A small molecule autophagy inducer exerts cytoprotection against α-synuclein toxicity

Journal Pre-proof A small molecule autophagy inducer exerts cytoprotection against α-synuclein toxicity S.N. Suresh, Ravi Manjithaya PII:

S0014-2999(19)30587-4

DOI:

https://doi.org/10.1016/j.ejphar.2019.172635

Reference:

EJP 172635

To appear in:

European Journal of Pharmacology

Received Date: 19 May 2019 Revised Date:

25 August 2019

Accepted Date: 2 September 2019

Please cite this article as: Suresh, S.N., Manjithaya, R., A small molecule autophagy inducer exerts cytoprotection against α-synuclein toxicity, European Journal of Pharmacology (2019), doi: https:// doi.org/10.1016/j.ejphar.2019.172635. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

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A small molecule autophagy inducer exerts cytoprotection against α-

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synuclein toxicity

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S. N. Suresh1,3, Ravi Manjithaya1,2*

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Affiliations

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1

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Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Jakkur,

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Bangalore, India.

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3

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Bangalore, India.

Molecular Biology and Genetics Unit (MBGU), and 2Neuroscience Unit (NSU),

Present address: Centre for Brain Research (CBR), Indian Institute of Science (IISc),

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*

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Correspondence to: Ravi Manjithaya ([email protected])

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Corresponding author

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Abstract

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α-synucleopathies are protein-misfolding disorders occur primarily due to aggregation

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and toxicity of α-synuclein. This study characterized the small molecule AGK2 as a

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modifier of α-synuclein mediated toxicity in an autophagy dependent manner in both

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yeast and mammalian cell line models. In yeast system, AGK2 enhances autophagy to

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clear toxic α-synuclein aggregates in an autophagy dependent manner. Autophagy

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flux analyses revealed that AGK2 induces autophagy especially autolysosomes.

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Importantly, AGK2 induces autophagy in an mTOR independent manner. These

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features enable AGK2 to exert cytoprotective potential against α-synuclein mediated

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toxicity in different model systems.

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Keywords

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Protein misfolding diseases; AGK2; autophagy; small molecule screen; yeast;

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neuronal models; toxic protein aggregates; neurodegeneration

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1. Introduction

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Protein misfolding diseases such as α-synucleopathies are lethal and drastically affect

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the quality of life with no disease modifying medications. The cause of

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neurodegeneration can be of genetic or of sporadic in nature. At intracellular level,

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resulting protein aggregates cause toxicity by perturbing essential cell survival

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pathways resulting in a cell death (Suresh et al., 2018b). These protein aggregates are

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toxic as they sequester the key cellular proteins including transcription factors that are

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necessary to maintain the upkeep of essential cellular pathways (Stefani and Dobson,

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2003). Thus, in this protein aggregation driven neurodegeneration, most of such

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cellular pathways become awry thereby perturbing cellular homeostasis (Stefani and

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Dobson, 2003). Plethora’s of studies have highlighted the beneficial effects of

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clearing these disease associated protein aggregates in terms of curbing proteotoxicity

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and enhancing cell viability (Rajasekhar et al., 2015; Suresh et al., 2017; Suresh et al.,

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2018a).

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Proteostatic machineries such as chaperones, ubiquitin-proteasome system (UPS) and

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autophagy related pathways helps in maintaining cellular as well as organismal

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homeostasis (Labbadia and Morimoto, 2015). Under steady state conditions of a cell,

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the proteins tend to misfold and these are mainly recognized by chaperones that refold

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them ensuring functionality of such proteins is not lost due to misfolding (Klaips et

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al., 2018). If chaperones fail to repair the misfolded protein or get or are overwhelmed

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due to increased misfolded proteins numbers in certain conditions, pathways such as

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Endoplasmic Reticulam Associated protein Degradation (ERAD) and UPS degrade

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them to prevent intracellular aggregation (Klaips et al., 2018). In spite of these

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available measures, due to various reasons, the toxic protein oligomers or aggregates

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are seen to accumulate inside cells. Such protein oligomers and aggregates are cleared

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by autophagy related pathways (Nixon, 2013).

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Autophagy is an evolutionarily conserved intracellular pathway that clears

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superfluous organelles, long-lived proteins, protein aggregates (Suresh et al., 2018b).

