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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. Author summary: NGLY1 deficiency is a rare disease with no effective treatment. We conducted a drug repurposing screen and used the Connectivity Map, a transcriptional-based computational approach, to identify compounds that may serve as therapeutics for NGLY1 deficient individuals. The drug repurposing screen identified FDA-approved compounds acting through the serotonin and dopamine pathway that partially rescued lethality in an NGLY1 deficiency fly model. We also found that expressing dNGLY1 (the Drosophila ortholog of NGLY1) exclusively in serotonin neurons, in an otherwise dNGLY1 deficient fly, partially rescued lethality. These data indicate the importance of the serotonin and dopamine systems in NGLY1 deficiency. The Connectivity Map analyses found GSK3 inhibitors as potential therapeutic compounds, which were validated in vivo in the fly. Furthermore, knockdown of sgg (the Drosophila ortholog of GSK3) partially rescued lethality in dNGLY1 deficient flies, suggesting GSK3 as a therapeutic target for NGLY1 deficiency. Taken together, this work identifies therapeutic strategies for NGLY1 deficiency. [ABSTRACT FROM AUTHOR]

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 causes CDGs. While both in vivo models ostensibly cause cellular 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. Author summary: Sugar modifications are important to proper protein folding and function. The biological pathways that control these sugar modifications are called glycosylation pathways. When these pathways are disrupted, such as through genetic mutation, it can cause rare developmental disorders called Congenital Disorders of Glycosylation (CDGs). Most CDGs lack any treatment options, and little is known about interacting genes or pathways that might improve these disorders. Here, we study DPAGT1-CDG which involves mutations in the enzyme-encoding gene, DPAGT1, by using the fruit fly model organism, Drosophila melanogaster. Using inhibitory drugs, we perform two cell-based screens with CRISPR technology to determine which genes are important under DPAGT1 inhibition. We then used a living fly, that models DPAGT1-CDG in the eye, in order to determine which of the top screened genes can improve its impaired eye development. Surprisingly, we found that inhibiting other CDG-causing genes, such as our top hit Dpm1, could improve our DPAGT1-CDG cell and fly models. In addition, loss of certain metabolism genes, and changes in diet, also improved the DPAGT1-CDG model. This study finds multiple new genes and pathways implicated in DPAGT1-CDG and lays the groundwork for new CDG therapies. [ABSTRACT FROM AUTHOR]