Biology subjects

Bhat, M. A.

Publications and source records attributed to Bhat, M. A..

10 recordsLinked to original sources

bioRxiv2026

Cigarette smoke extract and palmitic acid exposed patient-derived iPS- RPE cells as a model for AMD

Purpose: Age-related macular degeneration (AMD) is a multifactorial disease in which dysfunction and loss of retinal pigment epithelial (RPE) cells contribute to disease progression. Besides genetics, cigarette smoking and high fat diet are important environmental and metabolic risk factors for AMD and are associated with RPE dysfunction. However, the effects of combined environmental and metabolic stress on AMD-derived RPE cells remain poorly understood. In this study, we used patient-derived induced pluripotent stem cell-derived RPE (iPSC-RPE) cells to examine the effects of cigarette smoke extract (CSE) and palmitic acid (PA) on RPE cell function. Methods: iPSCs derived from non-AMD and AMD donors were differentiated into RPE cells and characterized based on morphology, RPE-specific marker expression, polarity, gene expression, and barrier function. Mature iPSC-RPE cells were exposed to different concentrations of CSE and PA to determine experimental conditions. Cell death, epithelial-to-mesenchymal transition (EMT)-like morphological changes, stress-related gene expression, RPE polarity, TEER, VEGF-A and PEDF secretion, and intracellular lipid accumulation were then evaluated following CSE, PA, or combined CSE and PA exposure. Results: Differentiated iPSC-RPE cells developed typical pigmented, hexagonal morphology, expressed RPE-specific markers, and formed polarized monolayers. Both CSE and PA increased cell death in a dose-dependent manner in the control and AMD-iPSC-RPE, with AMD-iPSC-RPE exhibiting greater susceptibility, particularly those from patients with dry AMD. CSE and PA also induced also induced EMT-like morphological changes and increased the expression of ZEB1, NQO1, and IL8. Furthermore, CSE and PA disrupted the polarized distribution of ZO-1 and Best1, significantly reduced the polarity ratio, decreased PEDF and VEGF-A secretion and increased intracellular lipid accumulation. Combined CSE and PA treatment produced more pronounced effects than either treatment alone in AMD lines. Conclusions: AMD- iPSC-RPE cells, particularly those derived from dry AMD donors, exhibit increased susceptibility to environmental and metabolic stress. Exposure to CSE and PA revealed this vulnerability through alterations in cell survival, stress responses, epithelial morphology, polarity, secretory function, and intracellular lipid accumulation. These findings highlight the effects of CSE and lipid stress on RPE metabolism and underscore the utility of patient-derived iPSC-RPE cells as a model for investigating how environmental and metabolic factors contribute to RPE dysfunction and AMD pathogenesis.

cell biology↗

bioRxiv2026

A chemigenetic fluorescence lifetime-based biosensor for imaging of ERK activity

Extracellular signal-regulated kinase (ERK) governs many cellular processes, and its spatiotemporal activity dynamics are important for downstream signaling specificity. Existing fluorescent ERK activity biosensors, however, fall short in enabling spectrally multiplexed, spatially resolved, or deep tissue measurements. Here, we introduce a chemigenetic, single-fluorophore biosensor, WHaloEKAR, that reports ERK activities through changes in fluorescence lifetime. WHaloEKAR combines large lifetime changes (-0.81 ns) with the spectral versatility of the self-labeling protein HaloTag giving access to orange, red, and far-red ERK biosensors. The latter enabled the investigation of heterogeneous ERK activity at distinct subcellular locations in cultured cell lines and primary cortical rat neurons via fluorescence lifetime imaging microscopy (FLIM). Moreover, multiplexed FLIM measurements allowed to study ERK activity in parallel to the second messengers calcium and cyclic adenosine monophosphate. Finally, WHaloEKAR enabled the investigation of ERK activity in 3D samples as well as in live animals.

cell biology↗

bioRxiv2025

Tbx1 heterozygosity in the oligodendrocyte lineage has distinct effects on myelinated axons in the fimbria and on behaviors related to neurodevelopmental disorders in mice.

