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• W2953454103 abstract "•aSyn sequesters HSP10 in the cytosol and prevents it from acting in the mitochondria•Overexpression of HSP10 delays aSyn pathology in vitro and in vivo•HSP10 modulates aSyn pathology in synaptic terminals Alpha-synuclein (aSyn) accumulates in intracellular inclusions in synucleinopathies, but the molecular mechanisms leading to disease are unclear. We identify the 10 kDa heat shock protein (HSP10) as a mediator of aSyn-induced mitochondrial impairments in striatal synaptosomes. We find an age-associated increase in the cytosolic levels of HSP10, and a concomitant decrease in the mitochondrial levels, in aSyn transgenic mice. The levels of superoxide dismutase 2, a client of the HSP10/HSP60 folding complex, and synaptosomal spare respiratory capacity are also reduced. Overexpression of HSP10 ameliorates aSyn-associated mitochondrial dysfunction and delays aSyn pathology in vitro and in vivo. Altogether, our data indicate that increased levels of aSyn induce mitochondrial deficits, at least partially, by sequestering HSP10 in the cytosol and preventing it from acting in mitochondria. Importantly, these alterations manifest first at presynaptic terminals. Our study not only provides mechanistic insight into synucleinopathies but opens new avenues for targeting underlying cellular pathologies. Alpha-synuclein (aSyn) accumulates in intracellular inclusions in synucleinopathies, but the molecular mechanisms leading to disease are unclear. We identify the 10 kDa heat shock protein (HSP10) as a mediator of aSyn-induced mitochondrial impairments in striatal synaptosomes. We find an age-associated increase in the cytosolic levels of HSP10, and a concomitant decrease in the mitochondrial levels, in aSyn transgenic mice. The levels of superoxide dismutase 2, a client of the HSP10/HSP60 folding complex, and synaptosomal spare respiratory capacity are also reduced. Overexpression of HSP10 ameliorates aSyn-associated mitochondrial dysfunction and delays aSyn pathology in vitro and in vivo. Altogether, our data indicate that increased levels of aSyn induce mitochondrial deficits, at least partially, by sequestering HSP10 in the cytosol and preventing it from acting in mitochondria. Importantly, these alterations manifest first at presynaptic terminals. Our study not only provides mechanistic insight into synucleinopathies but opens new avenues for targeting underlying cellular pathologies. A major pathological hallmark of Parkinson’s disease (PD) and other synucleinopathies is the presence of intraneuronal inclusions known as Lewy bodies (LBs) and Lewy neurites (LNs). These inclusions are composed mainly of α-synuclein (aSyn) but are known to also contain mitochondrial proteins (Leverenz et al., 2007Leverenz J.B. Umar I. Wang Q. Montine T.J. McMillan P.J. Tsuang D.W. Jin J. Pan C. Shin J. Zhu D. Zhang J. Proteomic identification of novel proteins in cortical Lewy bodies.Brain Pathol. 2007; 17: 139-145Crossref PubMed Scopus (157) Google Scholar, Xia et al., 2008Xia Q. Liao L. Cheng D. Duong D.M. Gearing M. Lah J.J. Levey A.I. Peng J. Proteomic identification of novel proteins associated with Lewy bodies.Front. Biosci. 2008; 13: 3850-3856Crossref PubMed Scopus (112) Google Scholar). Moreover, mitochondrial dysfunction has been implicated in the pathogenesis of PD: complex I deficiency was detected in the substantia nigra (SN) (Schapira et al., 1989Schapira A.H.V. Cooper J.M. Dexter D. Jenner P. Clark J.B. Marsden C.D. Mitochondrial complex I deficiency in Parkinson’s disease.Lancet. 