Biology subjects

Chow, C. Y.

Publications and source records attributed to Chow, C. Y..

At least 19 recordsLinked to original sources

bioRxiv2026

A drug repurposing screen reveals dopamine signaling as a candidate therapeutic pathway for PIGA-CDG

PIGA-CDG is a congenital disorder of glycosylation caused by pathogenic partial loss-of-function variants in the PIGA gene. PIGA encodes an enzyme responsible for the catalytic transfer of N-acetylglucosamine to phosphatidylinositol during the first step of glycosylphosphatidylinositol anchor biosynthesis. Loss of this enzyme has a widespread phenotypic impact, but primarily results in neurological symptoms including seizures, intellectual disability, and developmental delay. Currently, treatments are limited and focus on symptom management. We developed an eye model of PIGA-CDG that has a reduced eye size. We screened a library of 98% 1,520 FDA/EMA-approved compounds to find drugs that improved the small eye phenotype. This screen revealed numerous drugs that improved eye size, including those that targeted dopamine signaling and cyclooxygenases. Using pharmacological and genetic approaches, we show that modulating dopamine signaling improves the eye size. Genetic inhibition of dopamine 2 receptor signaling and dopamine reuptake improve both the eye model and neurologically relevant PIGA-CDG phenotypes, including seizures and locomotor deficits. We also pharmacologically and genetically validate cyclooxygenase targeting drugs in the eye model. These findings reveal novel biology underlying PIGA-CDG and point towards candidate therapeutic approaches. AUTHOR SUMMARYPIGA-CDG is a rare neurodevelopmental disorder caused by pathogenic variants in the gene PIGA. Patients primarily display neurological symptoms, including seizures, developmental delay, and intellectual disability. Fewer than 100 patients have been identified, and treatment strategies are limited. In the context of rare diseases, de novo drug development is difficult due to the high cost, lengthy development times, and often too small of a patient population to conduct a clinical trial. Our lab leverages drug repurposing screening to circumvent many of the hurdles associated with de novo drug development. Here, we develop and screen FDA- or EMA-approved compounds on a Drosophila model of PIGA-CDG, uncovering novel biology underlying PIGA-associated pathophysiology. We use pharmacological and genetic tools to demonstrate that modifying dopamine signaling and abundance, as well as cyclooxygenase-mediated pathways, contribute to PIGA associated phenotypes. This work highlights promising therapeutic targets for PIGA-CDG.

genetics↗

bioRxiv2026

The stress-induced transcription factor ATF4 has multiple conserved retrocopies that can alter gene expression

The cell must defend against various stressors from internal and external sources that disrupt cell homeostasis. The integrated stress response (ISR) is a highly conserved pathway that helps restore this homeostasis through upregulating the transcription factor, ATF4. Despite its importance to cell health and human disease, ATF4 has several duplications in humans that have never been studied. Here, we characterize three retroduplications (retrocopies) of ATF4 in humans for the first time. Evolutionary analysis demonstrates that these retrocopies are present and intact in many primate species over the past 37 million years, including several independent copies. We also find evidence of positive selection among primates. Human ATF4 retrocopies show basal transcription in healthy, unstressed cells and can be upregulated by the ISR. When translated in human cells, ATF4 retrocopy proteins are regulated by the proteasome in the same way as the parent ATF4 protein. Remarkably, each retrocopy can also alter the expression of several canonical ATF4 target genes, demonstrating that they can impact ISR-ATF4 stress signaling. Overall, ATF4 retrocopies are conserved, biologically functional, and should be considered in future studies of the ISR and ATF4.

genetics↗

bioRxiv2025

Natural SEL1L variants modify ERAD, proteasome function, and survival in a Drosophila model of NGLY1 deficiency

