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All Theses and Dissertations (ETDs)

Summer 9-1-2014

The Role of Sleep in Memory Consolidation with an Induced The Role of Sleep in Memory Consolidation with an Induced

Sleep-Like State in Drosophila AND The Role of Alpha-synuclein in Sleep-Like State in Drosophila AND The Role of Alpha-synuclein in

Fatty Acid Metabolism Fatty Acid Metabolism

Yasuko Suzuki Washington University in St. Louis

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WASHINGTON UNIVERSITY IN ST. LOUIS

Division of Biology and Biomedical Sciences

Neurosciences

Dissertation Examination Committee:

Paul T Kotzbauer, Chair

John R Cirrito

Erik Herzog

David M Holtzman

Jin-Moo Lee

Steven J Mennerick

Timothy M Miller

The Role of Sleep in Memory Consolidation with an Induced Sleep-like State in Drosophila

AND

The Role of Alpha-synuclein in Fatty Acid Metabolism

by

Yasuko Suzuki

A dissertation presented to the

Graduate School of Arts & Sciences

of Washington University in

partial fulfillment of the

requirements for the degree

of Doctor of Philosophy

August 2014

St. Louis, Missouri

Copyright by

Yasuko Suzuki

2014

ii

Table of Contents

Acknowledgements

iv

Chapter 1. The Role of Sleep in Memory Consolidation with an Induced Sleep- like

State in Drosophila

1

Preface 2

Abstract 4

Introduction 5

Materials and Methods 8

Results

PART I : Pharmacological method to induce a sleep-like state in flies 13

1.1 Increased quiescence in Cs flies following Gaboxadol treatment

meets all of the behavioral criteria of a spontaneous sleep

13

1.2 Knockdown the expression of GABA receptors in the Mushroom

Bodies and Clock cells alters the response in flies following

Gaboxadol treatment

17

1.3 Increased quiescence following Gaboxadol treatment facilitates a

long-term memory formation

19

1.4 Increased quiescence following Gaboxadol treatment does not

enhance a short-term memory in Cs flies, but improves a short-

term memory in memory mutants

20

1.5 Increased quiescence following Gaboxadol treatment may

improve short-term memory formation in Hydroxyurea (HU)

treated Cs flies

25

1.6 Increased quiescence following Gaboxadol improves long-term

memory deficits in period (per01

) and blistered (bs1348

) flies

26

PART II: Study the role of sleep in memory consolidation using

sleep-induction

28

2.1 a short duration of an induced sleep-like state (<4hr) following

learning is sufficient to facilitate memory consolidation

28

iii

2.2 Sleep that is induced 24 hours after the training also facilitates

memory consolidation

30

2.3 Sleep plays an “active” role in memory consolidation: 4 hours of

induced sleep compensates the memory interference caused by sleep

deprivation

31

Discussion 33

Reference

37

Chapter 2. The Role of Alpha-synuclein in Fatty Acid Metabolism

47

Abstract 48

Introduction 49

Materials and Methods 54

Results

1.1 Alpha-synuclein cooperatively interacts with monomeric fatty acid 61

1.2 Alpha-synuclein point mutants influence the binding of the

protein to oleic acids

64

1.3 Wild type (WT) human alpha-synuclein enhances incorporation

of free fatty acid into phospholipids in HEK293FT cells

67

Discussion 71

Future directions 81

References 86

iv

Acknowledgements

I would like to express my deepest gratitude to all individuals listed below. Without your

continuous support and advice, this dissertation would not have been possible.

Dr. David M Holtzman

Dr. Jin-Moo Lee

Dr. Steven J Mennerick

Dr. John R Cirrito

Dr. Timothy M Miller

Dr. Paul T Kotzbauer and Kotzbauer lab: Dr. Chandana Buddhala, Dr. Dhruva Dhavale, Dr.

Frank Liu and Ms. Christina Tsai

Dr. Erik D Herzog

Dr. Lawrence H Snyder

Ms. Sally L Vogt

I also would like to express my appreciation to the McDonnell Center for Cellular and Molecular

Biology for providing the financial support for my last year work in Dr. Kotzbauer’s lab.

Lastly, I thank Washington University in St. Louis for providing me a wonderful learning

experience throughout this chapter of my life.

1

Chapter 1

The Role of Sleep in Memory Consolidation with an Induced Sleep-like state in Drosophila

2

Preface

I changed lab one year ago. The following chapter summarizes a portion of works I did in that

lab. However, I now have only limited access to those data. As a result, the first chapter of this

thesis is necessarily incomplete in its description of the data and analysis. More details can be

found in my co-authored publications.

1. Foraging alters resilience/vulnerability to sleep disruption and starvation in Drosophila.

Donlea J, Leahy A, Thimgan MS, Suzuki Y, Hughson BN, Sokolowski MB, Shaw PJ.

Proc Natl Acad Sci USA. 2012 Feb 14;109(7):2613-8. Doi:10.1073/pnas.1112623109.

Epub 2012 Jan30. PMID:22308351

2. Inducing sleep by remote control facilitates memory consolidation in Drosophila.

Donlea JM, Thimgan MS, Suzuki Y, Gottschalk L, Shaw PJ. Science. 2011 Jun

24;332(6073) ):1571-6. doi: 10.1126/science.1202249. PMID:21700877

3. Notch signaling modulates sleep homeostasis and learning after sleep deprivation in

Drosophila.

Seugnet L, Suzuki Y, Merlin G, Gottschalk L, Duntley SP, Shaw PJ. Curr Biol. 2011

May 24;21(10):835-40. doi: 10.1016/j.cub.2011.04.001. Epub 2011 May 5.

PMID:21549599

4. Sleep deprivation during early-adult development results in long-lasting learning deficits

in adult Drosophila.

Seugnet L, Suzuki Y, Donlea JM, Gottschalk L, Shaw PJ. Sleep. 2011 Feb 1;34(2):137-

46. PMID: 21286249

5. The perilipin homologue, lipid storage droplet 2, regulates sleep homeostasis and

prevents learning impairment following sleep loss.

Thimgan MS, Suzuki Y, Seugnet L, Gottschalk L, Shaw PJ. PLoS Biol. 2010 Aug

31;8(8). doi:pii: e1000466. 10.1371/journal.pbio.1000466. PMID: 20824166

6. Persistent short-term memory defects following sleep deprivation in a drosophila model

of Parkinson disease.

Seugnet L, Galvin JE, Suzuki Y, Gottschalk L, Shaw PJ. Sleep. 2009 Aug;32(8):984-92.

PMID: 19725249

7. Identifying sleep regulatory genes using a Drosophila model of insomnia.

Seugnet L, Suzuki Y, Thimgan M, Donlea J, Gimbel SI, Gottschalk L, Duntley SP, Shaw

PJ. J Neurosci. 2009 Jun 3;29(22):7148-57. doi: 10.1523/JNEUROSCI.5629-08.2009.

PMID: 19494137

3

8. Aversive phototaxic suppression: evaluation of a short-term memory assay in Drosophila

melanogaster.

Seugnet L, Suzuki Y, Stidd R, Shaw PJ. Genes Brain Behav. 2009 Jun;8(4):377-89. doi:

10.1111/j.1601-183X.2009.00483.x. Epub 2009 Feb 11. PMID:19220479

9. D1 receptor activation in the mushroom bodies rescues sleep-loss-induced learning

impairments in Drosophila.

Seugnet L, Suzuki Y, Vine L, Gottschalk L, Shaw PJ. Curr Biol. 2008 Aug

5;18(15):1110-7. doi: 10.1016/j.cub.2008.07.028. PMID:18674913

4

Abstract

Introduction: Sleep plays an important role in memory consolidation. However, the underlying

mechanism remains elusive. The importance of sleep in the memory consolidation process has

been demonstrated by sleep deprivation studies in which sleep loss corresponded to poor

performance on memory tasks. A sleep induction approach, that allows us to study sleep-

mediated changes in the presence of sleep, provides further understanding of sleep’s role in the

memory consolidation process.

Method: We first developed a pharmacological method to induce a sleep-like state and

examined if the induced sleep-like state modulates memory consolidation in flies. Using sleep

induction, we then evaluated if changes in duration and timing of the induced sleep cause the

changes in memory outcomes to further understand the role of sleep in the memory consolidation

process.

Results: Gaboxadol, an extra-synaptic GABAA receptor agonist, induced a sleep-like state that

meets all the behavioral criteria of a spontaneous sleep-like state in flies. The induced sleep-like

state following Gaboxadol treatment facilitated the formation of long-term memory in Wild-type

flies. Inducing a sleep-like state in different strains of flies (ex. a series of memory mutants) and

under various experimental conditions (ex. following sleep deprivation) revealed the beneficial

role of induced sleep in memory processes, as supported by the observations that an increased

sleep-like state rescued memory deficits in memory mutants and may compensate memory

impairment caused by sleep-deprivation. By altering the duration and timing of sleep induction

following the training protocol that is only able to induce a short-term memory by itself, we

observed that a short-term memory was consolidated into a long-term memory even at when a

sleep-like state was induced for a short period of time (<4hrs) or induced at later time (>24hrs)

following the training.

Discussion: Incorporating Gaboxadol into food is a novel method to increase a sleep-like state

that was behaviorally and functionally equivalent to a spontaneous sleep-like state in flies. The

ability of an induced sleep to sustain the formation of long-term memory following interference

periods supports the active role of sleep in the memory consolidation process. Future studies

using sleep induction could complement studies from sleep deprivation and contribute to

identifying genes and neuronal circuits that link sleep with memory.

5

Introduction

Sleep plays an important role in memory consolidation (Payne et al., 2009; Saletin et al.,

2011; Stickgold and Walker, 2007; Walker, 2008; Walker et al 2005; Yoo et al., 2007). The link

between sleep and memory is adjoined by the observation that sleep disruption results in poor

performance of memory tasks (Graves et al., 2003; Guan et al., 2004). In addition, recent studies

have also suggested that sleep is implicated in several neurodegenerative disorders (Brown,

2002; Hayflick et al., 2013; Iranzo, 2013; Ju et al., 2014; Ju et al., 2013; Lehmann et al., 2013;

Malow, 2004; Sixel-Doring et al., 2014). However, how sleep affects the memory consolidation

process remains unclear. Additional experimental approaches, such as sleep induction, could aid

in a better understanding of the molecular mechanism that underlies how sleep affects memory

consolidation.

We used the fruit fly Drosophila melanogaster as a model to study the role of sleep in

memory for a few simple reasons. Its small size, rapid reproduction rates, approximate 15000

gene number and short life cycle make studies efficient and easily replicated. Its well

characterized biology and genetic tools such as the GAL4-UAS system allow easy manipulation

of gene expression in multiple ways (Hendricks et al., 2000). Moreover, although flies are

relatively simpler organisms when compared to mammals, the neural circuits that control

complex biological processes such as sleep and memory are well developed. Flies exhibit a

sleep-like state (Hendricks et al., 2000a; Hendricks et al., 2000b; Nitz et al., 2002; Shaw et al.,

2000) and are able to form memory (Blum et al., 2009; Tully et al., 1994; van Swinderen, 2009).

A sleep-like state in flies is also directly correlated with performance in memory task, as sleep

loss in flies corresponds to poor performance (Seugnet et al., 2009a; Seugnet et al., 2011a;

Seugnet et al., 2011b; Seugnet et al., 2009b; Seugnet et al., 2009c; Seugnet et al., 2008). Since

6

the ability of flies to recapitulate the effect of sleep on memory and many aspects of sleep appear

to be controlled genetically, fly does present an useful model for identification of the genes and

neuronal circuits that link sleep and memory.

Previously, taking advantage of fly genetics, we developed a method to induce a sleep-

like state in flies (Donlea et al., 2011). This sleep-induction approach allowed us to determine

changes in memory in the presence of sleep, apart from the sleep-deprivation approach in which

the function of sleep is proposed based on the negative consequences of losing sleep.

Beneficially, correlating findings from both the sleep-induction and sleep-deprivation approaches

could help in the process to determine the function of sleep; the sleep induction approach

provides additional experimental strategies that would otherwise be difficult with the sleep-

deprivation approach. While the use of genetics to stimulate a group of neurons in the fly’s brain

is one way to induce a sleep-like state, developing an additional approach which is independent

of genetics could open the door to a wide range of applications.

In fact, sleep-inducing drugs have been developed to increase amount of sleep for treating

sleep abnormalities and disturbances. Although these drugs increase inactive states, whether

medication-induced inactive states resemble to the spontaneous occurring sleep remains a

question. Since the levels of endogenous melatonin correlates with sleep-wake cycle, the

regulatory effect of increased melatonin level on sleep was studied. Although melatonin

treatment decreased time for falling sleep, melatonin treatment had no effect on maintaining

sleep (van den Heuvel et al., 2005). In contrast, Gaboxadol, an agonist for the δ-subunit of

extrasynaptic GABAA receptors, increases slow wave sleep and to maintain sleep in rats and

human (Dijk et al., 2012; Lancel, 1997; Lundahl et al., 2012; Lundahl et al., 2007).

