MITOL prevents ER stress-induced apoptosis by IRE1α ubiquitylation at ER-mitochondria contact sites.

Unresolved endoplasmic reticulum (ER) stress shifts the unfolded protein response signaling from cell survival to cell death, although the switching mechanism remains unclear. Here, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) inhibits ER stress-induced apoptosis through ubiquitylation of IRE1α at the mitochondria-associated ER membrane (MAM). MITOL promotes K63-linked chain ubiquitination of IRE1α at lysine 481 (K481), thereby preventing hyper-oligomerization of IRE1α and regulated IRE1α-dependent decay (RIDD). Therefore, under ER stress, MITOL depletion or the IRE1α mutant (K481R) allows for IRE1α hyper-oligomerization and enhances RIDD activity, resulting in apoptosis. Similarly, in the spinal cord of MITOL-deficient mice, ER stress enhances RIDD activity and subsequent apoptosis. Notably, unresolved ER stress attenuates IRE1α ubiquitylation, suggesting that this directs the apoptotic switch of IRE1α signaling. Our findings suggest that mitochondria regulate cell fate under ER stress through IRE1α ubiquitylation by MITOL at the MAM.

MITOL promotes cell survival by degrading Parkin during mitophagy.

Parkin promotes cell survival by removing damaged mitochondria via mitophagy. However, although some studies have suggested that Parkin induces cell death, the regulatory mechanism underlying the dual role of Parkin remains unknown. Herein, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) regulates Parkin-mediated cell death through the FKBP38-dependent dynamic translocation from the mitochondria to the ER during mitophagy. Mechanistically, MITOL mediates ubiquitination of Parkin at lysine 220 residue, which promotes its proteasomal degradation, and thereby fine-tunes mitophagy by controlling the quantity of Parkin. Deletion of MITOL leads to accumulation of the phosphorylated active form of Parkin in the ER, resulting in FKBP38 degradation and enhanced cell death. Thus, we have shown that MITOL blocks Parkin-induced cell death, at least partially, by protecting FKBP38 from Parkin. Our findings unveil the regulation of the dual function of Parkin and provide a novel perspective on the pathogenesis of PD.

Identification of highest neurotoxic amyloid-ß plaque type showing reduced contact with astrocytes.

Amyloid-ß (Aß) plaques are strongly associated with the development of Alzheimer's disease (AD). However, it remains unclear how morphological differences in Aß plaques determine the pathogenesis of Aß. Here, we categorized Aß plaques into four types based on the macroscopic features of the dense core, and found that the Aß-plaque subtype containing a larger dense core showed the strongest association with neuritic dystrophy. Astrocytes dominantly accumulated toward these expanded/dense-core-containing Aß plaques. Previously, we indicated that deletion of the mitochondrial ubiquitin ligase MITOL/MARCH5 triggers mitochondrial impairments and exacerbates cognitive decline in a mouse model with AD-related Aß pathology. In this study, MITOL deficiency accelerated the formation of expanded/dense-core-containing Aß plaques, which showed reduced contacts with astrocytes, but not microglia. Our findings suggest that expanded/dense-core-containing Aß-plaque formation enhanced by the alteration of mitochondrial function robustly contributes to the exacerbation of Aß neuropathology, at least in part, through the reduced contacts between Aß plaques and astrocytes.

Overview of Mitochondrial E3 Ubiquitin Ligase MITOL/MARCH5 from Molecular Mechanisms to Diseases.

The molecular pathology of diseases seen from the mitochondrial axis has become more complex with the progression of research. A variety of factors, including the failure of mitochondrial dynamics and quality control, have made it extremely difficult to narrow down drug discovery targets. We have identified MITOL (mitochondrial ubiquitin ligase: also known as MARCH5) localized on the mitochondrial outer membrane and previously reported that it is an important regulator of mitochondrial dynamics and mitochondrial quality control. In this review, we describe the pathological aspects of MITOL revealed through functional analysis and its potential as a drug discovery target.

Daily and short-term application of joint movement for the prevention of infrapatellar fat pad atrophy due to immobilization.

[Purpose] To mobilize the knee joint during cast fixation and to determine whether infrapatellar fat pad changes can be prevented. [Materials and Methods] We randomly allocated Wistar rats into 3 groups as follows: normal group, raised in normal conditions (n=5); contracture group, immobilized with cast fixation (n=5); and prevention group, treated with joint movement during immobilization (n=5). We immobilized the right hindlimb using cast fixation. Joint movement in the prevention group was accomplished by repeatedly pulling the right hindlimb caudally and then returning the leg to the bent position for 10 minutes every day for 2 weeks. We used a metronome to maintain a constant speed, with one set lasting 2 seconds (1-second traction and 1-second return). [Results] The contracture group had adipose cells of various sizes and fibrosis in the infrapatellar fat pad. These changes were also found in milder forms in the prevention group. We found significant differences in the cross section of adipose cells and in knee extension restriction between the groups. [Conclusion] Promoting joint movement may not only have a therapeutic effect on adipose cells but also a preventative effect.

