y cells have been previously

demonstrated to possess several neuronal phenotypes such as microtubule associated protein

65

(MAP), growth-associated protein (GAP-43), neuronal nuclei (NeuN) and synaptophysin (Xie,

Hu & Li, 2010). However, retinoic acid (RA) induced differentiation of SH-SY5Y cells resulted

in abnormal mitochondrial morphology (data not shown), suggesting the differentiation process

is toxic to the mitochondria. Given that the focus of this study is on mitochondrial functions, the

RA induced differentiation model would have not been suitable since it appears to have

detrimental effects on the organelle. Moreover, RA differentiated SH-SY5Y cells have been

shown to increase the expression of Bcl-2 (anti-apoptotic protein) and decrease the expression of

p53 (pro-apoptotic) (Tieu, Zuo & Yu, 1999), suggesting that it would be less sensitive to toxins

compared to real neurons.

In conclusion, SIRT3 presents itself as an attractive molecule to fight against mitochondria-

dysfunction related diseases such as PD due to its cytoprotective properties. It has the ability to

reduce ROS and stabilize ∆Ψm and ATP levels the dopaminergic SH-SY5Y cell line against

toxins that mimic cell death mechanisms of PD. Thus, SIRT3 is certainly a molecule worth

further investigating since it has the potential to be a protective treatment in PD.

66

Future directions:

1. The link between SIRT3 and uncoupling protein (UCP) remains elusive. However, Shi et al.

(2005) have demonstrated SIRT3 levels are upregulated under caloric restriction and

thermogenesis that result in UPC1 activation. UCP2, UCP4 and UCP5 have been shown to play

a protective role in Parkinson’s disease (Chu et al., 2009; Ho et al., 2010; Kwok et al., 2010) thus

it will be interesting to investigate further if SIRT3 can regulate and target the other UPC

homologs in the mitochondria.

2. Since mitochondrial dysfunction is believed to be central to Parkinson’s disease, the next

important variable to assess is effects of SIRT3 on mitochondrial dynamics. Mutation in leucine-

rich repeat kinase 2 (LRRK2) is the most common cause of autosomal dominant PD. Wild-type

LRRK2 has been previously demonstrated by Wang et al. (2012) results in increased

mitochondrial fragmentation. Given how important a balanced fusion and fission for cellular

functioning, it is important to investigate if SIRT3 can have protective effects on any of fusion or

fission proteins to maintain a healthy fusion and fission balance. SIRT3’s direct or indirect

interaction with fission protein Drp1, Fis1 and fusion protein Mfn1, Mfn2 will be an exciting

question to address in the future.

67

References

Ahn, B., et al. (2008). A role for the mitochondrial deacetylase Sirt3 in regulating energy

homeostasis. Proceedings of the National Academy of Sciences of the United States of

America, 105, 14447-52.

Ahuja, N., et al. (2007). Regulation of insulin secretion by SIRT4, a mitochondrial ADP-

ribosyltransferase. The Journal of Biological Chemistry, 282(46), 33583-33592.

Alvarez-Erviti, L. et al. (2011). Lysosomal dysfunction increases exosome-mediated alpha-synuclein

relase and transmission. Neurobiological Disorder, 42(3), 360-367.

Andrews, Z., Diano, S & Horvath, T. (2005). Mitochondrial uncoupling proteins in the CNS: in

support of function and survival. Nature Reviews Neuroscience, 6, 829-840.

Ballesteros, M et al. (2013). Effect of neuroprotective therapies (hypothermia and cyclosporine a) on

dopamine-induced apoptosis in human neuronal SH-SY5Y cells. Brain Injury, 27(3), 354-360.

Bao, J., et al. (2010). SIRT3 is regulated by nutrient excess and modulates hepatic susceptibility to

lipotoxicity. Free Radical Biology & Medicine, 49, 1230–1237.

Bell, E., et al. (2011). SirT3 suppresses hypoxia inducible factor 1alpha and tumor growth by

inhibiting mitochondrial ROS production. Oncogene, 72, 507-527.

Betarbet, B., et al. (2000). Chronic systemic pesticide exposure reproduces features of Parkinson's

disease. Nature Neuroscience, 3(12), 1301–1306.

Bonifati, V., Rizzu, P., Van Baren, M., et al. (2003). Mutations in the DJ-1 gene associated with

autosomal recessive early-onset parkinsonism. Science, 299(5604), 256–259.

