Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

The sirtuins are highly conserved nicotinamide adenine dinucleotide (NAD+)-dependent enzymes that play a broad role in cellular metabolism and aging. Mitochondrial sirtuin 3 (SIRT3) is downregulated in aging and age-associated diseases such as cancer and neuro-degeneration and plays a major role in maintaining mitochondrial function and preventing oxidative stress. Mitochondria dysfunction is central to the pathogenesis of Parkinson disease with mutations in mitochondrial-associated proteins such as PINK1 and parkin causing familial Parkinson disease. Here, we demonstrate that the presence of alpha-synuclein (αsyn) oligomers in mitochondria induce a corresponding decrease in mitochondrial SIRT3 activity and decreased mitochondrial biogenesis. We show that SIRT3 downregulation in the presence of αsyn accumulation is accompanied by increased phosphorylation of AMP-activated protein kinase (AMPK) and cAMP-response element binding protein (CREB), as well as increased phosphorylation of dynamin-related protein 1 (DRP1) and decreased levels of optic atrophy 1 (OPA1), which is indicative of impaired mitochondrial dynamics. Treatment with the AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) restores SIRT3 expression and activity and improves mitochondrial function by decreasing αsyn oligomer formation. The accumulation of αsyn oligomers in mitochondria corresponds with SIRT3 down-regulation not only in an experimental cellular model, but also in vivo in a rodent model of Parkinson disease, and importantly, in human post mortem brains with neuropathologically confirmed Lewy body disease (LBD). Taken together our findings suggest that pharmacologically increasing SIRT3 levels will counteract αsyn-induced mitochondrial dysfunction by normalizing mitochondrial bioenergetics. These data support a protective role for SIRT3 in Parkinson disease-associated pathways and reveals significant mechanistic insight into the interplay of SIRT3 and αsyn.


Introduction
kinson's disease and related alpha-synucleinopathies. Despite predominant localization in the 54 cytosol, αsyn is found localized to mitochondria in post-mortem Parkinson's disease brain 55 (Devi et al., 2008). Mitochondrial accumulation of αsyn has been associated with impairment 56 of complex-I dependent respiration, decreased mitochondria membrane potential, and in-57 creased levels of mitochondrial reactive oxygen species (mtROS) in multiple cellular models 58 (Hsu et al., 2000;Devi et al., 2008;Reeve et al., 2015;Ludtmann et al., 2018). The evidence 59 supporting a contribution of abnormal accumulation of αsyn to disruption of mitochondrial 60 processes is compelling and indicates a crucial role for αsyn-induced mitochondrial dysfunc-61 tion in Parkinson's disease pathogenesis and alpha-synucleopathies.

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Statistical analysis 216 All data were analyzed by the Graph Pad Prism 7 software (San Diego, CA) and statistical 217 significance was determined by one-way ANOVA analysis of variance with Tukey's multiple 218 comparisons test. Results presented as mean ± standard error of the mean (S.E.M.). For iso-219 lated mitochondria studies in vivo, a Mann-Whitney U test was used to analyze the Western 220 blots, Differences were considered to be statistically significant with *P< 0.05, **P< 0.01, 221 # P< 0.05, ## P< 0.01, and n.s, not statistically significant (p > 0.05).

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Data sharing is not applicable to this article as no new data were created or analyzed in this 224 study.

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Increased αsyn oligomers in mitochondria correlate with decreased SIRT3 protein levels 228 Although it has been described previously that αsyn localizes to mitochondria and αsyn over-229 expressing cells exhibit mitochondrial dysfunction (Devi et al., 2008;Marongiu et al., 2009;230 Nakamura et al., 2011) the relationship between αsyn oligomers and mitochondria in patho-231 logic conditions and the mechanisms whereby αsyn induces mitochondrial dysfunction are 232 still poorly understood. Herein, we use a previously described inducible cell model of human 233 αsyn overexpression that results in formation of intracellular oligomeric species over time 234 (Moussaud et al., 2015). This tetracycline-off (Tet-off) stable cell line facilitates monitoring 235 of αsyn oligomerization in situ via a split luciferase protein-fragment complementation assay.