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The specific form of autophagy that clears protein aggregates is called aggrephagy

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(Suresh et al., 2018b). Aggrephagy pathway is essentially helps the cells to cope up

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with aggregate burden by clearing them and thus ameliorate the cytotoxicity. As

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neurons seldom divide, they depend heavily on autophagy to maintain proteostasis

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(Nixon, 2013). Neuron specific genetic ablation of autophagy as done by knocking

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out the key autophagy genes such as Atg5 and Atg7, lead to the manifestation of

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neurodegenerative symptoms such as accumulation of ubiquitin positive aggregates,

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neuronal loss, behavioral deficits and reduced survivability (Hara et al., 2006;

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Komatsu et al., 2006). Such studies highlight the importance of basal autophagy in

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attaining cellular proteostasis and eventually organismal homeostasis (Hara et al.,

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2006). On the other hand, upregulation of autophagy by genetic intervention by

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overexpressing core autophagy proteins such as Atg 5 or beclin 1 protein enhanced

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lifespan of transgenic mice (Fernandez et al., 2018; Pyo et al., 2013). We and others

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show that genetic and pharmacological upregulation of autophagy is beneficial in

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curbing the pathogenesis of neurodegeneration (Sarkar et al., 2007; Suresh et al.,

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2018a).

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Previous work from our laboratory identified novel small molecule regulators of

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autophagy through screening performed in a yeast model of proteotoxicity (Suresh et

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al., 2017). In this screen, we identified AGK2 (2-Cyano-3 -[5-(2,5-dichlorophenyl)-2-

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furanyl]-N-5-quinolinyl-2-propenamide) as one of the hits. α-synuclein

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overexpression in the yeast, Saccharomyces cerevisiae, perturbs its growth and such

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cells eventually die, a phenomenon similar to that seen in case of aggregate mediated

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cytotoxicity in neurons (Khurana and Lindquist, 2010). Using this α-synuclein

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mediated proteotoxicity model, we previously identified the novel small molecule

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aggrephagy modulators such as 6-Bio and XCT790 (Suresh et al., 2017; Suresh et al.,

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2018a). In this study, we characterized AGK2, one of the hits identified from our

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previous screen as a potential aggrephagy modulator in a two model systems such as

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yeast and mammalian cells.

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2. Materials and methods

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2.1. Plasmids, antibodies and chemical reagents

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Plasmids used in this study are as follows; GFP-Atg8 [pRS316, a kind gift from Prof.

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Yoshinori Oshumi (Tokyo Institute of Technology)], SNCA-GFP (pRS 306, a gift

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from Prof. Paulo Ludovico) GFP-SNCA(Furlong et al., 2000) (a kind gift from Prof.

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David Rubinzstein; Addgene, 40822).

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From HiMedia we purchased dextrose (GRM077), galactose (RM101) peptone

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(RM667), yeast extract (RM027), and amino acids [uracil (RM264), histidine

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(RM051), leucine (GRM054), lysine (L5501) and methionine (GRM200)]. From

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Sigma-Aldrich we purchased 3-MA (M9281), AGK2 (A8231), DMEM F-12

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(D8900), DMEM (D5648), penicillin and streptomycin (P4333), DAB (3, 3′-

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Diaminobenzidine, D3939), trypsin EDTA (59418C) and Atto 663 (41176).

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From Cell Signaling Technology purchased, total P70S6K antibody (9202), Anti-p-

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P70S6K T389 antibody (9862), anti-rabbit IgG horseradish peroxidase (HRP; 7074)

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and total 4EBP1 antibody (9452) were purchased. From Sigma-Aldrich, anti-LC3B

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antibody (L7543) was purchased. From Thermo Scientific, anti β-Tubulin (MA5-

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16308) antibody was purchased. From Bio-Rad, anti-mouse IgG, HRP (172-1011)

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antibody was purchased. CMAC-Blue (C2110) was purchased from Life

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Technologies. Bafilomycin A1 (11038) was purchased from Cayman chemical.

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2.2. Yeast culture

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To culture WT GFP and autophagy mutant strains, YPD (yeast extract, peptone,

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dextrose) media was used. To culture SNCA-EGFP and EGFP-Atg8 strains,

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SD-Ura [synthetic dextrose without uracil] medium was used. To induce SNCA-

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EGFP protein aggregates, cells were grown in SG-Ura (synthetic galactose without

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uracil) medium.

