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.

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.

Evidence that phosphorylated ubiquitin signaling is involved in the etiology of Parkinson's disease.

The ubiquitin (Ub) kinase PINK1 and the E3 Ub ligase Parkin, two gene products associated with young-onset Parkinson's disease (PD), participate in mitochondrial quality control. The phosphorylation of mitochondrial polyUb by PINK1, which is activated in a mitochondrial membrane potential (ΔΨm)-dependent manner, facilitates the mitochondrial translocation and concomitant enzymatic activation of Parkin, leading to the clearance of phospho-polyUb-tagged mitochondria via mitophagy. Thus, Ub phosphorylation is a key event in PINK1-Parkin-mediated mitophagy. Here, we examined the role of phospho-Ub signaling in the pathogenesis of PD using fly PD models, human brain tissue and dopaminergic neurons derived from induced pluripotent stem cells (iPSCs) containing Parkin or PINK1 mutations, as well as normal controls. We report that phospho-Ub signaling is highly conserved between humans and Drosophila, and that phospho-Ub signaling and the relocation of axonal mitochondria upon ΔΨm reduction are indeed compromised in human dopaminergic neurons containing Parkin or PINK1 mutations. Moreover, phospho-Ub signaling is prominent in tyrosine hydroxylase-positive neurons compared with tyrosine hydroxylase-negative neurons, suggesting that PINK1-Parkin signaling is more required for dopaminergic neurons. These results shed light on the particular vulnerability of dopaminergic neurons to mitochondrial stress.

Lysosomal Storage of Subunit c of Mitochondrial ATP Synthase in Brain-Specific Atp13a2-Deficient Mice.

Kufor-Rakeb syndrome (KRS) is an autosomal recessive form of early-onset parkinsonism linked to the PARK9 locus. The causative gene for KRS is Atp13a2, which encodes a lysosomal type 5 P-type ATPase. We recently showed that KRS/PARK9-linked mutations lead to several lysosomal alterations, including reduced proteolytic processing of cathepsin D in vitro. However, it remains unknown how deficiency of Atp13a2 is connected to lysosomal impairments. To address this issue, we analyzed brain tissues of Atp13a2 conditional-knockout mice, which exhibited characteristic features of neuronal ceroid lipofuscinosis, including accumulation of lipofuscin positive for subunit c of mitochondrial ATP synthase, suggesting that a common pathogenic mechanism underlies both neuronal ceroid lipofuscinosis and Parkinson disease.

Lysosomal defects in ATP13A2 and GBA associated familial Parkinson's disease.

Genes encoding lysosomal proteins, such as ATP13A2 and GBA, are associated with familial Parkinson's disease (PD). Heterozygous mutations in GBA are strongly associated with familial PD. ATP13A2, which encodes a lysosomal P-type ATPase, has been identified as the causative gene for Kufor-Rakeb syndrome. While lysosomal dysfunction due to these mutations exhibited early onset Parkinsonism, each animal model demonstrated different pathological mechanisms. Clinicogenetic and animal model studies recently identified several lysosomal alterations that play a role in the pathogenesis of PD.

Alterations of PINK1-PRKN signaling in mice during normal aging.

The ubiquitin kinase-ligase pair PINK1-PRKN identifies and selectively marks damaged mitochondria for elimination via the autophagy-lysosome system (mitophagy). While this cytoprotective pathway has been extensively studied in vitro upon acute and complete depolarization of mitochondria, the significance of PINK1-PRKN mitophagy in vivo is less well established. Here we used a novel approach to study PINK1-PRKN signaling in different energetically demanding tissues of mice during normal aging. We demonstrate a generally increased expression of both genes and enhanced enzymatic activity with aging across tissue types. Collectively our data suggest a distinct regulation of PINK1-PRKN signaling under basal conditions with the most pronounced activation and flux of the pathway in mouse heart compared to brain or skeletal muscle. Our biochemical analyses complement existing mitophagy reporter readouts and provide an important baseline assessment in vivo, setting the stage for further investigations of the PINK1-PRKN pathway during stress and in relevant disease conditions.

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.

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.

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.

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.

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.

Molecular pathogenesis of Parkinson's disease: update.

Parkinson disease (PD) is a neurodegenerative disease characterised by progressive disturbances in motor, autonomic and psychiatric functions. Much has been learnt since the disease entity was established in 1817. Although there are well established treatments that can alleviate the symptoms of PD, a pressing need exists to improve our understanding of the pathogenesis to enable development of disease modifying treatments. Ten responsible genes for PD have been identified and recent progress in molecular research on the protein functions of the genes provides new insights into the pathogenesis of hereditary as well as sporadic PD. Also, genome wide association studies, a powerful approach to identify weak effects of common genetic variants in common diseases, have identified a number of new possible PD associated genes, including PD genes previously detected. However, there is still much to learn about the interactions of the gene products, and important insights may come from chemical and genetic screens. In this review, an overview is provided of the molecular pathogenesis and genetics of PD, focusing particularly on the functions of the PD related gene products with marked research progress.

