Role of Sporadic Parkinson Disease Associated Mutations A18T and

Subscriber access provided by JAMES COOK UNIVERSITY LIBRARY

Article

Role of sporadic Parkinson disease associated mutations A18T and A29S in enhanced alpha synuclein fibrillation and cytotoxicity Sanjay Kumar, Deepak Kumar Jangir, Roshan Kumar, Manisha Kumari, Neel Bhavesh, and Tushar Kanti Maiti ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00430 • Publication Date (Web): 25 Aug 2017 Downloaded from http://pubs.acs.org on August 26, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Neuroscience is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

1

Role of sporadic Parkinson disease associated mutations A18T and A29S in enhanced

2

alpha synuclein fibrillation and cytotoxicity

3

4

Sanjay Kumar1,2*, Deepak Kumar Jangir1,4*, Roshan Kumar1,2, Manisha Kumari1, Neel

5

Sarovar Bhavesh 3 and Tushar Kanti Maiti1#

6

1

Functional Proteomics Laboratory, Regional Centre for Biotechnology, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad-121001, India

7

2

8 3

9

12

Transcription Regulation Group, International Centre for Genetic Engineering and

Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi, 110067, India.

10 11

Manipal University, Manipal, Karnataka-576104, India

4

Present address: Centre for Biotechnology, Maharshi Dayanand University, Rohtak-124001, India

13

14

15

16

17

18

19

20

21

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

22

ABSTRACT

23

Deposition of pre-synaptic protein α-synuclein in Lewy bodies and Lewy neurites in the

24

substantia nigra region of brain has been linked with the clinical symptoms of the Parkinson’s

25

disease (PD). Proteotoxic stress conditions and mutations that cause abnormal aggregation of

26

α-synuclein have close association with onset of PD and its progression. Therefore, studies

27

pertaining to α-synuclein mutations play important roles in mechanistic understanding of

28

aggregation behaviour of the protein and subsequent pathology. Herein, guided by this fact,

29

we have studied the aggregation kinetics, morphology and neurotoxic effects of the two

30

newly discovered sporadic PD associated mutants A18T and A29S of α-synuclein. Our

31

studies demonstrate that both of the mutants are aggregation prone and undergo rapid

32

aggregation compared to wild type α-synuclein. Further, it was found that A18T mutant

33

followed faster aggregation kinetics compared to A29S substitution. Additionally, we have

34

designed three point mutations of α-synuclein for better understanding of the effects of

35

substitutions on protein aggregation and demonstrated that substitution of alanine at 18th

36

position is highly sensitive compared to adjacent positions. Our results provide better

37

understanding on the effects of α-synuclein mutations on its aggregation behaviour that may

38

be important in development of PD pathology.

39

Keywords: α-synuclein, aggregation, atomic force microscopy, mutation, neurodegeneration,

40

Parkinson’s disease

41

42

43

44

2


Page 2 of 41

Page 3 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


45

INTRODUCTION

46

Improper folding or misfolding of proteins that leads to aggregation is implicated in various

47

diseases all of which are referred as protein conformational disorders.1 This diverse group

48

includes type II diabetes, different amyloidosis, some types of cancer and a number of

49

neurodegenerative diseases including PD, Huntington’s disease, Alzheimer’s disease,

50

multiple

51

Synucleinopathy is a common term to refer to a subset of conformational disorders,

52

characterized by abnormal deposition of α-synuclein inclusions and fibrillar aggregates in

53

neurons, nerve fibres or glial cells in diverse parts of the brain.3 This class of

54

neurodegenerative disorders mainly comprises PD, multiple system atrophy (MSA) and

55

dementia with Lewy bodies (DLB). Among these, PD is the most common form of

56

synucleinopathy.3 Neurodegenerative disorders are thought to occur via either loss of

57

function or gain of toxic function of the causative protein.4 In PD, mutant α-synuclein has

58

been reported to induce pathology by gain of toxic function.3,5,6 Exact cause of PD is not well

59

understood but its onset, severity and progression directly correlate with the conditions that

60

cause aberrant aggregation of α-synuclein.7-9 α-synuclein is widely expressed in different

61

brain regions and accounts for 1% of total cytosolic neuronal proteome.3 Though, functional

62

assignments to α-synuclein is ambiguous and its exact role(s) in the cell remains to be

63

established. It might be involved in neuronal plasticity, trafficking of synaptic vesicles at the

64

neuronal ends and interaction with different proteins, associated with signal transduction.10-12

65

Predominant accumulation of α-synuclein at the presynaptic terminals even indicates its

66

possible role in synaptic transmission.3,13 This protein has a natural tendency to form amyloid

