Biology subjects

Kumari, R.

Publications and source records attributed to Kumari, R..

29 records · Page 2Linked to original sources

bioRxiv2023

VCD-induced menopause mouse model reveals reprogramming of hepatic metabolism

Menopause adversely impacts systemic energy metabolism and increases the risk of metabolic disease(s) including hepatic steatosis, but the mechanisms are largely unknown. Dosing female mice with vinyl cyclohexene dioxide (VCD) selectively causes follicular atresia in ovaries, leading to a murine menopause-like phenotype. In this study, we treated female C57BL6/J mice with VCD (160mg/kg i.p. for 20 consecutive days followed by verification of the lack of estrous cycling) to investigate changes in body composition, energy expenditure (EE), hepatic mitochondrial function, and hepatic steatosis across different dietary conditions. VCD treatment induced ovarian follicular loss and increased follicle-stimulating hormone (FSH) levels in female mice, mimicking a menopause-like phenotype. VCD treatment did not affect body composition, or EE in mice on a low-fat diet or in response to a short-term (1-week) high-fat, high sucrose diet (HFHS). However, the transition to a HFHS lowered cage activity in VCD mice. A chronic HFHS diet (16 weeks) significantly increased weight gain, fat mass, and hepatic steatosis in VCD-treated mice compared to HFHS-fed controls. In the liver, VCD mice showed suppressed hepatic mitochondrial respiration on LFD, while chronic HFHS diet resulted in compensatory increases in hepatic mitochondrial respiration. Also, liver RNA sequencing revealed that VCD promoted global upregulation of hepatic lipid/cholesterol synthesis pathways. Our findings suggest that the VCD- induced menopause model compromises hepatic mitochondrial function and lipid/cholesterol homeostasis that sets the stage for HFHS diet-induced steatosis while also increasing susceptibility to obesity.

physiology↗

bioRxiv2023

Mitochondrial dsRNA from B-ALL cells stimulates mesenchymal stromal cells to become cancer associated fibroblasts

Cancer associated fibroblasts (CAF) arising from bone marrow-derived mesenchymal stromal cells (MSC) are prominent in B-precursor acute lymphoblastic leukaemia (B-ALL). We have previously shown that CAF formation is triggered by exposure to reactive oxygen species-inducing chemotherapy and that CAF support chemoresistance by donating mitochondria to the cancer cells, through tunnelling nanotubes. In the present study, we show that exposure of MSC to ALL cell lines, PDX and primary cells or their conditioned media can also trigger CAF formation, in an oncogene-dependent manner. Using bulk RNA sequencing in cell lines, we show that the MSC to CAF transition is accompanied by a robust interferon pathway response and we have validated this finding in primary cells. Using confocal microscopy and flow cytometry, we identify the take up of leukaemia cell-derived mitochondrial dsRNA by MSC as a proximate trigger for the MSC to CAF transition. We show that degradation of dsRNA in ALL cell conditioned media by DMSO ablates the ability of the conditioned media to stimulate MSC to CAF transition. Since we find that only specific primary driver genetic subtypes of B-ALL possess the property to directly generate CAFs, we propose this phenomenon as the first mechanistic insight into the strong relationship between acute lymphoblastic leukaemia genetic subtype and survival outcomes. Key pointsX Exposure of MSC to B-precursor ALL cell lines triggers cancer-associated fibroblast formation in an oncogene-dependent manner The proximate trigger for CAF formation is ALL-derived mitochondrial double stranded RNA

cancer biology↗

bioRxiv2023

Sympathetic NPY controls glucose homeostasis, cold tolerance, and cardiovascular functions in mice

