Multiple System Atrophy and Parkinson’s

disease

Thesis submitted for the degree "doctor of philosophy"

By Haya Kisos

Submitted for the senate of Hebrew University

June 2013

This work was carried out by supervision of

Dr. Ronit Sharon and Prof. Tamir Ben Hur

Abstract:

The synucleinopathies are a diverse group of neurodegenerative disorders that share a

common pathologic intracellular lesion, composed primarily of aggregates of

insoluble α-Synuclein (α-Syn) protein in selectively vulnerable populations of

neurons and glia. The term, “synucleinopathy,” was introduced in 1998 after it was

recognized that filamentous α-Syn deposits might represent a link between Multiple

system atrophy (MSA), Parkinson’s disease (PD), and Dementia with Lewy bodies

(DLB). In this thesis I have focused on two of the synucleinopathies, PD and MSA.

-Syn is an abundant neuronal protein enriched in presynaptic nerve terminals, and it

has been critically implicated in the genetics and neuropathology of

synucleinopathies. α-Syn is a soluble protein, yet, under certain conditions it

gradually oligomerizes and deposits in intra-cellular insoluble inclusions. These

inclusions, when in neuronal cells, are called Lewy bodies. When in

oligodendrocytes, the inclusions are called glial cytoplasmic inclusions (GCIs), and

are characteristics of MSA histopatholgy. Specific point mutations in the α-Syn gene,

as well as duplications and triplications at the locus for wild-type -Syn, increase its

propensity to self-oligomerize, in addition to increasing its pathogenicity.

In recent years, extracellular -Syn became the focus of many studies due to its

potential role in disease initiation and progression While secretion of -Syn may

occur normally from healthy neurons, growing evidence indicates that enhanced

secretion is associated with -Syn pathogenesis. The mechanisms involved in -Syn

secretion are not yet fully understood.

The general aims of the thesis are:

1. To elucidate the mechanisms underlying the pathogenic accumulation of -Syn, a

neuronal protein in oligodendroglia, as observed in Multiple System Atrophy.

2. To elucidate the mechanisms by which -Syn expression affects catalase, the anti-

oxidative stress enzyme.

In the first part of the study, I tested the hypothesis that oligodendrocytes can take up

neuronal-secreted -Syn as part of the pathogenic mechanisms leading to MSA. I

reported that increases in the degree of soluble α-Syn oligomers and intracellular -

Syn levels, enhance its secretion from cultured MN9D dopaminergic cells, stably

expressing the protein. In accord, I showed that the oligodendroglial cell line, Oli-

Neu, can take-up neuronal-secreted or exogenously-added -Syn from their

conditioning medium. This uptake is concentration-, time-, and clathrin-dependent.

Utilizing the demonstrated effect of polyunsaturated fatty acids (PUFAs) to enhance

α-Syn neuropathology, I showed in vivo that increases in neuronal -Syn levels

enhance its localization to oligodendrocytes in the brains of a mouse model for

synucleinopathies, transgenic for the human A53T -Syn mutant form. Thus, the

pathogenic mechanisms leading to elevated levels of α-Syn in neurons underlie

neuronal secretion, and the subsequent uptake of -Syn by oligodendrocytes in MSA.

In the second part of the study I aimed at investigating the mechanisms by which -

Syn expression affects catalase, the anti-oxidative stress enzyme. Previous studies in

our lab showed evidence suggesting that -Syn specifically affects catalase

expression levels and enzymatic activity. Searching for a mechanism through which

-Syn expression affects catalase, I found that peroxisome proliferator-activated

receptor (PPAR which is known to control catalase transcription, has inhibited

transcription activity when -Syn is expressed. It is important to note that activating

PPARα, PPARγ and RXR, but not PPARβ/δ, restored and enhanced catalase activity.

Based on these results, I have suggested that through its involvement in fatty acid

metabolism, -Syn expression affects the pool of fatty acid metabolites, which

normally act as activating ligands for PPAR. The affected PPAR activity inhibits

catalase expression and results in the accumulation of oxidative damage that

characterizes PD. I concluded that α-Syn down-regulates catalase expression and

activity by interfering with the complex and overlapping network of nuclear receptor

dimerization and transcription activation.

In summary, my results may contribute to the understanding of the pathogenic

mechanisms involved in -Syn toxicity in the synucleinopathies. A role for brain

lipids, and specifically for PUFAs, has been demonstrated.

List of abbreviation:

1-methyl-4-phenylpyridinium ion (MPP+).

2,4-dinitrophenylhydrazine (DNPH)

3′-Cyclic Nucleotide 3′-Phosphodiesterase (CNPase)

3-nitropropionic acid (3-NP)

4-hydroxynonenal (4-HNE)

6-hydroxydopamine (6-OHDA)

carbonic anhydrase II (CAII)

cerebrospinal fluid (CSF)

clathrin-mediated endocytosis (CME)

conditioned medium (CM)

deep brain stimulation (DBS)

Dementia with Lewy bodies (DLB)

docosahexaenoic acid (DHA, 22:6)

docosohexaenoic acid (DHA)

eicosapentaenoic acid (EPA, 20:5)

genome-wide association (GWA)

glial cytoplasmic inclusions (GCI)

Glial fibrillary acidic protein (GFAP)

glial nuclear inclusions (GNIs)

glutathione (GSH)

hydrogen peroxide (H2O2)

hydroxyeicosatetraenoic acid (HETE)

Immunocytochemistry (ICC)

Immunohistochemistry (IHC

levodopa (L-dopa)

Lewy bodies (LB),

Lewy neurites (LN)

linolenic acid (ALA, 18:3)

Multiple System Atrophy (MSA)

Muno unsaturated fatty acid (MUFA)

myelin basic protein (MBP)

neuronal cytoplasmic inclusions (NCIs)

neuronal nuclear inclusions (NNIs)

Neuronal Nuclei (NeuN)

nuclear receptor related 1 (NURR1)

Olivopontocerebellar Atrophy (OPCA)

paraformaldehyde (PFA)

Parkinson’s disease (PD)

peroxisome proliferator- activated receptor coactivator (PGC-1-)

peroxisome proliferator-activated receptor gamma(PPAR)

polyunsaturated fatty acids (PUFAs)

prostaglandin J2 (PGJ2)

proteolipid protein (PLP)

quinolinic acid (QA)

reactive oxygen species (ROS)

Retinoid X Receptor (RXR)

Reverse transcription polymerase chain reaction (RT-PCR)

saturated fatty acids (SFAs)

Shy-Drager Syndrome (SDS)

single nucleotide polymorphisms (SNPs)

small hairpin RNA (shRNA)

Striatonigral Degeneration (SND)

substania nigra pars compacta (SNpc)

succinate dehydrogenase inhibitor

superoxide dismutase (SOD)

superoxide dismutase (SOD1)

transgenic (tg)

tubulin polymerization promoting protein (TPPP/P25)

wild type (wt)

α-Synuclein (α-Syn)

List of publication:

1. The clathrin-dependent localization of dopamine transporter

to surface membranes is affected by α-Synuclein" Haya Kisos, Tziona Ben-Gedalya and Ronit Sharon. Submitted.

2. -Synuclein expression inhibits catalase activity in brains of mice modeling Parkinson's disease.

Eugenia Yakunin, Haya Kisos, Willem Kulik,; Ronald J. A Wanders,and Ronit

Sharon. Submitted.

3. Enhanced neuronal -Synuclein pathology associates with its

accumulation in oligodendrocytes in mice modeling -Synucleinopathies. Haya Kisos, Katharina Pukaß, Tamir Ben-Hur, Christiane Richter-Landsberg and

Ronit Sharon. PLoS One,2012

4. Docosahexaenoic acid controls α-synuclein neuropathology in a mouse model. Eugenia Yakunin ,Virginie Loeb ,Haya Kisos ,Yoav Biala ,Shlomo Yehuda, Yoel

Yaari, Dennis J. Selkoe and Ronit Sharon. Brain Pathol. 2012

5. Alpha-synuclein expression selectively affects tumorigenesis in mice modeling Parkinson's disease. α-Synuclein Expression Selectively Affects Tumorigenesis in Mice Modeling Parkinson's Disease Eitan Israeli,Eugenia Yakunin,Yonaton Zarbiv,Amir Hacohen-Solovich,Haya Kisos,Virginie Loeb,Michal Lichtenstein,Tziona Ben-

Gedalya,Ofra Sabag,Eli Pikarsky,Haya Lorberboum-Galski,Ronit Sharon

PLoS One. 2011

Introduction .................................................................................................................. 1

1. Synucleinopathies ...................................................................................................... 1

2. Multiple System Atrophy ........................................................................................... 1

2.1 Clinical presentation and diagnosis................................................................. 1

2.3 Histopathology ................................................................................................. 2

2.4. α-Synuclein ..................................................................................................... 3

2.5 Animal models of MSA ..................................................................................... 6

3. Parkinson's disease ................................................................................................... 6

3.1 Pathology ........................................................................................................ 7

3.2 Therapeutic approaches for Parkinson's disease ........................................... 7

3.3 Etiology of PD .................................................................................................. 8

3.4 Models of Parkinson's disease ....................................................................... 11

3.5 Parkinson’s disease and oxidative stress ...................................................... 11

4. Alpha-synuclein: ..................................................................................................... 11

4.1 -Syn in PD pathology .......................................................................................... 11

4.2 Protein structure .................................................................................................. 12

4.3 Post-translational modifications ...................................................................... 13

4.5 Oligomerization and aggregation ..................................................................... 14

4.6 Physiological function ....................................................................................... 15

5. Catalase ................................................................................................................... 16

6. Nuclear receptors .................................................................................................... 16

6.1 Peroxisome proliferator-activated receptors (PPARs)................................... 16

6.2 RXR ................................................................................................................. 17

7. Working Hypothesis ................................................................................................ 18

8. Aims and Objectives ................................................................................................ 18

Materials and Methods: ............................................................................................ 19

Results ......................................................................................................................... 27

Chapter I ...................................................................................................................... 27

Increased neuronal -Synuclein pathology is associated with its accumulation in

oligodendrocytes in mice modeling -Synucleinopathies. ...................................... 27

Syn Secretion from Stably-Transfected Neuronal Cells ............................. 27

2. Oligodendroglial cell lines take-up α-Syn from their growth medium. ........... 33

3. Evidence for affected oligodendrocytes in the brains of A53T α-Syn tg mice modeling neuronal synucleinopathies ................................................................. 39

Chapter 2: .................................................................................................................... 66

Synuclein and Oxidative Stress .............................................................................. 66

1. Evidence for oxidative damage in brains of A53T -Syn tg mice. ................... 66

2. -Syn specifically inhibits catalase activity by affecting PPAR transcription activity .................................................................................................................. 67

Discussion ................................................................................................................... 60

Concluding remarks .................................................................................................... 69

Bibliography….............................................................................................................62

Introduction

1. Synucleinopathies The synucleinopathies are a diverse group of neurodegenerative disorders that share a

common pathologic intracellular lesion, composed primarily of aggregates of

insoluble α-Synuclein (α-Syn) protein in selectively vulnerable populations of

neurons and glia (Goedert, 1999; Spillantini et al., 2000; Galvin et al, 2001;

Trojanowski et al., 2003; Wakabayashi et al., 1998a). The term, “synucleinopathies,”

was introduced after it was recognized that filamentous α-Syn deposits might

represent a link between Multiple System Atrophy (MSA), Parkinson’s disease (PD),

and Dementia with Lewy bodies (DLB) (Spillantini et al., 1998).

2. Multiple System Atrophy Multiple System Atrophy (MSA) was first described by Graham and Oppenheimer in

1969 as a general term for three disorders that had “neuronal atrophy in a variety of

overlapping combinations” (Graham et al., 1969). MSA combines together the

following three distinct clinicopathological disorders: Olivopontocerebellar Atrophy

(OPCA) (Geary et al, 1956), Striatonigral Degeneration (SND) (Adams et al,

1964) and Shy-Drager Syndrome (SDS) (Shy et al., 1960). Pathologically, MSA is

characterized by filamentous glial cytoplasmic inclusions (GCIs) (Papp et al, 1989).

The finding of misfolded, hyper-phosphorylated, fibrillar α-Syn as a main component

of GCIs has classified MSA as a synucleinopathy (Wakabayashi et al, 1998b). At

present, therapy for MSA only treats symptoms, mainly targeting Parkinsonism and

autonomic failure (Stefanova et al, 2009).

2.1 Clinical presentation and diagnosis

An accurate and final diagnosis of MSA is obtained at autopsy, with characteristic

pathology consisting of widespread and abundant GCIs in association with

neurodegenerative changes in striatonigral or olivopontocerebellar structures.

According to clinical signs, MSA is divided into two types: MSA-P with predominant

Parkinsonism, and MSA-C with dominant cerebellar features (MSA-C).

Cases of probable or possible MSA are diagnosed based on the occurrence of the

following:

1. Autonomic failure involving urinary incontinence (with erectile dysfunction in

males), or orthostatic decrease of blood pressure within 3 minutes of standing.

2. Poorly-levodopa-responsive Parkinsonism (bradykinesia with rigidity, tremor, or

postural instability) for MSA-P, or a cerebellar syndrome (gait ataxia with cerebellar

dysarthria, limb ataxia, or cerebellar oculomotor dysfunction) for MSA-C.

In addition, possible MSA-P is diagnosed upon rapidly-progressive Parkinsonism;

postural instability within 3 years of motor onset; dysphasia within 5 years of motor

onset; atrophy (as visualized with MRI) of the putamen, middle cerebellar peduncle,

pons, or cerebellum; and hypo-metabolism (as visualized with FDG-PET imaging) in

the putamen, brainstem, or cerebellum. Atrophy (as visualized with MRI) of the

putamen, middle cerebellar peduncle, or pons; hypometabolism (as visualized with

FDG-PET imaging) in the putamen; and presynaptic nigrostriatal dopaminergic

denervation (as visualized with SPECT or PET imaging) are often signs of possible

MSA-C (Gilman et al., 1998; Gilman et al., 2008).

2.3 Histopathology

The histopathology of MSA includes glial cytoplasmic inclusions (GCI) and gliosis,

neuronal cell loss and axonal degeneration, and a reduction in myelin content.

The neuropathological hallmark of MSA is the presence of α-Syn in the GCIs (Papp

et al., 1989). Several studies have described the regional distribution and severity of

GCIs, with pyramidal, extrapyramidal, corticocerebellar and preganglionic autonomic

systems being particularly vulnerable. The density of GCIs is lower in white matter

structures with severe myelin pallor, and higher in those with only mild to moderate

myelin loss (Ishizawa et al, 2008). Importantly, a study focusing on the nigrostriatal

and olivopontocerebellar regions, found a correlation between the frequency of GCIs,

the severity of neuronal cell loss, and disease duration, suggesting a link between

neurodegeneration and GCIs in these particular regions (Ozawa et al., 2004).

In addition, pathogenic α-Syn accumulations have been detected in the nuclei of

oligodendrocytes, forming glial nuclear inclusions (GNIs) (Papp et al., 1992); in the

cytoplasm and nuclei of neurons, forming neuronal cytoplasmic inclusions (NCIs);

and neuronal nuclear inclusions (NNIs); and in cell processes, forming threads and

neurites. These pathogenic lesions are detectable using α-Syn immunohistochemistry

and Gallyas silver staining, but are less frequently observed than GCIs (Arima et al.,

1998; Papp et al., 1992). The neuronal inclusions, as seen in cases of MSA, are found

in the cortex, subcortex, brainstem and cerebellar nuclei (Arima et al., 1992), and are

particularly prevalent in the pontine base and inferior olive (Nishie et al., 2004). The

neuronal inclusions are ultrastructurally similar to GCIs, but show some differences in

their immunohistochemical staining profile (Wakabayashi et al., 2006).

Neuronal cell loss and axonal degeneration

Neuronal cell loss was documented in the substantia nigra, dorsolateral zone of the

caudal putamen, vermis, cerebellar hemisphere and inferior olivary nucleus in brains

with MSA (Ozawa et al., 2004). In addition, the locus coeruleus, intermediolateral

cell column and Onuf's nucleus were shown to be affected (Wenning et al., 1997).

The loss of neurons in the autonomic system nuclei of the hypothalamus, brainstem

and spinal cord is thought to be responsible for the autonomic dysfunction seen in

MSA patients (Ozawa, 2007).

Myelin pallor

White matter tracts associated with the striatonigral and Olivopontocerebellar regions,

such as the external capsule, striatopallidal fibers, transverse pontine fibers and

cerebellar white matter have shown a significant reduction in myelin staining (Papp et

al., 1989)

Gliosis

Reactive astrocytes (Song et al., 2009) and activated microglia are common

histological findings. In MSA, the degree of astrogliosis is correlated with the

severity of neurodegeneration (Ozawa et al., 2004).