7

Since the ability of Gaboxadol to increase a sleep-like state in flies remains unknown, in

the first part of this study, we tested if Gaboxadol induces quiescence in flies and characterized if

the increased quiescence following treatment had the same properties as spontaneous sleep.

In the second part of this study, we evaluated the effect of additional sleep-like states on

memory consolidation in flies. Since the sleep induction method allows temporal control of

induced sleep, we determined if modification in the duration and timing of additional sleep-like

states are associated with changes in memory.

In sum, results from this study provide a new method using drugs to induce a sleep-like

state in flies. Together with the previously demonstrated genetic method, the pharmacological

method could validate sleep induction as a method effectively to study the function of sleep. In

addition, the results from this study have also contributed to a better understanding of the effect

of additional sleep on memory consolidation. Future studies in sleep induction could

complement studies from sleep deprivation and contribute to identifying genes and neuronal

circuits that link sleep with memory.

8

Materials and Methods

Flies

Flies were maintained on a diet (yeast, dark corn syrup and agar) at 25°C with approximately 50-

60% humidity under light (8am-8pm): dark (8pm-8am) cycle. Flies used in this study: Canton-S

(CS) flies were used as Wild-type flies throughout this study, 104y-GAL4, 30y-GAL4, c929-

GAL4, UAS-NachBac, UAS-Lcch3-RNAi, UAS-GRD-RNAi, UAS-RDL-RNAi, UAS-

GABABR1-RNAi, UAS-GABABR2-RNAi, UAS-GABABR3-RNAi, Blistered

(w*;P{GAL4}bs1348

), Period ( per01). Flies used in experiment 1.4 (figure 9): rut2080>104y/+,

rut2080>UAS-NachBac, rut2080;104y-GAL4>UAS-NachBac, dnc1>104y/+, dnc1>UAS-

NachBac, dnc1;104y>UAS-NachBac; dnc1 line was kindly provided by Dr. Bruno Van

Swinderen.

Memory mutant flies listed were ordered from Bloomington Fly Center. Dunce (dnc1), Latheo

(al[1]b[1]lat[6]c[1]sp[1]/SM5), Leonardo (P{ry[+t7.2]=IArB}14-3-3 zeta[P1188]/CyO;ry[506]),

Lio2 (w[1118];Df(2R)lio[2],pigeon[2]drl[2]), volado (scb[Vol-2]/CyO)

Pharmacology

Both Gaboxadol (sigma-aldrich CAS#85118-33-8) and SKF97541 were incorporated into fly

diet (yeast, dark corn syrup and agar) to reach final concentrations of 0.1mg/mL or 0.5mg/mL for

Gaboxadol and 40μM for SKF. Gaboxadol targets GABAA receptors whereas SKF targets

GABAB receptors.

Hydroxyurea (HU) was used to chemically ablate mushroom bodies in fly larvae. Briefly, flies

were allowed to lay eggs on the freshly prepared apple juice plates. Hatched larvae was picked

and transferred to either the starvation plate (10% agar in dH2O) with yeast paste with HU (HU

9

group) or the starvation plate with yeast paste only (Vehicle control group) for 4 hours. After 4

hours of the treatment, larvae were transferred to a vial containing normal food (yeast, dark corn

syrup and agar).

Measurement of sleep-like state in flies

Sleep and locomotor activity patterns of flies were measured continuously. Briefly, flies were

individually placed into a 65mm glass tube that contains food at one end. Each tube containing

fly was then placed into Drosophila Activity Monitor (TriKinetics). Fly locomotor activity was

calculated from the number of times a fly crosses the midline of tube. Sleep was defined as a

period of quiescence that lasts for at least 5 minutes. Total sleep time, average sleep bout

duration and other sleep parameters were calculated using the Microsoft Excel.

Sleep deprivation and measurement of sleep rebound

Flies were individually placed into a 65mm glass tube that contains food at one end. Each tube

was then inserted into a vertical Drosophila Activity Monitor (TriKinetics). The sleep-nullifying

apparatus (SNAP) was used to sleep-deprive flies without activating stress response. The SNAP

tilts asymmetrically from -60° to +60° such that each fly is displaced during the downward

movement 10 times/minute. Following sleep deprivation, flies were allowed to recover. Sleep

rebound was calculated as % of sleep (min) gained during recovery periods in relative to baseline

sleep (min) divided by the total sleep (min) lost during sleep deprivation.

10

Measurement of arousal threshold

Flies were individually placed into a 65mm glass tube containing food. Each tube was inserted

into a vertical Drosophila Activity Monitor (TriKinetics) to continuously record locomotor

activity and sleep. The sleep-nullifying apparatus (SNAP) was used to perturb flies.

Arousal threshold was defined as the number of flies awakened when they were exposed to a

mechanical perturbation using the SNAP.

Aversive phototaxic suppression (APS) test

APS test is a learning assay that tests the ability of flies to learn to inhibit their affinity to light in

the presence of aversive stimulus, as previously described (Le Bourg and Buecher, 2002;

Seugnet et al., 2009b). Briefly, this assay uses a learning apparatus that contains two transparent

polystyrene vials vertically positioned at the end of the arms of the T-maze. One vial was lighted

by a fiber-optic light source (the lighted vial) while the other vial was covered by a gray plastic

cap such that no light can pass through (the darkened vial). During each learning test, a filter

paper of the lighted vial was wetted by 320μL of 1M Quinine hydrochloride (the aversive

stimulus in this assay) whereas that of the darkened vial was kept dry. Each fly was transferred to

the entrance of the T-maze, allowed to walk through the T-maze and choose between the lighted

and darkened vial. After each fly made a choice, the fly was removed from the vial and re-

transferred to the entrance of the maze for the subsequent trials. Each fly was tested in 4 blocks

of 16 trials. To avoid the effect of the right/left bias or the remaining odorous traces in the vial

choice, the location of lighted vial was altered as RRLLRRLLRRLLRRLL, where R and L

represent the lighted vial was on the right and left side of the maze, respectively. Time of

completion for 16 trials and their choice for each trial were recorded. Photopositive (P) was

11

recorded when the fly chose the lighted vial whereas Photonegative (N) was recorded when the

fly chose the darkened vial. The leaning score was indicated by the number of photonegative

choices and calculated for 4 blocks of 4 trials. Microsoft Excel was used to calculate the average

and standard error of the mean (SEM). A higher learning score indicates good learning. Flies

(n>8) were tested for each conditions. Flies chose the darkened vial during the first block (first 4

trials) were excluded from further experiment since their low light sensitivity may affect the

choice in this assay.

Courtship conditioning assay

Courtship conditioning is an associative learning assay in which naïve male flies learn to inhibit

courtship behaviors by negative experience of rejection (Ganguly-Fitzgerald et al., 2006). This

assay is consisted of two components: training and memory evaluation. Depending on the

experimental design, generally, memory was evaluated at 48 hours following the training.

Briefly, a naïve male fly was exposed to a phenomenally feminized male fly (Tai2) in a 65mm

glass tube for a period of time (in total of 3 hours). During this “training” period, a naïve male

attempts to court with unreceptive Tai2 male but receives repeated rejections from Tai2 male.

Following the training period, a “trained” male fly was separated from the tai2 male and placed

to a new 65mm glass tube until the memory evaluation. During memory evaluation period, a

“trained” naïve was again exposed to a new Tai2 male in a 65mm tube for 10 minutes under the

video recording. The courtship index (CI), the time that the “trained” male spent in courtship

behaviors during the 10 minutes, was recorded. CI of “trained” male was compared to age and

genotype matched “naïve” male. The reduction in CI indicates memory.

12

Modification of training methods in the “training” component of the courtship conditioning assay

induces different types of memory. 3 hours of consecutive training (a massed training) only

produces a short-term memory (STM) that cannot be detected at 48 hours following the training.

In contrast, three 1 hour- training that has 1 hour-rest in-between each training (a spaced

training) induces a long-term memory (LTM) that can be detected at 48 hours following the

training.

Statistics

We analyzed data using student-t test (two-tailed, unpaired). In all the experiments, P values less

than 0.05 are considered to be statistically significant.

13

Results

PART I: Pharmacological method to induce a sleep-like state in flies.

1.1 Increased quiescence in CS flies following Gaboxadol treatment meets all of the

behavioral criteria of a spontaneous sleep.

Because the effect of Gaboxadol on sleep-like state in flies remains unknown, we

incorporated Gaboxadol (sigma-aldrich CAS#85118-33-8) into the flies food and measured

changes following treatment. We observed that quiescence in flies was increased in a Gaboxadol

dose-dependent manner following treatment (Figure 1a). In more detail, the observed increases

in quiescence were mostly during the day (Figure 1 b) than at night (Figure 1c) following

Gaboxadol treatment. Since flies sleep most of the night, less change in quiescence at night could

simply be due to the saturation effect. We also observed a slight increase in the average day and

night bout duration following Gaboxadol treatment (Figure 1 d and e). Gaboxadol has been

reported to increase slow wave sleep and maintain sleep in rats and human (Lancel, 1997;

Lundahl et al., 2007). Since the values for bout duration represent the length of an episode of a

sleep-like state, our observation that average night bout duration was increased following

0.1mg/mL Gaboxadol suggests the ability of Gaboxadol to maintain a sleep-like state in flies.

14

15

In addition to EEG studies that dissect sleep into different stages, spontaneous sleep can

also be behaviorally defined (Hendricks et al., 2000a; Hendricks et al., 2000b). Behaviorally,

sleep is a state during which the quiescence and arousal threshold are increased. Sleep is a

rapidly reversible state, which distinguishes sleep from coma. Finally, sleep is homeostatically

regulated, that losing sleep increases the sleep needed to gain back the lost sleep. To address the

question that increased quiescence in CS flies following Gaboxadol treatment is equivalent to

spontaneous sleep, we further evaluated arousal threshold, rapid reversibility and homeostatic

response during increased quiescence following Gaboxadol treatment. To measure the arousal

threshold, we counted the number of sleeping flies that woke up by a mechanical disturbance.

We found similar levels of increased arousal threshold in flies during spontaneous sleep as

during the increased quiescence following 0.1mg/mL Gaboxadol treatment (Figure 2). Also, a

further increase in arousal threshold was observed during the increased quiescence following

0.5mg/mL Gaboxadol (Figure 2).

16

The observation that flies respond to a mechanical disturbance during increased quiescence

following Gaboxadol treatment indicates that increased quiescence was rapidly reversible. To

determine if losing the increased quiescence mediates a homeostatic response, we sleep deprived

flies during Gaboxadol treatment and measured the presence of sleep rebound following sleep

deprivation. We found that when spontaneous sleep (Figure 3a) or increased quiescence (Figure

3b) following Gaboxadol in flies was lost by overnight sleep deprivation, flies exhibited an

increased sleep-like state following the sleep deprivation (Figure 3c and d).

Collectively, our results indicate that while quiescence and arousal threshold was

increased during the increased quiescence following Gaboxadol treatment, the increased

17

quiescence was rapidly reversible. The increased quiescence following 0.1mg/mL Gaboxadol

was regulated by homeostasis, as evidenced by the observation that flies exhibited sleep rebound

to recover a decrease in quiescence. Therefore, the increased quiescence following Gaboxadol

met all the behavioral criteria of spontaneous sleep.

1.2 Knockdown the expression of GABA receptors in the Mushroom Bodies and Clock cells

alters the response in flies following Gaboxadol treatment.

Gaboxadol targets δ-units of extra-synaptic GABAA receptors in mammals (Lancel,

1999; Winsky-Sommerer et al., 2007) . To determine if the increased quiescence following

Gaboxadol treatment is mediated through the activation of GABA receptors in flies

(Buckingham et al., 2005; Hosie et al., 1997), we used RNAi to knockdown the expression of six

GABA receptors; Lcch3, GRD, Rdl, GABABR1, GABABR2 and GABABR3, in the Mushroom

bodies (MB: 30y-) and Clock cells (c929) and evaluated the response in flies following

Gaboxadol treatment. We observed that knocking down the expression of Lcch3, GRD or

GABABR1 subunits in the MB (Figure 4 a) and clock cells (Figure 4 c) significantly reduced the

amount of increased quiescence following 0.1mg/mL Gaboxadol treatment. In addition, because

a GABAB agonist, SKF also increases quiescence in flies, we also evaluated the effect of the

GABA receptor knockdown on the response following SKF treatment. We observed that the

increased quiescence following SKF treatment was reduced if the expression of GABAB

receptors (R1, R2 and R3) were abolished in the MB (Figure 4 b). Knocking down expressions

of any six GABA receptors (Lcch3, GRD, Rdl, GABABR1, GABABR2 or GABABR3), in clock

cells had no effect on the response following SKF treatment (Figure 4d).