Brenner-Lavie, H., Klein, E. & Ben-Shachar, D. (2009). Mitochondrial complex I as a novel target for

intraneuronal DA: modulation of respiration in intact cells. Biochemistry Pharmacology,

78(1), 85-95.

Brunk, U., Zhang, H., Dalen, H. et al. (1995). Exposure of cells to nonlethal concentrations of

hydrogen peroxide induces degeneration-repair mechanisms involving lysosomal

destabilization. Free Radical Biology and Medicine, 19(6), 813-822.

Caudle, W., Richardson, J., Wang, M et al. (2007). Reduced vesicular storage of dopamine causes

progressive nigrostriatal neurodegeneration. Journal of Neuroscience, 27(30), 8138-8148.

Chen, et al. (2008). VMAT and dopamine neuron loss in a primate model of Parkinson’s disease.

Journal of Neurochemistry, 105, 78-90.

Chen, et al. (2011). Tumor suppressor SIRT3 deacetylates and activates manganese superoxide

dismutase to scavenge ROS. EMBO Reports, 12, 534-541.

68

Chu, et al. (2009). Mitochondrial UCP4 attenuates MPP+ - and dopamine-induced oxidative stress,

mitochondrial depolarization, and ATP deficiency in neurons and is interlinked with UCP2

expression. Free Radical Biology and Medicine, 46, 810-820.

Cooke, M., Evans, M., Dizdaroglu et al. (2003). Oxidative DNA damage mechanisms, mutation, and

disease. FASEB Journal, 17(10), 1195-1214.

Devi, L., Raghavendran, B., Prabhu, N et al. (2008). Mitochondrial import and accumulation of

alpha-synuclein impair complex I in human dopaminergic neuronal cultures and Parkinson

disease brain. The Journal of Biological Chemistry, 283(14), 9089-9100.

Dickson, D., Braak, H. & Duda, D. (2009). Neuropathological assessment of Parkinson’s disease

refining the diagnostic criteria. The Lancet Neurology, 8(12), 1150-1157.

Duchen, M. (2000). Mitochondria and calcium: from cell signalling to cell death. The Journal of

Physiology, 15, 57-68.

Ekstrand, M., Terzioglu, M., Galter, D. et al. (2007). Progressive parkinsonism in mice with

respiratory-chain-deficient dopamine neurons. Proceedings of the National Academy of

Sciences of the United States of America, 104(4), 1325–1330.

Elmore, S. (2007). Apoptosis: a review of programmed cell death. Journal of Toxicologic Pathology,

35, 495-516.

Exner, N., Treske, B., Paquet, D et al. (2007). Loss-of-function of human PINK1 results in

mitochondrial pathology and can be rescued by parkin. Journal of Neuroscience, 27(45),

12413–12418.

Finley, et al. (2011). Succinate dehydrogenase is a direct target of sirtuin 3 deacetylase activity. PloS

One, 6, 8.

Friedman, J., et al. (2011). Oral contraceptive use, iron stores and vascular endothelial function in

healthy women. Contraception, 84, 285-290.

Fu, et al. (2012). Trans-(-)-e-Viniferin increases mitochondrial sirtuin 3 (SIRT3) activates AMP-

activated protein kinase (AMPK) and protects cells in models of Huntington Disease. The

Journal of Biological Chemistry, 287, 24460-24472.

Glozak, M et al. (2005). Acetylation and deacetylation of non-histone proteins. Gene, 363, 15-23

Grummt, I & Pikaard , C. (2003). Epigenetic silencing of RNA polymerase I transcription. Nature

Reviews Molecular Cell Biology, 4, 641–9.

Gunter, T., et al. (2004). Calcium and mitochondria. FEBS Letters, 567, 96-102.

69

Haas, R., Nasirian, F., Nakano, K et al. (1995). Low platelet mitochondrial complex I and complex

II/III activity in early untreated Parkinson’s disease. Annals of Neurology, 37(6), 714-722.

Hafner, A. V., et al. (2010). Regulation of the mPTP by SIRT3-mediated deacetylation of CypD at

lysine 166 suppresses age-related cardiac hypertrophy. Aging, 2(12), 914-923.

Haigis et al. (2006). SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie

restriction in pancreatic beta cells. Cell. 126(5), 941-954.

