The Parkinson's Disease-Associated Protein Kinase LRRK2 Modulates Notch Signaling through the Endosomal Pathway.

Leucine-rich repeat kinase 2 (LRRK2) is a key molecule in the pathogenesis of familial and idiopathic Parkinson's disease (PD). We have identified two novel LRRK2-associated proteins, a HECT-type ubiquitin ligase, HERC2, and an adaptor-like protein with six repeated Neuralized domains, NEURL4. LRRK2 binds to NEURL4 and HERC2 via the LRRK2 Ras of complex proteins (ROC) domain and NEURL4, respectively. HERC2 and NEURL4 link LRRK2 to the cellular vesicle transport pathway and Notch signaling, through which the LRRK2 complex promotes the recycling of the Notch ligand Delta-like 1 (Dll1)/Delta (Dl) through the modulation of endosomal trafficking. This process negatively regulates Notch signaling through cis-inhibition by stabilizing Dll1/Dl, which accelerates neural stem cell differentiation and modulates the function and survival of differentiated dopaminergic neurons. These effects are strengthened by the R1441G ROC domain-mutant of LRRK2. These findings suggest that the alteration of Notch signaling in mature neurons is a component of PD etiology linked to LRRK2.

PINK1 and Parkin complementarily protect dopaminergic neurons in vertebrates.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by selective dopaminergic cell loss in the substantia nigra, but its pathogenesis remains unclear. The recessively inherited familial PD genes PARK2 and PARK6 have been attributed to mutations in the Parkin and PTEN-induced kinase 1 (PINK1) genes, respectively. Recent reports suggest that PINK1 works upstream of Parkin in the same pathway to regulate mitochondrial dynamics and/or conduct autophagic clearance of damaged mitochondria. This phenomenon is preserved from Drosophila to human cell lines but has not been demonstrated in a vertebrate animal model in vivo. Here, we developed a medaka fish (Oryzias latipes) model that is deficient in Pink1 and Parkin. We found that despite the lack of a conspicuous phenotype in single mutants for Pink1 or Parkin, medaka that are deficient in both genes developed phenotypes similar to that of human PD: late-onset locomotor dysfunction, a decrease in dopamine levels and a selective degeneration of dopaminergic neurons. Further analysis also revealed defects in mitochondrial enzymatic activity as well as cell death. Consistently, PINK1 and Parkin double-deficient MEF showed a further decrease in mitochondrial membrane potential and mitochondrial complex I activity as well as apoptosis compared with single-deficient MEF. Interestingly, these mitochondrial abnormalities in Parkin-deficient MEF were compensated by exogenous PINK1, but not by disease-related mutants. These results suggest that PINK1 and Parkin work in a complementary way to protect dopaminergic neurons by maintaining mitochondrial function in vertebrates.

[A case of dysferlinopathy asymptomatic for 10 years after an episode of transient muscle weakness].

We report a 28-year-old male with dysferlinopathy, who has remained asymptomatic for 10 years from a rhabdomyolysis-like episode. He had been in good health since birth, but felt difficulty in walking after a month and a half of manual labor at 18 years old (at the year 2000). Rhabdomyolysis was suspected because of muscle weakness and elevated serum CK of 28,094U/L. He was hospitalized and his muscle weakness improved. He was referred to us, because his serum CK remained around 2,000U/L. Histological analysis of muscle, when anti-dysferlin antibody was unavailable, was not informative but later analysis at the age of 23 using preserved specimen showed loss of dysferlin immunoreactivity. Subsequently, a missense mutation (c.2997G>T) and a deletion (c.3373delG) of the dysferlin gene, both of which are common in Miyoshi myopathy in Japanese, were identified. He continuously showed hyper-CKemia, but no apparent muscle weakness emerged for more than ten years. Reports on asymptomatic dysferlinopathy over such a long duration are rare. This case may suggest that genetic factors, environmental factors such as intensity of work-load, or both, might affect the clinical course of dysferlinopathy. Further follow-up is necessary.

The loss of PGAM5 suppresses the mitochondrial degeneration caused by inactivation of PINK1 in Drosophila.

