Role of Sporadic Parkinson Disease Associated Mutations A18T and

Aug 25, 2017 - Sanjay Kumar†‡, Deepak Kumar Jangir†⊥, Roshan Kumar†‡, Manisha Kumari†, Neel Sarovar Bhavesh§, and Tushar Kanti Maiti†...
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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

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

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alpha synuclein fibrillation and cytotoxicity

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

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Sarovar Bhavesh 3 and Tushar Kanti Maiti1#

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

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Transcription Regulation Group, International Centre for Genetic Engineering and

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

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Manipal University, Manipal, Karnataka-576104, India

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

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ABSTRACT

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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be important in development of PD pathology.

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

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Parkinson’s disease

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INTRODUCTION

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

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

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

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

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multiple

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

system

atrophy

(MSA)

and

different

spinocerebellar

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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therapeutic intervention.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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RESULTS AND DISCUSSION

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A18T and A29S accelerate α-synuclein aggregation

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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from ANS binding that A29S displayed intermediate exposure of hydrophobic surfaces

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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mutant.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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aggregation and detail structural studies are necessary in this direction.

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Secondary structure analysis of A18T and A29S mutants during aggregation

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

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

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

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

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

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

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

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

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

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

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~120 h of incubation.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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synuclein.

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

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A19

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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constant of aggregation (tlag

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

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

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

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

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

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A18P mutation results in rapid aggregation of the protein

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

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

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

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

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

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its aggregation behaviour.

(A17T)

=16.31±0.89 h, tlag

(A19T)

=16.16±1.10 h and Kapp(A17T)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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aggregation.

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

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synuclein

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

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

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

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

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

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

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

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

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

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

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

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

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Cytotoxicity of A18T and A29S fibrils and oligomers

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

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

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

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

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

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

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

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

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

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

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

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

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Conclusions

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

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

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

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

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

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

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

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and A29S mutants.

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METHODS

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Protein expression and mutagenesis

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

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

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

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

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

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

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analytical grade. 13

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In vitro aggregation of α-synuclein

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

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

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

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

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

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

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

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

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

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

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different time intervals for the experiments.

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ThT and ANS fluorescence spectroscopy

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

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

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

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

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

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

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

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

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

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Xmin)/(Xmax - Xmin).

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

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

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

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

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

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

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

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[θ]rmw= (

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Where

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g/ml.

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AFM Imaging

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

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

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

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

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

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

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13-77 N/m.

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Solution-state NMR spectroscopy

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

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

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

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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

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

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

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

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

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

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

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following equation.52

 ∆δ 15 N H =   5

2

  + (∆δ H N )2 

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∆δ 15 N H ,H N

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

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for 1HN and 15N respectively.

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Fibrillar and oligomeric cytotoxicity

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

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

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

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

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

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

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

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

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

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

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

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

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

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Sunnyvale, CA, USA).

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Protein secondary structure analysis

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

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

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analysis.53

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

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

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

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

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

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

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

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

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

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

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

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

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AUTHOR INFORMATION

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#Corresponding Author: Tushar Kanti Maiti

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E-mail: [email protected]

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* Both authors contributed equally to this work

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

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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

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

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

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

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

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

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

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

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New Delhi.

414 415

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

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ACKNOWLEDGMENTS

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

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

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

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reading.

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Figure Legends

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

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

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

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

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along with CD spectra are shown (d).

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

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

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

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

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in lower panels.

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

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

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

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

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of mutant position on α-synuclein aggregation are clear.

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

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

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

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

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aggregation of A18P is evident from these results.

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

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

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

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

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

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

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

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presented for quantitative comparision.

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

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

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

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bar: 20 µm.

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

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

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

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n ≥ 3; *P