Shin-ichiro Kubo

Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase

Autosomal recessive juvenile parkinsonism (AR–JP), one of the most common familial forms of Parkinson disease, is characterized by selective dopaminergic neural cell death and the absence of the Lewy body, a cytoplasmic inclusion body consisting of aggregates of abnormally accumulated proteins. We previously cloned PARK2, mutations of which cause AR–JP (ref. 2), but the function of the gene product, parkin, remains unknown. We report here that parkin is involved in protein degradation as a ubiquitin-protein ligase collaborating with the ubiquitin-conjugating enzyme UbcH7, and that mutant parkins from AR–JP patients show loss of the ubiquitin-protein ligase activity. Our findings indicate that accumulation of proteins that have yet to be identified causes a selective neural cell death without formation of Lewy bodies. Our findings should enhance the exploration of the molecular mechanisms of neurodegeneration in Parkinson disease as well as in other neurodegenerative diseases that are characterized by involvement of abnormal protein ubiquitination, including Alzheimer disease, other tauopathies, CAG triplet repeat disorders and amyotrophic lateral sclerosis.

Immunohistochemical and subcellular localization of parkin protein: Absence of protein in autosomal recessive juvenile parkinsonism patients

Autosomal recessive juvenile parkinsonism (AR‐JP) is a distinct clinical entity characterized by a selective degeneration of nigral neurons. Recently, the parkin gene responsible for AR‐JP has been identified. Now, we report the subcellular localization of Parkin protein in patients with AR‐JP or Parkinsons disease (PD) and in controls by immunoblotting and immunohistochemistry using antibodies raised against the Parkin molecule. Parkin protein was absent in all regions of the brains of patients with AR‐JP. Parkin protein was not decreased in the brains of sporadic PD patients. Immunoreactivity was detected in a few Lewy bodies. Parkin protein was located in both the Golgi complex and cytosol. Ann Neurol 1999;45:668–672

Parkin is associated with cellular vesicles

We recently identified a novel gene, parkin, as a pathogenic gene for autosomal recessive juvenile parkinsonism. Parkin encodes a 52‐kDa protein with a ubiquitin‐like domain and two RING‐finger motifs. To provide a insight into the function of parkin, we have examined its intracellular distribution in cultured cells. We found that parkin was localized in the trans‐Golgi network and the secretory vesicles in U‐373MG or SH‐SY5Y cells by immunocytochemical analyses. In the subsequent subcellular fractionation studies of rat brain, we showed that parkin was copurified with the synaptic vesicles (SVs) when we used low ionic conditions throughout the procedure. An immunoelectromicroscopic analysis indicated that parkin was present on the SV membrane. Parkin was readily released from SVs into the soluble phase by increasing ionic strength at neutral pH, but not by a non‐ionic detergent. To elucidate its responsible region for membrane association, we transfected with green fluorescent protein‐tagged deletion mutants of parkin into COS‐1 cells followed by subcellular fractionation. We demonstrated the ability of parkin to bind to the membranes through a broad region except for the ubiquitin‐like domain. The significance of SV localization of parkin is discussed.

Pathogenesis of familial Parkinson's disease: new insights based on monogenic forms of Parkinson's disease

Parkinson’s disease (PD) is one of the most common movement disorders caused by the loss of dopaminergic neuronal cells. The molecular mechanisms underlying neuronal degeneration in PD remain unknown; however, it is now clear that genetic factors contribute to the pathogenesis of this disease. Approximately, 5% of patients with clinical features of PD have clear familial etiology, which show a classical recessive or dominant Mendelian mode of inheritance. Over the decade, more than 15 loci and 11 causative genes have been identified so far and many studies shed light on their implication in not only monogenic but also sporadic form of PD. Recent studies revealed that PD‐associated genes play important roles in cellular functions, such as mitochondrial functions, ubiquitin‐proteasomal system, autophagy‐lysosomal pathway and membrane trafficking. Furthermore, the proteins encoded by PD‐associated genes can interact with each other and such gene products may share a common pathway that leads to nigral degeneration. However, their precise roles in the disease and their normal functions remain poorly understood. In this study, we review recent progress in knowledge about the genes associated with familial PD.

Clinicogenetic study of mutations in LRRK2 exon 41 in Parkinson's disease patients from 18 countries

We screened LRRK2 mutations in exon 41 in 904 parkin‐negative Parkinsons disease (PD) patients (868 probands) from 18 countries across 5 continents. We found three heterozygous missense (novel I2012T, G2019S, and I2020T) mutations in LRRK2 exon 41. We identified 11 (1.3%) among 868 PD probands, including 2 sporadic cases and 8 (6.2%) of 130 autosomal dominant PD families. The LRRK2 mutations in exon 41 exhibited relatively common and worldwide distribution. Among the three mutations in exon 41, it has been reported that Caucasian patients with G2019S mutation have a single‐founder effect. In the present study, Japanese patients with G2019S were unlikely to have a single founder from the Caucasian patients. In contrast, I2020T mutation has a single‐founder effect in Japanese patients. Clinically, patients with LRRK2 mutations had typical idiopathic PD. Notably, several patients developed dementia and psychosis, and one with I2020T had low cardiac 123I‐metaiodobenzylguanidine (MIBG) heart/mediastinum ratio, although the ratio was not low in other patients with I2020T or G2019S. Clinical phenotypes including psychosis, dementia, and MIBG ratios are also heterogeneous, similar to neuropathology, in PD associated with LRRK2 mutations.