1989; 333: 1269Abstract Scopus (1154) Google Scholar), muscle (Blin et al., 1994Blin O. Desnuelle C. Rascol O. Borg M. Peyro Saint Paul H. Azulay J.P. Billé F. Figarella D. Coulom F. Pellissier J.F. et al.Mitochondrial respiratory failure in skeletal muscle from patients with Parkinson’s disease and multiple system atrophy.J. Neurol. Sci. 1994; 125: 95-101Abstract Full Text PDF PubMed Scopus (147) Google Scholar), and platelets (Parker et al., 1989Parker Jr., W.D. Boyson S.J. Parks J.K. Abnormalities of the electron transport chain in idiopathic Parkinson’s disease.Ann. Neurol. 1989; 26: 719-723Crossref PubMed Scopus (922) Google Scholar) of PD patients, and mutations in several mitochondria-related genes are associated with familial forms of the disease (Corti et al., 2011Corti O. Lesage S. Brice A. What genetics tells us about the causes and mechanisms of Parkinson’s disease.Physiol. Rev. 2011; 91: 1161-1218Crossref PubMed Scopus (397) Google Scholar, Bose and Beal, 2016Bose A. Beal M.F. Mitochondrial dysfunction in Parkinson’s disease.J. Neurochem. 2016; 139: 216-231Crossref PubMed Scopus (355) Google Scholar, Chang et al., 2017Chang D. Nalls M.A. Hallgrímsdóttir I.B. Hunkapiller J. van der Brug M. Cai F. Kerchner G.A. Ayalon G. Bingol B. Sheng M. et al.International Parkinson’s Disease Genomics Consortium23andMe Research TeamA meta-analysis of genome-wide association studies identifies 17 new Parkinson’s disease risk loci.Nat. Genet. 2017; 49: 1511-1516Crossref PubMed Scopus (524) Google Scholar). aSyn is considered primarily a cytoplasmic protein, but recent evidence suggests that it may also accumulate in mitochondria (Hu et al., 2019Hu D. Sun X. Liao X. Zhang X. Zarabi S. Schimmer A. Hong Y. Ford C. Luo Y. Qi X. Alpha-synuclein suppresses mitochondrial protease ClpP to trigger mitochondrial oxidative damage and neurotoxicity.Acta Neuropathol. 2019; 137: 939-960Crossref PubMed Scopus (35) Google Scholar, Wang et al., 2019Wang X. Becker K. Levine N. Zhang M. Lieberman A.P. Moore D.J. Ma J. Pathogenic alpha-synuclein aggregates preferentially bind to mitochondria and affect cellular respiration.Acta Neuropathol. Commun. 2019; 7: 41Crossref PubMed Scopus (63) Google Scholar) and associate with mitochondria-associated endoplasmic reticulum (ER) (Chinta et al., 2010Chinta S.J. Mallajosyula J.K. Rane A. Andersen J.K. Mitochondrial α-synuclein accumulation impairs complex I function in dopaminergic neurons and results in increased mitophagy in vivo.Neurosci. Lett. 2010; 486: 235-239Crossref PubMed Scopus (275) Google Scholar, Guardia-Laguarta et al., 2014Guardia-Laguarta C. Area-Gomez E. Rüb C. Liu Y. Magrané J. Becker D. Voos W. Schon E.A. Przedborski S. α-Synuclein is localized to mitochondria-associated ER membranes.J. Neurosci. 2014; 34: 249-259Crossref PubMed Scopus (306) Google Scholar). Whether aSyn plays a physiological role in mitochondria is unclear, but increased aSyn levels inhibit cellular respiration (Chinta et al., 2010Chinta S.J. Mallajosyula J.K. Rane A. Andersen J.K. Mitochondrial α-synuclein accumulation impairs complex I function in dopaminergic neurons and results in increased mitophagy in vivo.Neurosci. Lett. 2010; 486: 235-239Crossref PubMed Scopus (275) Google Scholar, Wang et al., 2019Wang X. Becker K. Levine N. Zhang M. Lieberman A.P. Moore D.J. Ma J. Pathogenic alpha-synuclein aggregates preferentially bind to mitochondria and affect cellular respiration.Acta Neuropathol. Commun. 2019; 7: 41Crossref PubMed Scopus (63) Google Scholar, Zambon et al., 2019Zambon F. Cherubini M. Fernandes H.J.R. Lang C. Ryan B.J. Volpato V. Bengoa-Vergniory N. Vingill S. Attar M. Booth H.D.E. et al.Cellular α-synuclein pathology is associated with bioenergetic dysfunction in Parkinson’s iPSC-derived dopamine neurons.Hum. Mol. Genet. 2019; 28: 2001-2013Crossref PubMed Scopus (52) Google Scholar), induce mitophagy and mitochondrial fragmentation (Nakamura et al., 2008Nakamura K. Nemani V.M. Wallender E.K. Kaehlcke K. Ott M. Edwards R.H. Optical reporters for the conformation of alpha-synuclein reveal a specific interaction with mitochondria.J. Neurosci. 