The etiology of Parkinson’s disease (PD) converges on a common pathogenic pathway of mitochondrial defects in which α-Synuclein (αSyn) is thought to play a role. However, the mechanisms by which αSyn and its disease-associated allelic variants cause mitochondrial dysfunction remain unknown. Here, we analyzed mitochondrial axonal transport and morphology in human-derived neurons overexpressing wild-type (WT) αSyn or the mutated variants A30P or A53T, which are known to have differential lipid affinities. A53T αSyn was enriched in mitochondrial fractions, inducing significant mitochondrial transport defects and fragmentation, while milder defects were elicited by WT and A30P. We found that αSyn-mediated mitochondrial fragmentation was linked to expression levels in WT and A53T variants. Targeted delivery of WT and A53T αSyn to the outer mitochondrial membrane further increased fragmentation, whereas A30P did not. Genomic editing to disrupt the N-terminal domain of αSyn, which is important for membrane association, resulted in mitochondrial elongation without changes in fusion-fission protein levels, suggesting that αSyn plays a direct physiological role in mitochondrial size maintenance. Thus, we demonstrate that the association of αSyn with the mitochondria, which is modulated by protein mutation and dosage, influences mitochondrial transport and morphology, highlighting its relevance in a common pathway impaired in PD.
Parkinson’s disease (PD), the second most prevalent neurodegenerative disorder, is pathologically characterized by progressive neuronal loss and the accumulation of eosinophilic intracellular inclusions, termed Lewy bodies1. The major component of these inclusions is α-Synuclein (αSyn)2, the first protein linked with dominant familial PD3. So far, more than 20 loci are known to contribute to familial PD, many of which are implicated in the regulation of mitochondrial homeostasis4, 5. Although genetic mutations account for a small proportion of PD cases, there is both pathological and pharmacological evidence to support a key role for mitochondrial dysfunction in the progression of sporadic PD6, 7. Recent evidence supports a direct link between αSyn function and mitochondrial pathologies8,9,10; however, the precise molecular mechanisms by which αSyn may induce mitochondrial defects remain unclear.
Mitochondrial-related proteins govern both fusion-fission rates and transport, leading to a direct effect on mitochondrial size and homeostasis. Impairments in these processes have been associated with many neurodegenerative diseases11. In PD, familial mutations in PTEN-induced putative kinase 1 (PINK1), Parkin, DJ-1, leucine-rich repeat kinase (LRRK), ATP13A2, and recently, vacuolar protein sorting-associated protein 35 (VPS35) have been found to control mitochondrial size and transport through interaction with fusion-fission proteins and molecular motors12,13,14,15,16,17,18.
Patients carrying αSyn gene (SNCA) triplications display earlier disease onset compared with patients carrying SNCA duplications, indicating that intracellular levels of αSyn determine the development of pathology in a dose-dependent manner19, 20. In addition to the aggregation properties of αSyn, in vitro membrane-binding studies suggest that αSyn adopts an α-helix conformation at the N-terminal domain when interacting with lipids21. Disruption of this conformation via the A30P αSyn mutation leads to a reduced affinity for lipids, whereas the A53T αSyn mutation produces the opposite effect22. This difference in lipid affinity correlates with an earlier onset and more severe manifestations in patients with A53T mutation, while A30P induces late age onset and milder symptoms23, 24. However, it is unclear how these two distinct mutations, which have opposing effects on membrane affinity, result in mitochondrial defects.
Differential associations of αSyn mutants with mitochondria have been proposed, with some studies reporting the presence of αSyn inside the mitochondria, whereas others report localization to the outer mitochondrial membrane (OMM)25,26,27. Interestingly, changes in mitochondrial morphology were described as common features in several animal models of αSyn mutation or overexpression28,29,30 as well as in human cellular models26, 31. Moreover, mitochondrial fission has been linked to αSyn function29, 31. However, robust evidence demonstrating a direct regulation of mitochondrial size by αSyn-dependent mechanisms in human neurons is lacking.
Here, we sought to determine whether αSyn protein plays a direct role in the regulation of mitochondrial homeostasis, both under physiological and pathological conditions, in a human cell model. Using human neurons derived from embryonic stem cells (hESC) or induced pluripotent stem cells (hiPSC), we studied mitochondrial axonal transport and morphology in the presence of overexpressed wild-type or mutant αSyn. αSyn overexpression was found to induce mitochondrial transport defects and fragmentation. Forced delivery of αSyn to the OMM resulted in a strong reduction in mitochondrial size, suggesting that αSyn variants can induce differential effects in a common pathological pathway. Moreover, disruption of the α-helix conformation in the N-terminal domain of αSyn in human neurons uncovered a physiological function for endogenous αSyn in the maintenance of neuronal mitochondrial size. Our findings, in a relevant human neuronal model, demonstrate a key role for αSyn in the development of mitochondrial pathology associated with PD, and highlight a potential target for early therapeutic intervention prior to neuronal loss and clinical manifestation of PD.