Hallows, W. C., Lee, S. & Denu, J. M. (2006). Sirtuins deacetylate and activate mammalian acetyl-

CoA synthetases. Proceedings of the National Academy of Sciences of the United States of

America, 103(27), 10230-10235.

Hallows, W., Yu, W & Denu, J. (2011). SIRT3 promotes the urea cycle and fatty acid oxidation

during dietary restriction. Molecular Cell, 41, 139-149.

Harman, D (1956). Aging: a theory based on free radical and radiation chemistry. Journal of

Gerontology, 11(3), 298-300.

Hassa, P et al. (2006). Nuclear ADP-ribosylation reactions in mammalian cells: Where are we today

and where are we going? Microbiology and Molecular Biology Reviews, 70(3), 789-829.

Hirschey, M et al. (2010). SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme

acetylation. Nature, 464(7285), 121-5.

Imamura, K., Takeshima, T., Kashiwaya et al. (2006). D-beta-hydroxybutyrate protects dopaminergic

SH-SY5Y cells in a rotenone of Parkinson’s disease. Journal of Neuroscience Research,

84(6), 1376-1384.

Irrcher, I., Aleyasin, H., Seifert, E. et al. (2010). Loss of the Parkinson's disease-linked gene DJ-1

perturbs mitochondrial dynamics. Human Molecular Genetics, 19(19), 3734–3746.

Ishihara, N., Eura, Y. & Mihara, K. (2004). Mitofusion 1 and 2 play distinct roles in mitochondrial

fusion reactions via GTPase activity. Journal of Cell Science, 117(26), 6535-6546.

Jing, et al. (2011). Sirtuin-3 (SIRT3) regulates skeletal muscle metabolism and insulin signaling via

altered mitochondrial oxidation and reactive oxygen species production. Proceedings of the

National Academy of Sciences of the United States of America, 108, 14608-14613.

Joza, et al. (2001). Essential role of the mitochondrial apoptosis-induced factor in programmed cell

death. Nature, 410, 549-554.

Kawahara, et al. (2009). SIRT6 links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene

expression and organismal life span. Cell, 136, 62-74.

Keane, P., Kurzawa, M., Blain et al. (2011). Mitochondrial dysfunction in Parkinson’s disease.

Parkinson’s Disease, 2011, 1-18

70

Kendrick, A et al. (2010). Fatty liver is associated with reduced SIRT3 activity and mitochondrial

protein hyperacetylation. Biochemical Journal, 433(3), 505–514

Kim, S., Lu, H & Alano, C. (2011). Neuronal Sirt3 protects against excitotoxic injury in mouse

cortical neuron culture. PloS one, 6(3).

Kim et al. (2010). SIRT3 is a mitochondria-localized tumor suppressor required for maintenance of

mitochondrial integrity and metabolism during stress. Cancer cell, 17(1), 41-52.

Kitada, T., Asakawa, S., Hattori, N et al. (1998). Mutations in the parkin gene cause autosomal

recessive juvenile parkinsonism. Nature, 392(6676), 605–608.

Kowall, N., Hantraye, P., Brouillet, E et al. (2000). MPTP induces alpha-synuclein aggregation in the

substantia nigra of baboons. Neuroreport, 11(1), 211-213.

Kuroda Y, et al. (2006). Parkin enhances mitochondrial biogenesis in proliferating cells. Human

Molecular Genetics, 15:883–895.

Kwok, et al. (2010). Mitochondrial UCP5 is neuroprotective by preserving mitochondrial membrane

potential, ATP levels, and reducing oxidative stress in MPP+ and dopamine toxicity. Free

Radical Biology & Medicine, 49, 1023-1035.

Lass, A., Agarwal, S & Sohal, R. (1997). Mitochondrial ubiquinone homologues, superoxide radical

generation, and longevity in different mammalian species. The Journal of Biological

Chemistry, 272(31), 19199-19204.

Lee, S. et al. (2007). Mitochondrial fission and fusion mediators, hFis1 and OPA1, modulate cellular

senescence. The Journal of Biological Chemistry, 282, 22977-22983.

Li, N et al. (2003). Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing

mitochondrial reactive oxygen species production. The Journal of Biological Chemistry,

278(10), 8516-8525.

