Resolving the relationship between mitochondrial dysfunction and the impact of non-syndromic autism spectrum disorder-related risk genes on neuronal structure and function

Despite great progress has been made in identifying hundreds of risk genes in autism spectrum disorders (ASD), we know little about modifiers that may exacerbate or ameliorate disease severity and thus can explain this highly heterogenous and extremely complex condition. Here we put forward the innovative “two-hit” hypothesis that posits that coexpression of molecular variants in individual risk genes associated with non- syndromal autism (“hit one”) and molecular variants underlying suboptimal mitochondrial function i.e., impaired neuronal bioenergetics (“hit two”) leads to impaired neurodevelopment, synaptic plasticity, and neuronal network phenotypes explaining the etiology of ASD. First, we will establish “two-hit” hiPSC lines, presenting with impaired neuronal bioenergetics caused by 30% of mtDNA heteroplasmy and deficiency in PPP2R5D or SHANK3 or GRIN2B autism relevant risk genes. Next, we will test our hypothesis in two specific aims. In specific aim 1, we will resolve the relationship between suboptimal mitochondrial function and the impact of molecular genetic defect in PPP2R5D, SHANK3 or GRIN2B genes on neuronal development and synaptic plasticity. We will image “two hit” iNeurons and 3D brain organoids to reconstruct neurons and neuronal networks as well as quantify soma size and dendritic morphology. We will also assess the number of synapses by quantifying presynaptic Synapsin 1/2 puncta on MAP2-positive dendrites. Finally, in 3D brain organoids, we will assess the production of radial glial cells and the production of cortical neuron subtypes expressing markers found in cortical layers. In specific aim 2, we will delineate that neuronal network-level phenotypes observed when disease-associated autism risk variants (PPP2R5D, SHANK3 or GRIN2B ) are expressed in neurons with suboptimal mitochondrial function. We will first examine and compare the spontaneous activity of neuronal networks, assess difference in the level and pattern of synchronous activity, neuronal network burst firing rate, mean firing rate, burst duration, inter-burst intervals, and the percentage of random spikes using multiple electrode array assays. This innovative hypothesis reinterprets the complexity of the genetics and pathophysiology of ASD in the context of neuronal bioenergetics. The proposed studies will also establish improved disease relevant in vitro neuronal models that will (A) overcome current limitations, (B) facilitate the development of novel testable etiological theories and (C) provide insights into disease modifiers, a knowledge that is currently lacking. Project Number: 1R21HD113986-01 | Fiscal Year: 2025 | NIH Institute/Center: Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) | Principal Investigator: Laszlo Kozicz | Institution: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI, NEW YORK, NY | Award Amount: $253,500 | Activity Code: R21 | Study Section: Developmental Brain Disorders Study Section[DBD] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21HD11398601