Basal activity of PINK1 and PRKN in cell models and rodent brain.

The ubiquitin kinase-ligase pair PINK1-PRKN recognizes and transiently labels damaged mitochondria with ubiquitin phosphorylated at Ser65 (p-S65-Ub) to mediate their selective degradation (mitophagy). Complete loss of PINK1 or PRKN function unequivocally leads to early-onset Parkinson disease, but it is debated whether impairments in mitophagy contribute to disease later in life. While the pathway has been extensively studied in cell culture upon acute and massive mitochondrial stress, basal levels of activation under endogenous conditions and especially in vivo in the brain remain undetermined. Using rodent samples, patient-derived cells, and isogenic neurons, we here identified age-dependent, brain region-, and cell type-specific effects and determined expression levels and extent of basal and maximal activation of PINK1 and PRKN. Our work highlights the importance of defining critical risk and therapeutically relevant levels of PINK1-PRKN signaling which will further improve diagnosis and prognosis and will lead to better stratification of patients for future clinical trials.

Gene therapy for alopecia in type II rickets model rats using vitamin D receptor-expressing adenovirus vector.

Type II rickets is a hereditary disease caused by a mutation in the vitamin D receptor (VDR) gene. The main symptoms of this disease are bone dysplasia and alopecia. Bone dysplasia can be ameliorated by high calcium intake; however, there is no suitable treatment for alopecia. In this study, we verified whether gene therapy using an adenoviral vector (AdV) had a therapeutic effect on alopecia in Vdr-KO rats. The VDR-expressing AdV was injected into six 7-week-old female Vdr-KO rats (VDR-AdV rats). On the other hand, control-AdV was injected into 7-week-old female rats (control-AdV rats); non-infected Vdr-KO rats (control rats) were also examined. The hair on the backs of the rats was shaved with hair clippers, and VDR-AdV or control-AdV was intradermally injected. Part of the back skin was collected from each rat after AdV administration. Hair follicles were observed using hematoxylin and eosin staining, and VDR expression was examined using immunostaining and western blotting. VDR-AdV rats showed significant VDR expression in the skin, enhanced hair growth, and low cyst formation, whereas control-AdV and non-infected rats did not show any of these effects. The effect of VDR-AdV lasted for nearly 60 days. These results indicate that gene therapy using VDR-AdV may be useful to treat alopecia associated with type II rickets, if multiple injections are possible after a sufficient period of time.

Involvement of casein kinase 1 epsilon/delta (Csnk1e/d) in the pathogenesis of familial Parkinson's disease caused by CHCHD2.

Parkinson's disease (PD) is a common neurodegenerative disorder that results from the loss of dopaminergic neurons. Mutations in coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) gene cause a familial form of PD with α-Synuclein aggregation, and we here identified the pathogenesis of the T61I mutation, the most common disease-causing mutation of CHCHD2. In Neuro2a cells, CHCHD2 is in mitochondria, whereas the T61I mutant (CHCHD2T61I ) is mislocalized in the cytosol. CHCHD2T61l then recruits casein kinase 1 epsilon/delta (Csnk1e/d), which phosphorylates neurofilament and α-Synuclein, forming cytosolic aggresomes. In vivo, both Chchd2T61I knock-in and transgenic mice display neurodegenerative phenotypes and aggresomes containing Chchd2T61I , Csnk1e/d, phospho-α-Synuclein, and phospho-neurofilament in their dopaminergic neurons. Similar aggresomes were observed in a postmortem PD patient brain and dopaminergic neurons generated from patient-derived iPS cells. Importantly, a Csnk1e/d inhibitor substantially suppressed the phosphorylation of neurofilament and α-Synuclein. The Csnk1e/d inhibitor also suppressed the cellular damage in CHCHD2T61I -expressing Neuro2a cells and dopaminergic neurons generated from patient-derived iPS cells and improved the neurodegenerative phenotypes of Chchd2T61I mutant mice. These results indicate that Csnk1e/d is involved in the pathogenesis of PD caused by the CHCHD2T61I mutation.