Li,W. et al. (2007). Sirtuin2, a mammalian homologofyeastsilentinformation regulator-2 longevity

regulator, is an oligodendroglialprotein that decelerates cell differentiation through

deacetylating alpha- tubulin. The Journal of Neuroscience, 27, 2606–2616.

Liszt, G., Ford, E., Kurtev, M., & Guarente, L. (2005). Mouse Sir2 homolog SIRT6 is a nuclear ADP-

ribosyltransferase. The Journal of Biological Chemistry, 280(22), 21313-21320

Lombard, D., et al. (2007). Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine

acetylation. Molecular and cellular biology, 27(24), 8807-14.

Lombard, D., Tishkoff, D & Bao, J. (2011). Mitochondrial sirtuins in the regulation of mitochondrial

activity and metabolic adaptation. Handbook of Experimental Pharmacology, 206, 163-188.

71

Madsen, et al. (1999). Mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase and carnitine

palmitoyltransferase II as potential control sites for ketogenesis during mitochondrion and

peroxisome proliferation. Biochemical Pharmacology, 57, 1011-1019.

Malena, A., et al. (2009). Inhibition of mitochondrial fission favours mutant over wild type

mitochondrial DNA. Human Molecular Genetics, 18, 3407-3416.

Manning-Bog, A et al. (2002). The herbicide paraquat causes up-regulation and aggregation of α-

synuclein in mice: paraquat and α-synuclein. The Journal of Biological Chemistry, 277(3),

1641–1644.

Martin, L. et al. (2006). Parkinson’s disease alpha-synuclein transgenic mice develop neuronal

mitochondrial degeneration and cell death. Journal of Neuroscience. 26: 41–50.

Martin, H. & Teismann, P (2009). Glutathione—a review on its role and significance in Parkinson’s

disease. FASEB Journal, 10, 3263-3272.

Mastroberardino, P et al. (2009). A novel transferrin/TfR2-mediated mitochondrial iron transport

system is disrupted in Parkinson’s disease. Neurobiology Disorder, 34(3), 417-431.

Mattiasson et al. (2003). Uncoupling protein-2 prevents neuronal death and diminishes brain

dysfunction after stroke and brain trauma. Nature Medicine, 9, 1062-1080.

McNaught, K., Perl, D., Brownell, A et al. (2004). Systemic exposure to proteasome inhibitors causes

a progressive model of Parkinson’s disease, Annals of Neurology. 56(1), 149-162.

Meredith, G., Totterdell, S., Beales, M. et al. (2009). Impaired glutamate homeostasis and

programmed cell death in a chronic MPTP mouse model of Parkinson's disease. Experimental

Neurology, 219 (1), 334-340.

Michan, S., & Sinclair, D. (2007). Sirtuins in mammals: insights into their biological function. The

Biochemical Journal, 404(1), 1-13.

Michishita, E., et al. (2008). SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric

chromatin. Nature, 452(7186), 492-496.

Miller, J., Koc, H & Koc, E. (2008). Identification of phosphorylation sites in mammalian

mitochondrial ribosomal protein DAP3. Protein Science, 17, 251-260.

Mizuno, Y., Ohta, S., Tanaka, M et al. (1989). Deficiencies in complex I subunits of the respiratory

chain in Parkinson’s disease. Biochemical and Biophysical Research Communications, 163(3),

1450-1455.

Moon, Y., Lee, K., Park, J et al. (2005). Mitochondrial membrane depolarization and the selective

death of dopaminergic neurons by rotenone: protective effect of coenzyme Q10. Journal of

72

Neurochemistry, 93(5), 1199-1208.

Morais et al. (2009). Parkinson’s disease mutations in PINK1 result in decreased Complex I activity

and deficient synaptic function. EMBO Molecular Medicine, 1(2), 99-111.

Müftüoglu, M., Elibol, B., Dalmizrak, O et al. (2004). Mitochondrial complex I and IV activities in

leukocytes from patients with parkin mutations. Movement Disorders, 19(5), 544–548.

Murray et al. (1995). Damage to dopamine system differs between Parkinson’s disease and

Alzheimer’s disease with Parkinsonism. Annals of Neurology, 37, 300-312.

Nakagawa, T., Lomb, D., Haigis, M. et al. (2009). SIRT5 deacetylates carbamoyl phosphate

synthetase 1 and regulates the urea cycle. Cell, 137(3), 560-570.