Constitutive heterozygosity of Tbx1, a T-box transcription factor gene located within the 22q11.2 deletion region, results in behavioral deficits and altered composition of myelinated axons in the fimbria, together with reduced levels of an oligodendrocyte precursor cell (OPC) marker, in mice. However, the cellular origins of these effects and the extent to which axonal changes causally contribute to behavioral impairments remain unclear. We hypothesized that Tbx1 deficiency specifically within the oligodendrocyte lineage contributes to myelin and behavioral phenotypes. To test this hypothesis, we first demonstrated through in vitro siRNA knockdown that Tbx1 regulates both OPCs and mature oligodendrocytes. Subsequently, we assessed the impact of Tbx1 heterozygosity initiated in OPCs on behavioral and myelin phenotypes in male conditional PdgfrCre;Tbx1+/flox mice. These mice exhibited Cre-mediated recombination in Pdgfr-expressing brain regions and in the OPC progeny within the fimbria. At one month of age, the mutants displayed a higher rate of spontaneous alternation at the longest inter-trial interval in the T-maze compared to their wild-type littermates--an effect that was dissipated at two months. No significant phenotypic abnormalities were observed in conditional PdgfrCre;Tbx1+/flox mice regarding neonatal ultrasonic vocalizations, social interaction, novel object approach, anxiety-like behavior (elevated plus maze), or open-field locomotion and thigmotaxis. Electron microscopic analysis revealed a compositional shift in myelinated axons within the fimbria of adult male mutants, characterized by an increased number of myelinated axons in the 300-800 nm diameter range and a decreased number in the [~]1,200 nm and [~]1,400 nm ranges, with myelin thickness remaining unchanged across diameters. These findings indicate that Tbx1 heterozygosity in the oligodendrocyte lineage leads to a selective shift towards smaller myelinated axons in the fimbria and a transiently higher level of capacity for working memory and cognitive flexibility. However, it does not replicate the full spectrum of myelination abnormalities or the broader cognitive and social deficits observed in constitutive Tbx1 heterozygotes, suggesting that Tbx1 deficiency in non-oligodendrocyte lineage cells may lead to altered myelination and neurodevelopmental behavioral impairments.

neuroscience↗

bioRxiv2025

Tmem117, an oligodendrocyte-enriched regulator of NCX activity, links myelin homeostasis to counterregulation and metabolic health.

The counterregulatory response (CRR) to hypoglycemia is a fundamental, evolutionarily conserved homeostatic mechanism orchestrated by the central nervous system (CNS) to ensure survival during glucose scarcity. In individuals with diabetes, this response is frequently impaired, contributing to life-threatening episodes of hypoglycemia. Tmem117 was previously identified in a genetic screen as a promising hypothalamic regulator of CRR. Our previous work highlighted its contribution to CRR through regulation of vasopressin secretion. Here, we reveal that Tmem117 is also enriched in cells of the oligodendrocytic lineage and we characterize the contribution of oligodendrocytic Tmem117 in CRR. We show that depletion of Tmem117 from either all oligodendrocyte lineage cells or only mature oligodendrocytes leads to myelin deficits and male-specific defects in CRR. Furthermore, we reveal that transient, adult-onset depletion of Tmem117 in mature oligodendrocytes is sufficient to induce long-lasting metabolic imbalances in male mice, suggesting that defects in oligodendrocytes and myelin can affect peripheral glucose homeostasis. Mechanistically, we provide for the first-time insights on the function of Tmem117 showing that it regulates intracellular calcium dynamics through its interaction with the sodium-calcium exchanger NCX1. Together, these results redefine our understanding of the cellular contributors to the CRR, highlight the importance of oligodendrocytes in systemic glucose regulation, and position Tmem117 as a promising molecular target for cell-specific manipulation of NCX activity.

neuroscience↗

bioRxiv2025

Cyclin-dependent kinase 9 inhibitors as oncogene signaling modulators in combination with targeted therapy for the treatment of colorectal cancer