Neuropeptide Y (NPY) is best known for its effects in the brain as an orexigenic and anxiolytic agent and in reducing energy expenditure. NPY is also co-expressed with Norepinephrine (NE) in sympathetic neurons. Although NPY is generally considered to modulate noradrenergic responses, its specific roles in autonomic physiology remain under-appreciated. Here, we show that sympathetic-derived NPY is essential for metabolic and cardiovascular regulation in mice. NPY and NE are co-expressed in 90% of prevertebral sympathetic neurons and only 43% of paravertebral neurons. NPY-expressing neurons primarily innervate blood vessels in peripheral organs. Sympathetic-specific deletion of NPY elicits pronounced metabolic and cardiovascular defects in mice, including reductions in insulin secretion, glucose tolerance, cold tolerance, pupil size, and an elevation in heart rate, while notably, however, basal blood pressure was unchanged. These findings provide new knowledge about target tissue-specific functions of NPY derived from sympathetic neurons and imply its potential involvement in metabolic and cardiovascular diseases.

neuroscience↗

bioRxiv2023

Investigating the ATP binding pocket of CX3CL1 binding protein 2 using in silico approach

Plasmodium falciparum (Pf) causes the most fatal form of malaria owing to its ability to cytoadhere in the microvasculature of various organs in the body. In addition to the Pf erythrocyte membrane protein 1 (PfEMP1) family that binds diverse host receptors, CX3CL1 binding proteins 1 and 2 (CBP1 and 2) also bind the endothelial chemokine CX3CL1 to effect cytoadhesion of parasite infected erthrocytes. CBP2 is a multifaceted protein that binds nucleic acids, Pf skeleton binding protein (PfSBP1) and ATP. ATP binding to the cytoplasmic domain of CBP2 (cCBP2) induces structural changes in the protein, and hints at its role in cell signaling. In this study, we have attempted to identify the ATP binding pocket of CBP2 using an in silico approach. We have also delineated the type of interactions and amino acid residues that are likely to bind ATP. As CX3CL1 binding proteins are central to parasite biology, the obtained information is likely to form the basis for inhibitor and drug design against this molecule.

bioinformatics↗

bioRxiv2023

PTEN knockout using retrogradely transported AAVs restores locomotor abilities in both acute and chronic spinal cord injury.

Restoring function in chronic stages of spinal cord injury (SCI) has often been met with failure or reduced efficacy when regenerative strategies are delayed past the acute or sub-acute stages of injury. Restoring function in the chronically injured spinal cord remains a critical challenge. We found that a single injection of retrogradely transported adeno-associated viruses (AAVrg) to knockout the phosphatase and tensin homolog protein (PTEN) in chronic SCI can effectively target both damaged and spared axons and restore locomotor functions in near-complete injury models. AAVrgs were injected to deliver cre recombinase and/or a red fluorescent protein (RFP) under the human Synapsin 1 promoter (hSyn1) into the spinal cords of C57BL/6 PTENFlox{Delta}/{Delta} mice to knockout PTEN (PTEN-KO) in a severe thoracic SCI crush model at both acute and chronic time points. PTEN-KO improved locomotor abilities in both acute and chronic SCI conditions over a 9-week period. Regardless of whether treatment was initiated at the time of injury (acute), or three months after SCI (chronic), mice with limited hindlimb joint movement gained hindlimb weight support after treatment. Interestingly, functional improvements were not sustained beyond 9 weeks coincident with a loss of RFP reporter-gene expression and a near-complete loss of treatment-associated functional recovery by 6 months post-treatment. Treatment effects were also specific to severely injured mice; animals with weight support at the time of treatment lost function over a 6-month period. Retrograde tracing with Fluorogold revealed viable neurons throughout the motor cortex despite a loss of RFP expression at 9 weeks post-PTEN-KO. However, few Fluorogold labeled neurons were detected within the motor cortex at 6 months post-treatment. BDA labeling from the motor cortex revealed a dense corticospinal tract (CST) bundle in all groups except chronically treated PTEN-KO mice indicating a potential long-term toxic effect of PTEN-KO to neurons in the motor cortex. PTEN-KO mice had significantly more {beta}-tubulin III labeled axons within the lesion when treatment was delivered acutely, but not chronically post-SCI. In conclusion, we have found that using AAVrgs to knockout PTEN is an effective manipulation capable of restoring motor functions in chronic SCI and can enhance axon growth of currently unidentified axon populations when delivered acutely after injury. However, the long-term consequences of PTEN-KO may exert neurotoxic effects.