Novel ultrasound-guided inter-semispinal plane block: a comparative pilot study in healthy volunteers.

We previously reported that a novel multifidus cervicis plane (MCP) block could anesthetize the dorsal rami of the cervical spinal nerves. While MCP sonoanatomy is easily detectable in most patients, it is sometimes difficult to recognize the MCP injection plane, especially in elderly patients. Thus, we proposed the inter-semispinal plane (ISP) block as an alternative for the MCP block. The aim of this study was to evaluate the utility of the ISP block by evaluating the area and duration of anesthesia, compared with that of the MCP block in eight healthy volunteers. Each participant underwent unilateral ultrasound-guided MCP block and ISP block. For each block, 20 ml of ropivacaine 0.2% was injected, and the area of anesthesia was determined using the pinprick test. The anesthetic area ranged from C4 to T2 (3/8; 37.5%), T3 (2/8; 25%), or T4 (3/8; 37.5%) in the MCP block, and from C4 to T1 (1/8; 12.5%), T2 (3/8; 37.5%), T3 (2/8; 25%), or T4 (1/8; 12.5%) in the ISP block. The mean (standard deviation) duration of sensory loss following MCP and ISP blocks was 329 (77) min and 349 (70) min, respectively. Thus, the ISP block may be a reliable alternative to the MCP block.

Rapid Access to Nitrogenous Heterobicycles via RhIII -Catalyzed Isomerization from Alkynes to Allenes.

We show that N-alkynylnitrones are efficiently converted to nitrogenous heterobicyclic compounds with a nitrogen atom at the bridgehead position by using a RhIII -catalyst. Our mechanistic studies suggest that the reaction proceeds via an allene intermediate, which is generated in situ through RhIII -catalyzed isomerization of the alkyne group.

Corepressive function of nuclear receptor coactivator 2 in androgen receptor of prostate cancer cells treated with antiandrogen.

BACKGROUND: Recruitment of cofactors in the interaction of the androgen receptor (AR) and AR ligands plays a critical role in determining androgenic/antiandrogenic effects of the AR ligand on signaling, but the functions of key cofactors, including nuclear receptor coactivator (NCOA), remain poorly understood in prostate cancer cells treated with AR ligands. METHODS: We examined prostate cancer cell lines LNCaP and VCaP expressing mutated and wild-type ARs, respectively, to clarify the significance of NCOAs in the effect of antiandrogens. Hydroxyflutamide showed antagonistic activity against VCaP and an agonistic effect on LNCaP. Bicalutamide served as an antagonist for both. We analyzed mRNA transcription and protein expression of NCOAs in these cells pretreated with dihydrotestosterone and thereafter treated with the mentioned antiandrogens. Transcriptional silencing of candidate NCOAs and AR was performed using small interfering RNA (siRNA). Cell proliferation was evaluated with MTT assay. RESULTS: LNCaP treated with bicalutamide showed an about four-fold increase in the expression of NCOA2 mRNA compared to those pretreated with dihydrotestosterone alone (P <0.01). In VCaP pretreated with dihydrotestosterone, transcriptions of NCOA2 and NCOA7 were slightly increased with bicalutamide (1.96- and 2.42-fold, respectively) and hydroxyflutamide (1.33-fold in both). With Western blotting, the expression of NCOA2 protein also increased in LNCaP cells treated with bicalutamide compared with that in control cells pretreated with dihydrotestosterone alone. Following silencing with siRNA for NCOA2, PSA levels in media with LNCaP receiving bicalutamide were elevated compared with those in non-silencing controls (101.6 ± 4.2 vs. 87.8 ± 1.4 ng/mL, respectively, P =0.0495). In LNCaP cells treated with dihydrotestosterone and bicalutamide, NCOA2-silencing was associated with a higher proliferation activity compared with non-silencing control and AR-silencing. CONCLUSION: NCOA2, which has been thought to be recruited as a coactivator, possibly plays a corepressive role in AR of prostate cancer cells when treated with antiandrogens, suggesting its potential as a therapeutic target.

Efficient synthesis of eight-membered nitrogen heterocycles from o-propargylic oximes by rhodium-catalyzed cascade reactions.

