Investigating Autism-Related Gut Dysfunction with Human Enteric Neurons and Intestinal Organoids

About This Grant

Project Summary Gastrointestinal (GI) disorders are among the most common comorbidities in patients with Autism Spectrum Disorder (ASD). The Enteric Nervous System (ENS), composed of neurons (ENs) and glia, is crucial in regulating various aspects of gut physiology. Animal models show GI motility impairments linked to altered expression of ASD-associated genes. However, recent advancements in single-cell genomic technologies have revealed remarkable molecular diversity among ENs and highlighted significant differences in ENS gene expression patterns across species. These findings underscore the need for human-specific models to recapitulate the human ENS molecular heterogeneity and dissect the cell type-specific contribution to the GI endophenotype in ASD. Under the mentorship of Dr. Giorgia Quadrato and Dr. Jason Spence, leaders in the field of the human neural and intestinal organoids, respectively, Dr. Birtele will use a human induced pluripotent stem cell (iPSC)- derived model that includes both ENs and intestinal organoids (HIOs). Using a mix-and-match approach, patient- derived neurons co-cultured with healthy intestinal cells will isolate ENS-specific contributions to GI dysfunction. Conversely, healthy neurons cultured with patient-derived intestinal organoids will reveal non-neuronal contributions. Aim 1 (K99 phase) will study the role of SYNGAP1, a top ASD gene, in GI dysfunction. ENs will be derived from a SYNGAP1 haploinsufficient-patient derived and isogenic control iPSCs line under the mentorship of Dr. Martin Garcia-Castro, expert in neural crest differentiations. Under the guidance of Dr. Jason Spence, Dr. Birtele will generate mixed and matched ENs-HIOs. Dr. Birtele will analyze mixed and matched ENs-HIOs to determine cellular and transcriptional changes caused by SYNGAP1 haploinsufficiency. In Dr. Spence's lab, Dr. Birtele will transplant ENs and ENs-HIOs in vivo to assess GI motility and peristaltic function. Additionally, under the mentorship of Dr. Unmesh Jadhav, an expert in epigenomics and intestinal stem cells, Dr. Birtele will examine the effect of SYNGAP1 haploinsufficiency on intestinal stem cell chromatin accessibility profiles by performing single-cell ATAC-seq on mixed and matched ENs-HIOs. Given the high comorbidity of GI dysfunction across many genetic forms of ASD and the enrichment in expression of these genes in ENs, Aim 2 (R00 phase) I will perform an high-throughput screening for molecular and functional impairments in ENs cultures by applying gapmer antisense oligonucleotides (ASOs) under the guidance of Dr. Justin Ichida, leader in the field of ASOs, to knock-out 35 top ASD-associated genes.Top candidates identified in this initial screen will be validated using patient-derived lines differentiated into ENs and HIOs and cultured following the mix-and-match approach. By applying a similar pipeline of experimental procedures as in Aim1, I will compare the functional and molecular profiles of in vitro and transplanted organoids to dissect possible convergent molecular mechanisms through which ASD-associated genes contribute to GI dysfunction This research will uncover molecular mechanisms governing ENs function and provide critical insights into ASD-related GI dysfunction. 1