Research ArticleParkinson’s Disease

α-Synuclein binds to TOM20 and inhibits mitochondrial protein import in Parkinson’s disease

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Science Translational Medicine  08 Jun 2016:
Vol. 8, Issue 342, pp. 342ra78
DOI: 10.1126/scitranslmed.aaf3634

α-Synuclein disrupts the mitochondrial protein import business

α-Synuclein accumulation and mitochondrial dysfunction are central to the pathogenesis of most forms of Parkinson’s disease and appear to intersect, but how the two are related to each other has remained elusive. Now, Di Maio and colleagues report that specific forms of wild-type α-synuclein, such as oligomeric and dopamine-modified forms, but not the monomeric or fibrillar forms, bind with high affinity to the mitochondrial receptor TOM20. This results in impaired import of proteins required for mitochondrial function and leads to senescence of mitochondria, which show reduced respiration and increased production of reactive oxygen species. This study also highlights potential ways to prevent this deleterious interaction and its downstream consequences.

Abstract

α-Synuclein accumulation and mitochondrial dysfunction have both been strongly implicated in the pathogenesis of Parkinson’s disease (PD), and the two appear to be related. Mitochondrial dysfunction leads to accumulation and oligomerization of α-synuclein, and increased levels of α-synuclein cause mitochondrial impairment, but the basis for this bidirectional interaction remains obscure. We now report that certain posttranslationally modified species of α-synuclein bind with high affinity to the TOM20 (translocase of the outer membrane 20) presequence receptor of the mitochondrial protein import machinery. This binding prevented the interaction of TOM20 with its co-receptor, TOM22, and impaired mitochondrial protein import. Consequently, there were deficient mitochondrial respiration, enhanced production of reactive oxygen species, and loss of mitochondrial membrane potential. Examination of postmortem brain tissue from PD patients revealed an aberrant α-synuclein–TOM20 interaction in nigrostriatal dopaminergic neurons that was associated with loss of imported mitochondrial proteins, thereby confirming this pathogenic process in the human disease. Modest knockdown of endogenous α-synuclein was sufficient to maintain mitochondrial protein import in an in vivo model of PD. Furthermore, in in vitro systems, overexpression of TOM20 or a mitochondrial targeting signal peptide had beneficial effects and preserved mitochondrial protein import. This study characterizes a pathogenic mechanism in PD, identifies toxic species of wild-type α-synuclein, and reveals potential new therapeutic strategies for neuroprotection.

INTRODUCTION

Parkinson’s disease (PD) is a common neurodegenerative disorder that results in motor impairment, cognitive and psychiatric symptoms, and autonomic dysfunction (1). Both genetic and environmental factors have been implicated in PD pathogenesis, and it appears that mitochondrial defects and accumulation of the synaptic protein α-synuclein are common to most forms of the disease (2). Moreover, there is evidence of a bidirectional interaction between mitochondrial dysfunction and α-synuclein accretion and aggregation. Inhibition of mitochondrial complex I leads to accumulation and oligomerization of α-synuclein (35), and increased levels of α-synuclein cause mitochondrial impairment and production of reactive oxygen species (ROS) (6). The nature of the interaction between α-synuclein and mitochondria remains obscure, as does the basis for the vulnerability of dopamine neurons of the nigrostriatal tract. Furthermore, it is unclear whether unmodified monomeric α-synuclein is responsible for these effects or whether posttranslational modifications that have been implicated in pathogenesis, such as oligomerization, dopamine modification, phosphorylation, or nitration, are important.

Although mitochondria contain their own genome, it encodes only 13 proteins (7). Because mitochondria may contain up to 1500 distinct proteins (8), they must import roughly 99% of these. The mitochondrial protein import and sorting machinery is complex and highly regulated (9, 10), and its components and mechanisms vary by the compartment to which a protein is to be sorted. The best-characterized system is one by which nuclear-encoded, matrix-targeted proteins that contain a mitochondrial targeting signal (MTS; presequence) are recognized by the translocase of the outer membrane (TOM) receptors and translocated through the outer membrane to the translocase of the inner membrane (TIM) and into the matrix, where the MTS is cleaved to yield the mature protein (10). In this system, import of a presequence-containing preprotein starts with recognition of the MTS via interaction of its hydrophobic face with the TOM20 receptor (10). Subsequently, the hydrophilic side of the presequence is recognized by TOM22. Given the normally close proximity of TOM20 and TOM22, it is also possible that both receptors recognize the MTS simultaneously. However, recent cryo-EM (electron microscopy) studies suggest that, in addition to the central TOM complex core, there are “peripheral” TOM20 components, which are in a dynamic equilibrium with the preassembled TOM complex (11).

Although monomeric α-synuclein is an intrinsically disordered protein in solution, in association with anionic lipids in membranes, it forms an amphipathic helix (12) similar to known MTS motifs. In this context, we hypothesized that, under some conditions, α-synuclein might interact with TOM20 and interfere with import of mitochondrially targeted proteins. As such, this may represent a new pathogenic mechanism and a potential target for therapeutic intervention in PD.

RESULTS

α-Synuclein interacts with TOM20 in vivo

Systemic defects in mitochondrial complex I have been described repeatedly in PD, and systemic inhibition of complex I with rotenone in rodents reproduces many features of PD, including Lewy pathology and accumulation and oligomerization of α-synuclein in the substantia nigra (4, 5, 13). We have also found that in vivo rotenone treatment increases S129 phosphorylation of α-synuclein in nigrostriatal neurons (213% of control; P < 0.0001, two-tailed unpaired t test; fig. S1). Moreover, occupational exposure to rotenone is a risk factor for developing the disease (14). Therefore, we used the rotenone model to assess potential interactions between α-synuclein and mitochondrial protein import machinery. Using proximity ligation assays (PLAs) (15), we demonstrated a marked increase in the interaction between α-synuclein and TOM20 in nigrostriatal dopamine neurons of rats treated with rotenone relative to rats treated with vehicle (n = 6 per group; P < 0.001, two-tailed unpaired t test, or P < 0.005 with Welch’s correction for unequal variances; Fig. 1A). There was no such interaction between α-synuclein and TOM22 or TOM40 or a component of the translocase of the inner membrane, TIM23 (fig. S2). As an initial assessment of whether this apparent interaction between α-synuclein and TOM20 resulted in loss of mitochondrially targeted protein, we examined levels of the nuclear-encoded, mitochondrially targeted subunit of complex I, NADH (reduced form of nicotinamide adenine dinucleotide)–ubiquinone oxidoreductase core subunit S3 (Ndufs3), and found a decrease in this imported protein after rotenone treatment. Furthermore, the remaining Ndufs3 immunoreactivity was more diffuse than punctate, as verified by a drop in the TOM20-Ndufs3 Pearson index from 0.77 ± 0.02 in control animals to 0.23 ± 0.02 in rotenone-treated rats (P < 0.0001, two-tailed unpaired t test; fig. S3).