2.4. α-Synuclein

α-Syn is primarily a presynaptic neuronal protein. It was originally localized to

presynaptic nerve terminals (Jakes et al., 1994; Georgeet et al., 1995). However, it is

now confirmed that -Syn expression is present in a variety of tissues, including in

the muscles, kidneys, liver, lungs, heart, testis, colon, blood vessels, lymphocytes and

platelets (Askanas et a.l, 2000; Barbour et al., 2008; Nakai et al., 2007; Ltic et al.,

2004; Tamo et al., 2002). Evidence from genetic studies supports a role for α-Syn in

MSA. Specifically, analysis of single nucleotide polymorphisms (SNPs) in the α-Syn

gene has identified an association between certain α-Syn SNPs and an increased risk

for the developing MSA (Al-Chalabi et al., 2009; Scholz et al., 2009).

2.4.1 α-Synuclein secretion

In recent years, extracellular -Syn has received a lot of scientific attention due to its

potential role in disease initiation and progression

-Syn is present in biological fluids, such as cerebrospinal fluid (CSF) and blood

plasma, of both PD and normal subjects (El-Agnaf et al., 2003; Lee et al., 2006;

Tokuda et al., 2006). -Syn was shown to be secreted into the cultured medium in

various cell lines over-expressing -Syn (El-Agnaf et al., 2003; Sung et al., 2005; Lee

et al , 2005). Moreover, secretion of endogenous Syn from rat embryonic cortical

neurons was also demonstrated (Lee et al., 2005). Both monomeric and aggregated

forms of -Syn were found to be secreted (Danzer et al., 2012; Lee et al., 2005). The

mechanisms of -Syn secretion are not fully understood. It has been suggested that -

Syn can be secreted by the non-classical ER/Golgi-independent protein export

pathway (Lee et al., 2005). Alternatively, -Syn secretion was shown to be mediated

by exosomal secretion, in a calcium-dependent mechanism (Emmanouilidou et al.,

2010.). Moreover, exosomal -Syn release was found to be increased as a result of

lysosomal dysfunction (Alvarez-Erviti et al., 2010.). In contrast, a recent study found

no evidence for the enrichment of -Syn in exosomal pellets of neuronal cells over-

expressing -Syn. In addition, no signal for -Syn was detected in exosomal fractions

derived from the CSF of PD patients and control subjects. This study further shows

that in neuronal cells, -Syn is present in endosomal compartments, and that -Syn

secretion and lysosomal targeting is regulated by vacuolar protein sorting (Hasegawa

et al., 2011). It was suggested that the recycling pathway regulated by Rab11 has

functional relevance in -Syn secretion (Hasegawa et al., 2011). Another study

demonstrates that α-Syn oligomers can be found in at least two extracellular fractions,

either associated with exosomes or free-floating, α-Syn oligomers are present on the

outside as well as the inside of exosomes. Notably, the pathway of secretion of α-Syn

oligomers is strongly influenced by autophagic activity (Danzer et al, 2012). -Syn

uptake was reported in microglia (Park et al, 2008; Lee et al, 2008b) and in astrocytes

(Lee et al, 2010b.), activating the cells to induce neuroinflammatory processes, and

also in oligodendrocytes. -Syn uptake by oligodendrocytes and neurons was

markedly decreased by the genetic suppression and pharmacological inhibition of the

dynamin GTPases (Konno et al, 2012).

Recent findings suggest that -Syn pathogenicity share similarities with the prion

protein. This hypothesis is supported by observations describing the presence of -

Syn-positive Lewy-body-like inclusions in long-term mesencephalic transplants in PD

patients (Li et al., 2008, Kordower et al., 2008). These findings suggest host-to-graft

propagation of -Syn-associated pathology. Subsequent in vitro experiments have

shown inter-neuronal transmission of Syn (Desplats et al., 2009 Danzer et al,

2012). Moreover, the host-to-graft transmission of -Syn has also been observed in

vivo in -Syn transgenic mouse models (Desplats et al., 2009; Hansen et al.,

2011). This transmission process seems to rely on endocytic uptake of -Syn by the

recipient neurons (Hansen et al., 2011; Angot et al., 2012; Emmanouilidou et al.,

2010; Lee et al., 2008a). Furthermore, a recent study demonstrated that a single intra-

striatal injection of synthetic misfolded α-Syn into WT mice, initiates a

neurodegenerative cascade characterized by the accumulation of intracellular -Syn

pathology, selective loss of DA neurons, and impaired motor coordination. Thus,

misfolded α-Syn is sufficient to induce the cardinal behavioral and pathological

features of sporadic PD (Luk et al., 2012).

Together, these findings indicate that -Syn can be secreted from neurons and also

transmitted between neuronal and potentially glial cells.

2.4.2. The origin of -Syn in oligodendrocytes in MSA

-Syn mRNA expression was not detected in the oligodendrocytes of MSA or healthy

human brains. Specifically, using in situ hybridization for α-Syn and proteolipid

protein (PLP), an oligodendrocyte marker , no signal for α-Syn mRNA was detected

(Miller et al., 2005). On the other hand, cultured rat brain oligodendrocytes have been

shown to express α-Syn mRNA and protein, but its expression was developmentally

regulated with peak expression at 2–3 days in culture, with limited expression before

and after this time (Richter-Landsberg et al., 2000). It is possible that α-Syn

expression is up-regulated and/or not degraded efficiently in cultured

oligodendrocytes. Nevertheless, the reason for pathogenic accumulation of -Syn

within oligodendrocytes is not known. It is therefore of interest to find out whether

oligodendrocytes may potentially take-up neuronal-secreted α-Syn, in a mechanism

leading to the development of MSA.

2.5 Animal models of MSA

Animal models of MSA were generated in mice, rats and primates using three

different approaches including, lesioning striatonigral structures via toxins, over-

expression of human α-Syn in oligodendrocytes, and combining toxin-caused

lesioning and α-Syn over-expression.

The toxins causing lesioning are: 6-hydroxydopamine (6-OHDA), quinolinic acid

(QA), succinate dehydrogenase inhibitor, 3-nitropropionic acid (3-NP) or 1-methyl-4-

phenylpyridinium ion (MPP+). Striatal and nigral neuronal cell loss and motor

impairment are features seen in the toxin models, but none of these models were able

to recapitulate GCI formation, thus limiting their use as MSA models. Transgenic

mouse models, where over-expression of human α-Syn was driven using an

oligodendroglial-promoter, were able to reproduce GCI-like inclusions in

oligodendrocytes, yet, limited neurodegeneration and motor deficits were observed

(Kahle et al., 2002 ;Yazawa et al., 2005 ). Currently there is no good model for MSA

which accurately reflects the human manifestation of the disease.

3. Parkinson's disease Parkinson’s disease (PD) is a progressive, age-dependent neurodegenerative disorder.

The major clinical manifestations of PD include slowness of movement, rigidity and

tremor. These motor symptoms result from a deficiency in dopamine levels, due to the

degeneration of dopaminergic neurons in the substantia nigra. In addition to the motor

symptoms, PD patients may also suffer from non-motor symptoms which are

progressive during the course of the disease and impair quality of life (for recent

reviews see (Meissner et al., 2011; Obeso et al., 2010; Shulman et al., 2011). A

prominent non-motor symptom is dementia. It has been estimated that at least 50% of

PD patients suffer mild cognitive impairment in addition to the motor symptoms, and

approximately 20-40% of patients may suffer severe dementia (called Parkinson’s

disease Dementia, PDD).

3.1 Pathology

The cytopathological hallmark of PD is the occurrence of round, hyaline neuronal

cytoplasmic inclusions called, Lewy bodies (LB), and enlarged aberrant neurites

called, Lewy neurites (LN), found in the substania nigra pars compacta (SNpc) as

well as in additional brain regions, including the locus coeruleus, reticular nuclei of

the brain stem, dorsal motor nucleus of the vagus, as well as the basal nucleus of

Meynert, the amygdala and the CA2 area of the hippocampus (Forno, 1996; Braak,

et al,1999; Dickson et al., 2001; Goedert, 2001; Braak et al., 2003).

A progressive conversion of the soluble α-Syn protein, into insoluble, aggregated,

forms of the protein, and its deposition in Lewy bodies, underlie the pathogenic

process of -Syn in PD and the synucleinopathies (Spillantini et al., 1997). Braak and

coworkers (Braak et al, 2004) proposed a staging system for PD that is based on the

progressiveness of Lewy pathology in PD brains. According to which, the earliest

Lewy pathology affects the enteric and peripheral autonomic nervous system as well

as the olfactory bulb, and subsequently spreads in a stereotyped, caudal-to-rostral

wave from the lower brainstem (stage 1, presymptomatic) to diffuse involvement of

the neocortical ribbon (stage 6, severely affected). According to the Braak paradigm,

α-Syn pathology is not observed in the midbrain SN until stage 3, consistent with the

hypothesis that a substantial prodromal syndrome precedes the development of PD

motor symptoms, and the subsequent clinical recognition of the disorder (Hawkes,

2008). Therefore, the Braak staging paradigm supports the theory of extracellular

-Syn as a pathogenic ‘prion-like’ agent.

3.2 Therapeutic approaches for Parkinson's disease

PD is an incurable and progressive condition. Presently, symptomatic treatments are

available with some indications for neuroprotective effects (Weinreb et al, 2011) . For

more than 40 years, levodopa (L-dopa, a precursor of dopamine), has been regarded

as the gold standard for the treatment of PD and may well treat many of the cardinal

symptoms of PD. However, motor fluctuations and dyskinesias appear in most

patients following years of treatment with L-dopa, either as part of the disease

progression or as a result of chronic dopaminergic therapy, or both (Fahn et al., 2004;

Lew, 2007; Meissner et al., 2011). Other agents, including dopamine agonists and

drugs preventing dopamine degradation, such as monoamine oxidase-B inhibitors and

catechol-O-methyltransferase inhibitors, may be added to treat the motor fluctuations.

In more advanced stages of the disease, deep brain stimulation (DBS) of the

subthalamic nucleus or globus pallidus is another option that may offer definitive

efficacy in treating the intractable tremor, dyskinesia, and "off" time (Bergman et al,

1990; Limousin et al., 1998). This can produce a dramatic improvement in symptoms;

however, there is concern regarding about the increased incidence of psychiatric side

effects, particularly depression, following DBS (Davie, 2008). There is no evidence

that DBS slows the progression of PD or that it is neuroprotective (Volkmann, 2007).

Another rapidly expanding therapeutic approach is stem cell technology, which

focuses on the replacement of lost dopaminergic neurons. Initial cellular therapies for

PD utilized fetal ventral midbrain tissue implantations, as a source of dopaminergic

neurons. Clinical trials have had varying degrees of success, and overall have

supported the cellular therapies for PD (Brundin et al, 2010). Potential limitations of

utilizing fetal tissue, however, include ethical concerns and the ability to obtain

adequate amounts of tissue for treatment. Alternatively, stem cells offer sources for

large-scale production of neurons that acquire a midbrain DA phenotype (Kim, 2011;

Morizane et al., 2010). A drawback of the cellular replacement therapy is that

autopsies from patients 9-22 years post-grafting have shown severe PD pathology

within the grafted neurons, including dopaminergic neuronal cell loss and the

presence of mature Lewy bodies in the grafted neurons, suggesting that the grafted

cells had not escaped the mechanisms of the disease (Kordower et al, 2008; Mendez

et al., 2008).

3.3 Etiology of PD

PD is considered a sporadic disorder, in which a single cause of the disease has not

been found. Numerous epidemiologic studies have shown that old age, genetic

predisposition and environmental factors contribute to the occurrence of the disease.

Age

PD is the second most common age-related neurodegenerative disease after

Alzheimer’s disease, with a prevalence of 1-2% of the population over the age of 65,

and 3% of the population over the age of 75 years. A higher prevalence is found

among males (19.0 per 100,000) than females (9.9 per 100,000) (de Lau et al., 2006).

With the aging of the population, it is anticipated that

the prevalence of PD will

increase dramatically in the coming decades (Lilienfeld et al., 1993).

Environmental factors

The environmental hypothesis posits that PD-related neurodegeneration results from

exposure to a dopaminergic neurotoxin. This hypothesis was supported by the

discovery of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- induced

parkinsonism (Langston et al., 1983) .The insecticide, rotenone, also acts to induce

Parkinsonism (Dauer et al., 2003). Human epidemiologic studies have implicated that

residence in rural environments, and the related exposure to herbicides and pesticides,

correlates with an elevated risk of PD (Tanner, 1992). So far, however, there is no

convincing data to implicate any specific toxin as a cause of sporadic PD.

On the other hand, cigarette smoking and coffee drinking are inversely associated

with the risk for the development of PD (Hernan et al., 2002), reinforcing the concept

that some environmental factors do modify PD susceptibility.

Genetic factors

While the majority of PD cases (90%) are sporadic and their etiology is unknown, a

number of mutated genes have been identified as causing familial PD. Utilizing two

genetic approaches, linkage and association analyses, some genes with the strongest

PD-association signals have been identified, among them are:

1) SNCA (α-synuclein)

2) LRRK2, encoding leucine-rich repeat kinase2

3) GBA-1, encoding glucocerebrosidase

4) MAPT2, encoding microtubule-associated protein tau

5) BST-1

6) PARK 16 (Shulman et al, 2011).

In addition, a large group of loci, including parkin, PINK-1 and DJ-1 are associated

with Parkinsonian syndrome, but show additional clinical features, which are atypical

for PD, and/or a more restricted pathology (Klein et al. 2007; Shulman et al., 2011).

The identification of new loci in association with the disease is an ongoing effort.

Recently, it was found that variants of VPS35, encoding a subunit of the retromer

complex, causes Parkinsonism (Vilarino-Guell et al., 2011; Zimprich et al., 2011).

Additionally, mutations in the gene EIF4G1, a translation initiator, were recently

associated with familial PD (Chartier-Harlin et al., 2011). Moreover, a homozygous

NURR1 polymorphism (a single base pair insertion in intron 6) (N16P), has been

reported to be associated with PD. NURR1 is a member of the nuclear receptor family

and plays a key role in mesencephalic dopaminergic neuron development and survival

(Xu et al., 2002). Furthermore, the results from a recent genetic analysis have

suggested that PGC-1- responsive genes are under-expressed in PD (Zheng et al.,

2010). PGC-1- is a coactivator of nuclear receptors and is a master regulator of

mitochondrial biogenesis and oxidative metabolism, as well as regulating the

maintenance of glucose, lipid and energy metabolism (reviewed in (Lin et al., 2005)).

Functional studies of these PD-linked genes have supported the hypothesis that

protein misfolding and aggregation, oxidative stress, and mitochondrial dysfunction

are major contributors to PD pathogenesis.

3.4 Models of Parkinson's disease

Several experimental models of PD were generated, either in vitro classic models,

using dopaminergic cell lines and primary mesencephalic cultures, or genetic animal

models, based on PD-associated gene mutations. None of the current PD models

completely recapitulate key clinical and neuropathological features of PD. The

requirements for the ideal model include age-dependency and progressiveness of

disease symptoms; there should be typical motor dysfunction and characteristic

histopathology, including the presence of LBs and LNs that contain α-Syn (Dawson et

al., 2010).

The commonly used models, consisting of administration of MPTP or other

mitochondrial or proteasome inhibitors, act acutely, spare non-dopaminergic areas

that degenerate in PD, and do not display Lewy bodies (Olanow et al., 2009). More

promising genetic animal models recapitulate the majority of PD features, but fail to

precisely replicate the neurodegenerative pattern or behavior of PD.

3.5 Parkinson’s disease and oxidative stress

Oxidative stress can be defined as a condition in which the cellular antioxidant

defense mechanisms are insufficient to keep the level of reactive oxygen species

(ROS) below the toxic threshold. This may be due to either an overproduction of

reactive free radicals or a failure of cell buffering mechanisms.

Since PD is primarily an age-related disorder, the oxidative stress theory of aging may

explain, in part, the etiology of Parkinson’s disease (PD).

The contribution of reactive oxygen species (ROS) to aging was first suggested by the

free radical theory of aging, which postulates that aging results from damage caused

by ROS that accumulate over time, leading to cellular dysfunction and an increased

probability of death ( Harman DJ, 1956). To date, the role of ROS in aging is

controversial. The oxidative stress theory of aging has been seriously challenged in

the last decade by numerous studies. It was shown that in the nematode C.elegans ,

the deletion of all SOD encoding genes increases the worm sensitivity to oxidative

stress but has no shortening effect on lifespan ( Van Raamsdonk JM et al. , 2012).

The lack of correlation between SOD activity and lifespan was also described in mice

(Pérez VI et al., 2009). Recent studies claim that ROS are actually required for

lifespan extension (Lee SJ et al.,2010) and for associating the mitochondrial unfolded

protein response with the longevity phenotype that stems from reduced activity of

the electron transport chain (Durieux J et al., 2011).