2008; 28: 12305-12317Crossref PubMed Scopus (150) Google Scholar, Nakamura et al., 2011Nakamura K. Nemani V.M. Azarbal F. Skibinski G. Levy J.M. Egami K. Munishkina L. Zhang J. Gardner B. Wakabayashi J. et al.Direct membrane association drives mitochondrial fission by the Parkinson disease-associated protein alpha-synuclein.J. Biol. Chem. 2011; 286: 20710-20726Crossref PubMed Scopus (395) Google Scholar), affect mitochondrial Ca2+ homeostasis (Calì et al., 2012Calì T. Ottolini D. Negro A. Brini M. α-Synuclein controls mitochondrial calcium homeostasis by enhancing endoplasmic reticulum-mitochondria interactions.J. Biol. Chem. 2012; 287: 17914-17929Crossref PubMed Scopus (189) Google Scholar) and protein import (Di Maio et al., 2016Di Maio R. Barrett P.J. Hoffman E.K. Barrett C.W. Zharikov A. Borah A. Hu X. McCoy J. Chu C.T. Burton E.A. et al.α-Synuclein binds to TOM20 and inhibits mitochondrial protein import in Parkinson’s disease.Sci. Transl. Med. 2016; 8: 342ra78Crossref PubMed Scopus (254) Google Scholar) and turnover (Hu et al., 2019Hu D. Sun X. Liao X. Zhang X. Zarabi S. Schimmer A. Hong Y. Ford C. Luo Y. Qi X. Alpha-synuclein suppresses mitochondrial protease ClpP to trigger mitochondrial oxidative damage and neurotoxicity.Acta Neuropathol. 2019; 137: 939-960Crossref PubMed Scopus (35) Google Scholar), and reduce mitochondrial membrane potential (Banerjee et al., 2010Banerjee K. Sinha M. Pham Cl.L. Jana S. Chanda D. Cappai R. Chakrabarti S. α-synuclein induced membrane depolarization and loss of phosphorylation capacity of isolated rat brain mitochondria: implications in Parkinson’s disease.FEBS Lett. 2010; 584: 1571-1576Crossref PubMed Scopus (82) Google Scholar), suggesting a pathological role that culminates with mitochondrial dysfunction. Here, we hypothesized that aSyn-dependent mitochondrial dysfunction might initiate in the synapse, because aSyn is likely to start accumulating in the cytoplasm of the presynaptic terminals (Kramer and Schulz-Schaeffer, 2007Kramer M.L. Schulz-Schaeffer W.J. Presynaptic alpha-synuclein aggregates, not Lewy bodies, cause neurodegeneration in dementia with Lewy bodies.J. Neurosci. 2007; 27: 1405-1410Crossref PubMed Scopus (394) Google Scholar) and then progress toward the perinuclear region to induce additional cellular pathologies. First, we screened for aSyn-interacting proteins in nerve terminals (striatal synaptosomes) and identified the 10 kDa heat shock protein (HSP10) as an aSyn interactor and mediator of aSyn-associated mitochondrial dysfunction. Importantly, we obtained detailed insight into the mechanisms through which the accumulation of aSyn, a predominantly cytosolic protein, can induce cellular pathologies by affecting mitochondrial functions. We found that accumulation of aSyn in middle-age transgenic animals sequesters HSP10 in the cytosol, reducing its mitochondrial function in the HSP10/HSP60 complex, which resulted in a reduction in the levels of the client protein superoxide dismutase 2 (SOD2). This led to increased mitochondrial oxidative stress and impaired respiration. Interestingly, improving mitochondrial health by restoring the levels of HSP10 delayed aSyn-induced mitochondrial pathology both in vitro and in vivo. Our findings provide novel mechanistic information on the molecular underpinnings of PD and might help in designing novel therapeutic strategies for PD and other synucleinopathies. aSyn interacts with several synaptic and mitochondrial proteins (Betzer et al., 2015Betzer C. Movius A.J. Shi M. Gai W.P. Zhang J. Jensen P.H. Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein.PLoS ONE. 2015; 10: e0116473Crossref PubMed Scopus (46) Google Scholar) and affects both synaptic and mitochondrial functions (Guardia-Laguarta et al., 2014Guardia-Laguarta C. Area-Gomez E. Rüb C. Liu Y. Magrané J. Becker D. Voos W. Schon E.A. Przedborski S. α-Synuclein is localized to mitochondria-associated ER membranes.J. Neurosci. 2014; 34: 249-259Crossref PubMed Scopus (306) Google Scholar, Calo et al., 2016Calo L. Wegrzynowicz M. Santivañez-Perez J. Grazia Spillantini M. Synaptic failure and α-synuclein.Mov. Disord. 2016; 31: 169-177Crossref PubMed Scopus (92) Google Scholar, Di Maio et al., 2016Di Maio R. Barrett P.J. Hoffman E.K. Barrett C.W. Zharikov A. Borah A. Hu X. McCoy J. Chu C.T. Burton E.A. et al.α-Synuclein binds to TOM20 and inhibits mitochondrial protein import in Parkinson’s disease.Sci. Transl. Med. 2016; 8: 342ra78Crossref PubMed Scopus (254) Google Scholar, Faustini et al., 2017Faustini G. Bono F. Valerio A. Pizzi M. Spano P. Bellucci A. Mitochondria and α-synuclein: friends or foes in the pathogenesis of Parkinson’s disease?.Genes (Basel). 2017; 8: E377Crossref PubMed Scopus (33) Google Scholar, Hu et al., 2019Hu D. Sun X. Liao X. Zhang X. Zarabi S. Schimmer A. Hong Y. Ford C. Luo Y. Qi X. Alpha-synuclein suppresses mitochondrial protease ClpP to trigger mitochondrial oxidative damage and neurotoxicity.Acta Neuropathol. 2019; 137: 939-960Crossref PubMed Scopus (35) Google Scholar, Wang et al., 2019Wang X. Becker K. Levine N. Zhang M. Lieberman A.P. Moore D.J. Ma J. Pathogenic alpha-synuclein aggregates preferentially bind to mitochondria and affect cellular respiration.Acta Neuropathol. Commun. 2019; 7: 41Crossref PubMed Scopus (63) Google Scholar, Zambon et al., 2019Zambon F. Cherubini M. Fernandes H.J.R. Lang C. Ryan B.J. Volpato V. Bengoa-Vergniory N. Vingill S. Attar M. Booth H.D.E. et al.Cellular α-synuclein pathology is associated with bioenergetic dysfunction in Parkinson’s iPSC-derived dopamine neurons.Hum. Mol. Genet. 2019; 28: 2001-2013Crossref PubMed Scopus (52) Google Scholar). However, the effects of aSyn in striatal synaptosomal mitochondria have not been addressed in detail. To assess the molecular underpinnings of aSyn-mediated toxicity in striatal mitochondria, we performed a proteomics-based unbiased screen for aSyn-interacting partners in nerve terminals (synaptosomal component). We identified aSyn-interacting proteins in preparations from transgenic overexpressing human aSyn (wild-type aSyn [WTaSyn]) or from control mice. In total, we identified 67 proteins (Table S1). Of those 67 proteins, we selected the HSP10 for further validation, because it forms, together with HSP60, a major folding complex in mitochondria (David et al., 2013David S. Bucchieri F. Corrao S. Czarnecka A.M. Campanella C. Farina F. Peri G. Tomasello G. Sciumè C. Modica G. et al.Hsp10: anatomic distribution, functions, and involvement in human disease.Front. Biosc. (Elite Ed.). 2013; 5: 768-778Crossref PubMed Google Scholar). HSP10 is a major hub protein for cellular interactions (Rizzolo et al., 2018Rizzolo K. Kumar A. Kakihara Y. Phanse S. Minic Z. Snider J. Stagljar I. Zilles S. Babu M. Houry W.A. Systems analysis of the genetic interaction network of yeast molecular chaperones.Mol. Omics. 2018; 14: 82-94Crossref PubMed Google Scholar) and regulates several mitochondrial functions, including respiration, membrane potential, and reactive oxygen species (ROS) removal. Therefore, even subtle changes in its concentration may affect the functionality of several proteins and cellular functions. The regulation of HSP10 levels in neurological diseases is not well understood (Hickey et al., 2000Hickey R.W. Zhu R.L. Alexander H.L. Jin K.L. Stetler R.A. Chen J. Kochanek P.M. Graham S.H. 10 kD mitochondrial matrix heat shock protein mRNA is induced following global brain ischemia in the rat.Brain Res. Mol. Brain Res. 2000; 79: 169-173Crossref PubMed Scopus (14) Google Scholar, Bross et al., 2007Bross P. Li Z. Hansen J. Hansen J.J. Nielsen M.N. Corydon T.J. Georgopoulos C. Ang D. Lundemose J.B. Niezen-Koning K. et al.Single-nucleotide variations in the genes encoding the mitochondrial Hsp60/Hsp10 chaperone system and their disease-causing potential.J. Hum. Genet. 