Fig. 1. Ex vivo PL between α-synuclein and TOM20 is associated with decreased mitochondrial import of the complex I subunit Ndufs3 in nigrostriatal neurons in vivo in the rotenone and α-synuclein overexpression rat models of PD.

(A) In a vehicle-treated rat (top row), there is little α-synuclein (α-syn)–TOM20 PL signal, and there is intense punctate staining of Ndufs3 in mitochondria of nigrostriatal neurons. In contrast, in a rotenone-treated rat (bottom row), there is a strong α-synuclein–TOM20 PL signal, which is associated with loss of mitochondrial Ndufs3 staining. In the box plot, mean nigrostriatal cellular PL fluorescence values for individual animals (vehicle- or rotenone-treated) are indicated by black circles. In each animal, PL signal was measured in 35 to 50 nigrostriatal neurons per hemisphere. P < 0.005 by two-tailed unpaired t test with Welch’s correction. TH, tyrosine hydroxylase. (B) In a rat that received a unilateral injection of AAV-shSNCA, the rotenone-induced α-synuclein–TOM20 PL signal was largely prevented, and mitochondrial Ndufs3 staining was preserved. In the box plot, mean nigrostriatal cellular PL fluorescence values for each hemisphere (control or AAV-shSNCA–injected) of individual animals are indicated by black circles. For each animal, PL signal was measured in 50 to 70 nigrostriatal neurons per hemisphere. P < 0.0005 by two-tailed paired t test with Welch’s correction. (C) In a rat that received unilateral injection of an α-synuclein overexpression vector (AAV-hSNCA), the α-synuclein–injected hemisphere showed a strong α-synuclein–TOM20 PL signal with an associated loss of Ndufs3 staining. In the box plot, mean nigrostriatal cellular PL fluorescence values for each hemisphere (control AAV-GFP or AAV-hSNCA–injected) of individual animals are indicated by black circles. For each animal, PL signal was measured in 50 to 70 nigrostriatal neurons per hemisphere. P < 0.05 by two-tailed unpaired t test with Welch’s correction. Scale bar, 30 μm. GFP, green fluorescent protein.

α-Synuclein in nigrostriatal dopamine neurons increases with normal aging, in PD, and in rats treated with rotenone (4, 16). Because the amount of oligomer formation and other posttranslational modifications are dependent on the concentration of α-synuclein, we examined the in vivo effects of reducing α-synuclein on its interaction with TOM20 in rotenone-treated rats. Three weeks before rotenone treatment, rats received a unilateral injection into substantia nigra of adeno-associated virus type 2 (AAV2) virus containing a short hairpin RNA specifically targeting rat α-synuclein (17). Quantitative confocal immunofluorescence showed a 30 to 40% knockdown of endogenous α-synuclein protein in transduced dopamine neurons 3 weeks after injection (17). Using tissue from this previous study, we found that after rotenone treatment, in the untransduced hemisphere, there was a strong PL signal between α-synuclein and TOM20, which was almost completely prevented by the modest reduction in α-synuclein in the AAV2-injected hemisphere (n = 4; P < 0.0001, two-tailed paired t test, or P < 0.0005 with Welch’s correction; Fig. 1B). Consistent with the decreased interaction of α-synuclein with TOM20, knockdown was associated with a preservation of punctate (mitochondrial) Ndufs3 staining, suggesting normal protein import. Knockdown of endogenous α-synuclein provided behavioral protection and preservation of nigrostriatal neurons, terminals, and dendrites (17), possibly indicating the importance of reduced mitochondrial protein import.

To validate our finding of an in vivo interaction between α-synuclein and TOM20, we used another model of PD that does not rely on a neurotoxin, namely, AAV2 viral-mediated overexpression of human α-synuclein (hSNCA) in nigrostriatal neurons. Quantitative immunofluorescence indicated a threefold overexpression of α-synuclein in the transduced nigral neurons (P < 0.01) along with a marked increase in S129 phosphorylation (394% of control; P < 0.005, paired t test; fig. S1). In this model, which induces α-synuclein oligomerization (18) and causes delayed and progressive neurodegeneration (19), we also found a strong PL signal between α-synuclein and TOM20 with a parallel loss of mitochondrial Ndufs3 in dopamine neurons in the vector-injected hemisphere, but not in the control hemisphere (n = 4; P < 0.005, two-tailed paired t test, or P < 0.05 with Welch’s correction; Fig. 1C).

α-Synuclein interacts with TOM20 in dopaminergic neurons in the PD brain

Our results from rotenone-treated and hSNCA-overexpressing rats demonstrated an interaction between α-synuclein and TOM20, which was associated with reduced import and a decrease in mitochondrially localized Ndufs3. To determine whether this process was relevant to idiopathic human PD, we performed PLAs assays (α-synuclein–TOM20) and immunocytochemistry for the Ndufs3 subunit in blinded postmortem substantia nigra sections from individuals with PD (n = 5) and from controls (n = 4). Compared to controls, nigrostriatal dopamine neurons from all PD cases had a strong PL signal (P < 0.0001), indicating an interaction between α-synuclein and TOM20 (Fig. 2). Furthermore, relative to the controls, there was a prominent loss of mitochondrial Ndufs3 staining (P < 0.02 or P < 0.05 with Welch’s correction), and the staining that remained tended to be diffuse rather than punctate. This suggested that α-synuclein–induced impairment of mitochondrial protein import may occur in the human disease.

Fig. 2. Evidence of impaired mitochondrial protein import in human dopaminergic substantia nigra neurons in PD.

(A) In TH-positive dopamine neurons from postmortem brain tissue of PD patients, there was an intense α-synuclein–TOM20 PL signal and a marked loss of Ndufs3 immunoreactivity. In PD cases, remaining Ndufs3 staining was rather diffuse instead of punctate. (B) Quantification of the α-synuclein–TOM20 PL signal in control versus PD dopamine neurons. (C) Quantification of Ndufs3 immunoreactivity in control versus PD dopamine neurons. The Ndufs3 signal was normalized to the TH signal, which tended to minimize the apparent differences. ***P < 0.0001; *P < 0.05, two-tailed unpaired t test with Welch’s correction for unequal variances.

