Biology subjects

Sehgal, A.

Publications and source records attributed to Sehgal, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Macrophage calcium signaling dynamics revealed using genetically-encoded sensors

Macrophages (M{Phi}s) inhabit all mammalian organs, adapting to and influencing the tissues in which they reside. These versatile cells rapidly respond to tissue-specific signals, clean up debris and serve as the first line of defense against pathogens. Their ability to act quickly relates to their abundance and specific arrangement in tissues, as well as the systems they possess for detecting, processing and integrating emergent signals. Ca2+ is a fast second messenger that has been linked to many of the core functions of M{Phi}s. However, spatiotemporal features of physiologically-relevant Ca2+ signals in M{Phi}s remain largely uncharacterized. Using mouse models expressing genetic biosensors and fluorescently tagged channels, we visualize M{Phi} Ca2+ signal dynamics and heterogeneity, uncover a remarkable degree of mechanosensitivity in these cells, and characterize the physiological consequences of genetic ablation of Piezo1 channels via analysis of knockout models across their lifetime. Our in-depth investigation of M{Phi} Ca2+ signaling dynamics has broad relevance for the field of M{Phi} biology and the tissues that these cells support.

immunology↗

bioRxiv2026

Gut-derived serine protease reduces sleep but extends lifespan

Sleep has historically been viewed through a brain-centric lens with little known about the contribution of the periphery. Through a targeted screen of secreted peptides from the fat body, gut, and body wall muscle in Drosophila, we identified CG11037, a trypsin-like serine endopeptidase secreted from midgut enterocytes, as a previously unrecognized regulator of sleep. Loss of CG11037 reduces daily sleep, as well as sleep following injury/infection, although recovery is enhanced suggesting reduced need for sleep during sickness. Proteomic analysis of flies with reduced CG11037 revealed altered oxidative stress response pathways including cytochrome P450-related proteins. Indeed, gut-specific knockdown of Cyp6a2 or Cyp28d1 phenocopies CG11037 sleep defects, and loss of CG11037 or P450 elevates reactive oxygen species (ROS) selectively in the gut without affecting brain ROS. Rescue of sleep by antioxidant treatment demonstrates that peripheral ROS accumulation drives the behavioral phenotype. Strikingly, despite physiological dysfunction and reduced sleep, CG11037 or Cytochrome P450 knockdown extends lifespan in a ROS-dependent manner, suggesting that stress adaptation of these knockdowns allows them to live longer. Together, these findings uncover a mechanism for the unexpected association of reduced sleep and extended lifespan.

genetics↗

bioRxiv2026

Profiling and Targeting of Regulatory RNAs to Upregulate Gene Expression

Transcription of long noncoding RNAs (lncRNAs), including enhancer RNAs (eRNAs) and promoter-associated RNAs (paRNAs), collectively termed regulatory RNAs (regRNAs), is a hallmark of active gene expression, yet it remains unknown whether regRNAs can be targeted to selectively enhance transcription in cis. We developed regRNA Capture-seq, a high-throughput method to profile regRNAs, and applied it to primary human hepatocytes, annotating thousands of regRNAs at [~]2,000 enhancers and promoters. Using this approach, we interrogated a genetically validated enhancer of the ornithine transcarbamylase (OTC) gene, mutations of which cause OTC deficiency (OTCD), the most common urea cycle disorder. Antisense oligonucleotides (ASOs) targeting enhancer-derived regRNAs led to dose-dependent upregulation of OTC in hepatocytes. Mechanistically, ASOs altered regRNA structure, elevated regRNA levels, displaced transcriptional repressors, and increased H3K27 acetylation at the targeted enhancer. This work establishes a potential therapeutic strategy for addressing haploinsufficiency and highlights regRNAs as actionable targets for ASO-mediated upregulation of gene expression.

genomics↗

bioRxiv2026

Endocytosis at the mouse blood brain barrier is elevated during sleep

Sleep is thought to be important for the clearance of brain waste, but exactly how it does so is still debated. Here, we demonstrate that endocytosis in brain endothelial cells (BECs) of the blood-brain barrier (BBB) is enhanced during sleep, facilitating the removal of brain-derived waste, including amyloid-{beta}, into the circulation. Using proteomics, in vivo tracer imaging, and endothelial-specific genetic perturbations in mice, we demonstrate that sleep enhances endocytic vesicle formation and cargo transcytosis in brain endothelial cells (BECs). Conversely, blocking endocytosis through endothelial Dnm2 knockout suppresses BEC-mediated transport and elevates sleep need, revealing a causal feedback loop between sleep and vascular endocytosis. These findings identify BBB endocytosis as a key sleep-dependent clearance pathway with implications for neurodegenerative disease.

