Defining and characterizing neuronal senescence, 'neurescence', as GX arrested cells.

Cellular senescence is a cell state characterized by resistance to apoptosis and stable cell cycle arrest. Senescence was first observed in mitotic cells in vitro. Recent evidence from in vivo studies and human tissue indicates that postmitotic cells, including neurons, may also become senescent. The quiescent cell state of neurons and inconsistent descriptions of neuronal senescence across studies, however, have caused confusion in this burgeoning field. We summarize evidence demonstrating that exit from G0 quiescence may protect neurons against apoptosis and predispose them toward senescence. Additionally, we propose the term 'neurescent' for senescent neurons and introduce the cell state, GX, to describe cell cycle arrest achieved by passing through G0 quiescence. Criteria are provided to identify neurescent cells, distinguish them from G0 quiescent neurons, and compare neurescent phenotypes with classic replicative senescence.

FGF23 and Cell Stress in SaOS-2 Cells-A Model Reflecting X-Linked Hypophosphatemia Dynamics.

Our study investigates the impact of FGF23 overexpression on SaOS-2 cells to elucidate its role in cellular stress and morphology, contributing to the understanding of skeletal pathologies like X-linked hypophosphatemia (XLH). Using transmission electron microscopy and protein analysis (Western blot), we analyzed the rough endoplasmic reticulum (rER) and mitochondria in SaOS-2 cells with FGF23 overexpression compared to controls. We found significant morphological changes, including enlarged and elongated rER and mitochondria, with increased contact zones, suggesting enhanced interaction and adaptation to elevated protein synthesis and secretion demands. Additionally, we observed higher apoptosis rates of the cells after 24-72 h in vitro and upregulated proteins associated with ER stress and apoptosis, such as CHOP, XBP1 (spliced and unspliced), GRP94, eIF2α, and BAX. These findings indicate a robust activation of the unfolded protein response (UPR) and apoptotic pathways due to FGF23 overexpression. Our results highlight the critical role of ER and mitochondrial interactions in cellular stress responses and provide new insights into the mechanistic link between FGF23 signaling and cellular homeostasis. In conclusion, our study underscores the importance of analyzing UPR-related pathways in the development of therapeutic strategies for skeletal and systemic diseases and contributes to a broader understanding of diseases like XLH.

Mechanisms underlying age-associated exacerbation of pulmonary veno-occlusive disease.

Pulmonary veno-occlusive disease (PVOD) is a rare but severe form of pulmonary hypertension characterized by the obstruction of pulmonary arteries and veins, causing increased pulmonary artery pressure and leading to right ventricular (RV) heart failure. PVOD is often resistant to conventional pulmonary arterial hypertension (PAH) treatments and has a poor prognosis, with a median survival time of 2-3 years after diagnosis. We previously showed that the administration of a chemotherapy agent mitomycin C (MMC) in rats mediates PVOD through the activation of the eukaryotic initiation factor 2 (eIF2) kinase protein kinase R (PKR) and the integrated stress response (ISR), resulting in the impairment of vascular endothelial junctional structure and barrier function. Here, we demonstrate that aged rats over 1 year exhibit more severe vascular remodeling and RV hypertrophy than young adult rats following MMC treatment. This is attributed to an age-associated elevation of basal ISR activity and depletion of protein phosphatase 1, leading to prolonged eIF2 phosphorylation and sustained ISR activation. Pharmacological blockade of PKR or ISR mitigates PVOD phenotypes in both age groups, suggesting that targeting the PKR/ISR axis could be a potential therapeutic strategy for PVOD.

Neuronal and glial cell alterations involved in the retinal degeneration of the familial dysautonomia optic neuropathy.

Familial dysautonomia (FD) is a rare genetic neurodevelopmental and neurodegenerative disorder. In addition to the autonomic and peripheral sensory neuropathies that challenge patient survival, one of the most debilitating symptoms affecting patients' quality of life is progressive blindness resulting from the steady loss of retinal ganglion cells (RGCs). Within the FD community, there is a concerted effort to develop treatments to prevent the loss of RGCs. However, the mechanisms underlying the death of RGCs are not well understood. To study the mechanisms underlying RGC death, Pax6-cre;Elp1loxp/loxp male and female mice and postmortem retinal tissue from an FD patient were used to explore the neuronal and non-neuronal cellular pathology associated with the FD optic neuropathy. Neurons, astrocytes, microglia, Müller glia, and endothelial cells were investigated using a combination of histological analyses. We identified a novel disruption of cellular homeostasis and gliosis in the FD retina. Beginning shortly after birth and progressing with age, the FD retina is marked by astrogliosis and perturbations in microglia, which coincide with vascular remodeling. These changes begin before the onset of RGC death, suggesting alterations in the retinal neurovascular unit may contribute to and exacerbate RGC death. We reveal for the first time that the FD retina pathology includes reactive gliosis, increased microglial recruitment to the ganglion cell layer (GCL), disruptions in the deep and superficial vascular plexuses, and alterations in signaling pathways. These studies implicate the neurovascular unit as a disease-modifying target for therapeutic interventions in FD.