67

aggregates that is greatly enhanced in PD and other synucleinopathies.14,15

68

At structural level, α-synuclein is a low molecular weight protein composed of 140 amino

69

acid residues and is classified as natively disordered protein.16,17 It contains three distinct

system

atrophy

(MSA)

and

different

spinocerebellar

3


ataxias

(SCA).2


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

70

regions, amphipathic N-terminus (1-60), fibrillation core or non-amyloid-β component

71

(NAC) region (61-95) and negatively charged C-terminus (96-140).9 Different regions of α-

72

synuclein have roles in retaining protein’s native disordered state that seems to be a requisite

73

to fulfil its normal cellular functions. Genetic factors like mutations and multiplications of

74

SNCA gene (encodes for α-synuclein) resulting in abrupt aggregation of the protein, have

75

been implicated in PD.7-9 Essentially, studies pertaining to mutations of α-synuclein have

76

shaped much of our understanding about its aggregation kinetics, oligomerization and

77

fibrillation processes that contribute to the pathological conditions. For instance, A30P

78

mutation is associated with early onset of PD and enhances α-synuclein oligomerization but it

79

impedes subsequent fibrillation process.18,19 This remarkable observation provided early clues

80

that apart from protofibrils and mature fibrillar species, soluble oligomers of α-synuclein are

81

indeed cytotoxic entity.19 Furthermore, established familial mutations A53T, H50Q and

82

E46K greatly accelerate the aggregation of α-synuclein hence cause early onset of PD.20,21

83

Because different mutations affect aggregation of α-synuclein and development of PD in

84

different ways, therefore, studies pertaining α-synuclein mutations are crucial towards a

85

better understanding of PD and other synucleinopathies as well as to further pave the way of

86

therapeutic intervention.

87

In 2013, studies by Hoffman-Zacharska et al. on SNCA gene repository retrieved from brains

88

of 629 PD patients of Polish origin, discovered two novel point mutations, A18T and A29S,

89

both predicted to be potentially pathogenic and deleterious.22 These two substitutions are

90

associated with sporadic late onset of PD and were absent in healthy controls. Interestingly,

91

all SNCA substitutions reported till date are confined within the N-terminal amphipathic

92

region of α-synuclein which is critical for α-synuclein aggregation.23 These two newly

93

discovered mutations are also located in the same N-terminus region. This region largely

94

exists as disordered in native free monomeric conditions, though, it readily adopts α-helical 4


Page 4 of 41

Page 5 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


95

conformation in vicinity of lipid membranes.24,25 This α-helix is not intact rather it is a broken

96

helix in which two helical stretches (positions 9-36 and 41-65) are separated by 4 amino acid

97

residues.24 While earlier mutations are confined within the second helical segment (except

98

A30P), both the newly discovered mutations are located in the first α-helical segment. A18T

99

and A29S substitutions replace the highly conserved amino acid of the N-terminal region.

100

Overall, it seems reasonable that A18T and A29S substitutions can significantly affect the

101

pathophysiological properties of α-synuclein in ways that may profoundly contribute to PD

102

pathology. Given the importance of these mutations and their association with PD, we

103

undertook the present study to characterize the structural, morphological and cytotoxic

104

properties of these α-synuclein mutants. Such studies play crucial roles in the better

105

understandings on development and progression of PD and other related synucleinopathies.

106

RESULTS AND DISCUSSION

107

A18T and A29S accelerate α-synuclein aggregation

108

A53T mutation of α-synuclein is linked to early onset of PD and its biophysical properties are

109

well characterized. Therefore, we used this mutant protein along with wild type α-synuclein

110

for comparison of aggregation behaviour of A18T and A29S substitutions. Thioflavin T

111

(ThT) kinetics of wild type and A53T mutant of α-synuclein were essentially similar to the

112

earlier reported data.26 Both A18T and A29S mutant proteins followed sigmoidal course of

113

aggregation but they differed in their lag phase and rate of aggregation (Figure 1a, SI Figure

114

S1, SI table 1). Interestingly, A18T was highly aggregation prone and its rapid aggregation

115

was evident by the apparent lack of lag phase in ThT assay (tlag~4.45±0.57 h). Its aggregation

116

pattern was fairly comparable to the established familial mutant A53T (Figure 1a, SI Table

117

1). Both of these mutant proteins showed plateau at ~40 h of incubation with A18T attained

118

saturation a bit faster than A53T (Kapp 0.150±0.007 and 0.110±0.001 h-1 for A18T and A53T

5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

119

respectively SI Table 1). Aggregation of A29S was also rapid compared to wild type protein

120

but was slower than A18T and A53T mutants (Kapp 0.070±0.004 and tlag 13.46±0.36 for

121

A29S, SI Table 1). To complement the ThT results, we performed 1-Anilinonaphthalene-8-

122

sulfonic acid (ANS) fluorescence binding assay for these samples. Results of ANS binding

123

were in good agreement with the ThT data (Figure 1b, SI Figure S2). Similar to ThT assay,

124

A18T exhibited rapid hydrophobic surface exposure compared to wild type α-synuclein.