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All the above-mentioned strains were incubated at 250 g and 30°C.

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2.3. Mammalian cell culture

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HeLa cells were grown in DMEM medium containing 10% FBS (fetal bovine serum,

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Pan-Biotech, P03-6510). We cultured and maintained undifferentiated neuroblastoma

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SH-SY5Y cells in DMEM-F12 containing 10% FBS (fetal bovine serum, Life

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Technologies, 12500062). All the cell lines were maintained at CO2 (5%) and 37°C.

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2.4. Growth assays

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Growth assays were performed using a multiplate reader in a high-throughput format.

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Yeast strains (A600~0.06) were seeded in a 384-well plate and incubated (30°C, 420 g

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and 80 µl) in a multiplate reader (Varioskan Flash, Thermo Scientific, USA) that

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recorded absorbance (A600) automatically every 30 min for 72 h. Using GraphPad

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Prism, the data were analysed and plotted the appropriate growth curves.

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2.5. α-synuclein-EGFP protein induction

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SNCA-EGFP strains were inoculated in a SD-Ura medium. From this, secondary

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culture was inoculated (30°C, 250 g) till the absorbance (A600) reaches 0.6 - 0.8/ml.

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Then, the cells were washed with autoclaved water and then incubated them (30°C,

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250 g) with SG-Ura medium for 16 h for the induction of SNCA-EGFP protein

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aggregates.

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2.6. Yeast lysate preparation

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The yeast strains (A600=3) were resuspended in a trichloroacetic acid (12.5%) and then

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stored at -80°C for minimum of 30 min. Samples were thawed on ice and then

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centrifuged (16,000 x g, 15 min). The pellets were washed twice using ice-cold

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acetone (80%). Pellets were air dried and then resuspended in lysis (0.1 N NaOH and

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1% SDS) solution, followed by the addition of Laemmli buffer and boiled it for 15

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min.

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2.7. Mammalian lysate preparation

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The mammalian cells were collected in a Laemmli buffer (1X) to perform the LC3B

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processing assay and other autophagy related signaling immunoblotting. For EGFP-

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SNCA clearance assay, we scraped the cells in a growth medium. Cells were washed

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twice with PBS (1X, phosphate buffer saline, 4°C, 2000 g) and lysed them using

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Laemmli buffer and boiled for 15 min.

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2.8. Immunoblotting

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The appropriated yeast lysates were electrophoresed on SDS-PAGE (8-15%) and

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transfered the proteins to PVDF membrane (Bio-Rad, 162-0177) using Transblot

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turbo (Bio-Rad). Membranes were probed with appropriate primary antibodies

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overnight at 4°C and then incubated with HRP-conjugated secondary antibody at

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room temperature for 2 h. The signals from blots were developed using enhanced

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chemiluminescence substrate (Clarity, Bio-Rad) and image were captured using a gel

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documentation system (G-Box, Syngene, UK). The bands were quantified using

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ImageJ software (NIH).

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2.9. Microscopy To image the yeast cultures, after appropriate treatments, the cells were washed with autoclaved water and mounted on an agarose pad (2%).

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To image the mammalian cells, after appropriate treatments, the cells in

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coverslips were fixed using paraformaldehyde (4%, PFA; Sigma, 158127),

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permeabilized using triton X-100 (0.2%; HiMedia, MB031) and mounted using

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Vectashield (Vector laboratories, H-1000). For immunofluorescence, coverslips were

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incubated with appropriate primary antibody [in 5% BSA (Sigma, RM105)] at 4°C

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for overnight. Then, the coverslips were incubated with fluorescent-conjugated

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antibody room temperature for 2 h. Then, the coverslips were mounted using

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Vectashield (Vector laboratories, H-1000).

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The images were acquired using DeltaVision Elite widefield microscope (API,

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GE, USA) with following filters: Cy5 (632/22 and 676/34), TRITC (542/27 and

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594/45), FITC (490/20 and 529/38) and DAPI (390/18 and 435/48). The images were

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processed using DV SoftWoRX or ImageJ software for further analysis.

