Oligodendrocytes (OLs) and their precursor cells (OPCs) express specific receptors to GABA; however, the direct functional consequences of their activation remain not fully determined. It has been shown that expression of functional γ-aminobutyric acid (GABA) type A receptors (GABAARs) in OPCs and OLs is controlled by their contact with neurons. This suggests a role for GABAergic signaling in OPC-neuron dialogue establishment and their differentiation toward mature OLs. Here, OPCs (NG2+) isolated from rat (postnatal day 11) optic nerve, maintained in vitro, were used to directly study the functional role of the GABA signal. The oligodendroglial GABAAR activity was specifically enhanced using N-butyl-β-carboline-3-carboxylate (β-CCB), a potent positive allosteric modulator (PAM). β-CCB effects were compared with those of ganaxolone (Gx), a synthetic allopregnanolone analog and unspecific PAM of GABAARs. It was found that medium supplemented with one or both PAMs increased cell survival. This effect was eliminated in the presence of GABAAR antagonists, such as gabazine or bicuculline, and it was dependent on intracellular Ca2+ increase, which activated the AKT pathway through Ca2+-calmodulin-dependent kinases (CaMKs) acting upstream. In addition, a morphometric analysis showed that both PAMs promoted an increase in cellular complexity commonly associated with cell maturation, increasing the average cell area, number, and length of branches per cell, as well as the branch point number. These direct actions on NG2+ cells would explain, at least in part, the positive effects that GABAergic signaling stimulation has on the myelination process in several experimental models, both in vitro and in vivo.NEW & NOTEWORTHY Oligodendrocyte precursor cells express GABAARs and release GABA into the extracellular milieu; however, direct consequences of receptor activation remain unclear. In this work, we demonstrate that positive allosteric modulation of the GABAAR increases cell survival in vitro and promotes morphological changes associated with maturation. Both effects are generated by an increase in intracellular Ca2+ and activation of Ca2+-CaM-dependent kinases signaling pathway, which subsequently promote AKT phosphorylation, triggering antiapoptotic signals.
Lithium (Li) salts have been widely used to treat bipolar disorder and unipolar depression for more than 50 yr. However, up to 40% of people taking Li develop nephrogenic diabetes insipidus (NDI). NDI is associated with cellular remodeling in the rodent kidney collecting duct (CD) and reduced aquaporin-2. Patients taking Li for more than 10-20 yr are at risk of chronic kidney disease, which ultimately can lead to end-stage renal disease, hemodialysis, transplantation, or death. The purpose of this study was to investigate whether the rat urinary proteome can be used as an indicator of Li-induced changes in the kidney. Extracellular vesicles were isolated from the urine of rats treated with Li for 2 or 4 wk, followed by LC-MS/MS analysis. The results showed a limited correlation between protein changes in the urine and the kidney. However, the urine contained markers of mitochondrial dysfunction. The cytoskeletal protein, keratin 8, showed a tendency to be higher in the urine and was greatly increased in collecting duct principal cells in response to Li, suggesting a potential role in cellular remodeling. Canonical histone H4 was increased in the urine and was observed in the nuclei of collecting duct principal and intercalated cells following Li. Moreover, histone H4-positive cells colocalized with the proliferation marker, proliferating cell nuclear antigen (PCNA). This correlates with the known increased proliferation in the collecting duct in response to Li. Thus, replication-dependent histone H4 is a possible urinary marker for the Li-induced cellular remodeling of the collecting duct.NEW & NOTEWORTHY Lithium (Li) increases replication-dependent histone H4 in urine and in principal and intercalated cells, so histone H4 is a possible urinary marker for Li-induced cellular remodeling.