N-glycanase 1 (NGLY1) deficiency is an ultra-rare disease caused by autosomal recessive loss-of-function mutations in the NGLY1 gene. NGLY1 removes N-linked glycans from glycoproteins in the cytoplasm and is thought to help clear misfolded proteins from the endoplasmic reticulum (ER) through the ER associated degradation (ERAD) pathway. Despite this, the physiological significance of NGLY1 in ERAD is not understood. The best characterized substrate of NGLY1 is NRF1, a transcription factor that upregulates proteasome expression and the proteasome bounce-back response. We previously performed a genetic modifier screen using a Drosophila model of NGLY1 deficiency and identified potential modifiers that alter the lethality of the model. We identified two protein-coding variants in Hrd3/SEL1L: S780P and{Delta} 806-809. Both variants are localized to the SEL1L cytoplasmic tail, an uncharacterized domain. SEL1L is a component of the ERAD complex that retrotranslocates misfolded proteins from the ER to the cytoplasm for degradation. We used CRISPR to generate fly lines carrying these SEL1L variants in a common genetic background and tested them with our model of NGLY1 deficiency. Validating our previous screen, the SEL1LP780 and SEL1L{Delta}806-809 variants increase the survival of the NGLY1 deficiency model, compared to the SEL1LS780 variant. To determine how these SEL1L variants were modifying lethality in NGLY1 deficiency, we interrogated the ERAD and NRF1 signaling pathways. We found that the SEL1LP780 and SEL1L{Delta}806-809 variants improve ERAD function in an NGLY1- dependent manner, further implicating NGLY1 in general ERAD function. We also found that these variants protect against changes in larval size and survival caused by proteasome inhibition in heterozygous NGLY1 null flies. These results provide new insights into the role of SEL1L in the disease pathogenesis of NGLY1 deficiency. SEL1L is a strong candidate modifier gene in patients, where variability in presentation is common. AUTHORS SUMMARYNGLY1 deficiency is a debilitating rare genetic disorder. There are currently no treatment options for NGLY1 deficiency and NGLY1 biology remains poorly understood. We previously performed a genetic modifier screen in a Drosophila model of NGLY1 deficiency and identified a number of candidate modifier genes that impacted the survival of our model. Modifier genes can help reveal NGLY1 biology and NGLY1 deficiency disease pathogenesis. In this study, we follow-up on two natural protein-coding variants of Hrd3 (the fly version of the human gene, SEL1L) that increased the survival of our NGLY1 deficiency model. SEL1L is a critical component of an important quality control pathway called the endoplasmic reticulum associated degradation (ERAD) pathway. We discovered that these SEL1L variants enhance ERAD and modify NGLY1 deficiency sensitivity to proteasome inhibition. This study confirms SEL1L as an important modifier gene of NGLY1 deficiency. Further study of the ERAD and proteasome degradation pathways may reveal additional candidate modifier genes of NGLY1 deficiency and potential targets for therapeutic development.

genetics↗

bioRxiv2024

Identification of CNTN2 as a genetic modifier of PIGA-CDG through pedigree analysis of a family with incomplete penetrance and functional testing in Drosophila

Loss of function mutations in the X-linked PIGA gene lead to PIGA-CDG, an ultra-rare congenital disorder of glycosylation (CDG), typically presenting with seizures, hypotonia, and neurodevelopmental delay. We identified two brothers (probands) with PIGA-CDG, presenting with epilepsy and mild developmental delay. Both probands carry PIGAS132C, an ultra-rare variant predicted to be damaging. Strikingly, the maternal grandfather and a great-uncle also carry PIGAS132C, but neither presents with symptoms associated with PIGA-CDG. We hypothesized genetic modifiers may contribute to this reduced penetrance. Using whole genome sequencing and pedigree analysis, we identified possible susceptibility variants found in the probands and not in carriers and possible protective variants found in the carriers and not in the probands. Candidate variants included heterozygous, damaging variants in three genes also involved directly in GPI-anchor biosynthesis and a few genes involved in other glycosylation pathways or encoding GPI-anchored proteins. We functionally tested the predicted modifiers using a Drosophila eye-based model of PIGA-CDG. We found that loss of CNTN2, a predicted protective modifier, rescues loss of PIGA in Drosophila eye-based model, like what we predict in the family. Further testing found that loss of CNTN2 also rescues patient-relevant phenotypes, including seizures and climbing defects in Drosophila neurological models of PIGA-CDG. By using pedigree information, genome sequencing, and in vivo testing, we identified CNTN2 as a strong candidate modifier that could explain the incomplete penetrance in this family. Identifying and studying rare disease modifier genes in human pedigrees may lead to pathways and targets that may be developed into therapies.