There are several pieces of evidence for the occurrence of oxidative stress in PD.

Among the genes linked to familial forms of PD are genes that appear to be involved

in the protection against or in the propagation of oxidative stress. These are,

PINK1 (Chien et al., 2012; Gautier et al., 2008), Parkin (Palacino et al., 2004; Itier et

al., 2003), DJ-1 (Guzman et al., 2010) and SNCA (Miller et al., 2007; Martin et al.,

2006). Data from postmortem brains with PD indicate a profound loss of glutathione

(GSH) levels, a reduction in mitochondrial complex I activity, increased oxidative

damage to lipids, proteins, and DNA, increased superoxide dismutase (SOD) activity,

and elevated free iron levels (Mythri et al., 2011; Venkateshappa et al., 2012a;

Venkateshappa et al., 2012b; Youdim et al., 2001). Moreover, several markers of

oxidative stress are altered in the cerebrospinal fluid (CSF) and blood samples of PD

patients (Buhmann et al., 2004; del Hoyo et al., 2010) . In accord with the occurrence

of oxidative damage, the activity levels of catalase and glutathione peroxidase, two

enzymes responsible for the elimination of reactive oxygen species (ROS), are

reduced in PD brains (Ambani et al., 1975; Kish et al., 1985).

It is currently unknown whether oxidative damage in brains with PD results from

increased production or decreased clearance of oxidants (for review see (Andersen,

2004; Zhou et al., 2008; Varcin et al., 2012).

4. Alpha-synuclein:

-Syn protein was first associated with PD, when the first PD mutation was identified

(Polymeropoulos et al., 1997). Two additional mutations in -Syn were found, A30P

and E46K.

4.1 -Syn in PD pathology

Two seminal findings clearly linked -Syn to PD: the discovery of point mutations in

-Syn and duplications and triplications in the -Syn locus, in association with

familial forms of PD (Polymeropoulos et al., 1997; Singleton et al., 2003), and the

demonstration that α-Syn is the major protein component of Lewy bodies and Lewy

neurites in idiopathic or sporadic PD (Spillantini et al., 1997).

Three missense mutations (A53T, A30P and E46K) in the SNCA gene (PARK1 and

PARK4 loci) were identified (Choi et al., 2004; Kruger et al., 1998; Polymeropoulos

et al., 1997). The A53T mutation is the most frequent mutation. Most patients with

point mutations have prominent dementia, as in Dementia with Lewy

bodies (DLB),

with an earlier onset than those with sporadic PD (Cookson et al., 2005).

Genomic duplications and triplications in the -Syn gene locus were identified in

families with inherited PD, suggesting that over-expression of -Syn is toxic

(Singleton et al., 2003). Except for a few cases with dementia, early-stage patients

with duplications resemble those with ‘idiopathic’ PD; those with

triplications have

earlier onset, faster disease progression, marked dementia and frequent dysautonomia

(Lesage et al., 2009). -Syn multiplications

have age-dependent or incomplete

penetrance, since they were also found in asymptomatic carriers who were older than

the onset age of the affected carriers. It is therefore possible that sporadic patients may

have -Syn duplications. Polymorphisms in α-Syn’s promoter region were also

associated with PD (Wang et al., 2006). Moreover, recent findings from two genome-

wide association (GWA) studies performed on sporadic PD patients, demonstrate a

strong association between common single nucleotide polymorphisms (SNPs) within

the SNCA locus and the disease, in two populations of different ancestry (Satake et

al., 2009; Simon-Sanchez et al., 2009).

4.2 Protein structure

At least three -Syn isoforms are produced in humans by alternative splicing (Beyer,

2006). The best-known isoform is -Syn-140, which is the complete and major

transcript of the protein. Two other isoforms, -Syn-126 and -Syn-112, are

produced by alternative splicing, resulting in an in-frame deletion of exons 3 and 5,

respectively. The complete transcript isoform, -Syn-140, can be divided into three

regions:

(I) The N-terminal region, residues 1–60, includes the sites of three familial PD

mutations and contains four imperfect repeats of 11 amino acids, consisting of a

highly conserved hexameric motif (KTKEGV). The N-terminal region is predicted to

form amphipathic α-helices (Clayton et al., 1998; George et al., 1995).

(II) The central region, residues 61–95, is enriched with hydrophobic residues (Han

et al, 1995).

(III) The C-terminal region, residues 96–149, is enriched with acidic residues. This

region may mediate α-Syn's interactions with other proteins (Fernandez et al., 2004;

Murray et al., 2003), metal ions (Brown, 2007) and other ligands like dopamine and

the polyamines (Hoyer et al, 2004). The C-terminal region contains sites for various

post-translational modifications and plays a critical role in modulating the stability,

structure, aggregation and function of the protein in vivo (Choi et al., 2011).

4.3 Post-translational modifications Several post-translational covalent modifications have been described for α-Syn,

including phosphorylation, ubiquitination, nitration, sumoylation, enzymatic cross-

linking and C-terminal truncation, some of which correlate with PD. For example,

phosphorylation at serine 129 (S129-P) is a defining hallmark of PD and

synucleinopathies (Anderson et al., 2006; Fujiwara et al., 2002), while

phosphorylation at tyrosine residues Y125, Y133 and Y135 suppresses S129-P-

induced aggregation and toxicity (Chen et al., 2009). At least five types of -Syn

C-terminal truncations were detected in the normal brain and formed aggregates with

the full-length α-Syn in PD and related disorders. It was found that certain C-terminal

truncated forms that accumulate selectively in LBs or insoluble fractions, aggregate

more readily and could act as effective seeds to accelerate the aggregation of the full-

length protein (Li et al., 2005; Liu et al., 2005).

4.5 Oligomerization and aggregation

The finding that -Syn appears in aggregated forms within LBs and LNs (Baba et al.,

1998; Spillantini et al., 1998; Spillantini et al., 1997), led to extensive investigation

focusing on the misfolding, oligomerization and aggregation of α-Syn. Mutant forms

of α-Syn, specifically A53T, have higher aggregation rates than the WT form

(Conway et al, 1998; El-Agnaf et al., 1998a; El-Agnaf et al, 1998b). Furthermore, the

aggregation of the protein is accompanied by a conformational transition from

random coil or α-helical structure to cross β-pleated sheet (El-Agnaf et al., 1998a;

Serpell et al, 2000; Weinreb et al, 1996; Serpell et al., 2000), similar to the structure

described for other amyloidal fibrils (Han et al., 1995). It was shown in vitro that the

fibrillation rate is enhanced by higher protein concentration (Uversky et al., 2001a),

lower pH and higher temperature (Uversky et al., 2001b). In addition, the

oligomerization and fibril formation of α-Syn were shown to be affected by post-

translational modifications (as detailed above), oxidative/nitrative challenges,

interactions with specific metals (Brown, 2007; Paik et al., 1999), fatty acid or

phospholipid vesicles (Jo et al., 2004; Perrin et al., 2001; Sharon et al., 2003b),

inhibitors of mitochondrial Complex I (Manning-Bog et al., 2002; Sherer et al., 2003)

and proteasome inhibitors (Uversky, 2007). Previous studies in our laboratory have

shown that α-Syn normally occurs in soluble oligomers (Sharon et al., 2001), and that

the enhanced levels of soluble oligomers and insoluble aggregates of α-Syn correlate

with disease pathology (Assayag et al., 2007; Sharon et al., 2003a). We have further

shown that soluble -Syn oligomers precede the formation of aggregated -Syn

(Assayag et al., 2007). Moreover, studies in our laboratory have indicated that the

degree of soluble oligomers and insoluble aggregates is affected by the cellular fatty

acid content. Specifically, saturated fatty acids (SFAs) inhibit, and polyunsaturated

fatty acids (PUFAs) enhance, the formation of α-Syn soluble oligomers, insoluble

aggregates, and α-Syn cytotoxicity, culminating in the formation of Lewy-like

cytoplasmic inclusions (Assayag et al., 2007; Sharon et al., 2003a)

The general concept identifying -Syn misfolding and aggregation as a neurotoxic

event leading to neurotoxicity in PD, was challenged by a recent finding indicating

that -Syn normally occurs in a tetrameric form (Bartels et al., 2011). However, this

conclusion was criticized by several groups of investigators who presented evidence

that α-Syn existed predominantly as a disordered monomer (Fauvet et al., 2012).

Therefore, it is important to develop the means to distinguish in vivo between the

physiologically-active and pathogenic Syn forms.

4.6 Physiological function Although α-Syn plays a crucial role in the pathogenesis of several neurodegenerative

disorders, its precise function remains undefined, though a number of suggestions

have been proposed. -Syn has structural homology with the fatty acid-binding

protein (FABP) family (Sharon et al., 2001).

Recent studies have suggested that -Syn has a role in membrane trafficking. It has

been reported that -Syn expression specifically inhibits ER-to-Golgi trafficking,

resulting in cytotoxicity, which can be prevented with Rab1 expression (Cooper et al.,

2006). It was further shown that -Syn expression affected vesicle docking or fusion

to the Golgi apparatus after an efficient budding from the ER (Gitler et al., 2008).

Other studies have suggested an indirect role for -Syn in promoting the assembly of

the SNARE complex in vivo and in vitro (Burre et al., 2010; Chandra et al., 2004).

SNAREs catalyze the fusion of vesicles with their target membranes to enable the

release of cargo from the vesicle (reviewed in (Ungar et al., 2003; Sudhof et al.,

2011). A study performed in our lab demonstrated that -Syn activates synaptic

vesicle recycling through activation of clathrin-mediated endocytosis (Ben Gedalya et

al., 2009). Collectively, growing evidence implicates -Syn in membrane trafficking,

including endocytosis and exocytosis.

Genome-wide screening in yeast showed that nearly one-third of the genes that

enhance the toxicity of α-Syn are functionally related to lipid metabolism and vesicle

trafficking (Willingham et al., 2003). Expression profiling of transgenic flies revealed

that the expression of lipid and membrane transport genes were associated with α-Syn

expression (Scherzer et al., 2003). Over-expression of α-Syn in a neuronal cell line, as

well as homozygous deletions of α-Syn in mice, were both accompanied by noticeable

changes in membrane fluidity and in cellular fatty acid uptake and metabolism

(Sharon et al., 2003b; Castagnet et al., 2005; Golovko et al., 2005).

Despite its toxicity in PD and models thereof, α-Syn silencing by antisense

oligonucleotides in primary cultures of cerebellar granule cells causes the widespread

death of neurons, which suggests that it plays a critical role in neuronal survival

(Monti et al., 2007).

5. Catalase Catalase is an antioxidant enzyme that catalyzes the dismutation of H2O2 to oxygen

and water (Aebi, 1984). In most mammalian tissues, catalase is present predominantly

within the peroxisome.

Catalase transcription was recently shown to be regulated by peroxisome proliferator-

activated receptor gamma(PPAR), a member of the nuclear receptors family of

transcription factors putative functional PPAR response element (PPRE) was

identified at the promoter region of the rat catalase gene (Girnun et al, 2002).

Activation of PPAR by a specific agonist was shown to further enhance catalase

activity and protect neurons from oxidative stress (Gray et al, 2012).

6. Nuclear receptors Nuclear receptors (NRs) play crucial biological functions in development,

inflammation, reproduction and metabolism, including the regulation of lipid

homeostasis (Kliewer et al, 1999; Mangelsdorf et al., 1995). The human genome

encodes for 48 members of this transcription factor family. NRs are characterized by

two important properties: first, they are activated upon the binding of specific ligands,

generally lipophilic molecules, and second, they bind to specific DNA response

elements, mainly located within the promoters of their target genes.

Most of the NRs function as dimers, either homodimers, such as the glucocorticoid

receptor (GR) or the estrogen receptor (ER), or more often, as heterodimers, primarily

with Retinoid X Receptor (RXR). RXR provides essential structural and signaling

support to its partner receptors during transcriptional activation (Aranda et al., 2001).

6.1 Peroxisome proliferator-activated receptors (PPARs)

The PPARs act principally as "lipid sensors." PPARs control a variety of genes in

several pathways of lipid metabolism, including fatty acid transport, uptake by cells,

intracellular binding and activation, as well as catabolism or storage. Among PPARs

target genes are peroxisomal and nuclear-encoded mitochondrial genes (Desvergne et

al., 1999; Heneka et al., 2007; Willson et al., 2001).

Three different PPAR isotypes have been identified in various species and are

structurally homologous: PPARα, PPARβ/δ and PPARγ. Two PPARγ isoforms,

PPARγ1 and PPARγ2, are splice variants of their N-terminal domain.

Until now, different functions and different tissue expressions have been identified for

different PPAR members. The identification of PUFAs as PPAR ligands provides

firm evidence that at least part of the PPAR-dependent transcriptional activity of fatty

acids, results from a direct interaction of the nuclear receptor with these molecules.

Interestingly, compared to the PUFAs, SFAs are, in general, poor PPAR ligands

(Desvergne et al., 1999). PPAR binding to Peroxisome Proliferator Responsive

Element (PPRE) strictly depends on RXR, thus forming the PPAR/RXR heterodimer.

6.2 RXR

RXRs are expressed in virtually every tissue of the body and contain three subtypes:

α, β and . RXR is activated in vitro by the Vitamin A metabolite, 9-cis Retinoic

Acid (9-cis RA), which binds exclusively and with high affinity to the RXR ligand

binding domain. 9-cis RA isomerizes from All-trans Retinoic Acid (AT-RA), an

activator of both RAR and RXR (Heyman et al., 1992; Levin et al., 1992). However,

9-cis RA has been difficult to detect in vivo and therefore it is currently unclear what

the in vivo activating ligand is for RXR. Importantly, docosohexaenoic acid (DHA), a

22 carbon-6 double bond, PUFA, was identified as the in vivo activating ligand for

RXR, modulating its transcription activity (de Urquiza et al., 2000). Furthermore, it

was shown that other naturally-occurring PUFAs can activate RXR with similar

efficiency as DHA (Lengqvist et al., 2004). DHA and some of its derivatives were

also found to activate PPARα and PPARγ (Gani et al., 2008).

The finding that RXR binds a series of PUFAs reinforces the potential involvement of

this receptor in the regulation of lipid homeostasis through a complex feedback

mechanism in potent association with PPARs, and its role as an intracellular sensor of

the cell’s metabolic status.

7. Working Hypothesis

1. The pathogenic mechanisms in MSA involve the uptake of neuronal-secreted

-Syn by oligodendrocytes. The two events, e.g., the secretion of -Syn from neurons

and the uptake of -Syn by oligodendrocytes, are affected by cellular mechanisms

controlling -Syn expression, oligomerization, aggregation and pathology. Based on

this working hypothesis, we hypothesized that the effect of polyunsaturated fatty

acids, to enhance neuronal -Syn oligomerization aggregation and pathology, will

result in -Syn localization in oligodendrocytes and thus, MSA.

2. -Syn is normally involved with the cellular pool of fatty acid metabolites,

including, the activating fatty-ligands for nuclear receptor activation. Consequently,

the activity of retinoic x receptor (RXR) and peroxisome proliferating activated

receptors (PPARs) is affected by -Syn. This effect of -Syn on PPAR activation

leads to altered transcription profiles of target genes, which are regulated by the

transcription factors. One such target gene is catalase. We therefore hypothesize that

-Syn regulates catalase expression through its effect on the availability of fatty acid

metabolites, which act as activating ligands for PPAR.

8. Aims and Objectives

1. To elucidate the mechanisms underlying the pathogenic accumulation of -

Syn, a neuronal protein in oligodendroglia, as observed in Multiple System

Atrophy.

2. To elucidate the mechanisms by which -Syn expression affects catalase, the

anti-oxidative

Materials and Methods:

Animals and Diets. The A53T -Syn tg mouse line (Giasson et al., 2002) was

purchased from Jackson laboratories (Bar Harbor, Maine, USA) and bred to

homozygosity. Similar to the original description, the mice remained healthy up to the

age of about7 months. At 8-9 months of age, the colony began to develop motor and

behavioral phenotype as described previously (Yakunin et al., 2011). In parallel, we

maintained a non-transgenic (wt) mouse (C57Bl/6) colony (Jackson Laboratories, Bar

Harbor, Maine, USA) and an -Syn -/- (knock-out) (Specht et al., 2001) colony. This

study was carried out in strict accordance with the recommendations outlined in the

“Guide for the Care and Use of Laboratory Animals” of the National Institute of

Health. The protocol was approved by the Authority of Biological and Biomedical

Models of the Hebrew University of Jerusalem, NIH approval # OPRR-A01-5011

(Permit Number: MD-09-12084).