α-Synuclein inhibits import of mitochondrial proteins

To explore in more detail the functional significance of the α-synuclein–TOM20 interaction, we turned to cellular and in vitro assays of mitochondrial protein import in the presence or absence of various forms of monomeric, oligomeric, or otherwise posttranslationally modified recombinant α-synuclein. For this purpose, we used (i) confocal imaging of dopaminergic SH-SY5Y cells expressing mitochondrially targeted GFP (MTS-GFP), (ii) direct assays of mitochondrial protein import in isolated mitochondria, and (iii) confocal measurements of mitochondrial localization of endogenous, presequence-containing, nuclear-encoded, and imported proteins. α-Synuclein binds to lipid membranes and can easily cross the plasma membrane, a property we took advantage of in in vitro studies (20). Control experiments using fluorescently labeled α-synuclein confirmed that each species of α-synuclein used in subsequent experiments entered cells to an equivalent extent (fig. S4). Furthermore, when cells were treated with exogenous α-synuclein species (200 nM monomer equivalent), there was no detectable change in the total cellular content of α-synuclein, consistent with the data suggesting that endogenous intraneuronal concentrations are in the range of 2 to 5 μM (21), or even higher (22).

Treatment of SH-SY5Y cells with 200 nM monomeric α-synuclein for 24 or 48 hours had no effect on import (mitochondrially localized GFP), but the same amount of α-synuclein, in the form of small oligomers, potently inhibited import by about 50% [P < 0.0001, two-way analysis of variance (ANOVA); n = 3; Fig. 3, A and B]. Dopamine stably modifies α-synuclein, and although the molecular nature of this modification is controversial, this form of α-synuclein has been implicated in pathogenesis (23, 24). When applied to SH-SY5Y cells, dopamine-modified α-synuclein also potently inhibited import (P < 0.0001). Similarly, S129E α-synuclein, a phosphomimetic mutant, strongly inhibited MTS-GFP import (P < 0.0001). In contrast, unlike the other posttranslational modifications, nitrated α-synuclein behaved like the monomeric, unmodified protein and did not inhibit import. Additionally, thioflavin T–positive fibrils of α-synuclein had no effect on import (fig. S5, A to D).

Fig. 3. Posttranslationally modified α-synuclein binds to TOM20 and inhibits mitochondrial protein import.

(A) Mitochondrial GFP (mtGFP) import in intact wild-type (WT) and TOM20-overexpressing (OE) SH-SY5Y cells exposed to various forms of α-synuclein. In WT cells treated with oligomeric, dopamine (DA)–modified, or S129E α-synuclein, note the diffuse pattern of staining compared to vehicle. In TOM20-overexpressing cells, mtGFP maintained its mitochondrial localization despite α-synuclein treatment. (B) Quantification of mtGFP import in WT and TOM20-overexpressing cells. For each condition in each experiment, mtGFP localization was determined in 5 to 10 regions of interest in zoomed confocal images from 5 to 10 cells, and three or four independent experiments were performed. (C) Autoradiographs of in vitro import of pre-OTC (pOTC) into mitochondria isolated from rat brain (top), WT SH-SY5Y cells (middle), and TOM20-overexpressing SH-SY5Y cells (bottom) after exposure to various forms of α-synuclein (30 min at 4°C). The upper band represents 35S-labeled pre-OTC, and the lower band represents imported, cleaved (mature) OTC. Mitos, mitochondria. (D) Quantification of OTC import into rat brain mitochondria. Results were normalized to the vehicle-treated control, and trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) + oligomycin was used to collapse membrane potential and define zero import. n = 3 independent experiments. (E) Quantification of OTC import into mitochondria from WT and TOM20-overexpressing SH-SY5Y cells. n = 3 to 4 independent experiments. (F) Immunolocalization of TOM20 and Ndufs3 in WT and TOM20-overexpressing HEK293 cells exposed to various forms of α-synuclein. In WT cells exposed to oligomeric, dopamine-modified, or S129E α-synuclein, Ndufs3 localization was diffuse rather than mitochondrial (that is, Ndufs3 redistributed outside of mitochondria as defined by TOM20). This effect was prevented in TOM20-overexpressing cells. (G) Correlation (Pearson index) of the localizations of TOM20 and Ndufs3 in WT versus TOM20-overexpressing cells. TOM20 overexpression rescues the normal localization of Ndufs3. For each experimental condition, at least 100 cells were analyzed in each of three or four independent experiments. Statistical analyses were by one- or two-way ANOVA followed by pairwise testing and correction for multiple comparisons. aP < 0.0001 versus monomer; bP < 0.0001 versus WT cells; cP < 0.005 versus monomer; dP < 0.05 versus WT cells; eP < 0.002 versus WT cells. Scale bars, 5 μm.

Given the critical role of TOM20 in the import of mitochondrially targeted proteins containing an N-terminal targeting signal, we examined whether overexpression of TOM20 might ameliorate the deleterious effects of α-synuclein on protein import. In cells transiently or stably overexpressing TOM20 (about fourfold over endogenous), the inhibitory effects of oligomeric, dopamine-modified, and S129E α-synuclein on import of MTS-GFP were completely prevented (P < 0.0001; Fig. 3, A and B). Overexpression of another component of the TOM complex, TOM5, did not preserve import (fig. S6C).

Although α-synuclein inhibited mitochondrial protein import in cells, this effect might be indirect. Therefore, we used isolated rat brain mitochondria to examine direct effects of various forms of α-synuclein on import. After a 30-min preincubation of 200 nM α-synuclein with mitochondria at 4°C, we found that oligomeric, dopamine-modified, and S129E α-synuclein each inhibited import of radiolabeled pre-ornithine transcarbamylase (pre-OTC) by more than 50% (P < 0.0001, one-way ANOVA; n = 3 to 4), but monomeric and nitrated α-synuclein did not (Fig. 3, C and D). Fibrillar α-synuclein was without effect (fig. S5). A time course experiment revealed that the reduction in import was not simply a slowing of import rate but instead reflected a true decrease in import capacity (fig. S7). α-Synuclein had identical effects in mitochondria isolated from human dopaminergic SH-SY5Y cells (Fig. 3, C and E). To rule out a nonspecific effect of α-synuclein on mitochondrial membrane potential (ΔΨm), and hence, on import, we monitored ΔΨm with tetramethylrhodamine methyl ester (TMRM) under the same conditions and found that it was unchanged for the duration of the experiment (fig. S8). Thus, the effect of α-synuclein on mitochondrial import seemed to be direct and not because of nonspecific disruption of ΔΨm. Furthermore, unlike in control mitochondria, protein import in mitochondria isolated from TOM20-overexpressing SH-SY5Y cells was not inhibited in vitro by posttranslationally modified α-synuclein (Fig. 3, C and E).

To examine the effects of α-synuclein on import of an endogenous, nuclear-encoded mitochondrial protein, we treated human embryonic kidney (HEK) 293 cells with various forms of α-synuclein for 24 hours and then used confocal microscopy to localize the complex I subunit Ndufs3. Treatment with oligomeric, dopamine-modified, and S129E α-synuclein decreased the import of the Ndufs3 subunit. As a result, the mitochondrial localization (Pearson index with TOM20) of Ndufs3 decreased from about 0.9 to 0.3 (P < 0.0001, two-way ANOVA with Sidak correction for multiple comparisons; n = 4 experiments), with an obvious increase in the diffuse cytosolic localization of this mitochondrial protein (Fig. 3, F and G). The impaired import of endogenous Ndufs3, with redistribution to the cytosol, was prevented by overexpression of TOM20 (P < 0.0001; Fig. 3, F and G).

