Biology subjects

Farooqi, I. S.

Publications and source records attributed to Farooqi, I. S..

7 recordsLinked to original sources

bioRxiv2026

Magel2 deficiency drives microglial synaptic engulfment and alters neuron-microglia interactions in Prader-Willi syndrome

Prader-Willi syndrome (PWS) is a neurodevelopmental disorder characterized by hypothalamic dysfunction, developmental delay, hyperphagia, and cognitive impairment, yet the cellular mechanisms underlying circuit abnormalities remain unclear. Here, we show that loss of the imprinted gene Magel2 remodels microglial state and function in a mouse model of PWS. Three-dimensional morphometric analysis of hypothalamic microglia revealed reduced structural complexity and contracted territorial domains, emerging early in development and largely independent of obesity. Single-nucleus RNA sequencing of hypothalamic brain tissue identified extensive transcriptional reprogramming involving immune signaling, cellular communication, ribosome function, synaptic organization, and protein translation, with stronger effects in females. Magel2 deficiency also increased microglia-neuron contacts and synaptic engulfment, accompanied by reduced synaptic marker density, suggesting aberrant refinement of hypothalamic circuits. Analyses of human post-mortem hypothalamic tissue from individuals with PWS confirmed microglial structural alterations and increased complement engagement. Ligand-receptor inference further revealed reorganization of microglia-centered communication networks, including altered adhesion, trophic, and immune signaling consistent with enhanced phagocytic states. Together, these findings identify microglial remodeling and increased synaptic engagement as central features of Magel2 deficiency, implicate microglia in hypothalamic circuit dysfunction in PWS, and highlight excessive synaptic pruning as a novel dysfunctional mechanism of PWS.

neuroscience↗

bioRxiv2025

A human missense variant in BSX decouples circadian behaviour from metabolic rhythmicity while preserving lifespan in mice

Mammalian circadian organisation is orchestrated by a light-entrained master pacemaker in the suprachiasmatic nucleus (SCN) and SCN-independent, food- and metabolism-responsive oscillators in the brain and periphery; feeding can entrain these systems, enabling anticipation of feeding time. Here, we show that a human missense variant (Q159K) in the homeodomain of the brain-specific transcription factor BSX causes profound loss of circadian behavioural rhythms and food anticipatory activity (FAA) in mice. Whole-brain imaging reveals failure of the compact dorsomedial hypothalamus (DMHc) to form and the absence of histaminergic neurons in the tuberomammillary nucleus (TMN) in BsxQ159K/Q159K mice. Consequently, under light-dark (LD) and constant darkness (DD), sleep-wake cycles and circadian locomotor rhythmicity are profoundly lost, whereas total wake time and spontaneous locomotor activity are preserved, with daily caloric intake substantially shifted from the dark to the light cycle. Therefore, in DD, metabolic recordings reveal a rhythmic respiratory exchange ratio (RER) pattern that differs substantially from that of wild-type mice and is markedly attenuated under LD conditions. In addition, BsxQ159K/Q159K mice lack FAA during restricted feeding and fail to increase locomotor activity upon food deprivation. Despite the loss of circadian behavioural rhythms and altered daily feeding behaviour, BsxQ159K/Q159Kmice maintain metabolic homeostasis, a lean body composition into advanced age, and a normal lifespan under ad libitum feeding and controlled LD. These findings provide genetic evidence that circadian organization is not universally required for metabolic homeostasis, suggesting instead that circadian rhythmicity is an adaptive feature advantageous primarily under diurnal conditions.

genetics↗

bioRxiv2024

Structures of human PTP1B variants reveal allosteric sites to target for weight loss therapy

Protein Tyrosine Phosphatase 1B (PTP1B) is a negative regulator of leptin signaling whose disruption protects against diet-induced obesity in mice. We investigated whether structural characterization of human PTP1B variant proteins might reveal allosteric sites to target for weight loss therapy. To do so, we selected 12 rare variants for functional characterization from exomes from 997 people with persistent thinness and 200,000 people from UK Biobank. Seven of 12 variants impaired PTP1B function by increasing leptin-stimulated STAT3 phosphorylation in human cells. Focusing on the variants in and near the ordered catalytic domain, we ascribed structural mechanism to their functional effects using in vitro enzyme activity assays, room-temperature X-ray crystallography, and local hydrogen-deuterium exchange mass spectrometry (HDX-MS). By combining these complementary structural biology experiments for multiple variants, we characterize an inherent allosteric network in PTP1B that differs from previously reported allosteric inhibitor-driven mechanisms mediated by catalytic loop motions. The most functionally impactful variant sites map to highly ligandable surface sites, suggesting untapped opportunities for allosteric drug design. Overall, these studies can inform the targeted design of allosteric PTP1B inhibitors for the treatment of obesity. SignificanceObesity is a growing public health concern worldwide. The human enzyme PTP1B is a validated obesity drug target, but to date, effective PTP1B inhibitors have not been developed. Allosteric drugs, which target parts of a protein distant from the active site, offer advantages for specific inhibition -- but finding promising allosteric sites that bind ligands and convey allosteric signals remains challenging. To address this knowledge gap, we used human genetic studies in thin people to identify amino acid variants that diminish PTP1B function. We then used complementary structural biology methods to show that the variants exploit distinct allosteric wiring within PTP1B that includes ligand-enriched binding sites. This work demonstrates how a unique combination of genetics and biophysics can unveil promising allosteric sites in challenging drug target proteins.