αSyn overexpression in hESC-derived neurons
To study the physiological and pathological roles of αSyn in mitochondrial dynamics within the context of human PD, we obtained enriched, polarized, and functional human neurons derived from hESC according to a previously described protocol (Figs 1A–F, S1A)32. The differentiation stages from pluripotent colonies to terminally differentiated neurons were evaluated by quantification of mRNA and protein expression levels. Pluripotent markers (OCT4, SOX2, Nanog) were present in the undifferentiated stages (Figure S1B) and downregulated at final differentiation, whereas neuronal markers, including βIII tubulin, pan-NCAM, and tyrosine hydroxylase (TH), appeared upregulated in differentiated neurons (Figs 1D, S1B and S1C). Imaging of differentiated neurons indicated conspicuous expression of βIII tubulin but weak expression of nestin (Fig. 1A). A diffuse cytosolic pattern of endogenous αSyn was observed, with punctate distribution among neurites labeled with p-Tau (Fig. 1B and C). Furthermore, enriched neuronal cultures (55.17%+/− 2.2; Figure S3A) showed a time-dependent maturation of tau isoform splicing33 and progression of electrophysiological profiles, including repetitive spiking and increased amplitude of voltage-dependent Na+ and K+ currents (Figs 1E,F and S2), recapitulating key hallmarks in neuronal development. To examine the role of αSyn in mitochondria, neurons were transfected with pLV-CMV-αSyn vectors encoding wild-type (WT) αSyn or the familial PD mutations, A30P or A53T (Fig. 1G). Based on electrophysiological profile and robust neuronal marker expression indicative of neuronal differentiation, transfections were performed on day 14 in vitro (DIV14). Increased expression of αSyn was observed in transfected p-Tau-labeled neurons (Fig. 1H). To distinguish transfected αSyn from endogenous protein, neurons co-transfected with vectors encoding EGFP and WT, A30P, or A53T αSyn were analyzed by immunofluorescence (Fig. 1I). Quantification of αSyn fluorescent intensity revealed a 2- to 3-fold increase in αSyn signal in transfected neurons (WT, A30P, or A53T) compared with endogenous αSyn expression (Fig. 1J), characterizing αSyn overexpression profiles in our neuronal model. αSyn overexpression was further validated by flow cytometric analysis of neurons transfected with vectors encoding mCherry and WT, A30P, or A53T αSyn (Figure S3B). No neurotoxic effects were observed in response to overexpression of any of the αSyn variants (Figs 1K, S3C).
αSyn overexpression induces mitochondrial axonal transport defects
Neuronal homeostasis is reliant on the appropriate subcellular distribution of organelles, including mitochondria18, 34. αSyn expression has been shown to influence axonal transport, which in turn affects mitochondrial fusion-fission rates13, 17, 35, 36. To test the effects of αSyn overexpression on axonal transport of mitochondria in our human-derived neuronal model, we performed live-imaging analysis of cells co-transfected with a vector driving GFP fused to a mitochondrial-targeting sequence (mito-GFP) in addition to WT, A30P, or A53T αSyn-expressing vectors (Figs 2A, S4A). Time-lapse movies from transfected axons were transformed to kymographs to track and quantify mitochondrial movement (Fig. 2B, Movies M1–4). Mitochondrial transport from WT, A30P, or A53T αSyn (Fig. 2C) showed similar overall anterograde, stationary, and retrograde mitochondrial proportions when compared with control (mCherry-transfected) neurons (Fig. 2D). However, detailed analysis of mitochondrial kinetics, by discriminating segmental velocities and time during movement (Fig. 2E and F), revealed a difference in control neurons, which displayed a significant bias to anterograde flux compared with retrograde flux (Fig. 2G). This asymmetry, which is important for maintenance of distal distribution of axonal mitochondria, was lost in αSyn-overexpressing cells (Fig. 2G). WT and A53T αSyn-overexpressing neurons showed similar anterograde and retrograde mitochondrial segmental velocities (Fig. 2F), and A53T αSyn overexpression also resulted in similar time spent moving in retrograde and anterograde directions (Fig. 2E). These results reveal impairments in mitochondrial flux in transfected axons, with the most pronounced effect in A53T αSyn-overexpressing neurons, indicating that αSyn overexpression leads to an imbalance in the distribution of mitochondria throughout the axon.
To test whether mitochondrial flux impairments are related to an αSyn effect on mitochondrial homeostasis, we assessed mitochondrial membrane potential in neurons transduced with WT, A30P, or A53T pLV-CMV-αSyn (Figs 2H, S4B). JC1, a mitochondrial probe that changes its emission wavelength when oligomerized under hyperpolarizing membrane potential, was used to assess mitochondrial function. Colocalization analysis using Manders’ correlation coefficient for red versus green intensities showed no significant changes in mitochondrial membrane potential in any of the αSyn-overexpressing neurons (Figs 2H and S4C). Our results indicate that axonal flux impairments observed upon αSyn overexpression are not caused by depolarization of the mitochondria; thus, the effects seen are unlikely to be the result of short-term effects on mitochondrial health.
