Mitochondrial translocation of alpha-synuclein is promoted by intracellular acidification

Abstract

Mitochondrial dysfunction plays a central role in the selective vulnerability of dopaminergic neurons in Parkinson's disease (PD) and is influenced by both environmental and genetic factors. Expression of the PD protein alpha-synuclein or its familial mutants often sensitizes neurons to oxidative stress and to damage by mitochondrial toxins. This effect is thought to be indirect, since little evidence physically linking alpha-synuclein to mitochondria has been reported. Here, we show that the distribution of alpha-synuclein within neuronal and non-neuronal cells is dependent on intracellular pH. Cytosolic acidification induces translocation of alpha-synuclein from the cytosol onto the surface of mitochondria. Translocation occurs rapidly under artificially-induced low pH conditions and as a result of pH changes during oxidative or metabolic stress. Binding is likely facilitated by low pH-induced exposure of the mitochondria-specific lipid cardiolipin. These results imply a direct role for alpha-synuclein in mitochondrial physiology, especially under pathological conditions, and in principle, link alpha-synuclein to other PD genes in regulating mitochondrial homeostasis.

Figure 1. Oxidative and metabolic stresses induce synuclein translocation to mitochondria

(A) Representitive immunofluorescence images from SK-N-SH cells stably expressing α-synuclein. Cells were untreated or treated for 2 h with 250 µM H₂O₂, 30 mM 2-deoxyglucose and 0.05% sodium azide (DOG/Az), 1 µM CCCP, or 2 mM sodium dithionite, as indicated, and fixed with 3.7% formaldehyde in 0.1 M PO₄ buffer, pH 7.2. Synuclein is labeled with the anti-synuclein antibody 202 and Alexa 488 donkey anti-mouse secondary antibodies. Mitochondria are labeled with antibodies to the outer mitochondrial protein Bcl_XL and Alexa 594 donkey anti-rabbit secondary antibodies. Merged images are shown at the bottom. Arrows indicate synuclein clusters apparently not associated with mitochondria. Bar: 10 µm; inset bar for DOG/Az: 5 µm. (B) pHi measurements determined from treatments in (A) using SNARF 4F (See Materials and Methods). (C) Representitive images showing the effect of DFO and FeCl₃ on α-synuclein localization in the presence of H₂O₂. Note that in cells pretreated with DFO (1 mM for 4 h), synuclein remains diffusely cytosolic at 250 µM H₂O₂, whereas FeCl₃ pretreatment (30 µM for 4 h) sensitizes synuclein translocation to mitochondria at 50 µM H₂O₂. Not shown are merged images showing colocalization (or lack thereof) of synuclein with mitochondrial markers under these conditions. Bar: 10 µm.

Figure 2. Effects of oxidative stress on translocation of different synucleins to mitochondria

(A) Representative immunofluorescence images of SK-N-SH cells transiently expressing various synucleins. Cells were untreated (A) or treated with 250 µM H₂O₂ for 2 h (B), before being fixed as in Fig. 1. Note that A53T, β-synuclein, and Syn1-102 translocate to mitochondria with H₂O₂, whereas translocation was minimal with A30P and γ-synuclein. Mitochondria were labeled either with antibodies to the outer mitochondrial membrane protein Tom20 or to the intermembrane space protein cytochrome c, depending on the species of primary synuclein antibody used. Cells expressing low levels of various synucleins are depicted. Bar: 10 µm.

Figure 3. Synuclein translocation to mitochondria is directly influenced by pHi

Immunofluorescence images of SK-N-SH cells expressing α-synuclein clamped to various intracellular pHs with high K⁺ buffers/nigericin. Cells were incubated for 15 minutes before fixation with 3.7% formaldehyde in 0.1 M PO₄, pH 7.2. Cells were labeled with antibodies to synuclein and Bcl_XL. Note the partial redistribution of synuclein at pHi 6.0 and below; complete redistribution occurs at pHi 5.0. The green arrow shows one of several synuclein clusters that form at low pH. Bar: 10 µm.

Figure 4. Synuclein translocates to mitochondria during acidic fixation

(A) Representative fluorescence micrographs of SK-N-SH cells transiently transfected with α-synuclein. Cells were fixed at pH 7.2 (left panels) or 5.0 (right panels). Synuclein is labeled with the antibody 202 and Alexa 488 donkey anti-mouse secondary antibodies. Mitochondria are labeled with MitoTracker Red. Merged images are shown at the bottom. Nuclei are stained with DAPI (blue). (B) Rat hippocampal neurons (4 div) were fixed at pH 7.2 (left panels) or pH 5.0 (right panels) and stained with antibodies to synuclein or to the mitochondrial protein Bcl_XL. Short arrow indicates a synuclein-positive synaptic bouton; long arrow shows colocalization of synuclein and mitochondria in distal processes. Scale bar: (A) 10 µm; (B) 5 µm.

Figure 5. Synuclein redistributes to the outer membrane of mitochondria

(A) Immunoelectron micrographs of HEK 293 cells stably expressing α-synuclein. Cells were fixed in deionized formaldehyde in PBS (left panel) or 0.1 M acetate buffer, pH 5.0 (right panel) and labeled with the anti-synuclein antibody 202. Scale bar: 500 nm. (B) Quantitative analysis from immunoelectron microscopy in (A). (Left graph) The fraction of total gold particles on mitochondria (mito) versus the cytosol (cyto) is indicated for different fixation conditions. The sum is <100% due to the presence of low levels of gold particles on unidentified organelles. (Right graph) The numbers of gold particles per µm length of individual mitochondria under the indicated fixation conditions. Error bars represent the mean ± s.e.m. (n = ~20 images from 2 independent experiments). (C) Immunoblot of α-synuclein extracted from HEK 293 cells during various fixation conditions. Note that increasing amounts of synuclein are extracted as the pH of the fixative is reduced. Adding 0.01% formic acid to formaldehyde buffered in PBS reduced the pH to 4.5. Increasing the buffering capacity neutralized this effect. Form: formaldehyde. FA: formic acid.

Figure 6. Synuclein binds to purified mitochondria at acidic pH

Synuclein binding assays from native mitochondrial membranes (A–C) and liposomes (D–F). (A) Recombinant synuclein proteins were incubated with intact mitochondria at the indicated pH in the absence (top panel) or presence (bottom panel) of 3.7% deionized formaldehyde before pelleting. S, supernatant; p, pellet. Note that formaldehyde treatment enhances pH-dependent binding of wild-type and Syn1-102, but not A30P synucleins. (B) Effect of nonyl acridine orange (NAO) on Syn1-102 binding in the presence of formaldehyde. (C) Effect of alkaline carbonate/proteinase K treatment on Syn1-102 binding (right panels). Immunoblots show the presence or absence of the outer mitochondrial membrane protein Tom20 and membrane embedded VDAC. Note the presence of higher molecular mass forms of Tom20 cross-linked with formaldehyde. No effect of formaldehyde on the mobility of synuclein was observed. All cytochrome c was released from mitochondria during carbonate treatment (not shown). (D–F) Flotation gradients of Syn1-102 binding to liposomes composed of PC, PC/PA, PC/CL, or from purified mitochondria (Mito). T, top of gradient; b, bottom of gradient. (D) Binding was without or with a prior 10,000 × g spin to pellet large lipid aggregates. (E) Specificity of NAO with PC/PA versus mitochondrial liposomes. (F) Formaldehyde- and pH-independent binding to mitochondrial liposomes. NAO inhibits binding.