Bidirectional Control of Autophagy by BECN1 BARA Domain Dynamics.

Membrane targeting of the BECN1-containing class III PI 3-kinase (PI3KC3) complexes is pivotal to the regulation of autophagy. The interaction of PI3KC3 complex II and its ubiquitously expressed inhibitor, Rubicon, was mapped to the first ß sheet of the BECN1 BARA domain and the UVRAG BARA2 domain by hydrogen-deuterium exchange and cryo-EM. These data suggest that the BARA ß sheet 1 unfolds to directly engage the membrane. This mechanism was confirmed using protein engineering, giant unilamellar vesicle assays, and molecular simulations. Using this mechanism, a BECN1 ß sheet-1 derived peptide activates both PI3KC3 complexes I and II, while HIV-1 Nef inhibits complex II. These data reveal how BECN1 switches on and off PI3KC3 binding to membranes. The observations explain how PI3KC3 inhibition by Rubicon, activation by autophagy-inducing BECN1 peptides, and inhibition by HIV-1 Nef are mediated by the switchable ability of the BECN1 BARA domain to partially unfold and insert into membranes.

HKDC1, a target of TFEB, is essential to maintain both mitochondrial and lysosomal homeostasis, preventing cellular senescence.

Mitochondrial and lysosomal functions are intimately linked and are critical for cellular homeostasis, as evidenced by the fact that cellular senescence, aging, and multiple prominent diseases are associated with concomitant dysfunction of both organelles. However, it is not well understood how the two important organelles are regulated. Transcription factor EB (TFEB) is the master regulator of lysosomal function and is also implicated in regulating mitochondrial function; however, the mechanism underlying the maintenance of both organelles remains to be fully elucidated. Here, by comprehensive transcriptome analysis and subsequent chromatin immunoprecipitation-qPCR, we identified hexokinase domain containing 1 (HKDC1), which is known to function in the glycolysis pathway as a direct TFEB target. Moreover, HKDC1 was upregulated in both mitochondrial and lysosomal stress in a TFEB-dependent manner, and its function was critical for the maintenance of both organelles under stress conditions. Mechanistically, the TFEB-HKDC1 axis was essential for PINK1 (PTEN-induced kinase 1)/Parkin-dependent mitophagy via its initial step, PINK1 stabilization. In addition, the functions of HKDC1 and voltage-dependent anion channels, with which HKDC1 interacts, were essential for the clearance of damaged lysosomes and maintaining mitochondria-lysosome contact. Interestingly, HKDC1 regulated mitophagy and lysosomal repair independently of its prospective function in glycolysis. Furthermore, loss function of HKDC1 accelerated DNA damage-induced cellular senescence with the accumulation of hyperfused mitochondria and damaged lysosomes. Our results show that HKDC1, a factor downstream of TFEB, maintains both mitochondrial and lysosomal homeostasis, which is critical to prevent cellular senescence.

Rubicon prevents autophagic degradation of GATA4 to promote Sertoli cell function.

Autophagy degrades unnecessary proteins or damaged organelles to maintain cellular function. Therefore, autophagy has a preventive role against various diseases including hepatic disorders, neurodegenerative diseases, and cancer. Although autophagy in germ cells or Sertoli cells is known to be required for spermatogenesis and male fertility, it remains poorly understood how autophagy participates in spermatogenesis. We found that systemic knockout mice of Rubicon, a negative regulator of autophagy, exhibited a substantial reduction in testicular weight, spermatogenesis, and male fertility, associated with upregulation of autophagy. Rubicon-null mice also had lower levels of mRNAs of Sertoli cell-related genes in testis. Importantly, Rubicon knockout in Sertoli cells, but not in germ cells, caused a defect in spermatogenesis and germline stem cell maintenance in mice, indicating a critical role of Rubicon in Sertoli cells. In mechanistic terms, genetic loss of Rubicon promoted autophagic degradation of GATA4, a transcription factor that is essential for Sertoli cell function. Furthermore, androgen antagonists caused a significant decrease in the levels of Rubicon and GATA4 in testis, accompanied by elevated autophagy. Collectively, we propose that Rubicon promotes Sertoli cell function by preventing autophagic degradation of GATA4, and that this mechanism could be regulated by androgens.

