Extracellular Î±-Synuclein Disrupts Membrane Nanostructure and

Subscriber access provided by - Access paid by the | UCSB Libraries

Extracellular #-synuclein disrupts membrane nanostructure and promotes S-nitrosylation induced neuronal cell death Roshan Kumar, Raniki Kumari, Sanjay Kumar, Deepak Kumar Jangir, and Tushar Kanti Maiti Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01727 • Publication Date (Web): 14 Mar 2018 Downloaded from http://pubs.acs.org on March 15, 2018

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 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 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.

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 39 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

Biomacromolecules

89x36mm (300 x 300 DPI)

ACS Paragon Plus Environment

Biomacromolecules 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 2 of 39

Extracellular α-synuclein disrupts membrane nanostructure and promotes S-nitrosylation induced neuronal cell death Roshan Kumar1, 2 Raniki Kumari1, 3 Sanjay Kumar1, 2 Deepak Kumar Jangir1 Tushar Kanti Maiti1* 1

Functional Proteomics Laboratory, Regional Centre for Biotechnology (RCB), NCR Biotech

Science Cluster, 3rd Milestone Gurgaon-Faridabad Expressway, Faridabad, 121001, India. 2

3

Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India. KIIT University, Bhubaneswar, Odisha, India

*

To whom correspondence should be addressed. E-mail: [email protected]

1 ACS Paragon Plus Environment

Page 3 of 39 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

Biomacromolecules

Abstract α-Synuclein, a major constituent of proteinaceous inclusions named Lewy body, has been shown to be released and taken up by cells, which may facilitate its progressive pathological spreading and neuronal cell death in Parkinson’s disease. However, the pathophysiological effect and signalling cascade initiated by extracellular α-synuclein in cellular milieu are not well understood. Herein we have investigated the perturbations induced by low molecular weight α-synuclein and different types of α-synuclein oligomers in the neuroblastoma SH-SY5Y cells. Atomic force microscopy studies have revealed formation of nanopores and enhanced roughness in the cell surface leading to membrane disruption. The damaged membrane allows altered ionic homeostasis leading to activation of nitric oxide synthase (NOS) machinery releasing burst of nitric oxide. The elevated levels of nitric oxide induces S-nitrosylation of key proteins like Actin, DJ-1, HSP70 UCHL1, Parkin, and GAPDH that alter cytoskeletal network, protein folding machinery, ubiquitin proteasome system inducing apoptosis. Keywords: Neurodegeneration, Parkinson’s disease, Extracellular α-synuclein, Atomic force microscopy, Membrane pore, Protein S-nitrosylation



Page 4 of 39

Introduction Amyloid refers to the abnormal fibrous, proteinaceous deposits found in organs and tissues1. Amyloids share common properties such as specific staining pattern, higher resistance to proteolytic degradation and a fibrillary appearance when observed at nanoscopic resolution 2. There is a group of neurodegenerative diseases characterised by misfolding of proteins that turn into β-sheet structure and accumulate as amyloid deposits in affected tissue. Parkinson’s disease (PD) is one such neurodegenerative disorder, characterized by loss of dopaminergic neurons in the substantia nigra mostly due to the presence of Lewy bodies and Lewy neurites 3. Lewy bodies are complex proteinaceous aggregates containing more than 500 different proteins where α-synuclein oligomeric and fibrillar assemblies are predominant 4, 5. The amino acid sequence of α-synuclein, a small protein of 140 amino acids, is subdivided into three regions: N-terminal region (1-60 amino acid), central region (61-95 amino acids) and C-terminal region (96-140 amino acid)6. Three distinct regions in the protein serve different purposes. N-terminus contains KTKEGV repeat that forms amphipathic αhelices to anchor with cell membranes. Central hydrophobic NAC (non-Aβ component) region is responsible for beta sheet formation and C- terminus region is negatively charged region that protects α-synuclein from spontaneous aggregation 7. α-Synuclein is a cytosolic protein and it was assumed earlier that the pathogenic effects are restricted to the cellular environment. However, recent studies suggest that α-synuclein is also present outside the cell and exerts a toxic effect. A portion of αsynuclein has been identified in vesicles which is secreted out of cells via exocytosis 8. In humans, αsynuclein is present in blood plasma and cerebrospinal fluid in both Low molecular weight (LMW) and oligomeric forms 9. Blood level of α-synuclein monomers is doubled in familial PD patients with α-synuclein gene locus triplication

10

. Moreover, monomeric and soluble oligomeric species of α-

synuclein have been detected as early as 2 h followed by transient overexpression of human αsynuclein in differentiated SH-SY5Y cells11. Neuronal cells secrete α-synuclein, which is thought to be related to signal transduction at synapsis, but it substantially increases under various stress condi3 ACS Paragon Plus Environment