125

Similarly, aggregation of A29S was slower than A18T and A53T mutants. It was evident

126

from ANS binding that A29S displayed intermediate exposure of hydrophobic surfaces

127

compared to wild type and A18T mutant of α-synuclein during early hours of aggregation. It

128

should be noted here that A18T has been reported to be more pathogenic compared to A29S

129

and our results indicate that faster aggregation of A18T could be the reason behind this

130

phenomenon (SI Figure S5, SI Table 1).22 Although, one important point should be taken in

131

account here that A18T forms amyloid-like fibrils more rapidly than A53T, yet it is

132

associated with late-onset PD, whereas A53T mutant is associated with early-onset PD.22,27

133

These observations suggest that the ability to predict relative degrees of pathogenicity of

134

different α-synuclein variants based on their relative rates of fibrillation is limited.

135

Both A18T and A29S form morphologically similar fibrils to wild type protein

136

Atomic force microscopy (AFM) imaging was used in combination with ThT and ANS

137

binding assay to determine the morphology of intermediate species and final amyloid fibrils.

138

AFM images at 0 h time point showed absence of any large pre-aggregated species in the

139

samples (Figure 1c). It reflected the homogeneity of low molecular weight (LMW) species of

140

the protein samples prepared for the experiments. Rapid aggregation of A18T and A53T were

141

evident from early fibril formation at 24 h of incubation (Figure 1c). Both of these mutants

142

showed presence of mature fibrils making dense networks. Less number of fibrils were

143

present in the sample of wild type protein due to slow aggregation of wild type protein 6


Page 6 of 41

Page 7 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


144

compared to mutants. Short protofibrils and fibrils were the dominating species at 24 h time

145

point for A29S as revealed by AFM imaging. To examine the distribution of protofibrils and

146

fibrils, quantitative analysis of AFM results were carried out. At 24 h, mean height of wild

147

type fibrils/protofibrils was ~4.69±0.83 nm whereas it was 5.36±1.27, 6.45±1.31 and

148

~7.04±1.25 nm for A18T, A29S and A53T respectively (Figure 2 and Table1). The mean

149

length of these protofibrils/fibrils was measured 459±223, 477±144, 313±94 and 613±237 nm

150

for wild type, A18T, A29S and A53T respectively. Short protofibrils/fibrils of A29S were

151

converted into mature and long fibrils upon further incubation at 37o C for 48 h as revealed

152

by the increase in their persistent length (Figure 2 and Table 1). Morphology of fibrils of

153

A18T protein was similar to other fibrils (A53T, A29S and wild type protein) at 48 h of

154

incubation (Figure 2 upper panel). Our data confirmed high aggregation propensity of A18T

155

mutant.

156

A18T and A29S mutants of α-synuclein do not change global structure

157

Studies have suggested that the C-terminal of α-synuclein (around residue 120) interacts with

158

the N-terminus region.28-31 These long range interactions shield the middle hydrophobic core

159

(NAC region) domain and keep it devoid of solvent exposure.28,32,33 Observation of long

160

range interactions is further reinforced by studies which showed that polyamine binding to C-

161

terminus releases the N-terminus and subsequent inherent residual helical propensity and thus

162

results in extended conformation which readily aggregates.28,34 Earlier studies have shown

163

that mutations perturb long range interactions between C-terminal and N-terminal domain,

164

resulting in varied aggregation rate of α-synuclein.35,36 On the contrary, a number of studies

165

have suggested that, though, long range interactions are present in the α-synuclein, they are

166

not sufficient to explain the altered aggregation rates due to different mutations.37 For

167

example, E46K has been shown to enhance these long range interactions but this mutation

168

results in enhanced aggregation of α-synuclein compared to wild type.37 To ascertain whether 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

169

enhanced aggregation of mutants was due either to a loss in long range interaction or to

170

altered secondary structure preference due to the local structural perturbation, we carried out

171

solution-state NMR spectroscopy studies, as previously described.36 Under our experimental

172

conditions, we did not observe any significant global chemical shift perturbation in the

173

mutants except a minor change in chemical shift perturbation observed in the neighbouring

174

residues next to the mutation site (Figure 6). These results are consistent with earlier

175

observations in some other mutations of α-synuclein.37 Though our NMR data cannot provide

176

the direct evidence of mechanism of faster aggregation of A18T mutant compared to A29S

177

one can presumably consider that A18T may prefer beta structure which drives faster

178

aggregation and detail structural studies are necessary in this direction.