The 2023 iteration of the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) estimated prevalence, incidence, and health burden for 375 diseases and injuries, including 12 mental disorders. We assess past, current, and emerging trends in the prevalence and burden of mental disorders across sexes and age groups, for 21 regions, 204 countries and territories, and by Socio-demographic Index (SDI) quintile, from 1990 to 2023. Mental disorders included in GBD 2023 were anxiety disorders, major depressive disorder, dysthymia, bipolar disorder, schizophrenia, autism spectrum disorders, conduct disorder, attention-deficit hyperactivity disorder, anorexia nervosa, bulimia nervosa, idiopathic developmental intellectual disability, and a residual category of other mental disorders. A literature review identified epidemiological data for each disorder. These were analysed via a Bayesian meta-regression to estimate prevalence by disorder, sex, age, location, and year. Disorder-specific prevalence was multiplied by disability weights representing the severity of health loss associated with each disorder to estimate years lived with disability (YLDs). Deaths due to anorexia nervosa were assessed with a Cause of Death Ensemble modelling strategy to estimate deaths by sex, age, location, and year, and then multiplied by the standard life expectancy at age of death to estimate years of life lost (YLLs). YLDs equalled disability-adjusted life-years (DALYs) for all mental disorders except anorexia nervosa (the only mental disorder considered as an underlying cause of death in GBD), for which DALYs represented the sum of YLDs and YLLs. We presented prevalence, deaths, YLDs, YLLs, and DALYs as counts, age-specific rates per 100 000 population, and age-standardised rates per 100 000 population. We estimated 1·17 billion (95% uncertainty interval 1·06-1·31) prevalent cases of mental disorders globally in 2023, equivalent to an age-standardised prevalence rate of 14 210·7 cases (12 849·5-15 940·1) per 100 000 population. These estimates represented a 95·5% (75·0-121·2) increase in prevalent cases and 24·2% (11·4-41·4) increase in age-standardised prevalence rate between 1990 and 2023. All mental disorders showed increases in prevalent cases between 1990 and 2023, while notable increases were seen in age-standardised prevalence rates for anxiety disorders, major depressive disorder, dysthymia, anorexia nervosa, bulimia nervosa, schizophrenia, and conduct disorder. There were an estimated 171 million (127-228) DALYs due to mental disorders globally across sex and age in 2023, equivalent to an age-standardised DALY rate of 2070·5 DALYs (1519·1-2750·5) per 100 000 population. Mental disorders contributed to 6·1% (4·8-7·6) of all-cause DALYs in 2023, making them the fifth leading cause of global DALYs (up from 12th in 1990). DALYs were almost entirely composed of YLDs. Mental disorders were the leading cause of YLDs in 2023 (up from second in 1990), explaining 17·3% (14·8-20·6) of all-cause global YLDs. Leading causes of mental disorder DALYs were anxiety disorders (ranked 11th among the 304 diseases and injuries at Level 4 of the GBD cause hierarchy), major depressive disorder (15th), and schizophrenia (41st). Globally in 2023, mental disorder age-standardised DALY rates were higher among females (2239·6 [1643·7-3014·1] per 100 000) than among males (1900·2 [1399·8-2510·8] per 100 000), and peaked in the 15-19 years age group (2617·3 [1850·6-3696·8] per 100 000). All locations showed increased mental disorder DALY rates in 2023 compared with 1990, ranging across countries and territories from 1302·4 (952·7-1683·7) per 100 000 in Viet Nam to 3555·8 (2661·9-4715·0) per 100 000 in the Netherlands. Across SDI quintiles, DALY rates ranged from 1853·0 (1352·1-2469·3) per 100 000 for middle SDI to 2184·1 (1606·1-2890·3) per 100 000 for high SDI. A significant health burden was imposed by mental disorders in all countries and territories in 2023, irrespective of the health resources available. In some instances, this burden has increased over time and is unevenly distributed across populations. Stronger surveillance systems, particularly in low-income and middle-income countries, are required. Additionally, we need more coordinated and inclusive policies to reduce the burden through early treatment and prevention, tailored to sex and age differences across locations. Responding to the mental health needs of our global population, especially those most vulnerable, is an obligation, not a choice. Gates Foundation, Queensland Health, and University of Queensland.
Skeletal muscle maintains considerable capacity for regeneration following injury, but successful regeneration is limited in instances of volumetric muscle loss, advanced aging or muscular dystrophies. Considerable research has been done on muscle stem cell (MuSC) transplantation; however, proliferative exhaustion and donor cell dose requirements have slowed progress. Due to the paramount role of cellular metabolism in regenerative function of stem cells, the clinical potential for MuSC therapy may be improved by minimizing the isolation-induced metabolic perturbations experienced by MuSCs. This study uses a model of simulated cell sorting combined with untargeted, small molecule metabolomic profiling to outline sorting-induced metabolic perturbations in C2C12 myoblasts. We expand upon this by performing a time course of metabolomic profiling on myoblasts recovering from either fluorescence activated cell sorting (FACS) or magnetic-bead activated cell sorting (MACS)-based isolation procedures to determine the method and recovery timing for optimal redox and energetic status. Using this metabolism-informed method, we then performed primary MuSC transplantation studies in mice to demonstrate the generalizability from the in vitro system to in vivo MuSC transplantation during regeneration from BaCl2-induced injury. Our findings demonstrate metabolically favorable strategies to isolate MuSC for analysis of the quiescent-to-activated metabolic transition or enhance transplantation efficacy.