neuroscience↗

bioRxiv2023

Musashi-2 causes cardiac hypertrophy and heart failure by inducing mitochondrial dysfunction through destabilizing Cluh and Smyd1 mRNA

Regulation of RNA stability and translation by RNA-binding proteins (RBPs) is a crucial process altering gene expression. Musashi family of RBPs comprising Msi1 and Msi2 are known to control RNA stability and translation. However, despite the presence of MSI2 in the heart, its function remains entirely unknown. Here, we aim to explore the cardiac functions of MSI2. We confirmed the presence of MSI2 in the adult mouse, rat heart, and neonatal rat cardiomyocytes. Furthermore, Msi2 was significantly enriched in the hearts cardiomyocyte fraction. Next, using RNA-seq data and isoform-specific PCR primers, we identified, Msi2 isoforms 1, 4, and 5 and two novel putative isoforms labeled as Msi2 isoforms 6 and 7 to be expressed in the heart. Overexpression of Msi2 isoforms led to cardiac hypertrophy in cultured cardiomyocytes. Additionally, Msi2 was also found to be significantly increased in a pressure-overload model of cardiac hypertrophy. To validate the hypertrophic effects, we selected isoforms 4 and 7 due to their unique alternative splicing patterns. AAV9-mediated overexpression of Msi2 isoforms 4 and 7 in murine hearts led to cardiac hypertrophy, dilation, heart failure, and eventually early death, confirming a pathological function for Msi2. Using global proteomics, gene ontology, transmission electron microscopy, and transmembrane potential measurement assays increased MSI2 was found to cause mitochondrial dysfunction in the heart. Mechanistically, we identified Cluh and Smyd1 as direct downstream targets of Msi2. Overexpression of Cluh or Smyd1 inhibited Msi2-induced hypertrophy and mitochondrial dysfunction in cardiomyocytes. Collectively, we show that Msi2 induces hypertrophy, mitochondrial dysfunction, and heart failure.

cell biology↗

bioRxiv2023

Specialized actin nanoscale layers control focal adhesion turnover

Focal adhesions (FAs) connect inner workings of the cell to the extracellular matrix to control cell adhesion, migration, and mechanosensing1,2. Previous studies demonstrated that FAs contain three vertical layers, which connect extracellular matrix to the cytoskeleton3,4,5. However, cellular processes rely on precisely-regulated FA turnover, but the molecular machineries that control FA assembly and disassembly have remained elusive. By using super-resolution iPALM microscopy, we identified two unprecedented nanoscale layers within FAs, specified by actin filaments bound to tropomyosin isoforms Tpm1.6 and Tpm3.2. The Tpm1.6-actin filaments beneath the previously identified actin-regulatory layer are critical for adhesion maturation and controlled cell motility, whereas the Tpm3.2-actin filament layer towards the bottom of FA facilitates adhesion disassembly. Mechanistically, Tpm3.2 stabilizes KANK-family proteins at adhesions, and hence targets microtubule plus-ends to FAs to catalyse their disassembly. Loss of Tpm3.2 leads to disorganized microtubule network, abnormally stable FAs, and defects in tail retraction during cell migration. Thus, FAs are composed of at least three distinct actin filament layers, each having specific roles in coupling of adhesion to the cytoskeleton, or in controlling adhesion dynamics. In a broader context, these findings demonstrate how distinct actin filament populations can co-exist and perform specific functions within a defined cellular compartment.

cell biology↗

bioRxiv2022

ATF5 is a regulator of exercise-induced mitochondrial quality control in skeletal muscle

