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The effect of familial mutants of Parkinson's disease on membrane remodeling

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THE EFFECT OF FAMILIAL MUTANTS OF
PARKINSON’S DISEASE ON MEMBRANE
REMODELING

ANURI. N. SHAH

A thesis submitted in partial fulfillment of the requirements for the degree of
Master of Science in Molecular Pharmacology and Toxicology

University of Southern California
School of Pharmacy
Fall 2013

Effect of Familial Mutants of Parkinson’s Disease on Membrane Remodeling Anuri Shah
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ABSTRACT
The pathological hallmark of Parkinson’s disease (PD) is the presence of Lewy bodies or
cell inclusions in neurons, comprising an amyloid protein known as alpha synuclein. These
aggregates of protein are normally found with the presence of lipid fragments. Whether these
fragments of lipid are derived from disrupted cellular membranes is unknown but more and more
evidence corroborates the plausibility of this phenomenon.
A recent breakthrough in the realm of alpha synuclein research is suggestive of its
membrane binding and curvature inducing ability. Alpha synuclein has been shown to tubulate
negatively charged phospholipid vesicles and even remodel them into nanoparticles at higher
protein: lipid ratios in the oligomeric state. This property of alpha synuclein is similar to that of
apolipoproteins, which are lipid carriers found in the blood stream. Studies till date have been
targeted at understanding better how these properties of alpha synuclein play a physiological
role, likening it to a lipid carrier or chaperone in exo/endocytosis. On the other hand, if these
very same properties are out of control they may surpass physiological relevance and set in
motion hazardous events such as cellular permeabilization, having neurotoxic implications that
might abet the pathophysiology of PD.
The 3 familial mutants of alpha synuclein associated with PD i.e, A30P, E46K and A53T
have consistently over the years shown a range of different properties. The A30P and A53T
mutants are known to be more prone to aggregation. Previous literature suggests that it is this
property of theirs which confers them toxic. The E46K mutant however does not exhibit this
property and must therefore cause cellular toxicity by another mechanism. To gauge this
predicament better, I aimed to characterize the membrane remodeling properties of these familial
mutants using tools such as clearance assays, leakage assays and electron microscopy. The goal
was to see whether these mutants possessed enhanced or diminished membrane remodeling
abilities and whether this could be a trigger for neurodegeneration. The E57K mutant which is a
well- known artificial but toxic mutant was tested as well. Based on the results of my study, the
E46K mutant exhibited greater membrane leakage and clearance, possibly implying that it
generates its toxic effects by remodeling membranes to an exaggerated extent and disrupts their
nature. A30P and A53T on the other hand were not proficient in their membrane remodeling and
leakage causing abilities further consolidating that the major role they play is of enhanced
aggregation. Thus they could bring about their effects by enhanced misfolding. Therefore two
distinct mechanisms for toxicity seem plausible based on these results, which could then be used
as an ideal tool for studying physiological membrane remodeling gone awry and how that might
lead to disease.

Effect of Familial Mutants of Parkinson’s Disease on Membrane Remodeling Anuri Shah
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TABLE OF CONTENTS
ABSTRACT……………………………………………………………………………………….i
CHAPTER I: INTRODUCTION……………………………………………………………… 4
1.1 Parkinson’s disease: Overview………………………………………………………………. 4
1.2 Alpha synuclein: Structure & Function……………………………………………………… 6
1.3 Alpha synuclein: In vitro properties…………………………………………………………. 9
1.4 Alpha synuclein and membranes…………………………………………………………… 12
1.41 Alpha synuclein and phospholipids………………………………………………….... 13
1.42 Alpha synuclein: Curvature induction……………………………………………….... 14
1.43 Alpha synuclein: Membrane remodeling…………………………………………….... 17
1.5 Structure of membrane bound alpha synuclein……………………………………………... 20
1.6 Alpha synuclein and apolipoproteins……………………………………………………….. 23
1.7 Alpha synuclein mutants……………………………………………………………………. 24
CHAPTER II: MATERIALS AND METHODS …………………………………………… 28
2.1 Purification of alpha synuclein……………………………………………………………... 28
2.2 Gel electrophoresis…………………………………………………………………………. 29
2.3 Determination of concentration of proteins………………………………………………… 29
2.4 Clearance Assay…………………………………………………………………………….. 30
2.5 Circular Dichroism………………………………………………………………………….. 31
2.6 Leakage Assay……………………………………………………………...………………. 31
2.7 Electron Microscopy……………………………………………………………………...… 32
2.8 Buffers………………………………………………………………………………….…… 32

CHAPTER III: RESULTS……………………………………………………………………. 34

DISCUSSION………………………………………………………………………………….. 55

REFERENCES……………………………………………………………………………...…. 58

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TABLE OF FIGURES

CHAPTER I

Figure1. Lewy bodies…………………………………………………………………………..… 5
Figure2. Alpha synuclein: Structure………………………………………………………….….. 7
Figure3. Alpha synuclein: Repeats…………………………………………………………….… 8
Figure4. Possible fates of alpha synuclein…………………………………………………...…. 10
Figure5. Alpha synuclein: Prion like properties……………………………………………..…. 12
Figure6. Alpha synuclein with POPG vesicles……………………………………………….… 15
Figure7. Alpha synuclein: Membrane blebbing………………………………………………… 15
Figure8. Apolipoproteins and alpha synuclein with phospholipids…………………………….. 16
Figure9. Alpha synuclein: Curvature induction………………………………………………… 17
Figure10. Alpha synuclein: Tubule formation……………………………………………….…. 18
Figure11. Alpha synuclein: Nanoparticles……………………………………………………… 19
Figure12. Alpha synuclein: Nanoparticles……………………………………………………… 19
Figure13. Alpha synuclein: Alpha helix……………………………………………………..…. 21
Figure14. Alpha synuclein and micelles……………………………………………………...… 21
Figure15. Alpha synuclein: Helical wheel……………………………………………………… 22
Figure16. Alpha synuclein: Alpha helix on nanoparticles……………………………………… 22
Figure17. Alpha synuclein: Superhelical twist…………………………………………………. 23

CHAPTER III

Figure18. Clearance assay principle………………………………………………………….… 35
Figure19. Clearance assay of mutants………………………………………………………. 35-36
Figure20. Clearance assay summary…………………………………………………………… 37
Figure21. Circular dichroism of mutants…………………………………………………… 39-40
Figure22. Leakage assay principle……………………………………………………………… 41
Figure23. Leakage assay principle……………………………………………………………… 42
Figure24. Leakage assay of mutants………………………………………………………… 42-43
Figure25. Leakage assay summary…………………………………………………………...… 44
Figure26. Electron microscopy of POPG control…………………………………………….… 45
Figure27. Electron microscopy of alpha synuclein with POPG………………………………... 45
Figure28. Membrane blebbing………………………………………………………………….. 46
Figure29. Electron microscopy of A53T with POPG……………………………………….….. 47
Figure30. Electron microscopy of A30P with POPG………………………………………..…. 47
Figure31. Electron microscopy of E46K with POPG………………………………………...… 48
Figure32. Electron microscopy of E46K with POPG………………………………………..…. 49
Figure33. Electron microscopy of E57K with POPG………………………………………...… 49
Figure34. Electron microscopy of brain lipids control……………………………………….… 50
Figure35. Electron microscopy of alpha synuclein with brain lipids…………………………... 51
Figure36. Electron microscopy of A30P with brain lipids……………………………………... 51
Figure37. Electon microscopy of E46K with brain lipids…………………………………...…. 52
Figure38. Clearance assay summary with brain lipids……………………………………….… 53
Figure39. Circular dichroism of mutants with brain lipids…………………………………...… 53
Effect of Familial Mutants of Parkinson’s Disease on Membrane Remodeling Anuri Shah
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CHAPTER I: INTRODUCTION

1.1 PARKINSON’S DISEASE: OVERVIEW
Parkinson’s disease is known to affect seven to ten million people worldwide. In the
United States alone, the cost of Parkinson’s is an estimated $25 billion per year
1
. As of today,
only symptomatic relief for the disease is available, a breakthrough curative therapy option is
being worked on globally. Based on these statistics the impact of the disease simply cannot be
overstated.
Being the second most common neurodegenerative disease, after Alzheimer’s, a vast
array of reasons is known to be the cause of PD. Although in most cases the disease may be
idiopathic, environmental and genetic factors may also lead to this neurodegenerative process.
Environmental factors include chemicals such as pesticides and insecticides
2
, and even heavy
metal poisoning. Genome wide association studies are increasingly generating mutated alleles
which may be responsible as well. Some of these include SNCA, the gene coding for alpha-
synuclein, parkin (PRKN), leucine-rich repeat kinase 2 (LRRK2 or dardarin), and PTEN-induced
putative kinase 1 (PINK1)
3,4
.
The importance of alpha synuclein can be elucidated by the fact that the hallmark of PD
is the formation of Lewy bodies in neurons. Lewy bodies are the abnormal aggregates of
proteins, alpha synuclein in the case of PD (Figure 1). These Lewy bodies, which are found
intracellularly, are also known to contain fragments of lipids. Alpha synuclein has recently been
discovered to possess curvature inducing properties, wherein it can bind membranes and
subsequently remodel them into smaller highly curved structures
5
. The role this property of alpha
synuclein plays in forming these Lewy bodies is unknown, but the idea that these fragments of
lipids are derived from previously disrupted membranes seems plausible. Thus it could be a
membrane remodeling ability gone out of control that is responsible for the accumulation of
these bodies.
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Since the loss of brain cells is what is known to trigger the chain of undesirable
symptoms, several attempts have been made to understand how it occurs. One possible and
obvious explanation is correlating the gradual accumulation of Lewy bodies with cell death.
Whether these Lewy bodies are the reason for loss of neurons or evolve as a neuroprotective
mechanism is still unknown
6,7
. However the presence of these bodies, when diagnosed in a
patient is considered definitive proof that an individual suffers from the disease. It is the presence
of the fibrils of alpha- synuclein in these bodies that renders PD to be a form of synucleinopathy.
Synucleinopathy is a group of diseases that are characterized by the polymerization of alpha
synuclein into fibrils and get incorporated into hallmark inclusions such as lewy bodies (LB),
lewy neuritis (LN) and glial cytoplasmic inclusions (GCI). Other than PD some other forms of
synucleinopathies are dementia with lewy bodies (DLB) and multiple system atrophy.
The symptoms of PD are ascribed to a loss of dopaminergic neurons in the brain.
Symptoms begin to appear when the degeneration of these neurons reaches about 80%. As the
disease progresses, symptoms may evolve from simply motor to psychiatric. Thus a difficulty in
walking, abnormal posture, and constant shaking may worsen to conditions like dementia,
depression and cognitive disorders. Since PD is primarily a motor condition, its four cardinal
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symptoms are tremors, rigidity, bradykinesia and postural inability
8
. These can further lead to
speech disorders and difficulty with swallowing
9
, rest tremors, impaired balance
10
among others
causing a grave decline in the quality of life. Executive dysfunction is one of the primary
secondary features of PD. It leads to cognitive imbalance and thus affects tasks like planning,
organizing, and a decrease in inhibiting inappropriate behavior versus initiating appropriate
behavior. A patient with PD is two to six times more susceptible to developing dementia than an
unaffected individual
8,11
. Fluctuations in attention span and a difficulty with recollecting
information have also been observed. These being only a small pool of the entire gamut of
symptoms associated with the condition, it is no doubt that billions of dollars are being invested
in unraveling the obscurity of the disease.
The therapy for PD has been greatly effective in alleviating motor symptoms of the
disease. Since there is no cure for the disease, but symptoms are claimed to arise due a
deficiency of dopamine, most treatments aim at increasing dopamine levels back to normal.
Therefore dopamine agonists, such as bromocriptine and pramipexole or MAO-B inhibitors like
selegiline that prevent metabolism of dopamine are the most commonly prescribed drugs.
However, more efforts are now being made to work on curative treatments, for which a more
insightful study of the pathophysiology of the disease needs to be done. We saw the extent of
involvement alpha synuclein has with the progression of the disease. Therefore it is not
unexpected that a profuse amount of research is directed at understand the structure and function
of alpha synuclein. Pharmaceutical advances are also increasingly aimed at controlling this
formation of Lewy bodies and studying the spiral of events brought about by alpha synuclein
further. Some of the newer studies include development of anti-apoptotic agents and clinical
trials are being carried out to test vaccines against alpha synuclein
12
. Alpha synuclein is now
undeniably established as one of the major players in PD. Several attempts are being made today
to comprehend the impact alpha synuclein has on the disease so that appropriate therapy options
can be formulated, which might lead to the development of curative medicine for PD.

