Manabu Funayama

PINK1 autophosphorylation upon membrane potential dissipation is essential for Parkin recruitment to damaged mitochondria

Dysfunction of PINK1, a mitochondrial Ser/Thr kinase, causes familial Parkinsons disease (PD). Recent studies have revealed that PINK1 is rapidly degraded in healthy mitochondria but accumulates on the membrane potential (ΔΨm)-deficient mitochondria, where it recruits another familial PD gene product, Parkin, to ubiquitylate the damaged mitochondria. Despite extensive study, the mechanism underlying the homeostatic control of PINK1 remains unknown. Here we report that PINK1 is autophosphorylated following a decrease in ΔΨm and that most disease-relevant mutations hinder this event. Mass spectrometric and mutational analyses demonstrate that PINK1 autophosphorylation occurs at Ser228 and Ser402, residues that are structurally clustered together. Importantly, Ala mutation of these sites abolishes autophosphorylation of PINK1 and inhibits Parkin recruitment onto depolarized mitochondria, whereas Asp (phosphorylation-mimic) mutation promotes mitochondrial localization of Parkin even though autophosphorylation was still compromised. We propose that autophosphorylation of Ser228 and Ser402 in PINK1 is essential for efficient mitochondrial localization of Parkin.

Mitochondrial dysfunction associated with increased oxidative stress and α-synuclein accumulation in PARK2 iPSC-derived neurons and postmortem brain tissue

BackgroundParkinson’s disease (PD) is a neurodegenerative disease characterized by selective degeneration of dopaminergic neurons in the substantia nigra (SN). The familial form of PD, PARK2, is caused by mutations in the parkin gene. parkin-knockout mouse models show some abnormalities, but they do not fully recapitulate the pathophysiology of human PARK2.ResultsHere, we generated induced pluripotent stem cells (iPSCs) from two PARK2 patients. PARK2 iPSC-derived neurons showed increased oxidative stress and enhanced activity of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. iPSC-derived neurons, but not fibroblasts or iPSCs, exhibited abnormal mitochondrial morphology and impaired mitochondrial homeostasis. Although PARK2 patients rarely exhibit Lewy body (LB) formation with an accumulation of α-synuclein, α-synuclein accumulation was observed in the postmortem brain of one of the donor patients. This accumulation was also seen in the iPSC-derived neurons in the same patient.ConclusionsThus, pathogenic changes in the brain of a PARK2 patient were recapitulated using iPSC technology. These novel findings reveal mechanistic insights into the onset of PARK2 and identify novel targets for drug screening and potential modified therapies for PD.

Analysis of Lrrk2 R1628P as a risk factor for Parkinson's disease

Common genetic variants that increase the risk for Parkinsons disease may differentiate patient subgroups and influence future individualized therapeutic strategies. Herein we show evidence for leucine‐rich repeat kinase 2 (LRRK2) c.4883G>C (R1628P) as a risk factor in ethnic Chinese populations. A study of 1,986 individuals from 3 independent centers in Taiwan and Singapore demonstrates that Lrrk2 R1628P increases risk for Parkinsons disease (odds ratio, 1.84; 95% confidence interval, 1.20–2.83; p = 0.006). Haplotype analysis suggests an ancestral founder for carriers approximately 2,500 years ago. These findings support the importance of LRRK2 variants in sporadic Parkinsons disease. Ann Neurol 2008

A rotarod test for evaluation of motor skill learning.

The rotarod test is widely used to evaluate the motor coordination of rodents, and is especially sensitive in detecting cerebellar dysfunction. However, mice with striatal dopamine depletion show only mild or no motor deficit on the typical accelerating rotarod. This suggests that dopamine-depleted mice are useful as animal models for non-motor symptoms, because the influence of motor deficit is minimum and easy to discriminate from cognitive aspects of the behavioral change. The typical accelerating rotarod test is designed to evaluate maximal motor performance and is not optimized to detect motor skill learning. In an attempt to make the test more selective to motor skill learning rather than maximal gait performance, we modified the rotarod test by using a slowly rotating large drum to obtain a steep learning curve. Furthermore, administration of nomifensine, a dopamine uptake inhibitor, improved the learning. On the other hand, apomorphine, an agonist of dopamine autoreceptor, a dopaminergic toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) impaired the learning. These pharmacological profiles fit the involvement of the so-called phasic dopamine neurotransmission. Using our modified procedure, we found impaired learning of Parkin-deficit mice, which has not been detected in typical accelerating rotarod. The modified rotarod test would be useful for evaluation of dopamine involvement in the acquisition of motor skill learning.