BackgroundColorectal cancer (CRC) is the second deadliest cancer worldwide and new treatment options are urgently needed. Cyclin dependent kinase 9 (CDK9) promotes aberrant RNA transcription in cancer and is a promising target for cancer therapies. MethodsUsing CRC cell lines as well as newly established patient-derived organoid models of CRC, we studied the clinically promising CDK9 inhibitors (AZD4573, BAY1125152/VIP152/enitociclib, and NVP2) to determine their therapeutic potential. We investigated the efficacy and mechanisms of action through cell growth and cytotoxicity assays, RNAseq, immunoblotting, and IHC. ResultsWe found CDK9 inhibitors to be highly potent against CRC, through suppression of proliferation and induction of apoptosis. Our results demonstrated CDK9 inhibitors to be broadly active against a set of CRC models derived from a diverse patient population. Our mechanistic studies showed significant suppression of the Mitogen-active protein kinase (MAPK) signaling pathway due to CDK9 inhibitor treatment, suggesting that CDK9 inhibitor efficacy could be enhanced when combined with MAPK pathway inhibitors. As proof-of-concept, we found that CDK9 inhibitors and MEK inhibitors could be combined to synergistically suppress CRC growth and survival. ConclusionsCDK9 inhibitors show promising activity against patient-derived models of CRC. MAPK signaling is particularly suppressed by CDK9 inhibitors. Combining CDK9 inhibitors and targeted therapy against MAPK signaling pathway may be a viable strategy worthy of further investigation preclinically and clinically. State of translational relevanceNovel therapies for chemotherapy-refractory CRC remain urgently needed. Rapid validation of efficacy in appropriate translational models can provide the necessary rationale to test new therapies in clinical trials. Using newly established patient-derived organoids, we validate the preclinical efficacy of transcriptional CDK9 inhibitors against CRC. This is a therapeutic class that has been scarcely evaluated for efficacy against CRC. We found that CDK9 inhibitors potently inhibit patient-derived organoid models of CRC. Our mechanistic work also reveals that clinically achievable concentrations of CDK9 inhibitor treatment results in suppression of MAPK signaling. As proof-of-concept of the potential actionability of our findings, we demonstrate that CDK9 inhibitors can be combined with MEK inhibitors to synergistically suppress CRC growth. Our study provides new patient-derived CRC models as a resource available to others for therapeutic testing, as well as a roadmap for novel hypotheses about synergistic combination treatments using CDK9 inhibitors.

cancer biology↗

bioRxiv2024

Probing PAC1 receptor activation across species with an engineered sensor

Class-B1 G protein-coupled receptors (GPCRs) are an important family of clinically relevant drug targets that remain difficult to investigate via high-throughput screening and in animal models. Here, we engineered PAClight1P78A, a novel genetically-encoded sensor based on a class-B1 GPCR (the human PAC1 receptor, hmPAC1R) endowed with high dynamic range ({Delta}F/F0 = 1100%), excellent ligand selectivity and rapid activation kinetics ({tau}ON = 1.15 sec). To showcase the utility of this tool for in vitro applications, we thoroughly characterized and compared its expression, brightness and performance between PAClight1P78A transfected and stably-expressing cells. Demonstrating its use in animal models, we show robust expression and fluorescence responses upon exogenous ligand application ex vivo and in vivo in mice, as well as in living zebrafish larvae. Thus, the new GPCR-based sensor can be used for a wide range of applications across the life sciences empowering both basic research and drug development efforts.

neuroscience↗

bioRxiv2023

Development of a genetically-encoded sensor for probing endogenous nociceptin opioid peptide release

Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fibre photometry enabled direct recording of NOPLight binding to exogenous N/OFQ receptor ligands, as well as detection of endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA) during natural behaviors and chemogenetic activation of PNOC neurons. In summary, we show here that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely behaving animals.

neuroscience↗

bioRxiv2023

Optical tools for visualizing and controlling human GLP-1 receptor activation with high spatiotemporal resolution