ObjectivesThe Mitochondrial Unfolded Protein Response (UPRmt) is a compartment-specific mitochondrial quality control (MQC) mechanism that uses the transcription factor ATF5 to induce the expression of protective enzymes to restore mitochondrial function. Acute exercise is a stressor that has the potential to temporarily disrupt organellar protein homeostasis, however, the roles of ATF5 and the UPRmt in maintaining basal mitochondrial content, function and exercise-induced MQC mechanisms in skeletal muscle are not known. MethodsATF5 KO and WT mice were examined at rest or after a bout of acute endurance exercise. We measured protein content in whole muscle, nuclear, cytosolic and mitochondrial fractions, in addition to mRNA transcript levels in whole muscle. Using isolated mitochondria, we quantified rates of oxygen consumption and ROS emission to observe the effects of the absence of ATF5 on organelle function. ResultsATF5 KO mice exhibited a larger and less functional muscle mitochondrial pool, most likely a culmination of enhanced biogenesis via increased PGC-1 expression, and attenuated mitophagy. The absence of ATF5 resulted in a reduction in antioxidant proteins and increases in mitochondrial ROS emission, cytosolic cytochrome c, and the expression of mitochondrial chaperones. KO muscle also displayed enhanced exercise-induced stress kinase signaling, but a blunted mitophagic and UPRmt gene expression response, complemented by significant increases in the basal mRNA abundance and nuclear localization of ATF4. Instead of promoting its nuclear translocation, acute exercise caused the enrichment of ATF5 in mitochondrial fractions. We also identified PGC-1 as an additional regulator of the basal expression of UPRmt genes. ConclusionThe transcription factor ATF5 retains a critical role in the maintenance of mitochondrial homeostasis and the appropriate response of muscle to acute exercise for the optimization of mitochondrial quality control. Graphical abstractA modified version of the schematic shown in Fig. 8 will be supplied at a later time. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=170 SRC="FIGDIR/small/500448v1_fig8.gif" ALT="Figure 8"> View larger version (65K): org.highwire.dtl.DTLVardef@1713feeorg.highwire.dtl.DTLVardef@1c48f3org.highwire.dtl.DTLVardef@f98720org.highwire.dtl.DTLVardef@164a7a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 8.C_FLOATNO Working model illustrating the altered physiology of ATF5 KO muscle. The absence of ATF5 in skeletal muscle yields a more abundant mitochondrial pool composed of organelles that are less functional. This is characterized by reductions in oxygen consumption and enhanced ROS emission in comparison to WT mitochondria. Increases in ROS may be exacerbated by decreases in the expression of the antioxidant enzymes MnSOD and NQO1, inducing an increase in apoptotic cytochrome c release into the cytosol via the mPTP. The increased abundance of nuclear PGC-1 in the absence of ATF5 may be contributing to the transcription of nuclear genes encoding mitochondrial proteins (NuGEMPs) as well as increases in mitochondrial content observed, suggesting that ATF5 may be a negative regulator of PGC-1 in WT conditions. An enlarged mitochondrial pool in KO muscle may also be attributed to decrements in basal mitophagy indicated by reduced mitochondrial Parkin. ATF5 KO animals also exhibit a blunted mitochondrial quality control response to acute exercise stress, with an attenuated induction of mitochondrial LC3-II and the transcription of UPRmt mRNAs. However, the increased mRNA levels and nuclear localization of ATF4 may explain the enhanced expression of chaperones HSP60 and Cpn10 basally in ATF5 KO muscle. Despite the attenuated mitochondrial response (above), an increased stress kinase signaling was evident post-exercise in ATF5 KO muscle, represented by enhanced JNK phosphorylation. The activation of JNK could also result from mitochondrial proteotoxicity to induce the transcription of ATF4 and the increase in ATF4 expression. Finally, acute exercise appears to induce the import of ATF5 into mitochondria, rather than to the nucleus. Solid arrows indicate evidence that signaling is occurring. C_FIG

physiology↗

bioRxiv2021

Cytoplasmic labile iron accumulates in aging stem cells perturbing a key rheostat for identity control

