Reduced TOM40 protein levels associated with α-Syn accumulation are independent of transcriptional regulation.
The accumulation of α-Syn within mitochondria has been frequently linked to mitochondrial dysfunction. This includes depolarization of the mitochondrial membrane, reduced activity of OXPHOS complexes, and mtDNA damage. Previous research has also associated α-Syn accumulation in PD brains and transgenic mice expressing α-Syn with decreased TOM40 protein levels, a key component of the mitochondrial outer membrane translocase complex 27. Despite these observations, the functional relationship between α-Syn accumulation and TOM40 reduction had not been explored until now. To investigate the underlying mechanism behind TOM40 reduction, we assessed TOM40 protein levels in postmortem brain tissue samples from Guam PD, Guam ALS, and Guam control patients obtained from the Binghamton University Biospecimen Archive34. Immunoblot analysis showed a specific decline in TOM40 but not TOM20 (Fig. 1A, Lns 4-7) as well as increased α-Syn aggregates (Supplementary Fig. 1, Lns 3-4) in Guam PD brain tissue. No TOM40 protein level reduction was observed in Guam non-neurological controls or ALS samples (Fig. 1A, Lns 1-3, 8-10). Patient demographic details are provided in Supplementary Table 1. To test the possibility of transcriptional regulation behind TOM40 reduction, we measured TOM40 and TOM20 mRNA levels in Guam-PD postmortem brain tissues by qRT-PCR (Fig. 1B). The results showed that TOM40 mRNA level normalized to TOM20 mRNA level did not differ, indicating that TOM40 degradation occurs post-translationally. To investigate this further, we utilized a PD patient-derived SNCA-tri line containing four functional SNCA gene copies, three mutant alleles, and one wildtype allele (Fig. 1C). Notably, despite having a sixfold higher α-Syn mRNA level, the SNCA-tri line exhibited stable TOM40 mRNA levels compared to the control (Fig. 1D), but reduced TOM40 protein level compared to the control line (Fig. 1E, Ln 2). Additionally, downregulating α-Syn in the SNCA-tri line resulted in increased TOM40 protein levels (Fig. 1F, Ln 2) but did not lead to any changes in the mRNA levels (Fig. 1G).
Factors inducing α-Syn oligomerization exacerbate TOM40 loss.
In normal cells, α-Syn predominantly resides in the cytoplasm, but redistributes to the mitochondria under oxidative stress, with its impact on outer mitochondrial membrane (OMM) proteins yet to be explored 2, 44, 45. To investigate the impact of α-Syn accumulation under oxidative stress on OMM proteins, we evaluated the effects of PD-associated neurotoxins and pro-oxidant metals—known for generating mitochondrial and cytosolic ROS and fostering α-Syn aggregation in PD models—on the levels of TOM40 and TOM20 (Fig. 2A) 11, 46, 47. Immunoblot analysis revealed a significant decrease in TOM40 levels in SH-SY5Y cells overexpressing ectopic α-Syn and exposed to 6OHDA (Fig. 2A, Ln 3), GO (Fig. 2A, Ln 4), FeCl3 (Fig. 2A, Ln 6), or FeSO4 (Fig. 2A, Ln 8) treatments, while TOM20 levels remained unaltered. Interestingly, the reduction in TOM40 levels correlated strongly with decreased α-Syn monomer levels and an increase in α-Syn oligomer formation (Fig. 2A, Lns 3-8), as indicated by high-mobility bands in the immunoblots probed with the α-Syn antibody.
Prompted by reports indicating reduced viability and stress resistance in comparable cell lines exposed to toxicants such as rotenone and 6OHDA, we conducted a thorough examination to assess the impact on TOM40 levels in PD patient-derived NPSCs harboring SNCA gene triplication 48, 49. The reduction in TOM40 protein levels observed in 6OHDA-treated NPSCs, particularly in the SNCA-tri line (Fig. 2B, Lns 3, 6), mirrored the observations in SH-SY5Y cells overexpressing α-Syn. Importantly, exposure to rotenone did not affect TOM40 levels in these cell models (Fig. 2B, Lns 2, 5), suggesting that TOM40 degradation is more susceptible to cytoplasmic ROS inducers than mitochondrial ones. Furthermore, Mitotracker-PLA immunofluorescence studies indicated an enhanced interaction between α-Syn and TOM20 (Fig. 2C, Supplementary Fig. 2), consistent with prior findings of α-Syn's mitochondrial translocation under oxidative stress 44. Altogether our results provide new insights into how oxidative stress, coupled with α-Syn accumulation, impacts TOM protein activities and levels in PD.