The Relationship Between Fear-avoidance Beliefs in Employees with Chronic Musculoskeletal Pain and Work Productivity: A Longitudinal Study.

This study aimed to identify risk factors for chronic musculoskeletal pain (CMSP) and sought to examine the effect of fear-avoidance beliefs (FABs) on work productivity in workers with CMSP. We performed a longitudinal study using self-administered questionnaires given to employees in three different industries between April 2016 and March 2017. The questionnaire concerned background characteristics, work-related factors and musculoskeletal pain, the Work Functioning Impairment Scale (WFun), and the Japanese version of the Tampa Scale for Kinesiophobia (TSK-J). We performed logistic regression analysis to evaluate factors affecting CMSP and a multi-way analysis of variance to analyze the relationship between FABs and CMSP and the effect of FABs on the ability to function at work. Age (odds ratio [OR] = 1.02, 95% confidence interval [CI]: 1.00-1.03), mean working hours (OR = 1.18, 95% CI: 1.04-1.33), and changes in working hours (OR = 1.18, 95% CI: 1.02-1.37) were significantly associated with CMSP. Regarding FABs, we found that the stronger the FAB, the greater the WFun score, and that an increase in FABs resulted in a significant increase in WFun scores. This study demonstrated that long or increased working hours may be risk factors for CMSP, and that stronger FABs in those with CMSP are associated with decreased ability to function at work. In addition, measures to reduce FABs in workers with CMSP may be effective.

Relationships Between Chronic Musculoskeletal Pain and Working Hours and Sleeping Hours: A Cross-sectional Study.

Ten to twenty percent of the population of Japan has chronic pain. Although studies have confirmed a relationship between sleeping hours and chronic pain, it remains unclear whether there is an association between working hours and chronic pain, especially chronic musculoskeletal pain (CMP), in workers. A self-administered questionnaire that sought information regarding background characteristics and work-related factors was sent to 118 enterprises; finally, 1,747 participants were included in the analysis and were classified into CMP (n = 448) and non-CMP (n = 1299) groups. Logistic regression analysis revealed that age (odds ratio [OR] = 1.02, 95% confidence interval [CI]: 1.01-1.03), sex (reference: female, OR = 0.68, 95% CI: 0.52-0.88), working hours (OR = 1.11, 95% CI: 1.03-1.20), and sleeping hours (OR = 0.84, 95% CI: 0.75-0.95) were significantly associated with CMP. Participants were categorized into four groups according to working hours (long: ≥ 9 hours/day [long-work], short: < 9 hours/day [short-work]) and sleeping hours (long: ≥ 7 hours/day [long-sleep], short: < 7 hours/day [short-sleep]). Furthermore, logistic regression analysis showed that the CMP OR was 2.02 (95% CI: 1.46-2.78) times higher in 'long-work plus short-sleep workers' and 1.47 (95% CI: 0.94-2.30) times higher in 'long-work plus long-sleep workers' than in 'short-work plus long-sleep workers'. Thus, working hours are associated with CMP frequency, but sleeping sufficiently may prevent CMP in workers even if they work for long hours. In conclusion, adequate instructions on sleeping hours should be provided by occupational health staff, as this may be effective in preventing CMP.

Autophagy and kidney aging.

Autophagy is a highly conserved intracellular degradation system in eukaryotes that maintains cellular and tissue homeostasis. Upon autophagy induction, cytoplasmic components are engulfed by a double-membrane organelle called the autophagosome that fuses with a lysosome to degrade its contents. In recent years, it has become clear that autophagy becomes dysregulated with aging, which leads to age-related diseases. Kidney function is particularly prone to age-related decline, and aging is the most significant risk factor for chronic kidney disease. This review first discuss the relationship between autophagy and kidney aging. Second, we describe how age-related dysregulation of autophagy occurs. Finally, we discuss the potential of autophagy-targeting drugs to ameliorate human kidney aging and the approaches necessary to discover such agents.

