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Grb10-Interacting GYF Protein-2 (GIGYF2) is a candidate gene for PARK11 locus. To date, seven different GIGYF2 missense mutations have been identified in patients with familial Parkinson disease (PD) of European descent. To clarify the pathogenic role of GIGYF2 in PD, we analyzed the frequency of GIGYF2 mutations in 389 Japanese patients with PD (including 93 patients with late-onset familial PD, 276 with sporadic PD, and 20 with a single heterozygous mutation in the PD-associated genes), and 336 Japanese normal controls, by direct sequencing and/or high-resolution melting analysis. None of the reported GIGYF2 mutations or digenic mutations were detected. Two novel non-synonymous variants were identified (p.Q1211delQ and p.H1023Q), however, we could not determine their roles in PD. In summary, we found no evidence for PD-associated roles of GIGYF2 mutations. Our data suggest that GIGYF2 is unlikely to play a major role in PD in Japanese patients, similar to other populations.

DJ-1 mutations cause autosomal recessive parkinsonism (ARP). Although some reports of DJ-1 mutations have been published, there is lack of information on the prevalence of these mutations in large-scale studies of both familial and sporadic parkinsonism. In this genetic screening study, we analyzed the distribution and frequency of DJ-1 mutations by direct nucleotide sequencing of coding exons and exon-intron boundaries of DJ-1, in 386 parkin-negative parkinsonism patients (371 index cases: 67 probands of autosomal recessive parkinsonism families, 90 probands of autosomal dominant parkinsonism families, 201 patients with sporadic parkinsonism, and 13 with unknown family histories) from 12 countries (Japan 283, China 27, Taiwan 22, Korea 22, Israel 16, Turkey 5, Philippines 2, Bulgaria 2, Greece 2, Tunisia 1, USA 2, Ukraine 1, unknown 1). None had causative mutation in DJ-1, suggesting DJ-1 mutation is very rare among patients with familial and sporadic parkinsonism from Asian countries and those with other ethnic background. This is in contrast to the higher frequencies and worldwide distribution of parkin- and PINK1-related parkinsonism in ARP and sporadic parkinsonism. Thus, after obtaining clinical information, screening for mutations in (1) parkin, (2) PINK1, (3) DJ-1, (4) ATP13A2 should be conducted in that order, in ARP and sporadic parkinsonism, based on their reported frequencies. In addition, haplotype analysis should be employed to check for homozygosity of 1p36, which harbors a cluster of causative genes for ARP such as DJ-1, PINK1 and ATP13A2 in ARP and sporadic parkinsonism, especially in parkinsonism with consanguinity.

We and other workers found markedly increased levels of proinflammatory cytokines and apoptosis-related proteins in parkinsonian brain. Although the pathogenesis of Parkinson's disease (PD) remains enigmatic, apoptosis might be involved in the degeneration of dopaminergic neurons in PD. To investigate the possible presence of other inflammatory cytokines and/or apoptosis-related protein, the levels of p53 protein, interferon-gamma, and NF-kappaB were measured for the first time in the brain (substantia nigra, caudate nucleus, putamen, cerebellum, and frontal cortex) from control and parkinsonian patients by a highly sensitive sandwich enzyme-linked immunosorbent assay. The p53 protein level in the caudate nucleus was significantly higher in parkinsonian patients than in controls (P<0.05), whereas this protein in the substantia nigra, putamen, and cerebral cortex showed no significant difference between parkinsonian and control subjects. The interferon-gamma level was significantly higher in the nigrostriatal dopaminergic regions (substantia nigra, caudate nucleus, and putamen) in parkinsonian patients than in the controls (P<0.05), but was not significantly different in the cerebellum or frontal cortex between the two groups. In accordance with previous immunohistochemical analysis, the NF-kappaB level in the nigrostriatal dopaminergic regions was significantly higher in parkinsonian patients than in the controls (P<0.05). These data suggest that the significant increase in the levels of p53 protein, interferon-gamma, and NF-kappaB reflect apoptosis and the inflammatory state in the parkinsonian brain and that their elevation is involved in the degeneration of the nigrostriatal dopaminergic neurons.

Considering the involvement of ubiquitin-proteasome system (UPS) in Parkinson's disease (PD), the aim of the present study was to determine the distribution of proteasomes in PD brains. Immunohistochemical studies showed localization of 20S proteasome in the nuclei of neurons of the putamen and substantia nigra of PD. In contrast, no nuclear staining was observed in the same areas of brains of controls. Our results suggest that nuclear localization of 20S proteasome seems to be associated with the pathogenesis of PD.

Vesicular monoamine transporter 2 (VMAT2) is responsible for packing dopamine into vesicles, and reduces the effects of neurotoxins by sequestering them into vesicles. In this report, we tested the hypothesis that VMAT2 is associated with Lewy body (LB) formation by immunohistochemical staining of midbrain and cortical sections of autopsied brains of patients with Parkinson's disease (PD) and diffuse Lewy body disease (DLBD) for VMAT2 using a polyclonal antibody against VMAT2. LBs in the substantia nigra (SN) of PD and DLBD were immunoreactive for VMAT2, especially in the peripheral zone. Previous electron microscopic studies also revealed the presence of numerous dense core vesicles around the LBs, suggesting that these vesicles are related to LB formation. Indeed, the presence of a few vesicle-linked proteins such as synaptophysin and chromogranin A in LBs has been reported. Together with the low expression of VMAT2 in the SN of PD, the involvement of VMAT2 in LBs of the SN suggests the association of this protein in the neurodegeneration of nigral neurons in PD.