genetics↗

bioRxiv2024

A drug repurposing screen reveals dopamine signaling as a critical pathway underlying potential therapeutics for the rare disease DPAGT1-CDG

DPAGT1-CDG is a Congenital Disorder of Glycosylation (CDG) that lacks effective therapies. It is caused by mutations in the gene DPAGT1 which encodes the first enzyme in N-linked glycosylation. We performed a drug repurposing screen on a model of DPAGT1-CDG in Drosophila ("DPAGT1 model") using 1,520 small molecules that are 98% FDA/EMA-approved. We identified 42 candidate drugs that improved the DPAGT1-CDG model. Notably from this screen, we found that pharmacological and genetic inhibition of the dopamine D2 receptor rescued the DPAGT1 model. Loss of both dopamine synthesis and recycling rescued the model, suggesting that dopaminergic flux and subsequent binding to D2 receptors is detrimental under DPAGT1 deficiency. This links dopamine signaling to N-glycosylation and represents a new potential therapeutic target for treating DPAGT1-CDG. We also genetically validate other top drug categories including acetylcholine-related drugs, COX inhibitors, and an inhibitor of NKCC1. These drugs and subsequent analyses reveal novel biology in DPAGT1 mechanisms, and they may represent new therapeutic options for DPAGT1-CDG.

genetics↗

bioRxiv2024

Evolutionary rate covariation is pervasive between glycosylation pathways and points to potential disease modifiers

Mutations in glycosylation pathways, such as N-linked glycosylation, O-linked glycosylation, and GPI anchor synthesis, lead to Congenital Disorders of Glycosylation (CDG). CDGs typically present with seizures, hypotonia, and developmental delay but display large clinical variability with symptoms affecting every system in the body. This variability suggests modifier genes might influence the phenotypes. Because of the similar physiology and clinical symptoms, there are likely common genetic modifiers between CDGs. Here, we use evolution as a tool to identify common modifiers between CDG and glycosylation genes. Protein glycosylation is evolutionarily conserved from yeast to mammals. Evolutionary rate covariation (ERC) identifies proteins with similar evolutionary rates that indicate shared biological functions and pathways. Using ERC, we identified strong evolutionary rate signatures between proteins in the same and different glycosylation pathways. Genome-wide analysis of proteins showing significant ERC with GPI anchor synthesis proteins revealed strong signatures with ncRNA modification proteins and DNA repair proteins. We also identified strong patterns of ERC based on cellular sub-localization of the GPI anchor synthesis enzymes. Functional testing of the highest scoring candidates validated genetic interactions and identified novel genetic modifiers of CDG genes. ERC analysis of disease genes and biological pathways allows for rapid prioritization of potential genetic modifiers, which can provide a better understanding of disease pathophysiology and novel therapeutic targets. AUTHOR SUMMARYCongenital Disorders of Glycosylation (CDGs) are a group of rare disorders resulting from impaired protein glycosylation. Glycosylation is the addition of sugar chains onto proteins and is required for proper protein function. CDG patients typically present with seizures and hypotonia. However, they can have a large amount of clinical variability, which is likely influenced by modifier genes. Modifier genes are genes that affect a phenotype without causing the disease. Using an evolutionary method that examines proteins that evolve at similar rates, we identified proteins within glycosylation pathways and among other unexpected pathways, such as ncRNA modification and DNA repair, that could be potential genetic modifiers of CDG genes. We also tested top protein pairs using the Drosophila eye as a model and identified novel genetic modifiers of CDG genes. Broadening our understanding of CDG modifiers can help us to better understand why loss of glycosylation results in specific patient symptoms and could provide new treatment targets.

