The deposition of the abundant presynaptic brain protein alpha-synuclein as fibrillary aggregates in neurons or glial cells is a hallmark lesion in a subset of neurodegenerative disorders. These disorders include Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy, collectively referred to as synucleinopathies. Importantly, the identification of missense mutations in the alpha-synuclein gene in some pedigrees of familial PD has strongly implicated alpha-synuclein in the pathogenesis of PD and other synucleinopathies. However, specific post-translational modifications that underlie the aggregation of alpha-synuclein in affected brains have not, as yet, been identified. Here, we show by mass spectrometry analysis and studies with an antibody that specifically recognizes phospho-Ser 129 of alpha-synuclein, that this residue is selectively and extensively phosphorylated in synucleinopathy lesions. Furthermore, phosphorylation of alpha-synuclein at Ser 129 promoted fibril formation in vitro. These results highlight the importance of phosphorylation of filamentous proteins in the pathogenesis of neurodegenerative disorders.

Loss-of-function mutations in parkin are the predominant cause of familial Parkinson's disease. We previously reported that parkin؊/؊ mice exhibit nigrostriatal deficits in the absence of nigral degeneration. Parkin has been shown to function as an E3 ubiquitin ligase. Loss of parkin function, therefore, has been hypothesized to cause nigral degeneration via an aberrant accumulation of its substrates. Here we employed a proteomic approach to determine whether loss of parkin function results in alterations in abundance and/or modification of proteins in the ventral midbrain of parkin؊/؊ mice. Two-dimensional gel electrophoresis followed by mass spectrometry revealed decreased abundance of a number of proteins involved in mitochondrial function or oxidative stress. Consistent with reductions in several subunits of complexes I and IV, functional assays showed reductions in respiratory capacity of striatal mitochondria isolated from parkin؊/؊ mice. Electron microscopic analysis revealed no gross morphological abnormalities in striatal mitochondria of parkin؊/؊ mice. In addition, parkin؊/؊ mice showed a delayed rate of weight gain, suggesting broader metabolic abnormalities. Accompanying these deficits in mitochondrial function, parkin؊/؊ mice also exhibited decreased levels of proteins involved in protection from oxidative stress. Consistent with these findings, parkin؊/؊ mice showed decreased serum antioxidant capacity and increased protein and lipid peroxidation. The combination of proteomic, genetic, and physiological analyses reveal an essential role for parkin in the regulation of mitochondrial function and provide the first direct evidence of mitochondrial dysfunction and oxidative damage in the absence of nigral degeneration in a genetic mouse model of Parkinson's disease.

Loss-of-function mutations in parkin are the major cause of early-onset familial Parkinson's disease. To investigate the pathogenic mechanism by which loss of parkin function causes Parkinson's disease, we generated a mouse model bearing a germline disruption in parkin. Parkin؊/؊ mice are viable and exhibit grossly normal brain morphology. Quantitative in vivo microdialysis revealed an increase in extracellular dopamine concentration in the striatum of parkin؊/؊ mice. Intracellular recordings of medium-sized striatal spiny neurons showed that greater currents are required to induce synaptic responses, suggesting a reduction in synaptic excitability in the absence of parkin. Furthermore, parkin؊/؊ mice exhibit deficits in behavioral paradigms sensitive to dysfunction of the nigrostriatal pathway. The number of dopaminergic neurons in the substantia nigra of parkin؊/؊ mice, however, is normal up to the age of 24 months, in contrast to the substantial loss of nigral neurons characteristic of Parkinson's disease. Steady-state levels of CDCrel-1, synphilin-1, and ␣-synuclein, which were identified previously as substrates of the E3 ubiquitin ligase activity of parkin, are unaltered in parkin؊/؊ brains. Together these findings provide the first evidence for a novel role of parkin in dopamine regulation and nigrostriatal function, and a non-essential role of parkin in the survival of nigral neurons in mice. Parkinson's disease (PD)1 is an age-related movement disorder characterized by bradykinesia, rigidity, resting tremor, and postural instability. The neuropathologic hallmarks of PD are the loss of dopaminergic neurons in the substantia nigra (SN) and the presence of intraneuronal cytoplasmic inclusions known as Lewy bodies. The clinical manifestations of PD are due to progressive degeneration of dopaminergic neurons in the pars compacta of the SN that give rise to the nigrostriatal pathway, causing dopamine (DA) depletion in the striatum, where it is required for normal motor function. Little is known about the mechanisms of PD pathogenesis and nigral degeneration, although DA neurons have been shown to be susceptible to oxidative stress (1), mitochondrial defects (2), and environmental toxins (3).The recent identification of genes linked to familial forms of PD (FPD) makes it possible to investigate the pathogenic mechanism by employing genetic approaches (4 -6). Over fifty recessively inherited mutations, including deletion, frameshift, nonsense, and missense mutations, have been identified in parkin in large numbers of families, making parkin the major gene responsible for early-onset FPD (7-10). Although the first report linked parkin mutations to autosomal recessive juvenile parkinsonism (AR-JP) with atypical clinical features (5), many more cases identified subsequently were considered typical early-onset FPD with symptoms often indistinguishable from sporadic PD (9, 11). Autopsies of limited numbers of patients showed selective loss of dopaminergic neurons in the SN either in the absence (12-15) or in the ...