Azocine derivatives were successfully synthesized from O-propargylic oximes by means of a Rh-catalyzed 2,3-rearrangement/heterocyclization cascade reaction. Moreover, the chirality of the substrate was maintained throughout the cascade process to afford the corresponding optically active azocines.

Histological and immunohistochemical analyses of articular cartilage during onset and progression of pre- and early-stage osteoarthritis in a rodent model.

Early diagnosis and treatment of pre- and early-stage osteoarthritis (OA) is important. However, the cellular and cartilaginous changes occurring during these stages remain unclear. We investigated the histological and immunohistochemical changes over time between pre- and early-stage OA in a rat model of traumatic injury. Thirty-six male rats were divided into two groups, control and OA groups, based on destabilization of the medial meniscus. Histological and immunohistochemical analyses of articular cartilage were performed on days 1, 3, 7, 10, and 14 postoperatively. Cell density of proteins associated with cartilage degradation increased from postoperative day one. On postoperative day three, histological changes, including chondrocyte death, reduced matrix staining, and superficial fibrillation, were observed. Simultaneously, a compensatory increase in matrix staining was observed. The Osteoarthritis Research Society International score increased from postoperative day seven, indicating thinner cartilage. On postoperative day 10, the positive cell density decreased, whereas histological changes progressed with fissuring and matrix loss. The proteoglycan 4-positive cell density increased on postoperative day seven. These findings will help establish an experimental model and clarify the mechanism of the onset and progression of pre- and early-stage traumatic OA.

A New Strengthening Process for Carbon-Fiber-Reinforced Thermoplastic Polyphenylene Sulfide (CFRTP-PPS) Interlayered Composite by Electron Beam Irradiation to PPS Prior to Lamination Assembly and Hot Press.

Impact by hailstone, volcanic rock, bird strike, or also dropping tools can cause damage to aircraft materials. For maximum safety, the goal is to increase Charpy impact strength (auc) of a carbon-fiber-reinforced thermoplastic polyphenylene sulfide polymer (CFRTP-PPS) composite for potential application to commercial aircraft parts. The layup was three cross-weave CF plies alternating between four PPS plies, [PPS-CF-PPS-CF-PPS-CF-PPS], designated [PPS]4[CF]3. To strengthen, a new process for CFRP-PPS was employed applying homogeneous low voltage electron beam irradiation (HLEBI) to both sides of PPS plies prior to lamination assembly with untreated CF, followed by hot press under 4.0 MPa at 573 K for 8 min. Experimental results showed a 5 kGy HLEBI dose was at or near optimum, increasing auc at each accumulative probability, Pf. Optical microscopy of 5 kGy sample showed a reduction in main crack width with significantly reduced CF separation and pull-out; while, scanning electron microscopy (SEM) and electron dispersive X-ray (EDS) mapping showed PPS adhering to CF. Electron spin resonance (ESR) of a 5 kGy sample indicated lengthening of PPS chains as evidenced by a reduction in dangling bond peak. It Is assumed that 5 kGy HLEBI creates strong bonds at the interface while strengthening the PPS bulk. A model is proposed to illustrate the possible strengthening mechanism.

A case of autoimmune pulmonary alveolar proteinosis with severe respiratory failure treated with segmental lung lavage and oral statin therapy.

Pulmonary alveolar proteinosis (PAP) is a diffuse lung disease characterized by the accumulation of alveolar surfactants due to dysfunction of granulocyte-macrophage colony-stimulating factor-dependent cholesterol clearance. Whole-lung lavage is the current standard of care for PAP, but it can lead to the exacerbation of hypoxia. A medication targeting cholesterol homeostasis is a promising therapy for refractory PAP. We present a case of autoimmune PAP with severe hypoxia that was successfully treated with segmental lung lavage (SLL). Following SLL for disease relapse, statin treatment for dyslipidemia was started. After initiating statin treatment, the patient did not require bronchoalveolar lavage for 10 months.

Strengthening Process by Electron Beam to Carbon Fiber for Impact Strength Enhancement of Interlayered Thermoplastic-Polypropylene Carbon Fiber Composite.