179

Secondary structure analysis of A18T and A29S mutants during aggregation

180

Circular dichroism (CD) spectroscopy was carried out to determine change in secondary

181

structure content of α-synuclein mutants at different time points of aggregation (Figure 1d).

182

CD spectra of A53T and A18T exhibited alterations at ~24 h time point. A18T showed more

183

β-sheet content at 24 h while disordered content was more for A53T at this time point.

184

However, conformational transition was accelerated for A53T after 24 h and it achieved

185

similar aggregation stage of A18T at ~48 h time point (SI Figure S4). It further confirmed

186

that aggregation of A18T was inherently rapid. A29S displayed slow transition from

187

disordered to β-sheet rich amyloid fibrils. Its fibrillation completed at ~72 h. These CD

188

results confirmed that A29S was slow aggregating compared to A18T and A53T mutants.

189

Conformational transition for wild type α-synuclein was even slower and completed after

190

~120 h of incubation.

191

ThT and ANS data showed that perturbation of both the positions 18th and 29th by threonine

192

and serine respectively, resulted in aberrant aggregation of α-synuclein (Figure 1a, b). A18T

8


Page 8 of 41

Page 9 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


193

substitution exerted greater effect than A29S substitution. Differences in the aggregation rates

194

of these two mutants could be a result of altered preference for secondary structure.28,37 At the

195

initial time point (0 h) all the proteins have similar secondary structural distribution as

196

observed in the CD (SI Table 2). However, at 6 h time point A18T mutant showed faster

197

conversion of β-sheet than wild type, A29S and A53T ( SI Table 2). α-Synuclein contains six

198

imperfect K-T-K-E-G-V motifs, shown to be important for regulation of its aggregation.38,39

199

Alanine residues at 18th and 29th positions fall between two K-T-K-E-G-V repeat motifs and

200

hence may significantly affect helix turn helix structure of α-synuclein. The observed

201

differences in their aggregation propensities might be governed by differences in their

202

respective positions as well as type of substitutions. For example, serine differs from

203

threonine in Cβ-position where H is replaced by methyl group in threonine. Threonine is ‘Cβ

204

branched’ i.e. it contains two non-hydrogen substituent attached to its Cβ carbon. It creates

205

more constrains in the protein backbone, consequently making it harder for threonine to adopt

206

an alpha-helical conformation40,41 and shift the equilibrium towards β rich conformation of α-

207

synuclein.

208

Alanine at 18 is more sensitive for aggregation compared to adjacent positions A17 and

209

A19

210

Our solution NMR data of A18T, A29S and A53T in comparison to wild type α-synuclein

211

indicates that there is change in local environment near to the mutation site. These local

212

changes contribute varying aggregation rate as observed in our ThT binding studies. To

213

evaluate the effects of local environment (adjacent positions) on α-synuclein aggregation, we

214

generated A17T and A19T mutants of α-synuclein and all biophysical experiments were

215

performed. The alanine to threonine substitution at 17th and 19th positions resulted in faster

216

aggregation than wild type protein, though, these two mutants were less aggregation prone

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 41

217

than A18T mutant. The solution NMR structural comparison of A19T with wild type α-

218

synuclein showed similar changes in chemical shift perturbation like A18T. However, A17T

219

showed additional minor change in CSP near to 50-60 residues along with the local mutation

220

site. A17T might have altered conformation compared to other mutants and it may not induce

221

the aggregation process (SI Figure S6). Both A17T and A19T mutants exhibited similar

222

aggregation kinetics like A29S (Figure 3a, b, SI Table 1). However, thin protofibrils/fibrils

223

were abundant for A17T and A19T mutants (Figure 1c, 3c). The mean values of height

224

profiles of A29S, A17T and A19T fibrils/protofibrils were ~6.45±1.31, 5.66±0.98 and

225

4.83±1.38 nm respectively (Table 1) at 24 h. A17T and A19T mutants formed fibrils with

226

similar aggregation kinetics as indicated in the lag time of aggregation and apparent rate

227

constant of aggregation (tlag

228

=0.070±0.003, Kapp(A19T) = 0.060±0.004) (SI Table 1). The CD spectra analysis of both these

229

mutants revealed that the beta sheet conversion for A17T was more than A19T at 24 h time

230

period (A17T, 22% and A19T 9%) and this finding was consistent with AFM analysis

231

(Figure 3d). At 48 h time point, mean height values and overall fibril morphology were

232

similar for all the mutants (Figure 2, Table 1).