genetics↗

bioRxiv2024

NaV1.7 targeted fluorescence imaging agents for nerve identification during intraoperative procedures

Surgeries and trauma result in traumatic and iatrogenic nerve damage that can result in a debilitating condition that approximately affects 189 million individuals worldwide. The risk of nerve injury during oncologic surgery is increased due to tumors displacing normal nerve location, blood turbidity, and past surgical procedures, which complicate even an experienced surgeons ability to precisely locate vital nerves. Unfortunately, there is a glaring absence of contrast agents to assist surgeons in safeguarding vital nerves. To address this unmet clinical need, we leveraged the abundant expression of the voltage-gated sodium channel 1.7 (NaV1.7) as an intraoperative marker to access peripheral nerves in vivo, and visualized nerves for surgical guidance using a fluorescently-tagged version of a potent NaV1.7-targeted peptide, Tsp1a, derived from a Peruvian tarantula. We characterized the expression of NaV1.7 in sensory and motor peripheral nerves across mouse, primate, and human specimens and demonstrated universal expression. We synthesized and characterized a total of 10 fluorescently labeled Tsp1a-peptide conjugates to delineate nerves. We tested the ability of these peptide-conjugates to specifically accumulate in mouse nerves with a high signal-to-noise ratio in vivo. Using the best-performing candidate, Tsp1a-IR800, we performed thyroidectomies in non-human primates and demonstrated successful demarcation of the recurrent laryngeal and vagus nerves, which are commonly subjected to irreversible damage. The ability of Tsp1a to enhance nerve contrast during surgery provides opportunities to minimize nerve damage and revolutionize standards of care across various surgical specialties.

biochemistry↗

bioRxiv2023

Comparing virtual staining artificial intelligence model performance on same-tissue versus serial-tissue sections based on CD3+ T-cell ground truth

Immunophenotyping via multi-marker assays significantly contributes to patient selection, therapeutic monitoring, biomarker discovery, and personalized treatments. Despite its potential, the multiplex immunofluorescence (mIF) technique faces adoption challenges due to technical and financial constraints. Alternatively, hematoxylin and eosin (H&E)-based prediction models of cell phenotypes can provide crucial insights into tumor-immune cell interactions and advance immunotherapy. Current methods mostly rely on manually annotated cell label ground truths, with limitations including high variability and substantial labor costs. To mitigate these issues, researchers are increasingly turning to digitized cell-level data for accurate in-situ cell type prediction. Typically, immunohistochemical (IHC) staining is applied to a tissue section serial to one stained with H&E. However, this method may introduce distortions and tissue section shifts, challenging the assumption of consistent cellular locations. Conversely, mIF overcomes these limitations by allowing for mIF and H&E staining on the same tissue section. Importantly, the multiplexing capability of mIF allows for a thorough analysis of the tumor microenvironment by quantifying multiple cell markers within the same tissue section. In this study, we introduce a Pix2Pix generative adversarial network (P2P-GAN)-based virtual staining model, using CD3+ T-cells in lung cancer as a proof-of-concept. Using an independent CD3 IHC-stained lung cohort, we demonstrate that the model trained with cell label ground-truth from the same tissue section as H&E staining performed significantly better in both CD3+ and CD3- T-cell prediction. Moreover, the model also displayed prognostic significance on a public lung cohort, demonstrating its potential clinical utility. Notably, our proposed P2P-GAN virtual staining model facilitates image-to-image translation, enabling further spatial analysis of the predicted immune cells, deepening our understanding of tumor-immune interactions, and propelling advancements in personalized immunotherapy. This concept holds potential for the prediction of other cell phenotypes, including CD4+, CD8+, and CD20+ cells.