Page 5 of 39 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

Biomacromolecules

tions including mitochondrial, proteasomal and lysosomal dysfunction as well as oxidative stress 12. It has been suggested that stress condition not only affects α-synuclein expression but also enhances its translocation into vesicles. Furthermore, vesicular α-synuclein is more prone to aggregation than cytosolic α-synuclein

13

. Recently it has been shown that there is a brain-to-stomach transfer of α-

synuclein via vagal preganglionic projections indicating a new mechanism in transmission of PD pathology 14. In rats brains there is transmission of PD pathology from the gastrointestinal tract to the brain15. Gastric α-synuclein immunoreactive inclusions in Meissner's and Auerbach's plexuses in cases staged for Parkinson's disease-related brain pathology16. Different types of α-synuclein oligomer have been detected in brains with Lewy body pathology compared to brains from nondiseased individuals

17

. High resolution microscopic techniques such as transmission electron

microscopy and atomic force microscopy allow for direct visualization of the oligomers which in size are in range from 4 to 24 nm and have their distinct shapes including spherical, chain-like, annular, and tubular oligomeric structures18. The presence of fatty acids, ferric chloride, 3,4- dihydroxyphenyl acetaldehyde (DOPAL), and many other chemicals were shown to enhance αsynuclein oligomerisation. The formation of highly soluble oligomer of α-synuclein is regulated by cellular fatty acids and it enhances in PD

19

. FeCl3 induced oligomers are SDS-resistant and form

ion-permeable pores in a planar lipid bilayer

20

. The interaction of α-synuclein with DOPAL is

reported to lead to an increase of the levels of potentially toxic α-synuclein aggregates

21

. α-

Synuclein oligomers that are present in the nervous system of A53T α-synuclein transgenic mice show resistance to SDS, heat, and urea 22. These oligomeric intermediates in the aggregation process of α-synuclein have been shown to be the toxic species that cause neurotoxicity due to lipid membrane disruption 23, 24. Protofibrillar α-synuclein is able to induce vesicle permeabilization25. An increased permeability of the cellular membrane leads to neurodegeneration via an altered calcium homeostasis

26

. It has been shown in vitro that α-synuclein spherical oligomer can disrupt

phospholipid vesicles 27 . The proposed disruption mechanism is the formation of pore-like structures



within the SH-SY5Y cells using patch clamp method

Page 6 of 39

28

. This hypothesis has been supported by

electrophysiological single channel recording 29. Direct visualisation of pore on neuronal membrane has not been shown. The pore-like mechanism is still uncertain and other mechanisms such as bilayer thinning have been shown with the aid of techniques like X-ray scattering, fluorescence correlation spectroscopy and coarse-grained molecular dynamics on lipid bilayer composed of mixture of 1palmitoyl-2-oleoyl-sn-phosphatidyl-L-serine phosphocholineand (POPC)

30

(POPS)

and

1-palmitoyl-2-oleoyl-sn-glycero-3-

. The lipid extraction mechanism in the presence of α-synuclein was

also shown in the lipid bilayer made up of 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and 1,2-dioleoyl-sn-glycero-3-phosphocholine

(DOPC)

using

supercritical

angle

fluorescence

microscopy and fluorescence recovery after photo bleaching 24. It is well established that due to α-synuclein induced membrane disruption ionic homeostasis gets disturbed leading to excessive nitric oxide (NO) release 31. But molecular signaling events initiated by this gasotransmitter are still lacking. Excessive nitric oxide has potential to promote protein Snitrosylation. S-nitrosylation is a redox-mediated posttranslational modification that regulates protein function via covalent reaction of nitric oxide (NO) with a cysteine thiol group on the target protein 32. It is well documented that S-nitrosylated G-actin polymerizes less efficiently than native monomers leading to reduced motility and neutrophil β2 integrin function 33. DJ-1 and HSP 70 which regulate protein folding machinery are found to be S-nitrosylated in PD pathology. DJ-1 nitrosylation compromises its antioxidant functions

34

. S-nitrosylation of HSP reduces its ATPase activity in the

endothelial cells and compromises its chaperone function

35

. Recently, we have shown that S-

nitrosylation of UCHL1, a neuron specific deubiquitinating enzyme, induces structural instability and promotes α-synuclein aggregation 36. S-nitrosylation of Parkin, an E3 ubiquitin ligase regulates degradation of neuronal protein, mitochondrial quality control as well as p53 mediated neuronal cell death in PD

37

.