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2.10. EGFP-Atg8 processing assay

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Yeast strain expressing EGFP-Atg8 plasmid was grown in SD-U medium at 30°C

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and 250 g. From this, the secondary culture inoculated and grown under the above-

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mentioned conditions till their absorbance (A600) reaches 0.6-0.8. Cells were washed

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twice and incubated in starvation medium with or without AGK2. Samples were

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collected for 0, 2, 4 and 6 h time points. The yeast lysates were prepared as mentioned

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above and electrophoresed with SDS-PAGE (10%) and immunoblotting was

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performed.

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2.11. α-synuclein-EGFP degradation assay

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SNCA-EGFP aggregates were induced by incubating the cells with galactose for 16 h.

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The aggregate induction was turned-off by addition of dextrose medium and then

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treated with AGK2 for 24 h. We analyze the SNCA-EGFP levels using

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immunoblotting.

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2.12. Tandem RFP-EGFP-LC3 processing assay

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HeLa cells were seeded in 60-mm dishes, transfected them with tandem RFP-GFP-

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LC3B plasmid construct and allowed the protein to express for 24 h. Then, the cells

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were trypsinized and seeded them on cover slips placed either in 12-well or 24 well

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plates. Cells were treated with AGK2 for 2 h and processed the cover slips for

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imaging.

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2.13. LC3 processing and autophagy related signaling assays

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To perform these assays, equal numbers of HeLa cells were seeded in 6-well plates

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and allowed to attach overnight. Cells were treated with AGK2 and/or bafA1 (100

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nM), EBSS and LiCl (25 mM) in growth medium for 2 h. EBSS induced mTOR

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dependent autophagy and LiCl induced mTOR independent autophagy were used as

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controls for the same. Mammalian lysates were prepared as mentioned above and

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analysed the LC3 processing, levels of mTOR substrates using SDS-PAGE (8-15%)

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electrophoresis and immunoblotting.

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2.14. Cell viability assay

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SH-SY5Y cells were seeded on a 96-well plate and transfected them with a EGFP-

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SNCA plasmid. Cells were treated with AGK2 and/or 3-MA (5 mM) for 24 h after 48

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h of transfection. Then, the cell viability was measured using the CellTitre-Glo®

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(Promega, G7570) by measuring luminescence using a multiplate reader (Varioskan

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Flash, Thermo Scientific).

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2.15. Statistics

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The unpaired Student t test and ANOVA (one-way or two-way) followed by the post-

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hoc Bonferroni test in GraphPad Prism were used for statistical analyses. Error bars

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represent mean ± S.E.M.

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3. Results

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3.1. Cytoprotection potential of AGK2 is dependent on autophagy in a yeast

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model of α-synuclein proteotoxicity

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In an α-synuclein overexpressing wild-type yeast strain, we observed a growth lag

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(WT-EGFP versus WT α-syn-EGFP, P < 0.001, Fig. 2B). Upon AGK2 treatment to

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wild-type α-synuclein overexpressing strain, growth was significantly enhanced than

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untreated (P < 0.01, versus untreated, Fig. 2A). In addition, AGK2 (50 μM) did not

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perturb the growth of wild-type yeast cells and was not cytotoxic (Fig. S1).

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Autophagy is one of the cellular defense mechanism to combat the aggregate

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mediated toxicity (Nixon, 2013). To examine the dependency of AGK2 on autophagy,

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we performed the growth assay in an α-synuclein overexpressing, core autophagy

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mutant (atg1∆) strain.

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When α-synuclein was overexpressed in atg1∆ strain, its growth was significantly

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affected as observed in WT cells. Upon treatment of AGK2 to atg1∆ cells

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overexpressing α-synuclein, its growth was not significantly changed than untreated

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(untreated versus AGK2 treated, P > 0.05, Fig. 3A). The ability of AGK2 to

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ameliorate α-synuclein toxicity is lost in an autophagy mutant strain indicating the

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essentiality of autophagy for its mechanism of action.

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These results show that AGK2 cytoprotects yeast cells from the α-synuclein mediated

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toxicity in an autophagy dependent manner.

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3.2. AGK2 is an inducer of autophagy in yeast

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In the yeast system, the EGFP-Atg8 processing assay is employed to assess the

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autophagy status of cells (Klionsky et al., 2016). In starvation conditions, we noted

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significant increase in free EGFP accumulation in untreated cells over time,

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confirming that the nitrogen starvation induced autophagy. AGK2 treatment

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significantly increased release of free EGFP temporally than that of untreated (2, 4

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and 6 h time points, P < 0.001 versus untreated; Fig. 1). This result demonstrates that

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AGK2 augments starvation-induced autophagy.

