Diversity and function of TRIO isoforms during human synapse development

About This Grant

ABSTRACT Alternative splicing vastly expands the functional repertoire of genes, and is especially important during human synapse development. Human synapses have unique structures and compositions, but the mechanisms involved in their development remains elusive. Investigations into human-specific aspects of synaptic machinery have revealed a critical role for Rho guanine nucleotide exchange factor (GEF) signaling. TRIO is a large multi-domain RhoGEF with essential roles in neuronal and synaptic development. TRIO is also a genome-wide significant risk factor for neurodevelopmental disorders and glutamatergic synapses have been implicated by genomic, neuropathological, and functional studies as key sites of pathogenesis. Individuals with pathogenic TRIO variants are characterized by intellectual disability, developmental delay, and seizures. The TRIO gene produces multiple distinct isoforms that differentially incorporate TRIO’s functional domains including two GEF domains that having opposing functions on the actin cytoskeleton. Thus, precise control of TRIO’s GEF domains through expression and localization of isoforms is paramount for its function. However, the diversity and expression patterns of TRIO isoforms are poorly understood, and their impact on human synapse development is unknown. A complete catalogue of isoform diversity is necessary for a holistic view of gene function, and is critical for gene therapy designs, interpreting clinical variants, and revealing novel biological mechanisms. Here, we will leverage state- of-the-art long-read sequencing, iPSC-models and fluorescence imaging to characterize human TRIO isoforms. Our central hypothesis is that TRIO produces an array of isoforms with distinct expression patterns and domain architectures that assist with the precise timing and spatial control of synaptic development. In Aim 1, we will combine exon capture and long-read sequencing technologies to systematically profile full-length TRIO transcripts in the human brain. We will create comprehensive isoform maps at three postnatal time points, and analyze novel isoforms for differential expression, presence of clinical variants, and protein motifs. In Aim 2, human iPSC-derived neuron models will be utilized to assess the localization and functional roles of individual isoforms in synaptic compartments. Together, this work will reveal a high-resolution map of the structure, expression and function of TRIO isoforms in the human brain, providing mechanistic insight into human synapse development and TRIO-related neurodevelopmental disorders.