To determine whether the import of other presequence-containing proteins was also disrupted by α-synuclein, we assessed the distributions of endogenous succinate dehydrogenase complex flavoprotein subunit A (SDHA; a subunit of complex II), cytochrome c oxidase subunit 4 (COX4; a subunit of complex IV), and mitochondrial heat shock protein 70 (mtHSP70; a matrix chaperone). As with Ndufs3, there was a cytosolic redistribution of each of these endogenous proteins after treatment of cells with oligomeric, dopamine-modified, and S129E α-synuclein, but not after monomeric or nitrated species (fig. S9). Additionally, we monitored the expression and distribution of two proteins encoded by the mitochondrial genome (which do not require import). Mitochondrially encoded NADH–ubiquinone oxidoreductase core subunit 1 (ND1; a subunit of complex I) and mitochondrially encoded COX1 (mtCO1; a subunit of complex IV) were unaffected by α-synuclein during the time course of this experiment (fig. S9).

α-Synuclein binds to TOM20

To determine whether the inhibitory effects of posttranslationally modified forms of α-synuclein on mitochondrial protein import correlated with their interactions with TOM20, we performed PLAs in cells exposed to various forms of α-synuclein. There was a strong PL signal between TOM20 and oligomeric, dopamine-modified, and S129E α-synuclein, but not monomeric or nitrated α-synuclein (P < 0.0001, ANOVA; n = 3; Fig. 4A). Control experiments showed that the α-synuclein–TOM20 PL signal localized to mitochondria (fig. S10). Fibrillar α-synuclein did not produce a PL signal with TOM20 (fig. S5E). Expression of a “naked” MTS peptide, the presequence of COX8, prevented PL between posttranslationally modified α-synuclein and TOM20, suggesting that α-synuclein binds to a site on TOM20 that overlaps with the MTS receptor site (Fig. 4A). Moreover, the MTS was able to reverse the α-synuclein–TOM20 PL interaction when transfection occurred 24 hours after α-synuclein treatment (Fig. 4B).

Fig. 4. PL of posttranslationally modified α-synuclein and TOM20 and Ndufs3 localization in HEK293 cells.

(A) In untransfected cells, there was PL between TOM20 and oligomeric, dopamine-modified, and S129E α-synuclein, but not monomeric or nitrated species. This was associated with a cytosolic redistribution of Ndufs3. Fibrillar α-synuclein did not interact with TOM20 (fig. S5). When cells were transfected with an MTS expression vector before treatment with α-synuclein, the TOM20–α-synuclein interaction was blocked, indicating that the α-synuclein binding site overlaps with the MTS binding site on TOM20. MTS transfection also preserved the punctate (mitochondrial) distribution of Ndufs3. (B) When cells were transfected with the MTS expression vector 24 hours after α-synuclein treatment, the TOM20–α-synuclein interaction was reversed. Bar graphs show quantification of the α-synuclein–TOM20 PL signal in mock-transfected (black bars) and MTS-overexpressing cells (white bars). At least 100 cells were analyzed for each condition in every independent experiment (n = 3). aP < 0.0001 versus vehicle; bP < 0.0001 versus mock-transfected, two-way ANOVA. Scale bar, 5 μm.

To confirm that the α-synuclein–TOM20 PL signals represent true protein-protein interactions, we measured binding between recombinantly purified α-synuclein and TOM20 (C-terminal cytosolic domain) (25) using fluorescence spectroscopy and a “pseudo–wild-type” tryptophan mutant of α-synuclein to impart fluorescence (26). We found saturable, specific binding of oligomeric, dopamine-modified, and S129E α-synuclein, with a dissociation constant (Kd) of about 5 μM (Fig. 5A). Because binding of monomeric and nitrated α-synuclein was not saturable, we concluded that there was not a specific interaction in this assay, which is consistent with our PL and import results. Inclusion of an MTS peptide (COX8 presequence) at a concentration of 250 μM (~10 × Kd of a native MTS) (25) in this in vitro assay abolished specific, saturable binding of α-synuclein to TOM20 and thereby confirmed that the MTS and α-synuclein bind to the same site on TOM20 (Fig. 5B).

Fig. 5. Binding curves of TOM20 C-terminal cytosolic domain to various forms of α-synuclein.

(A) There was saturable binding of oligomeric, dopamine-modified, and S129E α-synuclein, but not the monomeric or nitrated species. n = 3. CTD, C-terminal cytosolic domain. (B) The COX8 MTS peptide inhibited binding of oligomeric α-synuclein to TOM20. When binding was performed in the presence of excess MTS (250 μM), specific binding was markedly reduced or abolished; nonlinear curve fitting yielded an affinity of >7 × 1014 μM when the MTS was present. Similar results were obtained with dopamine-modified and S129E α-synuclein. The overall effect of the MTS was significant (P < 0.02) by two-way ANOVA. n = 3.

α-Synuclein disrupts the normal interaction of TOM20 with TOM22

It is believed that the presequence of mitochondrially targeted proteins is first recognized by TOM20 in a hydrophobic interaction and subsequently by TOM22 in a hydrophilic interaction before the preprotein is imported (10). The existence of peripheral TOM20 receptors at some distance from the core TOM complex (11) raised the possibility that TOM20-bound preproteins may be trafficked from the periphery to TOM22 in the core translocase complex. In vehicle-treated cells in culture, we found a strong PL signal between TOM20 and TOM22, indicating that at least a portion of these two proteins is normally in close approximation (Fig. 6A). After treatment with forms of α-synuclein that bind to TOM20 (and thereby produce a TOM20–α-synuclein PL signal), the TOM20-TOM22 PL signal was lost (P < 0.0001, ANOVA; n = 3) (Fig. 6A). This result suggested that binding of α-synuclein to TOM20 prevented its association with TOM22, and this, together with specific blockade of presequence binding sites on TOM20 by α-synuclein, likely accounted for loss of import activity. The reasons for loss of association of TOM20 and TOM22 are unclear, but steric interference with the PLA is unlikely because control experiments showed that antibody binding to TOM20 and TOM22 was not occluded by α-synuclein binding. It is possible that the normal mobility of TOM20 in the outer membrane might be impeded by the affinity of α-synuclein for membrane lipids (27). In cells overexpressing a naked MTS, the TOM20-TOM22 PL signal was maintained even in the presence of oligomeric, dopamine-modified, and S129E α-synuclein (Fig. 6A). Moreover, expression of the MTS 24 hours after treatment with oligomeric α-synuclein was able to restore the TOM20-TOM22 PL signal (Fig. 6B), further indicating that the effects of α-synuclein on protein import machinery were reversible.