Expression of Rat Cyp27b1 in HepG2 Cells Using Adenovirus Vector and Its Application to Evaluation of Self-Made and Commercially Available Anti-Cyp27b1 Antibodies.

Rat Cyp27b1 was successfully expressed in HepG2 cells using an adenovirus vector. High vitamin D 1α-hydroxylation activity was detected in them, whereas no activity was observed in non-infected cells. Similarly, vitamin D 1α-hydroxylation activity was also observed in HepG2 cells expressing Cyp27b1-Flag, which is tagged with a Flag at the C-terminus of Cyp27b1. Western blot analysis using an anti-Flag antibody showed a clear band of Cyp27b1-Flag. Next, we screened three types of anti-Cyp27b1 antibodies, which consist of two commercially available antibodies and our self-made antibody using Cyp27b1- or Cyp27b1-Flag expressing HepG2 cell lysate as a positive control. Surprisingly, Western blot analysis revealed that two commercially available antibodies did not detect Cyp27b1 but specifically detect other proteins. In contrast, our self-made antisera specifically detected Cyp27b1 and Cyp27b1-Flag in the HepG2 cells expressing Cyp27b1 or Cyp27b1-Flag. These commercially available antibodies have been used for the detection of Cyp27b1 by Western blotting and immunohistochemistry. Our results suggest that those data should be reanalyzed.

Gait improvement with wearable cyborg HAL trunk unit for parkinsonian patients: five case reports.

Cybernic treatment involves the generation of an interactive bio-feedback loop between an individual's nervous system and the worn cyborg Hybrid Assistive Limb (HAL); this treatment has been applied for several intractable neuromuscular disorders. Thus, it is of interest to determine its potential for parkinsonian patients. This study confirmed the feasibility of using a HAL trunk unit to improve parkinsonian gait disturbance. HAL establishes functional and physical synchronization with the wearer by providing lateral cyclic forces to the chest in the form of somatosensory and motor cues. To confirm the feasibility of its use for improving parkinsonian gait disturbances, we conducted experiments with three Parkinson's disease patients and two patients with progressive supranuclear palsy. During the experiments, the immediate effect of the intervention was assessed; all participants exhibited improvements in gait disturbance while wearing the HAL unit, and this improvement effect persisted without the HAL unit in two participants. Afterward, based on the assessment, we conducted a continuous intervention for one participant. In this intervention, the number of steps in the final experiment was significantly decreased compared with the initial state. These findings suggest that the proposed method is an option for treating parkinsonian patients to generate somatosensory and motor cues.

Functional analysis of vitamin D receptor (VDR) using adenovirus vector.

Recently, we generated type II rickets model rats, including Vdr(R270L), Vdr(H301Q), Vdr(R270L/H301Q), and Vdr-knockout (KO), by genome editing. All generated animals showed symptoms of rickets, including growth retardation and abnormal bone formation. Among these, only Vdr-KO rats exhibited abnormal skin formation and alopecia. To elucidate the relationship between VDR function and rickets symptoms, each VDR was expressed in human HaCaT-VDR-KO cells using an adenovirus vector. We also constructed an adenovirus vector expressing VDR(V342M) corresponding to human VDR(V346M) which causes alopecia. We compared the nuclear translocation of VDRs after adding 1α,25-dihydroxyvitamin D3 (1,25D3) or 25-hydroxyvitamin D3 (25D3) at final concentrations of 10 and 100 nM, respectively. Both 25D3 and 1,25D3 induced the nuclear translocation of wild type VDR and VDR(V342M). Conversely, VDR(R270L) translocation was observed in the presence of 100 nM 25D3, with almost no translocation following treatment with 10 nM 1,25D3. VDR(R270L/H301Q) failed to undergo nuclear translocation. These results were consistent with their affinity for each ligand. Notably, VDR(R270L/H301Q) may exist in an unliganded form under physiological conditions, and factors interacting with VDR(R270L/H301Q) may be involved in the hair growth cycle. Thus, this novel system using an adenovirus vector could be valuable in elucidating vitamin D receptor functions.