Alveolar epithelial cells (AEC) are covered apically by the alveolar lining layer (ALL). The alveolar epithelial glycocalyx (AEGCX), which is part of the ALL, consists of a mixture of glycoproteins, proteoglycans and glycosaminoglycans. In comparison to the endothelial glycocalyx (GCX), much less is known about the composition and structure of the AEGCX although it is presumed to be involved in many cellular processes. The challenges of in vivo visualization of the AEGCX, coupled with the distorting effects of aldehyde fixation, make it difficult to accurately determine its structural organization and molecular composition. In this study, we aimed to improve the preservation of the ultrastructure of the AEGCX by using high-pressure freezing combined with freeze substitution (HPF/FS) for transmission electron microscopy (TEM) analysis. HPF is a type of cryopreservation that uses both liquid nitrogen and high pressure. By using HPF/FS to preserve commercially available human primary AECs, human alveolar-like organoids, monolayers of human organoid-derived alveolar-like epithelial cells, and ex vivo tissue from both mice and human lungs, we observed two distinct regions on the apical surface via TEM: an inner GCX-like layer along the cell membrane followed by a loosely arranged outer layer. Our data shows a bi-layered structure on the surface of AECs. To minimize freeze induced damage during HPF, a protein-rich cryoprotectant was introduced to stabilize the sample. By processing control samples of protein-rich cryoprotectants lacking cells, we found that HPF/FS preserves components of the cryoprotectant, as confirmed by TEM analysis. This implies that HPF/FS preserves not only cellular derived constituents but also components found in the cryoprotectant which may interfere with interpretation of the outer surface layer.
The sodium-dependent neutral amino acid transporter 2 (SNAT2) is a plasma membrane transporter that facilitates the uptake of small, aliphatic amino acids. Restricting SNAT2-mediated alanine uptake may be a promising therapeutic strategy for various diseases, including pancreatic ductal adenocarcinoma (PDAC). The posttranslational mechanisms regulating the distribution of SNAT2 to intracellular membranes and its turnover remain uncharacterized in the context of PDAC and may be useful to devise future strategies to inhibit SNAT2 function. Human SNAT2 contains three conserved extracellular N-linked glycosylation moieties at N254, N258, and N274. A screen of SNAT2 expression across several human and mouse PDAC cell lines revealed that plasma membrane SNAT2 is exclusively modified by N-linked glycosylation. Preventing SNAT2 N-glycosylation using pharmacological inhibitors or mutagenesis abolished its glycosylation and attenuated its plasma membrane, but not lysosomal, localization. Furthermore, overexpressing glycosylation-deficient SNAT2 in knockout cell lines fails to restore cell proliferative capacity relative to wild-type SNAT2, despite partially rescuing the metabolomic defect associated with SNAT2 deficiency. Using an inducible expression system, we also demonstrate that N-linked glycosylation-deficient SNAT2 mutants exhibit altered degradation kinetics but use similar pathways as wild-type SNAT2 to coordinate its turnover. Our results highlight the importance of N-linked glycosylation for regulating the stability and cell surface expression of nascent SNAT2 in PDAC cells.NEW & NOTEWORTHY This study provides evidence that the localization of human SNAT2 to the plasma membrane requires N-linked glycosylation in PDAC cell lines. To our knowledge, our report is the first to show that localization of SNAT2 to the cell surface is important for supporting PDAC cell proliferation. This work establishes tools to investigate the significance of lysosomal SNAT2 and supports the concept that targeting nutrient transporter glycosylation may represent a viable strategy to limit PDAC growth.
Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS), which produces nitric oxide (NO) to maintain vascular homeostasis. When BH4 is deficient, eNOS becomes uncoupled, generating superoxide (O2-) instead of NO, contributing to endothelial dysfunction and cardiovascular disease. The cellular BH4 concentration is determined by its de novo synthesis via GTP cyclohydrolase I (GTPCH), oxidation of BH4 to BH2, and the regeneration of BH4 from BH2 by dihydrofolate reductase (DHFR). A diminished BH4/BH2 ratio, often due to DHFR dysregulation, promotes eNOS uncoupling. This study investigates how hypoxia affects eNOS activity and NO bioavailability in human endothelial cells (ECs) derived from various vascular beds. We show that hypoxia downregulates eNOS and DHFR, impairs BH4 regeneration, and induces eNOS uncoupling in all human EC types tested. We also demonstrate that human ECs exhibit low basal BH4 levels, which may result from limited GTPCH expression; consequently, the BH4/BH2 ratio appears to depend substantially on DHFR activity. Importantly, we show for the first time that BH4-dependent regulation of eNOS uncoupling varies between ECs derived from distinct vascular beds. This variability is driven by cell-type-specific differences in the relative levels of eNOS and DHFR under hypoxia. In particular, human aortic endothelial cells (HAECs) display high eNOS expression and low DHFR levels, making them especially prone to hypoxic eNOS uncoupling. These findings suggest that certain vascular beds may be intrinsically more susceptible to hypoxia-induced endothelial dysfunction, driven by greater eNOS uncoupling that depends on DHFR activity, highlighting DHFR as a potential therapeutic target.