The glucagon-like peptide-1 receptor (GLP1R) is a broadly expressed target of peptide hormones with essential roles in energy and glucose homeostasis, as well as of the blockbuster weight-loss drugs semaglutide and liraglutide. Despite its large clinical relevance, tools to investigate the precise activation dynamics of this receptor with high spatiotemporal resolution are limited. Here we introduce a novel genetically-encoded sensor based on the engineering of a circularly-permuted green fluorescent protein into the human GLP1R, named GLPLight1. We demonstrate that fluorescence signal from GLPLight1 accurately reports the expected receptor conformational activation in response to pharmacological ligands with high sensitivity (max {Delta}F/F0 = 528%) and temporal resolution ({tau}ON = 4.7 sec). We further demonstrated that GLPLight1 shows comparable responses to GLP-1 derivatives as observed for the native receptor. Using GLPLight1, we established an all-optical assay to characterize a novel photocaged GLP-1 derivative (photo-GLP1) and to demonstrate optical control of GLP1R activation. Thus, the new all-optical toolkit introduced here enhances our ability to study GLP1R activation with high spatiotemporal resolution.

biochemistry↗

bioRxiv2021

Hypoxia induces early neurogenesis in human fetal neural stem cells by activating the WNT pathway.

Fetal neural stem cells (FNSCs) are present in the brain of human fetuses that differentiate into cells of neuronal and glial lineages. Difference in oxygen concentration between maternal and fetal circulation indicates that the developing fetus may be exposed to lower oxygen concentrations compared to adults. This physiological hypoxia may influence the growth and differentiation of the FNSCs. This study aimed to evaluate the effect of hypoxia on the differentiation potential of human FNSCs isolated from the sub-ventricular zone of aborted fetal brains (n=5). FNSCs were isolated, expanded, and characterized by Nestin and Sox2 expression, using immunocytochemistry and flowcytometry respectively. These FNSCs were exposed to 20% oxygen (normoxia) and 0.2% oxygen (hypoxia) concentrations for 48 hours, and hypoxia exposure (n=5) was evaluated by a panel of markers (CA9, PGK1 and VEGF). Whole human genome transcriptomic analyses (Genespring GX13) of FNSCs exposed to hypoxia (Agilent 4x44K human array slides), highlighted that genes associated with neurogenesis were getting enriched. The pathway analysis of these enriched genes (using Metacore) showed that WNT signaling played a role in determining the cell fate of FNSCs exposed to hypoxic environment. Microarray analyses was validated using neuronal and glial lineage commitment markers such as Ngn1, Ngn2, ASCL1, DCX, GFAP, Olig2 and Nkx2.2 using qPCR (n=9). This demonstrated upregulation of the neuronal commitment markers on hypoxia exposure, while no change was observed in astrocytic and oligodendrocyte lineage commitment markers. Increased expression of downstream targets of the WNT signaling pathway, TCF4 and ID2, by qPCR (n=9), indicated its involvement in mediating neuronal differentiation on exposure to hypoxia.

neuroscience↗

bioRxiv2021

Tbx1, a 22q11.2-encoded gene, is a link between alterations in fimbria myelination and cognitive speed in mice

Copy number variants (CNVs) have provided a reliable entry point to identify structural correlates of atypical cognitive development. Hemizygous deletion of human chromosome 22q11.2 is associated with impaired cognitive function; however, the mechanisms by which numerous genes encoded in this CNV contribute to cognitive deficits via diverse structural alterations in the brain remain unclear. This study aimed to determine the cellular basis of the link between alterations in brain structure and cognitive functions in a mouse model. The heterozygosity of Tbx1, a 22q11.2 gene, altered the composition of myelinated axons in the fimbria, reduced oligodendrocyte production capacity, and slowed the acquisition of spatial memory and cognitive flexibility. Our findings provide a cellular basis for specific cognitive dysfunctions that occur in patients with loss-of-function TBX1 variants and 22q11.2 hemizygous deletion. TeaserA risk gene for autism alters myelin composition in the hippocampal connection and slows cognitive speed.

neuroscience↗