CHCHD2 mutations in autosomal dominant late-onset Parkinson's disease: a genome-wide linkage and sequencing study

BACKGROUND Identification of causative genes in mendelian forms of Parkinsons disease is valuable for understanding the cause of the disease. We did genetic studies in a Japanese family with autosomal dominant Parkinsons disease to identify novel causative genes. METHODS We did a genome-wide linkage analysis on eight affected and five unaffected individuals from a family with autosomal dominant Parkinsons disease (family A). Subsequently, we did exome sequencing on three patients and whole-genome sequencing on one patient in family A. Variants were validated by Sanger sequencing in samples from patients with autosomal dominant Parkinsons disease, patients with sporadic Parkinsons disease, and controls. Participants were identified from the DNA bank of the Comprehensive Genetic Study on Parkinsons Disease and Related Disorders (Juntendo University School of Medicine, Tokyo, Japan) and were classified according to clinical information obtained by neurologists. Splicing abnormalities of CHCHD2 mutants were analysed in SH-SY5Y cells. We used the Fishers exact test to calculate the significance of allele frequencies between patients with sporadic Parkinsons disease and unaffected controls, and we calculated odds ratios and 95% CIs of minor alleles. FINDINGS We identified a missense mutation (CHCHD2, 182C>T, Thr61Ile) in family A by next-generation sequencing. We obtained samples from a further 340 index patients with autosomal dominant Parkinsons disease, 517 patients with sporadic Parkinsons disease, and 559 controls. Three CHCHD2 mutations in four of 341 index cases from independent families with autosomal dominant Parkinsons disease were detected by CHCHD2 mutation screening: 182C>T (Thr61Ile), 434G>A (Arg145Gln), and 300+5G>A. Two single nucleotide variants (-9T>G and 5C>T) in CHCHD2 were confirmed to have different frequencies between sporadic Parkinsons disease and controls, with odds ratios of 2·51 (95% CI 1·48-4·24; p=0·0004) and 4·69 (1·59-13·83, p=0·0025), respectively. One single nucleotide polymorphism (rs816411) was found in CHCHD2 from a previously reported genome-wide association study; however, there was no significant difference in its frequency between patients with Parkinsons disease and controls in a previously reported genome-wide association study (odds ratio 1·17, 95% CI 0·96-1·19; p=0·22). In SH-SY5Y cells, the 300+5G>A mutation but not the other two mutations caused exon 2 skipping. INTERPRETATION CHCHD2 mutations are associated with, and might be a cause of, autosomal dominant Parkinsons disease. Further genetic studies in other populations are needed to confirm the pathogenicity of CHCHD2 mutations in autosomal dominant Parkinsons disease and susceptibility for sporadic Parkinsons disease, and further functional studies are needed to understand how mutant CHCHD2 might play a part in the pathophysiology of Parkinsons disease. FUNDING Japan Society for the Promotion of Science; Japanese Ministry of Education, Culture, Sports, Science and Technology; Japanese Ministry of Health, Labour and Welfare; Takeda Scientific Foundation; Cell Science Research Foundation; and Nakajima Foundation.

Phenotypic spectrum of patients with PLA2G6 mutation and PARK14-linked parkinsonism