Fig. 6. α-Synuclein interaction with TOM20 prevents the normal interaction between TOM20 and TOM22 in HEK293 cells.

(A) Under basal conditions (vehicle), PL detects an interaction between TOM20 and TOM22. This was blocked by oligomeric, dopamine-modified, and S129E α-synuclein, but not monomeric or nitrated species. Loss of the TOM20-TOM22 PL signal was associated with relocalization of Ndufs3 to the cytosol. In cells overexpressing a naked MTS (COX8 presequence), the TOM20-TOM22 PL signal was maintained even after treatment with oligomeric, dopamine-modified, and S129E α-synuclein. (B) When cells were treated with α-synuclein and then transfected with the MTS 24 hours later, the TOM20-TOM22 PL signal was restored. Graphs show quantification of TOM20-TOM22 signal in mock-transfected (black bars) and MTS-overexpressing cells (white bars). aP < 0.0001 versus vehicle; bP < 0.0001 versus mock-transfected, two-way ANOVA. At least 100 cells were analyzed per condition in each independent experiment. n = 3. Scale bar, 5 μm.

α-Synuclein–induced impairment of protein import has downstream effects on mitochondrial function

Certain complex I subunits, such as Ndufs3, can normally turn over completely in less than 24 hours (28), and reduced levels of Ndufs3 may lead to metabolic deficits and excessive production of ROS (29). To determine the functional consequences of α-synuclein–induced impairment of mitochondrial protein import, we measured mitochondrial respiration. Results indicated that monomeric α-synuclein had no effect on respiration, but oligomeric and dopamine-modified species depressed both basal and FCCP-stimulated respiration by 30 to 40% (P < 0.01, one-way ANOVA; n = 3; Fig. 7A). These deleterious effects were prevented by overexpression of TOM20 (Fig. 7B) or a naked MTS (fig. S11E).

Fig. 7. Downstream effects of α-synuclein on mitochondria.

(A) In WT SH-SY5Y cells, a 24-hour exposure to oligomeric or dopamine-modified α-synuclein reduced basal and FCCP-stimulated mitochondrial respiration. Monomeric α-synuclein was without effect. *P < 0.01 versus vehicle, one-way ANOVA; n = 3. OCR, oxygen consumption rate. (B) In SH-SY5Y cells overexpressing TOM20, the deleterious effects of oligomeric and dopamine-modified α-synuclein were prevented. n = 3. (C) In WT HEK293 cells, a 24-hour exposure to oligomeric, dopamine-modified, or S129E α-synuclein induced oxidation of protein thiols, whereas exposure to monomeric or nitrated α-synuclein did not. (D and E) In HEK293 cells overexpressing TOM20, α-synuclein did not induce oxidative stress. At least 100 cells were quantified per condition in each experiment. aP < 0.001 versus vehicle; bP < 0.001 versus mock-transfected cells, two-way ANOVA; n = 3. (F) TMRM fluorescence in SH-SY5Y cells (as an index of ΔΨm) was reduced by oligomeric but not monomeric α-synuclein. (G and H) In SH-SY5Y cells overexpressing TOM20, oligomeric α-synuclein did not significantly affect ΔΨm. Thirty to 50 cells were analyzed for each treatment in each of three independent experiments. aP < 0.001 versus vehicle; bP < 0.001 versus WT cells, two-way ANOVA. Scale bars, 10 μm. Oligo, oligomycin; Rot, rotenone.

We next examined protein thiol oxidation (oxidative damage) after exposure to various forms of α-synuclein. Treatment with oligomeric, dopamine-modified, and S129E α-synuclein induced protein thiol oxidation (P < 0.001, two-way ANOVA; n = 3), but monomeric and nitrated α-synuclein did not (Fig. 7, C and E). α-Synuclein–induced oxidative damage was prevented by overexpression of TOM20 (Fig. 7, D and E) or a naked MTS (fig. S11, A and B). Similarly, after 24-hour exposure to oligomeric α-synuclein, there was a significant drop in ΔΨm (P < 0.001, two-way ANOVA; n = 4) that was not seen with monomeric α-synuclein (Fig. 7, F and H), and which was prevented by overexpression of TOM20 (Fig. 7, F and H) or a naked MTS (fig. S11, C and D).

It is notable that in cells overexpressing TOM20, its distribution was mitochondrial, not ectopic, and mitochondrial morphology was unchanged (Fig. 3F). Moreover, although such cells were protected against α-synuclein–induced import defects and resultant respiratory defects, oxidative stress, and depolarization, the mitochondrial α-synuclein–TOM20 PL signal remained intact (Fig. 3F). The mechanism by which overexpression of TOM20 was protective remains unclear, but it appears to enhance the efficiency of import by increasing the initial rate of mitochondrial protein import. Additionally, the protective effect was saturable because higher concentrations of toxic α-synuclein species (1.7 or 3.4 μM versus 200 nM) could overcome the beneficial effects of enhanced TOM20 expression (fig. S12).

Toxic species of α-synuclein may be trimers and tetramers

Evidence presented here suggests that oligomeric, dopamine-modified, and S129E α-synuclein species impair protein import, whereas monomeric, nitrated, and fibrillar forms do not. To look for possible structural explanations for these differences, we used circular dichroism spectroscopy to examine the α-synuclein species used in biological experiments (fig. S13, A and B). However, this did not reveal clear differences between the toxic and nontoxic species; all were predominantly in a random coil conformation with a similar small component of α-helix conformation. As expected, only the fibril preparation had a significant amount of β-sheet structure. In contrast, there were some apparent differences when the species were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) (fig. S13C). As intended, the nontoxic monomer preparation was predominantly monomeric, with a small component of dimer. The nitrated species was composed mostly of monomer and dimer and high–molecular weight material, with little trimer or tetramer. On the other hand, each of the toxic species—oligomeric, dopamine-modified, and S129E α-synuclein—had relatively large amounts of trimer and tetramer, which are about 25 to 35% of the total (P < 0.05 to 0.001 versus monomer or nitrated species, ANOVA; fig. S13D). Under conditions of our preparation, the fibril sample was a continuous “smear,” and individual bands could not be resolved. From these experiments, we tentatively concluded that a trimeric and/or tetrameric structure may be important for mitochondrial toxicity of α-synuclein. It is also important to note that each species of α-synuclein was used in our experiments at a concentration of 200 nM (monomer equivalent), so the actual concentration of oligomer was substantially lower.

