Biology subjects

Wilcox, S.

Publications and source records attributed to Wilcox, S..

11 recordsLinked to original sources

bioRxiv2026

Autonomic neurons are intrinsically hyperexcitable in a Friedreich ataxia iPSC model

Friedreich ataxia (FRDA) is an inherited multisystem disorder caused by a GAA repeat expansion in the FXN gene and consequent frataxin deficiency, causing progressive neurodegeneration and heart disease. Clinical studies have documented autonomic dysfunction in FRDA, including elevated resting heart rate, raised plasma catecholamines and a high burden of urinary, bowel and sexual symptoms. Consistent with this, autopsy studies show degeneration of the cardiac conduction system and its associated ganglia in FRDA. Whether these features are intrinsic to FRDA autonomic neurons has been untestable without a human cellular model. Here, a human model of FRDA autonomic neurons was established by differentiating induced pluripotent stem cells (iPSCs) from individuals with FRDA and from their CRISPR/Cas9-corrected isogenic controls. FRDA autonomic neurons expressed canonical autonomic lineage markers and retained reduced FXN mRNA and frataxin protein levels. Electrophysiological analysis revealed pronounced hyperexcitability, with an approximately five-fold increase in spike number, shortened interspike intervals, and longer bursts. FRDA autonomic neurons also exhibited increased cell death, elevated mitochondrial reactive oxygen species, and a shift from networked to tubular mitochondrial morphology. Bulk RNA sequencing of all three isogenic pairs revealed loss of extracellular matrix programs and activation of stress response pathways. The top differentially expressed gene was MEG3, a maternally imprinted long noncoding RNA, which was consistently upregulated across all three isogenic pairs. MEG3 sits within the imprinted 14q32 DLK1-DIO3 locus, whose maternally expressed noncoding transcripts were co-ordinately de-repressed in FRDA autonomic neurons. Competitive gene-set testing of 43 imprinted genes across 20 domains showed this to be specific to 14q32: no other imprinted domain was co-ordinately altered. Together, these findings establish hyperexcitability of autonomic neurons as a cell autonomous consequence of frataxin deficiency, accompanied by mitochondrial dysfunction. This provides a cellular basis for the increased sympathetic tone observed in clinical studies.

neuroscience↗

bioRxiv2025

The microbiota elicits compensatory adaptation in a seasonally-adapting animal host

Seasonal adaptation in Drosophila melanogaster is a model for understanding the evolutionary responses of organisms to cyclical environmental changes, including roles played by associated microorganisms ( microbiota). Here we examined how the microbiota influences D. melanogaster seasonal adaptation by rearing flies in outdoor mesocosms, fed diets inoculated with different bacterial strains that have distinct influences on the flies life history. The bacterial treatments influenced fly population dynamics and microbiota composition over a summer-to-fall season. The developmental phenotype of the treated flies initially differed but converged over time in flies reared with a complete microbial community. Conversely, rearing the flies free of their colonizing microorganisms revealed that the bacterial treatments led to evolution of distinct developmental phenotypes. The development time of flies from the different treatments consistently adapted to compensate for the direct influence of the bacteria on host development; e.g., flies evolved faster development times if they were inoculated with microbes that slowed development. This compensatory trend was apparent in flies reared in a second location and season, and is consistent with a previous report of wild-sampled flies whose development phenotype segregated with their microbiota composition. Together, these results reveal that microbiota-dependent selection consistently elicits compensatory adaptation in seasonally-evolving flies, which we conclude is a mechanism whereby horizontally-acquired, low-fidelity microbial partners can shape the evolution of their animal hosts. ImportanceUnderstanding how organisms adapt to seasonal change can model evolutionary responses in a broader changing world. Drosophila melanogaster is a powerful model for studying these dynamics, especially when considering the influence of transient yet impactful colonizing microorganisms. This study reveals that microbial partners can drive consistent, compensatory adaptation in host development across seasons and locations. By demonstrating that flies evolve faster development times in response to microbes that slow a model hosts period of growth and development, this work highlights one way that the microbiota can influence adaptation in their animal hosts. These findings also provide evidence that low-fidelity, horizontally-acquired microbes can exert selective pressures strong enough to shape host life history traits. These insights underscore the microbiotas role as an ecological and evolutionary force.

microbiology↗

bioRxiv2025

Frataxin deficiency drives cardiac dysfunction and transcriptional dysregulation in Friedreich ataxia iPSC model