Nasrin, N., et al. (2010). SIRT4 regulates fatty acid oxidation and mitochondrial gene expression in

liver and muscle cells. The Journal of Biological Chemistry, 285(42).

Nicholls, D & Budd, S. (1998) Neuronal excitotoxicity: the role of mitochondria, BioFactors, 8(3-4),

287-299.

Nicklas, W., Vyas, I., & Heikkila, R. (1985). Inhibition of NADH-linked oxidation in brain

mitochondria by 1-methyl-4-phenyl-pyridine, a metabolite of the neurotoxin, 1-methyl-4-

phenyl-1,2,5,6-tetrahydropyridine. Life Sciences, 36(26), 2503–2508.

Nie et al. (2009). STAT3 inhibition of gluconeogenesis is downregulated by SIRT1. Nature Cell

Biology, 4, 492-500.

North, B., et al. (2003). The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase.

Molecullar Cell, 11, 437–444.

Novikova, L., et al. (2006). Early signs of neuronal apoptosis in the substantia nigra pars compacta of

the progressive neurodegenerative mouse 1-methyl-4-phenyl-1,2,3,6-

tetrahyropyrdine/probenecid model of Parkinson’s Disease. Neuroscience, 140(1), 67-76.

Ollinger, K. & Brunk, U. (1995). Cellular injury induced by oxidative stress is mediated through

lysosomal damage. Free Radical Biology and Medicine, 19(5), 635-641.

Oyarce, A & Fleming, P. (1991). Multiple forms of human dopamine beta-hydroxylase in SH-SY5Y

neuroblastoma cells. Archives of Biochemistry and Biophysics, 290, 503-510.

Palacino, J., Sagi, D., Goldberg, M. et al. (2004). Mitochondrial dysfunction and oxidative damage in

parkin-deficient mice. The Journal of Biological Chemistry, 279(18), 18614–18622.

Panov et al. (2005). Rotenone model of Parkinson disease: multiple brain mitochondria dysfunctions

after short term systemic rotenone intoxication. The Journal of Biological Chemistry,

280(51):42026-42035.

73

Parisiadou L., et al. (2009). Phosphorylation of ezrin/radixin/moesin proteins by LRRK2 promotes the

rearrangement of actin cytoskeleton in neuronal morphogenesis. The Journal of Neuroscience,

29, 13971–13980.

Peng, Y., et al. (2010). Binding of alpha-synuclein with Fe(III) and with Fe (II) and biological

implications of the resultant complexes. Journal of Inorganic Biochemistry,104, 365-370.

Plun-Favreau H, et al. (2007). The mitochondrial protease HtrA2 is regulated by Parkinson’s disease-

associated kinase PINK1. Nature Cell Biology, 9, 1243–1252.

Pridgeon, J., Olzmann, J., Chin, L. et al. (2007). PINK1 protects against oxidative stress by

phosphorylating mitochondrial chaperone TRAP1. PLOS Biology, 5, e172.

Puigserver, P., & Spiegelman, B. M. (2003). Peroxisome proliferator-activated receptor-gamma

coactivator 1 alpha (PGC-1 alpha): Transcriptional coactivator and metabolic regulator.

Endocrine Reviews, 24(1), 78-90.

Purushotham et al. (2009). Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and

results in hepatic steotosis and inflammation. Cell Metabolism, 4, 327-338.

Ramsay, R., Kowal, A., Johnson, M. et al. (1987). The inhibition site of MPP+, the neurotoxic

bioactivation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine is near the Q-binding

site of NADH dehydrogenase. Archives of Biochemistry and Biophysics, 259(2), 645-649.

Richardson, J. et al. (2005). Paraquat neurotoxicity is distinct from that of MPTP and rotenone.

Toxicological Sciences, 88(1), 193–201.

Roberg, K., Johansson, U & Ollinger, K. (1999). Lysosomal release of cathepsin D precedes

relocation of cytochrome c and loss of mitochondrial transmembrane potential during

apoptosis induced by oxidative stress. Free Radical Biology and Medicine, 27(11-12), 1228-

1237.

Salama, M. & Arias-Carrion. (2011). Natural toxins implicated in the development of Parkinson’s

disease. Therapeutic Advances in Neurology Disorder, 4(6), 361-373.

Scher, M., Vaquero, A., & Reinberg, D. (2007). SirT3 is a nuclear NAD+-dependent histone

deacetylase that translocates to the mitochondria upon cellular stress. Genes & Development,

21(8), 920-928.