1.2 ALPHA SYNUCLEIN: STRUCTURE & FUNCTION
The synuclein family includes three known proteins till date i.e., alpha, beta and gamma
synuclein, encoded by the SNCA, SNCB and SNCG genes respectively. Although the mystery of
their exact physiological role is still being unraveled, they are all known to show a pathological
presence. Gamma synuclein is known to be a marker for breast tumor progression
13
and is found
primarily in the peripheral nervous system and retina
14
. Beta synuclein on the other hand is found
mainly in brain tissue and more so in presynaptic termini. It has been shown to constitute some
of the neurofibrillary lesions in patients with Alzheimer’s but no association with the Lewy
bodies found in Parkinson’s has been shown. Beta synuclein is also known to inhibit the
aggregation of alpha synuclein.
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Alpha synuclein forms a major portion of the class of proteins known as intrinsically
disordered proteins or amyloid proteins. Others include β amyloid, Exon 1 of huntingtin, IAPP in
diabetes mellitus type II and tau protein associated with Alzheimer’s disease. The common
feature of all these proteins is that at neutral pH and in the physiological state they are all
natively unfolded or lack specific tertiary structure.
Alpha synuclein is a relatively small protein with a molecular weight of 14,400 kD and
has a primary structure of 140 amino acids. This primary sequence is further divided into 3
domains (Figure 2):
Residues 1-60: This is an amphipathic N- terminal region. This region is also known to show a
change in secondary structure, normally to α- helical when bound to membrane lipids. More
emphasis will be placed on this later.
Residues 61-95: Central region/ NAC domain, which includes the non-amyloid component,
putatively involved in protein aggregation. This is a hydrophobic region and was originally
thought to form the non- amyloid portion of Alzheimer’s disease plaques. This domain is also
known to contribute to the β -sheet conformation of the protein. It must be noted here, that β
synuclein does not contain this domain and thus has less aggregating power than alpha synuclein.
This might also be the reason for the inhibition of the aggregation of alpha synuclein in the
presence of beta synuclein
15
.
Residues 96-140: This is the acidic C- terminal region, rich in proline residues and shows no
structural relevance with respect to its role in the disease. Of the four tyrosine residues in alpha
synuclein, three of them are found in this region.

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The most interesting feature of the sequence of alpha synuclein is the presence of seven 11-
amino acid repeats in the first 95 residues (Figure 3). These amino acids contain the KTKEGV
sequence with an exception of four uncharged amino acids separating repeats 4 and 5. Domain 1
thus contains 5 of the repeats. The three familial point mutations that are commonly found
associated with PD are highlighted in the figure and are found at position 30, 46 and 53. No Cys
or Trp residues have been observed in the alpha synuclein sequence. It must however be noted
that residues 1-95 are alpha helical when bound to lipid and 34-95 form beta sheets.

Alpha synuclein constitutes ~1% of all cytosolic proteins
16
. Regions in the brain where it
has shown to be concentrated are the substantia nigra, neocortex, hippocampus and thalamus. Its
abundance especially in the substantia nigra has been shown by northern blotting and in situ
hybridization techniques
17
. Even though it is known to be a neuronal protein, some alpha
synuclein may also be found in glial cells. Alpha synuclein is known to be concentrated in the
presynaptic termini like beta synuclein and it has been shown that about 15% of all the alpha
synuclein is present bound to membranes at any given time
18
. A more recent study has also
demonstrated the localization of alpha synuclein in the mitochondria of neurons. The presynaptic
alpha synuclein may be in the free or bound form
19
. The significance of the bound form of alpha
synuclein and the implications of this membrane interaction on Parkinson’s disease are under
vigorous study and will be elucidated in greater detail later.
A range of functions can be attributed to alpha synuclein. It has been suggested that alpha
synuclein partakes in various different phenomena. These include maintenance of the synaptic
vesicle reserve pool
20
, it is known to act as a molecular chaperone in SNARE complexes and
leads to vesicle docking. It is hypothesized to do so by binding to the plasma membrane via the
N-terminal domain and Synaptobrevin, an R- SNARE protein via the C-terminal domain
21
. Thus
it seems to have a crucial role in vesicle trafficking, fusion and during synaptic activity. Related
studies have shown the role of alpha synuclein in exocytosis. Some studies have demonstrated a
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potential antioxidant activity of alpha synuclein in the brain
22
. Furthermore studies have shown
that alpha synuclein may play a role in memory and learning processes based on its increased
expression in the zebra finch during song learning
23
. The binding of alpha synuclein to
phospholipase D has also been shown although the clinical significance of this is not known
24
.
Alpha synuclein shows membrane binding, which can probably be deemed as its most
important role with relevance to lipids. The interaction of alpha synuclein with lipids is known to
have a correlation with PD. The ability of alpha synuclein to bind to negatively charged
membranes is a well- known property
25
. This binding of alpha synuclein to the membranes is
known to cause physiological changes in the membrane as well as a change in the secondary
structure of the protein itself
26
. Some studies have tried to elucidate the roles of alpha synuclein
in lipid metabolism and transport
27
, lipid organization
26
and some have even shown the potential
role of alpha synuclein in the prevention of oxidation of unsaturated lipids. However all this
being said, the exact physiological role that alpha synuclein plays in the brain remains
inconclusive and elusive.
The structure of alpha synuclein under normal, physiological conditions was studied
extensively and several conclusions were drawn. One of the first studies done by Weinreb et al.
showed by circular dichroism that alpha synuclein mainly showed a random coil structure (68%)
with traces of α-helicity (2%)
28
. The remainder of the protein was claimed to have a β- sheet
which was difficult to quantify by circular dichorism
28
. Furthermore, different conditions of
temperature, pH, presence of chemical and heat denaturants, difference in salt concentrations and
even difference in concentrations of protein itself do not affect this structural conformation of
alpha synuclein
28
. These results espouse previous findings that alpha synuclein is a highly
unstructured, unfolded protein.
Some NMR and paramagnetic relaxation enhancement experiments were done to study
the interactions between different regions of the alpha synuclein polypeptide chain
29
. It was
observed that residues 30-100 seemed to interact with the C- terminal residues 120-140 which
resulted in shielding of the hydrophobic NAC region
29
. Moreover, another study showed that a
hydrophobic cluster was formed between the C-terminal region and the NAC region which was
probably mediated by residues Met
116
, Val
118
, Tyr
125
and Met
127
.
30
Thus it has been observed that
these interactions may play a role in preventing alpha synuclein from oligomerizing.

1.3 ALPHA SYNUCLEIN: IN VITRO PROPERTIES
A crucial and extensively studied property of alpha synuclein is fibril formation. As
mentioned earlier, the fibrils of alpha synuclein have been correlated with the Lewy bodies found
in PD. A study has shown that the lipids present in Lewy bodies are derived from the
membranes previously disrupted by alpha synuclein
31
. Intense investigations into how these
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fibrils are formed and how they are included into Lewy bodies have put forth a bunch of theories.
Studies have shown that alpha synuclein can aggregate into species rich in a β-sheet
conformation. These structures are known as oligomers or protofibrils (Figure 4). Some
conditions that might potentially favor this aggregation are oxidative stress, genetic
predispositions and environmental toxins. Certain processes such as nitration could also expedite
this process of oligomer formation
32
. The oligomers in turn, have different fates, as highlighted
in the figure. Further aggregation of the protofibrils will lead to the formation of fibrils that form
the inclusions of Lewy bodies
32
. This particular study also showed the toxic effects of
protofibrils as compared to fibrils. The cytotoxicity was shown to be more pronounced with
protofibrils than fibrils indicating the impact of structural differences
31
. Thus the Lewy bodies
may be innocuous and might not play a role in the neurotoxicity of the disease. It has also been
shown in one particular study that the kinetics of the aggregate and fibril formation are
sigmoidal, indicating the requirement of nuclei
27
. The absence of nuclei in the initial stages
contributes to the lag phase, whereas the growth phase correlates to fibril elongation and plateau
correlates with a subsequent depletion of the soluble monomers. The validity of these hypotheses
however still remains ambiguous and the question of cytotoxicity vs neuroprotection of alpha
synuclein is still abstruse.

Recently alpha synuclein has been proposed to have prion like properties
33
. Prions are
non-living, infectious agents that transmit diseases via the induction of protein misfolding as
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opposed to conventional organisms such as bacteria and viruses which require DNA/ RNA or
both
34
. Prions have been known to misfold inherently present folded proteins, such as PrP found
in the human brain and transform it into the misfolded, diseased form PrP
Sc
. The PrP
Sc
acts as a
nucleus on which the normal PrP proteins aggregate
34
. Thus a chain reaction of sorts is created
and the prion acts as a template for this reaction. Prion diseases are known to be
neurodegenerative leading to accumulation of amyloid plaques
35
and aggregation of species rich
in beta sheets. A recent review explored the possible mechanisms that may liken the properties of
alpha synuclein to prions. Since no transmission of misfolded protein has been observed between
individuals suffering from PD, evidence has been shown to support the claim that alpha
synuclein can transmit from one cell to another
36,37,38,39
. This is in agreement with the Braak
hypothesis which states that as PD stages progress, the Lewy bodies are seen to move from the
region of the olfactory bulb into the brain stem and then into regions of the midbrain and
forebrain
40
. This hypothesis then continues to explain that alpha synuclein starts from the
peripheral nervous system and then spreads into the central nervous and this could occur via two
given routes; either from the enteric plexus of the stomach or the olfactory bulb
40
.
Investigations then aimed at highlighting the mechanism by which this cell to cell
transfer of alpha synuclein occurs (Figure 5). Alpha synuclein in the misfolded form could be
internalized into the cells either following endocytosis or an exosome mediated transport.
Whether alpha synuclein requires fusion with the plasma membrane is still unknown. Once
inside the cell, the misfolded alpha synuclein can either be degraded or partake in a seeding
process which causes aggregation of unfolded alpha synuclein. Once at the terminal, these
aggregates can further spread into adjacent neurons via exosomes and the cycle can repeat
again
40
.

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It has also been shown in some studies that alpha synuclein can cause vesicle
permeabilization by a pore-like mechanism. The protein thus induces pore formation that may
lead to rupture of vesicles. This phenomemon was especially noticed when aggregates of alpha
synuclein bind to the membrane. However, the most common and recently studied mechanism
by which alpha synuclein is known to bind and modify membranes is explained in the next
segment.

1.4 ALPHA SYNUCLEIN & MEMBRANES
The most impactful property of alpha synuclein with respect to my studies is the
membrane interaction ability of alpha synuclein. This is thought to be the basic premise for all
the functions, structural changes and roles that alpha synuclein might play in the
pathophysiology of PD. Studies have been going on over the years globally, to try and explicate
the interactions of alpha synuclein with various lipids the potential implications this could have.
The disruption of cell membranes that has been attributed to alpha synuclein continues to
confound scientists with a host of different explanations for this behavior.
In terms of normal physiological functions this property of alpha synuclein could have
implications in vesicular trafficking and vesicular budding. Furthermore, being a protein found in
presynaptic neurons, alpha synuclein membrane interactions might play a role in
neurotransmitter release as well. These very same properties however, could also lead to
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neurotoxicity in terms of disrupting membrane integrity, leading to organellar membrane
damage, Golgi and mitochondrial damage. To espouse these implications further is the
hypothesis that the lipids found in Lewy bodies are known to contain lipid fragments derived
from damaged membranes. Alpha synuclein- membrane interaction studies over the years have
highlighted pathways for us to deepen the understanding of how these roles of the protein might
come into play.

1.41 ALPHA SYNCULEIN & PHOSPHOLIPIDS
The membrane interacting abilities of alpha synuclein lead to a host of different studies.
These studies were aimed at understanding which kind of physiological membranes alpha
synuclein interacted with. Phospholipid vesicles have been used over the years to correlate this
interaction of alpha synuclein and its affinity towards phospholipid vesicles containing
negatively charged head groups was immediately noticed. Some of the phospholipids commonly
used during initial studies were the negative ones such as phosphotidyl serine (PS), phosphotidyl
glycerol (PG), phosphotidyl inositol (PI), and phosphotidic acid (PA). Neutral lipids such as
phosphotidyl choline (PC) and phosphotidyl ethanoloamine (PE) were also used. Studies using
small unilamellar vesicles (SUVs) containing the above phospholipids showed the increased
binding affinity of alpha synuclein to the acidic phospholipids. No effective binding is seen with
PC vesicles only, however when vesicles containing 30-50% acidic lipids along with PC/PE was
used binding was dramatically increased. As studies gradually progressed, interactions of alpha
synuclein with synthetic phospholipids were also shown, wherein POPC vesicles did not show
pronounced binding
20
. Another study showed the decreased binding of alpha synuclein to POPC
SUVs when compared to POPC/POPS vesicles. The protein was incubated with lipid and then
passed through a gel filtration column. It was then observed that the elution peaks contained a
mixture of the protein and lipid indicating binding affinities. It has also been demonstrated in
literature, using thin layer chromatography studies that alpha synuclein binds brain lipids
containing mainly PS, PE and PC. Interactions with cholesterol and sphingomyelin however are
almost zilch
41
. It must be noted here that alpha synuclein also seems to show binding and
structural modifications in physiologically relevant lipids such as those representing
mitochondrial and brain lipids
42
. Based on the binding affinities of alpha synuclein to different
lipids, and the occurrence of lipids in vivo, it has been concluded in literature that these binding
data cannot be holistic.
Another important consideration is that lipids are not evenly distributed in cells and
changes may be constantly occurring due to metabolism, diet, and allocation of lipids in different
types of cells. Thus a definitive in vivo- in vitro correlation cannot be drawn when alluding to
alpha synuclein. However since these basic studies were done, a lot more has been unraveled
about the interactions of alpha synuclein. It has been mentioned before that the binding of alpha
synuclein with lipids leads to a lot of changes in structure; of both, protein and lipid. More
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advanced techniques have since then been used to expound the binding effects of alpha synuclein
with different sized vesicles. I will next highlight some of the milestone findings with reference
to the mechanisms of interaction, structural changes in the lipids upon binding and the ensuing
conformational changes that occur in alpha synuclein itself.