233

A18P mutation results in rapid aggregation of the protein

234

Although α-synuclein is natively disordered in its monomeric form, previous studies indicate

235

that N-terminal segment of the protein is not completely disordered, rather it contains some

236

residual helical content (~5-8%).29 If this notion is true, proline residue at 18th position should

237

also result in rapid aggregation of α-synuclein because proline is a very good helix breaker

238

residue.42,43 We have investigated these possibilities by generating A18P mutant and studying

239

its aggregation behaviour.

(A17T)

=16.31±0.89 h, tlag

(A19T)

=16.16±1.10 h and Kapp(A17T)

10


Page 11 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


240

A18P mutant showed similar kind of aggregation that observed for A18T (Figure 4a, b).

241

Indeed, this substitution resulted in similar aggregation kinetics, indicating enhanced

242

aggregation for this mutant compared to wild type (SI table 1). The solution NMR structural

243

data of A18P in comparison to wild type also showed similar local change in CSP like A18T

244

mutant. The NMR data indicated the presence of similar conformation in both A18T and

245

A18P species in our experimental condition. In AFM imaging, A18P showed a large number

246

of short and thick fibrillar species at 24 h time point with height and length of 8.35±1.85 and

247

355±92 nm respectively, which converted into elongated fibrils upon further incubation

248

(Figure 4c, Table 1). Rapid aggregation of A18P was accompanied by sharp initial changes as

249

revealed by CD spectra (Figure 4d). To get further insight into conformational changes at

250

smaller time intervals, we conducted CD experiment at 6 h time intervals for A18P and

251

A18T, A53T and wild type α-synuclein (SI Figure S3a). Our CD results and rate analysis

252

revealed that conformational transition for A18T and A18P, were rapid compared to A53T

253

and wild type. The rate for β-sheet formation in A18T, A18P, A53T and wild type were 0.24

254

h-1, 0.21 h-1, 0.20 h-1 and 0.06 h-1 respectively (SI Figure S3a, b). Detailed analysis of

255

secondary structure analysis revealed that at 24 h time point, conformational transitions from

256

random coil to β-sheet were high for both A18T and A18P compared to A53T mutant (SI

257

figure S4). It further confirms that both A18T and A18P mutants prefer beta sheet structure

258

during progression of aggregation. Our results suggest that residue at the 18 position is

259

critical and thus affects α-synuclein folding and the fibrillation process. In contrast, a change

260

at adjacent sites (17th and 19th) is not that much robust, further illustrating importance of the

261

alanine residue at 18th position. Both A18T and A29S fall on the same helical position but

262

A18T shows crucial effects on α-synuclein aggregation compared to A29S (Figure 5a and

263

b).22 It reflects that both difference of position as well as type of substitution are crucial for

264

the aggregation propensity of the protein. These observations illustrate that a few amino acid

11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

265

positions in α-synuclein structure are highly sensitive towards substitution and for

266

aggregation behaviour of the protein. These positions can be regarded as hot spot for

267

aggregation.

268

Extent of fibrillation and cellular internalization of A18T and A29S mutants of α-

269

synuclein

270

We examined the differences in the extent of fibril formation induced by mutations (SI

271

methods). We found that A18T mutation resulted in slightly more fibrillar content compared

272

to A29S at 48 h time point (SI Figure S5). Other mutants also exhibited differences in their

273

extent of fibril formation at this time point. Recent reports suggest that similar to prions, cell

274

to cell transmission of α-synuclein may occur and thus it may contribute to initiation,

275

progression and spreading of PD pathology.44 Given the fact that α-synuclein is secreted in

276

extracellular space and interacts with neuronal cell membrane, it becomes more important to

277

evaluate the cytotoxicity of extra-cellular α-synuclein species.45 Internalization of wild type

278

α-synuclein fibrils is well documented.46 Hence, we examined the internalization efficiency

279

of fibrils of mutant proteins using cell based internalization assay. We found that when

280

incubated at the same fibrillar concentration, all the α-synuclein mutants internalized into the

281

neuroblastoma cells with similar efficacy (Figure 7a, b).