2007; 52: 56-65Crossref PubMed Scopus (21) Google Scholar, Kim and Lee, 2007Kim S.-W. Lee J.-K. NO-induced downregulation of HSP10 and HSP60 expression in the postischemic brain.J. Neurosci. Res. 2007; 85: 1252-1259Crossref PubMed Scopus (26) Google Scholar, Bie et al., 2016Bie A.S. Fernandez-Guerra P. Birkler R.I. Nisemblat S. Pelnena D. Lu X. Deignan J.L. Lee H. Dorrani N. Corydon T.J. et al.Effects of a mutation in the HSPE1 gene encoding the mitochondrial co-chaperonin HSP10 and its potential association with a neurological and developmental disorder.Front. Mol. Biosci. 2016; 3: 65Crossref PubMed Scopus (29) Google Scholar, Bross and Fernandez-Guerra, 2016Bross P. Fernandez-Guerra P. Disease-associated mutations in the HSPD1 gene encoding the large subunit of the mitochondrial HSP60/HSP10 chaperonin complex.Front. Mol. Biosci. 2016; 3: 49Crossref PubMed Scopus (29) Google Scholar, Fan et al., 2017Fan W. Fan S.S. Feng J. Xiao D. Fan S. Luo J. Elevated expression of HSP10 protein inhibits apoptosis and associates with poor prognosis of astrocytoma.PLoS ONE. 2017; 12: e0185563Crossref PubMed Scopus (16) Google Scholar), and to our knowledge, this has not been investigated in the context of synucleinopathies. To assess whether the level of HSP10 is altered in human brain tissue, we fractionated putamen samples of patients with PD and controls (Figures 1A and 1B ). We found that levels of synaptosomal HSP10 were reduced in samples from patients. In contrast, the levels of HSP60, the protein partner of HSP10 in the mitochondrial folding complex, and the levels of soluble aSyn were not altered. In parallel, the levels of synaptosomal SOD2 were also reduced in PD brains. Although we found no change in the levels of soluble aSyn in striatal synaptosomes, we detected the colocalization of HSP10 and phospho-aSyn (paSyn) in post-mortem tissue (putamen) from PD patients and from patients with dementia with LBs (DLBs) but not from control brains (Figure 1C; Figure S1). We could also co-immunoprecipitate HSP10 and aSyn from striatal synaptosomal samples of WT or aSyn transgenic mice (Figure 1D). In addition, we found colocalization of HSP10 and aSyn in primary cortical neurons using a proximity ligation assay (PLA; Figure S2). To further validate these results, we used surface plasmon resonance (SPR), a technique that enables estimation of the kinetics and binding constants for protein-protein interactions. We confirmed a high-affinity interaction between HSP10 and monomeric, oligomeric, and pre-formed fibrils (PFFs) of aSyn (Figure 1E). Interestingly, we found that monomeric and PFFs of aSyn showed similar binding affinities to HSP10 (monomeric: KD = 2.9 μM, ka = 11.92 M−1 s−1, kd = 3.46 × 10−5 s−1; PFF: KD = 5.27 μM, ka = 11.00 M−1 s−1, kd = 5.95 × 10−5 s−1) and that oligomeric aSyn had lower affinity to HSP10 (KD = 27.2 μM, ka = 6.8 M−1 s−1, kd = 1.85 × 10−4 s−1). Next, we assessed the levels of HSP10 in striatal synaptosomal mitochondria of aSyn transgenic and control mice (Figure 2). The levels of mitochondrial HSP10 were reduced in both young and middle-age transgenic animals (Figures 2A–2D), but the levels of total striatal HSP10 were not altered either by genotype or by age (Figure 2F). In parallel, the levels of cytosolic HSP10 were increased in both young and middle-age A30PaSyn mice (Figure 2E). To unequivocally confirm the identity