Copper is essential for cellular function but can become toxic in excess. Although its redox and enzymatic roles are well established, how copper availability affects cytoskeletal organization and cell mechanics remains unclear. Here, we show that elevated copper availability increases membrane tether force and F-actin anisotropy in HK-2 proximal tubule cells, consistent with actin cytoskeletal remodeling. Cotreatment with the membrane-permeable reactive oxygen species (ROS) scavenger Tiron reversed copper-induced ROS accumulation and mechanical changes without affecting cell viability, supporting a ROS-dependent mechanism. Quantitative proteomics and post-translational modification profiling (ProteomeXchange: PXD072220) identified coordinated changes in actin-regulatory proteins, including Rab35, methionine sulfoxide reductase B2, casein kinase 2 subunits, and Septin2, together with reduced actin methionine oxidation and copper-sensitive phosphorylation shifts. These findings identify copper-driven redox signaling as a modulator of renal epithelial cell mechanics associated with remodeling of actin-regulatory pathways.NEW & NOTEWORTHY Copper-driven redox signaling remodels the actin cytoskeleton and reprograms the mechanical properties of renal proximal tubule cells. Using optical tweezers, fluorescence imaging, quantitative proteomics, and PTM profiling, we link ROS-dependent mechanical changes to coordinated remodeling of the Rab35/MICAL1/MsrB2 and CK2/Septin2 regulatory axes, establishing copper availability as a modulator of renal epithelial cell mechanics with potential relevance to copper dyshomeostasis in kidney injury.
Acute pancreatitis (AP) is commonly linked to bile acid (BA) dyshomeostasis, yet the causal role of elevated circulating BAs and the functional significance of their uptake by pancreatic acinar cells in AP progression remain unclear and controversial. A cerulein (CER)-induced rat model of AP was established, and pathological elevation of circulating BAs was achieved via intraperitoneal injection of sodium taurocholate (50 mg/kg). Targeted metabolomics was used to quantify BA profiles in serum and pancreatic tissue. In vitro experiments on pancreatic acinar cells were conducted to assess BA uptake and its effects on cell survival. Mitochondrial function was evaluated via confocal microscopy, RNA sequencing, and biochemical assays for membrane potential, adenosine triphosphate (ATP), and reactive oxygen species. Systemic Na-TC administration increased serum and pancreatic total BA levels to concentrations comparable to biliary pancreatitis. Under these clinically relevant BA elevations, Na-TC treatment significantly attenuated CER-induced pancreatic histopathological damage, inflammation, and oxidative stress. In vitro, Na-TC (10-200 μM) reduced CER-induced acinar cell apoptosis/necrosis, but this protection was abrogated by the BA transporter inhibitor rifamycin sodium salt. RNA sequencing and functional analyses revealed that BA uptake upregulated mitochondrial electron transport chain subunits, enhanced oxidative phosphorylation, restored mitochondrial membrane potential, and increased ATP production. These findings challenge the traditional view of circulating BA toxicity in AP and indicate that at levels comparable to those observed in biliary pancreatitis, uptake of a certain amount of BAs by pancreatic acinar cells is beneficial rather than harmful.NEW & NOTEWORTHY This study challenges the view that elevated circulating bile acids (BAs) are toxic in acute pancreatitis. We demonstrate that at clinically relevant levels, uptake of BAs into pancreatic acinar cells via specific transporters is protective. It enhances mitochondrial oxidative phosphorylation, restores ATP production, and reduces cell death, revealing a novel beneficial role for intracellular BAs.