Friedreich ataxia (FRDA) is a progressive neuromuscular degenerative disorder caused by GAA repeat expansions in the FXN gene, leading to frataxin deficiency and multisystem pathology. Cardiomyopathy is the leading cause of mortality in individuals with FRDA. To investigate the cellular and molecular mechanisms underlying FRDA-associated cardiac dysfunction, we employed induced pluripotent stem cell (iPSC) lines derived from three individuals with FRDA, each paired with an isogenic control line generated through CRISPR/Cas9-mediated excision of the pathogenic GAA repeat expansion. Correction of the mutation restored FXN expression to levels comparable to healthy donor iPSCs, and all lines differentiated efficiently into cardiomyocytes. Functional analysis revealed significant contractile abnormalities in FRDA cardiomyocytes and multicellular cardiac microtissues, including prolonged contraction and relaxation times and faster beating rates, consistent with clinical observations of cardiac contractile dysfunction. FRDA cardiomyocytes also exhibited pathological features such as increased cell size, irregular calcium transients, elevated mitochondrial reactive oxygen species levels, increased mitochondrial fission and increased cell death. These phenotypes were exacerbated by pathological levels of iron supplementation in culture media, highlighting the heightened sensitivity of frataxin-deficient cardiomyocytes to iron-induced metabolic stress. RNA sequencing revealed a distinct transcriptional profile associated with frataxin deficiency. MEG3 and PCDHGA10 were consistently dysregulated across all three FRDA-iPSC lines and may represent early molecular markers of FRDA cardiomyopathy. Together, these findings establish a robust human iPSC model of FRDA cardiomyopathy that captures early disease phenotypes and reveals novel molecular targets. This preclinical human model provides valuable insight into the pathogenesis of FRDA and provides a platform for developing early-stage therapeutic interventions.

pathology↗

bioRxiv2025

β2AR Agonists Sustain Thermogenesis and Leanness via Sympathofacilitation

Human thermogenesis depends on {beta}2-adrenoceptors ({beta}2AR) expressed in thermogenic adipocytes, which are activated by norepinephrine released from sympathetic neurons. Whether {beta}2AR also modulates thermogenesis via direct presynaptic action within sympathetic neurons has remained unclear. Here, we identify Adrb2 expression in human and rodent cervical sympathetic neurons. {beta}2AR agonism exerts neurotrophic effects and facilitates cholinergic responsiveness in mouse sympathetic neurons, indicating a sympathofacilitatory role. Selective deletion of {beta}2AR in sympathetic neurons leads to impaired nerve activity in brown adipose tissue, sympathetic neuropathy, worsened fasting-induced hypothermia, and progressive obesity in chow-fed mice--without changes in food intake. These findings uncover a presynaptic role for {beta}2AR in sustaining thermogenesis and regulating adiposity, suggesting sympathofacilitation as a therapeutic avenue for obesity. HighlightsO_LIHuman and mouse cervical sympathetic neurons express adrenoceptor beta 2 (Adrb2) C_LIO_LI{beta}2-adrenoceptor ({beta}2AR) activation is neurotrophic and facilitates sympathetic neuronal excitability C_LIO_LILoss of {beta}2AR in sympathetic neurons leads to neuropathy in brown adipose tissue and reduced sympathetic activity C_LIO_LIDeletion of {beta}2AR in sympathetic neurons exacerbates fasting-induced hypothermia and promotes obesity independently of food intake C_LI

physiology↗

bioRxiv2024

Cortical layer 6b mediates state-dependent changes in brain activity and effects of orexin on waking and sleep

One of the most distinctive features of the mammalian cerebral cortex is its laminar structure. Of all cortical layers, layer 6b (L6b) is by far the least-studied, despite exhibiting direct sensitivity to orexin and having widespread connectivity, suggesting an important role in regulating cortical oscillations and brain state. We performed chronic electroencephalogram (EEG) recordings in mice in which a subset of L6b neurons was conditionally "silenced", during undisturbed conditions, after sleep deprivation (SD), and after intracerebroventricular (ICV) administration of orexin. While the total amount of waking and sleep or the response to SD were not altered, L6b-silenced mice showed a slowing of theta frequency (6-9 Hz) during wake and REM sleep, and a marked reduction of total EEG power, especially in NREM sleep. The infusion of orexin A increased wakefulness in both genotypes, but subsequent levels of EEG slow-wave activity during NREM sleep were lower in L6b-silenced animals in the occipital derivation. In summary, these results demonstrate a role for cortical L6b in state-dependent brain oscillations and in the response to orexinergic neurotransmission. Our findings provide new insights in functions of L6b neurons and could inform the understanding of abnormal regulation of brain states in neurodevelopmental and psychiatric disorders.