immunology↗

bioRxiv2023

Drosophila models of PIGA-CDG mirror patient phenotypes

Mutations in the phosphatidylinositol glycan biosynthesis class A (PIGA) gene cause a rare, X-linked recessive congenital disorder of glycosylation (CDG). PIGA-CDG is characterized by seizures, intellectual and developmental delay, and congenital malformations. The PIGA gene encodes an enzyme involved in the first step of GPI anchor biosynthesis. There are over 100 GPI anchored proteins that attach to the cell surface and are involved in cell signaling, immunity, and adhesion. Little is known about the pathophysiology of PIGA-CDG. Here we describe the first Drosophila model of PIGA-CDG and demonstrate that loss of PIG-A function in Drosophila accurately models the human disease. As expected, complete loss of PIG-A function is larval lethal. Heterozygous null animals appear healthy, but when challenged, have a seizure phenotype similar to what is observed in patients. To identify the cell-type specific contributions to disease, we generated neuron- and glia-specific knockdown of PIG-A. Neuron-specific knockdown resulted in reduced lifespan and a number of neurological phenotypes, but no seizure phenotype. Glia-knockdown also reduced lifespan and, notably, resulted in a very strong seizure phenotype. RNAseq analyses demonstrated that there are fundamentally different molecular processes that are disrupted when PIG-A function is eliminated in different cell types. In particular, loss of PIG-A in neurons resulted in upregulation of glycolysis, but loss of PIG-A in glia resulted in upregulation of protein translation machinery. Here we demonstrate that Drosophila is a good model of PIGA-CDG and provide new data resources for future study of PIGA-CDG and other GPI anchor disorders. Article SummaryPIGA-CDG is a rare genetic disorder. In order to study this rare disease, we generated and characterized several Drosophila models of PIGA-CDG. These models faithfully recapitulate different patient phenotypes, including movement disorder and seizures. Drosophila is a good model for PIGA-CDG and other GPI anchor disorders.

genetics↗

bioRxiv2023

A Drosophila screen identifies a role for histone methylation in ER stress preconditioning

Stress preconditioning occurs when transient, sublethal stress events impact an organisms ability to counter future stresses. Although preconditioning effects are often noted in the literature, very little is known about the underlying mechanisms. To model preconditioning, we exposed a panel of genetically diverse Drosophila melanogaster to a sublethal heat shock and measured how well the flies survived subsequent exposure to endoplasmic reticulum (ER) stress. The impact of preconditioning varied with genetic background, ranging from dying half as fast to four and a half times faster with preconditioning compared to no preconditioning. Subsequent association and transcriptional analyses revealed that histone methylation, transcriptional regulation, and immune status are all candidate preconditioning modifier pathways. Strikingly, almost all subunits (7/8) in the Set1/COMPASS complex were identified as candidate modifiers of preconditioning. Functional analysis of Set1 knockdown flies demonstrated that loss of Set1 led to the transcriptional dysregulation of canonical ER stress genes during preconditioning. Based on these analyses, we propose a model of preconditioning in which Set1 helps to establish an interim transcriptional memory of previous stress events, resulting in a preconditioned response to subsequent stress. Author SummaryStress preconditioning occurs when a history of previous stresses impacts an organisms response to subsequent stresses. There are many documented cases of stress preconditioning, but the specific genes and pathways involved in the process are not well understood. Here, we take advantage of the natural genetic variation in the Drosophila Genetic Reference Panel to examine the role genetic variants play in modifying preconditioning outcomes. Our goal is to identify genes that contribute to the underlying mechanisms of preconditioning. Specifically, we measured preconditioning outcomes as the change in death rates of Drosophila on constant endoplasmic reticulum (ER) stress with and without heat stress preconditioning for each strain. We demonstrate that preconditioning outcomes are highly dependent on genetic background. Through association and transcriptional analyses, we found that histone methylation, transcriptional regulation, and immune status are all candidate pathways impacting preconditioning. Functional studies utilizing Set1 knockdown flies demonstrated that Set1, a histone H3 lysine 4 (H3K4) methyltransferase enzyme, is critical for the proper expression of a subset of ER stress genes during preconditioning. Our data indicate that Set1 likely aids in creating a transient transcriptional memory following initial stress that impacts the response to subsequent stress.