Measurement of autophagy via LC3 western blotting following DNA-damage-induced senescence.

Senescent cells accumulation is associated with aging and age-related diseases, and recent findings suggest that autophagy, the activity of the intracellular degradation system, decreases during senescence. In this protocol, we detail steps to induce cellular senescence in response to DNA damage, evaluate the senescent state using SA-ß-gal staining and western blot for p21, LAMP1, and Lamin B1, and detect autophagy via LC3 western blotting. This protocol can be used in most cell lines and for various types of senescent cells. For complete details on the use and execution of this protocol, please refer to Yamamoto-Imoto et al. (2022).

Age-associated decline of MondoA drives cellular senescence through impaired autophagy and mitochondrial homeostasis.

Accumulation of senescent cells affects organismal aging and the prevalence of age-associated disease. Emerging evidence suggests that activation of autophagy protects against age-associated diseases and promotes longevity, but the roles and regulatory mechanisms of autophagy in cellular senescence are not well understood. Here, we identify the transcription factor, MondoA, as a regulator of cellular senescence, autophagy, and mitochondrial homeostasis. MondoA protects against cellular senescence by activating autophagy partly through the suppression of an autophagy-negative regulator, Rubicon. In addition, we identify peroxiredoxin 3 (Prdx3) as another downstream regulator of MondoA essential for mitochondrial homeostasis and autophagy. Rubicon and Prdx3 work independently to regulate senescence. Furthermore, we find that MondoA knockout mice have exacerbated senescence during ischemic acute kidney injury (AKI), and a decrease of MondoA in the nucleus is correlated with human aging and ischemic AKI. Our results suggest that decline of MondoA worsens senescence and age-associated disease.

A novel detection method of cleaved plasma high-molecular-weight kininogen reveals its correlation with Alzheimer's pathology and cognitive impairment.

INTRODUCTION: Accumulation of ß-amyloid is a pathological hallmark of Alzheimer's disease (AD). ß-Amyloid activates the plasma contact system leading to kallikrein-mediated cleavage of intact high-molecular-weight kininogen (HKi) to cleaved high-molecular-weight kininogen (HKc). Increased HKi cleavage is observed in plasma of AD patients and mouse models by Western blot. For potential diagnostic purposes, a more quantitative method that can measure HKc levels in plasma with high sensitivity and specificity is needed. METHODS: HKi/c, HKi, and HKc monoclonal antibodies were screened from hybridomas using direct ELISA with a fluorescent substrate. RESULTS: We generated monoclonal antibodies recognizing HKi or HKc specifically and developed sandwich ELISAs that can quantitatively detect HKi and HKc levels in human. These new assays show that decreased HKi and increased HKc levels in AD plasma correlate with dementia and neuritic plaque scores. DISCUSSION: High levels of plasma HKc could be used as an innovative biomarker for AD.

Abnormal clotting of the intrinsic/contact pathway in Alzheimer disease patients is related to cognitive ability.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by extracellular ß-amyloid (Aß) deposition. Although peripheral inflammation and cerebrovascular pathology are reported in AD, there is a lack of plasma biomarkers in this field. Because the contact system is triggered in patient plasma, we hypothesized that the hemostasis profile could be a novel biomarker in AD. Here, we assessed the clotting profile in plasma from AD patients and age-matched controls. Utilizing clinically relevant assays, thromboelastography and activated partial thromboplastin time, we found impaired clot initiation and formation rate in AD patient plasma. These coagulation end points correlated with cerebrospinal fluid neurofilament-light levels and cognition and were more profound in younger AD patients. Ex vivo intrinsic clotting of plasma from AD mice expressing human amyloid precursor protein (APP) was also delayed in an age-dependent manner, suggesting that this phenotype is related to APP, the parent protein of Aß. Further analysis of coagulation factors in human plasma indicated that endogenous inhibitor(s) of factors XII and XI in AD plasma contribute to this delayed clotting. Together, these data suggest that delayed clotting in young AD patients is a novel biomarker and that therapies aimed to correct this phenotype might be beneficial in this patient population. Follow-up studies in additional AD patient cohorts are warranted to further evaluate these findings.