Bone marrow resident and rarely dividing haematopoietic stem cells (HSC) harbour an extensive self-renewal capacity to sustain life-long blood formation;1-5 albeit their function declines during ageing.6, 7 Various molecular mechanisms confer stem cell identity, ensure long-term maintenance and are known to be deregulated in aged stem cells.8, 9 How these programs are coordinated, particularly during cell division, and what triggers their ageing-associated dysfunction has been unknown. Here, we demonstrate that HSC, containing the lowest amount of cytoplasmic chelatable iron (labile iron pool)10 among hematopoietic cells, activate a limited iron response during mitosis. Engagement of this iron homeostasis pathway elicits mobilization and {beta}-oxidation of arachidonic acid and enhances stem cell-defining transcriptional programs governed by histone acetyl transferase Tip60/KAT5. We further find an age-associated expansion of the labile iron pool, along with loss of Tip60/KAT5-dependent gene regulation to contribute to the functional decline of ageing HSC, which can be mitigated by iron chelation. Together, our work reveals cytoplasmic redox active iron as a novel rheostat in adult stem cells; it demonstrates a role for the intracellular labile iron pool in coordinating a cascade of molecular events which reinforces HSC identity during cell division and to drive stem cell ageing when perturbed. As loss of iron homeostasis is commonly observed in the elderly, we anticipate these findings to trigger further studies into understanding and therapeutic mitigation of labile iron pool-dependent stem cell dysfunction in a wide range of degenerative and malignant pathologies.

cell biology↗

bioRxiv2019

Mast cells are mediators of fibrosis and effector cell recruitment in dermal chronic graft-versus-host disease

Allogeneic hematopoietic stem cell transplant (allo-HSCT) is often used to treat acute leukemia or defects of hematopoiesis. Its widespread use is hampered by graft-versus-host disease (GVHD), which has high morbidity and mortality in both acute and chronic subtypes. Chronic GVHD (cGVHD) occurs most frequently in skin and often is characterized by pathogenic fibrosis. Mast cells (MCs) are known to be involved in the pathogenesis of other fibrotic diseases. In a murine model of cGVHD after allo-HSCT, C57BL/6J recipients of allogeneic LP/J donor cells develop sclerodermatous dermal cGVHD which is significantly decreased in mast cell-deficient B6.Cg-KitW-sh/HNihrJaeBsmGlliJ recipients. MCs survive conditioning and are associated with fibrosis, chemokine production, and recruitment of GVHD effector cells to the skin. Chemokine production by MCs is blocked by drugs used to treat cGVHD. The importance of MCs in skin cGVHD is mirrored by increased MCs in the skin of patients with dermal cGVHD. We show for the first time a role for MCs in skin cGVHD that may be targetable for preventive and therapeutic intervention in this disease.

immunology↗

bioRxiv2018

Regulation of ATM and ATR by SMARCAL1 and BRG1

The G2/M checkpoint is activated on DNA damage by the ATM and ATR kinases that are regulated by post-translational modifications. In this paper, the transcriptional co-regulation of ATM and ATR by SMARCAL1 and BRG1, both members of the ATP-dependent chromatin remodeling protein family, is described. SMARCAL1 and BRG1 co-localize on the promoters of ATM and ATR; downregulation of SMARCAL1/BRG1 results in transcriptional repression of ATM/ATR and therefore, overriding of the G2/M checkpoint leading to mitotic abnormalities. On doxorubicin-induced DNA damage, SMARCAL1 and BRG1 are upregulated and in turn, upregulate the expression of ATM/ATR.\n\nPhosphorylation of ATM/ATR is needed for the transcriptional upregulation of SMARCAL1 and BRG1, and therefore, of ATM and ATR on DNA damage. The regulation of ATM/ATR is rendered non-functional if SMARCAL1 and/or BRG1 are absent or if the two proteins are mutated such that they are unable to hydrolyze ATP, as in for example in Schimke Immuno-Osseous Dysplasia and Coffin-Siris Syndrome. Thus, an intricate transcriptional regulation of DNA damage response genes mediated by SMARCAL1 and BRG1 is present in mammalian cells.

molecular biology↗