genetics↗

bioRxiv2022

The voltage-gated sodium channel NaV1.7 underlies endometriosis-associated chronic pelvic pain

Chronic pelvic pain (CPP) is the primary symptom of endometriosis patients, but adequate treatments are lacking. Modulation of ion channels expressed by sensory nerves innervating the viscera have shown promise for the treatment of irritable bowel syndrome and overactive bladder. However, similar therapies have not been explored for endometriosis-associated CPP. Here we examined the role of the voltage-gated sodium (NaV) channel NaV1.7 in the sensitivity of vagina-innervating sensory afferents and investigated whether NaV1.7 inhibition reduces nociceptive signals from the vagina and ameliorates endometriosis-associated CPP. The mechanical responsiveness of vagina-innervating sensory afferents was assessed with ex vivo single unit recording preparations. Pain evoked by vaginal distension (VD) was quantified by the visceromotor response (VMR) in vivo. In control mice, pharmacological activation of NaV1.7 with OD1 sensitised vagina-innervating pelvic afferents to mechanical stimuli. Using a syngeneic mouse model of endometriosis, we established that endometriosis sensitized vagina-innervating pelvic afferents to mechanical stimuli. The highly selective NaV1.7 inhibitor Tsp1a revealed that this afferent hypersensitivity occurred in a NaV1.7-dependent manner. Moreover, in vivo intra-vaginal treatment with Tsp1a reduced the exaggerated VMRs to VD that is characteristic of mice with endometriosis. Conversely, Tsp1a did not alter ex vivo afferent mechanosensitivity or in vivo VMRs to VD in Sham control mice. Collectively, these findings suggest that NaV1.7 plays a crucial role in endometriosis-induced vaginal hyperalgesia. Importantly, NaV1.7 inhibition selectively alleviated endometriosis-associated CPP without the loss of normal sensation, suggesting that selective targeting of NaV1.7 could improve the quality of life of women with endometriosis.

cell biology↗

bioRxiv2021

A genome-wide CRISPR screen identifies the glycosylation enzyme DPM1 as a modifier of DPAGT1 deficiency and ER stress

Partial loss-of-function mutations in glycosylation pathways underlie a set of rare diseases called Congenital Disorders of Glycosylation (CDGs). In particular, DPAGT1-CDG is caused by mutations in the gene encoding the first step in N-glycosylation, DPAGT1, and this disorder currently lacks effective therapies. To identify potential therapeutic targets for DPAGT1-CDG, we performed CRISPR knockout screens in Drosophila cells for genes associated with better survival and glycoprotein levels under DPAGT1 inhibition. We identified hundreds of candidate genes that may be of therapeutic benefit. Intriguingly, inhibition of the mannosyltransferase Dpm1, or its downstream glycosylation pathways, could rescue two in vivo models of DPAGT1 inhibition and ER stress, even though impairment of these pathways alone usually cause CDGs. While both in vivo models ostensibly cause ER stress (through DPAGT1 inhibition or a misfolded protein), we found a novel difference in fructose metabolism that may indicate glycolysis as a modulator of DPAGT1-CDG. Our results provide new therapeutic targets for DPAGT1-CDG, include the unique finding of Dpm1-related pathways rescuing DPAGT1 inhibition, and reveal a novel interaction between fructose metabolism and ER stress.

genetics↗

bioRxiv2021

An in vivo drug repurposing screen and transcriptional analyses reveals the serotonin pathway and GSK3 as major therapeutic targets for NGLY1 deficiency

