P arkinson's disease (PD) is the second most prevalent neurodegenerative disorder, but the etiology remains poorly understood (1, 2). Patients with PD suffer from rigidity, slowness of movement, tremor, and disturbances of balance (1, 2). PD is characterized by the progressive loss of dopamine-containing neurons in the substantia nigra pars compacta (3, 4) and the accumulation of Lewy bodies, proteinaceous intracytoplasmic accumulations of eosinophilic material that stain for ubiquitin (5). Novel insights into the molecular mechanisms of the pathogenesis of PD have come from the identification of genes associated with rare forms of familial PD (6). Mutations in ␣-synuclein (A53T and A30P) are linked to autosomal dominant PD (7,8). This led to the discovery that ␣-synuclein is a major component of Lewy bodies and suggests that derangements in ␣-synuclein could be a major cause or contributor to the pathogenesis of sporadic PD (9, 10). Consistent with this notion are observations that overexpression of ␣-synuclein in transgenic fruit flies and mice causes a Parkinsonian phenotype and replicates many of the pathological features of PD (11-13).Other autosomal dominant genes or loci linked to PD have been described and include a mutation (I93M) in ubiquitin carboxyl-terminal-hydrolase-L1 (14), a member of the ubiquitin C-terminal hydrolase family that hydrolyzes small C-terminal adducts of ubiquitin to generate ubiquitin monomers and is involved in facilitating the degradation and processing of proteins through the 26 S proteasome. Thus, derangements in ubiquitin processing may be linked to the pathogenesis of PD. Linkages to chromosome 2P and 4P have been described, as well as yet to be identified loci, but the genes await identification (15-17).Mutations in the Parkin gene are responsible for autosomal recessive PD (18). Several Parkin-associated pedigrees have been described with both deletions and point mutations, as well as compound heterozygosity causing autosomal recessive PD (19)(20)(21). Recent studies suggest that mutations in Parkin are the major cause of autosomal recessive familial PD (19); thus, understanding the function of Parkin and how mutations interfere with the function of Parkin may provide novel insights into the pathogenesis of PD. The function of the Parkin protein remains unknown. However, Parkin shows mild homology to ubiquitin at the N terminus and contains two ring-finger motifs and an in-between ring-finger (IBR) domain at the C terminus (22). Recently, a few proteins with ring-finger motifs similar to Parkin were shown to be involved in E2-dependent ubiquitination (23-26). Ubiquitination requires the ATP-dependent activation of ubiquitin by the ubiquitin-activating enzyme E1. Ubiquitin is transferred to an E2 ubiquitin-conjugating enzyme, which works in conjunction with an E3 ubiquitin-protein ligase to ubiquitinate substrate proteins (23,24). The existence of two ring-finger motifs and the N-terminal homology to ubiquitin suggests that Parkin may be involved in the ubiquitination pathw...

Parkin is an E3 ubiquitin ligase involved in the ubiquitination of proteins that are important in the survival of dopamine neurons in Parkinson's disease (PD). We show that parkin is S-nitrosylated in vitro, as well as in vivo in a mouse model of PD and in brains of patients with PD and diffuse Lewy body disease. Moreover, S-nitrosylation inhibits parkin's ubiquitin E3 ligase activity and its protective function. The inhibition of parkin's ubiquitin E3 ligase activity by S-nitrosylation could contribute to the degenerative process in these disorders by impairing the ubiquitination of parkin substrates.

Parkinson disease is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons and the presence of intracytoplasmic-ubiquitinated inclusions (Lewy bodies). Mutations in alpha-synuclein (A53T, A30P) and parkin cause familial Parkinson disease. Both these proteins are found in Lewy bodies. The absence of Lewy bodies in patients with parkin mutations suggests that parkin might be required for the formation of Lewy bodies. Here we show that parkin interacts with and ubiquitinates the alpha-synuclein-interacting protein, synphilin-1. Co-expression of alpha-synuclein, synphilin-1 and parkin result in the formation of Lewy-body-like ubiquitin-positive cytosolic inclusions. We further show that familial-linked mutations in parkin disrupt the ubiquitination of synphilin-1 and the formation of the ubiquitin-positive inclusions. These results provide a molecular basis for the ubiquitination of Lewy-body-associated proteins and link parkin and alpha-synuclein in a common pathogenic mechanism through their interaction with synphilin-1.

