Macrocephaly and developmental delay caused by missense variants in RAB5C.

Rab GTPases are important regulators of intracellular vesicular trafficking. RAB5C is a member of the Rab GTPase family that plays an important role in the endocytic pathway, membrane protein recycling and signaling. Here we report on 12 individuals with nine different heterozygous de novo variants in RAB5C. All but one patient with missense variants (n = 9) exhibited macrocephaly, combined with mild-to-moderate developmental delay. Patients with loss of function variants (n = 2) had an apparently more severe clinical phenotype with refractory epilepsy and intellectual disability but a normal head circumference. Four missense variants were investigated experimentally. In vitro biochemical studies revealed that all four variants were damaging, resulting in increased nucleotide exchange rate, attenuated responsivity to guanine exchange factors and heterogeneous effects on interactions with effector proteins. Studies in C. elegans confirmed that all four variants were damaging in vivo and showed defects in endocytic pathway function. The variant heterozygotes displayed phenotypes that were not observed in null heterozygotes, with two shown to be through a dominant negative mechanism. Expression of the human RAB5C variants in zebrafish embryos resulted in defective development, further underscoring the damaging effects of the RAB5C variants. Our combined bioinformatic, in vitro and in vivo experimental studies and clinical data support the association of RAB5C missense variants with a neurodevelopmental disorder characterized by macrocephaly and mild-to-moderate developmental delay through disruption of the endocytic pathway.

Loss of circRNAs from the crh-1 gene extends the mean lifespan in Caenorhabditis elegans.

Accumulation of circular RNAs (circRNAs) during aging occurs on a genome-wide level for multiple organisms, but its significance is unknown. Generating circRNA loss-of-function mutants is difficult because the vast majority of these RNAs are comprised of exons shared with protein-coding mRNAs. In Caenorhabditis elegans, most circRNAs were previously found to accumulate during aging. Two of the most abundant, age-accumulating circRNAs are generated from exon 4 of the crh-1 gene (circ-crh-1). Here, we found that the biogenesis of circ-crh-1 was regulated by the double-stranded RNA-binding protein ADR-1. We identified Reverse Complementary Match (RCM) sequences in introns flanking circ-crh-1. Using CRISPR-Cas9, we deleted the downstream RCM and found that this completely eliminated expression of the circRNA without affecting linear mRNA expression from the crh-1 gene. Remarkably, worms lacking circ-crh-1 exhibited a significantly longer mean lifespan. Lifespan was partially restored to wild type by expression of circ-crh-1 in neural tissues. Widespread transcriptome alterations in circ-crh-1 mutants were identified using RNA-Seq. Moving forward, intronic RCM deletion using CRISPR should be a widely applicable method to identify lifespan-regulating circRNAs in C. elegans.

Sleep: AMPs Mediate Injury-Induced Lethargy.

Fatigue and sleepiness are widely observed but ill-understood responses to tissue injury. A new study in Caenorhabditis elegans illuminates how the innate immune system mediates injury-induced sleep, which may help in surviving the injury.

A salt-induced kinase is required for the metabolic regulation of sleep.

Many lines of evidence point to links between sleep regulation and energy homeostasis, but mechanisms underlying these connections are unknown. During Caenorhabditis elegans sleep, energetic stores are allocated to nonneural tasks with a resultant drop in the overall fat stores and energy charge. Mutants lacking KIN-29, the C. elegans homolog of a mammalian Salt-Inducible Kinase (SIK) that signals sleep pressure, have low ATP levels despite high-fat stores, indicating a defective response to cellular energy deficits. Liberating energy stores corrects adiposity and sleep defects of kin-29 mutants. kin-29 sleep and energy homeostasis roles map to a set of sensory neurons that act upstream of fat regulation as well as of central sleep-controlling neurons, suggesting hierarchical somatic/neural interactions regulating sleep and energy homeostasis. Genetic interaction between kin-29 and the histone deacetylase hda-4 coupled with subcellular localization studies indicate that KIN-29 acts in the nucleus to regulate sleep. We propose that KIN-29/SIK acts in nuclei of sensory neuroendocrine cells to transduce low cellular energy charge into the mobilization of energy stores, which in turn promotes sleep.

Increased food intake after starvation enhances sleep in Drosophila melanogaster.

Feeding and sleep are highly conserved, interconnected behaviors essential for survival. Starvation has been shown to potently suppress sleep across species; however, whether satiety promotes sleep is still unclear. Here we use the fruit fly, Drosophila melanogaster, as a model organism to address the interaction between feeding and sleep. We first monitored the sleep of flies that had been starved for 24 h and found that sleep amount increased in the first 4 h after flies were given food. Increased sleep after starvation was due to an increase in sleep bout number and average sleep bout length. Mutants of translin or adipokinetic hormone, which fail to suppress sleep during starvation, still exhibited a sleep increase after starvation, suggesting that sleep increase after starvation is not a consequence of sleep loss during starvation. We also found that feeding activity and food consumption were higher in the first 10-30 min after starvation. Restricting food consumption in starved flies to 30 min was sufficient to increase sleep for 1 h. Although flies ingested a comparable amount of food at differing sucrose concentrations, sleep increase after starvation on a lower sucrose concentration was undetectable. Taken together, our results suggest that increased food intake after starvation enhances sleep and reveals a novel relationship between feeding and sleep.

Cell-Autonomous and Non-Cell-Autonomous Regulation of a Feeding State-Dependent Chemoreceptor Gene via MEF-2 and bHLH Transcription Factors.

Food and feeding-state dependent changes in chemoreceptor gene expression may allow Caenorhabditis elegans to modify their chemosensory behavior, but the mechanisms essential for these expression changes remain poorly characterized. We had previously shown that expression of a feeding state-dependent chemoreceptor gene, srh-234, in the ADL sensory neuron of C. elegans is regulated via the MEF-2 transcription factor. Here, we show that MEF-2 acts together with basic helix-loop-helix (bHLH) transcription factors to regulate srh-234 expression as a function of feeding state. We identify a cis-regulatory MEF2 binding site that is necessary and sufficient for the starvation-induced down regulation of srh-234 expression, while an E-box site known to bind bHLH factors is required to drive srh-234 expression in ADL. We show that HLH-2 (E/Daughterless), HLH-3 and HLH-4 (Achaete-scute homologs) act in ADL neurons to regulate srh-234 expression. We further demonstrate that the expression levels of srh-234 in ADL neurons are regulated remotely by MXL-3 (Max-like 3 homolog) and HLH-30 (TFEB ortholog) acting in the intestine, which is dependent on insulin signaling functioning specifically in ADL neurons. We also show that this intestine-to-neuron feeding-state regulation of srh-234 involves a subset of insulin-like peptides. These results combined suggest that chemoreceptor gene expression is regulated by both cell-autonomous and non-cell-autonomous transcriptional mechanisms mediated by MEF2 and bHLH factors, which may allow animals to fine-tune their chemosensory responses in response to changes in their feeding state.