and integrity of HSP10, we used a parallel gel electrophoresis approach and subjected the gel band corresponding to immunodetected HSP10 to in-gel protein digestion followed by mass spectrometric protein identification (Ott et al., 2015Ott C. Martens H. Hassouna I. Oliveira B. Erck C. Zafeiriou M.P. Peteri U.K. Hesse D. Gerhart S. Altas B. et al.Widespread expression of erythropoietin receptor in brain and its induction by injury.Mol. Med. 2015; 21: 803-815Crossref PubMed Scopus (51) Google Scholar). Both mitochondrial and cytosolic HSP10 were identified with the same high sequence coverage (Figures 2G and 2H). Because the HSP60/HSP10 complex is an important player in mitochondrial folding, we next asked whether there were functional consequences of the age-associated decreased levels of mitochondrial HSP10. To better distinguish between synaptic and general effects, we fractionated striatal samples, prepared from young or middle-age animals, into synaptosome and total mitochondrial fractions (Figure S3A). We found reduced spare respiratory capacity (SRC; a measure of mitochondrial function) in synaptosomes prepared from young aSyn transgenic animals (Figure S3B) but not in total mitochondria fractions (Figure S3C). In middle-age transgenic mice, both synaptosomal (Figure S3D) and total mitochondrial SRC (Figure S3E) decreased compared with control animals. In addition, we found that mitochondrial ROS handling (Figure S3F), mitochondrial membrane potential (Figure S3G), and the opening of the mitochondrial permeability transition pore (mPTP; Figure S3H) were also compromised selectively in the striatal synaptic compartment but not in total mitochondrial fractions (Figures S3I and S3J). Because we found reduced levels of mitochondrial HSP10 in the brains of both PD patients and aSyn transgenic animals, we hypothesized that this might be associated with the functional deficits observed in mitochondria. Therefore, we next tested whether increasing the levels of HSP10 would ameliorate aSyn-associated mitochondrial dysfunction. First, we generated an in vitro model of synucleinopathies in primary striatal cultures, in which moderate overexpression of aSyn resulted in mild mitochondrial dysfunction. Neurons were infected with lentivirus encoding human WTaSyn, A30PaSyn, or GFP, as control protein. To test whether expression of HSP10 would modify aSyn-induced mitochondrial dysfunction, neuronal cultures were infected with lentivirus encoding for Flag-tagged HSP10 (HSP10-Flag) or GFP. Therefore, we generated six experimental groups: GFP+GFP, GFP+HSP10, WTaSyn+GFP, WTaSyn+HSP-10, A30PaSyn+GFP, and A30PaSyn+HSP10. We found that expression of human aSyn resulted in a ∼30% increase in the total levels (both rodent and human) of aSyn compared with control cells (GFP+GFP versus WTaSyn+GFP or A30PaSyn+GFP) (Figures 3A and 3B ; Figure S4A). Expression of human aSyn reduced the levels of endogenous HSP10 just moderately (Figure S4B) but did not affect the levels of HSP60 (Figure 3B; Figure S4C). Expression of human aSyn increased ER stress (elevated levels of growth arrest and DNA damage 153 [GADD153]) (Figures S4D and S4E) and reduced the levels of the mitochondrial serine protease high temperature-regulated A2 (HtrA2) (Figure S4F) and SOD2 (Figures 3B and 3C). mPTPs were more open in cells expressing A30PaSyn (Figure S4G). Interestingly, although basal mitochondrial ROS levels were not increased (Figure S4H), removal of mitochondrial ROS was less efficient in H2O2-treated cells (Figure 3D) when human aSyn was expressed. In contrast, basal cytoplasmic ROS levels were higher (Figure S4I), but H2O2-induced ROS elimination was identical in GFP-expressing and in human aSyn-expressing cells (Figure S4J). Expression of both WTaSyn and A30PaSyn