Abstract
Mutations in Chromodomain Helicase DNA Binding Protein 8 (CHD8) are the most common de novo mutations associated with autism spectrum disorder (ASD). CHD8 is a chromatin modifier that influences the transcription of many other ASD-risk genes; thus, it is regarded as a master regulator and defines a common ASD subtype characterized by macrocephaly and gastrointestinal (GI) problems. The GI phenotypes caused by CHD8 mutations have not been completely explored. In particular, how CHD8 impacts GI phenotypes at the cellular level, and if changes to the gut play a role in behavioral phenotypes associated with ASD. Our first goal with this project was to determine if CHD8 affects three specific GI phenotypes—gut microbiota composition, GI transit time, and gut length—in Drosophila melanogaster with a null allele of kismet (kis), a functionally conserved ortholog of CHD8. Our second goal was to determine if the kis mutant gut microbiome affects GI transit time and behavior. To compare the gut microbiomes of kis mutant and wild-type Drosophila, we isolated DNA from dissected midguts and used 16S rRNA sequencing. Our results indicate that the microbial composition of the guts are distinct in both anterior and posterior midguts, and the relative abundance of microbiota differs across the two genotypes at the genus and species level. This is the first research to our knowledge in which the gut microbiome of Drosophila with a mutation in an autism risk gene has been characterized. To study GI transit time, food containing a dye tracer was fed to wild-type and kis mutant flies with and without antibiotic-depleted gut microbiomes. We measured the time from consumption of food to excretion. We found that kis mutant flies had a significantly reduced GI transit time compared to wild-type and antibiotic depletion of the gut microbiome rescued mutant GI transit time. Since a difference in gut length could cause a difference in GI transit time, gut length was compared between kis mutant and wild-type flies and there was no significant difference. To assess behavior, we measured the feeding latency and naïve courtship behavior or kis mutant and wild-type flies with and without antibiotic-depleted microbiomes. The feeding latency assay revealed that kis mutants had a significantly shorter feeding latency than wild-type flies, and antibiotic depletion of the gut microbiomes significantly increased the feeding latency in both wild-type and kis mutant flies. The increased feeding latency in kis mutant flies with the antibiotic-depleted gut microbiome was significantly longer than the untreated wild-type flies; thus, we did not observe a rescue effect. In addition, the increased feeding latency in the antibiotic-treated wild-type flies was significantly longer than the antibiotic treated kis mutants. The kis mutants had significantly impaired courtship behavior compared to wild-type flies. Antibiotic depletion of the kis mutant gut microbiome rescued mutant courtship behavior, but significantly impaired wild-type courtship behavior. Our results demonstrate an ASD risk gene-specific impact of antibiotic depletion of the gut microbiome and implicate the kis mutant gut microbiome in impaired GI transit time, feeding latency, and courtship behaviors. These results suggest the gut microbiome may play a role in the pathophysiology of CHD8-associated ASD.