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.

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.

Mitochondrial membrane potential decrease caused by loss of PINK1 is not due to proton leak, but to respiratory chain defects.

Mutations in PTEN-induced putative kinase 1 (PINK1) cause a recessive form of Parkinson's disease (PD). PINK1 is associated with mitochondrial quality control and its partial knock-down induces mitochondrial dysfunction including decreased membrane potential and increased vulnerability against mitochondrial toxins, but the exact function of PINK1 in mitochondria has not been investigated using cells with null expression of PINK1. Here, we show that loss of PINK1 caused mitochondrial dysfunction. In PINK1-deficient (PINK1(-/-)) mouse embryonic fibroblasts (MEFs), mitochondrial membrane potential and cellular ATP levels were decreased compared with those in littermate wild-type MEFs. However, mitochondrial proton leak, which reduces membrane potential in the absence of ATP synthesis, was not altered by loss of PINK1. Instead, activity of the respiratory chain, which produces the membrane potential by oxidizing substrates using oxygen, declined. H(2)O(2) production rate by PINK1(-/-) mitochondria was lower than PINK1(+/+) mitochondria as a consequence of decreased oxygen consumption rate, while the proportion (H(2)O(2) production rate per oxygen consumption rate) was higher. These results suggest that mitochondrial dysfunctions in PD pathogenesis are caused not by proton leak, but by respiratory chain defects.

DJ-1 associates with synaptic membranes.

Parkinson's disease (PD) is a neurodegenerative disorder caused by loss of dopaminergic neurons. Although many reports have suggested that genetic factors are implicated in the pathogenesis of PD, molecular mechanisms underlying selective dopaminergic neuronal degeneration remain unknown. DJ-1 is a causative gene for autosomal recessive form of PARK7-linked early-onset PD. A number of studies have demonstrated that exogenous DJ-1 localizes within mitochondria and the cytosol, and functions as a molecular chaperone, as a transcriptional regulator, and as a cell protective factor against oxidative stress. However, the precise subcellular localization and function of endogenous DJ-1 are not well known. The mechanisms by which mutations in DJ-1 contributes to neuronal degeneration also remain poorly understood. Here we show by immunocytochemistry that DJ-1 distributes to the cytosol and membranous structures in a punctate appearance in cultured cells and in primary neurons obtained from mouse brain. Interestingly, DJ-1 colocalizes with the Golgi apparatus proteins GM130 and the synaptic vesicle proteins such as synaptophysin and Rab3A. Förster resonance energy transfer analysis revealed that a small portion of DJ-1 interacts with synaptophysin in living cells. Although the wild-type DJ-1 protein directly associates with membranes without an intermediary protein, the pathogenic L166P mutation of DJ-1 exhibits less binding to synaptic vesicles. These results indicate that DJ-1 associates with membranous organelles including synaptic membranes to exhibit its normal function.

p62/SQSTM1 cooperates with Parkin for perinuclear clustering of depolarized mitochondria.

PINK1 and Parkin were first identified as the causal genes responsible for familial forms of early-onset Parkinson's disease (PD), a prevalent neurodegenerative disorder. PINK1 encodes a mitochondrial serine/threonine protein kinase, whereas Parkin encodes an ubiquitin-protein ligase. PINK1 and Parkin cooperate to maintain mitochondrial integrity; however, the detailed molecular mechanism of how Parkin-catalyzed ubiquitylation results in mitochondrial integrity remains an enigma. In this study, we show that Parkin-catalyzed K63-linked polyubiquitylation of depolarized mitochondria resulted in ubiquitylated mitochondria being transported along microtubules to cluster in the perinuclear region, which was interfered by pathogenic mutations of Parkin. In addition, p62/SQSTM1 (hereafter referred to as p62) was recruited to depolarized mitochondria after Parkin-directed ubiquitylation. Intriguingly, deletion of p62 in mouse embryonic fibroblasts resulted in a gross loss of mitochondrial perinuclear clustering but did not hinder mitochondrial degradation. Thus, p62 is required for ubiquitylation-dependent clustering of damaged mitochondria, which resembles p62-mediated 'aggresome' formation of misfolded/unfolded proteins after ubiquitylation.

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.