significantly reduced the SRC of striatal neurons, with a greater decrease in the A30PaSyn-expressing cultures (Figures 3E and 3F). Expression of HSP10-Flag resulted in a ∼2-fold increase in the total levels of HSP10 protein (GFP+HSP10, WTaSyn+HSP10, and A30PaSyn+HSP10; Figure 3B; Figure S4B). In WTaSyn-expressing cells, overexpression of HSP10 resulted in increased SOD2 levels (Figures 3B and 3C), and consequently, removal of mitochondrial ROS upon H2O2 treatment was improved (Figure 3D). In addition, the mitochondrial HtrA2 levels (Figures S4D and S4F) and SRC (Figures 3E and 3F) were both normalized. In contrast, although basal cytoplasmic ROS decreased in cells co-expressing WTaSyn and HSP10 (Figure S4I), ER stress levels were not changed (Figures S4D and S4E). Overexpression of HSP10 in A30PaSyn-infected cells improved the elimination of mitochondrial ROS after oxidative stress (Figure 3D) but did not alter the levels of SOD2 or HtrA2. Similarly, SRC was not improved upon HSP10 overexpression (Figures 3E and 3F). The aggregation of aSyn is enhanced in oxidative environments (Hashimoto et al., 1999Hashimoto M. Hsu L.J. Xia Y. Takeda A. Sisk A. Sundsmo M. Masliah E. Oxidative stress induces amyloid-like aggregate formation of NACP/alpha-synuclein in vitro.Neuroreport. 1999; 10: 717-721Crossref PubMed Scopus (367) Google Scholar, Fernandes et al., 2014Fernandes J.T.S. Tenreiro S. Gameiro A. Chu V. Outeiro T.F. Conde J.P. Modulation of alpha-synuclein toxicity in yeast using a novel microfluidic-based gradient generator.Lab Chip. 2014; 14: 3949-3957Crossref PubMed Google Scholar, Scudamore and Ciossek, 2018Scudamore O. Ciossek T. Increased oxidative stress exacerbates α-synuclein aggregation in vivo.J. Neuropathol. Exp. Neurol. 2018; 77: 443-453Crossref PubMed Scopus (52) Google Scholar). Because increasing the levels of HSP10 improved mitochondrial ROS handling and decreased basal cytoplasmic ROS levels, we next tested whether HSP10 overexpression modulated aSyn aggregation. We compared the levels of aSyn phosphorylation on S129 (paSyn, used as a readout of aSyn pathology) in neurons expressing WTaSyn or A30PaSyn together with either HSP10 or GFP (see the six groups above) (Oueslati, 2016Oueslati A. Implication of alpha-synuclein phosphorylation at S129 in synucleinopathies: what have we learned in the last decade?.J. Parkinsons Dis. 2016; 6: 39-51Crossref PubMed Scopus (154) Google Scholar). After 2 weeks in culture, paSyn was found mostly in puncta along the neurites (Figures 4A and 4B ) and colocalized almost completely with synapsin (Figures 4F and 4G) in all cultures expressing human aSyn. Two weeks later, most neurons expressing human aSyn + GFP displayed paSyn in the soma, and the colocalization of paSyn and synapsin was reduced. Interestingly, HSP10 delayed the re-localization of paSyn-positive puncta from the neurites toward the soma, and the colocalization with synapsin remained relatively high even after 4 weeks in culture (Figures 4B and 4G). To further confirm the modulation of aSyn aggregation by HSP10, we assessed the solubility of aSyn in Triton X-100 in the older cultures (Figures 3C–3E). We found that the ratio of insoluble to soluble aSyn decreased in primary cells expressing WTaSyn+HSP10 compared with cells expressing WTaSyn+GFP. However, HSP10 expression had no effect in A30PaSyn-expressing cells (Figures 4C–4E). Next, we assessed the conversion of monomeric aSyn into fibrils in vitro, using the real-time quaking-induced conversion (RT-QuIC) assay (Candelise et al., 2019Candelise N. Schmitz M. Llorens F. Villar-Piqué A. Cramm M. Thom T. da Silva Correia S.M. da Cunha J.E.G. Möbius W. Outeiro T.F. et al.Seeding variability of different alpha synuclein strains in synucleinopathies.Ann. Neurol. 