Impact of Epstein-Barr Virus Nuclear Antigen 1 on Neuroinflammation in PARK2 Knockout Mice.

This study aimed to explore the intricate relationship between mitochondrial dysfunction, infection, and neuroinflammation, focusing specifically on the impact of pathogenic epitopes of the Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA1) in a mouse model of mitochondrial dysfunctions. The investigation included female middle-aged PARK2-/- and C57BL/6J wild-type mice immunized with EBNA1386-405 or with active experimental autoimmune encephalomyelitis (EAE) induction by the myelin oligodendrocyte glycoprotein (MOG)35-55 peptide. The PARK2-/- mice developed more severe EAE than the wild-type mice. Following immunization with EBNA1386-405, only PARK2-/- exhibited symptoms resembling EAE. During the acute phase, PARK2-/- mice immunized with either MOG35-55 or EBNA1386-405 exhibited a similar infiltration of the T cells and macrophages in the spinal cord and decreased glial fibrillary acidic protein (GFAP) expression in the brain. However, the EBNA1386-405 -immunized PARK2-/- mice showed significantly increased frequencies of CD8a+ T cells and CD11c+ B cells, and distinct cytokine profiles in the periphery compared to the wild-type controls. These findings highlight the role of EBV in exacerbating inflammation, particularly in the context of mitochondrial deficiencies.

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.

Comparison of changes in stress coping strategies between cognitive behavioral therapy and pharmacotherapy.

Background: Coping refers to conscious responses to negative circumstances, with the intention of ameliorating these situations. Few studies have compared the differences between psychotherapy and medication treatment for coping strategies for depression. In this study, we investigated the differences in coping strategies between cognitive behavioral therapy (CBT) combined with medication (CBT group) and medication alone (pharmacotherapy group) among outpatients with depression. Methods: A prospective observational study was conducted among 50 patients with major depression (24 and 26 in the CBT and pharmacotherapy groups, respectively). Stress coping strategies (Coping Inventory for Stressful Situations [CISS]) and depression severity (Beck Depression Inventory-Second Edition [BDI-II]) were assessed at baseline and 16 weeks later. Changes in the CISS and BDI-II scores in both groups were tested using repeated analysis of variance. Inverse probability weighting with propensity score analysis was applied to address potential selection bias. Results: At 16 weeks, the CBT group exhibited increased CISS task-oriented coping, distraction, and social diversion scores, which differed from those of the pharmacotherapy group. The CBT group exhibited a significantly greater reduction in depressive symptoms than the pharmacotherapy group. Limitations: This study was not a randomized controlled trial and thus may have selection bias. Conclusion: Gaining adaptive coping skills, including task-oriented coping, distraction, and social diversion skills, by combining CBT with medication may lead to greater improvement in depression symptoms. These findings suggest that clinicians should evaluate coping strategies and facilitate the acquisition of adaptive coping strategies in patients with depression to reduce their symptoms.

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.

Melancholic and reactive depression: a reappraisal of old categories.

BACKGROUND: The dominant diagnostic model of the classification of depression today is unitarian; however, since Kurt Schneider (1920) introduced the concept of endogenous depression and reactive depression, the binary model has still often been used on a clinical basis. Notwithstanding this, to our knowledge, there have been no collective data on how psychiatrists differentiate these two conditions. We therefore conducted a survey to examine how psychiatrists in Japan differentiate patients with major depressive disorder who present mainly with melancholic features and those with reactive features. METHODS: Three case scenarios of melancholic and reactive depression, and one-in-between were prepared. These cases were designed to present with at least 5 symptoms listed in the DSM-IV-TR with severity being mild. We have sent the questionnaires regarding treatment options and diagnosis for those three cases on a 7-point Likert scale (1 = "not appropriate", 4 = "cannot tell", and 7 = "appropriate"). Five hundred and two psychiatrists from over one hundred hospitals and community clinics throughout Japan have participated in this survey. RESULTS: The melancholic case resulted significantly higher than the reactive case on either antidepressants (mean ± SD: 5.9 ± 1.2 vs. 3.6 ± 1.7, p < 0.001), hypnotics (mean ± SD: 5.5 ± 1.1 vs. 5.0 ± 1.3, p < 0.001), and electroconvulsive therapy (mean ± SD: 1.5 ± 0.9 vs. 1.2 ± 0.6, p < 0.001). On the other hand, the reactive case resulted in significantly higher scores compared to the melancholic case and the one- in-between cases in regards to psychotherapy (mean ± SD: 4.9 ± 1.4 vs. 4.3 ± 1.4 vs. 4.7 ± 1.5, p < 0.001, respectively). Scores for informing patients that they suffered from "depression" were significantly higher in the melancholic case, compared to the reactive case (mean ± SD: 4.7 ± 1.7 vs. 2.2 ± 1.4, p < 0.001). CONCLUSIONS: Japanese psychiatrists distinguish between major depressive disorder with melancholic and reactive features, and thus choose different treatment strategies regarding pharmacological treatment and psychotherapy.

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.

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.

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.

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.

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.

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.

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.