neuroscience↗

bioRxiv2025

A paradoxical impact of alcohol on sleep-memory coupling

Sleep serves a fundamental role in memory consolidation, and yet it must adapt to the organisms physiological state. The latter is impacted profoundly by acute ethanol consumption, but whether intoxication affects the role of sleep in memory consolidation remains unexplored. Here, we demonstrate that acute ethanol inverts the canonical link between sleep and memory in Drosophila. Typically, satiated flies require sleep for appetitive memory consolidation, but starved flies that must forage for food switch to sleep-independent memory. Ethanol selectively impairs memory consolidation in satiated flies by causing a switch to sleep-independent memory, which then cant be supported because of ethanol-induced sedation. Under these conditions, sleep deprivation rescues memory and requires the upregulation of neuropeptide F, which signals through NPF receptors on PPL1 dopaminergic neurons. We suggest that reward-seeking behaviors, induced by starvation or ethanol, invoke sleep-independent memory. However, ethanol has a paradoxical impact, wherein it induces a switch in memory pathways but concurrently suppresses consolidation through the preferred pathway, thereby causing sleep to become detrimental for memory consolidation.

neuroscience↗

bioRxiv2025

Sleep controls peroxisomal abundance to reduce wake-induced brain oxidation

Sleep is increasingly linked to the regulation of Reactive Oxygen Species (ROS) and lipid metabolism. However, the mechanisms underlying this interaction are underexplored. Here, we use Drosophila melanogaster to report a bidirectional relationship between sleep and peroxisomes, cellular organelles that process lipids and alleviate ROS. Of the genes that change expression after sleep deprivation in the dorsal fan-shaped body, knockdown of the peroxisomal biogenesis factor Pex16 results in decreased sleep. Pex16 acts in several brain regions to modulate sleep amount, with ellipsoid body neurons (EB) producing the highest sleep reduction of the sleep-promoting regions. Consistent with a general role for peroxisomes, knockdown of other peroxisomal enzymes relevant for lipid import and synthesis also decreases sleep. Whole-brain peroxisomal numbers increase with wake, which is supported by lipidomic analysis indicating that peroxisomal-derived phospholipids are the major contributors to phospholipid changes after wake or sleep deprivation. Peroxisomal proliferation in the EB is driven by neuronal activity and increased oxidation, suggesting that these mediate the effect of wake/sleep loss. In turn, peroxisomes alleviate the oxidation accumulated during wake, such that loss of Pex16 in the EB works non-cell autonomously to increase lipid peroxidation brain-wide. This likely contributes to sleep loss, as sleep is rescued with an antioxidant. Together, these results position peroxisomes as key players in sleep, regulating ROS and thereby maintaining normal cycles.

neuroscience↗

bioRxiv2025

Sleep-dependent clearance of brain lipids by peripheral blood cells

Sleep is typically viewed through a brain-centric lens, with little known about the role of the periphery. Here, we identify a sleep function for peripheral macrophage-like cells (hemocytes) in the Drosophila circulation, showing that hemocytes track to the brain during sleep and take up lipids accumulated in cortex glia due to wake-associated oxidative damage. Through a screen of phagocytic receptors expressed in hemocytes, we discovered that knockdown of eater, a member of the Nimrod receptor family, reduces sleep. Loss of eater also disrupts hemocyte adhesion to the brain and lipid uptake, which results in increased brain levels of Acetyl CoA and acetylated proteins, including mitochondrial proteins PGC1 and DRP1. Dysregulation of mitochondria, reflected in high oxidation and reduced NAD+, is accompanied by impaired memory and lifespan. Thus, peripheral blood cells, which we suggest are precursors of mammalian microglia, perform a daily function of sleep to maintain brain function and fitness.

molecular biology↗

bioRxiv2025

Neutral lipid processing in glia is sexually dimorphic and promotes sleep through diacylglycerol catabolism

Sleep is thought to have a protective role in clearing toxic waste from the brain, which may include processing of damaged lipids. We recently showed that blocking endocytosis in glia increases sleep and report here that this block is associated with an increase in peroxidized lipids and glial lipid droplet accumulation, raising the possibility that accumulation of these lipids increases the need to sleep. Sleep gain induced by blocking glial transport is exaggerated by knockout of the lipid droplet coat protein, Lipid Storage Droplet 2 (Lsd2), suggesting that sleep-promoting lipids are not contained in lipid droplets. To identify lipids regulated by sleep state, we performed global, targeted lipidomics analysis on Drosophila neurons and glia, screening nearly 3,000 lipids across 11 major classes. This revealed that sex influences lipid composition in both cell types and lipid homeostasis following extended wakefulness. Female neurons and glia are enriched in ultra-long chain fatty acids, triacylglycerols, and diacylglycerols, with glial diacylglycerol enrichment correlating with elevated sleep need. Based on manipulations of neutral lipid metabolic pathways, we propose that monoacylglycerols, products of glial diacylglycerol catabolism, promote sleep.

