Project Summary Strong, longstanding evidence points to important roles for the cerebellum in autism spectrum disorders (ASDs), yet cerebellar mechanisms remain understudied in ASDs compared to neocortical circuits. Anatomical and functional studies have pointed, in particular, to changes in the synapses of Purkinje cells, the sole output neurons of the cerebellum. Purkinje cells were reduced both in number and size in ASD cases, particularly in the cerebellar vermis and other sub-regions involved in cognition and emotional control. Genes localized to Purkinje cell synapses were down-regulated. Purkinje cell-specific conditional knockout mice for several ASD risk genes suggest direct effects of these cerebellar neurons on ASD-related behaviors and synaptic structure and function. However, cell type-specific transcriptional and epigenomic changes in the cerebellum of individuals with ASDs remain poorly characterized, and it is unknown whether the synaptic features identified in mouse models translate to the human condition. Here, we propose comprehensive multi-omic and synaptic imaging studies to address these knowledge gaps, utilizing a unique post-mortem brain tissue resource from the University of Maryland Baltimore Brain and Tissue Bank. We will generate single-nuclei multi-omic profiles of gene expression and chromatin accessibility in ~1.5 million cells from 100 ASD cases and 100 controls. In the same brain tissue samples, we will perform super-resolution confocal imaging to quantify the density, size, and nanostructure of Purkinje cell synapses. ~50% of the donors in our cohort will have known causal mutations, including multiple cases with tuberous sclerosis complex (TSC), Rett syndrome, and Fragile X syndrome, enabling us to define shared vs. unique features of ASDs with mutations in different genes. Key findings will be validated in a mouse model of TSC, Tsc1 conditional knockout mice, enabling us to determine which transcriptomic and synaptic phenotypes ar