Axonal mitochondrial fragmentation in neurons overexpressing A53T αSyn
To test whether the changes in mitochondrial flux observed upon αSyn overexpression in human neurons promote mitochondrial fragmentation, as previously described in both cellular and animal systems28,29,30,31, we analyzed the morphology of axonal mitochondria after co-transfecting human-derived neurons with mito-GFP and αSyn vectors (Fig. 3A). Control, WT, and A30P αSyn-overexpressing neurons showed similar mean mitochondrial lengths per axon. However, transfection with A53T αSyn resulted in a significant decrease in mitochondrial length and an increased number of mitochondria per axon (Fig. 3A). To confirm this result, an immunofluorescent staining approach using mitochondrial cytochrome C oxidase I (COX-I) was performed in neurons transfected with EGFP, WT, A30P, or A53T αSyn vectors. Again, A53T αSyn overexpression resulted in a significant reduction in the size of axonal mitochondria while no changes were observed upon WT or A30P αSyn overexpression (Fig. 3B). In addition, total axonal mitochondria exhibited a significant size decrease in A53T and WT αSyn-transfected neurons (Fig. 3C). To test whether levels of αSyn expression correlated with fragmentation phenotype, co-transfected neurons were evaluated for mitochondrial size and αSyn intensity (Fig. 3D). Interestingly, low αSyn-expressing neurons showed significant fragmentation only in A53T αSyn (Fig. 3D), whereas high αSyn-expressing neurons showed significant mitochondrial fragmentation phenotypes in A53T and WT αSyn, but no difference in A30P compared with control (GFP, Fig. 3D). These results suggest that different αSyn variants induce a dose-dependent mitochondrial fragmentation effect. We then considered whether mitochondrial size correlated with movement, given the observed changes in mitochondrial flux and the possibility that smaller mitochondria may be more prone to movement. However, no association between mitochondrial size and type of mitochondrial movement was observed (Fig. 3E).
To determine whether the effect of A53T αSyn on mitochondrial size was specific to human-derived neurons, A53T αSyn was overexpressed in mouse primary neuronal cultures and in human neural stem cells (hNSCs). Consistent with our earlier results, co-transfection of mito-GFP and A53T αSyn into mouse hippocampal neurons resulted in a significant reduction in axonal mitochondrial length when compared with control (Fig. 3F). A significant decrease in mitochondrial size was also evident in hESC-derived NSCs upon A53T αSyn overexpression (Figs 3G and S5). Three-dimensional structural reconstruction analysis of mitochondria from transfected hNSCs showed that A53T αSyn not only led to a significant size reduction (surface and mean distance to surface), but also resulted in alterations in morphology, with mitochondrial shape becoming highly spherical (standard deviation of distance, Fig. 3G). Moreover, total mitochondrial mass was unaltered, indicating a compensatory increase in mitochondrial number (Fig. 3G). Taken together, these results reveal that A53T αSyn impairs normal mitochondrial homeostasis, inducing mitochondrial fragmentation in both neuronal and non-neuronal cells.
Targeted mitochondrial delivery of WT and A53T αSyn, but not A30P, reduces mitochondrial size
The location of disease-associated αSyn mutations in the N-terminal domain of αSyn combined with the known biophysical interaction of this domain with lipid membranes led us to consider whether A53T αSyn might display enhanced membrane binding affinity22, 37. To determine whether αSyn variants differentially associate with the mitochondria, subcellular fractions (total, cytosolic, and mitochondrial) were obtained from a human neuroblastoma SH-SY5Y cell line transfected with WT, A30P, or A53T αSyn-expressing vectors (Fig. 4A). Quantification of αSyn subcellular distribution, calculated as the normalized mitochondrial/cytosolic ratio, showed a mild association of WT αSyn with the mitochondria, a reduction of A30P αSyn, and high enrichment in A53T αSyn when compared with control (Fig. 4A). Taken together, these results suggest that structural features of αSyn variants differentially determine their association with mitochondria and, consequently, the degree of mitochondrial defect.
To test whether the effect exerted by αSyn on mitochondrial size requires a direct association of αSyn with mitochondrial membranes, we designed a quantitative and time-dependent mitochondrial delivery experiment using the FKBP-FRB dimerization system38. WT, A30P, or A53T αSyn subcloned in frame to the FRB domain (WT, A30P, or A53T αSyn-FRB) dimerize with the FKBP domain in the presence of a heterodimerizing agent (rapalog). GFP and the actin assembly-inducing protein (ActA) fused to FKBP then target the dimerization complex to the OMM (Fig. 4B). GFP-FKBP-ActA mitochondrial localization and αSyn-FRB distribution with or without rapalog was assessed (Figs 4C, S6A and B). Two rapalog concentrations (200 and 500 nM) were tested to take into account dose-responsive effects. Consistent with our previous results, only A53T αSyn-FRB led to a significant reduction in mitochondrial size in the absence of rapalog (Figs 4D,E, and S6C). Rapalog incubation in A53T αSyn-FRB resulted in further reductions in the length of axonal mitochondria. Noteworthy, in the presence of 500 nM rapalog, WT αSyn-FRB led to a strong and significant reduction in the size of mitochondria similar to that seen with A53T αSyn-FRB (Figs 4D,E and S6C). In contrast, no effect on mitochondrial size in A30P αSyn-FRB neurons was observed even in the presence of the highest rapalog concentration (Fig. 4D). Consistent with this, probability density function analysis of mitochondrial size showed similarities between WT-FRB and A30P-FRB αSyn in the absence of rapalog, and between WT-FRB and A53T-FRB αSyn after 6-h incubation with 500 nM rapalog (Fig. 4F). Our results therefore strongly suggest a biophysical effect of αSyn on axonal mitochondrial fragmentation that requires a direct interaction with the OMM in neurons.
