bioRxiv Science⌕ Search

Biology subjects

Gandhi, A.

Publications and source records attributed to Gandhi, A..

15 recordsLinked to original sources

HIRA-mediated H3.3 deposition preserves hepatocyte cell identity during liver aging

Age-associated functional decline is partly driven by progressive chromatin degeneration. Maintenance of chromatin integrity preserves cell identity and promotes healthy aging, but through different mechanisms in proliferating and non-proliferating cells. However, specific mechanisms of chromatin maintenance and their compensatory capacity in proliferating and non-proliferating cells are undefined. The histone chaperone HIRA deposits the histone variant H3.3 in a DNA replication-independent manner, leading to its accumulation in aging, non-proliferating cells. Here, we show that hepatocyte-specific loss of HIRA causes loss of cell identity, metabolic dysfunction, and accelerated fibrotic pathology with age. Transcriptomic and epigenomic analyses indicate that HIRA-H3.3 preserves chromatin integrity and sustains transcription of highly expressed genes, including cell identity genes. Partial hepatectomy, associated with induced proliferation, restores identity of HIRA knockout livers with compensatory deposition of canonical histones H3.1/2. Together, these results demonstrate that HIRA-mediated H3.3 deposition is essential for safeguarding cell identity and tissue function during aging of non-proliferating cells, but this function can be rescued by tissue regeneration and associated cell proliferation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/710643v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@a2a26aorg.highwire.dtl.DTLVardef@154e5aeorg.highwire.dtl.DTLVardef@b315a7org.highwire.dtl.DTLVardef@152bca4_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

Smooth Muscle Dysfunction Drives Cerebrovascular Reserve Failure and End-Organ Brain Injury

BackgroundFailure of cerebrovascular reserve is a fundamental determinant of ischemic vulnerability, yet the mechanisms by which vascular smooth muscle dysfunction compromises reserve and predisposes the brain to injury remain incompletely defined. We therefore tested whether a pathogenic smooth muscle mutation produces a baseline failure of cerebrovascular reserve sufficient to render the brain vulnerable to hypoperfusion, even in the absence of fixed arterial occlusion. MethodsWe examined cerebrovascular structure, hemodynamics, and reserve in a genetically defined mouse model of ACTA2-associated multisystemic smooth muscle dysfunction syndrome with systemic or brain-restricted expression of the mutant allele. Cerebral artery morphology was assessed using magnetic resonance angiography and black ink angiography. Vascular smooth muscle phenotype was evaluated by immunohistochemistry and proliferation assays. Blood pressure reactivity and cerebral blood flow (CBF) were measured simultaneously using femoral arterial catheterization and laser speckle flowmetry during vasoactive challenges and controlled hypotension. Cerebrovascular stress responses were tested using unilateral common carotid artery occlusion. Downstream brain effects were assessed by histology, resting state functional connectivity imaging, and behavioral testing. ResultsImpaired smooth muscle contractility drove rectification and narrowing of major cerebral arteries, downregulation of contractile markers, and increased vascular cell proliferation. These structural changes produced a distinct physiological phenotype: mutant mice exhibited blunted vasoreactivity, diminished spontaneous vasodynamic activity, and a downward shift in the blood pressure-CBF relationship across a wide range of arterial pressures, consistent with loss of cerebrovascular reserve. As a result, CBF was reduced at baseline and could not be maintained during hypotension or acute vascular stress. During carotid occlusion, mutant mice showed impaired compensatory perfusion, greater physiological instability, and worse behavioral outcomes. Chronic reserve failure coincided with white matter loss, reduced neuronal density, disrupted large-scale functional connectivity, and deficits in locomotion, anxiety-related behavior, and working memory. ConclusionsPathogenic smooth muscle dysfunction caused by ACTA2 mutation produces a baseline failure of cerebrovascular reserve that renders the brain vulnerable to hypoperfusion and stress-induced ischemic injury. These findings establish cerebrovascular reserve failure as a central physiological mechanism linking vascular dysfunction to end-organ brain injury and identify reserve preservation as a critical, potentially actionable determinant of brain health in hypotension-prone vascular disease. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABS- ACTA2 smooth muscle dysfunction produces baseline cerebrovascular reserve impairment, with reduced cerebral blood flow and a downward-shifted pressure-flow relationship in the absence of critical large-vessel occlusion. - Vascular tone dysregulation is coupled to end-organ brain injury, including white matter and neuronal loss, disrupted functional connectivity, and behavioral deficits. - The results support complementary disease mechanisms in ACTA2 vasculopathy: baseline reserve limitation and injury-provoked occlusive remodeling. Clinical Implications- Patients with ACTA2 vasculopathy may be vulnerable to ischemic brain injury during hypotension or systemic stress despite the absence of critical stenosis or occlusion on routine imaging. - Peri-procedural and acute-care management should emphasize preserving perfusion pressure and cerebrovascular reserve (e.g., during anesthesia, dehydration, or systemic illness). - More broadly, cerebrovascular reserve is a clinically relevant, potentially modifiable determinant of brain health in hypotension-prone vasculopathies and conditions characterized by impaired vascular reactivity.