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3.3. AGK2 induces autophagy in mammalian cells

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To understand if AGK2 regulates autophagy in mammalian cells, we undertook two

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assays: 1) tandem RFP-EGFP-LC3 assay (fluorescence based) and 2) LC3 processing

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immunoblotting experiment (Kimura et al., 2007).

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RFP-EGFP tandemly tagged LC3 allowed autophagosomes to visualize as yellow and

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autolysosomes as red, as lysosomal acidic pH quenches EGFP fluorescence (Klionsky

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et al., 2016). AGK2 (5 µM) treated HeLa cells showed increased number of

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autophagosomes (~2 fold, P < 0.001, compared to control; Fig. 4A) and

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autolysosomes (~4 fold, P < 0.001, compared to control; Fig. 4A) than that of

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untreated cells. The high numbers of autolysosomes suggested that AGK2 promoted

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autophagy flux.

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AGK2 treatment to HeLa cells enhanced the processing of LC3-I to LC3-II

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significantly than untreated (P < 0.001, versus untreated, Fig. 4B). Upon co-addition

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of bafilomycin A1 (flux inhibitor) and AGK2, LC3-II levels were significantly more

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than either bafilomycin A1 or AGK2 only treatments (P < 0.001, versus untreated,

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Fig. 4B). Notably, treatment of AGK2 to HeLa cells was not toxic (Fig. S3). These

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results confirm that AGK2 is indeed an autophagy enhancer.

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3.4. AGK2 induces autophagy in an mTOR independent manner

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Autophagy modulators can induce autophagy by either mTOR dependent or

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independent signaling pathways (Suresh et al., 2018a). We tested if AGK2 regulates

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autophagy by modulating mTOR by analyzing its substrate levels such as P70S6K

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and 4EBP1. When mTOR is active, its phosphorylated substrates levels will be

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enhanced (Suresh et al., 2018a). Upon AGK2 treatment to HeLa cells, we observed

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the presence of phosphorylated substrates with concomitant increase in LC3-II

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accumulation (Fig. 5A). Also, it was noted that AGK2 was not toxic to cells at 5 μM

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(Fig. S1). This result indicates that AGK2 is an mTOR independent autophagy

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enhancer.

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3.5. AGK2 degrades alpha-synuclein in an autophagy dependent manner

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One of the cellular defense mechanisms to ameliorate the aggregate mediated toxicity

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is by degrading the toxic α-synuclein protein aggregates through autophagy to elicit

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cyto/neuroprotection (Sarkar et al., 2007). We tested the efficacy of AGK2 in clearing

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α-synuclein protein aggregates by performing immunoblotting based α-synuclein-

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EGFP degradation assays.

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Upon AGK2 treatment to wild-type α-synuclein-EGFP overexpressing yeast strain,

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the level of α-synuclein-EGFP protein (P < 0.001, versus untreated, Fig. 3A) was

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significantly lesser than untreated. Also, the fluorescence microscopy showed that

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AGK2 significantly cleared α-synuclein-EGFP protein that resulted in the increased

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presence of free EGFP in vacuole (P < 0.001, versus untreated, Fig. S2).

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Does α-synuclein clearance potential of AGK2 is autophagy dependent? To answer

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this question, we performed the immunoblotting based α-synuclein-EGFP degradation

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assays in an α-synuclein-GFP overexpressing atg1∆ cells.

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Upon AGK2 treatment to α-synuclein-EGFP overexpressing atg1∆ cells, the level of

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α-synuclein-EGFP protein level was not significantly different from that of untreated

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(P > 0.05, versus untreated, Fig. 3B).

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These results confirm that AGK2 clears toxic α-synuclein-EGFP protein levels in an

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autophagy dependent manner.

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3.6. In mammalian cells, cytoprotection against α-synuclein toxicity by AGK2 is

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autophagy-dependent

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We analysed the ability of AGK2 in eliciting cytoprotection against α-synuclein

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toxicity model of SH-SY5Y (the human neuroblastoma) and HeLa cells.