αSyn N-terminal domain integrity is necessary for the control of neuronal mitochondrial morphology
To test whether the αSyn N-terminal domain controls the shape and morphology of axonal mitochondria, we disrupted the integrity of the α-helix structure in hiPSC. Directed CRISP-R/Cas9 genome editing of Craig Venter’s hiPSC line (CV) was performed to generate isogenic lines with disruptions in αSyn conformation using a specific short guide RNA directed to the exon 2 sequence39 (Fig. 5A). A PCR strategy using αSyn exon 2-specific primers was used to screen for positive clones with genomic insertions (Fig. 5A). One putative hiPSC-CV CRISPR/Cas9-modified clone (MS06) showed two modified alleles, one with a nucleotide insertion that shifted the reading frame, and the other with a net 18-amino acid insertion maintaining the downstream frame and giving rise to a higher molecular weight αSyn (Figure S7). αSyn expression in human-derived neurons and neural stem cells (NSC) from the MS06 clone revealed an increase of 2 kDa in αSyn molecular weight compared with isogenic controls or mouse hippocampal neurons (Fig. 5B). This confirmed the stable modification of αSyn protein with an insertion disrupting the proximity of the two α-helix N-terminal domains but with intact downstream sequence. Neuronal differentiation, polarity acquisition, and αSyn expression levels were tested in neurons differentiated from clone MS06 and compared with control hiPSC-CV derived neurons (Fig. 5C). After 14 days in culture, control and CRISP-R/Cas9-modified MS06 neurons were transfected with mito-GFP to assess mitochondrial morphology. MS06 neurons were found to display elongated morphologies of axonal mitochondria (Fig. 5E). Quantification of axonal mitochondria showed a significant 35% increase in average mitochondrial length with an abnormal distribution in MS06 neurons compared with control hiPSC-CV neurons (Fig. 5G). In addition, MS06 neurons contained abnormal mitochondrial morphologies, such as branched and interconnected mitochondria (Fig. 5F). To test whether these changes in mitochondrial morphology were dependent on a physiological role for αSyn in mitochondrial homeostasis, we overexpressed A53T αSyn in MS06 neurons. Interestingly, A53T αSyn overexpression led to significant reductions in mitochondrial length and distribution in MS06 neurons that resulted in even smaller mitochondria than in control hiPSC-CV neurons (Fig. 5E,G). To determine whether disruption of αSyn resulted in modification of fusion/fission-related protein levels, e.g., mitofusin (MFN) and Dynamin related protein 1 (DRP1), we performed Western blot analysis of CV control or MS06 neurons. Quantification of protein levels revealed similar amounts of MFN and DRP1 expression in MS06 and control neurons (Fig. 5D), suggesting that mitochondrial elongation in MS06 neurons is promoted without changes in expression of mitochondrial quality control proteins. However, this experiment did not rule out whether MFN or DRP1 activity may be driving the fragmentation phenotype. Together, these results strongly support a physiological role for αSyn in the control of mitochondrial morphology, which could be linked to a direct interaction of the N-terminal domain with the mitochondrial membrane.
Using a relevant human neuronal cell model, we provide novel evidence supporting the following conclusions: (i) αSyn variants have differential effects on mitochondrial transport and size, which is linked to levels of expression and mitochondrial localization; (ii) αSyn-dependent mitochondrial fragmentation requires direct interaction with the OMM; (iii) disruption of the N-terminal domain induces changes in mitochondrial morphology, supporting a physiological role for αSyn in mitochondrial size regulation; and (iv) this mechanism is not restricted to human-derived neuronal cells.
The role of αSyn in mitochondrial function has been the subject of extensive research in the context of neuronal pathology40. Mitochondrial fragmentation has been described in several PD models using different αSyn variants, although the specific effect of the different alleles has not been consistent29,30,31, 41,42,43. Comprehensive studies in relevant human cellular models to test the effect of αSyn mutation, dosage, and localization on mitochondrial fragmentation have been lacking. Here, we used human-derived neurons to characterize the effect of overexpression of three αSyn variants on axonal mitochondria. A53T αSyn overexpression led to significant reductions in axonal mitochondrial size. Milder size reductions were observed upon WT αSyn overexpression, but not in A30P overexpression. These differential αSyn effects on mitochondrial size were also dose dependent. The significant effect on mitochondrial size observed upon A53T overexpression suggests a gain-of-function mechanism, while the lack of effect of A30P, even upon high expression levels, suggests a loss of function. To determine whether the observed differences in mitochondrial size were related to a direct association of αSyn with the mitochondria, we performed subcellular fractionations in αSyn-overexpressing neuroblastoma cells. A53T αSyn localization within the mitochondrial fraction was enhanced compared with WT αSyn, while A30P αSyn showed reduced mitochondrial localization. These results suggest that αSyn might exert a functional regulation of mitochondrial morphology that can be modulated by different mutations.