Apolipoprotein E receptor 2 (apoER2) is a receptor for apolipoprotein E containing lipoprotein and also for Reelin. Apolipoprotein E-associated risk of developing Alzheimer's disease (AD) may be related to its binding to and clearance by cell surface receptors, including members of the low-density lipoprotein receptor family. Otherwise there is circumstantial evidence that the Reelin signaling pathway may contribute to neurodegeneration in AD. To investigate the role of apoER2 on amyloid deposition and neurodegeneration in vivo, we examined the presence of apoER2 in the brains of APP sw transgenic mice (Tg2576) using three apoER2 monoclonal antibodies. Our immunohistochemical study revealed that apoER2 was localized in fine granular structure and reactive astrocytes surrounding amyloid plaques. The double labeling immunohistochemistry revealed that this granular structure overlaps synaptophysin-positive dystrophic neurites. These findings indicate that neuronal apoER2 may play a role for amyloid deposition and neuronal degeneration in AD.

Nestin is an intermediate filament protein and mainly expressed during the development of the central nervous system. Recently, we reported that nestin is present in the cytoplasm, dendrites, and torpedoes of Purkinje cells in patients from Creutzfeldt-Jakob disease as the pathological change advances. In this study, we focused on the temporal and frontal lobes of the same patients in order to examine the ballooned neurons using anti-human nestin antibody. Although the ballooned neurons showed cytoplasmic immunoreactivity against nestin, the level of nestin immunoreactivity differed in each ballooned neuron. These findings could suggest that ballooned neurons are being reactivated to promote survival, although the role of nestin is not clear.

Vacuole-creating protein (VCP) is a member of the ATPases associated with diverse cellular activities and is a putative sensor protein for degenerative proteins. Immunohistochemical examinations demonstrated that VCP was observed in ubiquitin-positive intraneuronal inclusions in motor neuron disease with dementia, ballooned neurons in Creutzfeldt-Jakob disease, dystrophic neurites of senile plaque in Alzheimer's disease, and Lewy and Marinesco bodies and Lewy neurites in Parkinson's disease, while granules of granulovacuolar degeneration and neurofibrillary tangles in Alzheimer's disease were not positively stained for VCP. These results indicate that VCP reacts with abnormal or misfolded proteins and plays a role in accelerating the process of degeneration and cell death. The elucidation of an association between VCP and these degenerative proteins will provide an important clue for understanding common mechanisms underlying neurodegenerative diseases.

The mechanism of abnormal iron accumulation in the substantia nigra (SN) pars compacta of patients with Parkinson's disease (PD) is unknown. To explore this question, we made a hemiparkinsonism model in monkeys by injecting 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into the caudate or putamen on one side, and compared iron content in the SN and other basal ganglia structures by histochemistry. The injected side, especially the SN pars compacta, showed a marked increase in iron staining. Our study indicates that an injury to the nigrostriatal system by MPTP injection can induce iron accumulation in the SN.

Effects of long-term administration of 1,2,3,4-tetrahydroisoquinoline (TIQ) on the striatal dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) were investigated in mice and rats. Striatal dopamine content significantly increased and DOPAC decreased after repeated administrations of TIQ at a dose of 50 mg/kg/day s.c. for 70 days in mice. In contrast, dopamine and DOPAC content did not show any significant change in rats. The ratio DOPAC/dopamine decreased significantly in mice treated with TIQ for 70 days. The results presented suggest that TIQ reduces the turnover rate of the nigrostriatal dopamine neurons after repeated administration for a long period in mice.

Effect of 1-methyl-4-phenylpyridinium ion (MPP+) on adenosine triphosphate (ATP) synthesis was studied using isolated mitochondrial preparations from mouse brains. Oxidation of glutamate + malate in the mitochondria estimated by polarography was significantly inhibited by MPP+, and synthesis of ATP was inhibited to approximately 9% of that of control by 0.06 mM of MPP+. Oxidation of alpha-glycerophosphate and succinate was not inhibited, and ATP was synthesized normally. Energy crisis appears to be one of the most important mechanisms of neuronal degeneration in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced experimental parkinsonism.

Effects of tetrahydroisoquinoline (TIQ) on mitochondrial respiration, NADH-ubiquinone oxidoreductase (complex I) activity and on adenosine triphosphate (ATP) synthesis were studied using mitochondria prepared from mouse brains. Tetrahydroisoquinoline significantly inhibited mitochondrial respiration supported by glutamate + malate, pyruvate + malate or alpha-ketoglutarate. Activity of complex I and synthesis of ATP were also significantly inhibited by TIQ. Mitochondrial respiration supported by succinate and subsequent ATP synthesis were not inhibited at all by 5 mM of TIQ. Our study has revealed a novel action of TIQ, which has been proposed as a candidate for an endogenous substance that may induce Parkinson's disease.

This is the first report on in vivo effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on mitochondrial respiration in mouse brain. C57/BL mice, injected with 40 mg/kg of MPTP subcutaneously, were sacrificed by cervical dislocation 5-6 h after the injection. Mitochondrial suspensions were prepared from whole brains. Mitochondrial respiration was studied polarographically. The state 3 respiration, i.e., the active respiration in the presence of tricarboxylic acid (TCA) cycle substrates and ADP with coupled phosphorylation of ADP to ATP, was significantly inhibited in mice treated with MPTP. This inhibition was prevented by pretreatment of mice with pargyline. Activity of mitochondrial NADH-ubiquinone oxidoreductase was also inhibited in mice treated with MPTP.