Males and females (n=10-15) of A53T -Syn were randomly assigned into three

groups and were fed for 230 days either one of three diets: a standard mouse chow

(2018SC+F, Harlan Teklad, Madison, WI); a safflower-based diet depleted of DHA-

the "low-DHA diet” (TD 00522, Harlan Teklad, Madison, WI); or the “low-DHA

diet” (and low n-3 PUFA diet) supplemented with 0.69% w/w DHA- the “high diet”

(Martek Bioscience, Columbia, MD) (TD 07708) (Calon et al., 2005; Lim et al.,

2005). The percentages of total fat (6%), proteins (18%), and carbohydrate (49%)

were identical in all three diets. At termination of the experiment, mice were

anesthetized with an intraperitoneal overdose injection of sodium pentobarbitone

(1ml/1.5 kg), and were then perfused with PBS buffered formalin. Brains were

removed and fixed for another 24 hours in formalin.

Cell Cultures. The mesencephalic neuronal cell line with dopaminergic properties,

MN9D (Choi et al., 1991), and HeLa cells, were transfected with either WT α-Syn,

A53T human α-Syn, or -Syn cDNA in the pcDNA 3.1 vector. Since α-Syn

expression and oligomerization are dynamic in these clones (Assayag et al., 2007),

comparisons were made between WT and A53T clones that were maintained in

parallel from the point of DNA transfection and selection of stable clones to the actual

measurements (Assayag et al., 2007). Oli-neu (Jung et al., 1995) cell lines, derived

from oligodendrocytes, were cultured according to their original description.

α-Syn Uptake by Cultured Cells. Oli-Neu Cells were plated on cover slips

pretreated with PLL (1 mg/ml). Following incubation for 24 hours in standard growth

medium, cells were incubated for an additional 16 hours in medium supplemented

with purified human recombinant α-Syn protein. Alternatively, Oli-neu cells were

incubated in conditioned medium (CM) collected from MN9D cell (naïve or over-

expressing -Syn) for 16 hours. The CM was spun down to remove cell debris and

supplemented with apotransferrin (10 g/ml), bovine insulin (10 g/ml), and sodium-

selenite (220 nM), before adding it onto the oligodendroglial cell lines. Following the

incubation, cells were processed with Western blotting or immunocytochemistry

(ICC). The intracellular -Syn signal (obtained by choosing the image plane beneath

the cell surface) was quantified using pro/image j software (Media Cybernetics Inc.),

and represents fluorescence intensity across the entire cell area.

Immunocytochemistry (ICC). Cells were washed twice and fixed in

paraformaldehyde (PFA) (4%). The detection of -Syn within Oli-neu cells was

performed using a human specific monoclonal anti- -Syn antibody, LB509 (1:200

Invitrogen, Carlsbad, CA, USA) or 7071 (provided by Peter Lansbury, Harvard

Medical School, Boston, MA, USA). The secondary antibody was either anti- mouse

cy5 (Jackson laboratories, ME, USA) or anti- rabbit cy5 (Jackson laboratories, ME,

USA). Slides were sealed with mounting medium (cat# M1289 Sigma, Rehovot,

Israel) and analyzed using confocal microscopy (see below).

Western blotting. Detection of secreted -Syn or -Syn in CM. Naïve, -Syn, or -

Syn over-expressing MN9D cells were incubated to confluency in 100 mm dishes in

standard growth medium. The medium was then replaced with fresh medium or

serum-free medium supplemented with BSA and FA (at 50 and 250 M, respectively)

and cells were incubated for an additional 16 hours. The CM was collected and spun

at 10,000 xg for 10 minutes. Aliquots were kept in a 4º C refrigerator and analyzed

within 2-3 days.

Detection of Soluble -Syn in MN9D cells. WT or A53T mutant -Syn over-

expressing cells were cultured as indicated and were then harvested and fractionated

to collect the high speed cytosolic fraction (post- 280,000 Xg) as previously described

(Sharon et al., 2001). -Syn oligomer detection was performed following incubation

of protein samples at 65°C for 16–18 hours for optimal antigen retrieval (Sharon et

al., 2003a).

Samples of collected CM (equal volume) or the high-speed supernatant of MN9D

cells (equal protein amounts) were loaded into a 10% SDS-PAGE gel, and following

electrophoresis were transferred to a PVDF membrane (Biorad, Petach Tikva, Israel).

The membrane was fixed with 0.4% PFA and blotted with either one of the following

anti- α-Syn antibodies: H3C (obtained from Julia George, University of Illinois,

Urbana-Champaign, IL), LB509 (Invitrogen, Carlsbad, CA, USA). The blots were

reacted with the secondary antibody, HRP-conjugated, and visualized with the EZ-

ECL system (Biological Industries, Beit Haemek, Israel), scanned with a Umax Magic

Scan (Eastman Kodak, Rochester, NY, USA), and then analyzed for the density of the

α-Syn signal using UN-SCAN-IT GEL 3.1 software (Silk Scientific, Orem, UT,

USA). The signal obtained for -Syn in a specific sample of CM was normalized to

the total amount of protein in the particular cultured dish.

For the detection of oligodendroglial genes and PPAR in whole mouse brains: One

hemisphere of a young (4–6 week old) mouse brain was homogenized in RIPA-buffer

(Tris-HCl pH 7.6, 25 mM; NaCl, 150 mM; NP-40 1%; Sodium deoxycholate 1%; and

SDS 0.1%). The protein concentration was determined by the Bradford method

(Bradford, 1976). Protein samples (of 10 μg) were separated by 10% SDS-PAGE gel

electrophoresis and transferred to a PVDF membrane. Membranes were blocked in

5% non-fat milk in TBST. Thereafter, the membranes were immunoblotted with the

following primary antibodies: CNPase (1:1000, Sigma, Rehovot, Israel); PLP

(1:1000, Abcam, Tel-Aviv, Israel); CAII (1:5000, kind gift from Said Ghandour,

Louis Pasteur University, France); TPPP/P25 (1:500, kind gift from Paul Henning

Jensen, Aarhus University, Denmark); anti- PPAR-γ (1:100, Santa Cruz, sc-7273);

and anti- β-actin (1:5000, Sigma A3853).

Bands were further visualized using an appropriate HRP-conjugated secondary

antibody (Jackson ImmunoResearch Laboratories) and the EZ-ECL detection kit

(Biological Industries, Bet Haemek, Israel). The blots were scanned in an Umax

Magic Scan (Eastman Kodak, Rochester, NY, USA) and analyzed for the density of

the α-Syn signal using UN-SCAN-IT GEL 3.1 software (Silk Scientific, Orem, UT,

USA).

RT-PCR. Total RNA was isolated from one hemisphere each of WT and A53T α-Syn

mouse brains, and also from naïve MN9D, Oli-neu, N2A and HeLa cells using TRI

Reagent (MRC, Cincinnati, OH, USA). The generation of cDNA was performed

using High Capacity cDNA, Reverse Transcription Kit (Applied Biosystems, Foster

City, CA, USA). Primer pairs were designed to exon-exon boundaries by Primer3

(v.0.4.0 software).

For the detection of endogenous mouse α-Syn: 5' primer sequence

5'-GTCTCAAAGCCTGTGCATCT-3' and 3' primer sequence

5'- TCCACACTTTCCGACTTCTG-3';

For NG-2 mRNA detection: 5' primer sequence

5'-ACAGCTCCTGCCTCCTTCT- 3' and 3' primer sequence

5'- GCTGGGATGTGGAGAACTG -3';

For CNPase mRNA detection: 5' primer sequence

5'- GCCCCGGAGACATAGTACC-3' and 3' primer sequence:

5'-GCGGGTAAAGCTTGTGTTATG-3';

For MBP mRNA detection: 5' primer sequence

5'-CCAGCACCACTCTTGAACAC-3' and 3' primer sequence

5'-CTCCATCCTTACTGGCCTTCT-3';

For PLP mRNA detection: 5' primer sequence

5'-AGAACAGTGCCACTCCAAAGA-3' and 3' primer sequence

5'-CAAAGACATGGGCTTGTTAG-3';

For TPPP/P25 mRNA detection: 5' primer sequence

5'-CGA GGA GGC TGT ACG TGA G-3' and 3' primer sequence

5'-GAG GAC ACA GCT TTC GTG AC-3'

For CAII mRNA detection: 5' primer sequence

5'-CCTCTGCTGGAATGTGTGAC-3' and 3' primer sequence

5'-CAGCGTACGGAAATGAGACA-3'

For human CAT mRNA detection: 5' primer sequence

5'- GGG AGA AGG CAA ATC TGT GA-3' and 3' primer sequence

5'-CAG TGA TGA GCG GGT TAC AC-3'

For human PPAR mRNA detection: 5' primer sequence

5'- GCA GGA GAT CTA CAG GGA CAT-3' and 3' primer sequence

5'-TTG TAG TGC TGT CAG CTT CAGA-3'

For human PPARmRNA detection: 5' primer sequence

5'-GAT GGG AAC CAC CCT GTA GA-3' and 3' primer sequence

5'-CTG CTC CAT GGC TGA TCT C -3'

For human PPARmRNA detection: 5' primer sequence

5'- TGA TCA AGA AGA CGG AGA CAG A-3' and 3' primer sequence

5'- GCA GTG GCTCAG GACTCTCT-3'

For human RXRmRNA detection: 5' primer sequence

5'- GCC GGG CAT GAG TTA GTC-3' and 3' primer sequence

5'- GTT CAC CTG GGT GGA GAA AT-3'

Results were normalized against the expression levels of mouse 18S or human G6PD.

Analyses were performed using Applied Biosystems Prism 7300 Sequence Detection

system with SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA).

Clathrin Silencing. Two different shRNAs with a reported effect on clathrin heavy

chain expression were used (Ben Gedalya et al., 2009). Cells were transfected with

either one of the two clathrin shRNAs or a scrambled RNA sequence. 72 hours post-

DNA transfection, 15 g/ml of purified human α-Syn protein was added to the growth

medium of the cells and incubation lasted for an additional 16–18 hours. Cells were

then processed for ICC using anti- clathrin monoclonal antibody (1:200, BD

Biosciences) and anti- α-Syn polyclonal antibody 7071 (provided by Peter Lansbury,

Harvard Medical School, Boston, MA, USA), followed by secondary antibodies: anti-

mouse Alexa 488 (1:300, Invitrogene, USA) and anti- rabbit Cy5 (1:300, Jackson

laboratories, ME, USA)

Immunohistochemistry (IHC). Brain sections (of 6 μm) were deparaffinized in

xylene followed by graded alcohol in descending ethanol concentrations. For -Syn

staining, antigen retrieval was performed by incubating the slides in 100% formic acid

for 5 minutes, followed by extensive washes. Sections were blocked in 10% goat

serum in 0.1 M Tris-HCl pH 7.6. Slides were then reacted with anti -Syn antibody,

Syn303 (1:3000, obtained from Virginia M.-Y. Lee, see above), CAII (1:500,

provided by Said Ghandour, see above), TPPP/P25 (1:100, provided by Paul Henning

Jensen, see above), NeuN (1:200, clone A60, Millipore, MA, USA), GFAP (1:200

DAKO, Glostrup, Denmark), or synaptophysin (1:200, DAKO, Glostrup, Denmark).

Slides were then washed and reacted with a second antibody, anti- mouse Cy2 (1:300,

Jackson laboratories, ME, USA) and anti- rabbit Cy5 (1:300, Jackson laboratories,

ME, USA). The stained sections were observed using confocal microscopy (see

below). Quantifications were blinded for treatments. To reduce experimental error,

quantifications were performed in sets slides, representing each of the number of

groups, and comparisons were made only between slides that were stained and

handled in parallel. To sum up the different comparisons and compare between the

different staining events, we present the data as percent of the control standard diet or

the WT mice, set at 100% for each staining. The image series analyzed with Image

Pro Plus 6.3 program (Media Cybernetics, Bethesda, MD, USA).

Quantifying cell populations. The selection of objects was done automatically by

color definition. For object specification, we used the smoothing options. The selected

objects were counted based on the following estimations: above 200 pixels-

oligodendrocytes, 1000 pixels- neurons, and 400 pixels- astrocytes.

Confocal microscopy. Conducted using Zeiss LSM 710 Axio Observer.Z1 laser

scanning. The system is equipped with an argon laser 488 excitation with a 494-

630 nm pass barrier filter, Diode 405-30 laser excitation with 410-483 nm pass barrier

filter and HeNe 633 lasers, excitation with 638-759nm pass barrier filter.

Fluorescence was collected simultaneously with differential interference contrast

(DIC) images using a transmitted light detector. The fluorescence was collected by

employing a "Plan-Apochromat" 63x/1.40 Oil DIC M27 or "Plan-Neofluar" 40x/1.30

Oil DIC M27 (Zeiss). In each experiment, exciting laser, intensity, background

levels, photo multiplier tube (PMT) gain, contrast and electronic zoom size were

maintained at the same level. For each antibody, the background was subtracted

(determined by a negative control consisting of a secondary antibody alone). The

zoom of each picture was obtained by choosing the plane with the greatest fluorescent

signal.

Catalase activity assay. HeLa cells were scraped, washed three times in PBS and

stored at -80°C until needed. Cells were then resuspended in a buffer which consisted

of 50mM MOPS, pH 7.4, 250mM sucrose, 1mM EDTA, 0.1% ethanol (v/v), protease

inhibitors, and homogenized by hand using a homogenizer followed by seven

passages through a 27-gauge needle. Extracts were then centrifuged at 180,000g for 1

hour and supernatants were aliquoted and stored at -80°C. Catalase activity was

measured using Amplex Red Catalase Assay kit (Molecular Probes). This assay is

highly sensitive and specific, allowing detection of low levels of H2O2 in various

biological samples. After incubating the samples with 40μM H2O2 for 30 minutes, the

remaining H2O2 was measured to determine the catalase activity against a standard

curve ranging from 62.5 to 1000mU/mL. In the presence of horseradish peroxidase,

Amplex Red reacts stoichiometrically with H2O2 to generate the red fluorescent

oxidation product, resorufin. Resorufin has absorption and fluorescence emission

maxima of approximately 571 nm and 585 nm, respectively. Because the absorbance

is strong, the assay can be performed either fluorometrically or

spectrophotometrically. Catalase activity was determined by the measurement of

absorbance at 562 nm using a 96-well microplate reader, EL808 (Lumitron).

PPARreporter system: PPARγ transcription activity was determined by a GFP-

reporter plasmid assays, pGreenFire1-PPRE, (TR101PA-1, System Biosciences,

CA,USA). Naïve and -Syn over-expressing HeLa cells were transfected with the

reporter plasmid (3g/ 60 mm dish) using a ICAFectin™441 transfection reagent.

Following 48 hours after transfection, cells were either treated with troglitazone , or

left untreated, for 16 hours and the fluorescence intensity of GFP was measured for

10,000 cells using a FACScan System (BD Biosciences, NJ, USA). The average

intensity of the cell population was recorded for each treatment.

Protein carbonyl content. One hemisphere of a young (4–6 week old) mouse brain

was homogenized in RIPA-buffer (Tris-HCl pH 7.6, 25 mM; NaCl 150 mM; NP-40

1%; Sodium deoxycholate 1%; and SDS 0.1%). The protein concentration was

determined by the Bradford method (Bradford, 1976). For 1 mg of protein, 800 l of

12.5 mM 2,4 dinitrophenylhydrazine (DNPH) were added, incubated for 1 hour at

room temperature and mixed using a vortex every 15 minutes. The samples were

precipitated with 20% trichloroacetic acid (TCA, final concentration) and

subsequently washed with ethanol/Ethyl acetate1:1. The final pellets were dissolved

in 500 l of 6M Guadenidine HCl and incubated for 15 minutes at 37oC. Insoluble

materials were removed by centrifugation. The reactive carbonyl content was

calculated using a spectrophotometer at 375 nm absorbance. Protein content was

determined at 280 nm absorbance. The reactive carbonyl content was expressed as

nmol carbonyl/mg protein by using a molar absorption coefficient of 22,000M-1

cm-1

.

Statistical analysis. Correlation coefficients were calculated using Microsoft Excel.

P-values < 0.01 were considered significant. To compare between the three diets

groups, the non-parametric Kruskal-Wallis ANOVA test was applied with Bonfferoni

correction for multiple comparisons. Pair-wise comparisons between the low-DHA

and high-DHA groups for each mouse genotype were carried out using the non-

parametric Mann-Whitney test.

Results

Chapter I

Increased neuronal -Synuclein pathology is associated with its

accumulation in oligodendrocytes in mice modeling -

Synucleinopathies.

To elucidate the cellular mechanisms involved in the pathogenic expression of -Syn

in oligodendrocytes, I tested the hypothesis that oligodendrocytes can take-up

neuronal-secreted -Syn as part of the pathogenic mechanisms leading to MSA.