GAPDH which is an important enzyme in the glycolytic pathway has also

nitrosylated and induces an apoptotic cell death cascade 38. 5 ACS Paragon Plus Environment

Page 7 of 39 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

Biomacromolecules

Here, we report that extracellular α-synuclein alters membrane structure and forms a pore like structure in SH-SY5Y cells as indicated in our AFM studies. The membrane structure damage induces the cellular NO which subsequently S-nitrosylates regulatory proteins that are involved in neuronal function. This aberrant S-nitrosylation contributes to neuronal cell death and promotes disease pathology. Materials and Method Expression, mutagenesis and purification of α-Synuclein The A53T mutant of α-synuclein was generated by site directed mutagenesis using pT7-7-α- wild type as a template by quick change mutagenesis protocol (Stratagene). α-Synuclein and A53T mutant were over expressed and purified using established protocol 39. In vitro aggregation of α-Synuclein The low molecular weight species (LMW) of α-synuclein were prepared based on established literature with slight modifications40, 41. In brief, α-synuclein stock solution of 700 µM in 20 mM Tris HCl pH 7.4 was treated with 2 M NaOH for 10 min at 4o C to dissolve any preaggregated proteins and then pH was readjusted to 7.4 by dropwise addition of 6 M HCl. The protein solution was passed through 100 kDa Amicon Ultra 15 to remove aggregated proteins. The protein concentration was determined by spectroscopic method using molar extinction coefficient 5960 M-1cm-1 at 280 nm (Perkin-Elmer). α-Synuclein (550 µM) in Tris-HCl pH7.4 incubated at 37

o

containing 0.1% sodium azide was

C without stirring for homogenous aggregation for 22-24 h. The solution was

centrifuged at high speed to remove fibrillary content. The oligomeric fractions of α-synuclein were separated from monomers by passing through 30 kDa Amicon Ultra-15. The iron induced oligomer was prepared by incubating wild type monomeric α-synuclein with 20 µM ferric chloride and 20 % ethanol. The buffer exchange is performed with 20 mM Tris buffer pH 7.4 using 30 kDa Amicon



Page 8 of 39

Ultra-15. The cold induced oligomer was generated by incubating α-synuclein wild type monomeric α-synuclein at 4

o

C for 6 h at 300 rpm. Morphology of different oligomeric species were

characterized by AFM (Fig 1, Fig. S 2). Thioflavin T assay The monomeric and oligomeric α-synuclein (20 µM) were incubated with 20 µM of ThT for 5 min. Three independent measurements were performed and subsequently averaged for each sample. ThT fluorescence was recorded using Hitachi F-7000, fluorescence spectrophotometer with excitation at 442 nm and emission was recorded at 482 nm42. Circular dichroism spectroscopy Samples were diluted with 50 mM phosphate buffer, pH 7.2 to reach a final concentration of 10 µM. Circular dichroism spectra were recorded from 190 nm to 260 nm wavelength range using JASCO J815 CD spectrophotometer. The spectra were analyzed by DICHROWEB server for secondary structure content 36. Cell culture, treatment and fixation SH-SY5Y cells were grown in DMEM Glutamax containing 10% FBS (GIBCO, Thermo Fisher Scientific USA), antibiotics (pen strep 1%) in a humidified 5% CO2 atmosphere at 37°C and then cells were treated with monomeric and different oligomeric α-synuclein (10µM) for different time points. Sample fixation was done in 4% paraformaldehyde for 15 minutes. The cells were then rinsed three times with PBS and stored at room temperature. Atomic force microscopy of different α-synuclein preparations Samples of different α-synuclein oligomeric and LMW were placed on freshly cleaved mica and then air-dried. Samples were imaged in AC mode by JPK Nano Wizard III atomic force microscope (JPK instrument, Berlin, Germany). The drive frequency of silicon cantilever was between 300–


Page 9 of 39 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

Biomacromolecules

320 kHz and the scan rate was between 0.8–1 Hz with a spring constant of 13–77 N/m. The size of different species were measured from the topographic AFM images with JPK software. Dynamic Light Scattering DLS measurements of α-synuclein LMW and different oligomers were performed directly on a Zetasizer Nano ZS (Malvern Instruments) probing scattered light at 173° using a 630 nm light source. The instrument is equipped with a Peltier temperature controller set at room temperature. Disposable micro-cuvettes were used for size measurements. Every sample was measured ten times and the averaged intensity-size distribution was reported. Low force contact mode atomic force microscopy The imaging of surface property of SH-SY5Y cells were carried out by JPK Nano Wizard III atomic force microscope (JPK instrument, Berlin, Germany), which is equipped with AFM scanner and Zeiss optical microscope. The AFM samples were prepared by fixing the cells with 4 % PFA for 15 min. The fixed cells were washed thrice with water. The fixed SH-SY5Y cell images were measured with gold coated Hydra cantilever in contact mode (APPNANO, USA). The material properties and dimensions of the probe used in contact mode were as follows: resonance frequency of 17 kHz (±4 kHz), force constant of 0.1 N/m, cantilever length of 200 µm ,cantilever width of 40 µm, cantilever thickness of 0.6 µm, tip radius of

Extracellular Î±-Synuclein Disrupts Membrane Nanostructure and

Recommend Documents