biophysics↗

bioRxiv2024

A quantitative pipeline to assess secretion of human leptin coding variants reveals mechanisms underlying leptin deficiencies

The hormone leptin, primarily secreted by adipocytes, plays a crucial role in regulating whole-body energy homeostasis. Homozygous loss-of-function mutations in the leptin gene (LEP) cause hyperphagia and severe obesity, primarily through alterations in leptins affinity for its receptor or changes in serum leptin concentrations. Although serum concentrations are influenced by various factors (e.g., gene expression, protein synthesis, stability in the serum), proper delivery of leptin from its site of synthesis in the endoplasmic reticulum via the secretory pathway to the extracellular serum is a critical step. However, the regulatory mechanisms and specific machinery involved in this trafficking route, particularly in the context of human LEP mutations, remain largely unexplored. We have employed the Retention Using Selective Hooks (RUSH) system to elucidate the secretory pathway of leptin. We have refined this system into a medium-throughput assay for examining the pathophysiology of a range of obesity-associated LEP variants. Our results reveal that leptin follows the default secretory pathway, with no additional regulatory steps identified prior to secretion. Through screening of leptin variants, we identified three mutations that lead to proteasomal degradation of leptin and one mutant that significantly decreased leptin secretion, likely through aberrant disulfide bond formation. These observations have identified novel pathogenic effects of leptin variants, which can be informative for therapeutics and diagnostics. Finally, our novel quantitative screening platform can be adapted for other secreted proteins.

cell biology↗

bioRxiv2021

Visualization of Sympathetic Neural Innervation in Human White Adipose Tissue

Obesity, defined as an excess of adipose tissue that adversely affects health, is a major cause of morbidity and mortality. However, understanding the structure and function of human adipose tissue has been limited by the inability to visualize cellular components due to the innate structure of adipocytes, which are characterized by large lipid droplets. Combining the iDISCO and the CUBIC protocols for whole tissue staining and optical clearing, we developed a protocol to enable immunostaining and clearing of human subcutaneous white adipose tissue (WAT) obtained from individuals with severe obesity. We were able to perform immunolabeling of sympathetic nerve terminals in whole white adipose tissue and subsequent optical clearing by eliminating lipids to render the opaque tissue completely transparent. We then used light sheet confocal microscopy to visualize innervation of human WAT from obese individuals in a 3D manner.We demonstrate the visualization of sympathetic nerve terminals in human WAT. This protocol can be modified to visualize other structures such as blood vessels involved in the development, maintenance and function of human adipose tissue in health and disease.

neuroscience↗

bioRxiv2018

ProxECAT: Proxy External Controls Association Test. A new case-control gene region association test using allele frequencies from public controls

A primary goal of the recent investment in sequencing is to detect novel genetic associations in health and disease improving the development of treatments and playing a critical role in precision medicine. While this investment has resulted in an enormous number of sequenced genomes, individual studies of complex traits are often smaller and underpowered to detect rare variant genetic associations. Existing genetic resources such as the Exome Aggregation Consortium (>60,000 exomes) and the Genome Aggregation Database (~140,000 sequenced samples) could be used as controls in these studies. Fully utilizing these and other existing sequencing resources has the potential to increase power and could be especially useful in studies where resources to sequence additional samples are limited. However, to date, these large, publicly available genetic resources remain underutilized, or even misused, in large part due to the lack of statistical methods that can appropriately use this summary level data. We present a new method to incorporate external controls in case-control analysis called ProxECAT (Proxy External Controls Association Test). ProxECAT estimates enrichment of rare variants within a gene region using internally sequenced cases and external controls. We evaluated ProxECAT in simulations and empirical analyses of obesity cases using both low-depth of coverage (7x) whole-genome sequenced controls and ExAC as controls. We find that ProxECAT maintains the expected type I error rate with increased power as the number of external controls increases. With an accompanying R package, ProxECAT enables the use of publicly available allele frequencies as external controls in case-control analysis.

genetics↗

bioRxiv2018

Human POMC processing in vitro and in vivo revealed by quantitative peptidomics

Human obesity can result from the aberrant production or processing of proopiomelanocortin (POMC) in hypothalamic neurons, but it is unclear which human POMC-derived peptides are most relevant to body weight regulation. To address this question, we analysed both hypothalamic neurons derived from human pluripotent stem cells (hPSCs) and primary human hypothalamic tissue using quantitative liquid chromatography tandem mass spectroscopy (LC-MS/MS). In both in vitro- and in vivo-derived samples, we found that POMC was processed into {beta}-melanocyte stimulating hormone ({beta}-MSH), whose existence in the human brain has been controversial. {beta}-MSH and desacetyl -MSH (d--MSH) were produced at roughly equimolar concentrations and in vast excess to acetylated -MSH (5-to 200-fold), suggesting that the importance of both d--MSH and {beta}-MSH to human obesity has been underestimated. Since body weight is sensitive to changes in MSH concentration, we asked whether hPSC-derived hypothalamic neurons could provide mechanistic insights into the processing and secretion of MSH peptides. We found that cultured human hypothalamic neurons appropriately trafficked POMC and its derivatives, and robustly (P<0.0001) secreted them when depolarised. Furthermore, the adipocyte-derived hormone leptin significantly (P<0.01) promoted their production of both d--MSH and {beta}-MSH. These results establish hPSC-derived hypothalamic neurons as a model system for studying human-specific aspects of POMC processing that might be therapeutically harnessed to treat obesity.

neuroscience↗