In the first part, I investigated, in vitro, both the secretion of α-Syn from a neuronal

cell line as well as the uptake of α-Syn by an oligodendroglial cell line. In the next

part, I tested if A53T α-Syn tg mice could be models for MSA, specifically, if

oligodendrocytes have an effect in A53T α-Syn tg mice. Finally, I tested if enhanced

neuronal α-Syn pathology has an effect on α-Syn’s presence and accumulation in

oligodendrocytes.

Syn Secretion from Stably-Transfected Neuronal Cells

To investigate the secretion of α-Syn from neuronal cells, in particular, MN9D

neuronal cells that stably express human wt -Syn, a Western blot analysis was

performed for secreted -Syn in the conditioned medium (CM). There was a high and

clear signal for the Syn in the CM of wt -Syn MN9D cells. No signal was

detected in the CM of naïve cells, (Fig. 1A). In order to determine whether α-Syn

secretion from cells that over-express it is part of a specific mechanism or a result of

cell death, I compared cell viability and secretion between different clones of α-Syn

or -Syn over-expressing MN9D cells. The expression levels of the transfected

proteins were determined by Western blot analysis, comparing two to three different

clones with similar expression levels. The analysis resulted in no evidence for -Syn

secretion from over-expressing MN9D cells. Particularly, while the occurrence of α-

Syn was clearly detected in the CM (Fig. 1A), no signal for -Syn was detected in the

CM (Fig. 1B). The results were obtained with a specific anti- -Syn antibody (BD-

Transduction Laboratories, NJ, USA) and verified with a H3C antibody, which

recognizes both Synuclein homologs (not shown). It should be noted that I did not

detect significant differences in cell viability between the α-Syn and -Syn over-

expressing MN9D cells. I therefore concluded that the cells secrete α-Syn protein as

part of a specific mechanism.

Figure 1.Syn, but not its homolog Syn, is secreted from cultured neuronal cells.

A. Western Blot analysis of conditioned medium (CM) collected from naïve and wt -Syn over-

expressing MN9D cells, conditioned in standard serum-supplemented medium. A LB509 antibody was

used in the analysis. B. Western Blot analysis of naïve and two different -Syn over-expressing MN9D

clones, conditioned in standard serum-supplemented medium. An anti- -Syn antibody was used in the

analysis (BD-Transduction Laboratories). Arrows indicate non-specific bands.

1.1 Intracellular expression and soluble oligomer levels affect -Syn secretion from stably-transfected neuronal cells.

MN9D neuronal cells, stably-expressing human wt or A53T -Syn (eight and four

different clones respectively), were maintained in parallel from the point of stable

transfection to the final analyses. Each clone was analyzed by Western blot for

soluble α-Syn in the post-high-speed cytosolic fraction, and in parallel, for secreted -

Syn in the conditioned medium (CM). The signal obtained in the Western blot for

secreted -Syn was quantified, normalized to the amount of cells in the particular dish

(represented by protein concentration), and plotted against the signal obtained for total

soluble -Syn signal (the sum of monomers and oligomers), or to the degree of

soluble α-Syn oligomers (the ratio of oligomers to monomers) and to actin levels. As

expected, a higher degree of soluble -Syn oligomers was detected for the A53T than

A B

for the wt -Syn expressing clones (Fig. 2A). Plotting either total soluble -Syn or

degree of soluble -Syn oligomers against secreted -Syn resulted in a positive

correlation, with a correlation coefficient r=0.83, p<0.01 and r=0.81, p<0.01 for total

-Syn and degree of -Syn oligomers, respectively (Fig. 2B and C). Significantly,

such positive correlations were also calculated for the eight different wt α-Syn clones,

including high and low wt α-Syn expression levels (r=0.77, p<0.01 and r=0.78,

p<0.01 for total -Syn and degree of -Syn oligomers, respectively). I concluded that

both -Syn expression levels and oligomer levels within the soluble fraction, affect its

secretion from cultured neuronal cells. That is, higher α-Syn expression and a higher

degree of intracellular oligomerization enhance α-Syn’s secretion.

Fig. 2 -Syn secretion from MN9D neuronal cells positively correlates with its expression level

and the level of intracellular soluble oligomers.

A. Samples of the soluble fraction from different clones of MN9D cells stably expressing human wt or

A53T α-Syn (15 g protein) treated for oligomer detection at 65C, and the corresponding samples of

collected conditioned medium (CM, 100 l) were analyzed by Western blot analysis using the anti-

human α-Syn antibody, LB509. Equal protein loading was determined with an actin antibody in

samples of the soluble fraction. A representative blot. B. Graph representing the correlation of total

soluble α-Syn (represented by the entire immuno-reactivity throughout the lane) plotted against

secreted α-Syn. C. The correlation of the degree of soluble α-Syn oligomers (represented by the ratio of

α-Syn oligomers to monomers) plotted against secreted α-Syn.

As an alternative approach to alter the degree of -Syn oligomerization, I utilized the

enhancing effects of PUFAs on intracellular -Syn oligomerization (Assayag et al.,

2007; Sharon et al., 2003a; Sharon et al., 2003b; Sharon et al., 2001; Yakunin et al.,

2011). MN9D cells stably expressing wt α-Syn were cultured in a serum-free

medium, supplemented with BSA only (50 M) or with BSA (50M) bound to DHA

(250 M). 16 hours later, the CM and high-speed cytosolic fractions of the respective

A

C

B

cultures were collected and analyzed for secreted α-Syn and degree of -Syn

oligomers, as above. Consistent with our previous observations, α-Syn

oligomerization was not affected by 18:1 MUFA, however alpha linolenic acid (ALA,

18:3) and DHA (22:6) PUFAs enhanced -Syn oligomerization by approximately 3

and 6 fold, respectively (Fig. 3). In accord with PUFA-enhanced -Syn

oligomerization, we detected higher levels of secreted α-Syn in the CM of PUFA-

treated cells (Fig. 3). Specifically, α-Syn secretion from stably-transfected MN9D

cells, treated with 18:3 or 22:6 PUFAs, was enhanced between 2 to 5 fold. Although

there were high levels of variability between the different experiments, these

differences in secretion rates were significant (ANOVA p=0.023).

The enhancing effect of PUFA on -Syn release could potentially result from two

different effects, namely, enhanced -Syn oligomerization and pathology

oligomerization (Assayag et al., 2007; Sharon et al., 2003a; Sharon et al., 2003b;

Sharon et al., 2001; Yakunin et al., 2011) or enhanced membrane trafficking (Ben

Gedalya et al., 2009), including exocytosis of -Syn. To distinguish between the two

potential mechanisms, I utilized a pharmacological compound, HX531, which acts as

a specific antagonist for retinoic X receptor (RXR) (Suzuki et al., 2009). Recently, we

have successfully shown that HX531 eliminates the effect of DHA on -Syn

oligomerization through its effect on RXR activation (Yakunin et al, 2011.). Yet,

HX531 antagonist was not expected to affect membrane enrichment with PUFAs.

Therefore, HX531 was used here to differentiate between DHA’s effect on -Syn

oligomers and its effect on membrane trafficking.

HX531 (at both 1 and 2.5 M) was added to α-Syn over-expressing MN9D cells

cultured in medium which was supplemented with either BSA alone or BSA with

DHA (as above) for 16 hours. In accord with our previous observation, HX531 acting

as an antagonist inhibited the enhancing affect of DHA on intracellular -Syn

oligomerization, resulting in oligomer levels similar to those found in BSA-treated

control cells (Fig. 3a). Also in accord, lower levels of secreted -Syn were detected in

sister cultures treated with HX531. Yet while oligomer levels were reduced in the

control BSA- (or serum-) treated cells, the levels of secreted -Syn were still higher

than in the control cells. This result indicated to us that PUFA-induced -Syn

secretion is promoted by both mechanisms, e.g. enhanced intracellular -Syn

oligomerization and membrane trafficking.

Fig. 3 -Syn secretion from MN9D dopaminergic cells is associated with PUFA-enhanced -Syn

oligomerization

A. Western blot analysis (as in Fig. 2A) of wt α-Syn over-expressing MN9D cells. Sister cultures were

treated in parallel either with serum-free DMEM supplemented with BSA only (at 50 M) or together

with the indicated fatty acids (at 250 M), and the indicated concentrations of HX531, a specific

antagonist for retinoic acid receptor (RXR) for 16 hours. Control cells were conditioned in standard

conditioning medium. Bar graph represents quantification of signal obtained by Western blot analysis,

mean ± SD of 2-4 repeats. 18:1- oleic acid; 18:3 -linolenic acid; 22:6, docosahexaenoic acid.

B. Graph representing quantification of the signal obtained by Western blot. Blots were quantified

using UnScan It Gel 6.1. The results are presented as a percent of the 100% signal detected for BSA

treatment, mean ± SD of *, p<0.001 ANOVA.

2. Oligodendroglial cell lines take-up α-Syn from their growth medium.

To investigate whether oligodendroglial cells take-up -Syn from their growth

medium, a cell line with oligodendroglial properties was used, known as Oli-neu cells

(Jung et al., 1995). I first determined endogenous -Syn expression by RT-PCR and

A

B

ICC. A specific signal for -Syn mRNA was detected by RT-PCR in Oli-neu cells.

The α-Syn mRNA level detected in Oli-neu cells is similar to the level detected in

MN9D dopaminergic cells, while whole wt mouse brain has an approximately ten-

fold higher expression level (Fig. 4A). No signal for α-Syn mRNA was detected in

brain tissue of -Syn -/- mice (Specht et al., 2001). In accord with the presence of -

Syn mRNA, a low and antibody-specific signal for endogenous -Syn was obtained

in Oli-neu cells by ICC using the polyclonal antibody, 7071 antibody (Fig. 4B).

Similar results were obtained with the H3C antibody (not shown).

Fig. 4 -Syn is normally expressed in Oli-neu cells

A. Real-time PCR (RT-PCR) using specific primers designed to detect endogenous -Syn in naïve

MN9D and Oli-neu cells, non-transgenic (wt), and -Syn -/- whole mouse brain. B. ICC of cultured

naïve Oli-neu cells using anti- -Syn antibody, Syn-1. Scale bar=10 m

To determine whether Oli-neu cells take-up -Syn from their environment, I

incubated naïve Oli-neu cells in CM collected from naïve or α-Syn over-expressing

MN9D dopaminergic cells, or supplemented the growth medium of Oli-neu cells with

purified recombinant human -Syn.

A

B

The amounts of secreted α-Syn in CM collected from MN9D cells, naïve and those

over-expressing -Syn, were determined by Western blot analysis. Significantly,

although low -Syn mRNA levels were detected in naïve MN9D cells, I could not

detect secreted endogenous -Syn protein in the CM collected from these cells (Fig.

1A). Oli-neu cells were incubated for 16 hours in CM collected from naïve and -Syn

over-expressing MN9D cells, as specified. Cells were then processed for intracellular

-Syn detection by ICC, using the anti- human α-Syn antibody, LB509. Results were

verified with an additional anti- human -Syn antibody generated in our lab. The

presence of a specific immunoreactive signal for α-Syn, obtained with the anti- human

-Syn antibodies, was clearly demonstrated. The signal was present in the cytoplasm

and nuclei of Oli-neu cells cultured in CM collected from α-Syn over-expressing

MN9D cells. No signal for human -Syn was detected in Oli-neu cells cultured in

parallel, in CM collected from naïve MN9D cells (Fig. 5A).

Next I tested the uptake of exogenously-added human α-Syn by cultured Oli-neu

cells. The cells were cultured in standard growth medium supplemented with purified

recombinant human α-Syn (15µg/ml) for 16 hours, and then processed for the

detection of -Syn by ICC. A specific -Syn immunoreactive signal was detected in

Oli-neu cells cultured with the purified human -Syn, which was not seen in sister

cultures incubated in standard growth medium without -Syn (Fig. 5B).

Fig. 5. Oli-neu (oligodendroglial) cells take up -Syn from their environment.

A. ICC of Oli-neu cells incubated for 16 hours in CM collected from naïve or α-Syn over-expressing

MN9D cells using the LB509 ab. B. ICC as in (A), following 16 hours of incubation with or without 15

g/ml purified human α-Syn in the growth medium. Bar, 10 m

A B

2.1 α-Syn uptake by cultured oligodendrocytes is time and dose-dependent.

I investigated whether the uptake of purified human α-Syn by Oli-neu cells was

dependent on time and concentration. Uptake of purified human α-Syn by Oli-neu

cells was measured following 16 hours of incubation with 0-21 µg/ml purified -Syn,

or incubation with 15 µg/ml purified -Syn for 0-24 hours. Following these

incubations, cells were processed for ICC with the anti- human α-Syn antibody,

LB509. Quantifying the specific -Syn signal obtained within Oli-neu cells indicated

a time- and dose- dependent uptake of the exogenously-added -Syn protein (Fig. 6A

and B). Specifically, a linear graph representing -Syn uptake was obtained by

incubating the cells for 16 hours with 0.7-21 µg/ml (R2=0.945), indicating a dose-

dependent uptake. Similarly, a linear graph representing -Syn uptake was obtained

for Oli-neu cells incubated for 4-24 hours in the presence of 15 µg/ml -Syn

(R2=0.931), indicating a time-dependent uptake.

Fig. 6. Uptake of purified α-Syn by Oli-neu cells is both dose- and time-dependent.

A. Uptake of purified α-Syn by Oli-neu cells is dose–dependent. Cells were incubated with 0-21 g/ml

purified α-Syn in their growth medium for 16 hours and then processed for α-Syn detection by ICC

using anti- human α-Syn antibody, LB509 (Mean ±SD of n= 20 cells). B. Uptake of purified α-Syn by

Oli-neu cells is time-dependent. Oli-neu cells were incubated with 15 g/ml purified α-Syn in their

medium for 0-24 hours and then processed for intracellular α-Syn detection as in A.

A B

2.2 α-Syn uptake by cultured oligodendrocytes is clathrin-dependent.

To assess the involvement of clathrin in α-Syn uptake by oligodendroglial cells, I

transiently transfected naive Oli-neu cells with either one of two different shRNAs

designed to silence the expression of clathrin heavy chain or a third construct

containing a scramble sequence. These shRNAs were shown to effectively silence

clathrin expression (Ben Gedalya et al., 2009). 56 hours post-transfection, purified α-

Syn (15 g/ml) was added to the growth medium of the transfected cells and

incubation continued for an additional 16 hours. Cells were then processed for ICC to

detect α-Syn and clathrin using specific antibodies (Fig. 7). Quantifying the signal

obtained by ICC, I confirmed a significantly lower signal for clathrin in cells

transected with the clathrin shRNAs. That is, the measured signal for clathrin was

36.6±15.8% for shRNA1-transfected cells and 38.2±11.3% for shRNA2-transfected

cells of the signal obtained for control cells, transfected with the scramble sequence

and set at 100% (mean ± SD of n=20-30 cells). In accordance with the lower clathrin

expression levels, I measured a significantly lower α-Syn signal within Oli-neu cells,

representing lower uptake. Specifically, considering the α-Syn signal obtained in Oli-

neu cells transfected with the scrambled sequence as 100%, the signal measured for

-Syn was 17.5±6.3% and 37.7± 16.3% for shRNA 1 and shRNA 2, respectively

(mean ± SD of n=20-30 cells; P=0.0002 ANOVA) (Fig. 7A and B).

Fig. 7 -Syn uptake by Oli-neu cells is dependent on clathrin expression and enhanced by

exposure to PUFAs.

A. Naïve Oli-neu cells were transiently transfected either with a scramble sequence or two different

shRNAs designed to silence the expression of clathrin heavy chain. 56 hours post-DNA-transfection,

cells were cultured in standard growth medium supplemented with purified human α-Syn (at 15 µg/ml)

for an additional 16 hours and then processed for ICC with anti-clathrin and anti- α-Syn antibodies.

Bar, 10 m. B. Graph representing quantification of the signal obtained by ICC, mean ± SD of n=20-30

cells in each treatment. *, p<0.001

In previous studies, we have shown that membrane PUFA composition and clathrin-

mediated endocytosis (CME) are affected by exposure of cultured cells to

physiological concentrations of PUFAs (Ben Gedalya et al., 2009; Brand et al., 2010).

To find out whether PUFAs alter -Syn internalization into Oli-neu cells, I incubated

the cells in the presence of 15 g/ml exogenously-added, purified human -Syn for

16 hours in a serum-free medium supplemented either with BSA alone (at 50 M) or

together with FAs (at 250 M). Control cells were conditioned in standard serum-

B

A

supplemented medium. The signal detected within Oli-neu cells was quantified by

ICC with the anti- human -Syn antibody, LB509 (Fig. 8C). Setting the signal

obtained for control cells at 100%, I found that incubating Oli-neu cells in 18:3

PUFA-supplemented DMEM resulted in a higher degree of -Syn internalization.