18

In sum, our results suggest that the expressions of three GABA receptors; Lcch3, GRD,

and GABABR1, in the MB and clock cells are required to mediate the increased quiescence

following Gaboxadol treatment.

19

1.3. Increased quiescence following Gaboxadol treatment facilitates a long-term memory

formation.

Flies form a long-term memory when sleep-like state was increased by genetic method

following a massed training protocol of Courtship conditioning that itself does not induce LTM

(Donlea et al., 2011). To determine if the increased quiescence following Gaboxadol treatment

facilitates a long-term memory formation, quiescence was increased for 4 hours by 0.1mg/mL

Gaboxadol treatment immediately following the massed training protocol and memories were

evaluated. No long-term memory was observed in flies following a massed training in the

absence of increased quiescence (Figure 5a). In contrast, a long-term memory was observed in

flies that had increased quiescence by Gaboxadol treatment following a massed training (Figure

5b).

Like spontaneous sleep, our observations suggest that increased quiescence by Gaboxadol

treatment has the ability to facilitate long-term memory formation.

20

1.4 Increased quiescence following Gaboxadol treatment does not enhance a short-term

memory in CS flies, but improves a short-term memory in learning mutants.

A short-term memory (STM) in flies can be evaluated using an APS test (Seugnet et al.,

2009b). Although CS flies learn normally in the APS test, we asked if additional quiescence

following Gaboxadol could make CS flies even better learners. The quiescence was increased in

CS flies for 2 days by Gaboxadol treatment prior to the APS test and learning was evaluated.

Increased quiescence following Gaboxadol in CS flies had no effect on their learning, as

evidenced by the observation that the performance of Gaboxadol-fed flies in the APS test was

similar to that of their age-matched non-drug-fed controls (Figure 6). While quiescence was

increased in CS flies following Gaboxadol treatment, the observation that the increased

quiescence did not enhance learning prompts us to hypothesize that the beneficial effect of the

increased quiescence on STM may be demands-specific.

21

A series of memory mutant flies were identified previously using a classic olfactory

learning paradigm (Boynton and Tully, 1992; Dura et al., 2007; Grotewiel et al., 1998; Mery et

al., 2007; Philip et al., 2001; Pinto et al., 1999; Qiu and Davis, 1993; Skoulakis and Davis, 1996;

Tully and Quinn, 1985). Among these mutants, we found nine mutant flies; rut2080, dunce, pst-1,

latheo, Leonardo, volado, lio, forS2 and dumb2, that also present learning impairment in the APS

test. We measured quiescence in these mutants following Gaboxadol treatment. As in CS flies

(Figure 7a), quiescence was increased in each mutant following Gaboxadol treatment (Figure 7b-

j).

22

23

Except in dumb2 flies, the increased quiescence improved short-term memory in all the mutants

(rut2080, dunce, pst-1, latheo, Leonardo, volado, lio2) (Figure 8a and c-h). The beneficial effect

on short-term memory was mediated by increased quiescence, as supported by the observation

that no improvement in short-term memory was observed if flies were sleep-deprived during

Gaboxadol treatment (Figure 8a and c-h).

To further verify that the improvement of STM deficits is sleep-like state dependent,

sleep-like state was increased by genetic method (Donlea et al., 2011) using 104y>NachBac and

associated changes in memory were evaluated. We observed that STM deficits in rut2080 and

dunce were also reversed using 104y>NachBac (Figure 9a-b).

24

25

1.5 Increased quiescence following Gaboxadol treatment may improve short-term memory

formation in Hydroxyurea (HU) treated CS flies.

Since lio2 flies present structural abnormality (Moreau-Fauvarque et al., 1998) and

Gaboxadol induced a sleep-like state that improved their learning, we further evaluated the effect

of increased quiescence on memory deficits caused by structural abnormality. Mushroom Bodies

(MB) is a critical structure in the flies brain (Strausfeld et al., 1998) that regulates both sleep

(Guo et al., 2011) and learning and memory (Akalal et al., 2006; Blum et al., 2009). Treatment

with Hydroxyurea (HU) in larvae chemically ablates MB; therefore HU-treated flies have a

reduced sleep-like state and are impaired in short-term memory formation (de Belle and

Heisenberg, 1994). Based on the role of MB in a sleep-like state and memory, we first

determined if the quiescence is increased in HU-treated CS flies following Gaboxadol treatment

and then evaluated short-term memory. Quiescence was increased by Gaboxadol treatment (data

not shown). While the increased quiescence improved short-term memory deficit in HU-treated

flies (Figure 10a), the increased quiescence did not improve long-term memory (Figure 10b-c).

26

1.6 Increased quiescence following Gaboxadol improves long-term memory deficits in

period (per01

) and blistered (bs1348

) flies.

Since long-term memory formation following a spaced training is impaired in per01

and

bs1348

flies, we determined if increased quiescence following Gaboxadol treatment can rescue

LTM deficits in these flies. Quiescence in per01

and bs1348

flies were increased for 2 days by

Gaboxadol treatment prior to the spaced training, flies were removed from Gaboxadol food

during the training, and flies were placed back onto Gaboxadol food for 24 hours following the

27

training. We observed that increased quiescence in per01

and bs1348

flies following Gaboxadol

treatment improved long-term memory deficits (Figure 11c-d and f-g).

28

PART II: Study the role of sleep in memory consolidation using sleep-induction.

Since sleep is required to consolidate short-term memory, produced by massed training,

to a long-term memory that can be observed at 48 hours following the training, changing the

duration and timing of the induced sleep could further dissect the temporal relationship between

induced sleep and long-term memory formation. First, to address the question on how much

sleep is required to facilitate long-term memory formation, we determined the ability of a short

duration of induced sleep (<4hr) to consolidate memory (Result 2.1). Next, we evaluated if

modification in the timing of sleep induction is associated with changes in memory (Result 2.2).

Lastly, to challenge the hypothesis that sleep only passively protects memory from degradation

by simply acting as a shelter, we tested the ability of induced sleep to compensate sleep-

deprivation-mediated memory interference (Result 2.3).

2.1: a short duration of an induced sleep-like state (<4hr) following learning is sufficient to

facilitate memory consolidation.

To address the effect of sleep duration on memory consolidation, we induced a short-

duration (<4hr) of sleep using a genetic method (Donlea et al., 2011) in flies immediately

following the massed training protocol and then evaluated the memories at 48 hours after

training. Flies that slept for either 1 hour or 2 hours immediately following the massed training

were able to form a long-term memory (Figure 12 a-b), whereas flies that were only massed

trained without sleep exhibited no memory (data not shown). To further determine if the

beneficial effect on memory consolidation was sleep-dependent, we sleep-deprived flies while

the circuit was activated. We found that activation of the circuit alone produced no memory

(Figure 12c). Collectively, these data indicate that a short duration (<4hrs) of induced sleep was

29

sufficient to consolidate memory. Importantly, sleep, rather than the activation of Fan-shaped

body alone, mediates the beneficial effect on memory consolidation.

30

2.2 Sleep that is induced 24 hours after the training also facilitates memory consolidation.

In addition to determining the effect of sleep duration on memory consolidation, we also

examined the effect of sleep timing on memory consolidation. 4 hours of sleep were induced in

flies at 24 hours following massed training and memories were evaluated. We found that flies

that slept for 4 hours post massed training presented a long-term memory (Figure 13a). Notably,

we observed that 2 hours of sleep at 24 hour after the massed training also facilitated long-term

memory formation (Figure 13b). Collectively, these results suggest the ability of induced sleep to

facilitate memory consolidation even when it is introduced at a later time.

31

2.3 Sleep plays an “active” role in memory consolidation: 4 hours of induced sleep

compensates the memory interference caused by sleep deprivation.

A memory system contains three consecutive stages: encoding, consolidation and

retrieval. During encoding stage, information through sensory inputs is converted to a memory

trace which is susceptible to incoming interference. The newly formed memory trace is stabilized

during the consolidation process and is being stored until the retrieval stage at when the stored

memory is recalled. Despite the involvement of sleep in the memory consolidation process,

sleep’s role in memory consolidation remains the center of debate. Two hypotheses (Ellenbogen

et al., 2006a; Ellenbogen et al., 2006b)have been proposed for how sleep is involved in memory

consolidation. The “passive” hypothesis posits that sleep passively protects memory from

degradation by serving as a temporal shelter from any negative effect of ongoing cognitive

activity. In contrast, the “active” hypothesis argues that sleep can either directly or indirectly

trigger the memory consolidation process in addition to supplying the reduced interference

period.

Whether sleep facilitates memory consolidation by only supplying the reduced

interference period remains to be defined. Our observation that induced sleep at later time also

facilitates memory consolidation (section 2.2) supports the notion that sleep may have an “active”

role in memory consolidation, rather than sleep only acts as a shelter of interference. To address

this question, we determined the effect of induced sleep on a long-term memory formation

following the interference period caused by sleep-deprivation. It is quite established idea that

sleep deprivation has a negative impact in memory consolidation. A work in flies also

demonstrated that sleep deprivation after the training abolishes the ability to form a long-term

memory (Ganguly-Fitzgerald et al., 2006). Flies did not form a long-term memory when sleep-

32

like state was increased for 4 hours following 4hours of sleep deprivation (Figure 14a).

Increasing sleep-like state for 4 hours following 2 hours of sleep deprivation may permit the

expression of LTM (Figure 14b).

Together, these results suggest that memory consolidation during sleep is more likely an

active process rather than a passive state in which sleep only provides a time of reduced

interference.

33

Discussion

We observed that quiescence in flies was increased by Gaboxadol treatment. The increase

in quiescence was mediated by the action of Gaboxadol through GABA receptors in flies. The

increased quiescence following Gaboxadol treatment met all the behavioral criteria of a

spontaneous sleep-like state in flies. The function of increased quiescence in memory was

evidenced by the observation that LTM was formed in flies when the quiescence was increased

by Gaboxadol treatment following a massed training that itself does not induce LTM. Together,

our observations suggest that the increased quiescence observed in flies following Gaboxadol

treatment is behaviorally and functionally equivalent to the spontaneous sleep-like state.

Therefore, Gaboxadol can be used to induce a sleep-like state in flies. Sleep induction using

drugs does not require genetic crosses; thereby it is more technically convenient and allows

further application such as high-throughput screening.

Although drug treatments often cause anesthesia, our observations suggest that increased

quiescence following Gaboxadol treatment is a sleep-like state rather than an amnestic effect of

Gaboxadol. First, the increased quiescence was rapidly reversible as evidenced by the

observation that the ability of flies to respond to a mechanical SNAP was not altered by the

Gaboxadol treatment. External stimulus mediated rapid transition between a sleep and wake state

which is a distinguished feature found only in sleep not in coma state. Second, the increased

quiescence was regulated by homeostasis. Third, the ability of increased quiescence to facilitate

memory formation is sleep-dependent, as evidenced by the observation that memory formation

was disrupted when flies were sleep-deprived while they were on Gaboxadol. The ability of

anesthesia to enhance memory consolidation has not been reported.

34

The hypothesis that additional sleep-like state following Gaboxadol treatment mediates

beneficial effect on memory formation was further supported by our observation that increased

sleep-like state following sleep induction using genetics also facilitated memory formation.

Therefore, using both methods of sleep induction, we demonstrated the ability of sleep-like state

to facilitate memory formation in flies.

Furthermore, our observations that increased quiescence in flies following Gaboxadol

treatment rescued STM and LTM deficits in memory mutants suggest the therapeutic role of

additional sleep in memory deficit. In flies, most memory formation, if not for all, requires the

Mushroom bodies (MB), a structure in flies brain that is analogous to the hippocampus in

mammals (Davis, 1993). The excitatory cAMP/PKA signaling in MB is critical for learning and

memory and gene mutations that alter the cellular cAMP level lead to memory impairment as

observed in rut2080 and dunce flies (Davis et al., 1995). In addition, MB is also innervated by

GABAergic neurons (Yasuyama et al., 2002) and inhibitory GABAergic signaling is as

important as cAMP signaling in learning and memory (Busto et al., 2010; Liu and Davis, 2009).

The proper interaction and balance between the two signals may be important for memory

formation, as that alternation in cAMP signaling-mediated suppression of GABAergic inhibition

could underline the cognitive impairments in memory mutants. Because cAMP-PKA pathway in

MB also is implicated in sleep (Chen et al., 2013; Hendricks et al., 2001; Joiner et al., 2006), it is

possible that changes in sleep may modulate cAMP-PKA pathway. Therefore, reversal of STM

deficits in rut2080 and dunce flies could be mediated by the ability of increased sleep-like state

following Gaboxadol or genetic treatment to compensate the reduction in cAMP-mediated

suppression of GABAergic inhibition.

35

Memory in flies is also affected by genes that are not in the cAMP/PKA pathway, as flies

with mutations in these genes also present memory impairment (Kahsai and Zars, 2011).