Strong adhesion between recyclable thermoplastic (TP) polymer and carbon fiber (CF) has always been highly sought after. Therefore, for an interlayered CF reinforced TP polypropylene (CFRTPP) composite composed of 3 sized CF plies, alternating between 4 PP sheets, designated [PP]4[CF]3, a process of activating CF plies directly on both sides with homogeneous low energy electron beam irradiation (EBI) under N2 gas, prior to lamination assembly and hot press of 4.0 MPa at 493 K for 3 min was carried out. Experimental results showed EBI dose of 43.2, 129, or 216 kGy significantly raised Charpy impact values, auc at all fracture probabilities, Pf. The 129 kGy dose appeared to be at or near optimum increasing auc 103%, 83%, and 65% at low-, median-, and high-Pf = 0.07, 0.50, and 0.93; while raising statistically lowest impact value, as at Pf = 0 calculated by 3-dimensional Weibull equation about 110%, indicating increased safety and reliability. It is assumed dangling bonds generated by the EBI rapidly form covalent bonds CF:C:O:C:PP and CF:C:C:PP at the interface, along with cross-linking in the PP near the CF. This is by charge transfer from CF to PP.

Protective roles of MITOL against myocardial senescence and ischemic injury partly via Drp1 regulation.

Abnormal mitochondrial fragmentation by dynamin-related protein1 (Drp1) is associated with the progression of aging-associated heart diseases, including heart failure and myocardial infarction (MI). Here, we report a protective role of outer mitochondrial membrane (OMM)-localized E3 ubiquitin ligase MITOL/MARCH5 against cardiac senescence and MI, partly through Drp1 clearance by OMM-associated degradation (OMMAD). Persistent Drp1 accumulation in cardiomyocyte-specific MITOL conditional-knockout mice induced mitochondrial fragmentation and dysfunction, including reduced ATP production and increased ROS generation, ultimately leading to myocardial senescence and chronic heart failure. Furthermore, ischemic stress-induced acute downregulation of MITOL, which permitted mitochondrial accumulation of Drp1, resulted in mitochondrial fragmentation. Adeno-associated virus-mediated delivery of the MITOL gene to cardiomyocytes ameliorated cardiac dysfunction induced by MI. Our findings suggest that OMMAD activation by MITOL can be a therapeutic target for aging-associated heart diseases, including heart failure and MI.

Reduction of knee joint load suppresses cartilage degeneration, osteophyte formation, and synovitis in early-stage osteoarthritis using a post-traumatic rat model.

The purpose of this study was to clarify the histological effect of reducing the loading to knee on cartilage degeneration, osteophyte formation, and synovitis in early-stage osteoarthritis (OA) using a post-traumatic rat model. Ten male rats were randomly allocated into two experimental groups: OA induction by surgical destabilization of medial meniscus (DMM, OA group) and hindlimb suspension after OA induction by DMM (OAHS group). The articular cartilage, osteophyte formation, and synovial membrane in the medial tibiofemoral joint were analyzed histologically and histomorphometrically at 2 and 4 weeks after surgery. The histological scores and changes in articular cartilage and osteophyte formation were significantly milder and slower in the OAHS group than in the OA group. At 2 and 4 weeks, there were no significant differences in cartilage thickness and matrix staining intensity between both the groups, but chondrocytes density was significantly lower in the OA group. Synovitis was milder in OAHS group than in OA group at 2 weeks. Reducing knee joint loading inhibited histological OA changes in articular cartilage, osteophyte formation, and synovial inflammation. This result supports the latest clinical guidelines for OA treatment. Further studies using biochemical and mechanical analyses are necessary to elucidate the mechanism underlying delayed OA progression caused by joint-load reduction.

Mitochondrial ubiquitin ligase alleviates Alzheimer's disease pathology via blocking the toxic amyloid-ß oligomer generation.

Mitochondrial pathophysiology is implicated in the development of Alzheimer's disease (AD). An integrative database of gene dysregulation suggests that the mitochondrial ubiquitin ligase MITOL/MARCH5, a fine-tuner of mitochondrial dynamics and functions, is downregulated in patients with AD. Here, we report that the perturbation of mitochondrial dynamics by MITOL deletion triggers mitochondrial impairments and exacerbates cognitive decline in a mouse model with AD-related Aß pathology. Notably, MITOL deletion in the brain enhanced the seeding effect of Aß fibrils, but not the spontaneous formation of Aß fibrils and plaques, leading to excessive secondary generation of toxic and dispersible Aß oligomers. Consistent with this, MITOL-deficient mice with Aß etiology exhibited worsening cognitive decline depending on Aß oligomers rather than Aß plaques themselves. Our findings suggest that alteration in mitochondrial morphology might be a key factor in AD due to directing the production of Aß form, oligomers or plaques, responsible for disease development.

Significance of PSA Screening in Niigata, Japan: Survey of Actual Status of New Cases of Prostate Cancer.

