Proximity Analysis of Native Proteomes Reveals Interactomes Predictive of Phenotypic Modifiers of Autism and Related Neurodevelopmental Conditions

Abstract

One of the main drivers of autism spectrum disorder (ASD) are risk alleles within hundreds of genes, which may interact within shared biological processes through as-yet unclear mechanisms. Here we develop a high-throughput genome-editing-mediated approach to target 14 high-confidence ASD genes within the mouse brain for proximity-based proteomics of endogenous interactomes. The resulting interactomes are enriched for human genes dysregulated in the brain of ASD patients and reveal unexpected, but highly significant, interactions with other lower confidence ASD-risk gene products, positing new avenues to prioritize genetic risk. Importantly, the datasets are enriched for shared cellular functions and genetic interactions that may underlie the disorder. We test this notion by spatial proteomics and CRISPR-based regulation of expression in two ASD models, demonstrating new functional interactions that modulate mechanisms of their dysregulation. Together, our results reveal native protein-interaction networks in ASD, providing new inroads for understanding its cellular neurobiology.

Synaptic Signatures and Disease Vulnerabilities of Layer 5 Pyramidal Neurons

“…Short biotinylation windows also open new possibilities, for example to study how the composition of synaptic proteomes changes upon neuronal activity. For similar reasons, we limited the duration of expression of turboID constructs to 1 week (compared to multiple weeks or months in previous studies 37,83,[85][86][87] ), as low levels of protein biotinylation using endogenous biotin can occur while turboID is expressed.…”

“…This strategy is suitable for mature circuits but carries a risk of driving precocious postsynaptic maturation in developing neurons. CRISPR/Cas9-mediated knock-in approaches 86 might be one alternative. These approaches avoid the overexpression of exogenous proteins, however, the low percentage of successful recombination makes it challenging to target single neuronal populations in specific cortical regions 86,92 .…”

Synaptic proteomics decode novel molecular landscape in the brain

“…Therefore, a comparative analysis of cell-type-specific synaptic proteomes is becoming increasingly essential in understanding the diverse pathogenesis of many neurological disorders. A large-scale interactome analysis of 14 risk genes including synaptic genes associated with ASD, using TurboID, has revealed a wealth of common cellular functions and molecular interactions related to the pathomechanisms of ASD ( Gao et al, 2023 ). This dataset is expected to contribute to the establishment of new diagnostic and therapeutic methods targeting common molecules in ASD pathology.…”

“…Recently, the proteome of axon initial segment (AIS) has been detected using Neurofascin-HRP ( Ogawa et al, 2023 ) ( Figure 1B ; Table 1 ). iBioID has also been applied to extensive interactome analyses of molecules associated with autism spectrum disorders (ASD) ( Gao et al, 2023 ) ( Table 1 ). In this study, a high-throughput genome-editing approach was used to target 14 high-confidence ASD genes ( Anks1b , Syngap1 , Shank2 , Shank3 , Nckap1 , Nbea , Ctnnb1 , Lrrc4c , Iqsec2 , Arhgef9 , Ank3 , Scn2a , Scn8a , and Hnrnpu ) for the insertion of a highly active mutant biotin ligase TurboID.…”

“…Comparative interactome analysis revealed a significant overlap of protein interactions among the targeted genes, with enrichment for ASD-related cellular functions. Additionally, this genome-editing mediated iBioID approach enables the investigation of native proteomes without relying on any overexpression methods in vivo ( Gao et al, 2023 ). Therefore, these approaches will enable comprehensive analyses of synaptic-related molecules in physiological states and pathological conditions that lead to neurological disorders.…”