The intrinsic biophysical properties of αSyn allow interaction with lipids, mediating a physiological function in synaptic vesicle docking to the plasma membrane44, 45. αSyn association with mitochondria has been proposed to depend on the helical conformation of the N-terminal domain of αSyn46. Disease-associated mutations in αSyn, which are all located within this domain, impact αSyn membrane binding affinity, with the A53T mutation having the highest membrane affinity and A30P having a lower membrane affinity compared with WT αSyn22. These differential properties correlate with our results on mitochondrial size and with reports suggesting mutation-dependent effects on mitochondrial fragmentation and defects in import machinery47. The association of αSyn with the OMM has been initially reported in mouse neurons48. This association seems to be increased under artificially-induced low intracellular pH conditions in cell lines overexpressing αSyn variants49. To determine whether a specific mitochondrial interaction mediated fragmentation, we utilized a dimerization system to target the different αSyn variants to the OMM, while maintaining availability of the N-terminal domain to interact freely with membrane molecules. Interestingly, WT and A53T αSyn delivery to the OMM resulted in a rapid and dose-dependent mitochondrial fragmentation phenotype, more pronounced than by their overexpression alone. In contrast, A30P did not alter mitochondrial size, even at high concentrations of dimerizing agent. Our results with A30P are consistent with previous reports that indicate an inability to induce fragmentation, little impact on mitochondrial import machinery, and decreased induction of micelle fusion in vitro 29, 50. A relationship between membrane curvature and rates of fusion is thought to occur in mitochondria as a result of changing lipid and protein composition50, 51. When overexpressed, the enhanced association of αSyn to the OMM either by protein-protein or protein-lipid interaction could lead to a decrease in fusion rate29. Consequently, mitochondrial fragmentation is favored leading in some cases to mitophagy52, 53. Taken together, these results strongly suggest that the effect of αSyn on fragmentation depends on a direct association with the mitochondria.
Given the enriched expression of αSyn in neuronal cells, many endogenous roles have been proposed, including control of synaptic vesicle release54, modulation of secretory pathways55, and even vesicle transport regulation37. However, whether endogenous αSyn exerts a physiological role in neuronal mitochondria quality control has remained unclear. Some clues arise from αSyn knock-out mice, which display a higher resistance to mitochondrial neurotoxins used to model PD56. A previous observation that αSyn knockdown suppresses stress-induced mitochondrial fission phenotypes also suggests a physiological regulation of mitochondrial dynamics9. In addition, the N-terminal domain of αSyn has been implicated in the control of mitochondrial fragmentation31, 46. The morphology of mitochondria also depends on recovery of membrane-enriched fusion-fission proteins found in mitochondria-associated ER membranes (MAM)57. When overexpressed, α-Syn modulates the mitochondria-MAM interaction affecting the transfer of calcium from ER to mitochondria in cell lines58, 59. Pathogenic mutations in α-Syn when localized to MAMs reduced the mitochondria-ER interaction while induced mitochondrial fragmentation42. The mechanism exerted by α-Syn in MAMs hint at a possible, as yet unexplored compartment that might be relevant for understanding the role of α-Syn in PD pathogenesis. To further test the hypothesis that α-Syn N-terminal domain interact with membranes to regulate the shape of mitochondria, we performed CRISP-R/Cas9-mediated genome editing to disrupt the proximity of the α-helix domains of endogenous αSyn in hiPSC. A shift in mitochondrial morphology towards elongated sizes and branched mitochondria was observed in axonal projections of CRISP-R/Cas9-modified human-derived neurons, which was not dependent on changes in MFN or DRP1 proteins levels. In addition, the strong fragmentation elicited by A53T overexpression in these modified cells supports a physiological role for the N-terminal domain of αSyn in the regulation of mitochondrial dynamics.
The association of αSyn with molecular motors60, 61, and the accumulation of αSyn aggregation bodies suggest that αSyn plays a central role in the induction of axonal transport defects36, 62. Consistent with this view, we showed that overexpression of WT or mutated forms of αSyn in human-derived neurons resulted in significant defects in anterograde/retrograde mitochondrial flux associated with abnormal mitochondrial distribution in axons. Similar dynamic defects have been reported in cell lines and mouse neurons overexpressing WT or A53T αSyn overexpression; A30P was not analyzed30. Axonal mitochondrial mobility drives the fusion-fission rates that control mitochondria morphology63, and the same counterbalance regulation of mitochondrial transport has been suggested when fusion-fission is impaired64. This crosstalk between fusion-fission and axonal flux is further influenced by mitochondrial membrane potential, which acts as a sensor of mitochondrial integrity and favors retrograde over anterograde transport65. The axonal transport defects observed upon αSyn overexpression are consistent with the fragmentation phenotype we observed; however, mitochondrial membrane potential remained unchanged at the time of analysis. This opens the door to further investigation into the effects of long-term αSyn overexpression on mitochondrial transport and function.
Many genes associated with PD converge on the mitochondria as a crucial etiological factor in disease initiation. PINK1, Parkin, and DJ-1 interact within similar pathways that regulate mitophagy, transport, and mitochondrial fusion13, 16, 17. LRRK and VPS35 proteins control mitochondrial morphology by interacting with the fission protein DLP1, leading to an increase in mitochondrial fission12, 15. Our findings provide further evidence to suggest a physiological role for αSyn in the control of axonal mitochondrial transport and morphology in human-derived neurons, which can be modulated by mutation or gene dosage. The physiological and pathological role of αSyn in mitochondrial homeostasis uncovered here may lead to a common pathway underlying PD pathogenesis, which is ultimately relevant for understanding neurodegenerative diseases and for the future design of preventive or therapeutic strategies for PD.
