Mitochondrial associated metabolic proteins are selectively oxidized in A30P α-synuclein transgenic mice—a model of familial Parkinson's disease

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Abstract

Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized by the loss of dopaminergic neurons in the substantia nigra compacta. α-Synuclein is strongly implicated in the pathophysiology of PD because aggregated α-synuclein accumulates in the brains of subjects with PD, mutations in α-synuclein cause familial PD, and overexpressing mutant human α-synuclein (A30P or A53T) causes degenerative disease in mice or drosophila. The pathophysiology of PD is poorly understood, but increasing evidence implicates mitochondrial dysfunction and oxidative stress. To understand how mutations in α-synuclein contribute to the pathophysiology of PD, we undertook a proteomic analysis of transgenic mice overexpressing A30P α-synuclein to investigate which proteins are oxidized. We observed more than twofold selective increases in specific carbonyl levels of three metabolic proteins in brains of symptomatic A30P α-synuclein mice: carbonic anhydrase 2 (Car2), alpha-enolase (Eno1), and lactate dehydrogenase 2 (Ldh2). Analysis of the activities of these proteins demonstrates decreased functions of these oxidatively modified proteins in brains from the A30P compared to control mice. Our findings suggest that proteins associated with impaired energy metabolism and mitochondria are particularly prone to oxidative stress associated with A30P-mutant α-synuclein.

Introduction

Parkinson's disease (PD) is the second most common neurodegenerative disorder: approximately 1% of the population by age 65 is affected with PD (Eriksen et al., 2003). Loss of dopaminergic neurons in the substantia nigra compacta results in clinical symptoms of PD, such as bradykinesia, resting tremor, cogwheel rigidity, and postural instability. Although the majority of PD is sporadic, approximately 10% of PD are familial cases (Giasson and Lee, 2003). Mutations in α-synuclein, parkin, DJ-1, and PINK1 are all linked to early onset familial PD (Dawson and Dawson, 2003). In the case of α-synuclein, 4 different mutations have been identified in kindreds associated with familial PD: A53T, A30P, E46A, and genomic duplication (Kruger et al., 1998, Polymeropoulos et al., 1997).

Oxidative damage is a prominent pathological change in PD brains (Alam et al., 1997, Floor and Wetzel, 1998, Yoritaka et al., 1996). Toxicity associated with oxidative stress is exacerbated by overexpression of wild type or mutant α-synuclein (Ostrerova-Golts et al., 2000). Increasing evidence suggests that oxidative stress in PD might also be linked to mitochondrial dysfunction, excitotoxicity, and the toxic effects of nitric oxide, all of which are believed to be associated with cell death in PD brains (Jenner, 2003). Although the relationships of oxidative stress to PD are still unknown, basal protein oxidation is high in the substantia nigra of PD patients, and the levels of reactive carbonyls are increased in PD brains (Alam et al., 1997, Floor and Wetzel, 1998). Together, these data suggest that oxidative stress contributes significantly to the pathophysiology of PD.

Wild-type α-synucleins can bind to lipid membranes, inhibit enzymes such as phospholipase D2, protein kinase C and the dopamine transporter (reviewed in Goedert, 2001). However, α-synuclein has a strong tendency to aggregate, and most studies suggest that the accumulation of aggregated α-synuclein is toxic. Expression of mutant α-synuclein cells produces increased levels of 8-hydroxyguanine, protein carbonyls, lipid peroxidation, and 3-nitrotyrosine, and markedly accelerated cell death in response to oxidative insult (Lee et al., 2001). These studies suggest that accelerated aggregation of mutant α-synucleins stimulates oxidative stress by loss of protective effect of the soluble α-synuclein (Li et al., 2001, Li et al., 2002). In order to gain insight into the mechanism(s) by which loss of antioxidant ability caused by overexpression of A30P mutant α-synuclein leads to cell death, we used proteomics to identify the brain proteins that are significantly oxidatively modified in symptomatic mice with overexpression of a A30P mutation in α-synuclein compared to the brain proteins from the non-transgenic mice.

Section snippets

Animals

The animals used in this study were previously described. (Kahle et al., 2000, Neumann et al., 2002). The mice develop symptoms between 6 and 14 months. The symptoms begin with a tremor and progress to an end-stage phenotype characterized by muscular rigidity, postural instability and ultimately paralysis. Mice that progressed to end stage symptoms were sacrificed by cervical dislocation and their brains hemi-sectioned. Non-transgenic control mice were C57Bl/6 mice, and were also sacrificed at

Results

We used a parallel approach to investigate the effect of an A30P α-synuclein mutation on specific protein oxidation. Fig. 1 shows representative 2D-electrophoresis gels of brains homogenates from symptomatic A30P α-synuclein transgenic mice (A30P) and non-transgenic mice after Sypro Ruby staining. Fig. 2 shows representative 2D Western blots of the brains of symptomatic non-transgenic mouse and symptomatic A30P-α-synuclein transgenic mice. The specific carbonyl levels indicate the carbonyl

Discussion

A30P mutation accelerates synuclein aggregation (Conway et al., 1998, Li et al., 2001, Li et al., 2002, Narhi et al., 1999) and aggregation increases vulnerability to oxidative insult (Lee et al., 2001). Moreover, the aggregation of α-synuclein occurs in parallel with symptoms in A30P α-synuclein transgenic mice (Neumann et al., 2002). Transgenic mice overexpressing α-synuclein develop an age-dependent accumulation of α-synuclein in neurons of the brain stem (Giasson et al., 2002, Kahle et al.,

Acknowledgments

The authors would like to thank Susan Debusca and Marcia Butterfield for assistance in the preparation of the manuscript. This work was supported in part by grants from NIH [AG-105119; AG-10836] to D.A.B. and from NIH [NS41786] and DAMD [17-01-1-0781] to B.W.

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    Current address: Department of Pharmacology, Boston University School of Medicine, Boston, MA 02118.

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