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Upon EGFP-α-synuclein overexpression, the cellular viability was affected

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significantly than either untransfected or vector control cells. AGK2 treatment to

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EGFP-α-synuclein overexpressing cells significantly increased the viability of cells

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than untreated (P < 0.001, versus untreated, Fig. 5B and Fig. S4). The cell viability

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in AGK2 and 3-methyladenine (a pharmacological autophagy inhibitor) co-treatment

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in EGFP-α-synuclein overexpressing cells was significantly lesser compared to

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AGK2 treatment (P > 0.5, versus 3-MA, Fig. 5B and Fig. S4). Thus, we demonstrate

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that AGK2 cytoprotects neuronal cells from EGFP-α-synuclein toxicity in an

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autophagy dependent mechanism.

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4. Discussion

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We identified and characterized the AGK2 as an aggrephagy modulator in diverse

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models such as yeast and mammalian cells. In yeast, small molecules enhanced the

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starvation-induced autophagy and also cleared α-synuclein aggregates in an

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autophagy dependent manner. In mammalian cells, AGK2 induced autophagy

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precisely by enhancing the autolysosome formation. AGK2 cytoprotects neuronal

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cells from α-synuclein mediated toxicity that was autophagy-dependent.

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Under cellular steady state conditions, the α-synuclein is acetylated to prevent it from

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attaining the toxic oligomer conformation (de Oliveira et al., 2017). Upon various

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factors such as ageing and pathological conditions, sirtuin2 activity is upregulated to

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generate the deacetylated forms of α-synuclein (de Oliveira et al., 2017). AGK2, the

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sirtuin2 inhibitor has been shown to be neuroprotective in a Drosophila model of

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Parkinson’s disease (PD) (Outeiro et al., 2007). Sirtuin2 is a NAD+ dependent

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deacetylase that is predominantly expressed in brain and its levels are known increase

322

with ageing (de Oliveira et al., 2012). In PD, increased sirtuin2 activity aggravates

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proteotoxicity by deacetylating α-synuclein at various lysine residues (Singh et al.,

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2017). Acetylation of α-synuclein prevents its aggregation and thus is shown to be

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neuroprotective in primary cortical neurons (de Oliveira et al., 2017). On the other

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hand, dopaminergic neuronal loss is observed when acetylation of α-synuclein is

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blocked. Genetic down regulation of sirtuin2 suppresses the α-synuclein aggregation

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and its toxicity. Transgenic sirtuin2 knock out mice are protected from either α-

329

synuclein or MPTP mediated toxicities. In addition, sirtuin2 knock down cells show

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increased accumulation of LC3-II with concomitant degradation of p62 suggesting

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enhanced autophagic activity (de Oliveira et al., 2017). In addition to the post-

332

translational aspects of this protein, we investigated how sirtuin2 inhibition using a

333

small molecule (AGK2) regulates autophagy and we also tested its cytoprotection

334

potential. Our results, in particular show that in mammalian cells, the AGK2

335

enhanced autophagosome lysosome fusion.

336

α-synuclein is an intrinsically disordered protein (IDP) and upon overexpression

337

forms aggregates (Ghosh et al., 2017). These aggregates a) sequester essential cellular

338

proteins, b) get glued on to the endocytic vesicles perturbing their function, when

339

misfolded to impart cytotoxicity (Ghosh et al., 2017; Lautenschlager et al., 2017).

340

Thus, we and others demonstrated that degrading the toxic α-synuclein protein

341

aggregates exerts neuroprotection in various models such as yeast, mammalian cells

342

and mice. This clearance potential is dependent on autophagy as we demonstrated by

343

both pharmacological and genetic means.

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AGK2 induces autophagy in an mTOR independent manner. mTOR is an essential

345

player that regulates vital cellular survival pathways and inhibiting it would result in

346

evident side effects such as immune suppression and so on (Ruiz-Torres et al., 2018).

347

So, pharmacologically mTOR independent autophagy enhancers are preferred to

348

conduct the clinical trials.

349

In our study, we showed that small molecule AGK2 cleared toxic α-synuclein protein

350

aggregates in an autophagy dependent manner to exert cytoprotection potential in two

351

model systems such as yeast and mammalian cell lines such as HeLa and SH-SY5Y.