Importantly, sleep-like state in flies is also regulated by various genes, pathways and brain

regions (Bushey and Cirelli, 2011; Ishimoto et al., 2012). Our observation that increased sleep-

like state reversed STM deficits in pst-1, latheo, Leonardo, volado, lio2 flies supports the ability

of sleep to induce global effect on memory, as a result of sleep-mediated changes in expression

level of genes and protein, and the changes in excitability within and the connection between

neuronal circuits that are implicated in memory. Therefore, further studies that identify the

changes in gene transcription level and excitability of neuronal circuit that are associated with

modification of sleep would further dissect the connection between sleep and memory.

In addition, dopamine neurons are involved in the presentation of aversive information to

the MB during olfactory learning (Mao and Davis, 2009). Our observation that STM deficit in

dumb2 mutant was not reversed by the increased sleep-like state supports this function of

dopamine neurons. The function of dDA1 receptor is abolished due to the gene mutation in

dumb2 mutant, STM deficit could not be reversed simply because there is no learning to begin

with.

Moreover, our observation additional sleep rescues the STM deficit in MB ablated flies

was intriguing, given the role of MB in learning and memory (Davis, 1993; Keene and Waddell,

2007; Zars, 2000). This observation may suggest that additional sleep can bypass MB and permit

the expression of memory by the activation of other process, such as transcriptional regulation of

genes that are involved in memory. However, our results could be misleading by the incomplete

ablation of MB following hydroxyl-urea treatment. MB contains approximately 5000 Kenyon

cells (2500 per hemisphere) that are divided into three lobes: α/β, α’/β’ and ϒ (Crittenden et al.,

36

1998). Each lobe is implicated in different process of memory (Blum et al., 2009; Davis, 2011;

Krashes et al., 2007; McGuire et al., 2001). Further studies that examine the correlation between

degree of MB ablation and memory performance in individual fly could address the role of sleep

in memory in the absence of MB.

Because the sleep induction method allows for a temporal control of additional sleep, we

evaluated if modifications to the duration and timing of additional sleep was associated with

changes in memory. 4 hours of increased spontaneous sleep seems to be required for a long-term

memory formation following a spaced training (Ganguly-Fitzgerald et al., 2006). Our

observation that even a short duration (<4 hour) of additional sleep facilitated memory suggest

the hypothesis that induced sleep may be better than spontaneous sleep. Studies that compare the

length that memory can last following induced sleep and spontaneous sleep is one approach to

test this hypothesis. Results from further studies could address the effect of sleep quality on

memory consolidation.

Finally, whether sleep passively or actively affects the memory consolidation process

remains to be the center of debate. We observed that additional sleep following an interference

period, either it occurred naturally or artificially by sleep deprivation, had ability to consolidate

memory. Therefore, our results support the active roles of sleep in memory consolidation. In line

with our observations, the active roles of sleep are also demonstrated in other biological

processes. Sleep regulates brain mechanism in metabolites clearance (Xie et al., 2013) and also

regulates transcription of genes including those involved in metabolism (Moller-Levet et al.,

2013).

37

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form new human memories without sleep. Nature neuroscience 10, 385-392.

Zars, T. (2000). Behavioral functions of the insect mushroom bodies. Current opinion in

neurobiology 10, 790-795.

47

Chapter 2

The Role of Alpha-synuclein in Fatty Acid Metabolism

48

Abstract

Introduction: Accumulation of aggregated alpha-synuclein protein is the defining pathologic

feature of Parkinson’s disease (PD). However, the molecular mechanisms underlying protein

aggregation remain to be defined. Because the physiological function of alpha-synuclein remains

unclear and previous studies have indicated a role in fatty acid metabolism and specifically acyl

CoA synthesis, further studies that examine the interaction of alpha-synuclein with free fatty

acids could provide a better understanding of the protein’s normal function.

Method: We examined the interaction of alpha-synuclein with oleic acid, a monounsaturated

free fatty acid, using a scintillation proximity assay (SPA). We characterized the regions of

alpha-synuclein protein that are required for oleic acid binding using mutagenesis studies. We

over-expressed alpha-synuclein in HEK293FT cells and measured its effect on the rate at which

oleic acid is incorporated into phospholipids, which is determined by the rate at which oleic acid

is converted to oleyl CoA by acyl CoA synthetase enzymes.

Results: We found that alpha-synuclein cooperatively binds to monomeric oleic acid. Mutations

that change either amino acids 30 or 46 in the alpha-synuclein protein disrupt the alpha-

synuclein-oleic acid interaction. Over-expression of WT alpha-synuclein, but not A30P alpha-

synuclein, in HEK293FT cells increased the rate of oleic acid incorporation into

phosphatidylcholine (PC).

Discussion: SPA is a novel approach to characterize the binding of alpha-synuclein to oleic acids.

Alpha-synuclein promotes an increase in the oleic acid incorporation rate in HEK293FT cells.

The absence of oleic acid binding by A30P alpha-synuclein and the corresponding absence of an

effect of A30P alpha-synuclein on the incorporation rate in cells further supports the hypothesis

that alpha-synuclein promotes acyl CoA synthesis by binding to free fatty acids.

49

Introduction

The defining pathologic change in Parkinson disease (PD) is the accumulation of aggregated

alpha-synuclein protein in Lewy bodies and neurites. Alpha-synuclein is a soluble 140-amino-

acid protein expressed throughout the central nervous system with predominant localization to

the presynaptic terminals of neurons (Iwai et al., 1995; Maroteaux et al., 1988). The structure of

the protein contains three distinct domains: the N-terminus domain, the internal hydrophobic

NAC domain and the C-terminus domain. The N-terminus (residues 1-60) contains seven

imperfect repeats (KTKEGV) that form alpha-helical secondary structure upon interaction with

phospholipid vesicles and micelles in vitro (Bisaglia et al., 2005; Chandra et al., 2003; Davidson

et al., 1998; Ulmer et al., 2005) The internal hydrophobic NAC domain (residues 61-95)

participates in self-aggregation of the protein to form fibrils that are rich in β-sheet secondary

structure (Ueda et al., 1993). The C-terminus (residues 96-140) provides structural flexibility to

the protein.

While alpha-synuclein gene mutations, including gene multiplication and point mutations,

cause rare familial forms of PD (Chartier-Harlin et al., 2004; Ikeuchi et al., 2008; Kiely et al.,

2013; Kruger et al., 1998; Polymeropoulos et al., 1997; Proukakis et al., 2013; Singleton et al.,

2003; Zarranz et al., 2004), most cases of PD are sporadic and related to a combination of

genetic and environmental risk factors. The self-aggregation of alpha-synuclein to form fibrils is

observed in all PD cases and several other neurodegenerative disorders (REF). The molecular

mechanisms underlying protein aggregation remain to be defined. It is possible that alteration in

normal function of the protein could change protein structure to the conformational state that is

prone to aggregate. Because the physiological function of the alpha-synuclein protein remains

50

elusive, further studies that identify the normal function of the protein could also aid the

understanding on the self-aggregation property of the protein.

Among many proposed normal functions of alpha-synuclein, the interaction of alpha-

synuclein with fatty acids has been observed in both in vitro (Assayag et al., 2007; Broersen et

al., 2006; De Franceschi et al., 2009; Karube et al., 2008; Lucke et al., 2006; Perrin et al., 2001;

Sharon et al., 2003; Sharon et al., 2001; Varkey et al., 2013) and in vivo studies (Golovko et al.,

2005; Golovko et al., 2006; Golovko et al., 2007). Alpha-synuclein hinders the formation of fatty

acid micelles in vitro (Broersen et al., 2006; De Franceschi et al., 2009). In vivo studies of alpha-

synuclein-KO mice revealed that fatty acid metabolism, specifically the incorporation rate of free

fatty acids into brain lipids, is altered in the absence of the alpha-synuclein gene (Golovko et al.,

2005; Golovko et al., 2007). Furthermore, a recent population-based study reported that

increased dietary intake of fatty acids lowers the risk of developing PD (de Lau et al., 2005).

Together these studies suggest that the regulation of fatty acid metabolism could be a normal

function of alpha-synuclein protein. Importantly, the notion that alpha-synuclein regulates fatty

acid metabolism could explain our recent observation that alpha-synuclein overexpression

improves motor deficit of pla2g6-KO mice, an animal model of Infantile neuroaxonal dystrophy

(INAD).

Infantile neuroaxonal dystrophy (INAD) is a fatal progressive neurodegenerative disorder

for which there is currently no treatment. Onset of INAD typically occurs in early childhood and

INAD patients display progressive motor, sensory and cognitive impairment (Farina et al., 1999;

Nardocci et al., 1999). The defining histopathologic changes in INAD include the accumulation

of alpha-synuclein in Lewy bodies and neurites, and the presence of neuroaxonal spheroids

containing tubulovesicular membranes in axons and dendrites throughout the nervous system

51

(Newell et al., 1999; Riku et al., 2013; Simonati et al., 1999; Wakabayashi et al., 2000). Notably,

mutations in the PLA2G6 gene (chromosome 22q12-q13) which encodes Ca2+ independent

phospholipase A2β (iPLA2β) are identified in most INAD patients (Gregory et al., 2008; Morgan

et al., 2006). This enzyme was originally identified in the Chinese hamster ovary (CHO) cell line

based on its ability to hydrolyze sn-2 ester bond of phospholipids to release free fatty acids and

lysophospholipids (Wolf and Gross, 1996).

The molecular mechanisms underlying neurodegeneration in INAD remain to be defined.

Because pla2g6 mutations abolish the ability of the enzyme to produce free fatty acids from the

hydrolysis of phospholipids (Engel et al., 2010), and because fatty acids are involved in many

metabolic processes, the deficiency in fatty acid production caused by Pla2g6 mutations may be

central to the pathogenesis of INAD. To better understand the mechanism by which Pla2g6

dysfunction causes neurodegeneration, Pla2g6-KO mice were generated by Neo gene insertion at

exon 9 (Bao et al., 2004). Pla2g6-KO mice replicate both behavioral and histopathological

features of human INAD including progressive motor impairment and neuroaxonal spheroid

formation throughout the nervous system (Malik et al., 2008). Compared to WT mice, Pla2g6-

KO mice present an age-dependent impairment in rotarod test (Malik et al., 2008). To further

investigate the role of alpha-synuclein protein aggregation observed in INAD, human WT alpha-

synuclein was overexpressed in Pla2g6-KO mice. Interestingly, overexpression of human-WT-

alpha-synuclein significantly improved motor deficits in Pla2g6-KO mice (unpublished data).

Fatty acid metabolism is essential to many downstream metabolic processes, including

storage of energy in the form of triglycerides, production of energy through beta-oxidation, new

lipid synthesis and remodeling and signaling (Black et al., 2000; Grintal et al., 2009; Miller et al.,

1987; Rapoport, 2001; Tol, 1975). The sources of the free fatty acids come from both outside

52

and inside of the cells. Free fatty acids from dietary intake enter cells either by passive diffusion

through the cell membrane or via flip-flop mechanism, which may be aided by FABP mediated

transport (Hamilton and Brunaldi, 2007). Inside cells, free fatty acids are generated by a variety

of lipase enzymes that release free fatty acids from phospholipids and other lipid classes. Finally,

free fatty acids, or specifically acyl CoAs are produced by fatty acid synthase through the de

novo fatty acid synthesis pathway that includes desaturation and elongation reactions. In order to

be utilized in downstream metabolic processes, almost all free fatty acids must be activated by

acyl CoA synthetase (ACS) enzymes to form Acyl-CoAs (Soupene and Kuypers, 2008). Pla2g6

mutations that impair catalytic activity may alter fatty acid metabolism by reducing the free fatty

acid pools that are available for activation. Therefore, approaches that increase the efficiency of

fatty acid activation could correct the altered fatty acid metabolism by compensating for

impaired free fatty acid production.

Based on the previous observation that fatty acid incorporation rates were reduced in the

absence of alpha-synuclein gene (Golovko et al., 2005; Golovko et al., 2007), and our

observation that human WT-alpha-synuclein increases the catalytic rate of ACS enzymes in vitro

(unpublished), we hypothesized that the protective effect of alpha-synuclein in Pla2g6-KO mice

is mediated by increased conversion of fatty acids to fatty-acyl-CoAs, which compensates for

impaired fatty acid production caused by Pla2g6 mutations. Further studies that examine the

alpha-synuclein-fatty acid interaction could begin to address this hypothesis.