1.42 ALPHA SYNUCLEIN: CURVATURE INDUCTION
Previous findings from our lab demonstrate the membrane curvature inducing ability of
alpha synuclein. A commonly demonstrated property of alpha synuclein shown over time was its
curvature sensing ability, or ability to bind curved membranes. Furthermore, along with this it
has also been consistently shown that when alpha synuclein is bound to these membranes it is
found in the α- helical conformation
42,43,44,
. However some data also suggested that the β- sheet
rich fibrils may be responsible for the changes caused in membranes upon binding
45,46,47
. Thus in
order to resolve this conflicting issue our lab did some comparative investigations to analyze the
curvature inducing property of alpha synuclein as well as try and gauge what was responsible for
this.
When compared to amphiphysin, a well- known curvature inducing protein involved in
endocytosis, it was observed that similar structures were formed upon membrane binding.
Proteins like endophilin and amphiphysin which are commonly involved in endocytosis are
established as curvature inducing proteins that bind membranes via curved scaffolding domains
and lead to the transformation of curved bilayers into small vesicles and tubes
5
. In these studies,
the structural changes observed in lipids were investigated upon binding of alpha synuclein.
There observations were made using different lipids i.e, non -physiological and physiological
lipids and at different protein: lipid ratios. Indeed, it was observed that alpha synuclein has the
ability to transform large POPG containing vesicles into smaller structures (Figure 6). As a
comparison, apolipoproteins, or fatty acid carrying proteins, which are known to contain the 11-
amino acid repeats were also used. Optical imaging techniques were used to observe these
interactions and it was seen that upon addition of alpha synuclein to the spherical vesicles, the
spherical nature was lost (Figure 6). Moreover this loss of spherical integrity seemed to increase
with concentration of protein. Thus a ratio 1:50 which took about 15 minutes for the process to
complete was now a meager 1.5s above a certain threshold of protein. A noteworthy observation
made here was the formation of budded vesicles, or circular structures found breaking off from
the end of tubules that were indicative of membrane blebbing when alpha synuclein was added to
vesicles containing POPG/POPC (1:1) at a P/L ratio of 1:20 (Figure 7). When POPG/POPC (1:4)
was used at a P/L ratio of 1:10 the structures formed appeared as though smaller tubules were
breaking off from larger ones (Figure 8). β synuclein did not exhibit such efficient tubulation.
However some vesicles, such as those containing PS, normally found in cell membranes did
show tubulation as well, indicating that alpha synuclein may play a role in the remodeling of
physiologically relevant lipids
5
.
Effect of Familial Mutants of Parkinson’s Disease on Membrane Remodeling Anuri Shah
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Clearance assays were used to indicate that large multilamellar vesicles (MLVs) were
being transformed into smaller structures upon the addition of alpha synuclein, as a
consequential decrease in scatter was observed. This scatter was further pronounced at higher
protein: lipid ratios of 1:20 and 1:10. Transmission electron microscopy was then used to
investigate the nature of the resulting structures formed. It was observed that the large vesicles
had been converted into thick tubules, along with the presence of few smaller vesicles as well. At
ratios of 1:10, small circular structures of diameter 25nm were seen as opposed to tubules.
Apolipoproteins were found to tubulate vesicles containing traces of anionic phospholipids.
Leakage assay then done with all the proteins mentioned above showed increased vesicle leakage
indicating that not only were smaller species being formed but the membrane was being
disrupted and remodeled, thus losing its integrity. However, the mechanism of what seemed like
a curvature inducing property still seemed unresolved. Thus the structure of alpha synuclein after
it was bound to vesicles was observed using circular dichroism studies and found to be mostly α-
helical rather than random coil. This was consistent with data shown previously in literature.
Since alpha synuclein does not have a scaffolding domain to wedge into the lipid bilayer, it was
hypothesized that the amphipathic helix of alpha synuclein, that contains the 11 amino acid
repeats was in some way responsible for this (Figure 9). In summary this study successfully
indicated the ability of alpha synuclein to induce membrane curvature in a concentration
dependent manner, since the highest P/L ratio (1:10) showed formation of the most efficiently
curved structures. Furthermore the membrane blebbing like phenomenon observed might be
indicative of the vesicle trafficking role that has been attributed to alpha synuclein
5
. Other
studies have concurrently been going to investigate other potential roles that alpha synuclein
could play in PD. These have been highlighted briefly in the next section.
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1.43 ALPHA SYNUCLEIN: MEMBRANE REMODELING
Studies since then have been intensified in order to procure data related to the different
species formed from the large vesicles after the binding of alpha synuclein. Using cryo-EM the
nature of the tubes formed as mentioned above was further investigated. It was found that with
short acyl chain lipids such as POPG micellar tubes were formed with an average width of about
50 Ȧ51. These were found at lower P/L ratios (1:40, 1:20 and 1:10). It must be noted here that at
the highest P/L ratio (1:5) it was observed that instead of tubes, the main species formed were
small disk like structures (70-100Ȧ) as compared to tubes
48
. Indeed the conformation of alpha
synuclein accompanying this phenomenon of tubule formation also changed into an extended
alpha helix. Similar observations were also made with other short acyl chain lipids such as
DMPG. As the length of the acyl chain was gradually increased, as with DOPG, DLPG and
DAPG, more bilayer tubes were seen to form in addition to the cylindrical micelles (all showing
varying widths between 45-50Ȧ). Thus, the ratio of cylindrical micelles to bilayer tubes differed
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from lipid to lipid based on length of acyl chain and degree of unsaturation present, when they
were all compared at the same P/L ratio (1:20)
48
(Figure 10). Moreover, as the degree of
unsaturation was increased, the width of the bilayer tubes was seen to increase too. The
explanation for this phenomenon however seemed inexplicable. Based on this data certain
inferences were made about the orientation of alpha synuclein in the membrane surface, as well
as the mechanism of curvature induction.

Since the length of the amphipathic helix is known to be about 140Ȧ and the diameter
of the micellar tubes observed was about 50Ȧ, the authors made a logical claim that alpha
synuclein when bound orients itself parallel to the tubes. In the case of bilayer tubes however,
since the acyl chain has bulkier tubes it is probably more difficult for the protein to wedge into
the tube surface. Thus when membrane remodeling occurs, the tubes formed retain the bilayer.
When the protein can penetrate deeper into the surface however, it is more favorable for tubes
with a micellar nature to form
48
. This process of wedging into the lipid surface gives rise to
membrane curvature. It was hypothesized that since the lipids are oriented with their bulky acyl
chains facing inwards, when the protein gets inserted on the lipid surface, it pushes the outer
head groups apart, leading to induction of tubular structures.
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The succeeding study was based on studying the other disk like structures that were
observed at high P/L ratios (1:5) with SUVs and MLVs. Upon addition of alpha synuclein to
lipid vesicles a host of different structures are formed, including tubules and smaller vesicles.
Thus the disk like structures had to be separated from them using gel filtration. When cryo- EM
was further done to study these particles it was observed that they had a roundish structure with
an average size of 7nm, thus suggesting they are nanoparticles
49
. The nanoparticles showed a
more oval than roundish structure with a thicker density appearing around the rim suggesting
accumulation of alpha synuclein on the surface (Figure 11& 12). SUVs however show less tube
formation than MLVs probably attributing to the lower availability of lipids
49
.

The structure and conformation of alpha synuclein bound to these nanoparticles was
examined next. It was found that alpha synuclein existed as oligomers on the nanoparticles as
indicated with the help of FRET analysis. However it must be noted that these oligomers do not
exist on intact vesicles but on nanoparticles exclusively. When four- pulse DEER was done in
order to estimate whether alpha synuclein had the same extended helical conformation as on
tubes it was noted that nanoparticles yielded distances of ~27Ȧ as opposed to the 46-50Ȧ found
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in an extended helix. Thus might indicate that in the presence of nanoparticles alpha synuclein
forms a double helical structure instead of an extended structure
49
.
This property of alpha synuclein was then tested with physiologically relevant lipids
such as lipids mimicking mitochondrial lipids. As hypothesized, alpha synuclein did form
nanoparticles with these lipids and was found in an oligomeric state
49
. Similar data was also
observed with fatty acids such as oleic acid. All of the above mentioned properties of alpha
synuclein indicate similarities to apolipoproteins. This may give us an indication of the
physiological relevance that alpha synuclein might have.

1.5 STRUCTURE OF MEMBRANE BOUND ALPHA SYNUCLEIN
As mentioned extensively earlier it has been observed that alpha synuclein upon
binding to membranes takes up mostly an α -helical structure. Thus the unfolded random coil
structure is now found to be an extended α-helix, with a length of 140 Ȧ. Rigorous
computational refinement and electron paramagnetic resonance experiments have been used to
validate these findings. Alpha synuclein is known to show a bent helix/ “horseshoe” like
conformation with two antiparallel helices when bound to non- bilayer or detergent micelles such
as SDS/SLAS
43,48,5,38,
. This property has been attributed to the smaller size of micelles (Figure
13&14). Via these studies it was shown that when alpha synuclein is bound to curved bilayer
membranes, it takes up an extended helical conformation, which supplements previous data.
These studies elucidate how all the 11- amino acid repeats form 3 turns of the α- helix each,
which probably indicates a modified periodicity of 3.67 residues per turn, as opposed to the
conventional 3.6. Further, this is divided into potential lipid facing and solvent facing sites which
are on opposite sides of then helix, as shown by continuous wave EPR. Residues at position 1, V
and IX in each repeat faced away from the membrane whereas III, VII and XI faced the interior
(Figure 15). Four pulse EPR studies then showed using double cysteine mutants that the
intramolecular distances were much higher in the case of alpha synuclein with unilamellar
vesicles made of POPG, than was observed with the bent helix with SDS micelles. Moreover
when gel filtration was used to separate the entities formed it was also observed that alpha
synuclein showed a bent structure when bound to certain species that were obtained after the
structural integrity of POPG vesicles was lost. This may be indicative of some species relatively
smaller in size being formed, such as the nanoparticles mentioned earlier
49
(Figure 16).

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In addition to this, structural refinement studies were used to predict the three
dimensional structures of membrane bound alpha synuclein. The results produced from these
experiments showed that alpha synuclein formed an extended, but curved structure that showed
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signs of a superhelical twist as well
34
. This curved nature of the helix could be attributed for the
modified 3.67 residues per turn ratio. Furthermore, this superhelical structure showed signs of
anisotropy, as it was arranged along the curvature of the vesicle surface (Figure 17). A top view
of this arranged also showed the arrangement of certain amino acids with the lysine residues
being aligned perpendicular to the axis and glutamate residues facing upwards. The helix is also
situated on the membrane surface in such a way that it is deep enough for the phosphate groups
in the lipid bilayer to be located just above the centre of the helix. The lysine residues can thus
interact with the phosphate groups, whereas anionic residues such as the glutamates face
outwards and at the length of the choline groups. All the non- polar residues are then evidently
exposed to lipid surfaces
46
. This study thus helped elucidate the importance of the bilayer
environment in influencing the structure taken up by alpha synuclein. Furthermore it brought out
principle things to consider such as the orientation of alpha synuclein and some peculiarities in
the helix so formed.