282

Cytotoxicity of A18T and A29S fibrils and oligomers

283

To evaluate the extent of cytotoxicity caused by fibrils and oligomers of different mutants,

284

we performed the MTT assay. Oligomer rich samples were prepared as described previously

285

(SI method) and fibrils were isolated from residual oligomers and monomers by

286

centrifugation method as described in SI method. The oligomers and fibrils concentrations

287

were measured according to the method described earlier (SI method). Same molar

288

concentration of oligomers and fibrils were taken in cytotoxicity assay. The percentage of cell

12


Page 12 of 41

Page 13 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


289

death induced by different fibrillar species of. wild type A18T, A29S, A53T, A18P, A17T

290

and A19T α-synuclein were 16.9, 25.5, 22.18, 24.95, 19.03, 17.35 and 22.16 respectively

291

(Figure 8a) and percentage of cell death induced by oligomeric species of wild type, A18T,

292

A29S, A53T, A18P, A17T and A19T were 16.33, 35.72, 29.68, 26.66, 35.41, 14.76 and

293

35.23 respectively (Figure 8b). These data suggest that both oligomeric and fibrillar species

294

are toxic. Though our present data cannot discriminate the toxicity of different mutants.

295

Conclusions

296

The results presented here indicate that A18T is highly aggregating in nature, while A29S

297

have similar albeit weaker tendency. This study illustrate that some positions could be treated

298

as hot spot for α-synuclein aggregation while others could be less sensitive towards

299

substitutions. Indeed, substitutions which result in the significant destabilization of native α-

300

synuclein structure may lead to aberrant aggregation of the protein and hence render

301

deleterious effects and thus may contribute to PD pathology. Overall, the present study shed

302

light on aggregation behaviour and differences in aggregation kinetics of PD associated A18T

303

and A29S mutants.

304

METHODS

305

Protein expression and mutagenesis

306

Plasmid 36046 (pT7-7) containing wild type α-synuclein, was a generous gift from Professor

307

Hilal Lashuel.47 α-synuclein was expressed in BL21 (DE3) E. coli strain. Site directed

308

mutagenesis was carried out using Stratagene, Quick change mutagenesis kit to generate all

309

the mutant proteins. Protein expression and purification were done using the established

310

protocol.48 Purified proteins were stored at -80 ºC for further experiments. ThT and ANS

311

were procured from Sigma-Aldrich, USA. Other reagents and chemicals used were of

312

analytical grade. 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

313

In vitro aggregation of α-synuclein

314

In vitro fibrillation assay was initiated with low molecular weight (LMW) species of α-

315

synuclein. For LMW species preparation, first 2 mM NaOH was added to α-synuclein stock

316

solution and incubated for 10 min on ice. Then pH was adjusted to 7.4 by adding

317

concentrated HCl solution. This process dissolved the pre-aggregates large species. Finally,

318

protein solution was passed through 100 kDa Amicon® Ultra-15 (Millipore) to get

319

monomeric species. Concentration of α-synuclein and mutant proteins were determined by

320

spectrophotometric method (Perkin-Elmer Lambda 35, CT, USA), using excitation

321

coefficient 5960 M-1cm-1 at 280 nm.45 Protein samples of concentration 300 µM in 20 mM

322

sodium phosphate buffer (pH 7.4, containing 0.1 % sodium azide) were incubated at 37 ºC

323

with constant stirring at 300 rpm for homogenous aggregation. Aliquots were withdrawn at

324

different time intervals for the experiments.

325

ThT and ANS fluorescence spectroscopy

326

To characterize the aggregation kinetics of A18T and A29S mutants, we applied optimized

327

conditions of ThT assay for α-synuclein fibrillation.45 At different time intervals, 6 µM

328

protein sample was incubated with 8 µM of ThT dye to check the amyloid aggregation of

329

wild type α-synuclein and mutant proteins. For ANS binding assay, 30 µM protein was

330

incubated with 20 µM of ANS. Fluorescence increment were measured on fluorescence

331

spectrophotometer (Hitachi F-7000 Tokyo, Japan). Excitation and emission wavelengths

332

were 442 and 482 nm for ThT while 372 and 475 nm for ANS assay respectively. Slit width

333

was 5 nm for excitation and emission. All experiments were done in triplicates and results are

334

the average of three sets. Kinetic curves were normalized according to the equation: Xn = (X-

335

Xmin)/(Xmax - Xmin).

14


Page 14 of 41

Page 15 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


336

CD spectroscopy Protein samples were diluted in sodium phosphate buffer to a final

337

concentration of 10 µM for CD spectroscopy. Measurements were performed in the Far-UV

338

range of 190-260 nm to reveal changes in secondary structure of protein during aggregation.

339

All measurements were carried out on CD spectrophotometer (Jasco-815, Tokyo, Japan). Six

340

accumulations were taken for each sample with a scan speed of 50 nm/min. For all the CD

341

experiments, cuvette used was 1 mm thick. All spectra were background subtracted with

342

buffer sample. The formula of mean residual ellipticity ([θ]rmw) has been given as49

343

[θ]rmw= (

344

Where

345

g/ml.