Since previous studies examining a direct interaction between alpha-synuclein and fatty

acids have generated inconsistent results (Golovko et al., 2005; Karube et al., 2008; Sharon et al.,

2001), in the first part of our study, we characterized the binding of alpha-synuclein to free fatty

acids using a scintillation proximity assay (SPA). Because the N-terminus of the protein has been

53

reported to mediate binding to phospholipid vesicles and micelles (Chandra et al., 2003;

Davidson et al., 1998; de Laureto et al., 2006; Eliezer et al., 2001; Ulmer et al., 2005; Varkey et

al., 2013) and because the N-terminus of the protein hosts the identified point mutants that are

associated with familial PD (Kiely et al., 2013; Kruger et al., 1998; Polymeropoulos et al., 1997;

Proukakis et al., 2013; Zarranz et al., 2004), in the second part of our study, we characterized the

regions of alpha-synuclein protein that are required for oleic acid binding using mutagenesis

studies. Lastly, to determine if alpha-synuclein overexpression promotes fatty acid metabolism in

cell culture, we measured the effect of alpha-synuclein overexpression on the fatty acid

incorporation rate in HEK293FT cells.

Together, findings from this study will provide a better understanding about the direct

interaction of alpha-synuclein with free fatty acids, and the effect of alpha-synuclein-fatty acid

interaction on fatty acid metabolism. Given that fatty acids are involved in many metabolic

processes, and that the normal function of alpha-synuclein is unknown, findings on the alpha-

synuclein-fatty acid interaction could ultimately contribute to a better understanding of the

physiological function of the protein.

54

Materials and Methods

Recombinant protein expression and purification

To produce each recombinant alpha-synuclein protein, BL21 (DE3) competent cell was

transformed with pRK172 plasmids containing human WT or mutant alpha-synuclein. Control

protein was generated from BL21 cells transformed with an empty expression plasmid as

described (Bagchi et al., 2013). The colony containing each construct of plasmid DNA was

picked and inoculated in TB media at 37°C for 16 hours prior to purification. The cells were

harvested by centrifugation at 3900 xg for 10 minutes at 25°C. The supernatant was discarded.

To lyse the cells, the pellet (cells) was resuspended in 20mL of osmotic shock buffer (30mM

Tris-HCl, 2mM EDTA, 40% sucrose pH7.2), incubated in the buffer at room temperature for 10

minutes and collected by centrifugation at 8000 xg for 10 minutes at 25°C. The outer cell

membrane was removed by resuspention and incubation (3 minutes on ice) of the pellet in ice-

cold MQ water and 2M MgCl2, followed by centrifugation at 20,000xg for 15 minutes at 4°C.

The supernatant (periplasmic) was collected and DNA was precipitated by addition of

streptomycin mixture and centrifugation at 20,000xg for 15 minutes at 4°C. The supernatant was

heated at 95°C water bath for 10 minutes to further separate alpha-synuclein protein from other

proteins. To further increase the purity of alpha-synuclein, the supernatant was filtered with

0.45µm filter and loaded onto a chromatography column (Bio-Rad Laboratories) containing 1mL

of DEAE sepharose beads (Sigma) in 20mM Tris-HCl and 1mM EDTA. Each column was

washed with 10mL of ice-cold wash buffer (20mM Tris-HCl pH8, 1mM EDTA and 1mM DTT)

and each alpha-synuclein protein was eluted with 0.3M NaCl elution buffer (20mM Tris-HCl

pH8, 1mM EDTA, 1mM DTT and 0.3M NaCl). The elutant was dialyzed overnight in 5L of

dialysis buffer. The protein concentration of each construct was measured using BCA assay.

55

Mutagenesis

Forward and reverse primers for each construct were designed using online program (Agilent

technologies) and ordered from integrated DNA technologies. Using the QuikChange site-

directed mutagenesis (Agilent technology Inc), each alpha-synuclein mutant was created by a

single amino acid mutation onto the His-tagged WT alpha-synuclein-pRK172 plasmid (Table 1).

Nucleotide

location

Amino acid

substitution

WT-alpha-syn pRK172

A30P-alpha-syn pRK172 88 G to C

E46K-alpha-syn pRK172 188 G to A

H50Q-alpha-syn pRK172 150 T to G

G51D-alpha-syn pRK172 152 G to A

A53T-alpha-syn pRK172 209 G to A

Each plasmid was transformed into DH5α competent cells for DNA expression. Plasmid DNAs

from the competent cells were purified using the QIAprep®Spin Miniprep Kit following the

manufacture’s protocol (Qiagen). The concentrations of purified plasmid DNA were determined

by measuring absorption at 260 nm using NanoDrop spectrophotometer. Nucleotide sequences of

all plasmids were confirmed by DNA sequencing.

Scintillation Proximity Assay (SPA)-binding assay

The binding interaction of alpha-synuclein with oleic acid was examined using Scintillation

Proximity Assay. Chelated copper PVT SPA beads (PerkinElmer) and purified his-tagged empty

56

vector/alpha-synuclein proteins were diluted in 30mM Tris-HCl pH7.4 buffer such that the final

concentrations of SPA bead and protein were 0.2mg/mL and 4µg/mL, respectively. 3H-labeled

oleic acid (American radiolabeled chmicals Inc) was mixed with unlabeled oleic acid at a ratio of

1:100. Oleic acid was first diluted in DMSO to 100x of each final concentration, and then diluted

in 30mM Tris-HCl pH7.4 buffer to the final concentration of 0.5, 2, 3.5, 5, 6.5, 7 and 8µM. To

reduce the non-specific binding of protein to the plastic plate, each well of the 96-well

microplate was coated with 200µL of 0.02% gelatin (Sigma) and incubated at 37°C for 30

minutes prior to each experiment. 0.02% gelatin was aspirated and each well was washed twice

with 200µL of MQ water. We first added 25µL of SPA beads to each well, and then we added

25µL of either control protein or alpha-synuclein protein. 50µL of oleic acid mixture at different

concentrations were added to each well as the last component. The plate was sealed with

adhesive plastic film and incubated at 37°C for 2 hours. The plate was cooled to room

temperature for 10 minutes before it was read in the MicroBeta scintillation counter

(Perkinelmer).

We plotted count per minutes (cpm) detected at the different concentrations of oleic acid. Bmax,

Kd, and hill constant were determined using nonlinear regression by fitting the data to a one-site

binding model with hill slope:

(y= Bmax*Xh/Kd

h+X

h).

Bmax = the concentration of oleic acid at which the binding sites on the receptors are 100%

occupied.

Kd = the concentration of oleic acid at which half of the binding sites on the receptors are

occupied.

57

Hill constant = the degree of co-operativity

Dynamic Light Scattering (DLS)

We determined critical micelle concentration (CMC) of oleic acid using Dynamic light scattering.

Various concentrations of oleic acid were prepared by dissolving oleic acid in 30mM Tris-HCl

pH7.4 buffer. The intensity of scattered light at each concentration was measured at 25°C using

Nano ZS (Malvern Instrument, UK) with a measurement angle of 173°.

Transfection in HEK293FT cells

HEK293 cells were maintained in complete DMEM media (DMEM, 10% FBS, L-Glutamine and

Penicillin/Streptomycin) in T-75 flasks. Cells in the flask were briefly washed with 5mL of PBS

and dislodged with 1mL of Trypsin. The action of trypsin was stopped by adding 4mL of

complete DMEM media (10% FBS, L-glutamine, NO antibiotics) into the flask. The cells were

resuspended thoroughly in order to obtain a single-cell suspension and were transferred into a

new 50mL Falcon tube (ALL tube). To prepare the cell sample for counting (COUNT tube),

0.5mL of cell suspension was added into a new 50mL Falcon tube containing 4.5mL complete

DMEM media (10%FBS, L-glutamine, NO antibiotics). Cell numbers were determined by

counting cells using a hemocytometer. Cells were transfected in 6-well plate. To enhance the

adhesion of cells onto the wells, each well was coated with 500µL of 1xPoly-D-Lysine

Hydrobromide (sigma #P1149) for 2 hours prior to the transfection. HEK293 cells were

transfected with either pcDNA3.1 Vector maxi or WT/mutant alpha-synuclein using

Lipofectamine2000. The cells were incubated in DNA/Lipofectamine mixture (state the amount

of DNA, lipofectamine, and number of cells per well) overnight at 37°C. The morning after

58

transfection, the media was changed to complete DMEM media with 10% FBS (DMEM,

10%FBS, L-glutamine, Penicillin/Streptomycin).

14C Oleic acid pulse-labeling

HEK 293 cells were maintained in complete DMEM media with 10% FBS (DMEM, 10%FBS,

L-glutamine, Penicillin/Streptomycin). 2 hours prior to the pulse-labeling, the media was

changed to the complete media with 2% dialyzed FBS (DMEM, 2% Dialyzed FBS, L-glutamine

and Penicillin/Streptomycin). The pulse-solution containing 1mM fatty-acid free BSA, 14

C-oleic

acid and TBS (10mM Tris and 150mM NaCl) was prepared fresh and incubated at 37°C for 30

minutes before pulsing the cells. To pulse-label the cells, 1mL of media was removed and 10µL

of pulse-solution was added to each well. The plate was incubated at 37°C for 15 minutes.

Cell harvesting and extraction

Immediately following the 15 minutes pulse-labeling, the cells were harvested in ice-cold 1xPBS.

The cells were removed from each well and transferred into respective 1.5mL Eppendorf tubes.

Cells were collected by centrifugation at 5000xg for 1minute at 4°C. The supernatant was gently

removed from each tube and remaining PBS was removed by additional centrifugation at 5000xg

for 30 second at 4°C. The cells were resuspended gently in 50µL ice-cold 1xPBS. 5µL of

resuspended cells were transferred to a new 1.5mL Eppendorf tube and saved for BCA and

ELISA assay. The remaining 45µL of resuspended cells was processed for extraction. To extract

lipid from the cells, we used chloroform/methanol/water extraction procedure (Bligh and Dyer,

1959). Briefly, 3.3 volumes (148.5µL) of 1:2 chloroform/methanol, 1.1 volume (49.5µL) of

chloroform and 1 volume (45µL) of MQ water was added to each tube containing 45µL of

resuspended cells in order. The tubes were vortexed after each addition. Following centrifugation

59

at 2000rpm at 25°C for 5minutes, separation into two phases (top aqueous and bottom organic

phase) was observed. Both aqueous and organic phases were carefully removed and transferred

into respective 1.5mL Eppendorf tubes. The bottom organic phase was used for TLC analysis.

Thin Layer Chromatography (TLC)

To separate each species in the extracted organic phase, 10μL of cell suspension from each

sample plus four lipid standards (14

C-PC, 14

C-LPC, 14

C-acyl-CoA and 14

C-Oleic acid) were

applied onto a silica gel coated glass plate. The TLC plate was developed in the

Chloroform/methanol/water (65/25/4) system and visualized using a FLA-7000 phosphoimager

(Fuji). The radioactive intensity of each spot was determined using Multigauge software (Fuji).

Micro BCA Protein Assay

5μL of resuspended cell extract were dissolved in 45μL of TBS (25mM Tris-HCl pH8 and

150mM NaCl) buffer+1%Triton X and sonicated in a water bath for 1 minute. We used a BCA

assay Kit (Thermo scientific) to quantify the total concentration of protein in each sample.

Sandwich Enzyme linked Immunosorbent assay (ELISA)

We used sandwich ELISA to detect the level of alpha-synuclein expression in each sample. Each

well of the microtiter plate was coated with 50µL of carbonate coating buffer containing capture

antibody (1µg/mL Asyn-211) and incubated overnight at 4°C. The following day, each well was

washed 5 times with 1xPBS-tween 20 (150µL) and patted on a paper towel to remove the

residual coating mixture. Protein-binding sites in each coated well were blocked with 150µL of

blocking buffer (2% BSA-PBS) and incubated for 2 hours at 37°C. During the incubation we

prepared samples for the standard curve by dilution of each purified recombinant alpha-synuclein

60

protein in the sample buffer (300mM Tris-HCl pH8, 0.25% BSA, 1X aprotinin, 1x Leupeptin

and 0.1% triton in PBS) to the final concentration of 0, 0.1, 0.3, 0.9, 2.7, 8.1, 24.3 and

72.8ng/mL. The 1000-fold diluted unknown sample and samples for the standard curve were

loaded to each respective well, and the plate was incubated overnight at room temperature. Next

day, each well was incubated with the detection antibody (1µg/mL FL-140) and then secondary

antibody (HRP-anti rabbit), followed by detection with TMB and measurement of absorbance at

650 nm.

Statistics

We analyzed data using student-t test (two-tailed, unpaired). In all the experiments, P values less

than 0.05 are considered to be statistically significant.