These findings also led us to delve deeper into understanding the mechanisms underlying synthesis-induced TOM40 dysfunction and to develop strategies to counteract the perturbations in TOM40 as potential therapeutic avenues for PD.
Mitochondrial localization of α-Syn and its influence on TOM40 levels.
While α-Syn does lack a conventional mitochondrial targeting signal (MTS), its presence in different mitochondrial compartments raises questions about its import mechanism into the mitochondria. Previous studies have shown that α-Syn’s N-terminal 32 amino acids are essential for its mitochondrial translocation, a process that is likely mediated through interaction with TOM40 2. To identify the residues contributing to this interaction, we conducted molecular docking studies using the AutoDock CrankPep protein-peptide docking method 50. Since the program reliably docks peptides raging in lengths from 16 to 20 aminoacids, we divided α-Syn’s 140 aminoacid sequence into 20 aminoacid fragments using the micelle-bound human α-Syn (PDB ID: 1XQ8) as a reference structure (Fig. 3A). The docking results using the TOM40 structure (PDB ID: 7CK6) as the receptor revealed that α-Syn fragment (21-40) exhibited the highest predicted binding affinity for TOM40 residues (Fig. 3B). Notably, α-Syn C-terminal residues demonstrate a lower binding affinity. Overall, these docking results suggest that α-Syn N-terminal residues are crucial for interacting with TOM40.
Building on the docking analysis and existing literature, we designed two inducible expression vectors: one containing a mitochondrial targeting signal (pCW MTS-α-Syn-Flag) and another with a 33 amino acid deletion of α-Syn's N-terminal region (pCW Δ1-33-α-Syn-Flag) (Fig. 3C). These vectors were used to establish stable SH-SY5Y cell lines as a proof-of-concept model to test whether inhibition of α-Syn translocation into the mitochondria affects TOM40 levels or if targeted mitochondrial accumulation of α-Syn exacerbates TOM40 loss. Utilizing these inducible SH-SY5Y lines enables post-differentiation induction of α-Syn expression, preventing non-physiological α-Syn levels during the differentiation process, known to impact neuronal differentiation, resulting in poor neuronal morphology and shorter neurites 51. Western blot analysis of Δ1-33 α-Syn SH-SY5Y cells showed no significant alteration in TOM40 protein levels (Fig. 3C, Lns 10-12). On the other hand, targeting the expression of α-Syn into the mitochondria led to an enhanced reduction in TOM40 levels after 24 hours of induction (Fig. 3C, Lns 6-8), while TOM20 protein levels exhibited no alterations despite the enforced expression of α-Syn in the mitochondria. No significant changes in TOM40 mRNA levels were observed at any tested time points during α-Syn induced expression (Supplementary Fig. 3).
Investigating the impact of oligomeric forms of α-Syn, especially small soluble α-Syn aggregates, is crucial for understanding key pathological aspects of PD development 52. Using the anti-oligomer A11 polyclonal antibody, we detected the accumulation of α-Syn oligomers (>25 kDa) in a dose-dependent manner upon induction of α-Syn expression, with MTS-α-Syn expressing cells showing a more pronounced accumulation (Fig. 3D, Lns 6-8). These results align with the reduced TOM40 levels observed in (Fig. 3C, Lns 6-8), suggesting a potential association between α-Syn oligomeric forms and TOM40 loss. Immunofluorescence images (Fig. 3E) confirmed the mitochondrial accumulation of ectopic MTS-α-Syn. Finally, PLA analysis demonstrated increased interaction of WT and MTS-α-Syn with TOM20 (Fig. 3F).
Overall, these findings suggest that TOM40 loss in the context of Guam-PD pathology is influenced by two key factors: α-Syn's interaction with OMM proteins via its N-terminal residues, and accumulation of α-Syn oligomers, particularly at the mitochondria.
Proteasomal degradation as a key mechanism in α-Syn-induced TOM40 loss.
In our investigation into the mechanisms underlying TOM40 loss in response to α-Syn accumulation, we explored three pathways (Fig. 4A) associated with the selective degradation of OMM proteins: the ubiquitin-proteasome system (UPS), mitochondrial-derived vesicles (MDVs), and mitophagy 53, 54, 55. Our findings revealed a significant stabilization of TOM40 protein levels in the presence of MG132 (Fig. 4B, Ln 3 red arrow), implicating the proteasomal degradation pathway in the regulation of TOM40 under α-Syn accumulation. Stabilization of TOM40 levels was unique to the proteasome inhibition and was not replicated with the other inhibitors, underscoring the specificity of the proteasomal degradation in this context.