neuroscience↗

bioRxiv2025

Integrated Respirometry and Metabolomics Unveil Circadian Metabolic Dynamics in Drosophila

Sleep and circadian rhythms shape organismal energy patterns, but how this timing connects to oxygen use and carbon dioxide production remains incompletely understood. We combined high-resolution respirometry with LC-MS-based metabolomics to characterize respiratory dynamics and metabolic states in Drosophila melanogaster, resolving genotype-specific impacts of sleep disruption and circadian regulation. Wild-type flies under light-dark cycles (WT-LD) showed rhythmic respiratory patterns reflective of anticipatory coordination of mitochondrial energy metabolism, amino acid turnover, and redox cycling. Short-sleep mutants (fmn, sss) exhibited elevated metabolic rates with reactive shifts of fuel preferences toward lipid and amino acid catabolism and altered mitochondrial respiration. The clock-mutant (per01) and flies under constant darkness (WT-DD) showed reactive and widespread metabolic dysregulation and impaired redox homeostasis. These findings demonstrate that both sleep and circadian systems contribute to aligning metabolic substrate selection with energy demands, offering mechanistic insights into how disruptions in behavioral states compromise metabolic health.

biochemistry↗

bioRxiv2025

Constant light impairs memory processing transgenerationally in D. melanogaster

Environmental perturbations can have profound effects on our physiology and behavior, but their long-lasting impact remains debatable. We discovered that changes in standard light-dark conditions, such as exposure to constant light or simulated chronic jetlag, causes loss of associative memory in appetitive and aversive conditioning paradigms in D. melanogaster, and this behavior persists through three generations despite transfer of progeny to a standard light-dark cycle. Impaired memory is transmitted through females and is independent of any changes in fitness, brain or mushroom body architecture, or sensing acuity. Mechanistically, effects of constant light on memory are mediated by increased PIWI/piRNA pathway expression in the germline and its H3K9me3 writing capabilities, acting through altered expression of the Dopamine-1-like Receptor 1 (Dop1R1) in the brain. These findings suggest that irregular exposure to light, such as nighttime light pollution, can have negative consequences across generations. HIGHLIGHTSO_LIConstant light impairs associative memory in Drosophila for three generations C_LIO_LIpiwi is required in the brain and germline for inheritance C_LIO_LIDopamine 1 Receptor 1 activity is dampened by constant light C_LI

neuroscience↗

bioRxiv2025

Tanycyte Bmal1 sex-specifically regulates weight gain and hypothalamic neurogenesis in female mice

The hypothalamic radial-glia-like tanycyte population plays important and intertwined roles in feeding and metabolism, reproduction, and seasonality. Although these processes are circadian-regulated and clock genes reportedly show robust cycling along the 3rd ventricle, the role of the clock in tanycytes has not yet been examined. We report here that clock genes cycle with much higher amplitude in ventral tanycytes compared to more dorsal ependymocytes of the 3rd ventricle, and that specific disruption of the tanycyte clock can be achieved by adult Bmal1 deletion using the RaxCreER driver. Adult tanycyte Bmal1 deletion did not affect circadian rhythms of wheel-running and sleep, but did inhibit weight gain on high-fat diet in female mice. Altered tanycyte-derived hypothalamic neurogenesis, which can regulate feeding and weight gain by contributing new neurons to nearby feeding-relevant nuclei, is one mechanism that likely contributes to this phenotype. Fate mapping studies showed that female mice have higher baseline tanycyte-derived neurogenesis than males, with many of the resulting neurons localizing to the feeding-relevant arcuate nucleus. Female but not male mice show reduced tanycyte-derived arcuate neurogenesis after adult tanycyte Bmal1 deletion and an increased percentage of newborn arcuate neurons take on a feeding-suppressing POMC neuropeptidergic fate. Thereby, skewing of feeding and satiety promoting fates link the weight homeostasis and neurogenesis effects. Together, our data establish tanycyte Bmal1 as a sexually dimorphic regulator of weight homeostasis, likely mediated at least in part by a female-specific neurogenesis effect in the feeding circuitry.