physiology↗

Aging-induced hepatocyte CD44 drives IL6/STAT3 signaling and associates with impaired neighboring T cell function.

Liver cancer incidences increase dramatically beyond 55 years of age, suggesting that age-associated changes contribute critically to tumor initiation. However, the mechanisms linking liver aging and cancer initiation are not well defined. This study investigates the role of CD44, a marker of liver tumor-initiating cells (TIC), in age-associated liver pathophysiology. Aged livers showed accumulation of CD44-expressing hepatocytes exhibiting enrichment of immune modulatory genes and activation of the immunosuppressive IL6/JAK/STAT3 pathway. Indeed, in adoptive transfer assays, antigen-exposed CD8+ T cells mounted a lower IFN-{gamma} response in aged livers than in young livers, indicating an immunosuppressive aged milieu. Concordantly, spatial analyses showed that the proximal neighbourhoods of Cd44-expressing hepatocytes are enriched in T cells exhibiting reduced cytokine and chemokine gene expression. Finally, hepatocyte-specific knock out of Cd44 mitigated the IL6/JAK/STAT3 gene signature in aged livers. Overall, these findings suggest that CD44 expression in aged hepatocytes promotes activation of the immunosuppressive IL6/JAK/STAT3 pathway and this is associated with impaired T cell effector function.

cell biology↗

Aging alters tumor cell - T cell crosstalk to promote breast cancer progression

Age is a dominant risk factor for all major breast cancer subtypes. However, the mechanisms by which aging influences tumor development remain unclear. Using a novel mouse model whereby breast cancer is induced in situ in young and old wild-type mice via intraductal delivery of a lentivirus encoding the HER2/neu oncogene, we found that old mice exhibited a higher oncogene-induced tumor burden than young mice. Old tumor cells showed reduced expression of interferon-related genes, particularly the T cell-recruiting chemokines Cxcl9 and Cxcl10, linked to their altered chromatin accessibility. CXCL9/10 expression also declined with age in human HER2+ tumors. Correspondingly, old tumors exhibited fewer T cells within tumor lesions. Targeted interventions showed that decreased expression of Cxcl9/10 is responsible for reduced T cell infiltration and weakened anti-tumor immunity. These results show how aged tumor cells are impaired in their recruitment of immune cells, leading to a defective anti-tumor immune response.

cancer biology↗

KneEZ Clear, an Effective Tissue Clearing Protocol to Study Musculoskeletal Tissues in the Mouse