The senescence-associated secretome of Hedgehog-deficient hepatocytes drives MASLD progression.

The burden of senescent hepatocytes correlates with the severity of metabolic dysfunction-associated steatotic liver disease (MASLD), but the mechanisms driving senescence and how it exacerbates MASLD are poorly understood. Hepatocytes experience lipotoxicity and become senescent when Smoothened (Smo) is deleted to disrupt Hedgehog signaling. We aimed to determine whether the secretomes of Smo-deficient hepatocytes perpetuate senescence to drive MASLD progression. RNA-Seq analysis of liver samples from human and murine cohorts with MASLD confirmed that hepatocyte populations in MASLD livers were depleted of Smo+ cells and enriched with senescent cells. When fed a choline-deficient, amino acid-restricted high-fat diet (CDA-HFD) to induce MASLD, Smo- mice had lower antioxidant markers and developed worse DNA damage, senescence, steatohepatitis, and fibrosis than did Smo+ mice. Sera and hepatocyte-conditioned medium from Smo- mice were depleted of thymidine phosphorylase (TP), a protein that maintains mitochondrial fitness. Treating Smo- hepatocytes with TP reduced senescence and lipotoxicity, whereas inhibiting TP in Smo+ hepatocytes had the opposite effect and exacerbated hepatocyte senescence, steatohepatitis, and fibrosis in CDA-HFD-fed mice. We conclude that inhibition of Hedgehog signaling in hepatocytes promoted MASLD by suppressing hepatocyte production of proteins that prevent lipotoxicity and senescence.

Quantitative Analysis of Rhodobacter sphaeroides Storage Organelles via Cryo-Electron Tomography and Light Microscopy.

Bacterial cytoplasmic organelles are diverse and serve many varied purposes. Here, we employed Rhodobacter sphaeroides to investigate the accumulation of carbon and inorganic phosphate in the storage organelles, polyhydroxybutyrate (PHB) and polyphosphate (PP), respectively. Using cryo-electron tomography (cryo-ET), these organelles were observed to increase in size and abundance when growth was arrested by chloramphenicol treatment. The accumulation of PHB and PP was quantified from three-dimensional (3D) segmentations in cryo-tomograms and the analysis of these 3D models. The quantification of PHB using both segmentation analysis and liquid chromatography and mass spectrometry (LCMS) each demonstrated an over 10- to 20-fold accumulation of PHB. The cytoplasmic location of PHB in cells was assessed with fluorescence light microscopy using a PhaP-mNeonGreen fusion-protein construct. The subcellular location and enumeration of these organelles were correlated by comparing the cryo-ET and fluorescence microscopy data. A potential link between PHB and PP localization and possible explanations for co-localization are discussed. Finally, the study of PHB and PP granules, and their accumulation, is discussed in the context of advancing fundamental knowledge about bacterial stress response, the study of renewable sources of bioplastics, and highly energetic compounds.

Divergent Processing of Cell Stress Signals as the Basis of Cancer Progression: Licensing NFκB on Chromatin.

Inflammation is activated by diverse triggers that induce the expression of cytokines and adhesion molecules, which permit a succession of molecules and cells to deliver stimuli and functions that help the immune system clear the primary cause of tissue damage, whether this is an infection, a tumor, or a trauma. During inflammation, short-term changes in the expression and secretion of strong mediators of inflammation occur, while long-term changes occur to specific groups of cells. Long-term changes include cellular transdifferentiation for some types of cells that need to regenerate damaged tissue, as well as death for specific immune cells that can be detrimental to tissue integrity if they remain active beyond the boundaries of essential function. The transcriptional regulator NFκB enables some of the fundamental gene expression changes during inflammation, as well as during tissue development. During recurrence of malignant disease, cell stress-induced alterations enable the growth of cancer cell clones that are substantially resistant to therapeutic intervention and to the immune system. A number of those alterations occur due to significant defects in feedback signal cascades that control the activity of NFκB. Specifically, cell stress contributes to feedback defects as it overrides modules that otherwise control inflammation to protect host tissue. NFκB is involved in both the suppression and promotion of cancer, and the key distinctive feature that determines its net effect remains unclear. This paper aims to provide a clear answer to at least one aspect of this question, namely the mechanism that enables a divergent response of cancer cells to critical inflammatory stimuli and to cell stress in general.