Detailed methods can be found in the SI appendix.
The human embryonic stem cell (hESC) line Hues9 (H9, WiCell Research Institute) and induced pluripotent stem cell line (Craig Venter: CV iPSC SD2010–125, UCSD) were differentiated into polarized and functional neurons following established protocols32 as described in the Supplementary Experimental Procedures. pLv-Bobi-CMV-αSyn (WT, A30P, A53T) vectors were introduced into differentiated neurons using either lipofectamine transfection or viral transduction at DIV14 for live or fixed experiments.
Live imaging of fluorescent mitochondria
hESC- or hiPSC-derived neurons were live-imaged in CO2 and temperature control for fluorescent axonal mitochondria visualization and dynamic tracking of mitochondrial movement and morphology as previously described18, 34. Acquisition details and analyses are described in the Supplementary Experimental Procedures.
αSyn targeting to mitochondria
WT, A30P, and A53T αSyn-FRB vectors were co-transfected into human neurons with GFP-FKBP(x2)-ActA to drive mitochondrial targeting of αSyn in the presence of 200 or 500 nM rapalog dimerizing agent for 6 h. Transfected and treated neurons were rinsed, fixed, and immunostained prior to analysis of mitochondrial morphology.
αSyn N-terminal domain disruption by Crisp-R/Cas9 genomic editing
CrispR/Cas9 disruption of the N-terminal domain of αSyn was performed using a guide RNA targeting the SNCA exon 2 sequence between both alpha helices of the N-terminal domain of αSyn in iPSCs (CV iPSC). Each clone was tested for insertions or deletions by PCR amplification with specific primers flanking the genomic site of the gRNA target. Clone MS06 was sequenced and selected for analysis of neural differentiation and mitochondrial morphology.
Goedert, M., Spillantini, M. G., Del Tredici, K. & Braak, H. 100 years of Lewy pathology. Nat Rev Neurol 9, 13–24 (2013).
Spillantini, M. G. et al. Alpha-synuclein in Lewy bodies. Nature 388, 839–840 (1997).
Polymeropoulos, M. H. et al. Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science 276, 2045–2047 (1997).
Schapira, A. H. V. Mitochondria in the aetiology and pathogenesis of Parkinson’s disease. Lancet Neurol 7, 97–109 (2008).
Bonifati, V. Genetics of Parkinson’s disease–state of the art, 2013. Parkinsonism Relat Disord 20(Suppl 1), S23–8 (2014).
Langston, J. W., Ballard, P., Tetrud, J. W. & Irwin, I. Chronic Parkinsonism in humans due to a product of meperidine-analog synthesis. Science 219, 979–980 (1983).
Banerjee, R., Starkov, A. A., Beal, M. F. & Thomas, B. Mitochondrial dysfunction in the limelight of Parkinson’s disease pathogenesis. Biochim Biophys Acta 1792, 651–663 (2009).
Di Maio, R. et al. α-Synuclein binds to TOM20 and inhibits mitochondrial protein import in Parkinson’s disease. Sci Transl Med 8, 342ra78 (2016).
Norris, K. L. et al. Convergence of parkin, PINK1 and α-synuclein on stress-induced mitochondrial morphological remodelling. J Biol Chem (2015).
Menges, S. et al. Alpha-synuclein prevents the formation of spherical mitochondria and apoptosis under oxidative stress. Sci Rep 7, 42942 (2017).
Burté, F., Carelli, V., Chinnery, P. F. & Yu-Wai-Man, P. Disturbed mitochondrial dynamics and neurodegenerative disorders. Nat Rev Neurol 11, 11–24 (2015).
Tang, F.-L. et al. VPS35 Deficiency or Mutation Causes Dopaminergic Neuronal Loss by Impairing Mitochondrial Fusion and Function. Cell Rep 12, 1631–43 (2015).
Krebiehl, G. et al. Reduced basal autophagy and impaired mitochondrial dynamics due to loss of Parkinson’s disease-associated protein DJ-1. PLoS ONE 5, e9367 (2010).
Park, J.-S., Koentjoro, B., Veivers, D., Mackay-Sim, A. & Sue, C. M. Parkinson’s disease-associated human ATP13A2 (PARK9) deficiency causes zinc dyshomeostasis and mitochondrial dysfunction. Hum Mol Genet 23, 2802–15 (2014).
Su, Y.-C. & Qi, X. Inhibition of excessive mitochondrial fission reduced aberrant autophagy and neuronal damage caused by LRRK2 G2019S mutation. Human Molecular Genetics 22, 4545–4561 (2013).
Ziviani, E., Tao, R. N. & Whitworth, A. J. Drosophila parkin requires PINK1 for mitochondrial translocation and ubiquitinates mitofusin. Proc Natl Acad Sci USA 107, 5018–5023 (2010).
Yang, Y. et al. Pink1 regulates mitochondrial dynamics through interaction with the fission/fusion machinery. Proc Natl Acad Sci USA 105, 7070–7075 (2008).
Falzone, T. L. et al. Axonal stress kinase activation and tau misbehavior induced by kinesin-1 transport defects. J Neurosci 29, 5758–5767 (2009).