NGLY1 deficiency, a rare disease with no effective treatment, is caused by autosomal recessive, loss-of-function mutations in the N-glycanase 1 (NGLY1) gene and is characterized by global developmental delay, hypotonia, alacrima, and seizures. We used an adult Drosophila model of NGLY1 deficiency to conduct an in vivo, unbiased, small molecule, repurposing screen of FDA-approved drugs to identify therapeutic compounds. Seventeen molecules rescued lethality in a patient-specific NGLY1 deficiency model, including multiple serotonin and dopamine modulators. Exclusive dNGLY1 expression in serotonin and dopamine neurons, in an otherwise dNGLY1-null fly, was sufficient to rescue lethality. Further, genetic modifier and transcriptomic data supports the importance of serotonin signaling in NGLY1 deficiency. Connectivity Map analysis identified glycogen synthase kinase 3 (GSK3) inhibition as a potential therapeutic mechanism for NGLY1 deficiency, which we experimentally validated with TWS119 and lithium. Strikingly, GSK3 inhibitors and a serotonin modulator rescued size defects in dNGLY1 deficient larvae upon proteasome inhibition, suggesting that these compounds act through NRF1, a transcription factor that regulates proteasome expression. This study reveals the importance of the serotonin pathway in NGLY1 deficiency, and serotonin modulators or GSK3 inhibitors may be effective therapeutics for this rare disease.

pharmacology and toxicology↗

bioRxiv2021

Non-invasive Imaging of Sense of Smell by Tracking the Voltage-Gated Sodium Channel NaV1.7

The sense of smell (olfaction) is one of the most important senses for animals including humans. Despite significant advances in the understanding mechanism of olfaction, currently, there are no objective non-invasive methods that can identify loss of smell. Covid-19-related loss of smell has highlighted the need to develop methods that can identify loss of olfaction. Voltage-gated sodium channel 1.7 (NaV1.7) plays a critical role in olfaction by aiding the signal propagation to the olfactory bulb. We have identified several conditions such as chronic inflammation and viral infections such as Covid-19 that lead to loss of smell correlate with downregulation of NaV1.7 expression at transcript and protein levels in the olfactory epithelium. Leveraging this knowledge, we have developed a novel fluorescent probe Tsp1a-IR800 that targets NaV1.7. Using fluorescence imaging we can objectively measure the loss of sense of smell in live animals non-invasively. We also demonstrate that our non-invasive method is semiquantitative because the loss of fluorescence intensity correlates with the level of smell loss. Our results indicate, that our probe Tsp1a-IR800, can objectively diagnose anosmia in animal and human subjects using infrared fluorescence. We believe this method to non-invasively diagnose loss of smell objectively is a significant advancement in relation to current methods that rely on highly subjective behavioral studies and can aid in studying olfaction loss and the development of therapeutic interventions.

neuroscience↗

bioRxiv2021

The dynamic effect of regulatory genetic variation on the in vivo ER stress transcriptional response

Genotype x Environment (GxE) interactions occur when environmental conditions drastically change the effect of a genetic variant. In order to truly understand the effect of genetic variation, we need to incorporate multiple environments into our analyses. Many variants, under steady state conditions, may be silent or even have the opposite effect under stress conditions. This study uses an in vivo mouse model to investigate how the effect of genetic variation changes with tissue type and cellular stress. Endoplasmic reticulum (ER) stress occurs when misfolded proteins accumulate in the ER. This triggers the unfolded protein response (UPR), a large transcriptional response which attempts to return the cell to homeostasis. This transcriptional response, despite being a well conserved, basic cellular process, is highly variable across different genetic backgrounds, making it an ideal system to study GxE effects. In this study, we sought to better understand how genetic variation alters expression across tissues, in the presence and absence of ER stress. The use of different mouse strains and their F1s allow us to also identify context specific cis- and trans-regulatory mechanisms underlying variable transcriptional responses. We found hundreds of genes that respond to ER stress in a tissue- and/or genotype-dependent manner. Genotype-dependent ER stress-responsive genes are enriched for processes such as protein folding, apoptosis, and protein transport, indicating that some of the variability occurs in canonical ER stress factors. The majority of regulatory mechanisms underlying these variable transcriptional responses derive from cis-regulatory variation and are unique to a given tissue or ER stress state. This study demonstrates the need for incorporating multiple environments in future studies to better elucidate the effect of any particular genetic factor in basic biological pathways, like the ER stress response. Author SummaryThe effect of genetic variation is dependent on environmental context. Here we use genetically diverse mouse strains to understand how genetic variation interacts with stress state to produce variable transcriptional profiles. In this study, we take advantage of the endoplasmic reticulum (ER) stress response which is a large transcriptional response to misfolded proteins. Using this system, we uncovered tissue- and ER stress-specific effects of genetic variation on gene expression. Genes with genotype-dependent variable expression levels in response to ER stress were enriched for canonical ER stress functions, such as protein folding and transport. These variable effects of genetic variation are driven by unique sets of regulatory variation that are only active under context-specific circumstances. The results of this study highlight the importance of including multiple environments and genetic backgrounds when studying the ER stress response and other cellular pathways.