Schlicker, C., et al. (2008). Substrates and regulation mechanisms for the human mitochondrial

sirtuins Sirt3 and Sirt5. Journal of Molecular Biology, 382(3), 790-801.

Schwer, B., et al. (2006). Reversible lysine acetylation controls the activity of the mitochondrial

enzyme acetyl-CoA synthetase 2. Proceedings of the National Academy of Sciences of the

United States of America, 103(27), 10224-10229.

74

Schwer, B., North, B., Frye, R. et al. (2002). The human silent information regulator (Sir)2

homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide-dependent

deacetylase. Journal of Cell Biology. 158(4), 647-657.

Sherer, T., Trimmer, P., Borland, K. et al. (2001). Chronic reduction in complex I function alters

calcium signaling in SH-SY5Y neuroblastoma cells. Brain Research, 891(1-2), 94-105.

Sherman, M. &Goldberg, A. (2001). Cellular defenses against unfolded proteins: a cell biologist

thinks about neurodegenerative diseases. Neuron, 29, 15-32.

Shi, T., et al. (2005). SIRT3, a mitochondrial sirtuin deacetylase,regulates mitochondrial function and

thermogenesis in brown adipocytes. The Journal of Biological Chemistry, 280(14), 13560-

13567.

Shimazu, T., et al. (2010). SIRT3 deacetylates mitochondrial 3-hydroxy-3-methylglutaryl CoA

synthase 2 and regulates ketone body production. Cell Metabolism, 12, 654–661.

Shinde, S & Pasupathy, K. (2006). Respiratory-chain enzyme activities in isolated mitochondria of

lymphocytes from patients with Parkinson’s disease: preliminary study. Neurology India,

54(4), 390-393.

Shulman, J., De Jager, P., Feany, MB. (2011). Parkinson’s disease: genetics and pathogenesis. Annual

Review of Pathology, 6, 193-222.

Sminorva, E., et al. (2001). Dynamin-related protein Drp1 is required for mitochondrial division in

mammalian cells. Molecular Biology of the Cell, 12, 2245-2256.

Snyder, A. & Connor, J. (2009). Iron, the substantia nigra and related neurological disorders.

Biochimica et Biophysica Acta, 1790, 606-614.

Someya, S., et al. (2010). Sirt3 mediates reduction of oxidative damage and prevention of age-related

hearing loss under caloric restriction. Cell, 143(5), 802-812.

Stichel, C. , Zhu, X., Bader, V et al. (2007). Mono- and double-mutant mouse models of Parkinson’s

disease display severe mitochondrial damage. Human Molecular Genetics 16: 2377–2393.

Takashi T, et al. (1994). Uptake of a neurotoxin-candidate, (R)-1,2-dimethyl-6, 7-dihydroxy-1,2,3,4-

tetrahydroisoquinoline into human dopaminergic neuroblastoma SH-SY5Y cells by dopamine

transport system. Journal of Neural Transmission General Section, 98,107-118.

Tanaka, Y et al. (2001) Inducible expression of mutant alpha-synuclein decreases proteasome activity

and increases sensitivity to mitochondria-dependent apoptosis. Human Molecular Genetics,

10, 919–926.

Tao, R., et al. (2010). Sirt3-mediated deacetylation of evolutionarily conserved lysine 122 regulates

MnSOD activity in response to stress. Molecular Cell, 40(6), 893-904.

75

Terman, A. and Brunk, U. T. (2005). The aging myocardium: roles of mitochondrial damage and

lysosomal degradation. Heart, Lung and Circulation, 14, 107-114.

Territo, P., French, S., Balaban. (2001). Stimulation of cardiac work transitions, in vitro: effects of

simultaneous Ca2+ and ATPase additions on isolated porcine heart mitochondria. Cell

Calcium, 30, 19-27.

Tieu, K., Zuo, D. & Yu, P. (1999). Differential effects of staurosporine and retinoic acid on the

vulnerability of the SH-SY5Y neuroblastoma cells: involvement of bcl-2 and p53 proteins.

Journal of Neuroscience Research. 58(3), 426-435.

Trancikova, A., Tsika, E., Moore, DJ. (2012). Mitochondrial dysfunction in genetic animal models of

Parkinson’s disease. Antioxidant Redox Signalling, 16, 896-919.