Fuchs, J. et al. Phenotypic variation in a large Swedish pedigree due to SNCA duplication and triplication. Neurology 68, 916–22 (2007).
Ibáñez, P. et al. Alpha-synuclein gene rearrangements in dominantly inherited parkinsonism: frequency, phenotype, and mechanisms. Arch Neurol 66, 102–8 (2009).
Ulmer, T. S., Bax, A., Cole, N. B. & Nussbaum, R. L. Structure and dynamics of micelle-bound human alpha-synuclein. J Biol Chem 280, 9595–9603 (2005).
Perlmutter, J. D., Braun, A. R. & Sachs, J. N. Curvature dynamics of alpha-synuclein familial Parkinson disease mutants: molecular simulations of the micelle- and bilayer-bound forms. J Biol Chem 284, 7177–7189 (2009).
Kruger, R. et al. Familial parkinsonism with synuclein pathology: Clinical and PET studies of A30P mutation carriers. Neurology 56, 1355–1362 (2001).
Puschmann, A. et al. A Swedish family with de novo alpha-synuclein A53T mutation: evidence for early cortical dysfunction. Parkinsonism Relat Disord 15, 627–32 (2009).
Zhang, L. et al. Semi-quantitative analysis of alpha-synuclein in subcellular pools of rat brain neurons: an immunogold electron microscopic study using a C-terminal specific monoclonal antibody. Brain Res 1244, 40–52 (2008).
Devi, L., Raghavendran, V., Prabhu, B. M., Avadhani, N. G. & Anandatheerthavarada, H. K. Mitochondrial import and accumulation of alpha-synuclein impair complex I in human dopaminergic neuronal cultures and Parkinson disease brain. J Biol Chem 283, 9089–9100 (2008).
Martin, L. J. et al. Parkinson’s disease alpha-synuclein transgenic mice develop neuronal mitochondrial degeneration and cell death. J Neurosci 26, 41–50 (2006).
Butler, E. K. et al. The mitochondrial chaperone protein TRAP1 mitigates α-Synuclein toxicity. PLoS Genet 8, e1002488 (2012).
Kamp, F. et al. Inhibition of mitochondrial fusion by α-synuclein is rescued by PINK1, Parkin and DJ-1. EMBO J 29, 3571–3589 (2010).
Xie, W. & Chung, K. K. K. Alpha-synuclein impairs normal dynamics of mitochondria in cell and animal models of Parkinson’s disease. J Neurochem 122, 404–414 (2012).
Nakamura, K. et al. Direct membrane association drives mitochondrial fission by the Parkinson disease-associated protein alpha-synuclein. J Biol Chem 286, 20710–20726 (2011).
Zhang, X.-Q., Zhang, S.-C. & Turksen, K. Differentiation of Neural Precursors and Dopaminergic Neurons from Human Embryonic Stem Cells. Methods in Molecular Biology Methods in Molecular Biology 584, 355–366 (2009).
Lacovich, V. et al. Tau Isoforms Imbalance Impairs the Axonal Transport of the Amyloid Precursor Protein in Human Neurons. J Neurosci 37, 58–69 (2017).
Falzone, T. L. & Stokin, G. B. Imaging amyloid precursor protein in vivo: an axonal transport assay. Methods Mol Biol 846, 295–303 (2012).
Volpicelli-Daley, L. A. et al. Formation of α-synuclein Lewy neurite-like aggregates in axons impedes the transport of distinct endosomes. Mol Biol Cell 25, 4010–4023 (2014).
Roy, B. & Jackson, G. R. Interactions between Tau and α-synuclein augment neurotoxicity in a Drosophila model of Parkinson’s disease. Hum Mol Genet 23, 3008–23 (2014).
Jensen, P. H., Nielsen, M. S., Jakes, R., Dotti, C. G. & Goedert, M. Binding of alpha-synuclein to brain vesicles is abolished by familial Parkinson’s disease mutation. J Biol Chem 273, 26292–26294 (1998).
van Spronsen, M. et al. TRAK/Milton motor-adaptor proteins steer mitochondrial trafficking to axons and dendrites. Neuron 77, 485–502 (2013).
Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8, 2281–2308 (2013).
Pozo Devoto, V. M. and Falzone, T. L. Mitochondrial dynamics in Parkinson's Disease: A role for Alpha-synuclein? Dis Model Mech. In Press (2017).
O’Donnell, K. C. et al. Axon degeneration and PGC-1α-mediated protection in a zebrafish model of α-synuclein toxicity. Dis Model Mech 7, 571–582 (2014).
Guardia-Laguarta, C. et al. α-Synuclein is localized to mitochondria-associated ER membranes. J Neurosci 34, 249–259 (2014).
Chinta, 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. Neuroscience Letters 486, 235–239 (2010).
Abeliovich, A. et al. Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system. Neuron 25, 239–252 (2000).
Nemani, V. M. et al. Increased expression of alpha-synuclein reduces neurotransmitter release by inhibiting synaptic vesicle reclustering after endocytosis. Neuron 65, 66–79 (2010).
Robotta, M. et al. Alpha-synuclein binds to the inner membrane of mitochondria in an α-helical conformation. Chembiochem 15, 2499–502 (2014).