It is widely accepted that the familial Parkinson's disease (PD)-linked gene product, parkin, functions as a ubiquitin ligase involved in protein turnover via the ubiquitin-proteasome system. Substrates ubiquitinated by parkin are hence thought to be destined for proteasomal degradation. Because we demonstrated previously that parkin interacts with and ubiquitinates synphilin-1, we initially expected synphilin-1 degradation to be enhanced in the presence of parkin. Contrary to our expectation, we found that synphilin-1 is normally ubiquitinated by parkin in a nonclassical, proteasomal-independent manner that involves lysine 63 (K63)-linked polyubiquitin chain formation. Parkin-mediated degradation of synphilin-1 occurs appreciably only at an unusually high parkin to synphilin-1 expression ratio or when primed for lysine 48 (K48)-linked ubiquitination. In addition we found that parkin-mediated ubiquitination of proteins within Lewy-body-like inclusions formed by the coexpression of synphilin-1, ␣-synuclein, and parkin occurs predominantly via K63 linkages and that the formation of these inclusions is enhanced by K63-linked ubiquitination. Our results suggest that parkin is a dual-function ubiquitin ligase and that K63-linked ubiquitination of synphilin-1 by parkin may be involved in the formation of Lewy body inclusions associated with PD.

Mutations in parkin are largely associated with autosomal recessive juvenile parkinsonism. The underlying mechanism of pathogenesis in parkin-associated Parkinson's disease (PD) is thought to be due to the loss of parkin's E3 ubiquitin ligase activity. A subset of missense and nonsense point mutations in parkin that span the entire gene and represent the numerous inheritance patterns that are associated with parkin-linked PD were investigated for their E3 ligase activity, localization and their ability to bind, ubiquitinate and effect the degradation of two substrates, synphilin-1 and aminoacyl-tRNA synthetase complex cofactor, p38. Parkin mutants vary in their intracellular localization, binding to substrates and enzymatic activity, yet they are ultimately deficient in their ability to degrade substrate. These results suggest that not all parkin mutations result in loss of parkin's E3 ligase activity, but they all appear to manifest as loss-of-function mutants due to defects in solubility, aggregation, enzymatic activity or targeting proteins to the proteasome for degradation.

Autosomal-recessive juvenile parkinsonism (AR-JP) is caused by loss-of-function mutations of the parkin gene. Parkin, a RING-type E3ubiquitin ligase, is responsible for the ubiquitination and degradation of substrate proteins that are important in the survival of dopamine neurons in Parkinson's disease (PD). Accordingly, the abnormal accumulation of neurotoxic parkin substrates attributable to loss of parkin function may be the cause of neurodegeneration in parkin-related parkinsonism. We evaluated the known parkin substrates identified to date in parkin null mice to determine whether the absence of parkin results in accumulation of these substrates. Here we show that only the aminoacyl-tRNA synthetase cofactor p38 is upregulated in the ventral midbrain/hindbrain of both young and old parkin null mice. Consistent with upregulation in parkin knock-out mice, brains of AR-JP and idiopathic PD and diffuse Lewy body disease also exhibit increased level of p38. In addition, p38 interacts with parkin and parkin ubiquitinates and targets p38 for degradation. Furthermore, overexpression of p38 induces cell death that increases with tumor necrosis factor-␣ treatment and parkin blocks the pro-cell death effect of p38, whereas the R42P, familial-linked mutant of parkin, fails to rescue cell death. We further show that adenovirus-mediated overexpression of p38 in the substantia nigra in mice leads to loss of dopaminergic neurons. Together, our study represents a major advance in our understanding of parkin function, because it clearly identifies p38 as an important authentic pathophysiologic substrate of parkin. Moreover, these results have important implications for understanding the molecular mechanisms of neurodegeneration in PD.

Parkinson's disease (PD) is a common neurodegenerative disorder marked by movement impairment caused by a selective degeneration of dopaminergic neurons. The mechanism for dopaminergic neuronal degeneration in PD is not completely clear, but it is believed that oxidative and nitrosative stress plays an important role during the pathogenesis of PD. This notion is supported by various studies that several indices of oxidative and nitrosative stress are increased in PD patients. In recent years, different pathways that are known to be important for neuronal survival have been shown to be affected by oxidative and nitrosative stress. Apart from the well-known oxidative free radicals induced protein nitration, lipid peroxidation and DNA damage, increasing evidence also suggests that some neuroprotective pathways can be affected by nitric oxide through S-nitrosylation. In addition, the selective dopaminergic neurodegeneration suggests that generation of oxidative stress associated with the metabolism of dopamine is an important contributor. Thorough understanding of how oxidative stress can contribute to the pathogenesis of PD will help formulate potential therapy for the treatment of this neurodegenerative disorder in the future.

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