Project summary / Abstract Autism spectrum disorder (ASD) is a neurodevelopmental disorder whose underlying mechanisms have been proposed to include abnormal excitatory-inhibitory balance in brain regions including the cerebral cortex. The ATP-dependent chromatin-remodeling factor chromodomain helicase binding protein 8 (CHD8) is one of the most commonly-mutated genes in sporadic ASD, producing an ASD subtype associated with a high prevalence of macrocephaly (Barnard et al., Front Neurosci., 2015). In prior work supported by the original grant, we optimized a human cortical organoid model we developed (Velasco et al., Nature, 2019; Uzquiano et al., Cell, 2022) and applied it to show that CHD8 and other ASD risk genes converge on an early developmental defect in the GABAergic neuron lineage, leading to asynchronous development of target neurons relative to the other cell types developing in the organoids (Paulsen et al., Nature, 2022). From this data, we hypothesized that asynchronous development of GABAergic neurons relative to the excitatory neurons they wire with could lead to later abnormalities in the activity and function of the cortical local circuit. In support of this hypothesis, we showed that mutation in another ASD risk gene, SUV420H1, also associated with accelerated development of GABAergic neurons, leads to abnormal activity of mutant circuits in organoids (Paulsen et al., Nature, 2022). Building on this data, in this renewal we will leverage our expertise in organoid biology and cortical development together with co-Investigator Mark Harnett's expertise in human cortical physiology to investigate the effect of heterozygous loss-of-function of CHD8 on neuronal development and circuit activity using newly-developed organoid models. Firstly, we will apply a chimeric organoid (“chimeroid”) model (Antón Bolaños and Faravelli et al., preprint at bioRxiv 2023, and in revision at Nature) to understand whether the genetic context of ASD patients is permissive for expressivity of the previously-identified CHD8+/- phenotypes (Aim 1). In parallel, we will apply a new human organoid model of the ventral telencephalon, which we validated to be able to produce GABAergic neurons of the caudal, medial, and lateral ganglionic eminences (Sartore et al., manuscript in preparation), to understand whether specific classes of GABAergic interneurons are preferentially affected by CHD8 mutation (Aim 2). Finally, with the aim of promoting circuits that reflect the endogenous neuronal composition, we will apply a new “dorsal-ventral chimeroid” organoid model that allows us to control development of the correct proportions of both excitatory and inhibitory cortical neurons, to investigate cell identity and circuit activity in the CHD8 heterozygous mutant in a more physiologically-relevant system, using calcium imaging, extracellular recording, and pharmacological manipulations (Aim 3). Taken together, this work will provide mechanistic understanding of the ro...