Wholemount, 3-dimensional (3D) tissue imaging holds significant promise for analyzing heterogeneous musculoskeletal tissues, such as knee joints, that demand time- and labor-intensive processing using traditional histological methods. Current musculoskeletal clearing protocols rely on either solvent-based tissue clearing, which substantially alters the size and architecture of cleared tissues, possibly compromising downstream quantification and perhaps more importantly reducing signal from endogenous fluorescent reporters, or on expensive and time-consuming hydrogel-based approaches that requires specialized equipment. While aqueous-based clearing overcomes these challenges, there is a clear need for a method that is optimized for clearing musculoskeletal tissues and that can easily be implemented in a standard lab environment. Here, we present KneEZ Clear, a simple, rapid, and flexible aqueous-based method that renders mineralized and non-mineralized tissues of murine knee joints optically transparent. We show that KneEZ Clear, which is based on the EZ Clear method, is highly flexible, demonstrating efficacy in a wide range of murine musculoskeletal tissues including the vertebral column, hindlimb, skull, and teeth. Critically, KneEZ Clear does not require specialized equipment and retains endogenous signal from fluorophores and fluorescent proteins. Additionally, following clearing and wholemount imaging, precious samples can still be processed for subsequent 2D histological analyses for validation or further study. Finally, we show that KneEZ Clear can be applied to samples of disease models to reveal alterations in tissue architecture and homeostasis. The simplicity, versatility, and efficiency of KneEZ Clear for optical clearing of musculoskeletal tissues will accelerate our understanding of cellular interactions and dynamics in homeostasis and disease.

physiology↗

Targeting CyclinD1-CDK6 to Mitigate Senescence-Driven Inflammation and Age-Associated Functional Decline

Cellular senescence contributes to aging and age-related diseases by driving chronic inflammation through the Senescence Associated Secretory Phenotype (SASP) and interferon-stimulated genes (ISGs). Cyclin D1 (CCND1), a key cell cycle regulator, is paradoxically upregulated in these non-proliferating cells. We show that CCND1 and its kinase partner CDK6 drive SASP and ISG expression in senescent cells by promoting DNA damage accumulation. This leads to the formation of cytoplasmic chromatin fragments (CCFs) that activate pro-inflammatory CGAS-STING signaling. The tumor suppressor p53 (TP53) and its target p21 (CDKN2A) antagonize this CCND1-CDK6-dependent DNA damage accumulation pathway to suppress the SASP. In aged mouse livers, senescent hepatocytes show increased Ccnd1 expression. Hepatocyte-specific Ccnd1 knockout or treatment with the Cdk4/6 inhibitor Palbociclib reduces DNA damage and ISGs in aged mouse liver. Notably, Palbociclib also suppresses frailty and improves physical performance of aged mice. These findings reveal a novel role for CCND1/CDK6 in regulating DNA damage and inflammation in senescence and aging, highlighting it as a promising therapeutic target.

cell biology↗

Stromal LOX/FAK/beta catenin pathway locks mammary fibroblasts into a tumor-promoting myCAF state

BackgroundCancer-associated fibroblasts (CAFs) sustain tumor progression, yet the soluble cues that maintain their myofibroblast (myCAF) state are poorly defined. Transforming growth factor beta (TGF-{beta}) is a canonical CAF activator. This study aims to identify TGF-{beta}-induced secreted mediators that reinforce the myCAF phenotype in breast cancer and map the downstream signaling cascade. Methods and ResultsSecretome profiling of primary patient-derived myCAFs and human mammary fibroblasts (HMF3s) engineered to over-express TGF-{beta}1 revealed 20 extracellular-matrix remodelers shared exclusively by both activated states; lysyl oxidase (LOX) was the top-ranked hit. LOX knockdown abrogated TGF-{beta}-driven -smooth-muscle actin (-SMA) induction, collagen-gel contraction and migration in HMF3s, and reduced constitutive -SMA and {beta}-catenin in myCAFs. Mechanistically, TGF-{beta} upregulated LOX, which activated focal-adhesion kinase (FAK), leading to p38 MAPK- and Akt-mediated Ser9 phosphorylation (inactivation) of GSK3{beta} and consequent {beta}-catenin stabilization. In HCC1806-luciferase orthotopic xenografts, CAFs accelerated tumor growth, whereas LOX-deficient CAFs lost this pro-tumoral effect. ConclusionLOX is a pivotal autocrine effector of TGF-{beta} that locks breast CAFs into a pro-tumoral myCAF state through a LOX/FAK/GSK3{beta}/{beta}-catenin axis. Targeting stromal LOX may disrupt CAF activation and curb breast cancer progression.