1.6 ALPHA SYNUCLEIN & APOLIPOPROTEINS
Apolipoproteins are molecules with amphipathic properties that are known to carry
physiological lipids such as cholesterol, through hydrophilic solutions such as blood. Due to their
amphipathic nature they can bind the lipids on one surface and carry them through the
bloodstream forming lipoproteins. They share a structural similarity with alpha synuclein in
terms of the seven 11-amino acid repeats in the amphipathic region. There are six classes of
apolipoproteins, all having spatial and functional specificity. We can see that the physiological
significance of apolipoproteins has a great impact in terms of disease, normal functioning of the
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body and homeostasis. Since alpha synuclein is known to bind lipids with such great efficiency a
potential role in lipid binding and transport, comparable to apolipoproteins cannot be refuted.
The most notable similarities that can be deduced between alpha synuclein and
apolipoproteins, besides the structure, lie in their ability to produce nanoparticle like structures
when bound to lipids. These structures in both cases are found to be more on the elliptical/oval
rather than roundish side
50,51
. Furthermore, apolipoproteins too are found in the oligomeric state
when bound to lipids
52.,53
. It has been observed that several apolipoprotein particles bundle up to
surround the hydrophobic core of the lipid molecule
48
. Apolipoproteins have also been
previously shown to disrupt vesicles into tubular structures which is a well- established property
of alpha synuclein
51
.
A significant difference however based on cellular location is seen between alpha
synuclein and apolipoproteins. Since apolipoproteins are found extracellularly they are known to
bind more neutral lipids with greater efficiency. Alpha synuclein however, which is an
intracellular, cytosolic protein has been shown to bind anionic lipids more drastically. This
observation is in accord with the fact that within the phospholipid bilayer of cell membranes, the
outer layer is mainly composed of neutral lipids such as PC, whereas the inner surface is
abundant in anionic lipids such as PS
52
.

1.7 ALPHA SYNUCLEIN MUTANTS
The most important aspects of my study are aimed at understanding the behavior of
alpha synuclein mutants, commonly found in the familial forms of the disease, with respect to
membranes. Over the years it has been observed that alpha synuclein mutants i.e., A30P, A53T
and E46K show differences with respect to binding affinity, aggregation, and vesicle disruption.
The structures of the resulting membrane bound proteins also vary from mutant to mutant. How
these differences in properties leads to development of the disease and whether they all act by
different mechanisms and if so, how is yet to be determined. While some of the mutants are
known to enhance misfolding and this is thought to be their arsenal with respect to toxicity, some
mutants while still known to be toxic have no specific mechanism as yet. Therefore, there could
be other players such as membrane binding that might be responsible for the neurotoxicity
brought about by these mutants. To understand how this could be brought about, some of the
well- known properties of each mutant have been highlighted first.
A gamut of studies has used different lipids in order to study the affinity of different
mutants. The A30P mutant has consistently shown to have a lesser binding affinity than the
E46K mutant which shows increased binding affinity. The A53T mutant has also consistently
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shown binding affinity comparable to the wild type (WT). Several different techniques have been
used to highlight these differences in binding between mutants of which some are described here.
Initial studies done by Lenson et al on brain lipids showed that the A30P mutant is devoid
of the ability to bind to brain vesicles as compared to the WT
54
. Axonal transport studies were
used to show that A30P was devoid of any vesicle binding ability. Another study by Woong et al
used the glutathione pull down assay to show that when negatively charged vesicles
(PC:PS:Cholesterol at ratios of 52.5:17.5:30 and 35:35:30) were used, the E46K increased the
binding as compared to WT by almost 88% at the higher ratio of PC whereas A30P decreased it
by almost 92%
55
. A53T was known to cause no significant change in binding as compared to
WT. Structural analysis of these proteins when bound to lipid were done using circular
dichroism, high resolution NMR and proteolysis, and it was found that A53T and WT had almost
the same helical content. The helical nature of A30P was found to be similar to the WT in the
presence of POPS/POPC vesicles with the exception of a kink in the alpha helix where the
proline residue had been added. It was then proposed that this change in helicity, however minor
it may seem might be responsible for the decreased binding, possibly due to a deficit in the
ability to wedge into the membrane. The role of these mutants in PD thus still remains a puzzle
as it does not have the ability to bind and thus potentially disrupt membranes. It was then
proposed that the A30P mutant probably caused toxicity due to enhanced aggregate formation
55
.
Perrin et al also demonstrated the ability of A30P to bind POPA/POPC vesicles as
effectively as the WT, but POPS/POPC vesicles with reduced effectiveness
56
. An interesting
study by Bax et al used NMR to elucidate the above results. However they also showed that the
kinetics of binding changed from mutant to mutant
57
. The authors show that the lipid association
ability of mutants is dependent on the helical content, thus proposing a reason for the low
binding of A30P. On the other hand, the membrane dissociating ability is contingent on
electrostatic forces which are enhanced in the E46K mutant
57
.
A recent study also proposed that alpha synuclein binds to lipid rafts in nerve cells
58
.
Thus cholesterol may play an important role in membrane binding in vivo. However it can be
seen clearly that all mutants show variability in their binding affinities for different lipid
combinations. This can be in lieu of the fact that there are a host of different phospholipid
combinations present in the actual physiological cell membrane.
I will now elaborate some of the differences that the alpha synuclein mutants show with
respect to aggregation, fibril formation and some structural differences. The self- aggregating
and fibril forming ability of alpha synuclein has been talked about in detail before. How this
exactly is related to PD is still unknown. However, we do see certain differences between the
disease mutants when it comes to this aspect.
An initial study done at Amgen was one of the first to highlight that aggregate formation
was faster and more pronounced in the A30P and A53T mutants as compared to the WT
59
. They
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showed that the secondary structure of all proteins in the monomeric state was identical.
However upon incubation at 37°C for 3 days, the rates of aggregation were different for all. WT
showed the highest lag phase, followed by A30P and finally A53T which aggregated the fastest.
These aggregates were rich in β-sheets as shown by FTIR, and when further analyzed by electron
– microscopy and atomic force microscopy, a fibrillar nature was observed. A study by Lansbury
et al then put forth a mechanism by which these aggregates or protofibrils damage the
membranes
60
. It was concluded that protofibrils of the WT as well as A30P and A53T disease
mutants which typically consisted of 20 monomers, permeabilized PG vesicles in a pore like
fashion. This was concluded based on the finding that only molecules of certain sizes were able
to permeate the vesicles. On the other hand, they claimed that the monomeric units
permeabilized vesicles in a detergent like mechanism. Also, the ability of the mutant protofibrils
to permeabilize these vesicles was greater than the WT
60
.
Fink et al also attempted to highlight the differences between the WT and the
A30P/A53T mutants
61
. They first tried to test the effect of pH, temperature, and solvent but
found that the monomeric forms of all these proteins exhibited the same structural properties, as
also seen before. Next, they used Thioflavin T (ThT) fluorescence to demonstrate the differences
in aggregate and fibril formation of all three. As expected, it was observed that A53T showed
increased aggregation and fibrillation followed by the A30P mutant and then the WT. When
hydrophobicity studies were done on the mutants, it was indeed observed that the hydrophobicity
was reduced in both the mutants within the region of the substitution. This analysis also showed
the higher propensity of both the mutants to form a β-sheet in the region of the substitution rather
than an α-helix, which correlates with the fact that aggregates and fibrils are rich in β- sheets
61
.
Another study by Rienstra showed that the rate of fibrillation of the A30P mutant was
slower than the WT
62
. They showed that even though the structures of the final fibrils formed
were identical, the A30P mutant showed slower kinetics while forming fibrils. This was also
demonstrated by Lansbury et al
63
.
The E46K mutant showed similar structural properties to the WT in monomeric state.
However fibril formation was seen to be faster in the E46K mutant. However, it was noted that
the protofibrillar structures formed by the E46K mutant showed lesser vesicle permeabilizing
power than the WT, which is in contrast with the A30 and A53T mutants
62
.
The above mentioned literature gives us an idea about how the mutants are known to
behave. The properties of each of these mutants may seem erratic and although they differ
considerably from each other, they all play a part in the progression of PD. It is thus certain that
exploring their properties and mechanisms further might give us an insight about how exactly
each mutant plays a role in the pathology of the disease. It remains inconclusive whether E46K
acts by virtue of its membrane destroying property, which is a property that seems to have been
lost in the A30P mutant. On the other hand the A30P mutant has been theorized to play an
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enhanced role in protofibril formation. We therefore cannot overlook the theory than some
mutants seemed to have gained some functions of the WT alpha synuclein and some others.
Based on the insights gained through the above studies, in my experiments I have aimed
to try and find a correlation between the membrane curvature inducing properties and membrane
binding properties of each of these mutants. The E57K mutant, which is a designed mutant and is
known to be highly toxic, was also used as a comparison. It has been found that this E57K
mutant shows increased cytotoxicity and membrane bound oligomer formation in rat models
64
.
Based on this known data I hypothesize that the E46K mutant leads to membrane destruction and
subsequent toxicity by virtue of its enhanced membrane binding and remodeling, whereas the
A30P and A53T mutants might use their misfolding property as a tool to initiate neurotoxicity. I
have attempted to use clearance assays to indicate that decrease in scatter is indicative of the
vesicles being remodeled into smaller structures. Leakage assays were used to study the
capability of the mutants to induce vesicle disruption. Circular dichroism (CD) studies give
further insight into the secondary structures of all the mutants before and after membrane
binding. Eventually, electron microscopy was used to study the nature of the structures formed
when the initial phospholipid vesicles were remodeled. With the help of the above techniques, I
could compare the abilities of all the mutants with respect to these properties and study their
kinetics as well.

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CHAPTER II: MATERIALS AND METHODS

2.1 Purification of alpha-synuclein
Transformation and subsequent purification of WT alpha synuclein and mutants:
DAY1 - Escherichia coli cells (BL21 DE3 pLysS), 50ul/ transform were taken from -80°C and
kept to thaw on ice for 30 minutes. PCR product/plasmid was added (2 ul) on ice and kept for
another 20 minutes. Mixture was then kept at 42°C for 1 minute, back on ice for 5 minutes,
temperature and time being very crucial. Meanwhile LB broth previously prepared was also
heated to 42°C and 500ul was added to the tube near the flame. The mixture was then kept in the
incubator shaker at 37°C for an hour at a speed of 225-250 rpm. The cells were then spun in the
table top centrifuge for 2 minutes at a speed of 3500 rpm. The supernatant was thrown, cells
dissolved in 100 ul LB. 50 ul of this LB was taken and plated into 2 plates and kept in the
incubator at 37°C for about 16 hours.
DAY 2 – A single colony was picked and inoculated in 5ml LB containing 5ul ampicillin (stock
100mg/ml and concentration required was 100ug/ml). It was kept for 3 hours in the incubator at
37°C at a speed of 225 rpm. Then it was centrifuged for 5 minutes at 3500rpm and consequently
transferred to 50 ml LB with 50ul ampicillin. Kept in incubator at 37°C overnight.
DAY 3 – Cells were removed from the incubator, centrifuged again at 3500 rpm for 20 minutes
at 4°C. Supernatant was thrown off and cells were re-suspended in LB. 500ul ampicillin was
then added to flasks containing LB previously autoclaved. Then 1 ml transferred to each of the
flasks and incubated at 37°C in shaker at 250 rpm, until A
600
= 0.7, to ensure cells were in the
optimum stage of growth. Absorbance was measured at 600nm. The cells were then induced with
0.5mM IPTG (1M stock therefore 250ul required for 500 ml) while still in the shaker and
incubated overnight at 25°C at a speed of 250 rpm.
DAY 4 - Cells were spun down in the table top centrifuge at 7000 rpm for 15 minutes at 4°C in
1000 ml bottles. They were then frozen at -80°C in lysis buffer and stored or used further.
DAY 5 – Cells were removed from the freezer and allowed to thaw. Only cells from 1 liter
culture at a time could be taken as the column volume for next step was only 5ml. After thawing,
20ul β-mercapto ethanol (BME) was added to 20ml of sample. It was transferred into lysis buffer
and nitrogen gas bubbled for 10 seconds. Cells were boiled for 10 minutes and then cooled to
room temperature, keep in ice and 1mM PMSF was added. (500ul of 0.1mM stock). Cells were
spun at 19,000 rpm in SS-34 rotor for 30 minutes. Acid precipitation was then done at pH= 3.5.
Mixture was incubated at 4°C for 10 minutes and spin again at 19,000 for 30 minutes.
Supernatant was then dialyzed with dialysis buffer, changing from the first buffer solution after 3
hours.
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DAY 6 - Weak anion exchange column (ANX) in FPLC was run. Loop was cleaned first with
water, then filled with protein sample, and program was run eluting first with 100% of buffer A
and then gradually washing out with buffer B at gradients of 15%, 30% and finally 100%.
Fractions were checked using gel electrophoresis. Appropriate fractions were pooled, and
dialyzed as done previously using lysis buffer.
DAY 7 - Strong anion exchange column (QXL) in FPLC was run using the same method as
before. Fractions were checked again using gel electrophoresis.
DAY 8 – Appropriate fractions were pooled and concentrated to approximately half the volume.
They were spun in 3K centrifuge tubes at 5000 rpm for 20 min at 2°C.When volume sufficiently
decreased, buffer exchange was done with HEPES pH= 7.4. Concentrations were checked using
the UV-VIS spectrophotometer and stored at -80°C, in the same buffer.