Valente, E. et al. (2004). Hereditary early-onset Parkinson’s disease caused by mutations in PINK1.

Science 304: 1158–1160, 2004.

Vaquero et al. (2006). SIRT2 is a histone deacetylase with preference for histone H4 Lys 16 during

mitosis. Genes & Development, 20, 1256-1261.

Voges, D., Zwickl, P & Baumeister, W. (1999). The 26S proteasome: a molecular machine designed

for controlled proteolysis. Annual Review of Biochemistry, 68, 1015-1068.

Wallace, D & Fan, W (2010). Energetics, epigenetics, mitochondrial genetics. Mitochondrion, 10, 12-

31.

Wang et al. (2012). Penetrance of LRRK2 G2385R and R1628P is modified by common PD-

associated genetic variants. Parkinsonism related disorder, 18(8), 958-963.

Wang, F. & Tong, Q. (2009). SIRT2 suppresses adipocyte differentiation by deacetylating FOXO1

and enhancing FOXO1’srepressive interaction with PPARgamma. Molecular Biology of the

Cell, 20, 801–808.

Weir et al. (2012). CNS SIRT3 expression is altered by reactive oxygen species and in Alzheimer’s

disease. PLoS One, 7, e48225.

Wohlgemuth, S. E., et al. (2010). Skeletal muscle autophagy and apoptosis during aging: Effects of

calorie restriction and life-long exercise. Experimental Gerontology, 45, 138-148.

Xie, Hu & Li. (2010). SH-SY5Y human neuroblastoma cell line: in vitro cell model of dopaminergic

neurons in Parkinson’s disease. Chinese Medical Journal, 123(8), 1086-1092.

Xilouri, M., Stefanis, L. (2011). Autophagic pathways in Parkinson disease and related disorders.

Expert Reviews in Molecular Medicine.

Yang et al. (1995). Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes

cell death. Cell, 80, 285-291.

76

Yang, B., et al. (2009). The sirtuin SIRT6 deacetylates H3 K56Ac in vivo to promote genomic

stability. Cell Cyle, 8, 2662-2663.

Yang, Y et al. (2010). NAD+ dependent deacetylase SIRT3 regulates mitochondrial protein synthesis

by deacetylation of the ribosomal protein MRPL10. The Journal of Biological Chemistry, 285,

7417-7429.

Yang, Y. H., et al. (2000). Cloning and characterization of two mouse genes with homology to the

yeast Sir2 gene. Genomics, 69(3), 355-369.

Yong-Kee, C., Sidorova, E., Hanif, A, et al. (2012). Mitochondrial dysfunction precedes other sub-

cellular abnormalities in an vitro model linked with cell death in Parkinson’s disease.

Neurotoxic research, 21(2), 185-194.

Yong-Kee., C., Salomonczyk, D & Nash, J. (2011). Development and validation of a screening assay

for the evaluation of putative neuroprotective agents in the treatment of Parkinson’s disease.

Neurotoxic research, 19(4), 519-526.

Yoon, Y. S., et al. (2006). Formation of elongated giant mitochondria in DFO-induced cellular

senescence: involvement of enhanced fusion process through modulation of Fis1. Journal of

Cellular Physiology, 209, 468-480.

Zeng, B et al. (2006). MPTP treatment of common marmosets impairs proteasomal enzyme activity

and decreases expression of structural and regulatory elements of the 26S proteasome. Eur J

Neuroscience, 23(7), 1766-1774.

Zhang, D & Gutterman, D. (2007). Mitochondrial reactive oxygen species-mediated signaling in

endothelial cells. Am J Physiology Heart Circ Physiology. 292(5), H2023-2031.

Zhang, L et al. (2005). Mitochondrial localization of the Parkinson’s disease related protein DJ-1:

implications for pathogenesis. Human Molecular Genetics 14, 2063–2073.

Zhong et al. (2010). The histone deacetylase SIRT6 regulates glucose homeostasis via H1Falpha.

Cell, 140, 280-293.

Zhu, H., et al. (2012). The QKI-PLP pathway controls SIRT2 abundance in CNS myelin. Glia, 60,

69–82.

Ziering, A., Berger, L., Heineman, S., et al. (1947). Piperidine derivatives. Part III. 4-

Arylpiperidines. Journal of Organic Chemistry, 12(6), 894–903.