346

AFM Imaging

347

Aliquots of protein samples at different time intervals (0, 24 and 48 h) were withdrawn and

348

diluted to a final concentration of 5 µM. Samples were applied on freshly cleaved mica (Ted

349

Pella) and air dried in clean environment. Subsequently, slides were washed with Milli-Q

350

water and dried again before measurements. Imaging was done on JPK Nano Wizard III

351

instrument (JPK Instrument, Berlin, Germany). Drive frequency of silicon cantilever was

352

between 300 to 320 kHz and the scan rate was between 0.8 to 1Hz with a spring constant of

353

13-77 N/m.

354

Solution-state NMR spectroscopy

355

HSQC 2D [15N,1H] spectra were measured as described previously.36,50 The NMR sample

356

contained 0.5 mM α-synuclein 15N-labeled in 10 mM Tris-HCl buffer pH 7.4, 100 mM NaCl,

357

5% D2O (v/v). All NMR spectra were measured at 298 K on Bruker Avance III spectrometers

358

equipped with 5 mm cryogenic triple resonance TCI probes, operating at field strengths of

) deg. cm2 .dmol-1 = observed ellipticity in degrees, d = pathlength in cm, c = concentration in

15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

359

500 MHz. The spectra were acquired with 16 scans per t1 increment in 15N dimension.

360

Acquisitions times for 15N (SW = 1520 Hz) and 1H (SW = 7002 Hz) dimension were 105.2 ms

361

(t1max) and 146.2 ms (t2max) respectively. All spectra were referenced to DSS, processed

362

with Topspin 2.1 (Bruker AG) and data was analyzed using CARA software. To identify the

363

possible structural changes in the protein due to mutations, chemical shift perturbations

364

(CSP) were calculated from the unbound and bound 2D [15N, 1H] HSQC spectra using the

365

following equation.52

 ∆δ 15 N H =   5

2

  + (∆δ H N )2 

366

∆δ 15 N H ,H N

367

Where, ∆δ (1HN) and ∆δ (15N) are the changes in backbone amide chemical shifts

368

for 1HN and 15N respectively.

369

Fibrillar and oligomeric cytotoxicity

370

To evaluate the cytotoxicity of matured fibrils and oligomeric samples, MTT assay was

371

carried out. SH-SY5Y neuroblastoma cells were routinely cultured in DMEM Glutamax

372

(Gibco, Denmark) containing 10% fetal bovine serum, and 1% penicillin-streptomycin, and

373

maintained at 37 °C in a humidified incubator with 5% CO2/95% room air. In brief, SH-

374

SY5Y cells were plated at a density of 4000 cells per well on Costar, 96 well cell culture flat

375

bottom plate (Corning, New York, USA). After 48 h incubation, media was changed and

376

treated with 20 µM of wild type and mutated α-synuclein fibrils and oligomer enriched

377

samples. Control samples were prepared with addition of identical volumes of buffer. Cells

378

were incubated for additional 20 h and afterwards MTT 2-(2-methoxy-4-nitrophenyl)-3-(4-

379

nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium) was added at a final concentration of 0.5 16


Page 16 of 41

Page 17 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


380

mg/ml with 200 µl of media. The MTT reduction was allowed for 4 h. Then media was

381

removed and the formazan crystals that formed were dissolved in DMSO. The absorbance of

382

the formazan was measured at 570 nm in a M5 micro plate reader (Molecular Devices,

383

Sunnyvale, CA, USA).

384

Protein secondary structure analysis

385

Dichroweb, a web based online analysis tool for protein CD spectra was used for secondary

386

structure analysis of proteins. CDSSTR method with reference set 7 was applied for data

387

analysis.53

388

Supporting Information Paragraph: Methodology; measurement of fibril height and length

389

for quantitative measurement, calculation of kinetic parameters tlag and Kapp,, determination of

390

extent of fibrillation, internalization assay, preparation of oligomer samples for MTT assay

391

and HSQC spectral measurement. Figures: (S1) ThT fluorescence of different α-synuclein

392

mutants, (S2) ANS fluorescence of different α-synuclein mutants, (S3) CD measurements for

393

short time intervals and calculated rate curve, (S4) Dichroweb analysis of secondary structure

394

content of wild type and different mutants of α-synuclein, (S5) Percent fibrillation of

395

different mutant proteins and (S6) Overlaid 1H-15N heteronuclear single quantum coherence

396

(HSQC) spectra of wild type α-synuclein and mutant proteins show local perturbation of

397

NMR signals. Table (1): Lag time of aggregation (tlag) and apparent rate constant (Kapp) for

398

different mutants of α-synuclein and Table (2): Dichroweb analysis of secondary structure

399

content of wild type and different mutants of α-synuclein.