PURPOSE: This study aims to investigate the utility of prostate-specific antigen (PSA) screening by conducting an all-case survey of newly diagnosed prostate cancer patients at Niigata Prefecture, Japan. PATIENTS AND METHODS: Depending on whether patients were subjected to screening, information was prospectively collected on all prostate cancer patients newly diagnosed between October 1, 2019, and September 30, 2020, at all institutions in Niigata Prefecture where urologists performing prostate biopsy routinely work and differences in clinical parameters were investigated. RESULTS: PSA was measured in 478 out of 1332 patients (35.8%) as part of a community health screening. The rate of metastatic carcinoma (M1) in all patients was 14.9%. When patients were divided into three categories of population-based screening (community health screening and workplace health screening), opportunistic screening (PSA measurements at complete medical check-ups or on patient request), and testing triggered by clinical symptoms or findings, the proportion of metastatic cancer was 4.5%, 3.7%, and 30.6%, respectively, demonstrating that the number of distant metastases was significantly lesser in all patients who underwent screening. CONCLUSION: The one-year all-case survey of newly diagnosed prostate cancer patients demonstrated that PSA screening significantly contributed to the early diagnosis of current prostate cancer in Japan.

Parathyroid hormone-related peptide-producing non-familial pheochromocytoma in a child.

We experienced a case of parathyroid hormone-related peptide (PTHrP)-producing pheochromocytoma, which was found in a 12-year-old boy with hypercalcemia. The leading symptom was abdominal pain, and severe hypertension and tachycardia were noticed at the initial visit. His medical and familial histories were unremarkable. Laboratory examinations showed hypercalcemia (3.3 mmol/L of serum-calcium). Computed tomography showed a heterogeneous mass measuring 5.0 cm in the right adrenal gland, which had abnormal uptake with 123-I metaiodobenzylguanidine scintigraphy. Serum/urine catecholamines were highly elevated, and serum PTHrP also increased (1.4 pmol/L). The patient underwent laparoscopic right adrenalectomy. The tumor was histologically diagnosed as typical pheochromocytoma and the expression of PTHrP was confirmed with immunohistochemistry. The serum PTHrP level was normalized after surgery. He was free of disease postoperatively for 12 months. There has been no described pediatric patient with PTHrP-producing pheochromocytoma. We showed evidence that the present tumor is a complex neoplasm involving various neuroendocrine activities with the dual-lineage differentiation.

MITOL dysfunction causes dwarfism with anterior pituitary hypoplasia.

In mitochondrial disorders, short stature and growth failure are common symptoms, but their underlying mechanism remains unknown. In this study, we examined the cause of growth failure of mice induced by nestin promoter-driven knockout of the mitochondrial ubiquitin ligase MITOL (MARCH5), a key regulator of mitochondrial function. MITOL-knockout mice have congenital hypoplasia of the anterior pituitary caused by decreased expression of pituitary transcript factor 1 (Pit1). Consistently, both mRNA levels of growth hormone (GH) and prolactin levels were markedly decreased in the anterior pituitary of mutant mice. Growth failure of mutant mice was partly rescued by hypodermic injection of recombinant GH. To clarify whether this abnormality was induced by the primary effect of MITOL knockdown in the anterior pituitary or a secondary effect of other lesions, we performed lentiviral-mediated knockdown of MITOL on cultured rat pituitary GH3 cells, which secrete GH. GH production was severely compromised in MITOL-knockdown GH3 cells. In conclusion, MITOL plays a critical role in the development of the anterior pituitary; therefore, mice with MITOL dysfunction exhibited pituitary dwarfism caused by anterior pituitary hypoplasia. Our findings suggest that mitochondrial dysfunction is commonly involved in the unknown pathogenesis of pituitary dwarfism.

Mitochondrial Dynamics Regulation in Skin Fibroblasts from Mitochondrial Disease Patients.

Mitochondria are highly dynamic organelles that constantly fuse, divide, and move, and their function is regulated and maintained by their morphologic changes. Mitochondrial disease (MD) comprises a group of disorders involving mitochondrial dysfunction. However, it is not clear whether changes in mitochondrial morphology are related to MD. In this study, we examined mitochondrial morphology in fibroblasts from patients with MD (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and Leigh syndrome). We observed that MD fibroblasts exhibited significant mitochondrial fragmentation by upregulation of Drp1, which is responsible for mitochondrial fission. Interestingly, the inhibition of mitochondrial fragmentation by Drp1 knockdown enhanced cellular toxicity and led to cell death in MD fibroblasts. These results suggest that mitochondrial fission plays a critical role in the attenuation of mitochondrial damage in MD fibroblasts.