Age related immune modulation of experimental autoimmune encephalomyelitis in PINK1 knockout mice.

Objective: Recent research has shown that Parkin, an E3 ubiquitin ligase, modulates peripheral immune cells-mediated immunity during experimental autoimmune encephalomyelitis (EAE). Because the PTEN-induced putative kinase 1 (PINK1) protein acts upstream of Parkin in a common mitochondrial quality control pathway, we hypothesized that the systemic deletion of PINK1 could also modify the clinical course of EAE, altering the peripheral and central nervous systems' immune responses. Methods: EAE was induced in female PINK1-/- mice of different age groups by immunization with myelin oligodendrocyte glycoprotein peptide. Results: Compared to young wild-type controls, PINK1-/- mice showed earlier disease onset, albeit with a slightly less severe disease, while adult PINK1-/- mice displayed early onset and more severe acute symptoms than controls, showing persistent disease during the recovery phase. In adult mice, EAE severity was associated with significant increases in frequency of dendritic cells (CD11C+, IAIE+), lymphocytes (CD8+), neutrophils (Ly6G+, CD11b+), and a dysregulated cytokine profile in spleen. Furthermore, a massive macrophage (CD68+) infiltration and microglia (TMEM119+) and astrocyte (GFAP+) activation were detected in the spinal cord of adult PINK1-/- mice. Conclusions: PINK1 plays an age-related role in modulating the peripheral inflammatory response during EAE, potentially contributing to the pathogenesis of neuroinflammatory and other associated conditions.

Impaired mitochondrial accumulation and Lewy pathology in neuron-specific FBXO7-deficient mice.

Parkinson's disease, the second most common neurodegenerative disorder, is characterized by the loss of nigrostriatal dopamine neurons. FBXO7 (F-box protein only 7) (PARK15) mutations cause early-onset Parkinson's disease. FBXO7 is a subunit of the SCF (SKP1/cullin-1/F-box protein) E3 ubiquitin ligase complex, but its neuronal relevance and function have not been elucidated. To determine its function in neurons, we generated neuronal cell-specific FBXO7 conditional knockout mice (FBXO7flox/flox: Nestin-Cre) by crossing previously characterized FBXO7 floxed mice (FBXO7flox/flox) with Nestin-Cre mice (Nestin-Cre). The resultant Fbxo7flox/flox: Nestin-Cre mice showed juvenile motor dysfunction, including hindlimb defects and decreased numbers of dopaminergic neurons. Fragmented mitochondria were observed in dopaminergic and cortical neurons. Furthermore, p62- and synuclein-positive Lewy body-like aggregates were identified in neurons. Our findings highlight the unexpected role of the homeostatic level of p62, which is regulated by a non-autophagic system that includes the ubiquitin-proteasome system, in controlling intracellular inclusion body formation. These data indicate that the pathologic processes associated with the proteolytic and mitochondrial degradation systems play a crucial role in the pathogenesis of PD.

PARKIN modifies peripheral immune response and increases neuroinflammation in active experimental autoimmune encephalomyelitis (EAE).

