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NGLY1 and Ncc69 interact genetically in Drosophila. (A) Proportion of flies surviving to eclosion in ubiquitous knockdowns. NGLY1 knockdown (KD) are UAS-Pngl RNAi /+; Tubulin-GAL4/+. Ncc69 KD are UAS-Ncc69 RNAi /+; Tubulin-GAL4/+. NGLY1 Ncc69 double knockdown (DKD) are UAS-Pngl RNAi /+ UAS-Ncc69 RNAi /Tubulin-GAL4/+. Four separate matings were performed for each cross with at least 40 offspring generated for the balancer control for each. Fraction surviving is calculated compared to balancer offspring. Chi-square analysis was performed for the total number of flies compared to expected Mendelian numbers. NGLY1 KD c 2 = 109.7, p<0.0001; Ncc69 KD c 2 = 1.002, p=0.3168, and NGLY1 Ncc69 DKD c 2 = 186, p<0.0001. (B) Bang sensitivity assay to assess seizures in glial knockdown flies. WT flies are attP2 and attP40. NGLY1 KD are UAS-Pngl RNAi /+; repo-GAL4/+. Ncc69 KD are UAS-Ncc69 RNAi /+; repo-GAL4/+. NGLY1 Ncc69 DKD are UAS-Pngl RNAi /+; UAS-Ncc69 RNAi / repo-GAL4. For each genotype, at least 45 4-to 7-day-old females were used to calculate the percent seizing at a given time after vortexing. Repeated measures ANOVA p-value=0.000176.
Source publication
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 gen...
Contexts in source publication
Context 1
... validate the genetic interaction observed between NGLY1 and Ncc69 in the GWA, we generated ubiquitous double knockdown (DKD) Drosophila and scored offspring that survived to eclosion ( Figure 3A). The fraction of KD flies was calculated from observed offspring of the balancer phenotype. ...
Citations
... Accelerating research has demonstrated the functional importance of NGLY1 in various cellular processes (10, T. Suzuki 12,[25][26][27][28][29][30][31][32][33][34][35], with various therapeutic target genes/pathways being proposed (9,15,(36)(37)(38). I hope that these studies will eventually lead to the development of additional therapeutic options for NGLY1 deficiency. ...
Absract
The cytosolic peptide:N-glycanase (PNGase; NGLY1 in human and PNG1 in budding yeast) is a deglycosylating enzyme widely conserved in eukaryotes. Initially, functional importance of this enzyme remained unknown as the png1Δ mutant in yeast did not exhibit any significant phenotypes. However, the discovery of NGLY1 deficiency, a rare genetic disorder with biallelic mutations in NGLY1 gene, prompted an intensification of research that has resulted in uncovering the significance of NGLY1 as well as the proteins under its influence that are involved in numerous cellular processes. A recent report by Rauscher et al. (Patient-derived gene and protein expression signatures of NGLY1 deficiency. J. Biochem. 2022; 171: 187-199) presented a comprehensive summary of transcriptome/proteome analyses of various cell types derived from NGLY1 deficient patients. The authors also provide a web application called “NGLY1 browser”, which will allow researchers to have access to a wealth of information on gene and protein expression signature for patients with NGLY1 deficiency.
... Gene set enrichment analysis (GSEA) analysis was performed as previously described (Subramanian et al. 2005;Palu et al. 2019;Talsness et al. 2020). All polymorphisms from the stress preconditioning GWA (Supplementary File 1) were assigned to a gene as described in the above section. ...
... Therefore, in this study, we put little emphasis on individual polymorphisms. Instead, we prioritized identifying potential modifier genes, which has been a very successful approach in previous DGRP screens (Chow et al. 2013(Chow et al. , 2016Palu et al. 2019;Talsness et al. 2020). If a SNP fell within an annotated gene, we assigned it to that gene. ...
... With a standard P-value cutoff of P ≤ 1 × 10 −5 used in most DGRP studies (Chow et al. 2013(Chow et al. , 2016Palu et al. 2019;Talsness et al. 2020), the GWA identified only 6 polymorphisms associated with stress preconditioning outcomes. Of the 6 polymorphisms, 5 were in a known gene: 1 in a 3′ UTR, 3 in introns, and 1 synonymous variant in a protein-coding exon. ...
Stress preconditioning occurs when transient, sublethal stress events impact an organism's 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 4 and a half times faster with preconditioning compared to no preconditioning. Subsequent association and transcriptional analyses revealed that histone methylation, and transcriptional regulation are both 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 preconditioning model in which Set1 helps to establish an interim transcriptional “memory” of previous stress events, resulting in a preconditioned response to subsequent stress.
