Elsevier

Experimental Cell Research

Volume 314, Issue 10, 10 June 2008, Pages 2076-2089
Experimental Cell Research

Research Article
Mitochondrial translocation of α-synuclein is promoted by intracellular acidification

https://doi.org/10.1016/j.yexcr.2008.03.012Get rights and content

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 α-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 α-synuclein to mitochondria has been reported. Here, we show that the distribution of α-synuclein within neuronal and non-neuronal cells is dependent on intracellular pH. Cytosolic acidification induces translocation of α-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 α-synuclein in mitochondrial physiology, especially under pathological conditions, and in principle, link α-synuclein to other PD genes in regulating mitochondrial homeostasis.

Introduction

Oxidative stress and metabolic dysfunction appear to be important factors in a number of neurodegenerative diseases, including Parkinson's disease (PD), although the cellular pathways through which the various stresses converge are only slowly being uncovered [1], [2]. Many of the pathological features of PD, including the selective loss of dopaminergic neurons and α-synuclein aggregation can be reproduced by environmental toxins that principally target mitochondria [3], [4]. Impairments in the electron transport chain are associated with increased levels of reactive oxygen species and decreased energy production [4], [5], and can be amplified by proteasome inhibition [6], [7]. In addition to environmental factors, multiple genes mediating familial forms of PD or affecting PD risk have been identified, several of which encode proteins that localize to mitochondria and/or are associated with mitochondrial homeostasis. These include PINK1 [8], DJ-1 [9], [10], parkin [11], [12], and Omi/HtrA2 [13]. Since mitochondrial complex I deficiencies have been reported in patients with sporadic PD [14], [15], a common cellular network likely links familial and sporadic forms of the disease.

There is increasing evidence that the first familial PD gene to be identified, α-synuclein, also influences cellular responses to mitochondrial stress. Wild-type α-synuclein protects or sensitizes cells to apoptotic stimuli, depending on the cell type and insult examined [16], [17], whereas mutant α-synucleins (A30P or A53T) generally increase neuronal vulnerability to mitochondria-associated toxicity [16], [18], [19]. Conversely, α-synuclein knockout mice show marked resistance to several mitochondrial toxins [20]. Mitochondrial morphology or physiology may also be affected by α-synuclein overexpression [21], [22], and mitochondrial abnormalities are observed in α-synuclein-expressing mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [23]. There is, however, little evidence that α-synuclein exerts these effects by a direct physical association with mitochondria.

We now report that, in response to reduced intracellular pH, α-synuclein translocates from the cytosol onto the surface of mitochondria. This occurs under a number of oxidative and/or metabolic stress conditions, and is likely mediated by pH-dependent exposure of mitochondria-specific lipids. Thus, α-synuclein may play a direct role in mitochondrial physiology, ostensibly establishing a link between mitochondrial dysfunction and α-synuclein-associated toxicity in PD pathogenesis.

Section snippets

Cell lines, plasmids, and antibodies

All cell lines were from the ATCC. Stable cells were selected in G418 (500 μg/ml for HEK293) and 200 μg/ml (for SK-N-SH). Primary rat hippocampal neurons were provided by C. Winters (NINDS). Plasmids for expression of various synucleins in eukaryotic and prokaryotic cells were as previously described [24], [25]. Antibodies to synucleins included 202 (1/100, for immunofluorescence and immunoelectron microscopy, from V. Lee, Univ. of Pennsylvania; this antibody recognizes α- and β-synuclein) and

Synuclein translocates to mitochondria in response to cellular stress

α-Synuclein is a presynaptic neuronal protein that, when expressed in most non-neuronal cells, is diffusely distributed throughout the cytosol with little accumulation at specific intracellular sites [24], [34], [35]. A number of reports have demonstrated an effect of α-synuclein expression on responses to various stress conditions. Alterations in the subcellular distribution of α-synuclein under these conditions, however, have not been carefully addressed. We therefore examined α-synuclein

Discussion

Oxidative stress and mitochondrial dysfunction are common features in virtually all neurodegenerative diseases, and there is increasing evidence for a causal role of these stresses in disease pathogenesis. In PD, identification of disease-specific genes that influence mitochondrial physiology, either directly or indirectly, have contributed greatly to our understanding of the role of mitochondrial impairment in the etiology of this disease [1], [2]. For example, mitochondrial pathology and

Acknowledgments

We thank the NINDS electron microscopy facility for sample preparation, C. Winters (NINDS) for providing rat primary hippocampal neurons, and C. Ellis (NHGRI) for purified mitochondria. We also thank John Church (Department of Physiology, Univ. of British Columbia) for advice on intracellular pH measurements, and Rod Levine (NHLBI) for many helpful discussions. Part of this work was funded by the NINDS intramural program (to N.B.C.).

While this manuscript was under revision, two papers [69], [70]

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    1

    Current Address: Division of Medical Genetics, University of California, San Francisco School of Medicine, San Francisco, CA, USA.

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