Bender, A. et al. TOM40 mediates mitochondrial dysfunction induced by α-synuclein accumulation in Parkinson’s disease. PLoS One 8, e62277 (2013).
Li, W.-W. et al. Localization of alpha-synuclein to mitochondria within midbrain of mice. Neuroreport 18, 1543–1546 (2007).
Cole, N. B., DiEuliis, D., Leo, P., Mitchell, D. C. & Nussbaum, R. L. Mitochondrial translocation of alpha-synuclein is promoted by intracellular acidification. Exp Cell Res 314, 2076–2089 (2008).
Auluck, P. K., Caraveo, G. & Lindquist, S. α-Synuclein: membrane interactions and toxicity in Parkinson’s disease. Annu Rev Cell Dev Biol 26, 211–233 (2010).
Chan, E. Y. L. & McQuibban, G. A. Phosphatidylserine decarboxylase 1 (Psd1) promotes mitochondrial fusion by regulating the biophysical properties of the mitochondrial membrane and alternative topogenesis of mitochondrial genome maintenance protein 1 (Mgm1). J Biol Chem 287, 40131–40139 (2012).
Choubey, V. et al. Mutant A53T alpha-synuclein induces neuronal death by increasing mitochondrial autophagy. J Biol Chem 286, 10814–24 (2011).
Itoh, K., Nakamura, K., Iijima, M. & Sesaki, H. Mitochondrial dynamics in neurodegeneration. Trends Cell Biol 23, 64–71 (2013).
Snead, D. & Eliezer, D. Alpha-synuclein function and dysfunction on cellular membranes. Exp Neurobiol 23, 292–313 (2014).
Wang, T. & Hay, J. C. Alpha-synuclein Toxicity in the Early Secretory Pathway: How It Drives Neurodegeneration in Parkinsons Disease. Front Neurosci 9, 433 (2015).
Dauer, W. et al. Resistance of alpha -synuclein null mice to the parkinsonian neurotoxin MPTP. Proc Natl Acad Sci USA 99, 14524–14529 (2002).
Friedman, J. R. et al. ER tubules mark sites of mitochondrial division. Science 334, 358–362 (2011).
Calì, T., Ottolini, D., Negro, A. & Brini, M. α-Synuclein controls mitochondrial calcium homeostasis by enhancing endoplasmic reticulum-mitochondria interactions. J Biol Chem 287, 17914–29 (2012).
Paillusson, S. et al. α-Synuclein binds to the ER-mitochondria tethering protein VAPB to disrupt Ca(2+) homeostasis and mitochondrial ATP production. Acta Neuropathol (2017).
Utton, M. A., Noble, W. J., Hill, J. E., Anderton, B. H. & Hanger, D. P. Molecular motors implicated in the axonal transport of tau and alpha-synuclein. J Cell Sci 118, 4645–54 (2005).
Prots, I. et al. α-Synuclein oligomers impair neuronal microtubule-kinesin interplay. J Biol Chem 288, 21742–54 (2013).
Saha, A. R. et al. Parkinson’s disease alpha-synuclein mutations exhibit defective axonal transport in cultured neurons. J Cell Sci 117, 1017–24 (2004).
Misko, A., Jiang, S., Wegorzewska, I., Milbrandt, J. & Baloh, R. H. Mitofusin 2 is necessary for transport of axonal mitochondria and interacts with the Miro/Milton complex. J Neurosci 30, 4232–4240 (2010).
Fukumitsu, K. et al. Mitochondrial fission protein Drp1 regulates mitochondrial transport and dendritic arborization in cerebellar Purkinje cells. Mol Cell Neurosci 71, 56–65 (2016).
Wang, X. et al. PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility. Cell 147, 893–906 (2011).
We thank Dr. Eliezer Masliah from UCSD for providing pLV-CMV-αSyn plasmids; Dr. Casper Hoogenraad from Utrecht University for providing FKBP and FRB dimerization backbone vectors used for subcloning. We also thank Dr Veronica Alvarez and Shermali Gunawardena for helpful discussion on data interpretation and presentation. We thank Roux-Ocefa Argentina for kindly contributing with basic reagents. T.L.F. acknowledges support from CONICET, the University of Buenos Aires, and the Argentinean Science Ministry. V.M.P.D., M.A., M.G.O., T.M.S. and L.E.C. acknowledge support from CONICET fellowships. V.M.P.D. acknowledges support from the International Brain Research Organization (IBRO) and Company of Biologist (COB). This work was supported by grants from MINCyT (PICT 2011–2027; 2013-0402, T.L.F.); the Alzheimer Association (NIRG10-172840, T.L.F.); UBA (UBACyT 2011/2014, T.L.F.); the European Social Fund and European Regional Development Fund-Project MAGNET (Number CZ.02.1.01/0.0/0.0/15_003/0000492). We acknowledge editing work from Kerry MacLaughlin, Insight Editing London.
The authors declare that they have no competing interests.
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Pozo Devoto, V.M., Dimopoulos, N., Alloatti, M. et al. αSynuclein control of mitochondrial homeostasis in human-derived neurons is disrupted by mutations associated with Parkinson’s disease. Sci Rep 7, 5042 (2017). https://doi.org/10.1038/s41598-017-05334-9
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