We further assessed the effectiveness of these inhibitors by observing a notable increase in microtubule-associated protein 1A/1B-light chain 3B (LC3B). Elevated LC3B levels were consistently observed across all tested inhibitors compared to vehicle-treated α-Syn expressing cells, with notable effects following MG132 and BafA1 treatments (Fig. 4B, Lns 3, 4). The observed decrease in LC3B levels in α-Syn expressing cells (Fig. 4B, Ln 2) aligns with the lower LC3B levels detected in transiently overexpressed α-Syn in neuronal cells and the cerebrospinal fluid of PD patients, suggesting an impairment in autophagy 56, 57.
In α-Syn expressing cells treated with MG132, the simultaneous increase in TOM40 and LC3B, in particular, suggests the activation of autophagy as a compensatory mechanism, in line with findings from other studies showing autophagy induction by MG132 58, 59. However, this observation indicates that neither TOM40 nor α-Syn undergo degradation through this compensatory autophagic mechanism. Conversely, BafA1 inhibits the later stages of autophagic flux, as evidenced by high LC3B levels, but interestingly, it does not prevent TOM40 degradation or α-Syn accumulation. Together, these findings not only identify the involvement of proteasomal degradation pathway in α-Syn-induced TOM40 loss, but also provide crucial insights into the understanding of the physiological mitochondrial quality control mechanisms, especially in neurodegenerative diseases where protein misfolding and aggregation are prevalent.
Impact of α-Syn pathology on mitochondrial genome integrity.
To assess the impact of α-Syn pathology on mitochondrial genome integrity, we used long amplification (LA-PCR) analysis. Separation of PCR amplicons in 1% agarose gel electrophoresis (Fig. 5A) revealed a significant reduction in mtDNA amplification in cells overexpressing WT α-Syn (Fig. 5A, Ln 2) and MTS α-Syn (Fig. 5A, Ln 3) compared to control (Fig. 5A, Ln 1) or Δ1-33 α-Syn (Fig. 5A, Ln 4) cells. Quantitation of PCR products using an independent, highly sensitive PicoGreen based plate reader method confirmed a marked reduction in mtDNA integrity in WT and MTS α-Syn overexpressing cells (Fig. 5B). The reduction in LA-PCR products is indicative of PCR DNA polymerase blocking lesions in DNA, predominantly involving DNA strand breaks, as well as DNA crosslinks and bulky base damages. Therefore, reduction in mtDNA amplification suggests that residues located in the N-terminal region not only facilitate α-Syn's translocation into the mitochondria but also contribute to its adverse effects on mtDNA integrity. Consequently, averting the buildup of mitochondrial α-Syn by N-terminal deletion not only safeguards TOM40 levels but also preserves the integrity of mtDNA.
To gain deeper insights into the potential link between mtDNA instability and α-Syn accumulation, we conducted mitochondrial DNA sequencing to quantify insertions, deletions, and mutations in WT α-Syn cells. Our analysis identified several unique mutations in the ND2, COX1, ND4, and ND5 genes. The severity assessment using the PolyPhen-2 online tool revealed the impact of these mutations (Fig. 5C). A comparison between Dox (-) and Dox (+) WT α-Syn cells revealed a total of 148 mutations with a rate of 8.9 mutations per 1kb in Dox (-), while 167 mutations were present in Dox (+) with a mutation rate of 10 mutations per 1kb. Specifically, 26 unique mutations were reported in Dox (+) WT α-Syn cells mitochondria at a rate of 1.5 mutations per 1kb. Analyzing the distribution of mutations in the coding region of the mitochondrial genome in Dox (+) WT α-Syn cells, we found varied rates across different genes: ND1 (2.1/kb), ND2 (1.9/kb), COX1 (1.9/kb), COX3 (1.2/kb), ND3 (2.8/kb), ND4 (0.72/kb), ND5 (2.1/kb), ND6 (3.9/kb), and Cytb (0.8/kb).