neuroscience↗

bioRxiv2024

Humidity determines penetrance of a latitudinal gradient in genetic selection on the microbiota by Drosophila melanogaster

The fruit fly Drosophila melanogaster is a model for understanding how hosts and their microbial partners interact as the host adapts to wild environments. These interactions are readily interrogated because of the low taxonomic and numeric complexity of the flies bacterial communities. Previous work has established that host genotype, the environment, diet, and interspecies microbial interactions can all influence host fitness and microbiota composition, but the specific processes and characters mediating these processes are incompletely understood. Here, we compared the variation in microbiota composition between wild-derived fly populations when flies could choose between the microorganisms in their diets and when flies were reared under environmental perturbation (different humidities). We also compared the colonization of the resident and transient microorganisms. We show that the ability to choose between microorganisms in the diet and the environmental condition of the flies can influence the relative abundance of the microbiota. There were also key differences in the abundances of the resident and transient microbiota. However, the microbiota only differed between populations when the flies were reared at humidities at or above 50% relative humidity. We also show that elevated humidity determined the penetrance of a gradient in host genetic selection on the microbiota that is associated with the latitude the flies were collected from. Finally, we show that the treatment-dependent variation in microbiota composition is associated with variation in host stress survival. Together, these findings emphasize that host genetic selection on the microbiota composition of a model animal host can be patterned with the source geography, and that such variation has the potential to influence their survival in the wild. ImportanceThe fruit fly Drosophila melanogaster is a model for understanding how hosts and their microbial partners interact as hosts adapt in wild environments. Our understanding of what causes geographic variation in the fruit fly microbiota remains incomplete. Previous work has shown that the D. melanogaster microbiota has relatively low numerical and taxonomic complexity. Variation in the fly microbiota composition can be attributed to environmental characters and host genetic variation, and variation in microbiota composition can be patterned with the source location of the flies. In this work we explored three possible causes of patterned variation in microbiota composition. We show that host feeding choices, the host niche colonized by the bacteria, and a single environmental character can all contribute to variation in microbiota composition. We also show that penetrance of latitudinally-patterned host genetic selection is only observed at elevated humidities. Together, these results identify several factors that influence microbiota composition in wild fly genotypes and emphasize the interplay between environmental and host genetic factors in determining the microbiota composition of these model hosts.

microbiology↗

bioRxiv2024

An ATP-Binding Cassette Transporter Gene Links Innate and Adaptive Immune Responses

Positive-strand RNA viruses and DNA viruses generate double-stranded RNA (dsRNA) during their replication processes and innate immune responses against viral infections are orchestrated by numerous interferon-stimulating genes, yet the detailed coordination of downstream signaling of anti-viral immune responses is not fully understood. Recent studies suggest 2-5- Oligoadenylate Synthetase 1 (OAS1) may have a protective role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections; however, the mechanism regulating OAS1 remains uninvestigated. Our aim is to understand the regulation of OAS1 and its modulation of RNaseL activity, as this has significant implications for responses to RNA viruses, including Vesicular stomatitis virus (VSV) and SARS-CoV-2. We explore the hypothesis that ABCF1 an ATP-binding cassette family member protein, a key regulator of innate immune responses and macrophage polarization and cytokine storm, play a role in regulating the antiviral responses and downstream dsRNA signaling revealed by measuring responses to the synthetic dsRNA analog termed poly (I:C). We utilize ABCF1 haplo-insufficient mice to discover that ABCF1 modulates the amplitude and frequency of VSV-specific Cytolytic T lymphocyte in anti-viral immune responses and suggests that innate immune responses underpin this process. To understand this mechanism, we describe that ABCF1 interacts with 2-5-oligoadenylate synthetase 1 (OAS1) which in turn modulates essential proteins that leads to the modulation of RNaseL activity via ABCE1. Furthermore, we find that ABCF1 also influences the production of interferon- (IFN-) and interferon-{beta} (IFN-{beta}) in bone marrow-derived macrophages. Overall,, we unexpectedly discovered that ABCF1 acts as a crucial link between innate and adaptive immunity, regulating the development of adaptive Cytolytic T lymphocyte responses and interacting with OAS1, a key regulator of innate immune responses against viral infections. Exploring pharmacological agents that target ABCE1 or ABCF1 may lead to the discovery of novel modalities for countering SARS CoV-2 and other viruses where OAS1 is a crucial innate immune response gene.

immunology↗

bioRxiv2023

A Risk-reward Examination of Sample Multiplexing Reagents for Single Cell RNA-Seq