The effects of endogenous α-synuclein on protein import machinery may explain the selective vulnerability of dopamine neurons

In cultured cells, there was a strong PL signal between TOM20-TOM22, which could be disrupted by posttranslationally modified α-synuclein. Similar TOM20-TOM22 interactions were seen in neurons in rat brain sections (Fig. 8A). However, there was a conspicuous absence of the TOM20-TOM22 PL signal in nigrostriatal dopamine neurons in control animals. In the midbrain, these neurons have, by far, the highest expression of SNCA mRNA (17), raising the possibility that endogenous α-synuclein (perhaps combined with dopamine modification) may be sufficient to impair import in these neurons at baseline. Consistent with this hypothesis, knockdown of endogenous α-synuclein in control rats “restored” a TOM20-TOM22 PL signal in nigrostriatal neurons relative to the untransduced hemisphere (Fig. 8A). Furthermore, we have reported previously that, under basal conditions, nigrostriatal neurons exist in a more oxidized state than cortical or other dopaminergic neurons (30). The results in Fig. 7 suggest that α-synuclein may contribute to this process. In a separate cohort of animals, we found that AAV-shRNA–mediated knockdown of endogenous α-synuclein decreased basal levels of protein thiol oxidation in nigrostriatal neurons relative to the contralateral hemisphere, which received AAV-shControl vector (P < 0.05; Fig. 8B). Thus, neuron-specific differences in the amount of endogenous α-synuclein (or its posttranslational state) may confer differences in mitochondrial protein import efficiency and, possibly, selective vulnerability.

Fig. 8. The normal TOM20-TOM22 PL signal seen in most neurons is absent in rat nigrostriatal dopamine neurons in vivo but is restored by knockdown of endogenous α-synuclein.

(A) In the untreated hemisphere (top row), MAP2+ (microtubule-associated protein 2)/TH nondopaminergic neurons (arrows) showed a strong TOM20-TOM22 PL signal, which was absent in TH+ dopaminergic cells (asterisks). In the hemisphere that received AAV2-shSNCA (bottom row), there was emergence of a strong TOM20-TOM22 PL signal in the TH+ dopaminergic neurons. Scale bar, 30 μm. (B) Consistent with the in vitro data (Fig. 7, C to E), α-synuclein knockdown was associated with decreased basal protein thiol oxidation in otherwise untreated rats. Filled circles, −S-S−/−SH ratio of nigral neurons in the control hemisphere; half-filled circles, −S-S−/−SH ratio of nigral neurons in the SNCA knockdown hemisphere. Lines connect the means from each hemisphere in each animal. *P < 0.05, Wilcoxon matched-pairs signed-rank test.

DISCUSSION

Our results tie together two central pathogenic mechanisms in PD: α-synuclein accumulation and mitochondrial impairment. We have found that certain species of α-synuclein bind specifically to TOM20, prevent its interaction with the co-receptor TOM22, and inhibit mitochondrial protein import. This leads to impairment of mitochondrial function, with reduced respiration and excessive ROS production. Our data suggest that this process is operative in human PD and may contribute to pathogenesis. These findings help to explain the deleterious effects of α-synuclein on mitochondria, and they suggest new therapeutic strategies.

There is a single published report showing that α-synuclein is a substrate for the import machinery and is imported into the mitochondrial matrix, where it binds to and inhibits complex I (31). To the best of our knowledge, this has not been replicated, and our results suggest that there is no specific interaction between monomeric α-synuclein and the TOM machinery. Another report suggests that TOM40 levels are reduced when α-synuclein is overexpressed, but import was not measured, and no mechanism was elucidated (32). Finally, it has been reported that efficient import prevents PTEN-induced putative kinase 1 (PINK1) from accumulating on the mitochondrial surface, where it can recruit parkin and promote autophagic mitochondrial clearance (33). We hypothesize that by inhibiting protein import, α-synuclein may also alter PINK1-parkin signaling, and this is under investigation.

Here, we used complementary techniques (PL, multiple assays of mitochondrial protein import, and fluorescence spectroscopy) to provide convergent evidence that certain posttranslationally modified forms of α-synuclein (soluble oligomers, dopamine-modified, and S129E) bind to TOM20 and impair mitochondrial import of endogenous or exogenous presequence-containing nuclear-encoded proteins. Our binding assays indicate that these species of α-synuclein bind to TOM20 with affinities of about 5 μM. By contrast, the affinity of a normal TOM20 substrate, the aldehyde dehydrogenase presequence, is lower—about 20 μM (25). The intraneuronal concentration of α-synuclein has been estimated to be 2 to 5 μM (21), and in our experiments, α-synuclein species were applied to cells or isolated mitochondria at a concentration equivalent to 200 nM monomeric protein. Control experiments confirmed that all forms of α-synuclein used here entered cells to an equivalent extent and did not alter total α-synuclein in treated cells. Thus, we conclude that local conditions that predispose to relatively minor oligomer formation, dopamine modification, or S129 phosphorylation (or some combination thereof) are likely to have pronounced effects on protein import into mitochondria.

Whereas binding assays provided strong evidence that α-synuclein interacts specifically with TOM20, the fact that overexpression of a naked MTS (COX8 presequence) blocked the α-synuclein–TOM20 PL signal indicates that the α-synuclein binding site overlaps with the MTS recognition site on TOM20. Direct inhibition of α-synuclein binding to TOM20 by the MTS peptide confirms this conclusion. Thus, one mechanism by which α-synuclein inhibits protein import appears to involve direct competition for TOM20 presequence receptor sites. Moreover, when the MTS was overexpressed after α-synuclein was already bound to TOM20, it was able to normalize the α-synuclein–TOM20 PL signal, indicating that the interaction of α-synuclein with TOM20 is reversible.

The presequence of mitochondrially targeted proteins must be recognized by both TOM20 and TOM22 (either sequentially or simultaneously) before translocation through the TOM40 pore. α-Synuclein binds to TOM20, but we found no evidence for binding to TOM22. Cryo-EM studies suggest that, in addition to the central TOM complex core, there are peripheral TOM20 components, which are in a dynamic equilibrium with the preassembled TOM complex (11). Whether these peripheral TOM20 receptors bind to the presequences of preproteins and traffic them to the central TOM complex (containing TOM22) for import is unclear. Under basal conditions, our PL studies indicate that at least some portion of TOM20 interacts with TOM22, but this interaction is prevented by toxic species of α-synuclein. Interference with the normal TOM20-TOM22 interaction may represent a second mechanism by which α-synuclein impairs import.

The mechanism by which α-synuclein prevents the TOM20-TOM22 interaction is unclear. However, if peripheral TOM20 components must normally traffic to the central TOM complex, it is possible that the affinity of TOM20-bound α-synuclein for lipid membranes may impede lateral movement of TOM20 in the outer mitochondrial membrane and reduce formation of a functional import complex. We found that overexpression of a naked MTS in the setting of exogenous α-synuclein, or knockdown of endogenous α-synuclein in nigrostriatal neurons in vivo, was able to restore this normal TOM20-TOM22 interaction.