Neuroinflammation plays an important role in the pathogenesis of several neurodegenerative disorders. To elucidate the effects of the mitophagy-related gene Parkin on neuroinflammation, we used a mouse model of experimental autoimmune encephalomyelitis (EAE). Female Parkin-/- and female wild type control mice were immunized with myelin oligodendrocyte glycoprotein to develop active EAE. Compared to the wild type controls, the Parkin-/- mice showed an earlier onset and greater severity of EAE with a greatly increased number of CD8αß+TCRαß+ T cells in the spleen and brain as well as a stronger T-cell proliferative response and an altered cytokine secretion in splenocytes. Furthermore, the Parkin-/- mice showed massive recruitment of monocytes/macrophages and activated microglia in the spinal cord during the acute phase of the disease. They also showed accumulation of microglia co-expressing M1 and M2 markers in the brain and a strong over-expression of A1 reactive astrocytes in the spinal cord. Furthermore, the Parkin-/- mice that developed persistent disease exhibited reduced glial cell numbers and abnormalities in mitochondrial morphology. Our study sheds light on the role of PARKIN protein in modulating peripheral immune cells-mediated immunity during EAE, highlighting its importance in neuroinflammation, and thus elucidating its potential in the development of novel neuroprotective therapies.

Homeostatic p62 levels and inclusion body formation in CHCHD2 knockout mice.

Inactivation of constitutive autophagy results in the formation of cytoplasmic inclusions in neurones, but the relationship between impaired autophagy and Lewy bodies (LBs) remains unknown. α-Synuclein and p62, components of LBs, are the defining characteristic of Parkinson's disease (PD). Until now, we have analyzed mice models and demonstrated p62 aggregates derived from an autophagic defect might serve as 'seeds' and can potentially be a cause of LB formation. P62 may be the key molecule for aggregate formation. To understand the mechanisms of LBs, we analyzed p62 homeostasis and inclusion formation using PD model mice. In PARK22-linked PD, intrinsically disordered mutant CHCHD2 initiates Lewy pathology. To determine the function of CHCHD2 for inclusions formation, we generated Chchd2-knockout (KO) mice and characterized the age-related pathological and motor phenotypes. Chchd2 KO mice exhibited p62 inclusion formation and dopaminergic neuronal loss in an age-dependent manner. These changes were associated with a reduction in mitochondria complex activity and abrogation of inner mitochondria structure. In particular, the OPA1 proteins, which regulate fusion of mitochondrial inner membranes, were immature in the mitochondria of CHCHD2-deficient mice. CHCHD2 regulates mitochondrial morphology and p62 homeostasis by controlling the level of OPA1. Our findings highlight the unexpected role of the homeostatic level of p62, which is regulated by a non-autophagic system, in controlling intracellular inclusion body formation, and indicate that the pathologic processes associated with the mitochondrial proteolytic system are crucial for loss of DA neurones.

Pathogenic insights to Parkin-linked model mice.

In 2018, we summarized Parkin mutation analysis over the 20 years since its discovery. As a strategy for treating Parkinson's disease (PD), disease-modifying therapies based on the overall picture of PD, including pathological studies of hereditary PD, have been developed. With the discovery of Parkin, research on PD accelerated explosively around the world. Several PD mouse models were generated to investigate the pathology of PD. Recently, we reported dopaminergic neuron-specific autophagy-deficient mice as a model of sporadic PD. These mice exhibit Lewy pathology and motor dysfunction, and provide in vivo evidence for Lewy body formation. In these animals, synuclein deposition is preceded by p62, resulting in the formation of inclusions containing both proteins. The number and size of these inclusions increase gradually with aging. Consequently, dopaminergic (DA) neuron loss and motor dysfunction are observed in 120-week-old mice. To assess the critical role of Parkin in vivo, we characterized Parkin-knockout mice over a long period of time. At the age of 110 weeks, Parkin-knockout mice exhibited locomotor impairments, including hindlimb defects and neuronal loss, and fragmented mitochondria with abnormal internal structures accumulated in their DA neurons. Age-related motor dysfunction and damaged mitochondria were observed in Parkin-deficient mice.

Aging-related motor function and dopaminergic neuronal loss in C57BL/6 mice.