... Drosophila has been employed to study epilepsy, and especially seizures resulting from disease-linked mutations in transporters and ion channels (Parker et al., 2011;Talsness et al., 2020;Jeong et al., 2021). Thus, we reasoned that hGAT-1 epilepsy variants ought to exhibit seizure-like activity in flies. ...
Mutations in the human γ-aminobutyric acid (GABA) transporter 1 (hGAT-1) can instigate myoclonic-atonic and other generalized epilepsies in the afflicted individuals. We systematically examined fifteen hGAT-1 disease variants, all of which dramatically reduced or completely abolished GABA uptake activity. Many of these loss-of-function variants were absent from their regular site of action at the cell surface, due to protein misfolding and/or impaired trafficking machinery (as verified by confocal microscopy and de-glycosylation experiments). A modest fraction of the mutants displayed correct targeting to the plasma membrane, but nonetheless rendered the mutated proteins devoid of GABA transport, possibly due to structural alterations in the GABA binding site/translocation pathway. We here focused on a folding-deficient A288V variant. In flies, A288V reiterated its impeded expression pattern, closely mimicking the ER-retention demonstrated in transfected HEK293 cells. Functionally, A288V presented a temperature-sensitive seizure phenotype in fruit flies. We employed diverse small molecules to restore the expression and activity of folding-deficient hGAT-1 epilepsy variants, in vitro (in HEK293 cells) and in vivo (in flies). We identified three compounds (chemical and pharmacological chaperones) conferring moderate rescue capacity for several variants. Our data grant crucial new insights into: (i) the molecular basis of epilepsy in patients harboring hGAT-1 mutations, and (ii) a proof-of-principle that protein folding deficits in disease-associated hGAT-1 variants can be corrected using the pharmacochaperoning approach. Such innovative pharmaco-therapeutic prospects inspire the rational design of novel drugs for alleviating the clinical symptoms triggered by the numerous emerging pathogenic mutations in hGAT-1.
... Those passing the validation will be characterized in greater detail for their role in regulating the degenerative process, and possibly as candidates for therapeutic targeting or prognostic markers. Such characterization has proven extremely fruitful in previous studies [1,[34][35][36][37]. ...
The retinal degenerative disease retinitis pigmentosa (RP) is a genetic disease that is the most common cause of blindness in adults. In 2016, Chow et. al. identified over 100 candidate modifier genes for RP through the genome-wide analysis of 173 inbred strains from the Drosophila Genetic Reference Panel (DGRP). However, this type of analysis may miss some modifiers lying in trans to the variation. In this paper, we propose an alternative approach to identify transcripts whose expression is significantly altered in strains demonstrating extreme phenotypes. The differences in the eye size phenotype will, therefore, be associated directly with changes in gene expression rather than indirectly through genetic variation that might then be linked to changes in gene expression. Gene expression data are obtained from the DGRP2 database, where each strain is represented by up to two replicates. The proposed algorithmic approach first chooses the strains’ replicate combination that best represents the relationship between gene expression level and eye size. The extensive correlation analysis identified several genes with known relationships to eye development, along with another set of genes with unknown functions in eye development. The modifiers identified in this analysis can be validated and characterized in biological systems.
... Previously, our laboratory found that N-acetylglucosamine supplementation rescued lethality in dNGLY1 deficient Drosophila without restoring cap'n'collar (Drosophila ortholog of NRF1) dependent changes in proteasome gene expression [12]. Additionally, we recently reported that NGLY1 regulates the glycosylation state and function of NKCC1, a Na-K-2Cl cotransporter [22], but this reduced function was unrelated to the proteasome defects, demonstrating a role for NGLY1 outside of ERAD and proteasome regulation. ...
... Our lab previously performed a genetic modifier screen for genes that impacted lethality caused by dNGLY1 RNAi knockdown in the Drosophila Genetic Reference Panel (DGRP) [22]. We identified 61 genes that were associated with survival upon knockdown of dNGLY1, and one of the top hits was the serotonin receptor 5-HT1A. ...
... Gene expression data was from Tubulin>dNGLY1-RNAi whole flies reported in a previous study [12]. B) Spearman correlation analysis between survival upon knockdown of dNGLY1 [22] and gene expression of serotonin and dopamine receptors at baseline in the Drosophila Genetic Reference Panel [26]. 25 μM, respectively. ...