cancer biology↗

Activated Interferon Signaling Suppresses Age-Dependent Liver Cancer

Age is a major risk factor for liver cancer, as is the case for most adult human cancers. However, the underlying mechanisms are not well defined. A better understanding of the role of aging in liver and other cancers can facilitate approaches for risk assessment, early detection and prevention. We hypothesize that age-driven changes render aged liver more sensitive to oncogenic stress and hence tumorigenesis. To investigate how the liver changes with age, we documented the immune profile, transcriptome and epigenome of healthy livers from both young and aged mice, revealing pronounced alterations with aging. Notably, in aged hepatocytes, we identified heightened interferon (IFN) signaling, as well as simultaneous tumor suppressor and oncogene signaling at both bulk and single cell level, suggestive of an aged liver that is poised for neoplasia. To challenge this seemingly poised state, we employed adeno-associated virus (AAV)-mediated expression of a c-Myc oncogene in young and aged mouse liver hepatocytes in vivo. Analysis of aged hepatocytes expressing c-Myc revealed further elevated expression of IFN Stimulated Genes (ISGs). This ISG upregulation was evident in multiple models of oncogenic stress and transformation in older mice and also observed in aged humans with Metabolic dysfunction-Associated Steatohepatitis (MASH). We determined that Stat1 is both necessary and sufficient for the age specific elevated ISG expression in old wild type mice. Remarkably, inhibiting Jak/Stat signaling alongside ectopic c-Myc expression led to high-grade hepatocyte dysplasia and tumor formation, selectively in aged mice. Together, these results suggest that an aged liver is in a state of "precarious balance", due to concurrent activation of oncogenic and tumor suppressor pathways, but protected against neoplastic progression by IFN-signaling. Age-dependent activation of IFN signaling has been observed in many tissues and recent studies have demonstrated its detrimental consequences on aging, raising the question as to why IFN-signaling is activated during aging. We propose that aged tissues are intrinsically at higher risk of cancer and age-dependent activation of IFN-signaling is an adaptive process to protect from tumorigenesis, but one that also has maladaptive consequences.

cancer biology↗

THE NEURONAL ARCHITECTURE OF AUTONOMIC DYSREFLEXIA

Autonomic dysreflexia is a life-threatening medical condition characterized by episodes of uncontrolled hypertension that occur in response to sensory stimuli after spinal cord injury (SCI)1-7. The fragmented understanding of the mechanisms underlying autonomic dysreflexia hampers the development of therapeutic strategies to manage this condition, leaving people with SCI at daily risk of heart attack and stroke8-18. Here, we expose the complete de novo neuronal architecture that develops after SCI and causes autonomic dysreflexia. In parallel, we uncover a competing, yet overlapping neuronal architecture activated by epidural electrical stimulation of the spinal cord that safely regulates blood pressure after SCI. The discovery that these adversarial neuronal architectures converge onto a single neuronal subpopulation provided a blueprint for the design of a mechanism-based intervention that reversed autonomic dysreflexia in mice, rats, and humans with SCI. These results establish a path for the effective treatment of autonomic dysreflexia in people with SCI.

neuroscience↗

Small molecule mediated stabilization of PP2A modulates the Homologous Recombination pathway and potentiates DNA damage-induced cell death