Overall, these studies provide compelling evidence for a specific interaction between certain species of wild-type α-synuclein and the TOM20 presequence receptor. Furthermore, we have delineated two plausible and related mechanisms by which this interaction impairs mitochondrial protein import. The relative loss of mitochondrial protein import caused by α-synuclein has several deleterious downstream effects. Basal and FCCP-induced mitochondrial respiration are reduced, and protein thiol oxidation is increased. Additionally, because of impaired electron transport complex activity, ΔΨm declines. The fact that each of these effects can be prevented by overexpression of TOM20 (or an MTS peptide) confirms that they result from defective import. Together, these findings indicate that α-synuclein–induced impairment of mitochondrial protein import has the potential to produce senescent, inefficient mitochondria that produce less energy and more ROS.

Our studies have used a wide variety of assays, including PL, direct import assays, fluorescence spectroscopic binding assays, and respiratory measurements, and these have provided consistent results about which species of α-synuclein are toxic to mitochondria. Monomeric, wild-type α-synuclein appears to have no effect, but oligomeric, dopamine-modified, and S129E phosphomimetic species potently impair import function. Although the phosphomimetic mutant behaved like oligomeric and dopamine-modified species, it is important to recognize that the S129E mutation does not always behave identically to bona fide phosphorylated α-synuclein (34). Nitrated α-synuclein and amyloid fibrils of α-synuclein were essentially inert in our assays. Our limited structural studies suggest that a trimeric or tetrameric conformation may be important for toxicity. If so, we would anticipate that these conformations are distinct from the endogenous tetramers posited by Dettmer and colleagues (35). Although the current study focused on posttranslationally modified wild-type α-synuclein in the context of idiopathic (sporadic) PD, it will be of interest to examine in future studies the impact of mutant α-synuclein, and posttranslational modifications thereof, on mitochondrial protein import.

To determine the relevance of our findings to the human disease, we examined postmortem brain specimens from controls and individuals who died with PD. Just as in rats treated with rotenone or injected with an AAV overexpression vector, we found that nigrostriatal dopamine neurons from PD cases showed a marked increase in the α-synuclein–TOM20 PL signal compared to controls. Similarly, this α-synuclein–TOM20 PL signal was associated with a relative loss and cytosolic redistribution of the endogenous, imported complex I subunit, Ndufs3. Notably, another recent study reported a large loss of a presequence-containing imported protein (Ndufb8) relative to a mitochondrially encoded protein (COX1) in nigral neurons in PD (36). This discovery of an apparent protein import defect in human PD adds to the list of findings that have been predicted by the rotenone rat model of PD (13, 37, 38).

The results of this study may also shed light on the basis of the selective vulnerability of nigrostriatal neurons to degeneration in PD. At baseline, these cells express more SNCA mRNA than surrounding midbrain neurons, and they exist in a higher state of oxidation (17, 30). We found that these neurons do not show the normal TOM20-TOM22 interaction seen in other neurons (and presumably needed for efficient protein import); however, when endogenous α-synuclein was knocked down in vivo, the TOM20-TOM22 PL signal emerged. Furthermore, the knockdown of endogenous α-synuclein decreased the oxidation state of these neurons. Thus, under basal conditions, it appears that endogenous α-synuclein levels may be sufficient to affect protein import function; this situation is likely to be exacerbated in aging as α-synuclein accumulates in these cells (16). Moreover, elevated α-synuclein increased the redistribution of dopamine from vesicles to the cytosol (39) where it may modify α-synuclein. Dopamine-modified α-synuclein is particularly potent at inhibiting mitochondrial protein import.

Although the results of our studies are unanticipated and provide new insights into pathogenesis, there are limitations and unknowns as well. For example, thus far, we have not identified the structural characteristics that define the toxic species of α-synuclein, although a trimeric or tetrameric structure may be important. We also found that nitrated and fibrillar species of α-synuclein were essentially inert in our system; however, this does not exclude the possibility that they might exert toxicity by other mechanisms. Moreover, our study focused on wild-type α-synuclein, so we do not know yet how pathogenic α-synuclein mutations affect mitochondrial protein import. Additionally, many of our experiments used cell lines rather than primary neurons for technical reasons; however, key aspects of our findings were verified ex vivo using rat or human brain tissue. Finally, although we showed that mitochondrial protein import impairment caused cellular toxicity in forms of reduced respiration, oxidative damage, and mitochondrial depolarization, the relative extent to which this specific mechanism contributes to the overall neurotoxicity of α-synuclein remains to be determined.

Despite these caveats, the results presented here have several important therapeutic implications. First, together with the recent report by Zharikov et al. (17), they suggest that even the modest reduction of endogenous α-synuclein may have beneficial effects on mitochondrial function and nigral redox state—and may be neuroprotective in PD. Given that multiple posttranslational modifications render α-synuclein toxic to mitochondria, it appears that strategies aimed at a general reduction of α-synuclein, rather than targeting specific modifications or aggregation states, may be most efficacious.

Additionally, we found that overexpression of TOM20 prevented α-synuclein–induced impairment of mitochondrial protein import, as well as its downstream consequences, such as respiratory defects, ROS production, and loss of ΔΨm. The mechanism by which a moderate (two- to threefold) increase in TOM20 protein levels is protective is not yet clear, but preliminary studies indicate that TOM20-overexpressing mitochondria have a faster initial rate of protein import. This result is consistent with previous work showing that TOM20 overexpression can increase import (40). Additionally, however, we found that mitochondria isolated from TOM20-overexpressing cells and assayed in vitro were resistant to the inhibitory effects of the otherwise toxic species of α-synuclein. In this context, it will be of interest to determine whether overexpression of TOM20 in vivo is protective in models of PD.

Finally, contrary to our expectations, overexpression of a naked MTS had beneficial effects. We had anticipated that expression of the MTS would be similar to α-synuclein, competing for TOM20 presequence binding sites and inhibiting import. However, whereas the MTS blocked the α-synuclein–TOM20 PL signal as expected, it also preserved the mitochondrial import of the endogenous Ndufs3 subunit. In addition, MTS overexpression was able to prevent or even reverse the loss of the TOM20-TOM22 interaction induced by α-synuclein. As a consequence, overexpression of the naked MTS prevented downstream toxic effects of α-synuclein, such as reduced respiration, increased ROS production, and mitochondrial depolarization. On this basis, examination of an MTS peptide as a neuroprotective strategy is warranted.

In conclusion, we have defined a mechanism by which α-synuclein impairs a critical mitochondrial function, protein import, and have shown that this likely occurs in human PD. Our findings suggest new therapeutic strategies that should be tested in future studies.