Background: PLA2G6 is the causative gene for infantile neuroaxonal dystrophy, neurodegeneration associated with brain iron accumulation, and Karak syndrome. Based on previous reports, patients with PLA2G6 mutations could show axonal dystrophy, dystonia, dementia, and cerebellar signs. Recently, PLA2G6 was also reported as the causative gene for early-onset PARK14-linked dystonia-parkinsonism. Methods: To clarify the role of PLA2G6 mutation in parkinsonism, we conducted mutation analysis in 29 selected patients with very early-onset (≤30, mean 21.2 ± 8.4 years, ±SD) parkinsonism. These patients had other clinical features (e.g., mental retardation/dementia [14/29], psychosis [15/29], dystonia [11/29], and hyperreflexia [11/29]). Results: Two novel compound heterozygous PLA2G6 mutations were detected (patient A: p.F72L/p.R635Q; patients B1 and B2: p.Q452X/p.R635Q). All 3 patients had early-onset l-dopa–responsive parkinsonism with dementia and frontotemporal lobar atrophy. Disease progression was relatively rapid. SPECT in patient B1 showed frontotemporal lobar hypoperfusion. MRI in patient A showed iron accumulation in the substantia nigra and striatum. Conclusions: Although the clinical presentation of PLA2G6-associated neurodegeneration was reported to be homogeneous, our findings suggest patients with PLA2G6 mutation could show heterogeneous phenotype such as dystonia-parkinsonism, dementia, frontotemporal atrophy/hypoperfusion, with or without brain iron accumulation. Based on the clinical heterogeneity, the functional roles of PLA2G6 and the roles of PLA2G6 variants including single heterozygous mutations should be further elucidated in patients with atypical parkinsonism, dementia, or Parkinson disease. PLA2G6 mutations should be considered in patients with early-onset l-dopa–responsive parkinsonism and dementia with frontotemporal lobar atrophy.

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.

PARK9-linked parkinsonism in eastern Asia: mutation detection in ATP13A2 and clinical phenotype.

PARK9, a form of autosomal recessive parkinsonism, or Kufor-Rakeb syndrome (KRS), is characterized by subacute or slowly progressive, juvenile-onset, levodopa-responsive parkinsonism, pyramidal signs, dementia, and supranuclear gaze palsy.1–5 Recently, ATP13A2 was identified as the causative gene for PARK9 in Chilean and Jordanian families.4 This gene contains 29 exons encoding a lysosomal type 5 P-type ATPase. Six mutations have been reported in only five probands so far.4,5 Here, we describe a Japanese patient with KRS with a novel mutation who developed early onset parkinsonism, dementia, and other features. We also describe PET findings of PARK9 -linked parkinsonism. ### Methods. Haplotype analysis was conducted in 117 (mainly Japanese) patients with early onset (≤50, 26.8 ± 11.7 years, mean ± SD) parkinsonism. Among them, 14 patients had dementia. Patients who exhibited homozygosity on PARK9 locus by haplotype analysis underwent direct sequencing for all 29 exons (e-Methods on the Neurology ® Web site at www.neurology.org); the remaining patients underwent direct sequencing for exons 13, 16, and 26, in which mutations …

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.

Mutation Analysis of the PINK1 Gene in 391 Patients With Parkinson Disease

OBJECTIVES To determine the frequency, distribution, and clinical features of Parkinson disease (PD) with PINK1 mutations. DESIGN Retrospective clinical and genetic review. SETTING University hospital. PATIENTS We performed extensive mutation analyses of PINK1 in 414 PD patients negative for parkin mutations (mean [SD] age at onset, 42.8 [14.3] years), including 391 unrelated patients (190 patients with sporadic PD and 201 probands of patients with familial PD) from 13 countries. RESULTS We found 10 patients with PD from 9 families with PINK1 mutations and identified 7 novel mutations (2 homozygous mutations [p.D297MfsX22 and p.W437R] and 5 single heterozygous mutations [p.A78V, p.P196QfsX25, p.M342V, p.W437R, and p.N542S]). No compound heterozygous mutations were found. The frequency of homozygous mutations was 4.26% (2 of 47) in families with autosomal recessive PD and 0.53% (1 of 190) in patients with sporadic PD. The frequency of heterozygous mutations was 1.89% (2 of 106) in families with potential autosomal dominant PD and 1.05% (2 of 190) in patients with sporadic PD. The mean (SD) age at onset in patients with single heterozygous mutations (53.6 [11.1] years; range, 39-69 years) was higher than that in patients with homozygous mutations (34.0 [20.3] years; range, 10-55 years). Myocardial iodine-123 metaiodobenzylguanidine uptake was low in patients with heterozygous mutations but not in those with homozygous mutations. CONCLUSIONS Our results suggest that homozygous PINK1 mutations tend to be diagnosed as the early-onset autosomal recessive form of PD. Single heterozygous mutations may contribute to the development of sporadic PD and also could be an additional genetic predisposition for developing familial PD. The reduced myocardial iodine-123 metaiodobenzylguanidine uptake observed in patients with single heterozygous PINK1 mutations is similar to that seen in patients with sporadic PD.