400

AUTHOR INFORMATION

401

#Corresponding Author: Tushar Kanti Maiti

402

E-mail: [email protected]

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

403

* Both authors contributed equally to this work

404

Author Contribution S.K., D.K.J., R.K., and T.K.M. designed the research; S.K., D.K.J.,

405

R.K. and M.K. performed the research; S.K., D.K.J., R.K., M.K., N.S.B and T.K.M. analyzed

406

data S.K., D.K.J., and T.K.M. wrote the paper.

407

Funding SK and RK thank Department of Biotechnology (DBT) for senior research

408

fellowship. MK thanks Department of Science and Technology (DST) for Inspire fellowship

409

DKJ thanks Indian Council of Medical Research (ICMR) for research associateship. TKM

410

thanks to Regional Centre for Biotechnology for funding. We thank Department of

411

Biotechnology, Government of India, ICGEB, New Delhi and NII New Delhi for providing

412

financial support for the High Field NMR spectrometers at the ICGEB, New Delhi and NII,

413

New Delhi.

414 415

Conflict of interest: The authors declare no conflict of interest.

416

ACKNOWLEDGMENTS

417

The authors thank Professor Hilal Lashuel for providing us the α-synuclein plasmid (pT7-7

418

asyn WT, Addgene Plasmid # 36046). The Technical assistance from RCB biophysical and

419

microscopy facilities are highly appreciated. We also thank Mrs Saswati Maiti for proof

420

reading.

421

Figure Legends

422

Figure 1. Aggregation kinetics and morphological features of A18T and A29S mutant α-

423

synuclein. Normalized ThT binding assay for wild type α-synuclein, A53T, A18T and A29S

424

mutants as a function of time is presented (a). ANS binding also shows similar results

425

observed for ThT assay (b). AFM imaging for wild type and mutant α-synuclein proteins (c)

426

along with CD spectra are shown (d).

18


Page 18 of 41

Page 19 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


427

Figure 2. Atomic force microscopic evaluations show similar morphological features of

428

different mutants of α-synuclein. High magnification images of fibril and protofibrils along

429

with their line profile show similarity among their morphology (upper panel). Distribution of

430

height and length of fibril and protofibril has been given as a function of time (24 h and 48 h)

431

in lower panels.

432

Figure 3. A17T and A19T follow similar aggregation kinetics. Normalized ThT (a) and

433

ANS binding assays (b) for A17T and A19T mutants and their comparison wild type α-

434

synuclein and A18T mutant have been shown as a function of time. Representative AFM

435

imaging (c) and CD spectra (d) are shown for these two mutant proteins. Differential effects

436

of mutant position on α-synuclein aggregation are clear.

437

Figure 4. Aggregation of A18P is analogous to A18T mutant α-synuclein. A18P exhibits

438

faster aggregation kinetics as shown by representative ThT (a) and ANS (b) binding assays.

439

AFM image shows presence of dense fibrils at different time points of incubation (c) while

440

conformation changes in the protein can be traced by CD spectroscopy (d.). Overall, rapid

441

aggregation of A18P is evident from these results.

442

Figure 5. Helical wheel representation showing locations of A18T and A29S on the helix.

443

Left panel is alpha-helical wheel representation (a) while model of alpha helix is shown in

444

right (b) indicating that both mutants fall on the same helical position on α-synuclein protein.

445

Figure 6. Overlaid 1H-15N heteronuclear single quantum coherence (HSQC) spectra of

446

wild type α-synuclein and mutant proteins show local perturbation of NMR signals.

447

HSQC Spectra of A18T (a), A29S (b), A18P (c) and A53T (d) are presented. Chemical shift

448

perturbation between wild type and A18T (e), A29S (f), A18P (g) and A53T (h) are also

449

presented for quantitative comparision.

450 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

451

452

Figure 7. Internalization of FITC labelled α-synuclein fibrils. Confocal microscopy

453

images show fibril internalization by SH-SY5Y neuroblastoma cells. Images represent

454

control, wild type, A18T, A29S (a); A53T, A18P, A17T and A19T (b) respectively. Scale

455

bar: 20 µm.

456

Figure 8. MTT assay for fibrils of wild type and mutant α-synuclein proteins. Bar graph

457

represents percent survival of SH-SY5Y cells incubated with fibrils (a) and oligomers (b) of

458

wild type α-synuclein and different mutants as shown in the image. Values are mean ± s.e.m.,

459

n ≥ 3; *P

Role of Sporadic Parkinson Disease Associated Mutations A18T and

Recommend Documents