61

Results

1.1 Alpha-synuclein cooperatively interacts with monomeric fatty acid.

We determined the binding of alpha-synuclein to oleic acid using a scintillation proximity

assay (SPA). Recombinant human WT alpha-synuclein protein was purified using previously

established method (Bagchi et al., 2013). The purified recombinant protein migrates as a single

peak with a relative molecular weight of 57 kD in size-exclusion chromatography under native

conditions. Using static light scattering (SLS), we also found the size of the purified recombinant

protein to be around 57±7.27 kDa, consistent with the previously reported size of monomeric

protein. To determine the interaction of alpha-synuclein with oleic acid, purified his-tagged-

human WT-alpha-synuclein protein was incubated with a range of different concentrations of 3H-

labeled oleic acid in the presence of chelated copper PVT beads. Because 3H oleic acid may

interact directly with SPA beads, nonspecific binding was determined in parallel reactions

containing oleic acid and beads without recombinant alpha-synuclein. We calculated the alpha-

synuclein dependent binding at each oleic acid concentration by subtracting the non-specific

binding from the total binding. A saturation binding curve was generated by plotting the specific

binding versus the oleic acid concentration (Figure 1). Interestingly, we observed a sigmoidal

relationship between specific binding values and oleic acid concentration, indicating cooperative

binding of alpha-synuclein to oleic acid. We analyzed the data by fitting the curve to the

equation using non-linear regression and obtained Kd, Bmax and Hill coefficient values. We

observed specific binding of alpha-synuclein to oleic acid with a Kd of 4.27 μM, a Bmax of

2599.25 cpm and a Hill coefficient of 4.83. Notably, these binding values were consistent across

multiple independent sets of experiment. Because the hill coefficient represents the degree of

cooperativity and values of hill coefficient >1 indicate positive cooperativity, these results

support cooperative binding of oleic acid to alpha-synuclein.

62

Figure 1. Alpha-synuclein cooperatively binds to oleic acid. The binding interaction of alpha-

synuclein with oleic acid was characterized using scintillation proximity assay (SPA) at a range

of different oleic acid concentrations (0-8μM).

Fatty acids are amphiphilic molecules that contain hydrophobic tails and hydrophilic

heads, which can aggregate to form micelles, similar to other surfactants. The concentration

above which fatty acids form micelles is defined as the critical micelle concentration (CMC).

Because oleic acid can form micelles and because alpha-synuclein has been observed to interact

with SDS micelles in vitro (Bisaglia et al., 2005; Ulmer et al., 2005), we investigated the

properties of oleic acid over the range of concentrations used in the binding assay, to further

understand the nature of the alpha-synuclein-oleic acid complex. To determine whether the

observed binding interaction was mediated by the binding of alpha-synuclein to monomeric oleic

acid or to oleic acid micelles, we evaluated the CMC of oleic acid using Dynamic Light

Scattering (DLS). Based on the principle that micelles can be detected by a change in light

scattering properties relative to monomeric oleic acid (Davis et al., 2011), we measured light

scattering intensity over a range of different concentrations of oleic acid. The estimated CMC of

oleic acid in our assay was 30 μM (Figure 2). This result indicates that at the concentrations

(<10µM) examined in the SPA assay, oleic acid molecules are in a monomeric state. Thus, the

63

binding observed in the SPA assay represents the interaction between alpha-synuclein and

monomeric oleic acid molecules rather than micelles.

Figure 2. Determination of critical micelle concentration (CMC) of oleic acid. The size of

oleic acid was measured using dynamic light scattering (DLS) across different oleic acid

concentrations (0-200μM). No significant light scattering was detected below 30μM, however, a

linear increase in light scattering was observed beyond 30μM (Figure 2a). A line was plotted

using the four oleic acid concentrations (50, 100, 150 and 200μM) at which changes in light

scattering were observed (Figure 2b). CMC of oleic acid was estimated by finding the x-

intercept of the line. Consistent results were obtained across multiple independent experiments.

Because we can mathematically calculate the mass of oleic acid at the concentration

where all the binding sites on alpha-synuclein were occupied (Bmax), we estimated the mass ratio

of alpha-synuclein and oleic acid in the complex. A critical factor in this calculation is the

determination of counting efficiency needed to determine disintegrations per minute (dpm) from

counts per minute (cpm). Due to the decay energy of the isotope, the counting efficiency of 3H

using scintillation fluid is 42%. The counting efficiency of PVT-SPA beads has been estimated

at 10% based on data in Dr. Kotzbauer’s lab comparing SPA to filter binding assays for 3H-

labeled ligands that bind to alpha-synuclein fibrils (Liu, J., Dhavale, D. and Kotzbauer, P.,

unpublished). Therefore, we estimated the overall counting efficiency of the assay to be 5%. The

average Bmax value from multiple experiments was 2599.25 cpm, Using our estimated counting

64

efficiency (= cpm/dpm) of 5%, we obtained the average Bmax value of 51985 dpm. The average

Bmax value (in dpm) was further converted to millimoles by first converting dpm to Curie (since

1Ci = 2.22x1012

, 51985 dpm / (2.22x1012

dmp/Ci) =2.34x10-8

Ci), and specific activity

(60Ci/mmole for 3H oleic acid 2.34x10-8

Ci / (60Ci/mmole) = 3.90x10-10 mmole) was used for

conversion to mmoles. Because 3H-labeled oleic acid was diluted 100 fold with unlabeled oleic

acid in the assay, we multiplied the value by 100 and found an average of 39 pmole oleic acid at

Bmax. Given that each reaction contains 28 pmole of WT alpha-synuclein, this represents an

approximately 1:1 mole ratio of oleic acid binding to alpha-synuclein in the SPA assay.

1.2 Alpha-synuclein point mutants influence the binding of the protein to oleic acids.

Since several lines of evidence from in vitro studies suggest that the N-terminus of alpha-

synuclein mediates binding to phospholipid vesicles and SDS micelles (Chandra et al., 2003;

Davidson et al., 1998; de Laureto et al., 2006; Eliezer et al., 2001; Ulmer et al., 2005; Varkey et

al., 2013), we asked if the binding of alpha-synuclein to oleic acid is also mediated by the N-

terminus of the protein. To address this question, we produced recombinant alpha-synuclein

protein containing familial PD-associated missense mutations that are all located in the N-

terminus of the protein, and tested their ability to interact with oleic acid in the SPA assay.

Briefly, each point mutation was created using site-directed mutagenesis. Recombinant alpha-

synuclein protein containing each mutation was purified as previously described. 0.28µM of

each purified recombinant protein was incubated with his-tagged PVT SPA beads and 3H-labeled

oleic acid at a range of different concentrations for 2 hours at 37°C. The curve was generated for

each mutant by plotting the specific binding versus the range of different concentrations of oleic

acid. Interestingly, we observed either little or no specific binding across the oleic acid

65

concentrations tested in the assay for both A30P and E46K mutants (Figure 3b-c), suggesting

that both A30P and E46K mutations disrupt the binding of alpha-synuclein to oleic acid. In

contrast, we observed sigmoidal relationships for A53T, H50Q and G51D mutants, indicating

cooperative binding of each protein to oleic acid that is very similar to WT protein (Figure 3d-f).

To determine Kd, Bmax and Hill coefficient values for each mutant, we analyzed the data with

non-linear regression. We found Kd values of 5.47μM±0.60, 4.35 μM±0.33, 6.19 μM±1.12; Bmax

values of 2409 cpm±488.4, 1553 cpm±94.0, 2619 cpm±941.9; and values of hill coefficient

5.59±2.12, 15.18±7.42, 5.02±2.31; for A53T, G51D and H50Q mutants, respectively. The

binding values of these three mutants are similar to the values of WT-alpha-synuclein (Kd =

4.77μM±0.30, Bmax = 2181cpm±198.5 and hill coefficient = 4.78±1.01), suggesting that

mutations at codon 50, 51 and 53 do not affect the binding interaction of the protein with oleic

acid. Together, these results indicate that binding of alpha-synuclein to oleic acid is also

mediated by the N-terminus of the protein. Specifically, codon 30 and 46 of N-terminus are

required for alpha-synuclein-oleic acid interaction, as evidenced by the absence of binding

observed in both A30P and E46K mutants.

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Figure 3. Alpha-synuclein mutations affect oleic acid binding. Oleic acid binding interactions

with human WT (Figure 3a), A30P (Figure 3b), E46K (Figure 3c), A53T (Figure 3d), G51D

(Figure 3e) and H50Q (Figure 3d) alpha-synuclein were characterized using scintillation

proximity assay (SPA). For both A30P and E46K alpha-synuclein proteins, no significant

binding was observed over the range of oleic acid concentrations tested in the assay (Figure 3b-

c). The oleic acid binding observed for A53T, G51D and H50Q alpha-synuclein proteins were

similar to that of WT alpha-synuclein (Figure 3a and b-f). Similar Bmax and Kd values were

observed across multiple independent experiments for each protein.

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1.3 Wild type (WT) human alpha-synuclein enhances incorporation of free fatty acid into

phospholipids in HEK293FT cells.

Although Murphy et al did not observe binding of alpha-synuclein to oleic acid using

titration microcalorimetry (Golovko et al., 2005), they observed reduction of fatty acid uptake

and incorporation into phospholipid in alpha-synuclein-KO mice (Golovko et al., 2005; Golovko

et al., 2007). Notably, they also reported that alpha-synuclein affects fatty acid metabolism

through modulation of acyl-CoA synthetases (ACS) enzymatic activity (Golovko et al., 2006).

Thus, it is likely that overexpression of alpha-synuclein may promote ACS enzymatic activity

and fatty acid incorporation rate into phospholipids.

To begin to address this idea, previous studies in Dr. Kotzbauer’s lab explored whether

alpha-synuclein modulates the activity of ACS enzymes in vitro. An in vitro enzymatic assay

was utilized to evaluate the catalytic activity of two isoforms of ACS enzyme (ACSL3 and

ACSL6) in a range of different concentrations of recombinant human WT alpha-synuclein

protein. Interestingly, they found that the catalytic activity of both isoforms increased with a

sigmoidal dose response relationship to the concentration of alpha-synuclein protein (Figure 4a

and b), indicating that the increased enzymatic activity was alpha-synuclein dependent.

Importantly, A30P mutation abolishes the effect of WT alpha-synuclein on catalytic activity of

the enzymes (Figure 4c), whereas the A53T mutation had no significant effect (Figure 4c).

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Figure 4. Over-expression of human WT, but not A30P, alpha-synuclein increases catalytic

activity of Acyl-CoA synthetases (ACS) enzymes. (Unpublished data from Dr. Kotzbauer lab)

The effect of alpha-synuclein on the catalytic activity of two isoforms of ACS enzyme: ACSL3

(Figure 4b and c) and ACSL6 (Figure 4a) were measured in an in vitro enzymatic assay. The

catalytic rates of ACSL6 (Figure 4a) and ACSL3 (Figure 4b) were increased in an alpha-

synuclein dose dependent manner. A53T alpha-synuclein increased ACSL3 activity similar to

WT alpha-synuclein, whereas A30P alpha-synuclein did not increase ACSL3 activity (Figure

4c).

Based on our finding that the A30P mutation disrupts the binding of alpha-synuclein to

oleic acid, it is possible that the A30P mutation does not promote catalytic activity of ACS

enzyme due to the absence of oleic acid binding. To determine the effect of WT and A30P alpha-

synuclein on ACS catalytic activity in vivo, we examined fatty acid incorporation rates in

HEK293 cells transfected with alpha-synuclein expression plasmids. Briefly, either human-WT

or human-A30P alpha-synuclein was over-expressed in HEK293FT cells using lipofectamine

mediated transfection. 14

C-labeled oleic acid was added to the cell media and incubated with the

cells at 37°C for 15 minutes to allow the cells to metabolize the fatty acid. We then harvested

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cells, extracted lipids, and separated individual lipid classes using thin layer chromatography

(TLC). To determine the rate of oleic acid incorporation, we compared the amount of 14

C-labeled

phosphatidylcholine (PC) in cells over-expressing human WT alpha-synuclein and cells over-

expressing human A30P alpha-synuclein to control cells over-expressing no alpha-synuclein.

Notably, we found that over-expressing human WT alpha-syuclein in the cells increased oleic

acid incorporation into PC by approximately 20% (p<0.02 from three independent sets of

experiment) (Figure 5a). In contrast, overexpressing human-A30P alpha-synuclein in the cells

had no effect on the oleic acid incorporation rate (Figure 5b), supporting the idea that oleic acid

binding and ACS enzymatic activity mediate the effect of alpha-synuclein on the fatty

incorporation rate.

Figure 5. Over-expression of human WT, but not A30P, alpha-synuclein increases oleic

acid incorporation into phosphatidylcholine (PC) in HEK293FT cells. Compared to the

control group (vector), HEK293FT cells over-expressing human WT alpha-synuclein exhibited

an average of 20% increase in the rate of oleic acid incorporation into PC (Figure 5a). Oleic acid

incorporation rate into PC was similar between vector and HEK293FT cells over-expressing

A30P alpha-synuclein (Figure 5b). The rate of oleic acid incorporation into PC was presented as

the % difference of control group (vector).

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Because the number of cells and the alpha-synuclein expression level in each sample can

affect the resulting amount of 14

C-labeled PC, we estimated cell numbers and alpha-synuclein

expression level using BCA and ELISA, respectively. We observed no difference in cell

numbers (Figure 6a-b) and expression level of alpha-synuclein between WT and A30P groups

(Figure 6c), further indicating that the increase in oleic acid incorporation is mediated by the

binding and enzymatic activity.