2019; 85: 691-703Crossref PubMed Scopus (54) Google Scholar, van Rumund et al., 2019van Rumund A. Green A.J.E. Fairfoul G. Esselink R.A.J. Bloem B.R. Verbeek M.M. α-Synuclein real-time quaking-induced conversion in the cerebrospinal fluid of uncertain cases of parkinsonism.Ann. Neurol. 2019; 85: 777-781Crossref PubMed Scopus (44) Google Scholar). Briefly, WTaSyn or A30PaSyn monomers were incubated in the presence or absence of HSP10, and the aggregation was monitored by measuring thioflavin T fluorescence. Alternatively, WTaSyn or A30PaSyn monomers were seeded with PFFs of WTaSyn in the presence of HSP10. Only freshly thawed aSyn monomers were used, and the quality of the preparations was assessed using immunoblot (Figure 5A). WTaSyn monomers aggregated even without PFF seeding (Figure 5B) and HSP10 increased aggregation. Interestingly, in the presence of PFF seeds, HSP10 decreased aSyn aggregation (Figure 5C). In contrast, A30PaSyn monomers formed almost no thioflavin T-positive species without PFF seeding, irrespective of the presence of HSP10 (Figure 5B). Although the presence of PFF seeds increased A30PaSyn aggregation, HSP10 had no effect (Figure 5C). Because we found increased levels of cytoplasmic HSP10 in middle-age transgenic mice, we hypothesized that cytosolic aSyn might bind to and, therefore, sequester HSP10 in the cytoplasm, reducing the amount of protein that would normally localize in mitochondria. As a corollary of this hypothesis, this should result in reduced folding capacity of the HSP60/HSP10 complex. To test this, we used two distinct approaches. One mimicked a situation in which different aSyn species (monomers, oligomers, and PFFs) would interact with Hsp10 in the cytosol (Figure 5D, pre-incubation with HSP10). The other mimicked a situation in which the different aSyn species would interact with the HSP60/HSP10 complex in mitochondria (Figure 5D, incubation with HSP10 and HSP60). Interestingly, while aSyn monomers increased the folding activity when pre-incubated with HSP10 (before adding HSP60), incubation of aSyn together with HSP10 and HSP60 did not change complex activity. Oligomeric aSyn species reduced the folding activity only when added together with HSP10 and HSP60. In contrast, aSyn PFFs pre-incubated with HSP10 significantly reduced the folding activity of the complex but had no effect when incubated together with HSP10 and HSP60 (Figure 5D). These findings suggest conformation-specific interactions between aSyn and HSP10, consistent with the SPR data, and suggest that aggregated aSyn in the cytosol impairs the activity of the HSP10/HSP60 complex. Next, to determine whether we could rescue aSyn-associated pathologies in vivo, we injected lentiviruses encoding either HSP10 or GFP in the SN of WT or aSyn transgenic mice. HSP10 and GFP were detected both in synaptosomes prepared from the striatum and in dopaminergic neurons of the SN (Figures 6A and 6D ). One month after viral injection, neither the number of tyrosine hydroxylase (TH)-positive (dopaminergic) neurons nor the total number of neurons (Nissl) changed compared with control mice (Figures S5A and S5B). We found no alterations in the levels of synaptophysin or PSD95 in striatal synaptosomes (Figure 6B). ER stress (GADD153) was increased, and SOD1 and SOD2 levels were decreased in transgenic mice (Figure 6C). Expression of HSP10 did not change the levels of SOD1. In contrast, it" @default.
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• W2953454103 creator A5090998741 @default.
• W2953454103 date "2019-07-01" @default.
• W2953454103 modified "2023-10-18" @default.
• W2953454103 title "Cytosolic Trapping of a Mitochondrial Heat Shock Protein Is an Early Pathological Event in Synucleinopathies" @default.
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