While circadian rhythms are critical regulators of cardiovascular physiopathology, their role in Takotsubo Syndrome (TTS) remains poorly understood. This study aimed to investigate the influence of time-of-day on cardiac hypertrophy and inflammation in a mouse model of TTS induced by isoproterenol (ISO) administration. Female mice were injected with saline (Sal) or ISO at the beginning of the light (ZT0) or dark phase (ZT12). Our data show that mice treated with ISO at ZT12 developed more prominent cardiac hypertrophy and exhibited worse cardiomyocyte calcium handling. This was accompanied by an enhanced accumulation of leukocytes in the hearts of ISO/ZT12 compared with ISO/ZT0 mice. Flow cytometry analysis revealed an exacerbation in the number CD64hi/intLy6Chi/loCCR2+ monocytes/macrophages at ZT12 indicating a time-of-day influence on the inflammatory response following ISO administration. Of note, these differences were not secondary to differences in initial tissue injury as assessed by Evans Blue uptake by necrotic cells. However, cardiac expression of Ccl2/7 was significantly higher in the hearts of ISO/ZT12 in comparison to ISO/ZT0, suggesting the involvement of the CCL2/CCR2 signaling axis in the enhanced recruitment of monocytes. Finally, pharmacological and genetic strategies used to prevent CCR2-dependent recruitment of monocytes ameliorated the cardiac hypertrophy induced by ISO at ZT12, indicating that the CCL2/CCR2 signaling axis is crucial to the temporal dependent effects of ISO. Taken together, our data show a previously unrecognized role of the time-of-day on cardiac inflammation following adrenergic overload.
Vascular smooth muscle cell (VSMC) dedifferentiation, a phenomenon found in virtually all vascular diseases, is characterized by a transcriptional switch from a contractile to a phenotypically modulated state. Myocardin (MYOCD) is a smooth muscle cell-restricted coactivator that is necessary and sufficient for the differentiation of VSMC through the transcriptional activation of SMC-restricted cytoskeletal and contractile genes. Despite 25 yr of research on MYOCD, the reliable expression of this protein continues to be poorly represented and understood. Accordingly, we generated a novel rat model carrying HA-tagged MYOCD to address pervasive disparities in the literature and elucidate MYOCD protein expression in vivo. Western blotting studies documented the highest MYOCD protein expression in aorta, bladder, and uterus, with low or undetectable expression in all other tissues of the rat, including heart. Predictive modeling supports the C-terminus of MYOCD to be most immunoreactive, where the HA tag and one other commercial immunogen reside. Of note, the latter immunogen was used to generate what appears to be the most trustworthy commercial antibody against MYOCD. In vitro transcription/translation and phosphatase treatment of ectopic and endogenous MYOCD protein reveal an intrinsically high molecular weight of MYOCD that has eluded prior reports. Importantly, we present the very first spatial expression profile of MYOCD protein in several mouse and rat tissues under baseline and vascular injury conditions. The results offer the SMC community new resources and insight into the reliable detection of MYOCD protein.NEW & NOTEWORTHY Endogenous MYOCD protein has eluded reliable detection due to pervasive commercial antibody failures, likely due to protein disorder. A CRISPR-generated 3xHA knock-in rat enabled the first in vivo spatial characterization of MYOCD, revealing its presence in SMC-rich tissues and, notably, within the microvasculature of SMC-poor organs. Phosphorylation largely accounts for the high molecular weight of MYOCD. Strikingly, MYOCD is detected in neointimal cells after vascular insult, suggesting persistence or reexpression during vascular remodeling.
Cardiac arrhythmias affect 1%-5% of the global population, with atrial fibrillation (AF) being the most common and clinically relevant. While AF is traditionally linked to electrical and structural remodeling of the atria, growing evidence highlights a critical yet underexplored contributor: dysfunction of the autonomic nervous system (ANS). The ANS regulates heart rate and rhythm through sympathetic and parasympathetic inputs, and its imbalance can initiate and sustain AF by promoting ectopic activity, shortening refractory periods, and enhancing reentry circuits. Autonomic dysregulation is further impacted by lifestyle and environmental influences. Excessive alcohol intake, chronic stress, sleep deprivation, and extreme physical exertion have all been shown to modulate autonomic activity and elevate the risk of AF. Additionally, social determinants such as socioeconomic status and healthcare access indirectly influence AF susceptibility through chronic activation of neurocardiac stress pathways. Mechanistically, emerging research implicates sympathetic hyperinnervation, neuroinflammation, and dysfunction of intrinsic cardiac ganglionated plexi as key contributors to arrhythmogenic remodeling. However, current animal models often fail to capture the complexity of human neuro-cardiac interactions due to species-specific differences in cardiac anatomy, innervation patterns, and immune responses. Human induced pluripotent stem cell (iPSC)-derived models offer an alternative, patient-specific platform to study ANS-driven mechanisms in AF. This review focuses on the role of the ANS in AF pathophysiology, examining the cellular and molecular mechanisms by which autonomic dysregulation promotes arrhythmia. We explore current therapeutic interventions of autonomic-driven AF and discuss the potential of new models to improve mechanistic insight and therapeutic development.