Furthermore, we assessed mtDNA integrity in brain tissue samples from Guam PD patients exhibiting α-Syn pathology and compared them to three non-neurological controls using LA-PCR. Both agarose gel electrophoresis densitometry analysis (Fig. 5D, Lns 4-7) and independent PicoGreen based quantitation of mtLA 7601-16401 PCR amplicon (Fig. 5E) revealed a significant increase in mtDNA damage within the Guam PD patient samples, contrasting with Guam non-neurological controls (Fig. 5D, Lns 1-3). Altogether these findings emphasize a distinct correlation between mtDNA instability and α-Syn proteinopathy.
TOM40 supplementation partially counters α-Syn-induced defects in mitochondrial bioenergetics.
Considering the impact of α-Syn on cellular health, particularly focusing on cell viability (Fig. 6A) and mitochondrial membrane potential (Fig. 6B), we aimed to assess the functional consequences of overexpressing WT α-Syn in the absence or presence of ectopic TOM40 supplementation. Utilizing the Seahorse XFe96 analyzer, we conducted a real-time assessment of mitochondrial respiratory function, quantifying oxygen consumption rates (OCR) to measure the mitochondrial bioenergetic efficiency.
Our findings reveal that WT α-Syn overexpression in SH-SY5Y cells, without concurrent TOM40 supplementation, significantly diminishes OCR alongside all tested parameters of respiratory function (Fig. 6C). This reduction highlights a clear compromise in mitochondrial efficiency attributable to α-Syn overexpression. In contrast, cells overexpressing WT α-Syn and supplemented with TOM40 expression exhibited a substantial enhancement in OCR (Fig. 6C), indicating an improvement in mitochondrial bioenergetics. This improvement extended across several respiratory parameters, including basal respiration (Fig. 6D), ATP production (Fig. 6E), and maximal respiration (Fig. 6F), where we observed significant improvements. Notably, non-mitochondrial oxygen consumption also showed marked enhancement (Fig. 6I).
However, the spare respiratory capacity, which acts as a buffer during increased energy demand, remained adversely affected in cells with elevated α-Syn levels, indicating that TOM40 supplementation, while beneficial, could not completely mitigate this specific deficit (Fig. 6G). Furthermore, our results showed that proton leak rates were not significantly altered by either α-Syn overexpression or TOM40 supplementation (Fig. 6H), suggesting that some aspects of mitochondrial function remain unaffected by these modifications.
Collectively, these outcomes underscore that while TOM40 supplementation does not completely reverse the mitochondrial impairments induced by α-Syn overexpression, it does significantly ameliorate several key aspects of mitochondrial bioenergetics. This partial but meaningful recovery suggests a promising avenue for addressing mitochondrial deficits linked to α-Syn pathology, while also suggesting a need for additional strategies to mitigate such deficits.
PARP inhibition as a strategy to restore TOM40 levels and mitigate α-Syn toxicity
Building upon recent findings that suggest that PARP inhibition promotes the degradation of α-Syn aggregates via the autophagy-lysosomal pathway in PD models 33, here, we explored the potential of PARP inhibition in countering α-Syn oligomers-mediated TOM40 protein loss resulting from exposure to 6OHDA and elevated α-Syn expression. To assess whether a PARP inhibitor (PARPi) treatment can restore TOM40 levels after 6OHDA treatment, we treated control NPSCs with 6OHDA, followed by replacement of fresh media supplemented with or without PARP inhibitor Veliparib. Veliparib, chosen for its established efficacy in inhibiting PARP activity and ability to cross the blood-brain barrier 60, led to a significant restoration of TOM40 protein levels compared to untreated cells (Fig. 7A, Ln 2 vs 3). The efficacy of Veliparib was further supported by reduced Poly/Mono-ADP ribosylated (ADP-R) proteins and decreased α-Syn protein levels following treatment, consistent with previous reports 31, 33. These findings not only validate Veliparib's successful inhibition of PARP, but also propose a mechanism by which PARP inhibition contributes to TOM40 stabilization by reducing pathological α-Syn forms.
Finally, we compared the effectiveness of TOM40 supplementation and PARPi treatment on the viability of two PD in vitro models: the 6OHDA-induced (Fig. 7B) and α-Syn overexpression model (Fig. 7C). Interestingly, both TOM40 supplementation and PARPi treatment demonstrated similar efficacy in enhancing cell viability. This finding is particularly significant as it highlights the potential of two independent therapeutic pathways for mitigating the deleterious effects of α-Syn-induced mitochondrial dysfunction and subsequent cell death (schematically illustrated in Fig. 7D, a and b).