352 353

Acknowledgements

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We thank David Rubinzstein, Tamotsu Yoshimori and Y. Ohsumi for the kind gift of

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plasmids. This work was supported by the Wellcome Trust/DBT India Alliance

356

Intermediate Fellowship (500159-Z-09-Z), DBT (BT/INF/22/SP27679/2018), DST-

357

SERB Grant (EMR/2015/001946), JNCASR intramural funds to RM.

358 359

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749

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841

Figure legends

842

Figure 1: AGK2 enhances starvation induced autophagy. A) Representative

843

immunoblots (n=3) of EGFP-Atg8 processing assay. Wild type cells expressing

844

EGFP-Atg8 were treated with AGK2 and samples were collected at various time

845

points (0,2,4 and 6 h) to analyse the autophagy flux. B) Quantitation indicating the

846

fold change of autophagy flux measured by the release of free EGFP from EGFP-

847

Atg8 protein. Statistical analysis was performed using one-way ANOVA and the

848

post-hoc Bonferroni test. Error bars, mean ± S.E.M. ***-P < 0.001.

849

Figure 2: AGK2 cytoprotects yeast cells against α-synuclein mediated toxicity.

850

Growth curves of α-synuclein overexpressing wild-type (A) and autophagy mutant

851

(B) yeast strains treated with or without AGK2. Statistical analysis was performed

852

using one-way ANOVA and the post-hoc Bonferroni test. n=6, Error bars, mean ±

853

S.E.M. ns-non significant, **-P < 0.01, ***-P < 0.001.

854

Figure 3: AGK2 clears α-synuclein-EGFP in an autophagy dependent manner.

855

Both wild type (A) and autophagy mutant (B) strains overexpressing α-synuclein-

856

EGFP treated with AGK2 and analysed for total α-synuclein-EGFP protein levels.

857

Statistical analysis was performed using one-way ANOVA and the post-hoc

858

Bonferroni test. n=6, Error bars, mean ± S.E.M. ns-non significant, ***-P < 0.001.

859

Figure 4: AGK2 induces autophagy in mammalian cells. (A) Microscopy images

860

of HeLa cells transiently expressing RFP-EGFP-LC3 were treated with AGK2 for 2 h.

861

Graphs indicating the fold change or number of puncta per cell of autophagosomes

862

and autolysosomes upon appropriate small molecule treatments. n=75 cells, three

863

independent experiments. Statistical analysis was performed using one-way ANOVA

864

and the post-hoc Bonferroni test. (B) Blots indicating LC3 processing upon AGK2

865

treatment with or without bafilomycin A1. LC3-II protein levels were quantified

866

(n=3). Statistical analysis was performed using one-way ANOVA and the post-hoc

867

Bonferroni test. Error bars, mean ± S.E.M. *-P < 0.05, ***-P < 0.001.

868

Figure 5: AGK2 is an mTOR independent autophagy inducer and cytoprotects

869

neuronal cells from α-synuclein mediated toxicity. (A) Western blots of autophagy

870

related protein levels such as LC3, phospho and total P70S6K and 4EBP1. (B) SH-

871

SY5Y cells were transiently overexpressed with α-synuclein for 48 h. We then treated

872

them with autophagy enhancer such as AGK2 with or without autophagy inhibitor (3-

873

MA) for 24 h. After 72 h, the cell viability was measured of all treatments and plotted

874

the same. Three independent experiments were carried out. Statistical analysis was

875

performed using one-way ANOVA and the post-hoc Bonferroni test. Error bars, mean

876

± S.E.M. ns-non significant, ***-P < 0.001.

877 878

Supplementary figure legends

879

Figure S1: AGK2 is not toxic to yeast cells. WT cells were treated with and without

880

AGK2 (50 µM) and absorbance (A600) was recorded at 72 h. Statistical analysis was

881

performed unpaired Student t-test. Error bars, mean ± S.E.M. ns-non significant.

882 883

Figure S2: α-synuclein clearance assay. Microscopic images of yeast expressing α-

884

synuclein-EGFP treated with and without AGK2 (50 µM). The cells with free EGFP

885

were quantitated (the measure of free EGFP in vacuole indicate the vacuole mediated

886

clearance of α-synuclein-EGFP). Statistical analysis was performed unpaired Student

887

t-test (n=75 cells). Error bars, mean ± S.E.M. ***-P < 0.001.