High-Grade Serous Carcinoma (HGSC) is the most common and lethal ovarian cancer subtype. PARP-inhibitors (PARPi) have become the mainstay of HGSC targeted therapy, given that these tumors are driven by a high degree of genomic instability and Homologous Recombination (HR) defects. Nonetheless, only [~]30% of patients initially respond to treatment, ultimately relapsing with resistant disease. Thus, despite recent advances in drug development and increased understanding of genetic alterations driving HGSC progression, mortality has not declined, highlighting the need for novel therapies. Using a Small Molecule Activator of Protein Phosphatase 2A (PP2A) (SMAP-061), we investigated the mechanism by which PP2A stabilization induces apoptosis in Patient-Derived HGSC cells and Xenograft (PDX) models alone or in combination with PARPi. We uncovered that PP2A genes essential for transformation (B56,B56{gamma} and PR72) and basal phosphatase activity (PP2A-A and -C) are heterozygously lost in the majority of HGSC. Moreover, loss of these PP2A genes correlates with worse overall patient survival. We show that SMAP-061 stabilization of PP2A inhibits the HR output by targeting RAD51, leading to chronic accumulation of DNA damage and ultimately apoptosis. Furthermore, combination of SMAP-061 and PARPi leads to enhanced apoptosis in both HR-proficient and -deficient cells and in patient-derived xenograft models. Our studies identify PP2A as novel regulator of HR and introduces PP2A activators as a potential treatment for HGSC tumors. Our studies further emphasize the potential of PP2A modulators to overcome PARPi insensitivity, given that targeting RAD51 has presented benefits in overcoming PARPi-resistance driven by BRCA1/2 mutation reversions.

cancer biology↗

CORK1, a LRR-Malectin Receptor Kinase for Cellooligomer Perception in Arabidopsis thaliana

Cell wall integrity (CWI) maintenance is central for plant cells. Mechanical or chemical distortions, pH changes, or breakdown products of cell wall polysaccharides activate plasma membrane-localized receptors and induce appropriate downstream responses. Microbial interactions alter or destroy the structure of the plant cell wall, connecting CWI maintenance to immune responses. Cellulose is the major polysaccharide in the primary and secondary cell wall. Its breakdown generates short-chain cellooligomers which induce Ca2+-dependent CWI responses. We have shown here that these responses require the malectin domain-containing CELLOOLIGOMER-RECEPTOR KINASE 1 (CORK1) in Arabidopsis. CORK1 is required for cellooligomer-induced cytoplasmic Ca2+ elevation, reactive oxygen species (ROS) production, mitogen associated protein kinase (MAPK) activation, cellulose synthase phosphorylation, and the regulation of CWI-related genes including those involved in biosynthesis of cell wall material, secondary metabolites and tryptophan. Phosphoproteome analyses identified early targets involved in signaling, cellulose synthesis, the endoplasmatic reticulum/Golgi secretory pathway, cell wall repair and immune responses. Two conserved phenylalanine residues in the malectin domain are crucial for CORK1 function. We propose that cellulose breakdown products bind to the malectin domain in CORK1, indicating its role as a novel receptor kinase for CWI maintenance.

plant biology↗

Substrate dependent homeostatic control of c-di-AMP synthase (MsDisA) and hydrolase (MsPDE) from Mycobacterium smegmatis

Cyclic-di-nucleotide based secondary messengers regulate various physiological processes including the stress responses in bacteria. In the past decade, cyclic diadenosine monophosphate (c-di-AMP) has emerged as a crucial second messenger, implicated in fatty acid metabolism, antibiotic resistance, biofilm formation, virulence, DNA repair, ion homeostasis, sporulation etc. The level of c-di-AMP is maintained in the cell by the action of two opposing enzymes, namely diadenylate cyclase (DAC) and phosphodiesterase (PDE). In mycobacteria, this molecule is essential for its regulatory role in bacterial physiology and host-pathogen interactions. However, such modulation of c-di-AMP remains to be explored in Mycobacterium smegmatis. Here, we systematically characterised the c-di-AMP synthase (MsDisA) and a hydrolase (MsPDE) from M. smegmatis at different pH and osmolytic conditions in vitro. Our biochemical assays show that the MsDisA activity is enhanced during the alkaline stress and c-di-AMP is readily produced without any intermediates. At pH 9.4, the MsDisA promoter activity in vivo increases significantly, strengthening this observation. However, under physiological conditions, the activity of MsDisA was moderate with the formation of intermediates. To get further insights into the structural characteristics, we determined the cryo-EM structure of the MsDisA, revealing some interesting features. Biochemical analysis of individual domains shows that the N-terminal minimal region alone can form a functional octamer. Altogether, our results reveal the biochemical and structural regulation of mycobacterial c-di-AMP in response to various environmental stress.

