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Cnpy3 2xHA mice reveal neuronal expression of Cnpy3 in the brain
Get PDF浜松医科大学Hamamatsu University School of Medicine博士（医学）Background: Identification of biallelic CNPY3 mutations in patients with epileptic encephalopathy and abnormal electroencephalography findings of Cnpy3 knock-out mice have indicated that the loss of CNPY3 function causes neurological disorders such as epilepsy. However, the basic property of CNPY3 in the brain remains unclear.
New method: We generated C-terminal 2xHA-tag knock-in Cnpy3 mice by i-GONAD in vivo genome editing system to investigate the expression and function of Cnpy3 in the mouse brain.
Results: 2xHA-tagged Cnpy3 was confirmed by immunoblot analysis using anti-HA and CNPY3 antibodies, although HA tagging caused the decreased Cnpy3 protein level. Immunohistochemical analysis of Cnpy3 2xHA knock-in mice showed that Cnpy3-2xHA was predominantly expressed in the neuron. In addition, Cnpy3 and Cnpy3-2xHA were both localized in the endoplasmic reticulum and synaptosome and showed age-dependent expression changes in the brain.
Comparison with existing methods: Conventional Cnpy3 antibodies could not allow us to investigate the distri-bution of Cnpy3 expression in the brain, while HA-tagging revealed the expression of CNPY3 in neuronal cells.
Conclusions: Taken together, we demonstrated that Cnpy3 2xHA knock-in mice would be useful to further elucidate the property of Cnpy3 in brain function and neurological disorders
Disruption of the endopeptidase ADAM10-Notch signaling axis leads to skin dysbiosis and innate lymphoid cell-mediated hair follicle destruction
Get PDF浜松医科大学博士（医学）doctoral医学系研究科thesi
A Left Ventricle Remodeling in Patients with Bicuspid Aortic Valve
Get PDFPurpose: We assessed the impact of bicuspid aortic valve (BAV), aortic stenosis (AS), and regurgitation (AR) on the metrics of left ventricular (LV) remodeling, as measured by electrocardiogram (ECG), transthoracic echocardiography (TTE), and cardiac magnetic resonance (CMR).
Methods: This retrospective CMR study included 11 patients with both AS and AR (BAV-ASR), 30 with AS (BAV-AS), 28 with AR (BAV-AR), 47 with neither AS nor AR (BAV-no_AS/AR), and 40 with trileaflet aortic valve (TAV-no_AS/AR). CMR analysis included the LV end-diastolic volume index (LVEDVi), mass index (LVMi), and extracellular volume fraction (ECV). The Sokolow-Lyon and Cornell products by ECG and TTE-derived E/e’ were measured.
Results: There were no differences in the ECG, TTE, and CMR parameters between BAV-no_AS/AR and TAV-no_AS/AR. However, the presence of aortic valve dysfunction resulted in an elevated Sokolow-Lyon product for BAV-ASR (p=0.017) and BAV-AR (p=0.001), as well as increased Cornell product (p=0.04) and E/e’ (p<0.001) for BAV-AS compared with BAV-no_AS/AR. LVEDVi and LVMi were elevated in patients with BAV-ASR and BAV-AR compared with those with BAV-no_AS/AR (LVEDVi: 101 ± 29ml/m² and 112 ± 32ml/m² vs. 74 ± 15ml/m², p=0.005 and p<0.001, LVMi: 75 ± 7g/m² and 64 ± 14g/m² vs. 47 ± 9g/m², respectively; p<0.001). There was no difference in ECV between the BAV and TAV-no_AS/AR subgroups.