2.2 Gel Electrophoresis
15ul of elutes collected from the anion exchange column were run on the SDS-PAGE 12%
NuPage Bistris gels obtained from Invitrogen. Running buffer used was the MES running buffer
obtained from Invitrogen as well. Gel was made to run for 36 minutes 200mV and 400mA. Gel
was then thoroughly washed out with purified water and stained using SimplyBlue safe stain
(Invitrogen) for about 30 minutes followed by subsequent destaining with purified water.
Fractions that showed bands around 15kD contained pure alpha synuclein and were collected.

2.3 Determination of concentration of proteins
Concentration of protein was checked using the UV-VIS spectrophotometer as well as
colorimetric assays:
Spectrophotometry- 400ul of protein was taken in a quartz micro- cuvette with a 1 cm path
length and absorbance was measured on the Jasco V-550 UV-VIS spectrophotometer at room
temperature using the following parameters:-
Scanning speed- 200nm/min
Photometric mode- absorbance
Response- median
Band width- 2nm
Starting wavelength- 300nm
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End wavelength- 2450nm
Data pitch- 2mm
The absorbance value obtained from the readings was converted into Micromolar concentrations,
using extinction coefficient of alpha synuclein as 5120 M
-1
cm
-1
(due to presence of 4 absorbing
Tyrosines)
Colorimetric Assay- This assay was done to measure protein concentration using the BCA
TM

Protein Assay Kit (Pierce). Varying concentrations of Diluted Albumin (BSA) were prepared as
standards as indicated, followed by a Working Reagent (WR). 2 ml of the WR was added to
0.1ml of each of the standard and sample to be measured, mixed well and incubated at 37° C for
30 minutes. Absorbances were then measured at 562nm on the Jasco V-550 spectrophotometer
and a standard curve was plotted of concentration v/s absorbance, using the standards. Sample
absorbances were then plotted onto this curve and thus concentrations obtained.
Concentrations of all proteins obtained by both methods were found to be similar.

2.4 Clearance Assay
Once the concentrations of proteins were measured, vesicles were prepared for the clearance
assay. 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-RAC-(1-glycerol)] (POPG), 10% & 20%
phosphotidyl inotsitol (PI) obtained from Avanti Polar Lipids Inc. and Brain extract, Folch
Fraction I from bovine brain containing ~10% PI and ~50% phosphotidyl serine (PS) from
Sigma Aldrich were dried overnight. MLVs were formed when 500ul of HEPES buffer pH=7.4
was added. The system was kept for about 15 minutes and then vortexed for 30 seconds. Final
concentrations of POPG vesicles were ~5188uM, Brain extract was 6250uM, 10% PI was
5140uM and 20% PI was 5090uM.
Once vesicles were prepared, clearance assay was done with POPG at protein: lipid ratios of
1:10, 1:20 and 1:40, concentration of POPG taken being 500um, and at a concentration of 1:20
with Brain extract, 10% PI and 20% PI (with 90% and 80% POPC respectively).The assay was
done as follows:
Enough phospholipid vesicles were taken in the quartz micro-cuvette to make the final ratio as
required. Volume was made up to 400 ul using HEPES buffer.
A time course measurement was done on the Jasco V-550 spectrophotometer keeping band width
as 5nm, at a wavelength of 500nm for 4000 seconds.
The calculated volume of protein was added at ~100 seconds, and immediately 10ul of solution
was withdrawn for electron microscopy.
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Subsequently, circular dichroism and electron microscopy were done on these solutions after the
experiment was complete at 4000 seconds.

2.5 Circular Dichroism
The Jasco J-810 spectropolarimeter was used to obtain the spectra, at room temperature. 200ul of
the protein solutions, adjusted to the same concentration as required in clearance and diluted with
HEPES buffer were added to a 1mm quartz cell. Spectrum measurement was run using the
following parameters:
Starting wavelength- 250nm
End wavelength- 200nm
Data pitch- 0.5nm
Mode- continuous
Speed- 50nm/min
Response- 1 second
Band width- 1mm
Blanks were also collected and subtracted from sample readings to obtain accurate spectra.
The above procedure was then repeated after the clearance assay was completed. 200ul of the
solution from the cuvette used in the clearance assay was immediately transferred to the 1mm
quartz cell and the spectra were obtained using the same parameters as above. Blanks however
were different and were done using appropriate amount of lipid. Blank readings were again
subtracted to get the final spectra. It must be noted that the CD spectra were obtained after the
same time point of completing the clearance assay for each protein to ensure accuracy.

2.6 Leakage Assay
Vesicles were prepared for the leakage assay using POPG. Enough lipid was dried overnight to
make a 100 um solution on rehydration. A 22.7mM solution of ANTS and a 45mM solution of
DPX were prepared using buffer I. This solution of ANTS, DPX and buffer II were added to the
dried lipid in that order. Thus the final concentration in the solution for ANTS was 9mM, DPX
was 25mm and for lipid was 100uM. This lipid solution was then vortexed and freeze-thawed
about 5 times. In order to obtain large unilamellar vesicles (LUVs), lipids were extruded using
Avestin 1000X Polycarbonate filters. In order to remove any ANTS/DPX present outside the
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vesicles gel filtration was done using the PD-10 column. Buffer II was used to pack and elute the
column. Elute containing vesicles had a shine.
Once the vesicles were prepared, the leakage assay was done using 500um of lipid at a protein:
lipid ratio of 1:20 as follows:
15ul of vesicles were added to 285ul of buffer II in a quartz micro-cuvette.
A time course measurement was run on the Jasco FP-6500 spectrofluorometer, with an emission
wavelength of 520nm, excitation wavelength of 380nm, emission slit width 10nm, and excitation
slit width 5nm for 4000 seconds
When steady fluorescence intensity was observed, at about 200 seconds, calculated amount of
protein was added.
Towards the end of the experiment, at about 3200 seconds, 5 ul of Triton X-100 was added to
the cuvette which was used as the maximum value for the experiment.

2.7 Electron Microscopy
Transmission electron microscopy was done on the JEOL 1400 transmission electron microscope
at 100kV. Carbon coated Formvar films mounted on 150 mesh size copper grids were used. The
grids were first placed on ~10ul of the solutions of protein and lipid as obtained during the
clearance assay mentioned above, for approximately 10 minutes. Solutions of only lipid vesicles
were used as a control. The excess liquid was the blotted off, and the grids were placed upon
10ul of 2% uranyl acetate which used as the negative stain for 5 minutes. The excess stain was
then blotted off, and the grids were dipped and blotted with water and dried. Images of the
vesicles with and without the protein were then captured.

2.8 Buffer solutions used:
1) LYSIS BUFFER
300 mM Nacl
100 mM Tris (pH= 8)
1 mM EDTA (pH= 8)
βME (20ul of stock ~ 18M if 20 ml of lysis buffer is used)

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2) DIALYSIS BUFFER
20mM Tris (pH= 8)
1mM EDTA (PH= 8)
1mM DTT

3) FPLC BUFFERS
BUFFER A- 20 mM Tris (pH= 8) & 1mM DTT
BUFFER B- 20 mM Tris (pH= 8), 1M Nacl & 1mM DTT

4) HEPES BUFFER
20mM HEPES
100mM Nacl
(pH= 7.4)

5) LEAKAGE ASSAY BUFFERS
BUFFER I
10mM HEPES
1mM EDTA
3mM NaAzide

BUFFER II
10mM HEPES
50mM KCl
1mM EDTA
3mM NaAzide

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CHAPTER III: RESULTS

3.1 Alpha synuclein mutants clear POPG vesicles, transforming them into
smaller entities:
The clearance assay is used as a qualitative method to determine the membrane remodeling
property of a protein or peptide. It is based on Rayleigh’s principle which states that the intensity
of scattered radiation increases as the ratio of particle size to wavelength increases. Therefore as
the size of phospholipid vesicles decreases, the scatter will decrease. When light of wavelength
500nm is used, initially it is scattered by MLVs which are of different sizes from 1000nm and
above. When the protein is added to these vesicles, in just a few seconds a decrease in scatter is
observed. The proteins which have the ability to remodel these vesicles into smaller structures
will therefore cause a dramatic decrease in scatter.
When protein: lipid ratios of 1:10, 1:20 and 1:40 were used for the clearance assay, I
consistently observed that all, alpha synuclein WT and mutants had the ability to clear large
multilamellar POPG vesicles. Initial scatter (before adding protein) was seen to be different at all
ratios as different batches of lipid were used each time. A decrease in scatter was observed in all
cases within seconds of adding protein to the vesicles. The initial milky suspension comprising
just the vesicles and buffer became clear upon adding protein within 4000 seconds (Figure 18).
The clearance observed for all mutants increased with an increase in protein: lipid ratio.
I also observed with all three ratios, that the A30P mutant had the weakest ability to clear
these vesicles, and exhibited the slowest rate of clearance. Furthermore, the A53T mutant
consistently showed very similar clearance kinetics as the WT with all three ratios as well. The
E46K familial mutant showed fastest kinetics and the highest % decrease in scatter in all three
cases. However the E57K mutant was the one to show the maximum effect in all cases, though
not too much of a pronounced difference was observed between the E46K and E57K mutants.
Thus the general order of vesicle clearing ability observed was E57K > E46K > A53T~ WT >
A30P (Figure19& 20). This behavior of A30P might support earlier reports that the A30P mutant
decreases the vesicle binding ability of alpha synuclein
53,54
. Furthermore these results concur
with previous findings hypothesizing that alpha synuclein induces membrane curvature upon
binding to phospholipid vesicles and causes tubulation
37
, which in this might be leading to the
smaller structures formed and thus the subsequent decrease in scatter. It can thus be concluded
that the binding efficiencies of alpha synuclein and its mutants may correlate with their ability to
transform vesicles into smaller structures.

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3.2 Alpha synuclein mutants take up an alpha helical structure when bound to
POPG vesicles:
Circular dichroism spectra are used to determine the secondary structure of proteins. A
transition from random coil to an α -helix in the case of alpha synuclein is known to be
responsible for wedging into the membrane surface and inducing membrane curvature. Thus the
operational ability of alpha synuclein is known to lie in its structural relevance, which can be
indicated using circular dichroism.
Ideally a spectrum between 195-260nm efficiently gives us an idea about whether the
protein is in a random coil, α- helix or beta sheet conformation based on the location of peaks. A
plot of θ
mdeg
v/s wavlelength is ideally obtained. For comparison between proteins this plot is
converted to a mean residue ellipticity plot using the formula:
[θ]
MRE
= [θ
mdeg
X 10
6
] / [ (concentration of protein in uM) X no. of amino acids present)]
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As shown earlier, WT alpha synuclein in the normal physiological state exhibits a
random coil structure. However when bound to membranes it shows an α- helical structure
especially in the amphipathic region, which may be bent or extended, depending on the resultant
structures formed
34
. When this property of alpha synuclein was tested in the case of mutants, it
was indeed observed that upon membrane binding, the initial random coil structure of all mutants
was effectively altered to an alpha helical structure, at all three ratios. However, it still remains to
be tested whether this structure is a bent or extended helix in each case. Circular dichroism was
thus effective in demonstrating that the transition in case of each mutant was to an α- helix, as
opposed to a misfolded beta sheet structure. Since it is known that alpha synuclein can take up
parallel beta sheet structures too, the possibility of this having an implication in the disease
cannot be ruled out. The observation of an α- helix in all cases however gives a clear channel of
understanding which conformation all the mutants take up when bound to lipid and the role the
α- helix plays.
Circular dichroism studies were done on each mutant before and after the clearance assay
at each ratio to compare the secondary structure of the proteins before and after membrane
interaction. I observed that the α- helical content varied proportionally with the ability of each
mutant to clear the vesicles. Thus at the end of the clearance assay, at all ratios when circular
dichroism studies were done, the α- helicity of the A30P mutant was found to be the least in all
cases. A plot of mean residual ellipticity v/s wavelength consistently showed that the E57K
mutant had the highest alpha helical content in most cases, followed by the E46K and A53T
mutants which were seen to be similar to the WT. The spectrum of A30P in addition to showing
the least α- helicity also showed a slight difference in spectrum with respect to a left hand side
shift in the first peak. This might coincide with a previous observation that the A30P circular
dichroism spectra showed a kink in the alpha helix, which was said to be responsible for its
lowered binding efficiency.
From the above results it can be seen that the A30P mutant, which is known to decrease
alpha synuclein binding affinity also shows decreased ability to clear POPG vesicles as well as
lowest alpha helicity. It could thus be concluded that the A30P mutant due to its low binding
ability cannot efficiently remodel POPG vesicles. Moreover, since this mutant is not greatly
bound to the vesicles, probably as the helical content is not as prominent as the rest. Based on the
above data however it could be concluded that of all familial mutants, the E46K mutant that is
known to increase vesicle binding
54
also displays greatest α- helicity which could be responsible
for its enhanced membrane interacting ability. Since the clearance assay already indicated the
heightened membrane remodeling functions of E46K, we see that clearance as well as circular
dichroism data are complementary and lead to similar conclusions.

