Systematic phenomics analysis of autism-associated genes reveals parallel networks underlying reversible impairments in habituation

Abstract

A major challenge facing the genetics of autism spectrum disorders (ASDs) is the large and growing number of candidate risk genes and gene variants of unknown functional significance. Here, we usedCaenorhabditis elegansto systematically functionally characterize ASD-associated genes in vivo. Using our custom machine vision system, we quantified 26 phenotypes spanning morphology, locomotion, tactile sensitivity, and habituation learning in 135 strains each carrying a mutation in an ortholog of an ASD-associated gene. We identified hundreds of genotype–phenotype relationships ranging from severe developmental delays and uncoordinated movement to subtle deficits in sensory and learning behaviors. We clustered genes by similarity in phenomic profiles and used epistasis analysis to discover parallel networks centered onCHD8•chd-7andNLGN3•nlg-1that underlie mechanosensory hyperresponsivity and impaired habituation learning. We then leveraged our data for in vivo functional assays to gauge missense variant effect. Expression of wild-type NLG-1 innlg-1mutantC. elegansrescued their sensory and learning impairments. Testing the rescuing ability of conserved ASD-associated neuroligin variants revealed varied partial loss of function despite proper subcellular localization. Finally, we used CRISPR-Cas9 auxin-inducible degradation to determine that phenotypic abnormalities caused by developmental loss of NLG-1 can be reversed by adult expression. This work charts the phenotypic landscape of ASD-associated genes, offers in vivo variant functional assays, and potential therapeutic targets for ASD.

Neuroligin dependence of social behaviour inC. elegansprovides a model to investigate an autism associated gene

“…Previous studies have used C. elegans morphology and locomotion as readouts of disrupted behaviour in response to mutation to ASD associated genes (31, 32). We have extended this analysis to inform on the effect of a neuroligin variant on a phenotype that relates to one of the triad of impairments that make up the diagnostic criteria of ASD.…”

Peel-1negative selection promotes screening-free CRISPR-Cas9 genome editing inCaenorhabditis elegans

“…For all Multi-Worm Tracker experiments 4-6 plates (20-100 worms/plate) were run for each strain. The animals were maintained in a 20°C incubator for 96 hours prior to testing [42].…”

“…Our behavioral paradigm consisted of a 5-minute period to recover from being placed on the tracker followed by a 5 min baseline period from which we computed multiple measures of morphology and baseline locomotion ( Fig 4E ) [42]. Beginning at 10 minutes we administered 30 mechanosensory stimuli to the Petri plate holding the animals at a 10 second interstimulus interval (ISI) using an automated push solenoid ( Fig 4A ).…”

“…With repeated stimulation there is a decrease in the likelihood of a reversal, as well as the duration, speed, and distance of reversals (habituation learning; S1 Fig ). Following habituation training, we allowed a 5-minute recovery period after which we administered a 31 st stimulus to gauge spontaneous recovery from short-term habituation - an assay of short-term memory retention [42].…”

“…Arousal was defined as the increased mean absolute movement speed in the period following mechanosensory stimulation and prior to the delivery of the spontaneous recovery stimulus ( Fig 4E & F ; 600-1189 seconds). Custom R scripts organized and summarized Choreography output files [42]. No blinding was necessary because the Multi-Worm Tracker scores behavior objectively.…”