Conclusion: Normally functioning BAV did not result in LV remodeling. However, concomitant AV dysfunction was associated with statistically significant morphological remodeling.journal articl
Association between social activity and development of dementia in hearing impairment: A cohort study in Japan from Japan Gerontological Evaluation Study
Get PDF浜松医科大学博士（医学）dotoral医学系研究科We aimed to clarify the association between social activity and the development of dementia in older adults by hearing-impaired (HI) status. We applied a community-based prospective cohort study over 6 years as part of the Japan Gerontological Evaluation Study. The study included 53,549 participants aged 65 years and older who did not require long-term care. A baseline questionnaire survey was conducted; explanatory variables included physical and social activities, and the objective variable was dementia onset assessed by standardized protocol. Cox regression models were used to calculate hazard ratios (HRs) for dementia stratified by HI status. During the follow-up period, 6013 (11.2%) participants developed dementia. Analyses revealed increased dementia risk for participants with HI who participated in the following activities less than once a month: sport groups (HR 2.17, 95% CI 1.53-3.08), hobby groups (HR 1.70, 95% CI 1.34-2.17), going out (HR 2.19, 95% CI 1.51-3.17), and meeting with friends (HR 1.27, 95% CI 1.06-1.53). HI and lack of social activity increase the risk of dementia. The study results indicate that there is an association between low social activity and the development of dementia in people with HI; the strongest associations were found for low participation in sports and hobby groups.doctoral thesi
Therapeutic effects of KCC2 chloride transporter activation on detrusor overactivity in mice with spinal cord injury
Get PDF浜松医科大学博士（医学）dotoral医学系研究科This study aimed to clarify whether the down regulation of K+-Cl−co-transporter 2 (KCC2) in the sacral parasympathetic nucleus (SPN) of the lumbosacral spinal cord, from which the efferent pathway innervating the bladder originates, causes cellular hyperexcitability and triggers detrusor overactivity (DO) in spinal cord injury (SCI). SCI was produced by the Th8-9 spinal cord transection in female C57BL/6 mice. At 4 weeks after SCI, CLP290, a KCC2 activator, was administered, and cystometry was performed. Thereafter, neuronal activity with c-fos staining and KCC2 expression in the cholinergic preganglionic parasympathetic neurons in the SPN were examined using immunohistochemistry. Firing properties of neurons in the SPN region were evaluated by extracellular recordings in spinal cord slice preparations. DO evident as non-voiding contractions (NVC) was significantly reduced by CLP290 treatment in SCI mice. The number of c-fos-positive cells and co-expression of c-fos in choline acetyltransferase (ChAT)-positive cells were decreased in the SPN region of SCI-CLP290 group vs. SCI-vehicle group. KCC2 immunoreactivity was present on the cell membrane of SPN neurons, and the normalized fluorescence intensity of KCC2 in ChAT-positive SPN neurons was decreased in SCI-vehicle group, vs. SI-vehicle group, but recovered in SCI-CLP290 group. Extracellular recordings showed that CLP290 suppressed the high frequency firing activity of SPN neurons in SCI mice. These results indicated that SCI-induced DO is associated with downregulation of KCC2 in preganglionic parasympathetic neurons, and that activation of KCC2 transporters can reduce DO, increase KCC2 expression in preganglionic parasympathetic neurons and decrease neuronal firing of SPN neurons in SCI mice.NEW & NOTEWORTHY
This study is the first report to suggest that activation of KCC2 chloride ion transporter may be a therapeutic modality for the treatment of SCI-induced DO by targeting bladder efferent pathways.doctoral thesi
Tubulin polyglutamylation by TTLL1 and TTLL7 regulate glutamate concentration in the mice brain
Get PDF浜松医科大学博士（医学）doctoral医学系研究科As an important neurotransmitter, glutamate acts in over 90% of excitatory synapses in the human brain. Its metabolic pathway is complicated, and the glutamate pool in neurons has not been fully elucidated. Tubulin polyglutamylation in the brain is mainly mediated by two tubulin tyrosine ligase-like (TTLL) proteins, TTLL1 and TTLL7, which have been indicated to be important for neuronal polarity. In this study, we constructed pure lines of Ttll1 and Ttll7 knockout mice. Ttll knockout mice showed several abnormal behaviors. Matrix-assisted laser desorption/ionization (MALDI) Imaging mass spectrometry (IMS) analyses of these brains showed increases in glutamate, suggesting that tubulin polyglutamylation by these TTLLs acts as a pool of glutamate in neurons and modulates some other amino acids related to glutamate.doctoral thesi
UBL3 interaction with α-synuclein is downregulated by silencing MGST3
Get PDF浜松医科大学Hamamatsu University School of Medicine博士（医学）doctoral医学系研究科Ubiquitin-like 3 (UBL3) is a membrane-anchored protein that plays a crucial role in sorting proteins into small extracellular vesicles. Aggregations of alpha-synuclein (α-syn) are associated with the pathology of neurodegenerative diseases such as Parkinson’s disease. Recently, the interaction between UBL3 and α-syn was discovered, with potential implications in clearing excess α-syn from neurons and its role in disease spread. However, the regulator that can mediate the interaction between UBL3 and α-syn remains unclear. In this study, using the split gaussian luciferase complementation assay and RNA interference technology, we identified that QSOX2, HTATIP2, UBE3C, MGST3, NSF, HECTD1, SAE1, and ATG3 were involved in downregulating the interaction between UBL3 and α-syn. Notably, silencing MGST3 had the most significant impact. Immunocytochemistry staining confirmed the impact of MGST3 silencing on the co-localization of UBL3 and α-syn in cells. MGST3 is a part of the antioxidant system, and silencing MGST3 is believed to contribute to oxidative stress. We induced oxidative stress with hydrogen peroxide, observing its effect on the UBL3-α-syn interaction, and showing that 800 μM of H₂O₂ downregulated this interaction. In conclusion, silencing MGST3 downregulates the interaction between UBL3 and α-syn.doctoral thesi