Specifically, the measured -Syn signal in Oli-neu cells treated with 18:3 (227.9 ±

42.5%) was significantly higher than the signal measured for cells treated with BSA

alone (112.1 ± 15.9%) or with 18:1 monounsaturated fatty acid (MUFA) (69.0 ±

2.9%). This signal represents mean ± SD of n= 10-15 cells with P= 0.0034

(ANOVA). A stronger signal for -Syn uptake was determined in cells conditioned in

the presence of DHA (22:6, 316.6 ± 37.5%) (P= 0.0022, ANOVA n= 20-30 cells).

The signal measured for cells treated with BSA only or BSA-18:1 was not

significantly different than that measured for serum-treated cells (Fig. 8C). I therefore

concluded that membrane enrichment of Oli-neu cells with PUFAs enhances -Syn

uptake and internalization.

Fig. 8 -Syn uptake by Oli-neu cells enhanced by exposure to PUFAs.

Naïve Oli-neu cells (sister cultures) were incubated in the presence of purified α-Syn (at 15 g/ml)

added to standard growth medium (with serum), control; serum-free DMEM supplemented with BSA

only (at 50 µM); or BSA together with the indicated FA (at 250 µM) for 16 hours. Cells were then

washed and processed for ICC for the detection of intracellular α-Syn using the anti-human α-Syn

antibody, LB509. The signal within the cells was quantified. The signal obtained for control cells at

100%. Mean ±SD of n=10-15 cells in each treatment. *, P= 0.0034 (ANOVA).

3. Evidence for affected oligodendrocytes in the brains of A53T α-Syn

transgenic mice modeling neuronal synucleinopathies.

To test in vivo the hypothesis that neuronal-secreted -Syn underlies its pathogenic

accumulation in oligodendrocytes in MSA, I first determined whether the A53T α-

Syn tg mouse line (Giasson et al., 2002) depicts oligodendroglial pathology, and thus

if it may serve as a model for MSA. These mice express α-Syn under the PrP

promoter and originally were described as a model for neuronal synucleinopathies.

While the prion protein is mostly expressed in neurons, a portion of it is also

expressed in glial cells, including oligodendroglia (Moser et al., 1995).

To this aim, the expression levels of specific oligodendrocyte-encoded genes were

analyzed in A53T α-Syn tg mice in comparison to non transgenic (wt) mice (C57Bl).

Total RNA was extracted from one mouse brain hemisphere (n=4-5 brains), and the

mRNA levels of the following oligodendrocyte-specific genes were evaluated by RT-

PCR in relation to 18S RNA levels: myelin basic protein (MBP) and proteolipid

protein (PLP) as major structural components of the myelin sheath; 2′, 3′-Cyclic

Nucleotide 3′-Phosphodiesterase (CNPase), a protein enriched in myelin and

oligodendrocytes; carbonic anhydrase II (CAII) expressed in the cytosols of

oligodendroglia; tubulin polymerization promoting protein (TPPP/P25), an

oligodendroglia-specific protein with its expression being initiated at the time of

myelination; and, chondroitin sulfate proteoglycan (NG-2), a marker for

oligodendroglial precursor cells. Setting the levels detected for wt mice at 100%, I

measured lower levels of expression for PLP (47%, p=1.53E-06), CNPase (61%,

p=1.45E-06), CAII (77%, p=0.002), TPPP/P25 (81%, p=6.46E-05) and NG-2 (52%,

p=1.07E-05) in A53T α-Syn mouse brains, yet no differences were detected in the

mRNA levels of MBP (Fig. 9A).

To compare expression at the protein level I utilized the second mouse brain

hemisphere to extract proteins, and quantified their amounts by Western blot analysis

using specific antibodies. Comparing the protein levels, I detected ~54-74%

significantly lower levels for CNPase, CAII, and TPPP/P25 in the A53T α-Syn brains

than in the wt control brains. No differences in the levels of the myelin proteins, PLP

and MBP, were detected in the A53T α-Syn tg mouse brains (n=4-6 mouse brains for

each genotype, p<0.01, ANOVA) (Fig. 9B).

Fig. 9 Oligodendrocytes are affected in A53T -Syn tg mouse brains.

A. Total RNA was extracted from one brain hemisphere (n=4-5 brains) of wt mice and A53T α-Syn tg

mice and analyzed by RT-PCR with specific primers as indicated in the Methods. B. Samples of

protein extracts from one brain hemisphere (n=4-5) of wt mice and A53T α-Syn tg mice, analyzed by

Western blot analysis using specific antibodies to the indicated proteins (as in A). Blots were quantified

using UnScan it gel 6.1. The results are presented as a percent of the 100% signal detected for each

gene in the wt mouse brains (represented by the vertical bar). MBP, myelin basic protein; PLP,

proteolipid protein; CNPase, 2′, 3′-Cyclic Nucleotide 3′-Phosphodiesterase; CAII, carbonic anhydrase

II; TPPP/P25, tubulin polymerization promoting protein; and NG-2 chondroitin sulfate proteoglycan. *

P< 0.01, ANOVA.

To determine whether the lower expression levels of specific oligodendrocyte genes

(described above) are accompanied by a loss of oligodendrocytes, I quantified

oligodendroglia, astroglia and neurons in paraffin sections of young wt and A53T

α-Syn tg mouse brains (n=4 for each genotype). Specifically, I stained in parallel, for

oligodendroglia (CAII or TPPP/P25), astroglia (GFAP), and neurons (NeuN). The

relative amounts of the different cell populations were quantified in three different

brain regions: the cortex, corpus callosum, and the gigantocellular nuclei in the brain

stem. Consistent with the original description of this mouse model, no difference in

A

B

the number of neurons, determined by NeuN staining, was detected between wt and

A53T α-Syn tg mice in the three specific brain regions tested (Giasson et al., 2002). In

addition, no evidence for astrocytic gliosis was found in these three brain regions. In

contrast, lower signals for either one of the oligodendrocyte-specific protein markers

tested, CAII or TPPP/P25, were detected in A53T α-Syn tg mice brains, but not in wt

mice brains. Specifically, the number of oligodendrocytes per slide stained with CAII

was 102±7.2 and 72±11.2 in the corpus callosum of wt and A53T α-Syn tg mice,

respectively, and 65.5±8 and 44.3±8 in the brain stem of wt and A53T α-Syn tg mice,

respectively (n=4-5 mice, p< 0.01, ANOVA) (Fig. 10A). The differences in the

number of oligodendrocytes in the cortex were not significant. To verify the results

obtained with CAII, I stained adjacent sections with the same antibody set-up but used

TPPP/P25 as the marker for oligodendrocytes and obtained very similar results (Fig.

10B).

In accord with the lower number of oligodendrocytes in the brain stem and corpus

callosum of young A53T α-Syn tg mice, I detected evidence of synaptic injury

represented by a lower signal for synaptophysin in these brain regions (Fig. 10A).

Specifically, the measured signal for synaptophysin in the brain stem and corpus

callosum of A53T α-Syn tg mice was 45.4 ±15.7% and 45 ± 3% (p<0.01 ANOVA)

respectively, of the signal obtained for wt mice (set at 100%). These results indicate

that the A53T α-Syn tg mice, originally described as a model for neuronal

synucleinopathies such as PD, also harbor insults characteristic of MSA, with affected

oligodendrocytes and synaptic injury.

Fig. 10 Oligodendrocytes cells number, GFAP and synaptophysin signals are affected in A53T

-Syn tg mouse brains.

A. Graph representing quantifications of GFAP, synaptophysin and CAII signals by IHC in paraffin

sections from wt (100%, bar) and A53T α-Syn tg mice brains. N=4-5 mouse brains with 3-5 fields for

each mouse brain, mean ± SD, *, P< 0.01, ANOVA. B. A representative image of gigantocellular

nuclei in the brain stem of wt and A53T α-Syn tg mice brains obtained by IHC. Paraffin sections

stained with TPPP/P25 (oligodendrocytes, red) and NeuN (neurons, green) and counter stained with

DAPI (blue). Bar, 50 m.

3.1 Dietary brain DHA levels affect neuronal and oligodendroglial -Syn pathology in A53T α-Syn tg mouse brains.

In a recent study, I have shown that dietary alterations in brain DHA levels affect the

degree of -Syn–related pathology in brains of A53T α-Syn tg mice (Yakunin et al.,

2011). I extended this study to investigate the effect of brain DHA levels on -Syn

localization to oligodendrocytes. For this aim, we utilized our recent protocol for

dietary manipulations in brain DHA composition (Yakunin et al., 2011).

A

B

A53T

-Syn

wt

Specifically, A53T -Syn tg mice were fed one of the following diets: a low-DHA

diet (and low in n-3 PUFA); a high-DHA diet (0.67% DHA); or a standard mouse

chow diet. The diets were administered for 230 days and the mice were sacrificed at

9-10 months of age. Comparisons were performed between the high- and low-DHA

diets after normalization to the standard mouse chow diet.

Following our recent findings, I attempted to determine the potential effect of

alterations in levels of neuronal -Syn pathology on its localization to

oligodendrocytes by IHC, measuring α-Syn signal co-localizing with

oligodendrocyte-specific protein markers in paraffin brain sections. For this aim, I

triple stained coronal brain sections with specific antibodies to α-Syn, CAII, and

synaptophysin. To verify the results, I repeated the stains with a similar set-up but

used TPPP/P25 instead as the antibody for the oligodendrocytes. I focused on two

brain regions, the brain stem and cortex, which contain high and low levels of α-Syn

cytopathology, respectively (Giasson et al., 2002; Yakunin et al., 2011.).

The results indicated a higher degree of -Syn immunoreactivity detected within

oligodendrocytes of A53T -Syn tg mice fed the high-DHA diet and lower -Syn

immunoreactivity with the low-DHA diet. A significant difference in the -Syn signal

detected by IHC and co-localized with the oligodendroglial markers, CAII, was

obtained by comparing brain stems of mice from the low- and high-DHA diet groups

(Fig. 11). Specifically, the relative -Syn signal in oligodendrocytes measured and

normalized to the standard chow group (designated as 100%) for the low- and high-

DHA diets was 55.5 ± 32.3% and 151 ± 30.2%, respectively (mean ± SE; n=6 mice

per group; p<0.01, ANOVA) (Fig. 11). Similar results were obtained in the cortex of

these mice. In particular, the relative -Syn signal measured and normalized to the

standard chow group (designated as 100%) for the low- and high-DHA diets was 45 ±

8.7% and 274 ± 120%, respectively (mean ± SE; n=4-6 mice per group; p<0.01,

ANOVA). The effects of DHA-enhanced -Syn neuropathology on α-Syn

localization to oligodendrocytes were verified with a second antibody, TPPP/P25,

with highly similar results (Fig. 11B).

Fig. 11 Dietary alterations in brain DHA levels affect -Syn accumulation in oligodendrocytes in

the brains of A53T -Syn tg mice modeling MSA.

A53T -Syn mice were fed for 230 days one of the three diets: a low-DHA diet (and low in n-3

PUFA); a high-DHA diet (0.67% DHA); or a standard (std) mouse chow diet. A. IHC of paraffin

sections showing the gigantocellular nuclei in the brain stem, stained in parallel for CAII

(oligodendrocytes, green); α-Syn (red); and synaptophysin (white). B. Graph representing the α-Syn

signal co-localized with TPPP/P25, but not with the synaptophysin signal. Relative results (normalized

to the standard chow group, designated as 100%) for the low- and high-DHA diets. Images were

quantified with the Image Pro Plus 6.3 program (Media Cybernetics, MD, USA). Mean ± SD of n=6;

*, p<0.001 ANOVA. Bar, 10 m.

To eliminate the possibility that dietary DHA affects -Syn expression in

oligodendrocytes through enhanced PrP promoter transcription activity, we measured

the mRNA levels of transgenic human -Syn and endogenous PrP using real-time

PCR. Specifically, total RNA was extracted from one hemisphere of A53T -Syn tg

mice, from each of the three tested diet groups (n=4 mice for each group). We found

no evidence for enhanced PrP promoter transcription activity in the brains of mice fed

A

B

the high- versus low-DHA diets. Likewise, no evidence for an effect from the diet on

mouse endogenous α-Syn expression was found (Yakunin et al., 2011).

Moreover, treating N2a cells with DHA at 50-250 M for 16-24 hours had no

enhancing effect on PrP mRNA levels (Fig.12). I therefore concluded that the effect

of dietary DHA on α-Syn localization to oligodendrocytes results from enhanced

neuronal α-Syn pathology and is not a result of enhanced transcription mediated by

the PrP promoter.

Fig. 12 DHA did not affect PrP mRNA levels.

N2A cell line treated with DHA at 50-250 M for 16-24 hours and analyzed by real time-PCR with PrP

specific primers. The results normalized to BSA treatment.

Chapter 2:

Synuclein and Oxidative Stress

There is ample evidence for the occurrence of oxidative stress in brains with PD. It is

currently unknown whether oxidative damage in brains with PD results from

increased production or decreased clearance of oxidants (Zhou et al., 2008). We

investigated the effect of α-Syn pathology on oxidative stress and if catalase is

involved.

1. Evidence for oxidative damage in brains of A53T -Syn tg mice.

The carbonyl content in proteins extracted from one hemisphere of ~8 month old non

transgenic (wt) and A53T -Syn tg mouse brains was measured as an indicator of

oxidative damage. The assay for protein carbonyl detection involves derivation of the

carbonyl group with 2,4-dinitrophenylhydrazine (DNPH), which leads to the

formation of a stable 2,4-dinitrophenyl (DNP) hydrazone (Levine et al., 1990). A

significantly higher degree of protein carbonyls was detected in the brains of A53T -

Syn tg mice than in the brains of wt mice. Specifically, I measured 24 ± 2.2 and 16±

3.9 nmol DNP/mg protein (mean ± SD of n=3 mouse brains) for A53T -Syn tg and

wt brains, respectively (Fig.13). Based on this result I concluded that enhanced

oxidative damage occurs in the brains of A53T -Syn tg mice.

Figure 13: enhanced oxidative stress in A53T -Syn tg mice.

Protein extracts were prepared as described (Materials and Methods), protein carbonyl tests were

performed in a spectrophotometer at absorbance of 375nm and normalized to protein quantification.

Quantification data for 8 month-old mice, representing nmol carbonyl protein /mg protein in A53T

α-Syn and control wt mouse brains. (n = 3 mice, 3 independent measurements, mean ± SD, *, p<0.01,

Student's t-test).

2. -Syn specifically inhibits catalase activity by affecting PPAR transcription activity

Catalase plays a key role in the scavenging of reactive oxygen species (ROS) and in

protecting the cell from oxidative stress. Previous experiments in the lab

demonstrated that α-Synuclein inhibits catalase activity in dopaminergic cells and in

the brains of A53T Syn mice. Specifically Dr. Eugenia Yakunin showed, using

the Amplex Red catalase assay kit (Invitrogen), that the measured catalase activity

was 50 ± 2.9% and 65 ± 2.6% for cells over-expressing wt or A53T mutant -Syn,

respectively, in comparison to naïve cells designated as 100%. In addition, a ~30 ±

6% lower activity level was detected in samples from one month old A53T Syn

mouse brains compared with wt mouse brains. Furthermore a significantly lower

level of catalase was detected in the brains of A53T -Syn mice.

Catalase transcription is controlled by PPAR (Girnun et al., 2002).

Syn expression affects PPAR transcription activity

To find out whether or not the lower catalase expression and activity levels detected

in brains of A53T -Syn tg mice result from affected PPAR transcription activity, I

utilized a PPAR reporter plasmid (pGreenFire1-PPRE, TR101PA-1, System

Biosciences, CA, USA). The plasmid, consisting of a PPAR response element

(PPRE) conjugated to GFP, was transiently transfected into -Syn over-expressing or

mock-transfected clones of HeLa cells, and the GFP signal was measured by FACS

analysis in live cells. A 60-77% lower transcription activity for PPARwas detected

in -Syn over-expressing cells compared with mock-transfected cells. The effect of

troglitazone, a specific PPAR agonist, on PPAR transcription activity was tested in

-Syn and mock-transfected cells. Interestingly, while troglitazone enhanced PPAR

transcription activity in control cells, it had no effect on the GFP signal in -Syn

over-expressing cells. (Fig. 14).

Fig. 14. α-Syn affects PPARγ activity

A. Naïve and α-Syn over-expressing HeLa cells were transfected with a PPARγ-GFP reporter plasmid.

56 hours post-DNA-transfection, cells were treated with troglitazone (at 5 and 30 μM) or left untreated

for 16 hours and the GFP signal was measured by FACScan. The graph summarizes three experiments.

*, p < 0,05, ANOVA.

