Functional assessment of homozygous ALDH18A1 variants reveals alterations in amino acid and antioxidant metabolism.

Mono- and bi-allelic variants in ALDH18A1 cause a spectrum of human disorders associated with cutaneous and neurological findings that overlap with both cutis laxa and spastic paraplegia. ALDH18A1 encodes the bifunctional enzyme pyrroline-5-carboxylate synthetase (P5CS) that plays a role in the de novo biosynthesis of proline and ornithine. Here we characterize a previously unreported homozygous ALDH18A1 variant (p.Thr331Pro) in four affected probands from two unrelated families, and demonstrate broad-based alterations in amino acid and antioxidant metabolism. These four patients exhibit variable developmental delay, neurological deficits and loose skin. Functional characterization of the p.Thr331Pro variant demonstrated a lack of any impact on the steady-state level of the P5CS monomer or mitochondrial localization of the enzyme, but reduced incorporation of the monomer into P5CS oligomers. Using an unlabeled NMR-based metabolomics approach in patient fibroblasts and ALDH18A1-null human embryonic kidney cells expressing the variant P5CS, we identified reduced abundance of glutamate and several metabolites derived from glutamate, including proline and glutathione. Biosynthesis of the polyamine putrescine, derived from ornithine, was also decreased in patient fibroblasts, highlighting the functional consequence on another metabolic pathway involved in antioxidant responses in the cell. RNA sequencing of patient fibroblasts revealed transcript abundance changes in several metabolic and extracellular matrix-related genes, adding further insight into pathogenic processes associated with impaired P5CS function. Together these findings shed new light on amino acid and antioxidant pathways associated with ALDH18A1-related disorders, and underscore the value of metabolomic and transcriptomic profiling to discover new pathways that impact disease pathogenesis.

The Drosophila brain on cocaine at single-cell resolution.

Whereas the neurological effects of cocaine have been well documented, effects of acute cocaine consumption on genome-wide gene expression across the brain remain largely unexplored. This question cannot be readily addressed in humans but can be approached using the Drosophila melanogaster model, where gene expression in the entire brain can be surveyed at once. Flies exposed to cocaine show impaired locomotor activity, including climbing behavior and startle response (a measure of sensorimotor integration), and increased incidence of seizures and compulsive grooming. To identify specific cell populations that respond to acute cocaine exposure, we analyzed single-cell transcriptional responses in duplicate samples of flies that consumed fixed amounts of sucrose or sucrose supplemented with cocaine, in both sexes. Unsupervised clustering of the transcriptional profiles of a total of 86,224 cells yielded 36 distinct clusters. Annotation of clusters based on gene markers revealed that all major cell types (neuronal and glial) as well as neurotransmitter types from most brain regions were represented. The brain transcriptional responses to cocaine showed profound sexual dimorphism and were considerably more pronounced in males than females. Differential expression analysis within individual clusters indicated cluster-specific responses to cocaine. Clusters corresponding to Kenyon cells of the mushroom bodies and glia showed especially large transcriptional responses following cocaine exposure. Cluster specific coexpression networks and global interaction networks revealed a diverse array of cellular processes affected by acute cocaine exposure. These results provide an atlas of sexually dimorphic cocaine-modulated gene expression in a model brain.

Functional Diversification, Redundancy, and Epistasis among Paralogs of the Drosophila melanogaster Obp50a-d Gene Cluster.

Large multigene families, such as the insect odorant-binding proteins (OBPs), are thought to arise through functional diversification after repeated gene duplications. Whereas many OBPs function in chemoreception, members of this family are also expressed in tissues outside chemosensory organs. Paralogs of the Obp50 gene cluster are expressed in metabolic and male reproductive tissues, but their functions and interrelationships remain unknown. Here, we report the genetic dissection of four members of the Obp50 cluster, which are in close physical proximity without intervening genes. We used CRISPR technology to excise the entire cluster while introducing a PhiC31 reintegration site to reinsert constructs in which different combinations of the constituent Obp genes were either intact or rendered inactive. We performed whole transcriptome sequencing and assessed sexually dimorphic changes in transcript abundances (transcriptional niches) associated with each gene-edited genotype. Using this approach, we were able to estimate redundancy, additivity, diversification, and epistasis among Obp50 paralogs. We analyzed the effects of gene editing of this cluster on organismal phenotypes and found a significant skewing of sex ratios attributable to Obp50a, and sex-specific effects on starvation stress resistance attributable to Obp50d. Thus, there is functional diversification within the Obp50 cluster with Obp50a contributing to development and Obp50d to stress resistance. The deletion-reinsertion approach we applied to the Obp50 cluster provides a general paradigm for the genetic dissection of paralogs of multigene families.