biophysics↗

Influence of visual feedback persistence on visuo-motor skill improvement

Towards the larger goal of understanding factors relevant for improving visuo-motor control, we investigated the role of visual feedback for modulating the effectiveness of a simple hand-eye training protocol. The regimen comprised a series of curve tracing tasks undertaken over a period of one week by neurologically healthy individuals with their non-dominant hands. Our three subject groups differed in the training they experienced: those who received Persistent visual-feedback by seeing their hand and trace evolve in real-time superimposed upon the reference patterns, those who received Non-Persistent visual-feedback seeing their hand movement but not the emerging trace, and a Control group that underwent no training. Improvements in performance were evaluated along two dimensions - accuracy and steadiness, to assess visuo-motor and motor skills, respectively. We found that persistent feedback leads to a significantly greater improvement in accuracy than non-persistent feedback. Steadiness, on the other hand, benefits from training irrespective of the persistence of feedback. Our results not only demonstrate the feasibility of rapid visuo-motor learning in adulthood, but more specifically, the influence of visual veridicality and a critical role for dynamically emergent visual information.

neuroscience↗

Structural basis of the dynamic human CEACAM1 monomer-dimer equilibrium

Human (h) carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) function depends upon IgV-mediated homodimerization or heterodimerization with host ligands, including hCEACAM5 and hTIM-3, and a variety of microbial pathogens. However, there is little structural information available on how hCEACAM1 transitions between monomeric and dimeric states which in the latter case is critical for initiating hCEACAM1 activities. We therefore mutated residues within the hCEACAM1 IgV GFCC face including V39, I91, N97 and E99 and examined hCEACAM1 IgV monomer-homodimer exchange using differential scanning fluorimetry, multi-angle light scattering, X-ray crystallography and/or nuclear magnetic resonance. From these studies, we describe hCEACAM1 homodimeric, monomeric and transition states at atomic resolution and its conformational behavior in solution through NMR assignment of the wildtype (WT) hCEACAM1 IgV dimer and N97A monomer. These studies reveal the flexibility of the GFCC face and its important role in governing the formation of hCEACAM1 dimers and potentially heterodimers.

immunology↗

JAK Inhibitors Suppress Colon Cancer Cachexia-Associated Anorexia and Adipose Wasting in Mice

BackgroundCachexia (CX), a syndrome of muscle atrophy, adipose loss, and anorexia, is associated with reduced survival in cancer patients. The colon adenocarcinoma C26c20 cell line secretes the cytokine leukemia inhibitor factor (LIF) which induces CX. We characterized how LIF promotes CX-associated weight loss and anorexia in mice through JAK-dependent changes in adipose and hypothalamic tissues. MethodsCX was induced in vivo with C26c20 colon adenocarcinoma cells or recombinant LIF administration in the absence or presence of JAK inhibitors. Blood, adipose, and hypothalamic tissues were collected and processed for cyto/adipokine ELISAs, immunoblot analysis, and quantitative RT-PCR. CX was induced in vitro by stimulating differentiated adipocytes with recombinant LIF or IL-6 in the absence or presence of lipase or JAK inhibitors. These activated adipocytes were processed for lipolysis, immunoblot analysis, and RT-PCR. ResultsTumor-secreted LIF induced changes in adipose tissue expression and serum levels of IL-6 and leptin in a JAK-dependent manner influencing CX-associated adipose wasting and anorexia. We identified two JAK inhibitors that block cytokine-mediated adipocyte lipolysis and IL-6 induction using an in vitro CX lipolysis assay. JAK inhibitors administered to in vivo colon cancer CX mouse models led to 1) a decrease in STAT3 phosphorylation in hypothalamic and adipose tissues, 2) a reverse in the CX serum cyto/adipokine signature, 3) a delay in colon cancer CX-associated anorexia and adipose loss, and 4) an improvement in overall survival. ConclusionsJAK inhibitors suppress cytokine-associated adipose loss and anorexia in multiple in vitro and in vivo models of cancer CX.

cancer biology↗