α-Synuclein is localized to mitochondria-associated ER membranes

Abstract

Familial Parkinson disease is associated with mutations in α-synuclein (α-syn), a presynaptic protein that has been localized not only to the cytosol, but also to mitochondria. We report here that wild-type α-syn from cell lines, and brain tissue from humans and mice, is present not in mitochondria but rather in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM), a structurally and functionally distinct subdomain of the ER. Remarkably, we found that pathogenic point mutations in human α-syn result in its reduced association with MAM, coincident with a lower degree of apposition of ER with mitochondria, a decrease in MAM function, and an increase in mitochondrial fragmentation compared with wild-type. Although overexpression of wild-type α-syn in mutant α-syn-expressing cells reverted the fragmentation phenotype, neither overexpression of the mitochondrial fusion/MAM-tethering protein MFN2 nor inhibition/ablation of the mitochondrial fission protein DRP1 was able to do so, implying that α-syn operates downstream of the mitochondrial fusion/fission machinery. These novel results indicate that wild-type α-syn localizes to the MAM and modulates mitochondrial morphology, and that these behaviors are impaired by pathogenic mutations in α-syn. We believe that our results have far-reaching implications for both our understanding of α-syn biology and the treatment of synucleinopathies.

Figure 1.

Subcellular localization of α-synuclein. A, In vitro mitochondrial import assays. Import of [³⁵S]Met/Cys-labeled α-syn and a control protein (Mdh2) into HeLa cell mitochondria for the indicated times. p, Precursor protein; m, mature processed protein. Imported proteins were separated by SDS-PAGE. Note that depletion of Δψ affected only the import of Mdh2. B, Western blot to detect the indicated proteins in standard subcellular fractionation of M17 and HeLa cells expressing WT α-syn and in normal human and mouse brain. The CM fraction was purified further into MAM and mitochondrial fractions. ATPase-α (complex V) was used as a mitochondrial marker, protein kinase C as a cytoplasmic marker, and ERLIN-2 as a MAM marker.

Figure 2.

Localization of α-syn in M17 cells and transgenic mice. A, B, Western blots to detect the indicated proteins in standard subcellular fractionation of M17 cells stably expressing the indicated α-syn proteins (A; quantitation of the ratio of α-syn in the CM:Cyto fractions at right) and in transgenic mice expressing the indicated α-syn species (B). C, Localization of α-syn species in DRM and non-DRM fractions. CM (containing mitochondria and MAM) from transgenic mouse brain expressing WT and mutant α-syn were treated with Triton X-100 and then loaded onto a continuous 5–35% sucrose gradient, and the indicated fractions were collected and subjected to Western blotting to detect and quantitate α-syn (see Materials and Methods). Note that α-syn from WT and A53T, but not A30P, mice comigrate with flotillin, a marker of DRMs/lipid rafts.

Figure 3.

Expression of α-syn in M17 and HeLa cells. A, Left, Example of a Western blot of the expression of α-syn in EV and stably transfected WT, A53T, and A30P α-syn-expressing M17 cells relative to that of control β-actin. Right, Quantitation of the total α-syn in the blots exemplified at left, in arbitrary units; n = 3; asterisk denotes significance versus WT. B, Expression and quantitation of α-syn in HeLa cells, as in (A). In both cases, note reduced expression in A53T cells.

Figure 4.

Apposition of ER and mitochondria in M17 cells. A, Examples of colocalization of ER labeled with GFP-Sec61-β (green), and mitochondria labeled with pDsRed2-mito (red), in WT- and A30P-expressing M17 cells. B, Quantitation of colocalization (as in A) by ImageJ analysis. Asterisk denotes significance versus EV.

Figure 5.

Apposition of ER and mitochondria in HeLa cells. A, Examples of colocalization of ER labeled with GFP-Sec61-β (green), and mitochondria labeled with pDsRed2-mito (red), in HeLa cells transiently expressing WT- and A30P-α-syn. B, Quantitation of colocalization (as in A) by ImageJ analysis, normalized to the baseline level of apposition value in EV cells (baseline = 100; average of 4 experiments ±SD). Asterisk denotes significance versus EV.

Figure 6.

Phospholipid synthesis in M17 cells. Synthesis of ³H-PtdSer and ³H-PtdEtn after labeling the indicated M17 cells with ³H-Ser for the indicated times; n = 3 independent experiments. *Significant difference versus EV (horizontal lines; p < 0.05). Note the imputed reduction in MAM function in the two point mutations.

Figure 7.

Mitochondrial fragmentation in M17 cells. A, Representative images of mitochondria in α-syn-expressing cells labeled with pDsRed2-Mito. Insets, Enlargements of the indicated regions (small boxes). B, Quantitation of fragmentation of the cells in A, measured as the percentage of all cells that contained predominantly fragmented (darker shading) versus tubular (lighter shading) mitochondria. Numbers within the boxes denote the average length (±SE) of the mitochondria in micrometers; n = number of mitochondria measured. *Significant difference versus EV (p < 0.05).

Figure 8.

Mitochondrial fragmentation in HeLa cells. A, Representative images of mitochondria in α-syn-expressing cells labeled with pDsRed2-Mito. Insets, Enlargements of the indicated regions (small boxes). B, Quantitation of fragmentation of the cells in A, measured as the percentage of all cells that contained predominantly fragmented tubular mitochondria. *Significant difference versus EV (p < 0.05); n = 5 independent experiments.

Figure 9.

Mitochondrial dynamics in M17 cells. A, DRP1 recruitment to mitochondria. Representative images of fluorescently labeled mitochondria (red) and anti-DRP1 (green) in M17 cells either untreated or treated with the DRP1 inhibitor mdivi-1. Arrowheads indicate examples of DRP1-positive foci. Other notation as in Figure 7. B, Inhibition of DRP1 with mdivi-1. Representative images of fluorescently labeled mitochondria (red) in M17 cells either untreated or treated with the DRP1 inhibitor mdivi-1. Note the persistence of fragmented mitochondria in the A53T and A30P cells even after mdivi-1 treatment.

Figure 10.

Mitochondrial dynamics in Drp1-null MEFs. A, Representative images of mitochondria in Drp1-WT and -KO MEFs labeled with MitoTracker Red (red) transiently expressing the indicated plasmid constructs. B, Quantitation of fragmented mitochondria from the experiments shown in A (n = 3). Note the persistence of fragmented mitochondria in the A53T and A30P cells even in the Drp1-KO cells. Asterisk denotes significance versus EV.

Figure 11.

Western blot to detect OPA1 in CM fractions from M17 cells stably expressing the indicated forms of α-syn. Mitochondrial ATPase-α was used as a loading control.

Figure 12.

Relationship of WT α-syn expression to mitochondrial fragmentation. A, M17 cells stably transfected with an EV plasmid or with the indicated plasmids expressing α-syn species were transiently cotransfected with plasmids expressing pDsRed2-Mito (red) and WT α-syn-GFP (green) and visualized by confocal microscopy. Cells were inspected visually and scored for whether they contained predominantly tubular or predominantly fragmented mitochondria (red). Note that, compared with Figure 7, the fragmented phenotype in the α-syn mutant cells is ameliorated by the expression of WT α-syn-GFP. B, Quantitation of percentage fragmented cells shown in A. *Significant difference versus corresponding untransfected cells (p < 0.05).