Aging is associated with a progressive decline in skeletal muscle mass and function, contributing to reduced physical capacity in older adults. Central is the deterioration of satellite cells, our tissue-resident muscle stem cells, which participate in adaptation, repair, and regeneration of skeletal muscle. Evidence from in vitro, murine, and human studies indicates an age-related reduction in satellite cell content, notably within type II muscle fibers, alongside impairments in myogenic potential. There is no single causative mechanism behind satellite cell age-related dysfunction, but a convergence of morphological changes and intrinsic and extrinsic factors that affect satellite cell dynamics and its niche. Intrinsic factors such as signaling pathways, cellular senescence, impaired autophagy, mitochondrial dysfunction, and epigenetic modifications can impact satellite cell function. Concurrently, extrinsic factors can impact the satellite cell niche and their function such as systemic circulating factors and vasculature and extracellular matrix remodeling. These age-related alterations can diminish regenerative capacity, blunt hypertrophic responses, and impair recovery from disuse or injury. Satellite cell dysfunction is a pivotal contributor to age-related skeletal muscle decline, frailty, and the quality of life in older adults. Despite growing insights from in vitro and animal models, the key mechanistic changes that underlie human satellite cell dysfunction with age are not fully understood. Improved characterization of age-related satellite cell changes in humans is essential to preserving muscle health across the lifespan.
Organic dust (OD) from animal production facilities contains a complex mixture of microbial products, metabolites, and particulates that engage airway epithelial signaling pathways. Here we investigated the mechanisms by which OD extract (ODE) activates immune signaling pathways in human bronchial epithelial cells. ODE rapidly stimulate reactive oxygen species (ROS) generation and a biphasic increase in intracellular Ca2+ concentration ([Ca2+]i), consisting of an early transient peak followed by a smaller sustained phase. Antioxidant scavenger pretreatment (glutathione, N-acetyl-cysteine) markedly attenuated both ROS production and Ca2+ mobilization, whereas induction of endogenous antioxidant defenses with bardoxolone abolished the response, indicating redox sensitivity. Pharmacologic inhibition of Gqα with YM-254890 suppressed both phases of the Ca2+ response, implicating Gq-coupled receptor activation. Consistent with an autocrine amplification mechanism, selective antagonists of histamine (H1), cysteinyl leukotriene (CysLT1 and CysLT2), leukotriene B4 (BLT1), and prostaglandin receptors (EP1) each reduced ODE-evoked Ca2+ mobilization. In parallel, inhibitors of histidine decarboxylase, 5-lipoxygenase, and cyclooxygenases (COX-1/COX-2) attenuated Ca2+ signaling, supporting rapid endogenous ligand production and secretion. Downstream of Ca2+ mobilization, ODE activated protein kinase C alpha/beta (PKCα/β) and protein kinase C delta (PKCδ) and induced robust transcription of proinflammatory cytokine and chemokine mRNAs (IL1β, IL6, IL8, IL33, TNFα) within 2 h of exposure. Enzyme-linked immunosorbent assay confirmed increased secretion of IL-1β, IL-6, IL-8, and IL-33, with differential sensitivity to PKC isoforms and NF-κB inhibition. These findings identify a redox-sensitive G-protein coupled receptor (GPCR) network that amplifies Gq-dependent Ca2+ signaling in airway epithelial cells and provides a mechanistic framework for epithelial inflammatory activation following ROS-inducing environmental exposures.NEW & NOTEWORTHY This study investigates how agricultural organic dust promotes airway inflammation. We show that organic dust extract rapidly induces ROS production along with a biphasic Ca2+ response through a redox-sensitive, Gq-coupled G-protein coupled receptor (GPCR) network involving histamine, leukotrienes, and prostaglandins. This signaling cascade activates PKC isoforms that trigger production and secretion of inflammatory cytokines, linking oxidative stress to Ca2+-dependent inflammatory responses by the airway epithelium following organic dust exposure.
Senescence is broadly considered an age-related phenomenon; however, it also been implicated in normal tissue repair and wound healing. Skeletal muscle repair is a complex process that requires the coordination of several different cell populations, but the role of senescence in skeletal muscle repair has yet to be fully elucidated. We hypothesize that senescence serves as a control mechanism throughout the regenerative process, and the removal of senescent cells through senolytics will negatively impact the repair process in young mice. Briefly, young mice were exposed to either 1) vehicle (VEH), receiving only a cardiotoxin (CTx) injection in one hindlimb, or 2) 7 days of senolytic treatment (SEN) pre-CTx and 3×/week for 4 wk post-CTx. Dasatinib + Quercetin (D + Q) was used to selectively eliminate senescent cells. There were no significant differences between groups in functional measures such as hindlimb grip strength and cross-sectional area. eMHC+ fibers remained elevated at D28 in the SEN group. Macrophage infiltration was twice as high in the SEN group compared with VEH at D7. Satellite cell quantity and fibrotic area were significantly increased at D14 in the SEN group compared with VEH. We conclude that reducing senescent cells during muscle repair in young mice significantly altered the kinetics of muscle repair. Therefore, senescent cells may act as a regulatory mechanism in skeletal muscle to orchestrate the activity of the different cell populations involved in repair and regeneration, such as immune cells, satellite cells, and fibrotic cells.NEW & NOTEWORTHY Senolytic treatment in young mice results in a transient delay in the repair kinetics of satellite cells, macrophages, and fibrosis without disrupting functional repair of skeletal muscle. Fibers associated with a p21+ nucleus were smaller in size than myofibers not associated with a p21+ nucleus, possibly signifying areas with delayed or incomplete repair or where greater senescence-associated signalling is needed to regulate nearby cell populations.