genetics↗

bioRxiv2020

A Drosophila natural variation screen identifies NKCC1 as a substrate of NGLY1 deglycosylation and a modifier of NGLY1 deficiency

N-Glycanase 1 (NGLY1) is a cytoplasmic deglycosylating enzyme. Loss-of-function mutations in the NGLY1 gene cause NGLY1 deficiency, which is characterized by developmental delay, seizures, and a lack of sweat and tears. To model the phenotypic variability observed among patients, we crossed a Drosophila model of NGLY1 deficiency onto a panel of genetically diverse strains. The resulting progeny showed a phenotypic spectrum from 0-100% lethality. Association analysis on the lethality phenotype as well as an evolutionary rate covariation analysis generated lists of modifying genes, providing insight into NGLY1 function and disease. The top association hit was Ncc69 (human NKCC1/2), a conserved ion transporter. Analyses in NGLY1 -/- mouse cells demonstrated that NKCC1 is misglycosylated and has reduced function, making it only the second confirmed NGLY1 enzymatic substrate. The misregulation of this ion transporter may explain the observed defects in secretory epithelium function in NGLY1 deficiency patients.

genomics↗

bioRxiv2019

Insulin potentiates JAK/STAT signaling to broadly inhibit flavivirus replication in insect vectors

The World Health Organization estimates that over half of the worlds population is at risk for vector-borne diseases, such as those caused by arboviral infection. Because many arboviruses are mosquito-borne, investigation of the insect immune response will help identify targets that could reduce the spread of these viruses by the mosquito. In this study, we used a genetic screening approach to identify insulin-like receptor as a novel component of the immune response to arboviral infection. We determined that vertebrate insulin reduces West Nile virus (WNV) replication in Drosophila melanogaster as well as WNV, Zika, and dengue virus titers in mosquito cells. Mechanistically, we showed that insulin signaling activates the JAK/STAT, but not RNAi, pathway to control infection. Finally, we validated that insulin priming of adult female Culex mosquitoes through a blood meal reduces WNV infection, demonstrating an essential role for insulin signaling in insect antiviral responses to emerging human pathogens.

microbiology↗

bioRxiv2019

Natural genetic variation screen in Drosophila identifies Wnt signaling, mitochondrial metabolism, and redox homeostasis genes as modifiers of apoptosis

Apoptosis is the primary cause of degeneration in a number of neuronal, muscular, and metabolic disorders. These diseases are subject to a great deal of phenotypic heterogeneity in patient populations, primarily due to differences in genetic variation between individuals. This creates a barrier to effective diagnosis and treatment. Understanding how genetic variation influences apoptosis could lead to the development of new therapeutics and better personalized treatment approaches. In this study, we examine the impact of the natural genetic variation in the Drosophila Genetic Reference Panel (DGRP) on two models of apoptosis-induced retinal degeneration: overexpression of p53 or reaper (rpr). We identify a number of known apoptotic, neural, and developmental genes as candidate modifiers of degeneration. We also use Gene Set Enrichment Analysis (GSEA) to identify pathways that harbor genetic variation that impact these apoptosis models, including Wnt signaling, mitochondrial metabolism, and redox homeostasis. Finally, we demonstrate that many of these candidates have a functional effect on apoptosis and degeneration. These studies provide a number of avenues for modifying genes and pathways of apoptosis-related disease.

genetics↗