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3.3 Alpha synuclein mutants cause membrane disruption as indicated by leakage
assay:
The leakage assay is used a quantitative tool to illustrate the ability each mutant has to
effectively disrupt and remodel vesicles. The leakage assay works on the principle of an increase
in fluorescence when the fluorophore (ANTS) and the quencher (DPX) move out of the vesicle
into a less confined, more dilute space (Figure 22). This can happen only when the contents of
the vesicles are leaked out. Thus the higher the shift in fluorescence intensity seen, the more the
protein is capable of causing leakage of vesicular contents. As seen from previous studies
31, 52
alpha synuclein is seen to remodel vesicles into a host of different structures. Thus LUVs that
were initially more than 1000nm in size are effectively remodeled into smaller vesicles, a
network of intrinsic tubules and nanoparticles of size ~50-60nm, 25-30nm and 7-10nm
respectively (Figure 23). This observation corroborates the principle of membrane leakage,
indicating that if vesicles are remodeled into such undersized structures, the contents of the
LUVs have to have been leaked out.
The rate of leakage induction was observed to be the highest in the case of the E46K
mutant at all ratios, whereas the E57K mutant showed the highest % leakage in all cases, with
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the leakage caused by adding Triton X-100 taken as 100%. This ability of E46K to cause the
fastest rate of leakage may coincide with previous observations indicating a greater binding
affinity of E46K as compared to the WT. the A30P mutant however in all cases showed the
lowest leakage rate as well as % leakage which may correlate with its low binding affinity
(Figure 25). A low binding affinity could thus result in poor membrane remodeling properties of
A30P.
Since we know from the clearance assay that alpha synuclein has the ability to transform
LUVs into smaller structures probably of different shapes such as tubules, it can also be
concluded from the leakage assay that this phenomenon is accompanied by a loss of membrane
integrity. Thus alpha synuclein mutants also have the ability to induce membrane disruption,
some more than others. Mutants such as E46K and E57K have enhanced abilities to cause this
membrane disruption which may be implied in their ability to contribute to the pathology of the
disease. However the role this membrane disruption plays in the disease still remains to be
tested. These properties however are indicative of the fact that all familial mutants do not act
similarly and do not share the same properties. It can however be concluded that the ability of
alpha synuclein mutants to remodel vesicles into different structures is a process that goes hand
in hand with membrane disruption. On a physiological level, this phenomenon could be
correlated with a loss of cellular contents that may have an implication in the disease.

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3.4 Alpha synuclein and its mutants remodel vesicles into smaller structures:
Transmission electron microscopy was done in order to study the nature of the structures
formed after remodeling. Images of just the vesicles were taken as a control and compared with
the structures formed after addition of protein. This technique was used as a tool to gain further
insight into what happens to the vesicles and potentially biological membranes in more structural
terms. When these results were correlated with clearance and leakage assay data a distinct
pattern was observed.
Electron microscopy revealed the different structures formed when each mutant was
added to the different phospholipid solutions. The WT as previously shown, with POPG vesicles
induced tubulation
37
. At a ratio of 1:20, abundant tubes of diameters ~30nm were observed to be
seen immediately after addition of the protein to the lipid solution along with the presence of few
vesicles intermittently (Figure 27). The diameters of these tubes decreased to about ~20nm by
the end of 4000 seconds. At a ratio of 1: 10 and 1: 20 the membrane blebbing effect was also
observed in addition to tubulation (Figure 28). The 1: 40 protein: lipid ratio showed more
presence of small vesicles of about 200nm in addition to tubes of ~ 40nm that decreased to ~
20nm. Thus these results were in accord with previous findings that showed the tubulating ability
of alpha synuclein along with blebbing effects.
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The A53T mutant upon electron microscopy showed tubes with similar diameters as with
the WT. The presence of smaller vesicles was also seen along with these tubes, at all three ratios.
At a ratio of 1:20 tubes of about 50nm were seen that went down to all ranges between 10-40nm
(Figure 29). At a ratio of 1:40 tubes of between 10-30 nm were seen with particles of ~ 10-30nm
as well. Finally, at the highest ratio tubes of ~ 20nm were prevalent. This observation of A53T
having similar behavior as the WT is in accordance with previous findings wherein the A53T
showed similar results to the WT in the clearance and leakage assays. Thus the notion that A53T
acts similar to the WT has been reiterated via a host of different assays.
Next, the A30P mutant was added to POPG vesicles and structures formed were
investigated using electron microscopy. Contrary to previous observations as in the case of WT
and A53T, the A30P mutant showed negligible signs of tubulation. Smaller vesicles of size 30-
70nm were seen at all three ratios with the A30P mutant (Figure 30). At the 1:20 ratio small
vesicles of 60nm and with 1:40 particles of ranges between 30-100nm were observed. Finally, at
the ratio of 1:10 as well, the A30P showed small vesicles that ranged between 10-100 nm as
well. It is unclear whether this phenomenon has any direct correlation with the fact that A30P
didn’t show any pronounced effects with the clearance and leakage assay as well. Since the
A30P mutant is known to be efficient in lowering the vesicle binding affinity of alpha synuclein,
the low binding might be responsible for its lack of tubulating ability as well.
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The E46K mutant, which seemed like the most toxic familial mutant based on clearance
and leakage assay data was then checked by electron microscopy to study the structures it
induced. Upon addition to POPG vesicles it was observed that the E46K mutant had the ability
to tubulate POPG vesicles as well. When the protein was just added to POPG vesicles at a ratio
of 1:20, copious amounts of tubes with diameter between 10-40nm were seen (Figure 31).
However, at all ratios, at the end of 4000 seconds it was observed that these tubes were
transformed into smaller structures between 30-50nm in size. With ratios of 1:40 and 1:10 as
well tubes showed a diameter of 10-50nm as well. One of the most interesting observations seen
with E46K was what appeared to be tubes breaking into smaller particles (Figure 32). Whether
this is similar to the membrane- blebbing effect, and whether this is how the nanoparticles are
formed, remains to be tested. Conversely, these particles could be joining and forming the tubes.
Whether the increased toxicity of the E46K mutant has anything to do with this ability of alpha
synuclein to rapidly remodel membranes into very small structures is obscure.
Lastly, the E57K mutant at all three ratios showed intense tubulation, with traces of
smaller vesicles at all ratios as well (Figure 33). With a 1:20 ratio tubes of 10-15 nm were
observed, whereas with 1:40 tubes of 5-12nm were seen. With 1:10 as well tubes showed a
diameter of ~ 10nm. Thus it seems clear that mutants exhibiting intense tubulation are the ones
that have been attributed to be more toxic based on previous literature.

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The same observations were made with all the mutants with brain extract as well. The
A30P mutant again showed severely reduced signs of tubulation, as compared to the others, but
mainly the presence of smaller vesicles of ~400-600nm (Figure 36), whereas all the other
mutants and the WT were adept inducers of tubulation. The WT showed signs of tubes having a
diameter of about 20nm and smaller vesicles of about 100-250nm. The A53T showed similar
results with tubes of ~30nm and vesicles of 50-150nm in size. The E46K and E57K mutants on
the other hand showed tubes ranging from 5-50nm and 5-25nm respectively along with the
presence of smaller vesicles (Figure 37).
A general pattern was seen thus with all the mutants and the structures they formed upon
remodeling. While the WT, A53T and E46K formed mostly tubes along with smaller vesicles
and certain smaller particles, the A30P showed mainly only the presence of smaller vesicles with
significantly reduced signs of tubes. The electron microscopy data correlates precisely with the
leakage data in that vesicles that were previously of the order of 1000nm were modified into
structures such as tubes, smaller vesicles and potentially nanoparticles. The very small size of
these new structures formed would imply that most vesicular contents i.e., buffer were leaked
outside into the system, only a fairly small amount of buffer is now retained in these smaller
structures.

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3.5 Alpha synuclein mutants with physiologically relevant lipids:
Since all the above experiments were done with POPG, which is a bacterial phospholipid,
they were repeated again with more physiologically relevant lipids such as brain extract and PI.
Even though the results obtained were seen to be varied a certain pattern could be deciphered.
When the above mentioned clearance assays were done with more human physiological
lipids such as brain extract and PI different results were obtained. With brain extract a similar
result as with POPG was seen. All the mutants were seen to clear vesicles made with brain
extract at a protein: lipid ratio of 1:20. Again, it was observed that the order of clearance
observed was E57K > E46K > A53T~ WT > A30P (Figure 38). These results may thus
reproduce findings as seen before indicating a lowering of binding affinity with the A30P mutant
with brain vesicles
53
. When circular dichroism was done on these solutions of lipid and protein
after clearance it was again observed that the random coil nature of all mutants previously seen
was modified to an alpha helix upon membrane binding (Figure 39). The A30P mutant again
showed the lowest alpha helical content. These results shown on brain lipids however suggest
that the processes of membrane remodeling and disruption may occur in vivo with physiological
lipids as well. Thus these results shown with POPG could be simulated with brain extract as well
revealing a more cellular nature of these phenomena.
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When the above mentioned experiments were done on 10% and 20% PI vesicles however,
no decrease in scatter was observed. Furthermore the CD spectra of all mutants remained in the
same random coil state even after addition to lipid. Whether this loss of ability to remodel PI
vesicles is simply due to the comparatively low content of PI and relatively higher levels of
POPC in these vesicles remains to be tested, but this explanation cannot be rejected. Electron
microscopy data with PI showed no major changes in the integrity of the vesicles (Data not
shown).

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DISCUSSION
The aim of my studies was to determine whether abnormal membrane remodeling plays
a role in the cascade of neurotoxicity. Based on the results different categories of toxic
mechanisms have been elucidated. One category is that of a loss of function mutant, wherein the
A30P seems to have a diminished ability to bind membranes. Whether this mutant could then
lead to neurotoxicity due to its enhanced propensity to aggregate is yet to be speculated. On the
other hand, the other explanation that deems fit for toxicity is that of E46K wherein there is a
greatly enhanced ability to bind and remodel membranes, probably more than physiologically
required. This may lead to a downward spiral of membrane permeabilization and subsequent
neuronal death. This phenomenon could also explain the presence of fragments of lipids in Lewy
bodies, which might be consequence of membrane remodeling spiraled out of control. However
further validation for these two mechanisms is yet to be established. Moreover, the A53T mutant,
which is known to show increased signs of aggregation