As has been known for many decades, oxaloacetate (OAA) is a very potent inhibitor of succinate dehydrogenase (SDH). However, the phenomenon has received little attention for several reasons to be discussed. Although the interaction between OAA and the structure of SDH has been scrutinized, there has been little attention to the mechanism underlying OAA inhibition of SDH in respiring mitochondria or to its functional implications. In recent years, we have used more advanced methodology to examine these issues. OAA is unstable and therefore very difficult to detect by mass spectroscopy. Hence, we used a novel NMR approach to assess OAA in mitochondria of muscle, brown adipose tissue, and liver under active respiratory conditions. We also used a modification of existing technology to assess mitochondrial respiration in states apart from the extremes of state 4 and state 3. We found strong evidence that mitochondrial OAA content and inhibition of SDH are dependent on inner mitochondrial membrane potential (ΔΨ) and the effects of ΔΨ on the NADH/NAD+ redox state. Furthermore, we examined the effects of perturbed OAA content by deleting glutamic-oxaloacetic transaminase, which metabolizes OAA and glutamate to aspartate and α-ketoglutarate. Such deletion enhanced mitochondrial OAA and impaired metabolism through SDH. Here, we review historical and recent studies addressing OAA inhibition of SDH. We also discuss the possible physiological role of OAA/SDH interaction and whole-body consequences. Furthermore, we describe a novel methodology for the detection of OAA and assessment of mitochondrial function under conditions of clamped mitochondrial inner membrane potential.
Renal fibrosis is a common pathogenic stage during the progression of acute injury to chronic kidney disease. Pathophysiological changes such as cellular remodeling, dysregulation of fibrogenic signaling, and extracellular matrix contribute to kidney fibrosis. Oxidative stress is a common feature of nephrotoxicants. However, the target molecules and mechanistic pathways dysregulated by oxidative stress during acute injury and fibrosis in kidney are not fully understood. Therefore, the objective of this study was to identify the target molecules altered by oxidative stress and determine whether the antioxidant N-acetyl cysteine can attenuate acute injury and long-term fibrosis in kidney. Both in vitro cell culture and in vivo mice models were used to address these questions. Multiple approaches, such as serum creatinine-albumin levels, histopathological, immunohistochemical, transcriptome analysis by RNA-sequencing, and target-specific expression of fibrogenic marker genes for cellular remodeling and fibrogenic signaling, were used to evaluate the role of oxidative stress in acute kidney injury (AKI) and kidney fibrosis. Novel findings of this study not only revealed that antioxidants can protect from acute injury and attenuate fibrosis in kidney by abrogating oxidative stress-induced cellular remodeling, activated profibrogenic genes, and signaling pathways, but also identified several target genes and signaling molecules, including those associated with kidney function and extracellular matrix regulation that were previously not known to be associated with AKI and kidney fibrosis. Findings of this study have significance in understanding the pathophysiological effects of nephrotoxicant-induced oxidative stress in kidney fibrosis and the roles of dysregulated genes in the etiology of this disease.NEW & NOTEWORTHY Using both in vitro and in vivo models, this study suggests that antioxidants can protect from acute injury and attenuate fibrosis in kidney by abrogating oxidative stress-induced partial-EMT, activated profibrogenic genes, and signaling pathways including TGF-β, Wnt, and Notch signaling. Transcriptome analysis identified several previously unknown fibrosis-associated gene transcripts, and further investigation is needed to understand their precise roles in the etiology of this disease.