neuroscience↗

bioRxiv2025

Effect of external cues on clock-driven protection from Influenza A infection

Influenza and other respiratory viral pathogens are leading causes of mortality and morbidity. We previously demonstrated that circadian rhythms confer temporal protection from influenza infection. Here, we investigated whether this protection requires rhythmic function after the initial infection by manipulating environmental cycles. We demonstrate that disrupting environmental lighting cues within a critical window of vulnerability abrogates time-of-day specific protection. This poor outcome is mediated by a dysregulated immune response, evidenced by the accumulation of inflammatory monocytes and CD8+ cells in the lungs and a transcriptomic profile indicative of an exaggerated immune response. Disruption of the light cycle does not affect outcomes in a clock mutant, indicating that it acts by compromising endogenous timekeeping. Importantly, rhythmic meal timing mitigates the adverse effects of disrupted light cycles, suggesting that external cycling cues, which act through different body clocks, can substitute for each other. Our findings highlight the crucial interplay between environmental factors and endogenous clocks in shaping influenza outcomes, offering significant translational potential for improving the care of critically ill patients with respiratory viral infections.

immunology↗

bioRxiv2024

Effects of Nf1 on sleep behavior are mediated through starvation caused by deficits in SARM1 dependent NAD+ metabolism.

Neurofibromatosis 1 (NF1) is a relatively common autosomal dominant disease which predisposes to the formation of tumors, and is also associated with behavioral phenotypes, including sleep disturbances. As loss of the NF1 protein has been recently associated with metabolic dysfunction, we explored the relationship between metabolic and behavioral phenotypes through metabolomic analysis of Drosophila Nf1-null mutants. Nf1-null mutants exhibit a metabolic signature indicative of starvation, with diminished metabolites related to glucose, glycogen, and fatty acid processing and increased mRNA of Akh, a hormone that promotes foraging during starvation. Reduced sleep in Nf1-null mutants was rescued by genetic manipulation of the AKH pathway and by a high-sucrose diet, which also partially corrected hypolipidemia, suggesting that sleep loss is due to starvation-induced foraging. Interestingly, behavioral phenotypes can be recapitulated by loss of NF1 only in the periphery and trace to mitochondrial defects that include elevated levels of the NADase SARM1. Indeed, inhibition of SARM1 activity rescues sleep behavior in Nf1-null flies. These findings suggest a novel connection between loss of NF1 and mitochondrial dysfunction caused by SARM1 hyperactivation, setting the scene for new pharmacological and dietary approaches that could provide relief to NF1 patients.

molecular biology↗

bioRxiv2024

Transcriptomic analysis of the functions of CSF1R-dependent macrophages in postnatal development in the rat

Adaptation to existence outside the womb is a key event in the life of a mammal. The absence of macrophages in rats with a homozygous mutation in the Csf1r gene (Csf1rko) severely compromises pre-weaning somatic growth and maturation of organ function. Transfer of wild-type bone marrow cells (BMT) at weaning rescues tissue macrophage populations permitting normal development and long-term survival. To dissect the phenotype and function of macrophages in postnatal development, we generated transcriptomic profiles of all major organs of wild-type and Csf1rko rats at weaning and selected organs following rescue by BMT. The transcriptomic profiles revealed subtle effects of macrophage deficiency on development of all major organs. Network analysis revealed a common signature of CSF1R-dependent resident tissue macrophages that includes the components of complement C1Q (C1qa/b/c genes). Circulating C1Q was almost undetectable in Csf1rko rats and rapidly restored to normal levels following BMT. Tissue-specific macrophage signatures were also identified, notably including sinus macrophage populations in the lymph nodes. Their loss in Csf1rko rats was confirmed by immunohistochemical localisation of CD209B (SIGNR1). By 6-12 weeks, Csf1rko rats succumb to emphysema-like pathology associated with the selective loss of interstitial macrophages and granulocytosis. This pathology was reversed by BMT. Along with physiological rescue, BMT precisely regenerated the abundance and expression profiles of resident macrophages. The exception was the brain, where BM-derived microglia-like cells had a distinct expression profile compared to resident microglia. In addition, the transferred BM failed to restore blood monocyte or CSF1R-positive bone marrow progenitors. These studies provide a model for the pathology and treatment of CSF1R mutations in humans and the innate immune deficiency associated with prematurity.

immunology↗

bioRxiv2024

The microbiome interacts with the circadian clock and dietary composition to regulate metabolite cycling in the gut