also shows membrane binding properties
which are not as pronounced as E46K but not as weak as A30P. Thus whether this mutant brings
about toxicity by both mechanisms is still to be investigated into. Since a putative scheme for the
toxicity of each mutant has now been elucidated, further investigations can be done
appropriately.
Based on previous literature, the effects of alpha synuclein on negatively charged lipids
such as POPG have been adequately shown over time. However recent advances such as the
curvature inducing and tubulating ability of alpha synuclein, which have been well established in
molecules such as endophilin, are related to the pathogenesis of PD are just being brought into
light. Alpha synuclein has been attributed to have a host of different functions that might relate
to its membrane binding ability. Furthermore, this very property of alpha synuclein if gone awry
could lead to series of events that may cause cellular fatality, as can be indicated by membrane
leakage.
Over the course of my studies I made attempts to try and gauge some of the similarities
and differences between the WT alpha synuclein and the familial as well as artificial mutant
which is known to be toxic. The differences and similarities helped in understanding which
properties of alpha synuclein have been exaggerated in the mutants and which have been
obliterated. These insights might help us in understanding which properties of alpha synuclein
might play a role in normal physiological function and which in the escalation of toxicity.
Therefore we see that such a correlation is fundamental in understanding the biology of alpha
synuclein.
The A30P mutant that throughout my studies consistently showed significantly different
behavior than the other mutants might indicate that uncontrolled membrane remodeling could be
just one of the mechanisms implicated in PD. A30P constantly seemed like a loss of function
mutant as properties with potential physiological implications such as membrane binding,
membrane remodeling and tubulation, commonly seen with alpha synuclein seemed to have been
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lost in this particular mutant only. The A30P mutant however is well known to be a neurotoxic
mutant implicated in PD. The distinctive behavior shown by this mutant may imply different
theories. The A30P mutant might thus play a different role in the pathogenesis of the disease and
act by a completely different mechanism as the rest of the proteins. It has been shown
extensively in literature that the A30P mutant has enhanced oligomer forming ability. Also, it has
been previously hypothesized that it is these oligomeric protofibrils that may lead to toxicity and
ultimate cell death. On the other hand, the properties that seem to have been lost in the A30P
mutant could probably be essential for normal functioning of the neurons, and thus a loss of
these properties may lead to toxicity by the A30P mutant. In both cases there are plausible and
contradictory explanations to consider.
The A53T mutant consistently showed data on a very similar scale as the WT. A53T on
the other hand also shows a great propensity to aggregate almost similar to A30P as seen earlier.
Since no pronounced membrane remodeling was observed A53T may lead to toxicity in a similar
fashion as A30P. In the case of these mutants we see that oligomers seem essential to contribute
to neuronal toxicity. It is only above a certain threshold of protein concentration that these
perilous effects could be seen.
When we looked at the behavior of the E46K mutant, which is well known to be a highly
toxic mutant, we saw that all the properties shown by the WT alpha synuclein were greatly
intensified in the case of this mutant. The E46K mutant showed similar vesicle leakage and
clearance results as the E57K mutant which was used due to its prominent deleterious effects.
Therefore results comparable to the E57K mutant would ideally imply a mutant that is highly
toxic and perilous. In this case the explanation that alpha synuclein leads to toxicity by damaging
membranes first by binding to them, subsequently leading to membrane remodeling and the
formation of other structures to an exaggerated extent and finally completely destroying the
nature of the membrane seems most plausible. Thus a potential lipid carrying function such as
that of apolipoproteins which would require membrane binding and tubulation might go awry in
the case of the E46K mutant and lead to a spiral of downward events ultimately leading to cell
death. Throughout literature there are several examples to demonstrate the toxicity of the E57K
mutant. One study showed in vivo in rat models that E57K oligomers lead to a decrease in the
dopaminergic cells
65
. Lentiviral injections were used as a tool to introduce the mutant oligomers
into rat models. Moreover this study also showed that these oligomers were found to be
associated with membranes and the cause of death of the neurons was a loss of membrane
integrity. We can thus conclude that the E57K mutant has a role to play in membrane
interactions, just like shown in my studies, and these interactions in turn have a role to play in
toxicity.
Based on membrane remodeling, the dilemma of how each mutants plays its role in PD
can be deciphered. It seems clear that while some mutants have the distinct ability to aggregate,
others did not. However based on leakage assay, clearance assay and electron microscopy we can
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conclude that unrestrained membrane remodeling is definitely a major player in this toxicity.
Mutants that do not necessarily aggregate can still bring about cell death by this mechanism.
On a more cellular level, there have been an increasing number of studies to show events
leading to cell death that coincide with alpha synuclein activity. Some studies have correlated an
increased caspase activity after being treated with alpha synuclein. Besides an increase in
Caspase 1
66
and Caspase 3
67
it was observed that alpha synuclein was localized in regions of
vesicular rupture. Further the cell to cell transfer to alpha synuclein lead to vesicular rupture as
well. These studies might further highlight my findings that cellular vesicle rupture plays a
significant role in disease.
Recent studies have now proposed that the native form of alpha synuclein is found to not
a random coil, but an alpha helical tetramer as seen endogenously
68,69
. Further studies expressed
the N-acetyl form of the protein and showed that it is known to occur in an oligomeric form
which is mainly tetramers of alpha helical protein
70
. This phenomenon is known to prevent the
protein from aggregating. N-acelyation is known to reduce the N-terminal charge of the protein,
possibly favoring protein-protein interactions, which may in turn favor tetramer formation.
However this increased hydrophobicity in the N-terminal region might also lead to reduced
membrane binding. Thus it remains to be tested whether this develops as a neuroprotective
mechanism. The functional relevance of this form of the protein is therefore not known. Based
on my findings I think an exciting avenue to explore next would be to express this N-acetyl form
of the protein and test it for its curvature inducing and membrane remodeling abilities. This
would give an indication of what enhanced/dimished properties the acetylation ascribes and how
this affects the physiological and functional abilities of alpha synuclein. Furthermore another
domain I would like to explore would be to test the oligomers of the A30P and A53T mutants.
Testing their effects on membrane binding and remodeling would give us an insight about how
exactly these oligomers and responsible for bringing about their chain of toxic effects.
In conclusion, the A30P and A53T mutants which have previously been shown to cause
pronounced aggregation, as well as diminished membrane binding properties, indeed did not
show enhanced membrane remodeling abilities. The E46K on the other hand which is not
aggregation prone did however exhibit membrane remodeling and disruption that seemed a lot
more unrestrained that normal. This could lead to permeabilization of membranes and eventual
leakage of cellular contents, imposing a great threat. Based on these data a new avenue has been
created to understand the different mechanisms of toxicity that could be implicated in PD. A
more insightful understanding of these mechanisms would give us a definitive mode of
understanding these pathways and research apposite treatment options.

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REFERENCES:
1) http://www.pdf.org/en/parkinson_statistics
2) Freire C, Koifman S (October 2012). "Pesticide exposure and Parkinson's disease:
epidemiological evidence of association". Neurotoxicology 33 (5): 947–71.
doi:10.1016/j.neuro.2012.05.011. PMID 22627180
3) Davie CA (2008). "A review of Parkinson's disease". Br. Med. Bull. 86 (1): 109–27.
doi:10.1093/bmb/ldn013. PMID 18398010.
4) IPDGC; Nalls, MA; Plagnol, V; Hernandez, DG; Sharma, M; Sheerin, UM; Saad, M;
Simón-Sánchez, J et al. (2011). "Imputation of sequence variants for identification of genetic
risks for Parkinson's disease: a meta-analysis of genome-wide association studies". Lancet 377
(9766): 641–649. doi:10.1016/S0140-6736(10)62345-8. PMID 21292315
5) Membrane curvature induction and tubulation are common features of synucleins and
apolipoproteins. Varkey J, Isas JM, Mizuno N, Jensen MB, Bhatia VK, Jao CC, Petrlova J, Voss
JC, Stamou DG, Steven AC, Langen R. J Biol Chem. 2010 Oct15;285(42):32486-93. doi:
10.1074/jbc.M110.139576. Epub 2010 Aug 6.
6) Obeso JA, Rodriguez-Oroz MC, Goetz CG, et al. (May 2010). "Missing pieces in the
Parkinson's disease puzzle". Nat. Med. 16 (6): 653–61. doi:10.1038/nm.2165. PMID 20495568.
7) "The synaptic pathology of alpha-synuclein aggregation in dementia with Lewy bodies,
Parkinson's disease and Parkinson's disease dementia". Acta Neuropathol. 120 (2): 131–43.
doi:10.1007/s00401-010-0711-0. PMC 2892607. PMID 20563819.
8) Jankovic J (April 2008). "Parkinson's disease: clinical features and diagnosis". J. Neurol.
Neurosurg. Psychiatr. 79 (4): 368–76.
9) Russell JA, Ciucci MR, Connor NP, Schallert T. (2010-06-23). "Targeted exercise therapy for
voice and swallow in persons with Parkinson's disease". Brain Res. 1341: 3–11.
doi:10.1016/j.brainres.2010.03.029. PMC 2908992. PMID 20233583.
10) Yao, S.C.; Hart, A.D.; Terzella, M.J. (May 2013). "An evidence-based osteopathic approach
to Parkinson disease". Osteopathic Family Physician 5 (3): 96–101.
doi:10.1016/j.osfp.2013.01.003.
11) Caballol N, Martí MJ, Tolosa E (September 2007). "Cognitive dysfunction and dementia in
Parkinson disease". Mov. Disord. 22 (Suppl 17): S358–66. doi:10.1002/mds.21677. PMID
18175397.
12) World's first Parkinson's vaccine is trialed". New Scientist. 2012-06-07.
Effect of Familial Mutants of Parkinson’s Disease on Membrane Remodeling Anuri Shah
59

13) Bruening, W, Giasson, BI, Klein-Szanto, AJ, Lee, VM, Trojanowski, JQ, Godwin, AK.
(2000). "Synucleins are expressed in the majority of breast and ovarian carcinomas and in
preneoplastic lesions of the ovary". Cancer 88 (9): 2154–2163.
14) "Entrez Gene: SNCG synuclein, gamma (breast cancer-specific protein 1)
15) Park, J.Y. & Lansbury, P.T. (2003). Beta-synuclein inhibits formation of alpha-synuclein
protofibrils: A possible therapeutic strategy against Parkinson's disease. Biochemistry, Vol.42,
No.13, (April 2003), pp. 3696-3700, ISSN 0006-2960.
16) Iwai A, Masliah E, Yoshimoto M, Ge N, Flanagan L, de Silva HA, Kittel A, Saitoh T
(February 1995). "The precursor protein of non-A beta component of Alzheimer's disease
amyloid is a presynaptic protein of the central nervous system". Neuron 14 (2): 467–75.
17) Abeliovich, A., Schmitz, Y., Farinas, I., Choi-Lundberg, D., Ho, W.H., Castillo, P.E.,
Shinsky, N., Verdugo, J.M.G., Armanini, M., Ryan, A., Hynes, M., Phillips, H., Sulzer, D. &
Rosenthal, A. (2000). Mice lacking alpha-synuclein display functional deficits in the nigrostriatal
dopamine system. Neuron, Vol.25, No.1, (January 2000), pp. 239-252, ISSN 0896-6273.
18) Lee HJ, Choi C, Lee SJ (January 2002). "Membrane-bound alpha-synuclein has a high
aggregation propensity and the ability to seed the aggregation of the cytosolic form". J. Biol.
Chem. 277 (1): 671–8.
19) McLean PJ, Kawamata H, Ribich S, Hyman BT (March 2000). "Membrane association and
protein conformation of alpha-synuclein in intact neurons. Effect of Parkinson's disease-linked
mutations". J. Biol. Chem. 275 (12): 8812–6.
20) Davidson, W.S., Jonas, A., Clayton, D.F. & George, J.M. (1998). Stabilization of
alphasynuclein secondary structure upon binding to synthetic membranes. Journal of Biological
Chemistry, Vol.273, No.16, (April 1998), pp. 9443-9449, ISSN 0021-9258.
21) Burré J, Sharma M, Tsetsenis T, Buchman V, Etherton MR, Südhof TC (September 2010).
"Alpha-synuclein promotes SNARE-complex assembly in vivo and in vitro". Science 329
(5999): 1663–7. doi:10.1126/science.1195227.
22) Zhu M, Qin ZJ, Hu D, Munishkina LA, Fink AL (July 2006). "Alpha-synuclein can function
as an antioxidant preventing oxidation of unsaturated lipid in vesicles". Biochemistry 45 (26):
8135–42. doi:10.1021/bi052584t.
23) George, J. M., Jin, H., Woods, W. S., and Clayton, D. F. (1995) Neuron 15, 361-72.
24) Jenco, J. M., Rawlingson, A., Daniels, B., and Morris, A. J. (1998) Biochemistry 37, 4901-
4909.
Effect of Familial Mutants of Parkinson’s Disease on Membrane Remodeling Anuri Shah
60

25) Zhu M, Qin ZJ, Hu D, Munishkina LA, Fink AL (July 2006). "Alpha-synuclein can function
as an antioxidant preventing oxidation of unsaturated lipid in vesicles". Biochemistry 45 (26):
8135–42. doi:10.1021/bi052584t.
26) Uversky VN (October 2007). "Neuropathology, biochemistry, and biophysics of alpha-
synuclein aggregation". J. Neurochem. 103 (1): 17–37. doi:10.1111/j.1471-4159.2007.04764.
27) Madine J, Doig AJ, Middleton DA (May 2006). "A study of the regional effects of alpha-
synuclein on the organization and stability of phospholipid bilayers". Biochemistry 45 (18):
5783–92. doi:10.1021/bi052151.
28) Weinreb, P.H., Zhen, W.G., Poon, A.W., Conway, K.A. & Lansbury, P.T. (1996). NACP, a
protein implicated in Alzheimer's disease and learning, is natively unfolded Biochemistry,
Vol.35, No.43, (October 1996), pp. 13709-13715, ISSN 0006-2960.
29) Dedmon, M.M., Lindorff-Larsen, K., Christodoulou, J., Vendruscolo, M. &. Dobson, C.M
(2005). Mapping long-range interactions in alpha-synuclein using spin-label NMR and ensemble
molecular dynamics simulations. Journal of the American ChemicalSociety, Vol.127, No.2,
(January 2005), pp. 476-477, ISSN 0002-7863.
30) Bertoncini, C.W., Jung, Y.S., Fernandez, C.O., Hoyer, W., Griesinger, C., Jovin, T.M. &
Zweckstetter, M. (2005)
31) Gai, W. P., Yuan, H. X., Li, X. Q., Power, J. T., Blumbergs, P. C., and Jensen, P. H. (2000)
Exp. Neurol. 166, 324–333
32) Mechanisms of neuronal death in synucleinopathy. Takeda A, Hasegawa T, Matsuzaki-
Kobayashi M, Sugeno N, Kikuchi A, Itoyama Y, Furukawa K. J Biomed Biotechnol.
2006;2006(3):19365.
33) Brain Pathol. 2013 May;23(3):350-7. doi: 10.1111/bpa.12046. α-Synuclein: the long distance
runner. George S, Rey NL, Reichenbach N, Steiner JA, Brundin P.
34) Biochemistry- Styer 2006 edition.
35) Robbins SL, Cotran RS, Kumar V, Collins T, ed. (1999). Robbins pathologic basis of
disease. Philadelphia: Saunders. ISBN 0-7216-7335-X
36) Brundin, P., Melki, R. and Kopito, R. (2010) Prion-like transmission of protein aggregates in
neurodegenerative diseases. Nat. Rev. Mol. Cell Biol. 11, 301-307.
37) Moreno-Gonzalez, I. and Soto, C. (2011) Misfolded protein aggregates: mechanisms,
structures and potential for disease transmission. Semin. Cell Dev. Biol. 22, 482-487.
Effect of Familial Mutants of Parkinson’s Disease on Membrane Remodeling Anuri Shah
61

