Kenny K.K. Chung

Parkin ubiquitinates the α-synuclein-interacting protein, synphilin-1 : implications for Lewy-body formation in Parkinson disease

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 α-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 α-synuclein–interacting protein, synphilin-1. Co-expression of α-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 α-synuclein in a common pathogenic mechanism through their interaction with synphilin-1.

Parkin Mediates Nonclassical, Proteasomal-Independent Ubiquitination of Synphilin-1: Implications for Lewy Body Formation

It is widely accepted that the familial Parkinsons 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, α-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.

Accumulation of the Authentic Parkin Substrate Aminoacyl-tRNA Synthetase Cofactor, p38/JTV-1, Leads to Catecholaminergic Cell Death

Autosomal-recessive juvenile parkinsonism (AR-JP) is caused by loss-of-function mutations of the parkin gene. Parkin, a RING-type E3 ubiquitin ligase, is responsible for the ubiquitination and degradation of substrate proteins that are important in the survival of dopamine neurons in Parkinsons 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-α 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.

The role of the ubiquitin-proteasomal pathway in Parkinson's disease and other neurodegenerative disorders

0166-2236/01/

Oxidative and nitrosative stress in Parkinson's disease

– see front matter ©2001 Elsevier Science Ltd. All rights reserved. PII: S0166-2236(00)01998-6 Most, if not all, neurodegenerative diseases are marked by the presence of protein aggregates or inclusion bodies1. These include the prion protein (PrP) plaques in Prion disease, amyloid plaques and neurofibrillary tangles in Alzheimer’s disease (AD), Lewy bodies in Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), nuclear inclusions in the poly-glutamine repeat diseases such as Huntington’s disease (HD), spinocerebellar ataxias (SCA), dentatorubral and pallidoluysian atrophy (DRPLA), as well as other neurodegenerative diseases (Table 1). The linkage of two genes within the ubiquitin-proteasomal pathway (UPP) in hereditary PD (Refs 2,3), and recent advances in other neurological disorders, clearly indicate that the UPP plays a crucial role in the pathogenesis of neurodegenerative diseases, and has elevated the importance of the UPP in these disorders. The close relationship between neurodegeneration and the ubiquitin system has long been implicated through the consistent findings of ubiquitin-positive protein aggregates in various neuropathological studies. In fact, the observation of ubiquitinated-protein inclusion bodies is one of the hallmarks of neurodegeneration. One general idea is that, under certain adverse conditions [including oxidative stress, protein misfolding during endoplasmic reticulum (ER) stress and aging], damaged proteins can accumulate in the cell. In addition, abnormal accumulation of proteins could occur owing to altered post-translational modification of newly synthesized proteins, abnormal proteolytic cleavage, diminished clearance of degraded protein and/or improper expression or altered gene splicing. The UPP might play a prominent role in the detoxification and targeting of damaged proteins for degradation. Under some conditions, the protein damage could be so severe that the clearance of damaged protein by the UPP and other degradative pathways might not be able to cope with the demand, resulting in the accumulation of damaged ubiquitin-tagged proteins and ultimately neuronal dysfunction and/or death.

SEPT5_v2 is a parkin-binding protein

Parkinsons 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.

S-nitrosylation of XIAP compromises neuronal survival in Parkinson's disease

Mutations in parkin are associated with various inherited forms of Parkinsons disease (PD). Parkin is a ubiquitin ligase enzyme that catalyzes the covalent attachment of ubiquitin moieties onto substrate proteins destined for proteasomal degradation. The substrates of parkin-mediated ubiquitination have yet to be completely identified. Using a yeast two-hybrid screen, we isolated the septin, human SEPT5_v2 (also known as cell division control-related protein 2), as a putative parkin-binding protein. SEPT5_v2 is highly homologous to another septin, SEPT5, which was recently identified as a target for parkin-mediated ubiquitination. SEPT5_v2 binds to parkin at the amino terminus and in the ring finger domains. Several lines of evidence have validated the putative link between parkin and SEPT5_v2. Parkin co-precipitates with SEPT5_v2 from human substantia nigra lysates. Parkin ubiquitinates SEPT5_v2 in vitro, and both SEPT5_v1 and SEPT5_v2 accumulate in brains of patients with ARJP, suggesting that parkin is essential for the normal metabolism of these proteins. These findings suggest that an important relationship exists between parkin and septins.

Alpha-synuclein impairs normal dynamics of mitochondria in cell and animal models of Parkinson's disease.

Inhibitors of apoptosis (IAPs) are a family of highly-conserved proteins that regulate cell survival through binding to caspases, the final executioners of apoptosis. X-linked IAP (XIAP) is the most widely expressed IAP and plays an important function in regulating cell survival. XIAP contains 3 baculoviral IAP repeats (BIRs) followed by a RING finger domain at the C terminal. The BIR domains of XIAP possess anticaspase activities, whereas the RING finger domain enables XIAP to function as an E3 ubiquitin ligase in the ubiquitin and proteasomal system. Our previous study showed that parkin, a protein that is important for the survival of dopaminergic neurons in Parkinsons disease (PD), is S-nitrosylated both in vitro and in vivo in PD patients. S-nitrosylation of parkin compromises its ubiquitin E3 ligase activity and its protective function, which suggests that nitrosative stress is an important factor in regulating neuronal survival during the pathogenesis of PD. In this study we show that XIAP is S-nitrosylated in vitro and in vivo in an animal model of PD and in PD patients. Nitric oxide modifies mainly cysteine residues within the BIR domains. In contrast to parkin, S-nitrosylation of XIAP does not affect its E3 ligase activity, but instead directly compromises its anticaspase-3 and antiapoptotic function. Our results confirm that nitrosative stress contributes to PD pathogenesis through the impairment of prosurvival proteins such as parkin and XIAP through different mechanisms, indicating that abnormal S-nitrosylation plays an important role in the process of neurodegeneration.

The Role of Alpha-Synuclein Oligomerization and Aggregation in Cellular and Animal Models of Parkinson’s Disease

J. Neurochem. (2012) 122, 404–414.

New insights into Parkinson’s disease

α-synuclein (α-syn) is a synaptic protein in which four mutations (A53T, A30P, E46K and gene triplication) have been found to cause an autosomal dominant form of Parkinson’s disease (PD). It is also the major component of intraneuronal protein aggregates, designated as Lewy bodies (LBs), a prominent pathological hallmark of PD. How α-syn contributes to LB formation and PD is still not well-understood. It has been proposed that aggregation of α-syn contributes to the formation of LBs, which then leads to neurodegeneration in PD. However, studies have also suggested that aggregates formation is a protective mechanism against more toxic α-syn oligomers. In this study, we have generated α-syn mutants that have increased propensity to form aggregates by attaching a CL1 peptide to the C-terminal of α-syn. Data from our cellular study suggest an inverse correlation between cell viability and the amount of α-syn aggregates formed in the cells. In addition, our animal model of PD indicates that attachment of CL1 to α-syn enhanced its toxicity to dopaminergic neurons in an age-dependent manner and induced the formation of Lewy body-like α-syn aggregates in the substantia nigra. These results provide new insights into how α-syn-induced toxicity is related to its aggregation.