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Abstract
Autism Spectrum Disorder (ASD) is a neurodevelopmental condition with a complex genetic basis. ASD is characterized by social impairments, repetitive behaviors, and sensory processing alterations that present as early as age 2 and develop across childhood and adolescence. While genomic studies consistently implicate synaptic biology, the molecular mechanisms underlying ASD remain elusive. This study employs proteomic analyses to investigate these synaptic protein networks across postnatal development in postmortem ASD cortical tissue. To account for potential postmortem degradation effects, we first conducted a deep and systematic postmortem interval study in the murine brain via quantitative mass spectrometry. We demonstrated that protein stability varies by domain but can be accounted for through linear modeling, ensuring the reliability of our human postmortem findings. We then analyzed bulk homogenate and synaptosome-enriched fractions from postmortem primary visual cortex samples of ASD and neurotypical (NT) individuals aged 4 to 32 years. Our findings reveal widespread differential protein expression by diagnosis, with distinct patterns of dysregulation between homogenate and synaptic fractions. ASD brains exhibited increased expression of proteins involved in synaptic vesicle cycling, lipid raft organization, and cell adhesion, while proteins associated with voltage-gated channels, mitochondrial metabolism, and proteostasis were downregulated. Developmental trajectory analyses revealed dynamic shifts in synaptic protein levels, with synaptic vesicle cycling proteins initially upregulated in early postnatal life before declining, whereas metabolic proteins followed an inverse trend. Notably, a cluster of synaptic vesicle-associated proteins was found to be tightly co-regulated with vesicular glutamate transporter 1 (vGlut1), suggesting excessive synaptic vesicle number or glutamate load as a potential mechanism of excitatory neurotransmission imbalance in early development of ASD subjects. Finally, to facilitate future proteomic studies of postmortem brain tissue with increased spatial resolution, we developed and validated an antibody-based proximity labeling method to map synaptic subcompartments in human postmortem tissue. We demonstrated this tools utility to capture the vGlut1 proximal proteome, providing a novel approach for high-resolution spatial proteomics. Collectively, our findings contribute to a deeper understanding of the molecular basis of ASD as well as facilitate spatially resolved proteomic investigations of autism and other psychiatric diseases at the compartment level in postmortem brain.