Aging-related dopaminergic neuronal loss and its motor phenotypes are well known. Excessive loss of dopaminergic neurons leads to Parkinson's disease (PD), the most common neurodegenerative disorder characterized by the loss of nigrostriatal dopamine-producing neurons. In mice, however, aging-related dopaminergic neuronal loss and its consequences for motor function are poorly understood. We observed the phenotype of wild-type C57BL/6 mice over an extended period of time. C57BL/6 mice exhibited age-dependent locomotor impairments, including hindlimb defects and the number of dopaminergic neurons decreased in aged mice, contributing to locomotor dysfunction. We observed a reduction in striatal dopamine levels in aged mice using high-performance liquid chromatography (HPLC). Thus, dopamine levels are affected by the loss of dopaminergic neurons. Furthermore, fragmented mitochondria were observed in dopaminergic neurons of aged mice but not in those of young mice. Aging-related dopaminergic neuronal loss and accumulation of damaged mitochondria may underlie the pathophysiology of aging.

Variants in saposin D domain of prosaposin gene linked to Parkinson's disease.

Recently, the genetic variability in lysosomal storage disorders has been implicated in the pathogenesis of Parkinson's disease. Here, we found that variants in prosaposin (PSAP), a rare causative gene of various types of lysosomal storage disorders, are linked to Parkinson's disease. Genetic mutation screening revealed three pathogenic mutations in the saposin D domain of PSAP from three families with autosomal dominant Parkinson's disease. Whole-exome sequencing revealed no other variants in previously identified Parkinson's disease-causing or lysosomal storage disorder-causing genes. A case-control association study found two variants in the intronic regions of the PSAP saposin D domain (rs4747203 and rs885828) in sporadic Parkinson's disease had significantly higher allele frequencies in a combined cohort of Japan and Taiwan. We found the abnormal accumulation of autophagic vacuoles, impaired autophagic flux, altered intracellular localization of prosaposin, and an aggregation of α-synuclein in patient-derived skin fibroblasts or induced pluripotent stem cell-derived dopaminergic neurons. In mice, a Psap saposin D mutation caused progressive motor decline and dopaminergic neurodegeneration. Our data provide novel genetic evidence for the involvement of the PSAP saposin D domain in Parkinson's disease.

Loss of Parkin contributes to mitochondrial turnover and dopaminergic neuronal loss in aged mice.

Parkinson's disease (PD), the second most common neurodegenerative disorder, is characterized by the loss of nigrostriatal dopamine neurons. PARK2 mutations cause early-onset Parkinson's disease (EO-PD). PARK2 encodes an E3 ubiquitin ligase, Parkin. Extensive in vitro studies and cell line characterization have shown that Parkin is required for mitophagy, but the physiological pathology and context of the pathway remain unknown. In general, monogenic Parkin knockout mice do not accurately reflect human PD symptoms and exhibit no signs of dopaminergic (DA) neurodegeneration. To assess the critical role of Parkin-mediated mitophagy in DA neurons, we characterized Parkin knockout mice over a long period of time. At the age of 110 weeks, Parkin knockout mice exhibited locomotor impairments, including hindlimb defects and neuronal loss. In their DA neurons, fragmented mitochondria with abnormal internal structures accumulated. The age-related motor dysfunction and damaged mitochondria pathology in Parkin-deficient mice suggest that impairment of mitochondrial clearance may underlie the pathology of PD.

Metabolomics-based identification of metabolic alterations in PARK2.