888

Figure S3: AGK2 is not toxic to mammalian cells. HeLa cells were treated with

889

AGK2 (5 µM) for 72 h and cell viability was measured (n=3). Statistical analysis was

890

performed unpaired Student t-test. Error bars, mean ± S.E.M. ns-non significant.

891

Figure S4: α-synuclein toxicity assay. HeLa cells were overexpressed with α-

892

synuclein for 48 h transiently. Cells were treated with AGK2 with or without 3-MA

893

for 24 h. After 72 h, the cell viability was assayed and plotted the same. Statistical

894

analysis was performed using one-way ANOVA and the post-hoc Bonferroni test

895

(Three independent experiments). Error bars, mean ± S.E.M. ns-non significant, ***-

896

P < 0.001.

897 898 899 900 901 902 903 904 905 906 907

Figure 1 A Starvation (h))

0

Untreated 2 4

6

AGK2 2 4

0

EGFP-Atg8

Free EGFP

Gapdh

Fold change

B

60

909 910 911 912 913 914 915 916

*** ***

40 ***

20 0

908

Control AGK2

0

2

4

Time (h)

6

6

Figure 2 A 1.0

***

0.8

A

A600

** 0.6 0.4 0.2 0.0

WT EGFP

WT α -syn-EGFP WT α -syn-EGFP + AGK2

B *** 1.0 0.8

A600

ns 0.6 0.4 0.2 0.0

917 918 919 920 921

atg1∆ ∆ EGFP

∆ α -syn-EGFP atg1∆ ∆ α -syn-EGFP atg1∆ + AGK2

A

Fold (α -syn-EGFP/Gapdh)

Figure 3 ***

1.5

*** 1.0

0.5

0.0

AGK2

0

24

24

-

-

+

α-syn EGFP Gapdh

Fold (α -syn-EGFP/Gapdh)

B ns 1.0

0.5

0.0

AGK2

α-syn EGFP Gapdh 922 923 924 925 926

ns

1.5

0

24

24

-

-

+

Figure 4 A MERGE

No. of puncta/cell

AGK2

Control

RFP LC3 EGFP LC3

AGK2

B

927 928 929 930 931 932 933 934

Autophagosomes Autolysosomes

120 100

***

80

***

60 40 20 0

-

+

-

+

Figure 5 A GM

EBSS AGK2

LC3-I LC3-II p-P70S6K t-P70S6K t- 4EBP1 β-tubulin

B ***

935

***

***

936 937

Figure S1 ns

1.0

A600

0.8 0.6 0.4 0.2 0.0

Control

938 939 940 941 942 943 944 945 946 947 948 949 950

AGK2

951 952

Figure S2 Control

AGK2

Fold change (cells with free EGFP)

8

*** 6 4 2 0

Control

953 954 955 956 957 958 959 960 961 962

AGK2

963 964

Figure S3

800000

ns

RLU

600000

400000

200000

0 Control

965 966 967 968 969 970 971 972 973

AGK2

974 975

Figure S4 ***

800000.0

***

***

RLU

600000.0

400000.0

200000.0

0.0

Vector control α -synuclein AGK2 3-MA

976 977 978 979 980 981 982 983 984

-

+ -

+ + -

+ +

+ -

+ +

+ + -

+ + +

Jawaharlal Nehru Center for Advanced Scientific Research (Autonomous Body under the Department of Science & Technology, Government of India)

Jakkur Campus, Jakkur Post Bengaluru 560 064, INDIA Email: [email protected]

Office Tel: +91 (80) 2208 2924 Office Fax: +91 (80) 2208 2766 +91 (80) 2208 2767

19th Aug 2019 The author contributions for this manuscript: SNS - Conceptualization; Data curation; Formal analysis; Investigation; Methodology; Validation; Visualization; Roles/Writing - original draft. RM - Funding acquisition; Investigation; Methodology; Project administration; Resources; Software; Supervision; Validation; Visualization; Roles/Writing - original draft; Writing - review & editing.

Best regards, Dr. Ravi Manjithaya Associate Professor Molecular Biology and Genetics Unit NeuroScience Unit Jawaharlal Nehru Centre for Advanced Scientific Research Jakkur, Bangalore 560 064 INDIA Office 91 80 2208 2924