The gut microbiome plays a key role in the maintenance of host metabolic homeostasis and health. Most metabolic processes cycle with a 24-hour rhythm, but the extent to which the microbiome influences metabolite cycling under different conditions, such as variations in dietary composition, remains largely unknown. In this study, we utilized high temporal resolution metabolite profiling of the Drosophila gut to investigate the role of the microbiome in metabolite cycling. We find that the microbiome increases the number of oscillating metabolites despite the previous finding that it dampens transcript cycling in the gut. Time restricted feeding (TF) also promotes metabolite cycling and does so to a larger extent in germ-free flies, thereby increasing cycling in these flies to levels comparable to those in microbiome containing flies. Enhancement of cycling by the microbiome depends upon a circadian clock, which also maintains phase in the face of changes in the microbiome. Interestingly, a high-protein diet increases microbiome- dependent metabolite cycling, while a high sugar diet suppresses it. Gene Ontology identifies amino acid metabolism as the metabolic pathway most affected by changes in the gut microbiome, the circadian clock and timed feeding, suggesting that it is subject to regulation by multiple inputs. Collectively, our observations highlight a key role of the gut microbiome in host metabolite cycling, and reveal a complex interaction with internal and external factors.

microbiology↗

bioRxiv2024

Neurovascular mitochondrial susceptibility impacts blood-brain barrier function and behavior

Maintenance of blood-brain barrier (BBB) integrity is critical to optimal brain function, and its impairment has been linked to multiple neurological disorders. A notable feature of the BBB is its elevated mitochondrial content compared to peripheral endothelial cells, although the functional implications of this phenomenon remain unknown. Here we studied BBB mitochondrial function in the context of the 22q11.2 deletion syndrome (22qDS), a condition associated with a highly increased risk for neuropsychiatric disease. As the 22q11.2 deletion includes 6 mitochondrial genes, and because we have previously identified BBB impairment in 22qDS, we addressed the hypothesis that mitochondrial deficits contribute to BBB dysfunction and impact behavior in this condition. We report mitochondrial impairment in human induced pluripotent stem cell (iPSC)-derived BBB endothelial cells from 22qDS patients, and in BBB endothelial cells from a mouse model of 22qDS. Remarkably, treatment to improve mitochondrial function attenuates mitochondrial deficits and enhances BBB function in both the iPSC and mouse 22qDS models. This treatment also corrected social memory in the mouse model, a deficit previously associated with BBB dysfunction. As BBB integrity correlated with social memory performance, together our findings suggest that mitochondrial dysfunction in the BBB influences barrier integrity and behavior in 22qDS.

neuroscience↗

bioRxiv2024

Genome-wide profiling of genetic variation at tandem repeat from long reads

Tandem repeats are frequent across the human genome, and variation in repeat length has been linked to a variety of traits. Recent improvements in long read sequencing technologies have the potential to greatly improve TR analysis, especially for long or complex repeats. Here we introduce LongTR, which accurately genotypes tandem repeats from high fidelity long reads available from both PacBio and Oxford Nanopore Technologies. LongTR is freely available at https://github.com/gymrek-lab/longtr.

bioinformatics↗

bioRxiv2023

Genome wide detection of somatic mosaicism at short tandem repeats

MotivationSomatic mosaicism, in which a mutation occurs post-zygotically, has been implicated in several developmental disorders, cancers, and other diseases. Short tandem repeats (STRs) consist of repeated sequences of 1-6bp and comprise more than 1 million loci in the human genome. Somatic mosaicism at STRs is known to play a key role in the pathogenicity of loci implicated in repeat expansion disorders and is highly prevalent in cancers exhibiting microsatellite instability. While a variety of tools have been developed to genotype germline variation at STRs, a method for systematically identifying mosaic STRs (mSTRs) is lacking. ResultsWe introduce prancSTR, a novel method for detecting mSTRs from individual high-throughput sequencing datasets. Unlike many existing mosaicism detection methods for other variant types, prancSTR does not require a matched control sample as input. We show that prancSTR accurately identifies mSTRs in simulated data and demonstrate its feasibility by identifying candidate mSTRs in whole genome sequencing (WGS) data derived from lymphoblastoid cell lines for individuals sequenced by the 1000 Genomes Project. Our analysis identified an average of 76 and 577 non-homopolymer and homopolymer mSTRs respectively per cell line as well as multiple cell lines with outlier mSTR counts more than 6 times the population average, suggesting a subset of cell lines have particularly high STR instability rates. AvailabilityprancSTR is freely available at https://github.com/gymrek-lab/trtools. DocumentationDetailed documentation is available at https://trtools.readthedocs.io/ Contactmgymrek@ucsd.edu

bioinformatics↗