Inhibition of PHLDA3 expression in human superoxide dismutase 1-mutant amyotrophic lateral sclerosis astrocytes protects against neurotoxicity.

Pleckstrin homology-like domain family A-member 3 (PHLDA3) has recently been identified as a player in adaptive and maladaptive cellular stress pathways. The outcome of pleckstrin homology-like domain family A-member 3 signalling was shown to vary across different cell types and states. It emerges that its expression and protein level are highly increased in amyotrophic lateral sclerosis (ALS) patient-derived astrocytes. Whether it orchestrates a supportive or detrimental function remains unexplored in the context of neurodegenerative pathologies. To directly address the role of pleckstrin homology-like domain family A-member 3 in healthy and ALS astrocytes, we used overexpression and knockdown strategies. We generated cultures of primary mouse astrocytes and also human astrocytes from control and ALS patient-derived induced pluripotent stem cells harbouring the superoxide dismutase 1 mutation. Then, we assessed astrocyte viability and the impact of their secretome on oxidative stress responses in human stem cell-derived cortical and spinal neuronal cultures. Here, we show that PHLDA3 overexpression or knockdown in control astrocytes does not significantly affect astrocyte viability or reactive oxygen species production. However, PHLDA3 knockdown in ALS astrocytes diminishes reactive oxygen species concentrations in their supernatants, indicating that pleckstrin homology-like domain family A-member 3 can facilitate stress responses in cells with altered homeostasis. In support, supernatants of PHLDA3-silenced ALS and even control spinal astrocytes with a lower pleckstrin homology-like domain family A-member 3 protein content could prevent sodium arsenite-induced stress granule formation in spinal neurons. Our findings provide evidence that reducing pleckstrin homology-like domain family A-member 3 levels may transform astrocytes into a more neurosupportive state relevant to targeting non-cell autonomous ALS pathology.

Small RNA-Seq and real time rt-qPCR reveal islet miRNA released under stress conditions.

Replacement of beta cells through transplantation is a potential therapeutic approach for individuals with pancreas removal or poorly controllable type 1 diabetes. However, stress and death of beta cells pose significant challenges. Circulating miRNA has emerged as potential biomarkers reflecting early beta cell stress and death, allowing for timely intervention. The aim of this study was to identify miRNAs as potential biomarkers for beta cell health. Literature review combined with small RNA sequencing was employed to select islet-enriched miRNA. The release of those miRNA was assessed by RT-qPCR in vivo, using a streptozotocin induced diabetes mouse model and in vitro, through mouse and human islets exposed to varying degrees of hypoxic and cytokine stressors. Utilizing the streptozotocin induced model, we identified 18 miRNAs out of 39 candidate islet-enriched miRNA to be released upon islet stress in vivo. In vitro analysis of culture supernatants from cytokine and/or hypoxia stressed islets identified the release of 45 miRNAs from mouse and 8 miRNAs from human islets. Investigation into the biological pathways targeted by the cytokine- and/or hypoxia-induced miRNA suggested the involvement of MAPK and PI3K-Akt signaling pathways in both mouse and human islets. We have identified miRNAs associated with beta cell health and stress. The findings allowed us to propose a panel of 47 islet-related human miRNA that is potentially valuable for application in clinical contexts of beta cell transplantation and presymptomatic early-stage type 1 diabetes.

Function of unconventional T cells in oral lichen planus revealed by single-cell RNA sequencing.