PTEN-induced kinase 1 (PINK1), which is required for mitochondrial homeostasis, is a gene product responsible for early-onset Parkinson's disease (PD). Another early onset PD gene product, Parkin, has been suggested to function downstream of the PINK1 signalling pathway based on genetic studies in Drosophila. PINK1 is a serine/threonine kinase with a predicted mitochondrial target sequence and a probable transmembrane domain at the N-terminus, while Parkin is a RING-finger protein with ubiquitin-ligase (E3) activity. However, how PINK1 and Parkin regulate mitochondrial activity is largely unknown. To explore the molecular mechanism underlying the interaction between PINK1 and Parkin, we biochemically purified PINK1-binding proteins from human cultured cells and screened the genes encoding these binding proteins using Drosophila PINK1 (dPINK1) models to isolate a molecule(s) involved in the PINK1 pathology. Here we report that a PINK1-binding mitochondrial protein, PGAM5, modulates the PINK1 pathway. Loss of Drosophila PGAM5 (dPGAM5) can suppress the muscle degeneration, motor defects, and shorter lifespan that result from dPINK1 inactivation and that can be attributed to mitochondrial degeneration. However, dPGAM5 inactivation fails to modulate the phenotypes of parkin mutant flies. Conversely, ectopic expression of dPGAM5 exacerbated the dPINK1 and Drosophila parkin (dParkin) phenotypes. These results suggest that PGAM5 negatively regulates the PINK1 pathway related to maintenance of the mitochondria and, furthermore, that PGAM5 acts between PINK1 and Parkin, or functions independently of Parkin downstream of PINK1.

Localization of HtrA2/Omi immunoreactivity in brains affected by Alzheimer's disease.

HtrA2/Omi is a mitochondrial serine protease that promotes apoptotic processes, and this study investigated whether the abnormal immunoexpression of HtrA2/Omi occurs in patients with Alzheimer's disease. We prepared autopsied brains from seven control individuals and seven patients with Alzheimer's disease, and then carried out immunohistochemical studies on HtrA2/Omi using formalin-fixed, paraffin-embedded sections from all of these cases. In the cerebral cortex and hippocampus from the cases of Alzheimer's disease, densely accumulated HtrA2/Omi immunoreactivity was scattered, both intracellularly and extracellularly. Double immunofluorescence analyses showed the partial localization of HtrA2/Omi immunoreactivity in amyloid ß-peptide-immunopositive senile plaques and phosphorylated τ-immunopositive neurofibrillary tangles. These results suggest that HtrA2/Omi may be partially associated with the pathogenesis of Alzheimer's disease.

Loss of PINK1 in medaka fish (Oryzias latipes) causes late-onset decrease in spontaneous movement.

Parkinson's disease is a neurodegenerative disease associated with the degeneration of dopaminergic neurons in the substantia nigra. The PTEN-induced kinase 1 gene (PINK1) is responsible for recessive inherited familial Parkinson's disease (PARK6). Neither the function of PINK1 nor its role in the prevention of Parkinson's disease is fully understood. Gene disruption of PINK1 causes remarkably different phenotypes in animal models such as Drosophila melanogaster, zebrafish, and mouse, none of which recapitulate Parkinson's-disease-like symptoms. We established PINK1-gene-disrupted medaka fish. These mutant fish grew normally at first, then developed significant decrease in the frequency of spontaneous swimming movements in the late-adult stage. Although the mutants did not show any dopaminergic cell loss, the amount of 3,4-dihydroxyphenylacetic acid, a major metabolite of dopamine, decreased. Thus, PINK1 contributes to the maintenance of dopamine metabolism, even before the selective death of dopaminergic neurons. Our animal model is therefore a valuable tool to detect pathogenesis in Parkinson's patients in the early stages.

Accumulation of HtrA2/Omi in neuronal and glial inclusions in brains with alpha-synucleinopathies.

HtrA2/Omi is a mitochondrial serine protease that is released into the cytosol and promotes apoptotic processes by binding to several members of the inhibitors of apoptosis protein family. HtrA2/Omi knockout mice show a parkinsonian phenotype, and mutations in the gene encoding HtrA2/Omi have been identified as susceptibility factors for Parkinson disease (PD). These results suggest that HtrA2/Omi may be involved in the pathogenesis of PD. We performed immunohistochemical studies of HtrA2/Omi on brains from patients with alpha-synuclein-related disorders, including PD, dementia with Lewy bodies (DLB), and multiple-system atrophy (MSA); patients with other neurodegenerative diseases; and controls. HtrA2/Omi is expressed in normal brain tissue, and there was some anti-HtrA2/Omi immunostaining of neurons in normal brains as well as those with other neurodegenerative diseases. In PD and DLB brains, both classic (i.e. brainstem-type) and cortical Lewy bodies were intensely immunostained; pale bodies were also strongly immunopositive for HtrA2/Omi. In MSA brains, numerous glial cytoplasmic inclusions, neuronal cytoplasmic inclusions, and dystrophic neurites were also intensely immunoreactive for HtrA2/Omi. These results suggest that widespread accumulation of HtrA2/Omi may occur in pathologic alpha-synuclein-containing inclusions in brains with PD, DLB, or MSA and that HtrA2/Omi may be associated with the pathogenesis of alpha-synucleinopathies.