Expression of 8-oxoguanine DNA glycosylase (OGG1) in Parkinson’s disease and related neurodegenerative disorders

Oxidative stress including DNA oxidation is implicated in Parkinson’s disease (PD). We postulated that DNA repair enzymes such as 8-oxoguanosine DNA glycosylase (OGG1) are involved in the PD process. We performed immunohistochemical and biochemical studies on brains of patients with PD and those of patients with progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) as disease controls, and control subjects. We found higher expression levels of mitochondrial isoforms of OGG1 enzymes in the substantia nigra (SN) in cases of PD. Furthermore, Western blot analysis revealed high OGG1 levels in the SN of the patients with PD. Our results indicate the importance of oxidative stress within the susceptible lesions in the pathogenesis of PD.

Parkin stabilizes PINK1 through direct interaction.

Parkinson disease (PD) is the most common movement disorder and is characterized by dopaminergic dysfunction. The majority of PD cases are sporadic; however, the discovery of genes linked to rare familial forms of the disease has provided crucial insight into the molecular mechanisms of disease pathogenesis. Multiple genes mediating familial forms of Parkinsons disease (PD) have been identified, such as parkin (PARK2) and phosphatase and tensin homologue deleted on chromosome ten (PTEN)-induced putative kinase 1: PINK1 (PARK6). Here, we showed that Parkin directly interacts with PINK1, but did not bind to pathogenic PINK1 mutants. Parkin, but not its pathogenic mutants, stabilizes PINK1 by interfering with its degradation via the ubiquitin-mediated proteasomal pathway. In addition, the interaction between Parkin and PINK1 resulted in reciprocal reduction of their solubility. Our results indicate that Parkin regulates PINK1 stabilization via direct interaction with PINK1, and operates through a common pathway with PINK1 in the pathogenesis of early-onset PD.

Progress in the pathogenesis and genetics of Parkinson's disease

Recent progresses in the pathogenesis of sporadic Parkinsons disease (PD) and genetics of familial PD are reviewed. There are common molecular events between sporadic and familial PD, particularly between sporadic PD and PARK1-linked PD due to α-synuclein (SNCA) mutations. In sporadic form, interaction of genetic predisposition and environmental factors is probably a primary event inducing mitochondrial dysfunction and oxidative damage resulting in oligomer and aggregate formations of α-synuclein. In PARK1-linked PD, mutant α-synuclein proteins initiate the disease process as they have increased tendency for self-aggregation. As highly phosphorylated aggregated proteins are deposited in nigral neurons in PD, dysfunctions of proteolytic systems, i.e. the ubiquitin–proteasome system and autophagy–lysosomal pathway, seem to be contributing to the final neurodegenerative process. Studies on the molecular mechanisms of nigral neuronal death in familial forms of PD will contribute further on the understanding of the pathogenesis of sporadic PD.

Association between a polymorphism of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) gene and sporadic Parkinson's disease

We found a novel polymorphism (S/Y18) of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) gene a mutation of which is expected to contribute to the etiology of a form of familial Parkinsons disease (PD). We report the frequency of this polymorphism in 313 patients with sporadic PD and 302 control subjects (Japanese and Caucasians). The frequency of the mutant allele (Y) was significantly higher in Japanese control subjects (51.2%) than in Japanese PD patients (43.4%) (chi(2)=3.917, p=0.048<0.05). It appears that this polymorphism has a weak protective factor against PD in at least the Japanese population. The frequencies of Y allele and S/Y and Y/Y genotypes in the PD patients and the controls were more significantly higher in Japanese than in Caucasian population (p<0.0001). It seems that the role of this polymorphism in PD may be different between Caucasian and Japanese populations.

Toxic effects of dopamine metabolism in Parkinson's disease

Levodopa is the most effective medication for Parkinsons disease (PD). In contrast, there is evidence that levodopa and its metabolites such as dopa/dopamine quinone are toxic for nigral neurons based on in vitro studies. Moreover, there is growing evidence that oxidative stress and mitochondrial dysfunction contribute the pathogenesis of PD. Thus, studies for oxidative stress give us good information for elucidating the pathogenesis of PD. In this regard, it is mandatory to develop markers such as 4-hydroxy-nonenal (HNE). HNE is a product of lipid peroxidation. Indeed, immunohistochemical studies have revealed that HNE-modified proteins accumulate within ragged red fibers (RRFs). This finding indicated that mitochondrial impairment may be linked to oxidative stress. Moreover, HNE-modified proteins accumulate in nigral neurons. In PD, mitochondrial dysfunction such as complex I deficiency has also been reported. In addition, HNE can modify alpha-synuclein (SNCA). Subsequently, this modification may trigger the aggregation of this protein. At a minimum, this modification could be associated with oligomer formation or fibrillation of SNCA.