OBJECTIVE: We intended to map the single-cell profile of OLP, explore the molecular characteristics of unconventional T cells in OLP tissues. METHODS: Buccal mucosa samples from OLP patients and healthy individuals were used to prepare single-cell suspension. Single-cell RNA sequencing was used to analyze the proportion of all the cells, and the molecular characteristics of unconventional T cells. Immunohistochemical staining was used to detect the expression of unconventional T cells marker genes. RESULTS: The cell clusters from buccal mucosa were categorized into immune cells, fibroblasts, endothelial cells, and epithelial cells. Unconventional T cells with phenotype of CD247+TRDC+NCAM1+ were identified. Immunohistochemical staining revealed higher expression of unconventional T cell marker genes in OLP tissue, predominantly in the lamina propria. In OLP, unconventional T cells are in a unique stress response state, exhibited enhanced NF-κB signaling and apoptosis inhibition, enhanced heat shock protein genes expression, weakened cytotoxic function. A large number of ligand-receptor pairs were found between unconventional T cells and other cells, particularly with fibroblasts and endothelial cells. CONCLUSIONS: This study mapped the single-cell profile of OLP, delineated the molecular characteristics of unconventional T cells in OLP, and uncovered that these unconventional T cells are in a stress response state.

Transcriptional analysis of primary ciliary dyskinesia airway cells reveals a dedicated cilia glutathione pathway.

Primary ciliary dyskinesia (PCD) is a genetic condition that results in dysmotile cilia. The repercussions of cilia dysmotility and gene variants on the multiciliated cell remain poorly understood. We used single-cell RNA-Seq, proteomics, and advanced microscopy to compare primary culture epithelial cells from patients with PCD, their heterozygous mothers, and healthy individuals, and we induced pluripotent stem cells (iPScs) generated from a patient with PCD. Transcriptomic analysis revealed unique signatures in PCD airway cells compared with their mothers' cells and the cells of healthy individuals. Gene expression in heterozygous mothers' cells diverged from both control and PCD cells, marked by increased inflammatory and cellular stress signatures. Primary and iPS-derived PCD multiciliated cells had increased expression of glutathione-S-transferases GSTA2 and GSTA1, as well as NRF2 target genes, accompanied by elevated levels of reactive oxygen species (ROS). Immunogold labeling in human cilia and proteomic analysis of the ciliated organism Chlamydomonas reinhardtii demonstrated that GSTA2 localizes to motile cilia. Loss of human GSTA2 and C. reinhardtii GSTA resulted in slowed cilia motility, pointing to local cilia regulatory roles. Our findings identify cellular responses unique to PCD variants and independent of environmental stress and uncover a dedicated ciliary GSTA2 pathway essential for normal motility that may be a therapeutic target.

Mitochondrial perturbation in the intestine causes microbiota-dependent injury and gene signatures discriminative of inflammatory disease.

Mitochondrial dysfunction is associated with inflammatory bowel diseases (IBDs). To understand how microbial-metabolic circuits contribute to intestinal injury, we disrupt mitochondrial function in the epithelium by deleting the mitochondrial chaperone, heat shock protein 60 (Hsp60Δ/ΔIEC). This metabolic perturbation causes self-resolving tissue injury. Regeneration is disrupted in the absence of the aryl hydrocarbon receptor (Hsp60Δ/ΔIEC;AhR-/-) involved in intestinal homeostasis or inflammatory regulator interleukin (IL)-10 (Hsp60Δ/ΔIEC;Il10-/-), causing IBD-like pathology. Injury is absent in the distal colon of germ-free (GF) Hsp60Δ/ΔIEC mice, highlighting bacterial control of metabolic injury. Colonizing GF Hsp60Δ/ΔIEC mice with the synthetic community OMM12 reveals expansion of metabolically flexible Bacteroides, and B. caecimuris mono-colonization recapitulates the injury. Transcriptional profiling of the metabolically impaired epithelium reveals gene signatures involved in oxidative stress (Ido1, Nos2, Duox2). These signatures are observed in samples from Crohn's disease patients, distinguishing active from inactive inflammation. Thus, mitochondrial perturbation of the epithelium causes microbiota-dependent injury with discriminative inflammatory gene profiles relevant for IBD.

Verification of Outer Hair Cell Motor Protein, Prestin, as a Serological Biomarker for Mouse Cochlear Damage.