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 a 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 partially 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 deficient fly, was sufficient to partially 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, lithium, and GSK3 knockdown. 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 is regulated by NGLY1 and 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.
... four of five proteins exhibit signatures of gene coevolution (27). Although these and other studies have demonstrated that signatures of coevolution are a powerful method to detect functional associations among genes in the absence of functional data (20,25,26,(28)(29)(30), the network biology principles of gene coevolution, especially between genes that have coevolved for hundreds of millions of years, remain unexplored. ...
The evolutionary rates of functionally related genes often covary. We present a gene coevolution network inferred from examining nearly 3 million orthologous gene pairs from 332 budding yeast species spanning ~400 million years of evolution. Network modules provide insight into cellular and genomic structure and function. Examination of the phenotypic impact of network perturbation using deletion mutant data from the baker's yeast Saccharomyces cerevisiae, which were obtained from previously published studies, suggests that fitness in diverse environments is affected by orthologous gene neighborhood and connectivity. Mapping the network onto the chromosomes of S. cerevisiae and Candida albicans revealed that coevolving orthologous genes are not physically clustered in either species; rather, they are often located on different chromosomes or far apart on the same chromosome. The coevolution network captures the hierarchy of cellular structure and function, provides a roadmap for genotype-to-phenotype discovery, and portrays the genome as a linked ensemble of genes.
... Based on lethality association and co-evolution analysis, a conserved Na/K/Cl ion transporter Ncc69 (human NKCC1/2, officially SLC12A1/2) was identified, ubiquitously expressed and associated genetically with Ngly1 in Drosophila [123]. In Ncc69 or Ngly1 knockdown (KD) Drosophila, more than 30% showed severe seizures [124]. In mouse embryonic fibroblasts (MEFs), the homolog of Ncc69, NKCC1 was highly expressed and N-glycosylated, which was important for the function [125]. ...
The cytosolic PNGase (peptide:N-glycanase), also known as peptide-N4-(N-acetyl-β-glucosaminyl)-asparagine amidase, is a well-conserved deglycosylation enzyme (EC 3.5.1.52) which catalyzes the non-lysosomal hydrolysis of an N(4)-(acetyl-β-d-glucosaminyl) asparagine residue (Asn, N) into a N-acetyl-β-d-glucosaminyl-amine and a peptide containing an aspartate residue (Asp, D). This enzyme (NGLY1) plays an essential role in the clearance of misfolded or unassembled glycoproteins through a process named ER-associated degradation (ERAD). Accumulating evidence also points out that NGLY1 deficiency can cause an autosomal recessive (AR) human genetic disorder associated with abnormal development and congenital disorder of deglycosylation. In addition, the loss of NGLY1 can affect multiple cellular pathways, including but not limited to NFE2L1 pathway, Creb1/Atf1-AQP pathway, BMP pathway, AMPK pathway, and SLC12A2 ion transporter, which might be the underlying reasons for a constellation of clinical phenotypes of NGLY1 deficiency. The current comprehensive review uncovers the NGLY1'ssdetailed structure and its important roles for participation in ERAD, involvement in CDDG and potential treatment for NGLY1 deficiency.
... Therefore, we are measuring the dominant effect of the DGRP background on the Sirt1 RNAi hyperglycemia phenotype. This experimental design is similar to a model of NGLY1 deficiency using RNAi that was also crossed to the DGRP (Talsness et al. 2020). ...
... Instead, the focus is on identification of candidate modifiers and pathways that can be validated through further study and that will provide the basis for future projects. This approach has been quite successful in previous studies (Chow et al. 2013(Chow et al. , 2015Lavoy et al. 2018;Palu and Chow 2018;Palu et al. 2019Palu et al. , 2020Talsness et al. 2020). ...
... We also see no significant difference in Sirt1 expression between experimental lines associated with high or low glucose, as determined by qPCR ( Supplementary Fig. 7b). Furthermore, the GAL4/UAS system is commonly used to model disease in the DGRP, and the candidates identified have always been unique to the disease and, at times, even the specific model in question (He et al. 2014;Chow et al. 2016;Lavoy et al. 2018;Palu et al. 2019;Talsness et al. 2020). All of this suggests that the candidate genes identified through this screen are modifying Sirt1-associated hyperglycemia directly rather than altering the degree of Sirt1 knockdown. ...