Objective: Parkin is the causative gene for autosomal recessive familial Parkinson's disease (PD), although it remains unclear how parkin dysfunction is involved with the general condition. Recently, serum and/or plasma metabolomics revealed alterations in metabolic pathways that might reflect pathomechanisms of idiopathic PD (iPD). Thus, we hypothesized that serum metabolomics of patients with homozygous or compound heterozygous parkin mutations (namely, PARK2) might reflect metabolic alterations due to parkin dysfunction. Methods: We enrolled 15 PARK2 patients (52 ± 17.6 years) confirmed with homozygous (seven cases) and compound heterozygous (eight cases) parkin mutations, along with 19 healthy age-matched controls (51 ± 11.5 years). We analyzed 830 metabolites from participants' serum using well-established metabolomics technologies, including ultra-high performance liquid chromatography/tandem mass spectroscopy. Results: Based on metabolic profiles, hierarchical matrix analysis can divide samples between control and PARK2 subjects. Profiles from PARK2 patients showed significantly higher levels of fatty acid (FA) metabolites and oxidized lipids, and significantly lower levels of antioxidant, caffeine, and benzoate-related metabolites. Interpretation: Metabolomics can identify specific metabolic alterations in PARK2 patients compared with controls. Alterations in FA metabolites suggest a relationship between parkin function and lipid metabolism. The elevation of oxidized lipids in combination with decreasing antioxidants may reflect general hyperoxidative stress. Decreasing benzoate-related metabolites might be due to the alteration in gut microbiota. Consequently, caffeine and its metabolites may be decreased due to malabsorption. These findings are similar to metabolic alterations in iPD. Thus, serum/plasma metabolomics may reflect the association between parkin dysfunction and parkinsonism.

Loss of nuclear REST/NRSF in aged-dopaminergic neurons in Parkinson's disease patients.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Lewy bodies and pale bodies in dopaminergic neurons in the substantia nigra are pathological hallmarks of PD. A number of neurodegenerative diseases demonstrate aggregate formation, but how these aggregates are associated with their pathogenesis remains unknown. It has been reported that repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is induced in the nuclei of aged neurons, preserves neuronal function, and protects against neurodegeneration during aging through the repression of cell death-inducing genes. The loss of REST is associated with Alzheimer's disease pathology. However, its function in dopaminergic neurons remains unknown. Here we demonstrated that REST enters the nucleus of aged dopaminergic neurons. On the other hand, REST is partially sequestrated in Lewy bodies and is mostly absent from the nucleus of neurons in brains with PD and dementia with Lewy bodies (DLB). Dopaminergic neuron-specific autophagy-deficient mice exhibit REST accumulation in aggregates. Defects in the protein quality control system induce REST mRNA expression; its gene product mainly appears in aggregates. Our results suggest that Lewy pathology disturbs normal aging processes in dopaminergic neurons by sequestering REST and the loss of REST may associate with the PD pathology.

Dopaminergic Neuron-Specific Autophagy-Deficient Mice.

None of the current genetic Parkinson's disease (PD) models in mouse recapitulates all features of PD. Additionally, only a few of these models develop mild dopamine (DA) neurodegeneration. And the most parsimonious explanation for the lack of DA neurodegeneration in genetic PD models is a compensatory mechanism that results from adaptive changes during development, making it hard to observe the degenerative phenotype over the life span of mice. Here, we characterize DA neuron-specific autophagy-deficient mice and provide in vivo evidence for Lewy body formation. Atg7-deficient mice demonstrate typical Lewy pathology, including endogenous synuclein and neuronal loss, which resembles PD. Furthermore DA levels are affected by dopaminergic neuronal loss. The age-related motor dysfunction and pathology in DA neurons suggest that impairment of autophagy is a potential mechanism underlying the pathology of PD.

Loss of autophagy in dopaminergic neurons causes Lewy pathology and motor dysfunction in aged mice.

Inactivation of constitutive autophagy results in the formation of cytoplasmic inclusions in neurons, but the relationship between impaired autophagy and Lewy bodies (LBs) as well as the in vivo process of formation remains unknown. Synuclein, a component of LBs, is the defining characteristic of Parkinson's disease (PD). Here, we characterize dopamine (DA) neuron-specific autophagy-deficient mice and provide in vivo evidence for LB formation. Synuclein deposition is preceded by p62 and resulted in the formation of inclusions containing synuclein and p62. The number and size of these inclusions were gradually increased in neurites rather than soma with aging. These inclusions may facilitate peripheral failures. As a result, DA neuron loss and motor dysfunction including the hindlimb defect were observed in 120-week-old mice. P62 aggregates derived from an autophagic defect might serve as "seeds" and can potentially be cause of LB formation.