The motor protein prestin, found in the inner ear's outer hair cells (OHCs), is responsible for high sensitivity and sharp frequency selectivity in mammalian hearing. Some studies have suggested that prestin could be a serological biomarker for cochlear damage, as OHCs are highly vulnerable to damage from various sources. However, the reported data are inconsistent and lack appropriate negative controls. To investigate whether prestin can be used as a serological biomarker for cochlear damage or stress, we measured prestin quantities in the bloodstreams of mice using ELISA kits from different companies. Wildtype (WT) mice were exposed to different ototoxic treatments, including noise exposure and ototoxic reagents that rapidly kill OHCs. Prestin-knockout (KO) mice were used as a negative control. Our data show that some ELISA kits were not able to detect prestin specifically. The ELISA kit that could detect the prestin protein from cochlear homogenates failed to detect prestin in the bloodstream, despite there being significant damage to OHCs in the cochleae. Furthermore, the optical densities of the serum samples, which correlate to prestin quantities, were significantly influenced by hemolysis in the samples. In conclusion, Prestin from OHCs is not a sensitive and reliable serological biomarker for detecting cochlear damage in mice using ELISA.

Inflammation primes the murine kidney for recovery by activating AZIN1 adenosine-to-inosine editing.

The progression of kidney disease varies among individuals, but a general methodology to quantify disease timelines is lacking. Particularly challenging is the task of determining the potential for recovery from acute kidney injury following various insults. Here, we report that quantitation of post-transcriptional adenosine-to-inosine (A-to-I) RNA editing offers a distinct genome-wide signature, enabling the delineation of disease trajectories in the kidney. A well-defined murine model of endotoxemia permitted the identification of the origin and extent of A-to-I editing, along with temporally discrete signatures of double-stranded RNA stress and adenosine deaminase isoform switching. We found that A-to-I editing of antizyme inhibitor 1 (AZIN1), a positive regulator of polyamine biosynthesis, serves as a particularly useful temporal landmark during endotoxemia. Our data indicate that AZIN1 A-to-I editing, triggered by preceding inflammation, primes the kidney and activates endogenous recovery mechanisms. By comparing genetically modified human cell lines and mice locked in either A-to-I-edited or uneditable states, we uncovered that AZIN1 A-to-I editing not only enhances polyamine biosynthesis but also engages glycolysis and nicotinamide biosynthesis to drive the recovery phenotype. Our findings implicate that quantifying AZIN1 A-to-I editing could potentially identify individuals who have transitioned to an endogenous recovery phase. This phase would reflect their past inflammation and indicate their potential for future recovery.

TTK inhibitor OSU13 promotes immunotherapy responses by activating tumor STING.

TTK spindle assembly checkpoint kinase is an emerging cancer target. This preclinical study explored the antitumor mechanism of TTK inhibitor OSU13 to define a strategy for clinical development. We observed prominent antitumor activity of OSU13 in melanoma, colon and breast cancer cells, organoids derived from patients with melanoma, and mice bearing colon tumors associated with G2 cell cycle arrest, senescence, and apoptosis. OSU13-treated cells displayed DNA damage and micronuclei that triggered the cytosolic DNA-sensing cGAS/STING pathway. STING was required for the induction of several proteins involved in T cell recruitment and activity. Tumors from OSU13-treated mice showed an increased proportion of T and NK cells and evidence of PD-1/PD-L1 immune checkpoint activation. Combining a low-toxicity dose of OSU13 with anti-PD-1 checkpoint blockade resulted in prominent STING- and CD8+ T cell-dependent tumor inhibition and improved survival. These findings provide a rationale for utilizing TTK inhibitors in combination with immunotherapy in STING-proficient tumors.

Characterisation of premature cell senescence in Alzheimer's disease using single nuclear transcriptomics.

Aging is associated with cell senescence and is the major risk factor for AD. We characterized premature cell senescence in postmortem brains from non-diseased controls (NDC) and donors with Alzheimer's disease (AD) using imaging mass cytometry (IMC) and single nuclear RNA (snRNA) sequencing (> 200,000 nuclei). We found increases in numbers of glia immunostaining for galactosidase beta (> fourfold) and p16INK4A (up to twofold) with AD relative to NDC. Increased glial expression of genes related to senescence was associated with greater ß-amyloid load. Prematurely senescent microglia downregulated phagocytic pathways suggesting reduced capacity for ß-amyloid clearance. Gene set enrichment and pseudo-time trajectories described extensive DNA double-strand breaks (DSBs), mitochondrial dysfunction and ER stress associated with increased ß-amyloid leading to premature senescence in microglia. We replicated these observations with independent AD snRNA-seq datasets. Our results describe a burden of senescent glia with AD that is sufficiently high to contribute to disease progression. These findings support the hypothesis that microglia are a primary target for senolytic treatments in AD.

Susceptibility to innate immune activation in genetically mediated myocarditis.