2.2 Lower levels of PPAR protein in brains of A53T -Syn mice and in

cells overexpressing -Syn

Since PPAR is a type II nuclear receptor, constitutively bound to DNA, I next sought

to find out whether the lower transcription activity detected in -Syn over-expressing

cells may represent lower PPAR protein levels. For this aim, I quantified the PPAR

signal obtained by Western blot analysis in naïve and -Syn over-expressing HeLa

cells and in the brains of wt and A53T -Syn tg mice. Two immuno-reactive bands

were detected upon reacting the blot with an anti- PPAR antibody (Santa Cruz, sc-

7273). According to their molecular mass, these bands represent PPAR1 and

PPAR2, migrating at 54 and 57 kDa, respectively. Importantly, both PPAR1 and

PPAR2 levels were affected by -Syn expression (Fig. 15).

Figure 15: -Syn expression affects PPAR protein levels.

Protein samples (20 µg protein) were boiled for 10 min at 100ºC and separated on a 10% SDS-PAGE

gel. The immunoblot was reacted with anti- PPAR antibodies. A. Representative immunoblot of

whole brain extracts of A53T -Syn tg and wt mice. B. Protein samples of naïve and α-Syn over-

expressing cells and non-transgenic. The signal specificity was determined using a samfple of mouse

liver protein extract analyzed in parallel.

A wt

2.3 Agonist-activation of PPAR or RXR eliminate the inhibiting effect

of -Syn on catalase activity

To verify the specific involvement of PPAR in catalase inhibition by -Syn, I treated

HeLa cells stably expressing -Syn with the specific PPAR agonist, troglitazone.

Since PPAR heterodimerizes with RXR for transcription regulation, I have also

tested the effect of 9-cis retinoic acid, an agonist for RXR. Cells were conditioned in

medium containing DMSO solvent or with the specific agonist for 16 hours. I found

that troglitazone added to cells at 5 μM, enhanced catalase activity by 130%.

Importantly, troglitazone added at and 30 μM significantly enhanced catalase activity

by 150%. In accordance with the effects of troglitazone, I found that 9-cis-RA at 5

μM significantly enhanced catalase activity by 210%. (Fig 16A) Importantly, catalase

activity was highly similar with and without -Syn expression in the agonist-treated

culture (not shown)

2.5 Over expression of PPAR, PPAR2 and RXR enhance catalase activity

Next, I sought to examine the potential involvement of additional PPAR members in

catalase inhibition by -Syn. For this aim I have transiently transfected HeLa cells

with either one of the following cDNAs: PPAR, PPAR; PPAR1; PPAR2 or

RXR. Each one of these transcription factors was transfected either with human

-Syn cDNA or a mock plasmid. 48 hours later, cells were lyzed and RNA and

proteins were collected. Following transfection, the mRNA levels of α-Syn were

higher by 20-75 folds in those cells transfected with a α-Syn cDNA. Accordingly, the

mRNA levels of PPARα, PPARβ/δ, PPARγ1, PPARγ2 and RXR were elevated by 7-

67 folds. Catalase activity was determined in protein extracts using Amplex Red

catalase assay kit (Invitrogen). The results suggest an enhancing effect for PPAR,

PPAR2 and RXR on catalase activity in the absence of -Syn expression. PPAR

and PPAR1, although expressed at high levels, did not significantly affect catalase

activity (Fig. 16B). In -Syn expressing cells all except PPAR have significantly

enhanced catalase activity (Fig. 16B) to the level detected without -Syn and higher.

We concluded that activation of certain PPAR members prevents the inhibiting effect

of -Syn on catalase activity.

Specifically, normalizing the results to the activity measured for cells transfected with

α-Syn and a mock plasmid for PPARs (16.1 ± 4.2 mU/ml/μg protein), we found that

catalase activity was increased 329.9 ± 107.9% by PPARα, 283 ± 75.6% by PPARγ1,

257 ± 75.0% by PPARγ2, and 366 ± 68.2% by RXR. We conclude that α-Syn

interferes with the complex and overlapping network of nuclear receptor dimerization

and transcription activation.

Fig 16. Catalase inhibition is eliminated by activation of PPARα, PPARγ or RXR

A. Catalase activity measured in α-Syn expressing-HeLa clonal cells (samples of 20 ng protein), treated

with troglitazone (5 and 30 μM) or 9-cis retinoic acid (5 μM) for 16 hours, determined by Amplex Red

Catalase Assay kit (Molecular Probes). Mean of n=2-3 different clones ± SD; *: p<0.05, one way

ANOVA. B. Catalase activity measured in HeLa cells, transiently transfected with cDNA for the

indicated nuclear receptors, together with (black bars) or without (white bars) α-Syn. Mean of n=2-3

experiments ± SD. *: p<0.05 ANOVA

A

B

* * *

*

*

*

*

Discussion

This thesis summarizes the investigation performed in order to elucidate two

questions relevant to the pathogenesis of -Syn in the synucleinopathies:

i. What are the cellular mechanisms that underlie the pathogenic

accumulations of -Syn in oligodendroglia, in Multiple System Atrophy?

ii. What are the cellular mechanisms that underlie -Syn expression affect

catalase, an anti-oxidative stress enzyme?

Neurodegenerative diseases are complex diseases and may result by more than one

mechanism. In my thesis I focused on the role played by α-Syn protein in the

pathogenesis of two synucleinopathies, Parkinson’s disease and Multiple systems

atrophy.

-Syn secretion is the focus of many recent studies. While secretion of -Syn may

occur normally, enhanced secretion is associated with -Syn pathogenesis. The

results generated in the frame of this thesis suggest an association between enhanced

neuronal -Syn secretion, and the subsequent uptake of -Syn by oligodendrocytes in

the pathogenic mechanism of MSA.

I have found that elevated intracellular levels of α-Syn lead to enhanced secretion of

-Syn from neuronal cells and subsequently, increases in -Syn levels within

oligodendrocytes. Specifically, utilizing cultured neuronal MN9D cells, over-

expressing WT or A53T -Syn, I was able to show that the intracellular levels of total

-Syn, as well as the degree of soluble α-Syn oligomers, are associated with its

secretion. In accordance, I have shown that oligodendroglial cells are capable of

taking-up -Syn from their environment, either from medium conditioned by

neuronal cells or from growth medium supplemented with recombinant human -Syn.

-Syn uptake by oligodendrocytes was dependent on both time and concentration,

and on clathrin expression.

To investigate the effect of intra-neuronal -Syn pathology on its accumulation in

oligodendrocytes in vivo, I measured -Syn localization in oligodendrocytes

following dietary alterations in brain docosahexaenoic acid (DHA) levels, in mice

transgenic for the A53T α-Syn mutant form. In accord with our recent report

describing enhanced levels of -Syn-related pathology following a high-DHA diet

and the reverse effect with a low-DHA diet (Yakunin et al., 2011), I have detected

higher amounts of -Syn immunoreactivity within oligodendrocytes in brain sections

of mice fed the high-DHA diet than those fed the low-DHA diet. Hence, the

conclusion that enhanced neuronal -Syn pathology is causally related to its

accumulation in oligodendrocytes in MSA.

The A53T -Syn Tg mouse-line was originally described as a model for

synucleinopathies (Giasson et al., 2002). These mice express human A53T -Syn

under the control of the PrP promoter. The mice develop an age-dependent disease,

characterized by motor impairment leading to paralysis and death. Along with

phenotypic expression, the mice develop neuronal intra-cytoplasmic -Syn–positive

inclusions. The -Syn pathology is abundant in the brain stem and in several nuclei

affected in PD and DLB, such as the locus coeruleus and raphe nucleus, and also in

the striatum. However, α-Syn pathology was not originally described in

oligodendrocytes in this mouse model. In addition, no evidence for neuronal cell loss,

including of tyrosine hydroxylase-positive neurons, was originally reported for this

A53T -Syn Tg mouse-line. Evidence for synaptic injury was detected, represented

by lower levels of synaptophysin immunoreactivity in the two brain regions tested,

the corpus callosum and brain stem. The lower expression levels of oligodendrocyte-

specific genes, and the lower density of oligodendrocytes detected in the brains of

young A53T -Syn Tg mice, suggest that oligodendrocytes are indeed affected in this

mouse model.

Several mouse models for MSA were recently generated, specifically directing the

expression of -Syn to oligodendrocytes using specific promoters of oligodendroglial

genes (see for example (Kahle et al., 2002; Yazawa et al., 2005; Stefanova et al.,

2005). These models were shown to recapitulate features of MSA and are very useful

in elucidating the pathways of -Syn toxicity in oligodendrocytes, yet these models

cannot explain the mechanisms involved in the pathogenic accumulation of -Syn

within oligodendrocytes. The advantages of the A53T -Syn Tg mouse model include

the age-dependent motor manifestations that reflect the age-dependent onset of MSA,

and the progressive nature of the disease. The occurrence of -Syn in

oligodendrocytes and the effect of -Syn expression under non-oligodendroglial

specific genes, make this model more closely resemble MSA, and can thus be used as

a model to investigate and elucidate the cellular mechanisms of MSA. Nevertheless,

this MSA model has some limits: 1) it does not distinguish between PD- and MSA-

associated symptoms. 2) The occurrence of -Syn in oligodendrocytes in this mouse

model may emanate from α-Syn over expression which is regulated by the PrP

ptomoter.

Of note, the findings presented herein indicate that DHA-enhanced α-Syn-pathology

in neuronal cells resulted in increases in -Syn levels whithin oligodendrocytes

without affecting α-Syn expression, mediated by the PrP promoter. Therefore, the

results support a mechanism of α-Syn toxicity in MSA, consisting of neuronal

increases in α-Syn protein levels as a cause for its accumulation in oligodendrocytes

and MSA. .

It is currently unclear whether -Syn secretion is part of a pathogenic process or

rather relates to the physiological function of this protein. This is represented by the

difficulty in distinguishing healthy individuals from PD patients based on CSF levels

of -Syn (Aerts et al, 2012; Bruggink et al., 2011; Foulds et al., 2012; Mollenhauer et

al., 2011; Reesink et al., 2010; Shi et al., 2011; Sierks et al., 2011; Tateno et al., 2011;

Tokuda et al., 2010) In this regard, the secretion of α-Syn appears to be a specific

mechanism and does not occur for the -Syn homolog. There was a posicorrelation

between levels of intra-neuronal -Syn oligomers and its subsequent secretion.

Higher oligomer levels and higher amounts of secreted α-Syn are detectable in

cultured cells stably-expressing the A53T mutant form, as compared with levels in

WT -Syn expressing cells. Furthermore, I found a positive correlation between the

total amount of intracellular -Syn and the amount of secreted -Syn, as detected in

the CM. -Syn dosage is strongly associated with the disease, and as such

duplications and triplications in the -Syn gene locus, translating to elevated levels of

-Syn expression, were shown to cause familial PD (Devine et al., 2011). It is

therefore of interest to determine whether -Syn secretion from neuronal cells is

enhanced in these patients with familial PD.

The mechanism of -Syn secretion was shown to involve vesicle trafficking that is

independent of the endoplasmic-reticulum-Golgi apparatus. -Syn was detected

within the lumen of vesicles (Lee et al., 2005) and was shown to be released by

exosome-like vesicles in a Ca2+

-dependent manner (Emmanouilidou et al., 2011.).

Secreted -Syn was shown to affect the recipient cells, specifically, uptake of

neuronal-secreted -Syn was shown to affect the viability of neighboring neuronal

cells and to alter the gene expression patterns in astrocytes, causing an inflammatory

response (Emmanouilidou et al., 2011; Lee et al., 2010a). The significance of -Syn

release and its uptake by neighboring cells, is not fully understood, but it may underlie

the mechanism for the pathogenic spread of -Syn as suggested by the Braak

hypothesis (Braak et al., 2004). Recently, it was suggested that -Syn pathology may

propagate throughout the brain by way of a prion-like mechanism (Luk et al.,2012).

Based on our findings, I suggest that the pathogenic spread of -Syn may also

underlie the mechanisms leading to MSA.

In this thesis, I focused on the effect of enhanced accumulation of neuronal -Syn on

its secretion and subsequent uptake by oligodendroglial cells in the context of the

cellular mechanisms of MSA. However, it is plausible that additional cell populations,

including astrocytes, microglia and certain neuronal cells, are similarly affected.

I utilized the effect of dietary brain DHA levels as a means to control the degree of

-Syn pathology in vivo, in the brains of A53T -Syn tg mice. Recently, we have

demonstrated that α-Syn-related pathology positively correlates with brain DHA

levels. The accumulation of soluble and insoluble neuronal -Syn, astrocytic gliosis,

and synaptic injury were affected by these dietary DHA alterations (Yakunin et al.,

2011). The results herein may represent an extension of the previous study, indicating

that dietary brain DHA levels control an additional aspect of -Syn pathology,

namely, its accumulation in oligodendrocytes in MSA. Together, our previous and

current results suggest that -Syn associations with PUFAs, and specifically, its

associations with DHA, are involved in a spectrum of mechanisms implicated in

-Syn-pathophysiology.

Two apparently different mechanisms may underlie the effect of DHA and additional

PUFAs on -Syn secretion: 1) their previously-shown effect of enhancing the levels

of intra-neuronal cytotoxic α-Syn forms through activation of nuclear receptors

(Yakunin et al.,2011); 2) their enhancing effect on membrane trafficking, including

endocytosis and exocytosis, and also enhanced synaptic vesicle recycling (Ben

Gedalya et al., 2009). To determine which of the two mechanisms is involved in

-Syn secretion I utilized the specific RXR antagonist, HX531. This antagonist

eliminates the DHA-enhancing effect on -Syn oligomerization (Yakunin et al.,

2011). Thus, -Syn secretion from cells treated with DHA together with HX531 is

potentially only affected by DHA-dependent membrane trafficking. The results

indicate that -Syn secretion is affected by both mechanisms, intracellular -Syn

oligomer levels in addition to membrane trafficking. -Syn secretion levels, from

cultured cells treated with DHA together with HX531, are lower than from cells

treated with DHA without HX531, yet higher than BSA-treated control cells.

The results indicate that -Syn uptake by cultured oligodendrocytes is inhibited by

silencing the expression of clathrin heavy chain, thereby indicating the potential

involvement of clathrin-mediated endocytosis in this process. The involvement of

endocytosis-dependent uptake of -Syn was previously shown in neuronal and glial

cells, suggesting specific rather than non-specific uptake. Yet, the mechanism of

endocytosis varies between cell types. -Syn internalization, affected by a dynamin

dominant negative, was previously suggested for the uptake of fibrillar and

aggregated, but not monomeric, -Syn in neuronal cells (Lee et al., 2008a). The

involvement of dynamin in -Syn uptake was also shown in astroglial and

oligodendrocytes cells (Lee et al.,2010b ; Konno et al., 2012). In microglia,

monomeric -Syn was shown to be internalized via GM1-mediated endocytosis, and

required intact lipid rafts. The uptake of -Syn by microglia was independent of

clathrin-, caveolae-, or dynamin-dependent endocytosis mechanisms (Park et al.,

2008). Others have shown that oligomeric -Syn is internalized by microglia more

efficiently than monomeric -Syn (Lee et al., 2008b).

Because Syn represents a major component of both oligodendroglial and neuronal

inclusions in MSA, some scientists have postulated two parallel disease processes:

one that is attributable to the degeneration of the oligodendroglia-myelin axis, as in a

primary oligodendrogliopathy; and the other that is caused by the accumulation of

Syn within nerve cells, as in a neuronal synucleinopathy. Currently, the main

hypothesis is that the pathogenesis of MSA primarily involves oligodendrogliopathy

featuring early myelin dysfunction, associated with axonal damage, thereby resulting

in secondary neurodegeneration (Wenning et al., 2008). The results in this thesis

suggest a complex network between neurons and oligodendrocytes. The Syn

neuropathology enhances the occurrence of Syn in oligodendrocytes, which causes

oligodendrogliopathy, as well as resulting in secondary neurodegeneration.

In conclusion, the results here raise a relevant question, which signals or down stream

pathogenic mechanisms that are activated upon neuronal secretion of -Syn,

differentiate between the development of PD or MSA.

In the second part of this thesis, I have investigated the cellular mechanisms of

oxidative stress occurring in PD, specifically the cellular mechanism by which -Syn

expression affects catalase, the anti-oxidative stress enzyme.

A previous study in the lab demonstrated an inhibitory effect for -Syn expression on

catalase mRNA and protein levels, resulting in lower enzymatic activity of catalase.

The finding that catalase expression and activity are unaffected in brains of mice

modeling Alzheimer’s disease provides evidence for a specific effect of -Syn on

catalase. Following-up on these findings and searching for a mechanism of oxidative

stress in PD, which would explain the role of -Syn in decreased catalase expression

and activity, I was interested in studying the potential involvement of Nuclear

Receptors (NRs).