MATERIALS AND METHODS

Study design

This study was designed to determine the potential role of various species of wild-type α-synuclein on mitochondrial protein import and downstream mitochondrial function. For this purpose, we used several in vivo manipulations, including rotenone-treated rats and rats with AAV2-mediated knockdown or overexpression of α-synuclein in the substantia nigra. To confirm relevance to the human disease, we examined measures of mitochondrial protein import and binding of α-synuclein to TOM20 in postmortem human brain tissue. Additional mechanistic experiments were conducted in intact cells (HEK293 and SH-SY5Y) and isolated mitochondria from brains and cultured cells. Most experiments used recombinant α-synuclein in the following forms: monomeric, oligomeric, dopamine-modified, S129E phosphomimetic mutant, nitrated, and fibrillar. Mitochondrial protein import was assessed by (i) direct import assays in isolated mitochondria, (ii) assessment of “mtGFP” uptake in transfected cells, and (iii) localization of endogenous presequence (MTS)–containing proteins in cells or brain sections. The binding of α-synuclein species to TOM20 was assessed by PL in intact cells or in brain sections and in vitro by fluorescence spectroscopy using recombinant proteins. In response to different species of α-synuclein, mitochondrial functions were assessed in intact cells by measurement of respiration (Seahorse), ΔΨm (TMRM fluorescence), and ROS production (thiol staining). Finally, to test potential protective therapeutic strategies, cells were transfected to overexpress TOM20 or the COX8 presequence (MTS).

α-Synuclein expression and purification. hSNCA complementary DNA (cDNA) (wild type or S129E) was transfected into BL21-DE3 Escherichia coli, and α-synuclein was purified as described by Volles and Lansbury (41).

α-Synuclein modification

For oligomers, monomeric α-synuclein was diluted to a concentration of 5 mg/ml and shaken at ~300 to 500 rpm at 37°C for 3 days. Treatment for dopamine modification was carried out as described by Martinez-Vicente et al. (23). For nitration, monomeric α-synuclein was diluted to a concentration of 5 mg/ml, and 50 μl of 1% tetranitromethane was added per 500 μl of α-synuclein solution. The solution was vortexed twice for 10 min. For S129 phosphomimetic, S129E mutant SNCA cDNA was transfected into E. coli as described above. All samples (monomers, oligomers, dopamine-modified, nitrated, and S129E) received the following treatment after modification: dialysis in the dark in 4 liters of phosphate-buffered saline with gentle stirring overnight (10-kD molecular weight cutoff). After dialysis, all samples were spun at 14,000g for 5 min to pellet any fibrils that formed. The supernatant contained soluble oligomers and monomers as confirmed by SDS-PAGE, and the pellet contained fibrils. Samples were prepared fresh weekly.

Proximity ligation assay. PLA was performed in 4% paraformaldehyde–fixed tissue or cells. Samples were incubated with specific primary antibodies to the proteins to be detected. Secondary antibodies conjugated with oligonucleotides were added to the reaction and incubated. Ligation solution, consisting of two oligonucleotides and ligase, was added. In this assay, the oligonucleotides hybridize to the two PLA probes and join to a closed loop if they are in close proximity. Amplification solution, consisting of nucleotides and fluorescently labeled oligonucleotides, was added together with polymerase. The oligonucleotide arm of one of the PLA probes acts as a primer for “rolling-circle amplification” (RCA) using the ligated circle as a template, and this generates a concatemeric product. Fluorescently labeled oligonucleotides hybridize to the RCA product. The PL signal was visible as a distinct fluorescent spot and was analyzed by confocal microscopy (Duolink, Sigma-Aldrich). Control experiments included routine immunofluorescence staining of proteins of interest under identical experimental conditions.

Fluorescence measurements. Quantitative fluorescence measurements were made with an Olympus upright three-laser scanning confocal microscope, taking care to ensure that images contained no saturated pixels. For quantitative comparisons, all imaging parameters (for example, laser power, exposure, and pinhole) were held constant across specimens.

Statistical analyses

Each result presented here was derived from three to six independent experiments. For simple comparisons of two experimental conditions, two-tailed unpaired t tests were used. Where variances were not equal, Welch’s correction was used. When virus was injected into one hemisphere of the brain and the other hemisphere was used as a control, two-tailed paired t tests or Wilcoxon matched-pairs signed-rank tests were used. For comparisons of multiple experimental conditions, one- or two-way ANOVA was used, and if significant overall, post hoc corrections (Bonferroni or Sidak) for multiple pairwise comparisons were made. P values less than 0.05 were considered significant. All bar graphs show means ± SEM.

SUPPLEMENTARY MATERIALS

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Materials and Methods

Fig. S1. Amounts of α-synuclein and S129-phosphorylated α-synuclein in vivo.

Fig. S2. Positive and negative PL interactions with α-synuclein in substantia nigra pars compacta.

Fig. S3. Rotenone induces a loss of mitochondrial localization of the nuclear-encoded, imported protein Ndufs3.

Fig. S4. All species of α-synuclein used in this study enter cells to an equivalent extent, and when added at 200 nM, they do not change intracellular concentrations of α-synuclein or its localization.

Fig. S5. Fibrillar α-synuclein does not affect mitochondrial protein import.

Fig. S6. Overexpression of TOM20 and TOM5.

Fig. S7. Time course of isolated brain mitochondrial protein import in the absence or presence of monomeric and oligomeric α-synuclein.

Fig. S8. Lack of mitochondrial depolarization by α-synuclein during import assays.

Fig. S9. Effects of α-synuclein on import and localization of other mitochondrial proteins.

Fig. S10. The α-synuclein–TOM20 PL signal colocalizes with mitochondria.

Fig. S11. Downstream effects of α-synuclein on mitochondria are blocked by MTS overexpression.

Fig. S12. The protective effects of TOM20 overexpression on mitochondrial protein import can be overcome by increased concentrations of α-synuclein.

Fig. S13. Structural analysis of the α-synuclein species used in this study.

REFERENCES AND NOTES

  1. Acknowledgments: We thank H. Yano for the pGEM-3Zf(+)-pOTC plasmid. Funding: This work was supported by research grants from the DSF Charitable Foundation, the Ri.MED Foundation, the Consolidated Anti-Aging Foundation, NIH (NS095387, NS059806, ES022644, ES020718, ES020327, NS065789, AG026389, and P50AG005133), the U.S. Department of Veterans Affairs (1I01BX000548), the Blechman Foundation, the American Parkinson Disease Association, and the Department of Biotechnology, Government of India. Author contributions: R.D.M. and P.J.B. designed, performed, and analyzed the PL and protein import experiments and edited the manuscript; E.K.H. was responsible for molecular biology and created and validated cell lines; C.W.B. also did some of the protein import experiments; J.M. did some of the PL experiments; A.Z. and A.B. did in vivo gene transfer experiments; X.H. was responsible for cell culture experiments; C.T.C. supervised human neuropathological studies; E.A.B. and T.G.H. designed and analyzed the experiments and edited the manuscript; and J.T.G. supervised the project, designed and analyzed the experiments, and wrote the paper. Competing interests: The authors declare that they have no competing interests. J.T.G. is a paid consultant for Biogen and FORMA Therapeutics.
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