Immune Response-Dependent Assembly of IMP Dehydrogenase Filaments

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the conversion of IMP to xanthosine monophosphate, the rate-limiting step in de novo guanosine monophosphate (GMP) synthesis. In cultured cells, IMPDH polymerizes into micron-scale filamentous structures when GMP synthesis is inhibited by depletion of purine precursors or by various drugs, including mycophenolic acid, ribavirin, and methotrexate. IMPDH filaments also spontaneously form in undifferentiated mouse embryonic stem cells and induced pluripotent stem cells, hinting they might function in various highly proliferative cell types. Therefore, we investigated IMPDH filament formation in human and murine T cells, which rely heavily on de novo guanine nucleotide synthesis to rapidly proliferate in response to antigenic challenge. We discovered extensive in vivo IMPDH filament formation in mature T cells, B cells, and other proliferating splenocytes of normal, adult B6 mice. Both cortical and medullary thymocytes in young and old mice also showed considerable assembly of IMPDH filaments. We then stimulated primary human peripheral blood mononuclear cells ex vivo with T cell mitogens phytohemagglutinin (PHA), concanavalin A (ConA), or antibodies to CD3 and CD28 for 72 h. We detected IMPDH filaments in 40–60% of T cells after activation compared to 0–10% of unstimulated T cells. Staining of activated T cells for the proliferation marker Ki-67 also showed an association between IMPDH filament formation and proliferation. Additionally, we transferred ovalbumin-specific CD4+ T cells from B6.OT-II mice into B6.Ly5a recipient mice, challenged these mice with ovalbumin, and harvested spleens 6 days later. In these spleens, we identified abundant IMPDH filaments in transferred T cells by immunofluorescence, indicating that IMPDH also polymerizes during in vivo antigen-specific T cell activation. Overall, our data indicate that IMPDH filament formation is a novel aspect of T cell activation and proliferation, and that filaments might be useful morphological markers for T cell activation. The data also suggest that in vivo IMPDH filament formation could be occurring in a variety of proliferating cell types throughout the body. We propose that T cell activation will be a valuable model for future experiments probing the molecular mechanisms that drive IMPDH polymerization, as well as how IMPDH filament formation affects cell function

Heart Failure-Inducible Gene Therapy Targeting Protein Phosphatase 1 Prevents Progressive Left Ventricular Remodeling

BACKGROUND: The targeting of Ca(2+) cycling has emerged as a potential therapy for the treatment of severe heart failure. These approaches include gene therapy directed at overexpressing sarcoplasmic reticulum (SR) Ca(2+) ATPase, or ablation of phospholamban (PLN) and associated protein phosphatase 1 (PP1) protein complexes. We previously reported that PP1β, one of the PP1 catalytic subunits, predominantly suppresses Ca(2+) uptake in the SR among the three PP1 isoforms, thereby contributing to Ca(2+) downregulation in failing hearts. In the present study, we investigated whether heart-failure-inducible PP1β-inhibition by adeno-associated viral-9 (AAV9) vector mediated gene therapy is beneficial for preventing disease progression in genetic cardiomyopathic mice. METHODS: We created an adeno-associated virus 9 (AAV9) vector encoding PP1β short-hairpin RNA (shRNA) or negative control (NC) shRNA. A heart failure inducible gene expression system was employed using the B-type natriuretic protein (BNP) promoter conjugated to emerald-green fluorescence protein (EmGFP) and the shRNA sequence. AAV9 vectors (AAV9-BNP-EmGFP-PP1βshRNA and AAV9-BNP-EmGFP-NCshRNA) were injected into the tail vein (2×10(11) GC/mouse) of muscle LIM protein deficient mice (MLPKO), followed by serial analysis of echocardiography, hemodynamic measurement, biochemical and histological analysis at 3 months. RESULTS: In the MLPKO mice, BNP promoter activity was shown to be increased by detecting both EmGFP expression and the induced reduction of PP1β by 25% in the myocardium. Inducible PP1βshRNA delivery preferentially ameliorated left ventricular diastolic function and mitigated adverse ventricular remodeling. PLN phosphorylation was significantly augmented in the AAV9-BNP-EmGFP-PP1βshRNA injected hearts compared with the AAV9-BNP-EmGFP-NCshRNA group. Furthermore, BNP production was reduced, and cardiac interstitial fibrosis was abrogated at 3 months. CONCLUSION: Heart failure-inducible molecular targeting of PP1β has potential as a novel therapeutic strategy for heart failure

Induction of Cytoplasmic Rods and Rings Structures by Inhibition of the CTP and GTP Synthetic Pathway in Mammalian Cells

Background: Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined. Methodology/Principal Findings: Distinct cytoplasmic rods (,3–10 mm in length) and rings (,2–5 mm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in.95 % of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (.95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin

The Association between Sleep and Bone Mineral Density: Cross‐Sectional Study Using Health Check‐up Data in a Local Hospital in Japan

ABSTRACT This study aimed to investigate the association between daily sleep duration of <7 hours and lower bone mineral density (BMD) using data from annual health check‐ups conducted in Japan between 2020 and 2022. Multivariate regression models were used, where BMD was the objective variable and daily sleep duration (<5 hours, 5 to <7 hours, 7 to <9 hours [reference], ≥9 hours) was the exposure variable adjusted for age, body mass index, physical activity, smoking status, and alcohol intake for men and women and further adjusted for menopausal status for women. The association between insomnia and BMD was also investigated. BMD was determined using calcaneal quantitative ultrasound and expressed as a percentage of the young adult mean (%YAM). In total, 896 men and 821 women were included. Median age was 54 years (interquartile range [IQR]: 46 to 64) for men and 55 years (IQR: 46 to 64) for women). Median BMD for men and women was 79%YAM (IQR: 71 to 89) and 75%YAM (IQR: 68 to 84), respectively. Approximately 80% of men and women slept <7 hours daily. Multivariate regression showed no association between sleep duration and BMD in men. However, women who slept 5 to <7 hours daily had significantly higher BMD by 3.9% compared with those who slept 7 to<9 hours (p = 0.004). No association between insomnia and BMD was found. Overall, a daily sleep duration of <7 hours was not independently associated with lower BMD compared to those who slept 7 to <9 hours in men and women. However, as there is evidence of both shorter and longer sleep durations being associated with an increased risk of adverse events, including cardiovascular events, our result needs to be interpreted with caution. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research