Myocarditis is clinically characterized by chest pain, arrhythmias, and heart failure, and treatment is often supportive. Mutations in DSP, a gene encoding the desmosomal protein desmoplakin, have been increasingly implicated in myocarditis. To model DSP-associated myocarditis and assess the role of innate immunity, we generated engineered heart tissues (EHTs) using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with heterozygous DSP truncating variants (DSPtvs) and a gene-edited homozygous deletion cell line (DSP-/-). At baseline, DSP-/- EHTs displayed a transcriptomic signature of innate immune activation, which was mirrored by cytokine release. Importantly, DSP-/- EHTs were hypersensitive to Toll-like receptor (TLR) stimulation, demonstrating more contractile dysfunction compared with isogenic controls. Relative to DSP-/- EHTs, heterozygous DSPtv EHTs had less functional impairment. DSPtv EHTs displayed heightened sensitivity to TLR stimulation, and when subjected to strain, DSPtv EHTs developed functional deficits, indicating reduced contractile reserve compared with healthy controls. Colchicine or NF-κB inhibitors improved strain-induced force deficits in DSPtv EHTs. Genomic correction of DSP p.R1951X using adenine base editing reduced inflammatory biomarker release from EHTs. Thus, EHTs replicate electrical and contractile phenotypes seen in human myocarditis, implicating cytokine release as a key part of the myogenic susceptibility to inflammation. The heightened innate immune activation and sensitivity are targets for clinical intervention.

The IRE1α/XBP1 pathway sustains cytokine responses of group 3 innate lymphoid cells in inflammatory bowel disease.

Group 3 innate lymphoid cells (ILC3s) are key players in intestinal homeostasis. ER stress is linked to inflammatory bowel disease (IBD). Here, we used cell culture, mouse models, and human specimens to determine whether ER stress in ILC3s affects IBD pathophysiology. We show that mouse intestinal ILC3s exhibited a 24-hour rhythmic expression pattern of the master ER stress response regulator inositol-requiring kinase 1α/X-box-binding protein 1 (IRE1α/XBP1). Proinflammatory cytokine IL-23 selectively stimulated IRE1α/XBP1 in mouse ILC3s through mitochondrial ROS (mtROS). IRE1α/XBP1 was activated in ILC3s from mice exposed to experimental colitis and in inflamed human IBD specimens. Mice with Ire1α deletion in ILC3s (Ire1αΔRorc) showed reduced expression of the ER stress response and cytokine genes including Il22 in ILC3s and were highly vulnerable to infections and colitis. Administration of IL-22 counteracted their colitis susceptibility. In human ILC3s, IRE1 inhibitors suppressed cytokine production, which was upregulated by an IRE1 activator. Moreover, the frequencies of intestinal XBP1s+ ILC3s in patients with Crohn's disease before administration of ustekinumab, an anti-IL-12/IL-23 antibody, positively correlated with the response to treatment. We demonstrate that a noncanonical mtROS-IRE1α/XBP1 pathway augmented cytokine production by ILC3s and identify XBP1s+ ILC3s as a potential biomarker for predicting the response to anti-IL-23 therapies in IBD.

Transient stimulation of TRPMLs enhance the functionality of hDPCs and facilitate hair growth in mice.

Autophagy is essential for eliminating aging and organelle damage that maintaining cellular homeostasis. However, the dysfunction of autophagy has been proven in hair loss such as AGA. Despite the crucial role of TRPML channels in regulating autophagy, their specific function in hair growth remains unclarified. To investigate the biological functions and associated molecular mechanisms of TRPMLs in hair growth, Animal experiments were conducted to confirm the function of TRLMLs activation in promoting hair growth. Subsequently, we analyzed molecular mechanisms in human dermal papilla cells (hDPCs) activated by TRPMLs through transcriptome sequencing analysis. MLSA1(a TRPML agonist) promoted hair regeneration and accelerated hair cycle transition in mice. The activation of TRPMLs upregulated calcium signaling inducing hDPCs to secrete hair growth promoting factors and decrease hair growth inhibiting factors. In addition, activation of TRPMLs triggered autophagy and reduced the generation of ROS, thereby delaying the senescence of hDPCs. All these findings suggested that TRPMLs activation could promote hair growth by regulating hDPCs secretion of hair growth-related factors. Moreover, it may play a prominent role in preventing hDPCs from ROS damage induced by H2O2 or DHT. Targeting TRPMLs may represent a promising therapeutic strategy for treating hair loss.