Pleiotropic fitness effects of a Drosophila odorant-binding protein.

Insect odorant-binding proteins (OBPs) are members of a rapidly evolving multigene family traditionally thought to facilitate chemosensation. However, studies on Drosophila have shown that members of this family have evolved functions beyond chemosensation, as evident from their expression in reproductive tissues and the brain. Previous studies implicated diverse functions of Obp56h, a member of the largest gene cluster of the D. melanogaster Obp repertoire. Here, we examined the effect of CRISPR/Cas9-mediated deletion of Obp56h on 2 fitness phenotypes, on resistance to starvation stress and heat stress, and on locomotion and sleep phenotypes. Obp56h-/- mutants show a strong sexually dimorphic effect on starvation stress survival, with females being more resistant to starvation stress than the control. In contrast, Obp56h-/- females, but not males, are highly sensitive to heat stress. Both sexes show changes in locomotion and sleep patterns. Transcriptional profiling of RNA from heads of Obp56h-/- flies and the wildtype control reveals differentially expressed genes, including gene products associated with antimicrobial immune responses and members of the Turandot family of stress-induced secreted peptides. In addition, differentially expressed genes of unknown function were identified in both sexes. Genes encoding components of the mitochondrial electron transport chain, cuticular proteins, gene products associated with regulation of feeding behavior (Lst and CCHa2), ribosomal proteins, lncRNAs, snoRNAs, tRNAs, and snRNAs show changes in transcript abundances in Obp56h-/- females. These differentially expressed genes are likely to contribute to Obp56h-mediated effects on the diverse phenotypes that arise upon deletion of this OBP.

Tracer metabolomics reveals the role of aldose reductase in glycosylation.

Abnormal polyol metabolism is predominantly associated with diabetes, where excess glucose is converted to sorbitol by aldose reductase (AR). Recently, abnormal polyol metabolism has been implicated in phosphomannomutase 2 congenital disorder of glycosylation (PMM2-CDG) and an AR inhibitor, epalrestat, proposed as a potential therapy. Considering that the PMM2 enzyme is not directly involved in polyol metabolism, the increased polyol production and epalrestat's therapeutic mechanism in PMM2-CDG remained elusive. PMM2-CDG, caused by PMM2 deficiency, presents with depleted GDP-mannose and abnormal glycosylation. Here, we show that, apart from glycosylation abnormalities, PMM2 deficiency affects intracellular glucose flux, resulting in polyol increase. Targeting AR with epalrestat decreases polyols and increases GDP-mannose both in patient-derived fibroblasts and in pmm2 mutant zebrafish. Using tracer studies, we demonstrate that AR inhibition diverts glucose flux away from polyol production toward the synthesis of sugar nucleotides, and ultimately glycosylation. Finally, PMM2-CDG individuals treated with epalrestat show a clinical and biochemical improvement.

Developmental Alcohol Exposure in Drosophila: Effects on Adult Phenotypes and Gene Expression in the Brain.

Fetal alcohol exposure can lead to developmental abnormalities, intellectual disability, and behavioral changes, collectively termed fetal alcohol spectrum disorder (FASD). In 2015, the Centers for Disease Control found that 1 in 10 pregnant women report alcohol use and more than 3 million women in the USA are at risk of exposing their developing fetus to alcohol. Drosophila melanogaster is an excellent genetic model to study developmental effects of alcohol exposure because many individuals of the same genotype can be reared rapidly and economically under controlled environmental conditions. Flies exposed to alcohol undergo physiological and behavioral changes that resemble human alcohol-related phenotypes. Here, we show that adult flies that developed on ethanol-supplemented medium have decreased viability, reduced sensitivity to ethanol, and disrupted sleep and activity patterns. To assess the effects of exposure to alcohol during development on brain gene expression, we performed single cell RNA sequencing and resolved cell clusters with differentially expressed genes which represent distinct neuronal and glial populations. Differential gene expression showed extensive sexual dimorphism with little overlap between males and females. Gene expression differences following developmental alcohol exposure were similar to previously reported differential gene expression following cocaine consumption, suggesting that common neural substrates respond to both drugs. Genes associated with glutathione metabolism, lipid transport, glutamate and GABA metabolism, and vision feature in sexually dimorphic global multi-cluster interaction networks. Our results provide a blueprint for translational studies on alcohol-induced effects on gene expression in the brain that may contribute to or result from FASD in human populations.