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To determine if αsyn oligomeric species are localized within mitochondria, cells were har-237 vested at various time points after tetracycline removal, mitochondrial-enriched fractions 238 were isolated, and luciferase activity was measured as a surrogate for αsyn oligomeric species.

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Luciferase activity increased in a time-dependent manner in both the mitochondrial (Fig. 1A   240 and B) and cytosolic fractions (Suppl. Fig. 1A) of the cells. Increased αsyn oligomers were 241 confirmed by the detection of increased high molecular weight species in both compartments 242 72h after removal of tetracycline (Suppl. Fig. 1B). GAPDH and COXIV immunoblotting confirmed purity of mitochondrial fractionation (Figs. 1A and B). Interestingly, the increase 244 in mitochondrial-localized αsyn oligomers was accompanied by a decrease in SIRT3 protein 245 levels beginning 12h after αsyn expression was turned on, and becoming significant by 24h 246 (Fig. 1A). Immunocytochemistry confirmed decreased SIRT3 immunofluorescence in cells 247 accumulating αsyn oligomers (Suppl. Fig. 1C, Tet-72h) compared to control (Suppl. Fig. 1C, 248 Tet+ 72h). In support of a αsyn-mediated effect on SIRT3 levels, knockdown of SIRT3 in-249 creased αsyn oligomers with a corresponding increase in αsyn protein levels (Figs. 1C and D). of αsyn in the mitochondria affects cellular bioenergetics we measured the oxygen consump-308 tion rate (OCR) in cell lysates using the Seahorse XF96 analyzer. The OCR was measured under basal conditions followed by the sequential addition of oligomycin (ATP synthase inhibitor), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; mitochondrial un-311 coupler), and rotenone plus antimycin A (Complex I and III inhibitor) to assess ATP produc-312 tion, maximal respiration, and spare capacity respectively. Cells overexpressing αsyn had 313 significantly decreased OCR in all paradigms tested when compared to control cells (Tet+) 314 (Figs. 5A -E). This is highly suggestive of a mitochondria respiratory deficit in the presence 315 of mitochondrial αsyn oligomers. We next analyzed the OCR of cells treated with AICAR 316 and found that AICAR treatment was able to significantly restore the OCR level of basal res- with DRP1 significantly increased in the injected SN (Fig. 6B, *P<0.05) and OPA1 signifi-cantly decreased (Fig. 6B, *P<0.05). Lastly, AMPKα-CREB signaling was downregulated in the SN of these animals (Suppl. Fig. 4B), mimicking once again our in vitro observation.

SIRT3 levels are decreased in human Lewy body disease brains.
345 Lastly, we assessed the level of SIRT3 in human post mortem brain with a confirmed neuro-346 pathological diagnosis of Lewy body disease (LBD) ( Table 1). Frozen striatal tissue from 10 347 LBD and 10 healthy controls was homogenized, run on SDS-PAGE, and probed with anti-348 bodies to detect SIRT3, OPA1, and DRP1. Western blot analyses showed significantly re-349 duced expression of SIRT3 in LBD brains compared to controls ( Fig. 7A and B). We also 350 detected reduced expression of OPA1 protein but no significant difference in the level of 351 DRP1 compared to controls ( Fig. 7A and B). Brains from both sexes were utilized but there 352 was no difference in the interpretation of the data when stratified by sex (data not shown).

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Together, these results are consistent with our findings from cell and rodent models with de-354 creased SIRT3 protein levels when αsyn accumulates and aggregates in neurons.      blots of whole brain lysates from ten LBD and ten control brains normalized to GAPDH 688 loading control. Error bars represent the mean ± S.E.M. **P < 0.01 compared to control 689 conditions. n.s: not significant.