Variation in the onset, progression, and severity of symptoms associated with metabolic disorders such as diabetes impairs the diagnosis and treatment of at-risk patients. Diabetes symptoms, and patient variation in these symptoms, is attributed to a combination of genetic and environmental factors, but identifying the genes and pathways that modify diabetes in humans has proven difficult. A greater understanding of genetic modifiers and the ways in which they interact with metabolic pathways could improve the ability to predict a patient's risk for severe symptoms, as well as enhance the development of individualized therapeutic approaches. In this study we use the Drosophila Genetic Reference Panel (DGRP) to identify genetic variation influencing hyperglycemia associated with loss of Sirt1 function. Through analysis of individual candidate functions, physical interaction networks, and Gene Set Enrichment Analysis (GSEA) we identify not only modifiers involved in canonical glucose metabolism and insulin signaling, but also genes important for neuronal signaling and the innate immune response. Furthermore, reducing the expression of several of these candidates suppressed hyperglycemia, making them potential candidate therapeutic targets. These analyses showcase the diverse processes contributing to glucose homeostasis and open up several avenues of future investigation.
... Indeed, the extreme sensitivity of the NGLY1 loss-of-function phenotypes to genetic background was shown in recent work from the Chow lab. By using a set of genetically heterogeneous Drosophila strains, they showed that depending on the genetic background, Pngl-knockdown flies show a wide range of survivability, from 100% lethal to fully viable [152]. Genome-wide association analysis on this panel identified a Drosophila Pngl modifier gene, Ncc69, which is the fly homolog of human NKCC1 and NKCC2 (official names: SLC12A2 and SLC12A1, respectively) [152]. ...
... By using a set of genetically heterogeneous Drosophila strains, they showed that depending on the genetic background, Pngl-knockdown flies show a wide range of survivability, from 100% lethal to fully viable [152]. Genome-wide association analysis on this panel identified a Drosophila Pngl modifier gene, Ncc69, which is the fly homolog of human NKCC1 and NKCC2 (official names: SLC12A2 and SLC12A1, respectively) [152]. Ncc69/NKCC1 encodes for a member of the SLC12 family of Na + /K + /2Cl − transporters. ...
N-Glycanase 1 (NGLY1) is a cytosolic enzyme involved in removing N-linked glycans of misfolded N-glycoproteins and is considered to be a component of endoplasmic reticulum-associated degradation (ERAD). The 2012 identification of recessive NGLY1 mutations in a rare multisystem disorder has led to intense research efforts on the roles of NGLY1 in animal development and physiology, as well as the pathophysiology of NGLY1 deficiency. Here, we present a review of the NGLY1-deficient patient phenotypes, along with insights into the function of this gene from studies in rodent and invertebrate animal models, as well as cell culture and biochemical experiments. We will discuss critical processes affected by the loss of NGLY1, including proteasome bounce-back response, mitochondrial function and homeostasis, and bone morphogenetic protein (BMP) signaling. We will also cover the biologically relevant targets of NGLY1 and the genetic modifiers of NGLY1 deficiency phenotypes in animal models. Together, these discoveries and disease models have provided a number of avenues for preclinical testing of potential therapeutic approaches for this disease.
... Biallelic NGLY1 mutation causes an ultrarare human disorder called Ngly1 deficiency, characterized by intellectual disability, hyperkinetic movement disorder, liver dysfunction, and alacrima (29). NGLY1 disruption has been associated with defects in mitochondrial homeostasis and inflammatory responses (30,31), 5 0 adenosine monophosphate-activated protein kinase signaling (32), plasma membrane transporter function (33,34), and BMP4 signaling (35). How altered NFE2L1 glycosylation and function may contribute to phenotypes observed in NGLY1mutant models and patients, such as increased oxidative stress (36), are not well-understood. ...
Significance
Ferroptosis is an oxidative form of cell death whose biochemical regulation remains incompletely understood. Cap’n’collar (CNC) transcription factors including nuclear factor erythroid-2–related factor 1 (NFE2L1/NRF1) and NFE2L2/NRF2 can both regulate oxidative stress pathways but are each regulated in a distinct manner, and whether these two transcription factors can regulate ferroptosis independent of one another is unclear. We find that NFE2L1 can promote ferroptosis resistance, independent of NFE2L2, by maintaining the expression of glutathione peroxidase 4 (GPX4), a key protein that prevents lethal lipid peroxidation. NFE2L2 can also promote ferroptosis resistance but does so through a distinct mechanism that appears independent of GPX4 protein expression. These results suggest that NFE2L1 and NFE2L2 independently regulate ferroptosis.