Single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for understanding cellular heterogeneity and function. However the choice of sample multiplexing reagents can impact data quality and experimental outcomes. In this study, we compared various multiplexing reagents, including MULTI-Seq, Hashtag antibody, and CellPlex, across diverse sample types such as human peripheral blood mononuclear cells (PBMCs), mouse embryonic brain and patient-derived xenografts (PDXs). We found that all multiplexing reagents worked well in cell types robust to ex vivo manipulation but suffered from signal-to-noise issues in more delicate sample types. We compared multiple demultiplexing algorithms which differed in performance depending on data quality. We find that minor improvements to laboratory workflows such as titration and rapid processing are critical to optimal performance. We also compared the performance of fixed scRNA-Seq kits and highlight the advantages of the Parse Biosciences kit for fragile samples. Highly multiplexed scRNA-Seq experiments require more sequencing resources, therefore we evaluated CRISPR-based destruction of non-informative genes to enhance sequencing value. Our comprehensive analysis provides insights into the selection of appropriate sample multiplexing reagents and protocols for scRNASeq experiments, facilitating more accurate and cost-effective studies.

genomics↗

bioRxiv2022

Genome-wide in vivo CRISPR screens identify GATOR1 as a TP53 induced tumour suppressor

Identifying tumor suppressor genes is predicted to inform on the development of novel strategies for cancer therapy. To identify new lymphoma driving processes that cooperate with oncogenic MYC (which is abnormally highly expressed in [~]70% of human cancers) we have used a genome-wide CRISPR knockout screen in E{micro}-Myc;Cas9 transgenic hematopoietic stem and progenitor cells in vivo. We discovered that loss of any of the GATOR1 complex components - NPRL3, DEPDC5, NPRL2 - significantly accelerated c-MYC-driven lymphoma development in mice. Low expression of the GATOR1 complex genes correlated with poor survival outcomes for human patients with high MYC- expressing cancers. Murine lymphomas lacking GATOR1 were highly sensitive to mTOR inhibitors as a single agent therapy, both in vitro and in vivo. These findings identify inhibition of mTORC1 as a potent tumor suppressive mechanism in c-MYC-driven lymphomagenesis and suggest a new avenue for therapeutic intervention in GATOR1-deficient lymphomas through mTOR inhibition. Key PointsO_LIIn vivo CRISPR/Cas9 whole genome knockout screens identified the GATOR1 complex as a potent suppressor of c-MYC-driven lymphomagenesis. C_LIO_LIGATOR1 deficiency in MYC-driven lymphomas confers sensitivity to mTOR inhibition, suggesting a novel therapeutic approach. C_LI

cancer biology↗

bioRxiv2019

The spread of resistance to imidacloprid is restricted by thermotolerance in natural populations of Drosophila melanogaster

Imidacloprid, the worlds most utilised insecticide1, has raised considerable controversy due to its harmful effects on non-pest species2-6 and there is increasing evidence showing that insecticides have become the primary selective force in many insect species7-14. The genetic response to insecticides is heterogeneous across population and environment15-17, leading to more complex patterns of genetic variation than previously thought. This motivated the investigation of imidacloprid resistance at different temperatures in natural populations of Drosophila melanogaster originating from four climate extremes replicated across two continents. Population and quantitative genomic analysis, supported by functional tests, demonstrated a polygenic basis to resistance and a major trade-off with thermotolerance. Reduced genetic differentiation at resistance-associated loci indicate enhanced gene flow at these loci. Resistance alleles showed stronger evidence of positive selection in temperate populations compared to tropical populations. Polygenic architecture and ecological factors should be considered when developing sustainable management strategies for both pest and beneficial insects.

evolutionary biology↗

bioRxiv2017

Development Of Amplicon Deep Sequencing Markers And Data Analysis Pipeline For Genotyping Multi-Clonal Malaria Infections

Amplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power for genotyping multi-clone Plasmodium falciparum infections. Such high resolution is needed for molecular monitoring of drug efficacy trials. Targeted sequencing of molecular marker csp and novel marker cpmp was conducted in duplicate on mixtures of parasite culture strains and 37 field samples. A protocol to multiplex up to 384 samples in a single sequencing run was applied. Software \"HaplotypR\" was developed for data analysis. Cpmp was highly diverse (He=0.96) in contrast to csp (He=0.57). Minority clones were robustly detected if their frequency was >1%. False haplotype calls owing to sequencing errors were observed below that threshold. To reliably detect haplotypes at very low frequencies, experiments are best performed in duplicate and should aim for coverage of >10000 reads/amplicon. When compared to length polymorphic marker msp2, highly multiplexed amplicon sequencing displayed greater sensitivity in detecting minority clones.

microbiology↗