Figure 6. Cell numbers and alpha-synuclein expression levels were similar between

HEK293 cells overexpressing Wild-type (WT) or A30P alpha-synuclein. The number of

HEK293FT cells overexpressing human WT (Figure 6a) and A30P (Figure 6b) alpha-synuclein

were estimated using BCA assay. Cell numbers in each group were similar to vector controls.

Protein expression level of alpha-synuclein was determined using ELISA (Figure 6c).

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Discussion

In this study, we demonstrated using a scintillation proximity assay (SPA) that alpha-

synuclein cooperatively binds monomeric oleic acid with a Kd of 5 μM. Amino acid residues 30

and 46 in the N-terminus of alpha-synuclein protein are required for oleic acid binding, as

evidenced by the observation that both A30P and E46K mutations disrupt binding. We also

demonstrated that alpha-synuclein increases the catalytic rate of Acyl-CoA synthetase (ACS)

enzymes in vitro, and increases the rate of oleic acid incorporation into phosphatidylcholine in

transfected HEK293FT cells. The disruptive effect of the A30P mutation on both fatty acid

binding and enhanced FA incorporation rate indicates that alpha-synuclein promotes conversion

of fatty acids to acyl CoAs by enhanced delivery of fatty acids to acyl CoA synthetase enzymes.

Thus, our results support the hypothesis that alpha-synuclein controls a critical step needed to

utilize fatty acids in multiple metabolic pathways.

SPA is a novel assay to characterize the binding of alpha-synuclein to oleic acid

We are the first to utilize SPA to characterize the binding of alpha-synuclein to fatty acid.

Our results indicate that SPA is a valuable approach to characterize the interaction of alpha-

synuclein with oleic acid. Because SPA does not require the separation of bound from unbound

ligand, the possibility of losing bound complex due to dissociation or inefficient capture on

filters is reduced. Importantly, the binding we observe using SPA is consistent with previous

studies (Karube et al., 2008; Sharon et al., 2001), confirming a direct interaction between alpha-

synuclein and oleic acid. The interaction of alpha-synuclein with oleic acid was not observed in

study using titration microcalorimetry (Golovko et al., 2005), possibly because it lacked the

sensitivity to detect an interaction based on thermodynamic measurements. Our results extend

those of Sharon et al by demonstrating cooperativity in binding, in part because we were able to

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detect the specific binding at lower concentration of alpha-synuclein (<0.28µM). However, our

SPA requires further determination of counting efficiency in order to further increase the

accuracy of Bmax calculations. Specifically, the efficiency of his-tagged alpha-synuclein capture

by PVT-SPA beads and the efficiency with which beads capture emissions from 3H-oleic acid

require further characterization.

Characterization of the nature of alpha-synuclein-oleic acid complex

Determination of the critical micelle concentration (CMC) of oleic acid revealed that the

binding we observed in SPA represents the interaction of alpha-synuclein with monomeric oleic

acid. There are two important reasons why we determined CMC of oleic acid. First, although the

CMC of oleic acid has been estimated to be around 6μM (Davis et al., 2011), the CMC can

change depending on the experimental conditions (pH, ionic strength or temperature). Second,

fatty acid, as a surfactant, aggregates to form micelles with different sizes and shapes, which

could exert different effects on its binding partner (Walde, 2006). Therefore, we specifically

determined CMC of oleic acid under the same experimental condition where the binding

interaction was observed. Since we found that the oleic acid concentrations we used in our

binding assay were lower than the estimated CMC of oleic acid (~30uM) in our system, we

concluded that the binding we observed was between alpha-synuclein and monomeric oleic acid.

Our finding of the interaction between alpha-synuclein and monomeric oleic acid has a

physiological significance in brain where the concentration of oleic acid in CSF is approximately

3.6μM (Schmid et al., 2012).

Our observation of an interaction between alpha-synuclein and monomeric oleic acid

could explain the observed effect of alpha-synuclein on fatty acid micelle formation in vitro

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(Broersen et al., 2006; De Franceschi et al., 2009) . Since CMC is determined by solubility of

monomeric fatty acid, the reduction in free fatty acid monomer concentration by formation of

alpha-synuclein-fatty acid complexes is likely to increase CMC. Because the effect of alpha-

synuclein on CMC of oleic acid has not been determined, we can further determine the ability of

alpha-synuclein to increase CMC of oleic acid. It is also of interest to determine the interaction

of alpha-synuclein with other fatty acid species. It has been previously reported that alpha-

synuclein affects micellar behavior of polyunsaturated fatty acids (AA and DHA) but not

saturated fatty acids (arachidic and palmitic acids) (Broersen et al., 2006). Brain palmitate

metabolism was decreased in the absence of alpha-synuclein, however, the binding of alpha-

synuclein to palmitic acid was not detected using titration microcalorimetry (Golovko et al.,

2005). Therefore, we could further characterize the binding of alpha-synuclein to palmitic acids

(saturated fatty acid) and DHA (polyunsaturated fatty acid) using SPA. DHA plays important

roles in brain function. Polyunsaturated fatty acids (PUFAs) can affect membrane properties and

functions when incorporated into phospholipids (Hagve et al., 1998; Hulbert, 2008), PUFAs also

have signaling roles (Bazan et al., 2011; Grintal et al., 2009). One study suggested high binding

affinity of alpha-synuclein toward PUFAs (DHA>AA) using a competition assay in which the

radiolabeled oleic acid binding was measured in the presence of unlabeled PUFA (Sharon et al.,

2001). Direct measurements of binding interactions using 3H-labeled PUFAs would provide

further information on the binding affinities for different fatty acid species. Together, both

binding results and the effect of alpha-synuclein on micellar behavior of fatty acids indicate

direct interactions of alpha-synuclein with several different free fatty acid species.

Importantly, we were also able to relate alpha-synuclein-fatty acid interactions to the

effect of human WT alpha-synuclein on fatty acid metabolism in cells. Our cell culture studies

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revealed that human WT, but not A30P, alpha-synuclein over-expression enhanced the rate of

oleic acid incorporation into phospholipid by approximately 20%. Together with the observation

that the A30P mutation disrupted oleic acid binding, the inability of A30P alpha-synuclein to

promote oleic acid incorporation into phospholipid supports the hypothesis that the regulatory

role of alpha-synuclein in fatty acid metabolism is mediated by the direct binding of alpha-

synuclein to fatty acids. This idea is further supported by our observations in the in vitro

enzymatic assay that recombinant human WT-alpha-synuclein increases the catalytic activity of

two ACS enzymes, ACSL3 and ACSL6, while recombinant A30P alpha-synuclein has no effect

(unpublished data in Kotzbauer lab). Furthermore, the effect of alpha-synuclein in fatty acid

metabolism observed in our alpha-synuclein over-expression studies are consistent with previous

findings reported in mice with a targeted alpha-synuclein gene mutation, in which fatty acid

incorporation into phospholipid was found to be reduced through reduced enzymatic activity of

acyl-CoA synthetases (Golovko et al., 2005; Golovko et al., 2006; Golovko et al., 2007).

The effect of fatty acid binding on the structural conformation of alpha-synuclein

The N-terminus of alpha-synuclein plays an important role in its interactions with

phospholipid vesicles and SDS micelles and adopts alpha-helical structure upon the bindings

(Chandra et al., 2003; Davidson et al., 1998; de Laureto et al., 2006; Eliezer et al., 2001; Perrin et

al., 2000; Ulmer et al., 2005; Varkey et al., 2013). While studies report alpha-helical structural

change of the protein in the presence of fatty acid (Broersen et al., 2006; Perrin et al., 2001),

whether monomeric fatty acids also induce the conformational changes in the protein is unclear.

Although further studies are needed to determine the changes in alpha-helical contents, our

binding studies using mutagenesis demonstrate that the amino acids at position 30 and 46 in the

N-terminus of protein are required for fatty acid binding. Here, we proposed two possible

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hypotheses of the effect of fatty acid binding on the alpha-synuclein structure based on our

results. First, since monomeric fatty acid is far smaller in size relative to phospholipid vesicles

and micelles, it is possible that fatty acid binding may not be associated with a conformational

change of the protein from unfolded to alpha-helical structure.

Alternatively, similar to binding interactions with phospholipid vesicles (Davidson et al.,

1998; Eliezer et al., 2001) and SDS micelles (de Laureto et al., 2006; Ulmer et al., 2005), alpha-

synuclein may also adopt alpha-helical structure upon interaction with monomeric fatty acid. The

proline substitution in the A30P mutant disrupts alpha-helix formation (Bussell and Eliezer,

2001) and disrupts binding interactions with vesicles (Bussell and Eliezer, 2004; Perrin et al.,

2000). The E46K mutation also causes local disruption of alpha-helical structure but retains its

ability to interact with phospholipid. Based on the previous reports that both A30P and E46K

mutations reduce the alpha-helical content and our observations in this study that both mutants

disrupted oleic acid binding, we propose that conformational transition to alpha-helical structure

may also be important for oleic acid binding. However, the effect of the E46K mutation on

interactions with fatty acids but not phospholipid vesicles may indicate distinct structural

requirements for fatty acid binding. Further determination of alpha-helical content upon oleic

acid binding using circular dichroism (CD) could provide a better understanding of the role of

alpha-synuclein structural conformation in the interaction with free fatty acids.

Possible roles of fatty acid binding in alpha-synuclein fibril formation

Natively unfolded alpha-synuclein (Weinreb et al., 1996) adopts alpha-helical structure

upon the interaction with phospholipid vesicles (Chandra et al., 2003; Davidson et al., 1998;

Ulmer et al., 2005), but the aggregated form of alpha-synuclein found in neurodegenerative

disorders is defined by beta-sheet structure. Although the normal function of alpha-synuclein is

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not understood, structural changes related to its normal function may affect protein mis-folding

and aggregation. For example, a previous study reported that the conformational transition from

unfolded to alpha-helical structure upon the association of alpha-synuclein with membrane

phospholipid inhibits fibril formation (Zhu and Fink, 2003). The effect of monomeric fatty acid

binding on alpha-synuclein fibril formation has not been studied. While the A30P mutation is

implicated in PD pathogenesis, A30P alpha-synuclein forms fibrils slowly relative to WT alpha-

synuclein in vitro. The different observations between in vitro and in vivo experimental systems

could be explained by an effect of fatty acids. In the absence of fatty acids in vitro, the rate of

fibril formation depends primarily on the intrinsic ability of the protein to adopt a conformational

change and self-associate. On the other hand, when fatty acids are present in vivo, disruption of

fatty acid binding due to the proline substitution in part could explain the accelerated alpha-

synuclein pathology in A30P mutant. Alpha-helical structure stabilized by fatty acid binding

could reduce the probability of the protein to adopt β-sheet structure, thereby hindering protein

aggregation. Furthermore, even in the absence of an alpha-helical shift, the formation of alpha-

synuclein-oleic acid complex could hinder protein aggregation by reducing the ability of free

alpha-synuclein monomers to self –associate, either in the nucleation or extension phases of fibril

formation.

It is also possible that fibril accumulation is regulated by multiple other mechanisms.

Increased intercellular levels of alpha-synuclein monomer may promote fibril formation, as

suggested by alpha-synuclein gene duplication and triplication mutations linked to rare familiar

PD (Singleton et al., 2003). Multiplication of the alpha-synuclein gene is predicted to increase

protein production; thereby increasing the concentration of alpha-synuclein monomers in

neurons. In addition to production, clearance of alpha-synuclein can also regulate the level of

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alpha-synuclein monomers. It has been shown that alpha-synuclein protein is degraded by both

the ubiquitin-proteasome pathways as well as lysosome pathways (Lee et al., 2004; Mak et al.,

2010) and therefore alteration in alpha-synuclein clearance may also contribute to fibril

formation.

Potential role of fatty acid metabolism in the pathogenesis of PD

Our findings in this study support the regulatory role of alpha-synuclein in fatty acid

metabolism. Our findings further suggest that altered fatty acid metabolism may also be

implicated in PD pathogenesis. In fact, a population-based Rotterdam study looked at the

relationship between dietary intake of fatty acids and the risk of developing PD. The study

reported that increased dietary intake of total fat, monounsaturated fatty acids and

polyunsaturated fatty acids lowered the risk of developing PD (de Lau et al., 2005), suggesting

that altered fatty acid metabolism could contribute to the pathogenesis of PD. Furthermore,

altered fatty acid metabolism is also associated with a genetic mutation that increases the risk of

developing PD. The glucocerebrosidase (GBA) gene encodes the enzyme β-glucocerebrosidase,

which hydrolyzes glucosylceramide to produce ceramide. While homozygous mutation of GBA

gene causes an autosomal recessive lysosomal storage disorder called Gaucher’s disease,

heterozygous GBA mutations are associated with an increased risk of PD (Mitsui et al., 2009;

Sidransky and Lopez, 2012). What causes the increased susceptibility to PD in patients with

heterozygous GBA mutations remains unclear. One study compared fatty acid levels in CSF of

GBA-PD patients with both idiopathic PD and healthy controls, and found significantly lower

fatty acid levels in CSF of GBA-PD patients (Schmid et al., 2012), further supporting the

correlation of altered fatty acid metabolism with increased risk of PD. Hydrolysis of

glucosylceramide is an important intermediate step in sphingolipid metabolism in which

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sphingolipid is broken down to produce free fatty acids and sphingosine. Although

glucosylceramidase is not directly involved in the hydrolysis reaction that produces free fatty

acids, it certainly could be the rate limiting step in the conversion of glucosylceramide to fatty

acids. Therefore, reduced enzymatic activity of glucosylceramidase caused by GBA mutations

could change the rate of free fatty acid production.