Kv3.1 voltage-gated potassium channels play a critical role in regulating neuronal excitability, and dysregulation driven by gain-of-function (GoF) mutations has been implicated in neurological disease. Although Kv3.1 potentiators have been at the forefront of drug development as a means to enhance neuronal firing, progress towards small-molecule Kv3.1 inhibitors has been limited. Here, we address this gap with the discovery of novel and structurally diverse Kv3.1 channel inhibitors identified through a high-throughput screening of over 50,000 compounds. Among these, VU426 emerged as the most potent compound with an IC50 of 4.3 μM. VU426 induces pronounced, state-dependent inhibition of outward K+ current with sustained depolarization, indicating stabilization of an inactivated channel conformation accessed from the open state. Functional characterization of four GoF mutations (V425M, M430I, V432M, and V434L) in the S6 pore lining domain demonstrated that VU426 exhibits 1.5- to 5-fold enhanced potency toward pathogenic GoF Kv3.1 mutants relative to wild-type channels. Automated patch clamp electrophysiological studies revealed that V432M and V434L mutations embedded deep in the S6 domain had the greatest sensitivity to VU426. Despite its potency, VU426 exhibited limited selectivity for Kv3.1 over related Kv channels. Together, these findings identify novel Kv3.1 inhibitors and highlight a pharmacological strategy for targeting clinically identified pathogenic variants.NEW & NOTEWORTHY This study identifies the first structurally diverse collection of small-molecule inhibitors of Kv3.1 potassium channels, highlighting VU426 as a moderately potent, state-dependent inactivator. Notably, VU426 displays enhanced potency against multiple pathogenic gain-of-function Kv3.1 mutants, revealing a mechanism-based strategy for selectively targeting disease-associated channel variants.
Iron is essential for cellular function, and lung cells are no exception. Previous studies have demonstrated an association between increased level of iron in the lung and aging and age-related lung diseases including pulmonary fibrosis and asthma in the elderly (AIE). However, the mechanisms underlying the accumulation of iron with aging or AIE, or the cell types involved, remain understudied. In the context of asthma, airway smooth muscle is a key cell type contributing to contractility as well as airway remodeling (proliferation, fibrosis). In this study we characterized iron level and regulation in human (hASM) from young (<45 yr), old (≥65 yr) and AIE (≥65 yr) male/female patients, and investigated the contribution of iron overload in hASM to airway remodeling. Cells were treated with ferric ammonium citrate (FAC) (100 μM; 72h) or iron chelator deferoxamine (DFO) (100 µM; 72h). Basal levels of intracellular ferrous iron (Fe2+) were determined using the fluorescent dye FerroOrange. Cell lysates were analyzed for iron accumulation, antioxidant, lipid peroxidation, and extracellular matrix (ECM) markers, and cell proliferation was assessed. We found that iron accumulates with aging, but surprisingly decreases with AIE. hASM from AIE patients showed activated antioxidant pathways and lipid peroxidation, while FAC-exposure impaired iron metabolism and enhanced ECM deposition. Iron depletion mitigated the antioxidant response, lipid peroxidation, cell proliferation, ECM production. These findings suggest that iron metabolism in hASM contributes to cell hyperplasia and ECM, while in aging and particularly AIE, counter-regulatory changes in iron metabolism and antioxidant pathways occur, overall promoting airway remodeling.
Pulmonary ionocytes are rare epithelial cells that bear the highest amount of mRNA for CFTR and whose function is necessary to regulate airway surface liquid homeostasis. Lineage induction is regulated by the transcription factor forkhead box i1 (Foxi1), but the temporal and spatial localization of Foxi1+ cells during airway development and the impact of CFTR mutations remain poorly understood. Here, we used immunofluorescence and RNAscope to detect Foxi1+ cells throughout postnatal airway development in male and female mice, from neonatal stages to 1 yr of age, including CftrΔF508 mutant animals at the adult stage. Foxi1+ cells were observed in higher density in the laryngeal subglottis region, decreasing toward the distal trachea, and principally located in the surface epithelium covering intercartilage zones. The Foxi1+ cells were observed at postnatal day 7.5, and their appearance correlated with the development of submucosal glands. The distribution of these cells was not altered in the CftrΔF508/ΔF508 mutant mouse. These findings demonstrate that pulmonary ionocytes display a highly regionalized distribution pattern in the mouse airway and emerge during a defined postnatal developmental window associated with submucosal gland maturation. Their localization near submucosal gland openings in the mouse trachea and coappearance during development suggest a potential role in regulating submucosal gland secretion and airway epithelial fluid homeostasis.NEW & NOTEWORTHY This study provides the first spatiotemporal map of Foxi1+ pulmonary ionocytes in mouse airways. These rare cells, which express the majority of CFTR mRNA, emerge postnatally near developing submucosal glands and concentrate in the laryngeal subglottis during adulthood. Their distribution remains unaltered in CftrΔF508/ΔF508 mice, suggesting CFTR dysfunction does not disrupt ionocyte patterning.