Experiments have shown reduced transcription activity and lower levels of PPAR a

nuclear receptor that controls catalase expression, in the brains of A53T -Syn Tg

mice, and in dopaminergic cells over-expressing -Syn Activating PPARα, PPARγ

and RXR, but not PPARβ/δ, restored and enhanced catalase activity. I suggested that

this lower PPAR transcription activity may explain the effects of -Syn on catalase,

as described previously.

There are 48 known human nuclear receptors (NRs) that form a complex network of

gene activation pathways. A high degree of overlap among NR-activated pathways

complicates the investigative efforts to understand and identify their distinct effects.

Although it has been well-established that NRs control diverse mechanisms, including

development, inflammation, reproduction and metabolism, their role in

neurodegeneration is less clear. Nevertheless, the involvement of NRs in

synucleinopathies was highlighted when rare mutations in the orphan NR, Nurr1,

were associated with familial late-onset PD (Xu et al., 2002; Zheng et al., 2010),

providing genetic evidence for the involvement of these transcription regulators in

PD. Nurr1 can form heterodimers with the retinoid X receptor alpha (RXRα) and

bind to certain retinoic acid-responsive elements. (Perlmann et al., 1995). I have

shown that α-Syn’s inhibition of catalase activity can be eliminated by activation of

certain NRs. It is plausible that activation of specific NRs may affect the fine balance

of this complex network. I therefore suggest that α-Syn interferes with the regulation

of catalase. Nonetheless, a comprehensive study is needed in order to define the

specific pathways that are involved in the regulation of catalase.

Beyond their physiological role in peripheral organs and tissues, PPARs play an

important role in the pathogenesis of various disorders in the CNS. The discovery that

PPARinhibits inflammation in vitro (Ricote et al., 1998) and in vivo (Su et al.,

1999), has raised hope that PPAR could become a drug target for the treatment of

neurological diseases with an inflammatory component, such as Alzheimer's disease.

Specifically, it was shown that stimulating PPAR activates clearance mechanisms for

Amyloid peptide (Camacho et al., 2004). PPARmediated anti-inflammation

mechanisms protected dopaminergic cells from MPTP-induced cell death in the SN of

mice (Breidert et al., 2002). In addition, it was shown that PPAR activation prevents

neuronal cell death and neuritic injury induced by the Apeptide (Santos et al.,

2005). Furthermore, a recent report suggests a role for PPARγ2 in

longevity (Argmann et al., 2009). The activation of NHRs, such as PPARγ, may

therefore link aging with neurodegeneration, in particular aging and α-Syn

neuropathology.

The possible roles of reactive oxygen species (ROX) as the driving force for aging

and age-related neurodegeneration is a matter of debate The results herein suggest

and the involvement of α-Syn pathophysiology in the regulation of catalase and

therefore, of oxidative stress by nuclear receptors.

Lower catalase activity results in elevated levels of its substrate, hydrogen peroxide

(H2O2), and is associated with aging and age-related diseases (Bonekamp et al., 2009).

At low concentrations, H2O2 is a critical signaling molecule, involved in carbohydrate

metabolism, cell proliferation and apoptosis (Rhee et al., 2005). At high

concentrations, H2O2 is toxic, mainly because it can be easily converted into reactive

oxygen species (ROS), like superoxide anion (O2-) and hydroxyl radical (OH).

Similar to H2O2, ROS have been shown to play a role both in pathogenic and

physiologic mechanisms, as well as in aging (Beckman et al., 1998; Kregel et al.,

2007). Hydroxyl radicals can attack the double bonds in the fatty acyl side chains of

phospholipids (primarily n-6 PUFA), forming the 4-hydroxynonenal (4-HNE)

oxidation product. 4-HNE is an abundant reactive carbonyl molecule produced in

neurodegeneration (Uchida et al., 2004). The chemical reaction of 4-HNE with lysine,

histidine, or cysteine residues (Schaur, 2003) results in protein carbonylation.

Thus, protein carbonyl content is the most general and well-used biomarker for

oxidative damage.

Mitochondria are a major source of reactive oxygen species, generated through the

reduction of oxygen by the respiratory chain and oxidative phosphorylation system.

Defects of respiratory chain function, whether inherent or induced by toxins, cause an

excess of free radical production (Schapira, 2012). Mitochondrial dysfunction is

indeed critically implicated in the pathogenesis of PD. Several pieces of evidence

support a role for mitochondrial dysfunction in the pathogenesis of PD: familial forms

of PD involve specific mutations in mitochondrial genes; mitochondrial Complex I

activity is inhibited in postmortem PD brains; and, several mitochondrial toxins were

shown to cause PD (recently reviewed in (Schapira, 2012). While the role of

mitochondria in the pathogenesis of PD is being extensively studied, the role of

catalase, an anti-oxidative stress enzyme, as a key player in PD and other relevant

age-dependent neurodegenerations, is still unknown. Here I have shown evidence

linking -Syn expression with the dysfunction of catalase, an enzyme residing

primarily in peroxisomes. Our results point at catalase inhibition as a potential source

for the oxidative stress that characterizes the pathogenic mechanisms leading to PD. It

is estimated that ~35% of cellular H2O2 is produced by peroxisomes (Boveris et al.,

1972). This figure exemplifies the importance in elucidating the potential role played

by peroxisomes in neurodegeneration and particularly, in PD.

PPAR is a ligand-activated transcription factor, belonging to the nuclear receptor

super-family. It preferentially dimerizes with retinoic X receptor (RXR) to control the

transcription of various genes involved in cellular energy metabolism. Among the

identified activating ligands for PPAR are: PUFAs, such as linolenic acid (18:3),

eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6); the

naturally occurring oxidized FA, (S)-hydroxy-octadecadienoic acid (HODE, 18:2);

and FA metabolites, prostaglandin J2 (PGJ2) and eicosanoids, such as

hydroxyeicosatetraenoic acid (HETE). Importantly, growing evidence suggests that

-Syn expression and pathology are associated with alterations in levels of certain

PUFAs and their metabolites (Ellis et al., 2005; Golovko et al., 2005; Golovko et al.,

2007; Rappley et al., 2009; Sharon et al., 2003b). It is therefore possible that -Syn

affects PPAR transcription activity by affecting the availability, or local

concentrations, of specific PPAR activating ligands.

A recent meta-analysis of 17 independent genome-wide gene expression microarray

studies has identified several gene sets that are strongly associated with PD (Zheng et

al., 2010). The gene sets with the strongest association indicated reduced expression

of genes that are involved in energy metabolism, primarily glucose metabolism and

nuclear genes encoding subunits of the electron transport chain proteins found in

mitochondria. It was found that genes whose expression is controlled by PPAR

coactivator-1 (PGC-1) are under-expressed in patients with PD and in accord,

activation of PGC-1 rescued dopaminergic neurons from -Syn–induced toxicity

(Zheng et al.,2010). Other studies have shown that PGC-1 is down-regulated in

Huntington’s disease (Weydt et al., 2006). Together, these studies provide a link

between neurodegeneration and impaired energy metabolism. Interestingly, PGC-1

is activated in response to ROS and RNS (Spiegelman, 2007), and was shown to

induce the expression of catalase and additional genes encoding ROS defense

enzymes, including copper/zinc superoxide dismutase (SOD1), manganese SOD

(SOD2) and glutathione peroxidase (St-Pierre et al., 2006).

It is currently unknown whether the oxidative damage is a result of increased

production or decreased clearance of oxidants (Zhou et al., 2008). The results herein

suggest a mechanism of decreased clearance of oxidants due to decreased activity and

expression of catalase under the control of NHs.

Concluding remarks

The great importance of the above findings stem from their relevance to the

mechanisms of human disease. MSA and PD are fatal diseases without long-term

treatment options. Current treatments alleviate symptoms however do not stop disease

progression. Elucidating the cellular mechanisms of the diseases can help to develop

better treatment options that can prevent their progression.

The recent discovery of the secretion of -Syn has been investigated thoroughly. In

this thesis, I have suggested that neuronal-secreted -Syn is the origin of the -Syn

found in oligodendrocytes in cases of MSA. Furthermore, I have suggested that the

elevated levels of α-Syn in neurons enhance its secretion, and that the amplified

amounts of secreted -Syn cause the elevated α-Syn occurrence in oligodendrocytes.

.

Another cellular mechanism that I was investigated in this thesis was the mechanism

of oxidative stress in PD.

The findings suggest that -Syn acts on catalase through its effect on PPAR activity.

Since PPAR activity is regulated by energy, and specifically, lipid metabolites, it is

possible that -Syn is involved in a metabolic loop, where its assembly in specific

forms is affected by lipid metabolites, and thus affects brain lipid and energy

metabolism.

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י אלפא "מנגנוני ניוון עצבי המתווכים ע

סינוקלאין במחלות פרקינסון

.ואטרופיה רב מערכתית

חיבור לשם קבלת תואר דוקטור לפילוסופיה

מאת

חיה קיסוס

הוגש לסנט האוניברסיטה העברית

ג"תמוז תשע

בודה זו נעשתה בהדרכתם של ע

ר רונית שרון"ד

תמיר בן חור' פרופ

,שלמי תודה

.תמיר בן חור על ההכוונה וההדרכה' ר רונית שרון ופרופ"ד, תודה למנחים שלי

. התמיכה והסיוע, על הליווי , תודה לצוות המעבדה

.הדרך תודה למשפחתי היקרה על התמיכה והנשיאה בעול לכל אורך

.ותודה לבורא עולם

תקציר

, ההיסטופתולוגית ברמה משותף מכנה עם המח של ניווניות מחלות של קבוצה הן הסינוקלאינופתיות

של מסיסיים בלתי צורונים של מהצטברות המורכבים, תאיים תוך הסגר גופפי יצירת של בצורה המופיע

לראשונה הוגדר סינוקלאינופתיות המונח. וגליה עצב בתאי( α-Syn) סינוקלאין אלפא החלבון

פרקינסון למחלות משותף מכנה מהווה α-Syn של האופינית הפתולוגיה כי התגלה כאשר, 1998 בשנת

PD ,מערכתית רב אטרופיה (MSA )לואי גופיפי עם ודמנציה (DLB .)בשתי התמקדתי זו בעבודה

.יתמערכת רב ואטרופיה פרקינסון, המחלות

α-Syn תאי בקצות סינפטי-הפרה באזור בעיקר ונמצא, המרכזית העצבים במערכת המתבטא חלבון הוא

, α-Syn. הסינוקלאינופטיות המחלות של ונוירופתולוגיה בגנטיקה קריטי תפקידα-Syn ל .העצב

שאינם ואגרגטים אוליגומרים יצירת של תהליך בהדרגה עובר מסוימים בתנאים, כלל בדרך סמסי חלבון

. Lewy bodies לואי גופפי הנקראים תאיים ךתו גופיפיםב יםשוקע דבר של פובסו אשר ,מסיסיים

נמצאים הגופיפים כאשר (Lewy bodies) יאלו גופיפי קרויים הם, עצב בתאי נמצאים הגופיפים כאשר

glial cytoplasmic inclusions (GCI .) נקראים הם באוליגודנדרוציטים

GCI של ההיסטופטולוגי המאפיין הם MSA .ל בגן מסוימות נקודתיות מוטציות-α-Syn ,כן וכמו

לאוליגומריזציה החלבון נטיית את מגבירות, סינוקלאין-אלפא של הלוקוס של וטריפליקציות דופליקציות

.החלבון של הפתוגנזה ואת

מתוך, העצב מתאי המופרשת, תאית החוץ α-Syn -ה במולקולת דווקא התמקדות ישנה האחרון בעשור

מופרש α-Syn. הסינוקלאינופטיות והתפתחות בהיווצרות תפקיד יש המופרש α-Syn ל כי מחשבה

-α של ההפרשה הגברת כי מצביעים רבים מחקרים זאת עם יחד, עצב תאי של הנורמאלי מהתפקוד כחלק

Syn דיו ברור אינו עדיין ההפרשה מנגנון. החלבון של יותהפתוגנ להגברת קשורה.

: היו הדוקטורט עבודת של העיקריות המטרות

כפי, באוליגודנטרוציטים, α-Syn העצבי החלבון של להופעה המוביל התאי המנגנון את לאפיין .א

.MSA חולי במוחות שמופיע

נזק המונע אנזים, בקטלאז לפגיעה גורם α-Syn ביטוי באמצעותו התאי המנגנון את לאפיין .ב

.חמצוני

-α לקלוט יכולים שהאוליגודנדרוציטים הטוענת המחקר השערת את בדקתי, העבודה של הראשון בחלק

Syn ל המוביל הפתולוגי מהמנגנון כחלק העצב מתאי המופרשMSA .כ"בסה שהגברה מצאתי בעבודתי

עצב תאי, MN9D מתאי α-Syn בהפרשת יהלעל גרמה α-Syn של האוליגומרים וברמת α-Syn כמות

, אולגודנדרוגליאלי ממקור תאים, Oli-Neu תאי כי מצאתי בנוסף. α-Syn המבטאים דופאמינרגים

תלויה האוליגודנדרוציטים לתאי α-Syn של זו כניסה. הגידול ממדיום, חיצוני ממקור α-Syn קולטים

.אנדוציטוזה בתהליכי המעורב חלבון, קלטרין י"ע תמתווכ כן וכמו הגידול במדיום החלבון וריכוז בזמן

רוויות בלתי רב שומן בחומצות עשירה דיאטה כי נמצא בהם, במעבדה קודמים למחקרים בהמשך

(PUFA )של הפתולוגיה להגברת גורמת α-Syn ,גם הראיתי in vivo של בפתולוגיה הגברה כי -Syn

מודל עכברי במוחות אוליגודנדרוציטיםב -Syn של בהופעה להגברה הובילה, העצב בתאי

המנגנון על מלמדות אלו תוצאות(. אנושי -Syn A53Tל טרנסגנים עכברים) לסינוקלאינופטיות

-Syn הצטברות והגברת העצב בתאי -Syn ברמת פתוגנית עליה בין המקשר, MSA בחולי הפתוגני

למדיום -Syn בהפרשת להגברה מובילה העצב בתאי -Syn ברמת עליה, כלומר. באוליגודנדרוציטים

. לאוליגודנדרוציטים הנכנס -Syn בכמות עליה ישנה מכך וכתוצאה תאי החוץ

עדויות. פרקינסון חולי במוחות חמצוני לנזק הגורמים המנגנונים את חקרתי, העבודה של השני בחלק

סובסטנציה כגון) פרקינסון מחלת חולי של במוחות הפתולוגיה באזורי נמצאו חמצוני נזקל

הגורם הוא חמצוניה הנזק האם ברור לא אולם. למחלה מודל חיות של ובמוחות( Substatia nigraניגרה

.ההתנוונות תהליך של תוצר או במחלה נוירונים לניוון הראשוני

, קטלאז ובפעילות בביטוי לירידה גורם ספציפי באופן -Syn שביטוי נמצא במעבדה קודמות בעבודות

, קטאלז על משפיע α-Syn ביטוי באמצעו מנגנון אחר בחיפוש. חמצוני נזק המונעת פעילות בעל זיםאנ

Peroxisome ובפעילות בכמות ירידה ישנה, ובעכברים תאים בשורת -Syn ביטוי בעקבות כי מצאתי

proliferator-activated receptor (PPAR יבדקת בנוסף. קטלאז לביטוי בקרה כגורם הידוע

פעילות על משפיעים -nuclear receptors (NR)ה במשפחת נוספים פקטורים של שפעול כיצד

עליהל םגר , PPARβ/δ לא אךRXR ו PPARα PPARγ , שיפעול כי הראו התוצאות. הקטלאז

. -Synביטוי ללא הפעילות לרמת וחזרה קטאלז פעילותב

משפיע -Syn ביטוי, השומן חומצות במטבוליזם ותהתערב דרך כי מציעה אני אלו תוצאות על בהתבסס

את המפעיל כליגאנד נורמלי באופן משמשות אשר, שומן. ח של מטבוליות נגזרות או השומן. ח הרכב על

. PPARγ בפעילות הפגיעה PPARγ חולי במוחות חמצוני נזק ולהיווצרות קטלאז בביטוי לירידה גורמת

בפעילות והחפיפה מהמורכבות מושפעת -Syn י"ע קטאלז ביטוי על הההשפע כי לציין חשוב. פרקינסון

. הגרעיניים הרצפטורים

,לסיכום

במחלות α-Syn רעילות המערב הפתוגני המנגנון להבנת תורמות זו בעבודה התוצאות

ספציפי ובאופן במוח לשומנים תפקיד ישנו הפתולוגי במנגנון כי מצביעות התוצאות. הסינוקלאינופטיות

( PUFA) רוויות בילתי רב שומן לחומצות