38) Garden, G. A. and La Spada, A. R. (2012) Intercellular (mis)communication in
neurodegenerative disease. Neuron 73, 886-901.
39) Lee, S.-J., Lim, H.-S., Masliah, E. and Lee, H.-J. (2011) Protein aggregate spreading in
neurodegenerative diseases: problems and perspectives. Neurosci. Res. 70, 339-348.
40) Brain Pathol. 2013 May;23(3):350-7. doi: 10.1111/bpa.12046. α-Synuclein: the long distance
runner. George S, Rey NL, Reichenbach N, Steiner JA, Brundin P.
41) Fink, A.L. (2006). The aggregation and fibrillation of alpha-synuclein. Accounts of
Chemical Research, Vol.39, No.9, (September 2006), pp. 628-634, ISSN 0001-4842.
42) α-Synuclein Oligomers with Broken Helical Conformation Form Lipoprotein Nanoparticles.
Varkey J, Mizuno N, Hegde BG, Cheng N, Steven AC, Langen R. J Biol Chem. 2013 Jun
14;288(24):17620-30. doi: 10.1074/jbc.M113.476697. Epub 2013 Apr 22.
43) Jo, E., McLaurin, J., Yip, C.M., George-Hyslop, P., Fraser, P.E. (2000). Alpha-synuclein
membrane interactions and lipid specificity. Journal of Biological Chemistry, Vol.27 No.44,
(November 2000), pp. 34328-34334, ISSN 0021-9258.
44) Jao, C. C., Der-Sarkissian, A., Chen, J., and Langen, R. (2004) Proc. Natl. Acad. Sci. U.S.A.
101, 8331–8336
45) Jao, C. C., Hegde, B. G., Chen, J., Haworth, I. S., and Langen, R. (2008) Proc. Natl. Acad.
Sci. U.S.A. 105, 19666–19671.
46) Membrane curvature sensing by amphipathic helices: a single liposome study using α-
synuclein and annexin B12. Jensen MB, Bhatia VK, Jao CC, Rasmussen JE, Pedersen SL, Jensen
KJ, Langen R, Stamou D. Source Bionanotechnology and Nanomedicine Laboratory,
Department of Neuroscience and Pharmacology, University of Copenhagen, Universitetsparken
5, 2100 Copenhagen, Denmar.
47) Necula, M., Chirita, C. N., and Kuret, J. (2003) J. Biol. Chem. 278, 46674–46680.
48) Remodeling of lipid vesicles into cylindrical micelles by α-synuclein in an extended α-
helical conformation. Mizuno N, Varkey J, Kegulian NC, Hegde BG, Cheng N, Langen R,
Steven AC. J Biol Chem. 2012 Aug 24;287(35):29301-11. doi: 10.1074/jbc.M112.365817. Epub
2012 Jul 5.
49) α-Synuclein Oligomers with Broken Helical Conformation Form Lipoprotein Nanoparticles.
Varkey J, Mizuno N, Hegde BG, Cheng N, Steven AC, Langen R. J Biol Chem. 2013 Jun
14;288(24):17620-30. doi: 10.1074/jbc.M113.476697. Epub 2013 Apr 22.
50) Yip, C. M., Darabie, A. A., and McLaurin, J. (2002) J. Mol. Biol. 318, 97–107. Drescher,
M., Godschalk, F., Veldhuis, G., van Rooijen, B. D., Subramaniam, V., and Huber, M. (2008)
Effect of Familial Mutants of Parkinson’s Disease on Membrane Remodeling Anuri Shah
62

Spin label EPR on α -synuclein reveals differences in the membrane binding affinity of the two
antiparallel helices. ChemBioChem. 9, 2411–2416
51) Drescher, M., Veldhuis, G., van Rooijen, B. D., Milikisyants, S., Subramaniam, V., and
Huber, M. (2008) Antiparallel arrangement of the helices of vesicle-bound α-synuclein. J. Am.
Chem. Soc. 130, 7796–7797
52) Gu, F., Jones, M. K., Chen, J., Patterson, J. C., Catte, A., Jerome, W. G., Li, L., and Segrest,
J. P. (2010) Structures of discoidal high density lipoproteins: a combined computational-
experimental approach. J. Biol. Chem. 285, 4652–4665.
53) Peters-Libeu, C. A., Newhouse, Y., Hall, S. C., Witkowska, H. E., and Weisgraber, K. H.
(2007) Apolipoprotein E*dipalmitoylphosphatidylcholine particles are ellipsoidal in solution. J.
Lipid Res. 48, 1035–1044.
54) Binding of a-Synuclein to Brain Vesicles Is Abolished by Familial Parkinson’s Disease
Mutation Poul H. Jensen‡§¶, Morten S. Nielsen‡, Ross Jakesi, Carlos G. Dotti§, and Michel
Goederti. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 41, Issue of October
9, pp. 26292–26294, 1998.
55) Mutation E46K increases phospholipid binding and assembly into filaments of human a-
synuclein Woong Choia, Shahin Zibaeeb,1, Ross Jakesa, Louise C. Serpellb,1, Bazbek
Davletova,R. Anthony Crowthera, Michel Goederta,* aMRC Laboratory of Molecular Biology.
56) Biochemistry 2010, 49, 862–871 DOI: 10.1021/bi901723p Differential Phospholipid Binding
of R-Synuclein Variants Implicated in Parkinson’s Disease Revealed by Solution NMR
Spectroscopy† Christina R. Bodner,‡,§ Alexander S. Maltsev,‡ Christopher M. Dobson,§ and Ad
Bax*,‡
57) Fortin, D.L., Troyer, M.D., Nakamura, K., Kubo, S., Anthony, M.D. and Edwards, R.H.
(2004) J. Neurosci. 24, 6715–6723.
58) Both Familial Parkinson’s Disease Mutations Accelerate a-Synuclein Aggregation Linda
Narhi‡, Stephen J. Wood‡, Shirley Steavenson‡, Yijia Jiang, Gay May Wu, Dan Anafi,
59) Stephen A. Kaufman, Francis Martin, Karen Sitney, Paul Denis, Jean-Claude Louis,
60) Jette Wypych, Anja Leona Biere, and Martin Citro THE JOURNAL OF BIOLOGICAL
CHEMISTRY Vol. 274, No. 14, Issue of April 2, pp. 9843–9846, 1999
61) The Impact of the E46K Mutation on the Properties of R-Synuclein in Its Monomeric and
Oligomeric States† Ross A. Fredenburg,‡ Carla Rospigliosi,§ Robin K. Meray,‡ Jeffrey C.
Kessler,‡ Hilal A. Lashuel, David Eliezer,§ and Peter T. Lansbury, Jr.
Effect of Familial Mutants of Parkinson’s Disease on Membrane Remodeling Anuri Shah
63

62) Effect of Familial Parkinson’s Disease Point Mutations A30P and A53T on the Structural
Properties, Aggregation, and Fibrillation of Human R-Synuclein† Jie Li,‡,§ Vladimir N.
Uversky,*,‡,§,| and Anthony Fink.
63) Mutant Protein A30P -Synuclein Adopts Wild-type Fibril Structure, Despite Slower
Fibrillation Kinetics* Luisel R. Lemkau‡1, Gemma Comellas§2, Kathryn D. Kloepper‡3,
Wendy S. Woods¶, Julia M. George¶, and Chad M. Rienstra. THE JOURNAL OF
BIOLOGICAL CHEMISTRY VOL. 287, NO. 14, pp. 11526–11532, March 30, 2012
64) Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset
Parkinson disease. Conway KA et al. Nat Med. (1998)
65) Konrad J. Böhm and Beate Winner Silvia Campioni, Katrin Buder, Roland Riek, Iryna Prots,
Vanesa Veber, Stefanie Brey, Microtubule-Kinesin Interplay. J. Biol. Chem. 2013, 288:21742-
21754
66) Alpha-synuclein induces lysosomal rupture and cathepsin dependent reactive oxygen species
following endocytosis, Campbell EM et al. PLoS One. 2013 Apr 25;8(4):e62143. doi:
10.1371/journal.pone.0062143
67) Matsuzaki-Kobayashi M, Hasegawa T, Kikuchi A, Takeda A, Itoyama Y. Role of iron in the
intracellular aggregation of α- synuclein. Movement Disorders. 2004;19(sup9):S36
68) Bartels T, Choi JG, Selkoe DJ (2011) Alpha-synuclein occurs physiologically as a helically
folded tetramer that resists aggregation. Nature 477:107–110.
69) Wang W, Perovic I, Chittuluru J, Kaganovich A, Nguyen LTT, Liao JL, Auclair JR, Johnson
D, Landeru A, Simorellis AK, Ju SL, Cookson MR, Asturias FJ, Agar JN, Webb BN, Kang CH,
Ringe D, Petsko GA, Pochapsky TC, Hoang QQ. (2011) A soluble alpha-synuclein construct
forms a dynamic tetramer. Proc Natl Acad Sci USA 108:17797–17802
70) N-terminal acetylation is critical for forming a-helical oligomer of a-synuclein Adam J.
Trexler1 and Elizabeth Rhoades., DOI: 10.1002/pro.2056 Published online 9 March 2012
proteinscience.org.

Abstract (if available)

Abstract The pathological hallmark of Parkinson’s disease (PD) is the presence of Lewy bodies or cell inclusions in neurons, comprising an amyloid protein known as alpha synuclein. These aggregates of protein are normally found with the presence of lipid fragments. Whether these fragments of lipid are derived from disrupted cellular membranes is unknown but more and more evidence corroborates the plausibility of this phenomenon. ❧ A recent breakthrough in the realm of alpha synuclein research is suggestive of its membrane binding and curvature inducing ability. Alpha synuclein has been shown to tubulate negatively charged phospholipid vesicles and even remodel them into nanoparticles at higher protein: lipid ratios in the oligomeric state. This property of alpha synuclein is similar to that of apolipoproteins, which are lipid carriers found in the blood stream. Studies till date have been targeted at understanding better how these properties of alpha synuclein play a physiological role, likening it to a lipid carrier or chaperone in exo/endocytosis. On the other hand, if these very same properties are out of control they may surpass physiological relevance and set in motion hazardous events such as cellular permeabilization, having neurotoxic implications that might abet the pathophysiology of PD. ❧ The 3 familial mutants of alpha synuclein associated with PD i.e, A30P, E46K and A53T have consistently over the years shown a range of different properties. The A30P and A53T mutants are known to be more prone to aggregation. Previous literature suggests that it is this property of theirs which confers them toxic. The E46K mutant however does not exhibit this property and must therefore cause cellular toxicity by another mechanism. To gauge this predicament better, I aimed to characterize the membrane remodeling properties of these familial mutants using tools such as clearance assays, leakage assays and electron microscopy. The goal was to see whether these mutants possessed enhanced or diminished membrane remodeling abilities and whether this could be a trigger for neurodegeneration. The E57K mutant which is a well- known artificial but toxic mutant was tested as well. Based on the results of my study, the E46K mutant exhibited greater membrane leakage and clearance, possibly implying that it generates its toxic effects by remodeling membranes to an exaggerated extent and disrupts their nature. A30P and A53T on the other hand were not proficient in their membrane remodeling and leakage causing abilities further consolidating that the major role they play is of enhanced aggregation. Thus they could bring about their effects by enhanced misfolding. Therefore two distinct mechanisms for toxicity seem plausible based on these results, which could then be used as an ideal tool for studying physiological membrane remodeling gone awry and how that might lead to disease.

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Creator Shah, Anuri N. (author)

Core Title The effect of familial mutants of Parkinson's disease on membrane remodeling

School School of Pharmacy

Degree Master of Science

Degree Program Molecular Pharmacology and Toxicology

Publication Date 11/07/2013

Defense Date 10/14/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag alpha synuclein,membrane remodeling,mutants,OAI-PMH Harvest,Parkinson's

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Advisor Haworth, Ian S. (committee chair), Langen, Ralf (committee member), Okamoto, Curtis Toshio (committee member)

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