The potential involvement of fatty acid metabolism in the pathogenesis of PD raises the

importance of studying dietary fatty acid intake and fatty acid levels in CSF in PD patients.

Future studies that relate changes in fatty acid levels and changes in alpha-synuclein levels in

CSF could further confirm the interaction of alpha-synuclein and fatty acids in brain and the

implication of alpha-synuclein-fatty acid interaction in the pathogenesis of PD.

Implication of alpha-synuclein-fatty acid interaction in pla2g6-KO mice

The underlying molecular mechanism of how PLA2G6 mutation causes

neurodegeneration remains to be defined. Since the conversion of free fatty acid to acyl-CoA is

an essential step that regulates downstream metabolic processes, deficiency in free fatty acid

production caused by PLA2G6 mutations is considered as one of the possible mechanisms

underlying neurodegeneration. Our results support the role of alpha-synuclein in the fatty acid

activation step. Specifically, alpha-synuclein directly binds to free fatty acids and increases the

enzymatic activity of acyl-CoA synthetase, resulting in increased acyl-CoA production. PLA2G6

mutations cause the disruption of Pla2g6 enzyme to produce free fatty acid through the

hydrolysis of phospholipid. Although free fatty acids are available from other sources, such as

dietary intake and breakdown by other lipases, the free fatty acid pools that are available for

conversion to acyl-CoA are likely to be significantly reduced in INAD. Based on our findings of

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the role of alpha-synuclein in the fatty acid activation step, we hypothesize that the alpha-

synuclein mediated increase in acyl-CoA production could compensate for the deficiency in free

fatty acid production in Pla2g6-KO mice. Therefore, the ability of alpha-synuclein to increase

acyl-CoA production could explain why over-expression of human WT alpha-synuclein

improves the neurologic impairment of Pla2g6-KO mice.

Potential role of tetrameric structure in the interaction of alpha-synuclein with fatty acids

The observation of cooperativity in binding is of special interest in the current debate

about the structure of native alpha-synuclein. Although it remains controversial, studies have

indicated that native alpha-synuclein exists as a tetramer with alpha-helical structure rather than

a natively unfolded monomer (Bartels et al., 2011; Wang et al., 2011). In this study, to

understand the cooperativity and the nature of alpha-synuclein-oleic acid complex observed in

SPA, we estimated the mole ratio of alpha-synuclein to oleic acid to be approximately 1:1.

Cooperative binding indicates that binding of a ligand to one binding site on a receptor increases

the binding affinity of the ligand to other sites on the same receptor. If the 1:1 ratio is correct, it

indicates that alpha-synuclein must exist as a multimer in order to produce cooperative binding,

and at least four alpha-synuclein monomers must be present in the complex in order to explain

the hill coefficient of 4 observed in our assays. Alternatively it is possible that we underestimated

the Bmax value for oleic acid in the assay, possible because we over-estimated the efficiency of

dpm detection in the assay, in which case it is possible that 4 or more oleic acid molecules bind

to a single alpha-synuclein monomer in a cooperative fashion. We estimated the molecular

weight of recombinant alpha-synuclein to be 57kD using static light scattering (SLS) which is

consistent with the size reported for both the disordered monomer as well as the alpha-helical

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tetramer. Further size measurements in the presence of fatty acid, perhaps in combination with

circular dichroism studies to measure alpha-helical content, could help determine structural

changes that occur when alpha-synuclein binds to fatty acids.

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Future Directions

In our study, we demonstrated the binding interaction of alpha-synuclein with oleic acid

using SPA assay. Although oleic acid is a common monounsaturated fatty acid, it is only one

type of many fatty acids that exist. Structurally different fatty acids (ex, saturated vs unsaturated)

play different roles in lipid metabolism. Previous studies suggest that alpha-synuclein interacts

with polyunsaturated fatty acid (ex. DHA, AA) but not with saturated fatty acids (ex. Palmitic

acid) although affinities were not measured or compared (Broersen et al., 2006). To further

understand the interaction of alpha-synuclein with fatty acids, we propose to evaluate the binding

of alpha-synuclein to other fatty acids species using the same method we used for oleic acid. If

the binding is observed, we can examine binding affinities (Kd value) of alpha-synuclein toward

different fatty acids in our SPA assay. Since each fatty acid may be involved in different

metabolic pathways, differences in binding affinity will be helpful to understand the potential

role of alpha-synuclein in these particular metabolic pathways.

Our results from in vitro and cell culture studies support the regulatory role of alpha-

synuclein in fatty acid metabolism by increasing acyl-CoA production. To further determine if

alpha-synuclein increases Acyl-CoA levels in vivo, we propose to study the effect of alpha-

synuclein over-expression on fatty acid incorporation in mice. Using the in vivo kinetic method

described by others (Golovko et al., 2006; Rapoport, 2001), we can measure the kinetics of brain

oleic acid metabolism in WT alpha-synuclein transgenic mouse line (M7 syn) in which human

WT alpha-synuclein is over-expressed in neurons (Giasson et al., 2002). Based on the results in

this study, we expect to detect a higher rate of oleic acid incorporation into phospholipids in M7

syn transgenic mice, compared to wild-type mice. Such observations will further support our

findings in this study. In addition, our results suggested that the fatty acid binding is required for

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alpha-synuclein to regulate fatty acid metabolism. To further examine the effect of binding

interaction on the ability of alpha-synuclein to promote acyl-CoA synthesis, we propose to

compare the kinetics of brain oleic acid metabolism in wild-type mice to that in wild-type mice

over-expressing either the A30P or E46K mutation.

Based on the ability of alpha-synuclein to increase acyl-CoA production, we predicted

that an alpha-synuclein-mediated increase in acyl-CoA synthesis compensates the deficiency in

free fatty acid production caused by PLA2G6 mutation in Pla2g6-KO mice. To further test if our

prediction underlies the protective effect of human WT alpha-synuclein over-expression on the

motor performance observed in Pla2g6-KO mice, we propose to measure the kinetics of brain

oleic acid metabolism in Pla2g6-KO/human WT alpha-syn transgenic (Pla2g6-/-syn+/+) mice

and appropriate controls; Pla2g6-KO/human WT alpha-syn non-transgenic (Pla2g6-/-syn-/-),

Pla2g6-WT/human WT alpha-syn transgenic (Pla2g6+/+syn+/-) and WT (Pla2g6+/+syn-/-) mice,

using the same in vivo kinetic method described previously (Golovko et al., 2006; Rapoport,

2001). If we detect a higher rate of oleic acid incorporation into phospholipid in Pla2g6-

KO/human WT alpha-syn transgenic (Pla2g6-/-syn+/+) mice compared to Pla2g6-KO/human

WT alpha-syn non-transgenic (Pla2g6-/-syn-/-), this observation will confirm our prediction that

the protective effect of alpha-synuclein on motor performance is mediated by increased acyl-

CoA synthesis which compensates for impaired free fatty acid production caused by PLA2G6

mutations.

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Currently, no cure is available for INAD. Based on our knowledge about the possible role

of fatty acid metabolism in pathogenesis of the disease and findings in our current study, further

studies can be proposed to test potential therapeutic approaches that target the fatty acid

activation pathway in INAD. For instance, if our hypothesis that an alpha-synuclein-mediated

increase in acyl-CoA synthesis underlies the observed improvement in motor performance of

Pla2g6-KO/human WT alpha-synuclein transgenic mice is confirmed, such observations will

support acyl-CoA synthesis as a therapeutic target for treating INAD. While the ability of alpha-

synuclein to promote acyl-CoA production is supported by our studies and others (Golovko et al.,

2005; Golovko et al., 2006), increasing the expression level of alpha-synuclein in INAD

patients is impractical approach due to the self-aggregation property of the protein. Instead, it is

appropriate to target ACS enzymes. Therefore, identifying and developing drugs that increase the

protein expression level of ACS enzymes or that increase enzymatic activity could increase acyl-

CoA production in INAD patients.

Retinoid X receptor (RXR) is a ligand-dependent nuclear receptor transcription factor

that can interact with other transcriptional activators (ex. liver X receptor and peroxisome

proliferator-activated receptor) to increase the expression of enzymes that regulate cholesterol

and fatty acid metabolism (Martin et al., 2000; Yoshikawa et al., 2001). Sterol response element

binding protein (SREBP-1c) is a transcription regulator that binds to sterol regulatory elements

of genes that encode enzymes for fatty acid synthesis; thereby up-regulation of SREBP-1c

expression increases the expression level of fatty acid synthetases that produce acyl CoAs

(Martin et al., 2000). For example, Bexarotene, a FDA approved RXR agonist, increases ACSL3

mRNA levels. Brain ACSL3 mRNA level was increased by 41% in mice treated with

Bexarotene diet compared to mice with control diet (Bagchi and Kotzbauer, unpublished results).

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Interestingly, one study also reported that nicotine increases mRNA and protein level of ACSL6

in mouse brain (Chen et al., 2011). Further studies could test whether these approaches to

increase brain acyl CoA levels reduce neurologic impairment in Pla2g6-KO mice.

A better understanding of therapeutic targets is helpful for probing the molecular

mechanisms of drug actions and for identifying features that guide new drug design. Therefore, a

better understanding of brain ACS enzymes is helpful in developing approaches to increase

activities of these enzymes. ACS enzymes play a critical role in fatty acid metabolism. Except

for eicosanoid metabolism, esterification of free fatty acid by ACS enzymes to acyl-CoA is

required for fatty acids to be utilized in downstream metabolic processes. In addition to their

enzymatic functions to activate free fatty acids, they also regulate cellular uptake and channeling

of fatty acids (Mashek et al., 2007). ACS enzymes are grouped into families depending on the

acyl-chain lengths of free fatty acids to which they interact (Soupene and Kuypers, 2008).

Isoforms in each ACS family differ in their substrate preferences and intracellular localization

(Lewin et al., 2001; Mashek et al., 2007; Soupene and Kuypers, 2008), suggesting the activity of

each enzyme to regulate fatty acid metabolism in a localization-dependent manner. Of the eleven

enzymes from the three gene families; long-chain acyl-CoA synthetases (ACSLs), Bubblegum

acyl-CoA synthetases (ACSBG) and Fatty acid transporter protein (FATPs), ACSL3, ACSL6,

FATP4 and ACSBG1 are highly expressed in the brain (Chen et al., 2011; Golovko et al., 2006;

Hall et al., 2005; Kee et al., 2003; Pei et al., 2003). However, the physiologic function and

localization of each enzyme within the brain have not been completely characterized. Since the

direct protein-protein interaction was not detected between alpha-synuclein and two isoforms

(ACSL3 and ACSL6) of ACS enzymes using co-immunoprecipitation (data not shown), we

hypothesized based on our results that alpha-synuclein binds to fatty acid and then delivers fatty

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acid to the enzyme sites for increased fatty acid activation. Further studies that examine the

localization patterns of ACS in neurons can test our hypothesis. The observation of ACS

enzymes in dendrites, axons and presynaptic terminals where alpha-synuclein and pla2g6 are

localized will further support our hypothesis.

The results of our binding studies also have potential implications in the pathogenesis of

PD. We speculate that the interaction of alpha-synuclein with fatty acids could reduce the

probability of the protein to self-aggregate, because of either the conformational changes to more

stable structure or the reduction of free monomer concentration. Using an in vitro fibril

formation assay, we can determine the effect of fatty acid on the rate of fibril formation for WT,

A30P, and E46K alpha-synuclein protein. An observed reduction in the fibril formation rate for

WT but not for A30P and E46K in the presence of fatty acid could support the notion that fatty

acid binding affects self-aggregation. Cell-based assays and transgenic mice have been

developed to study fibril formation by alpha-synuclein protein. To further study the relationship

between fatty acids and fibril formation, we could add fatty acid to a cell-based aggregation

assay and examine changes in the rate of fibril formation. We also could modulate the level of

fatty acids in the diet for transgenic mice and evaluate corresponding changes in the

accumulation of fibrillar alpha-synuclein.

86

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