PROJECT SUMMARY
Genetic variants in the TRIO gene increase risk for neurodevelopmental disorders (NDDs) including
schizophrenia, autism, and related disorders. TRIO encodes a large protein with two guanine nucleotide
exchange factor (GEF) domains for Rho family GTPases: GEF1 activates Rac1 and RhoG, and GEF2 activates
RhoA. We found a cluster of variants associated with autism and intellectual disability that selectively activate or
inhibit TRIO GEF1 activity. While our findings highlight the central importance of this enzyme activity for proper
brain development, the molecular mechanisms by which TRIO GEF1 activity is regulated, the downstream
targets of TRIO GEF1 signaling, and how these processes are disrupted by GEF1-targeting variants remain
fundamental, yet unresolved questions. Answering them will reveal how variants in TRIO lead to NDDs and may
inform new therapeutic interventions. Our proposal will address these questions in three Aims:
Aim 1. To elucidate the mechanism of TRIO GEF1 activation. We discovered that spectrin repeats 6-9 in
TRIO bind and autoinhibit its GEF1 activity and that NDD-associated variants in spectrin repeat 8 relieve this
autoinhibition. A short list of receptors and kinases has been identified as known or likely TRIO GEF1 regulators,
but the mechanisms by which these activators engage TRIO to activate GEF1 activity are unclear. We will use
purified recombinant proteins to test how these receptors’ cytoplasmic domains and kinases impact TRIO GEF1
activity. We will also use a FRET-based activity biosensor and morphological measurements to reveal how these
mechanisms contribute to Rac1/RhoG activation and neuronal development induced by receptor activation.
Aim 2. To identify and characterize the neuronal signaling events regulated by TRIO GEF1 activity. We
have generated mice bearing TRIO variant alleles with reduced (K1431M) or elevated (R1078Q) TRIO GEF1
activity. We will use comparative proteomics and phospho-proteomics in samples from wild-type mice versus
those bearing TRIO GEF1-inhibiting or activating alleles to identify proteins, signaling events, and TRIO-
interaction partners impacted by changes in TRIO GEF1 activity. We will systematically test how manipulation
of these GEF1-mediated events impacts neuronal development and synaptic connectivity.
Aim 3. To measure how selective changes in TRIO GEF1 activity impact neuronal development and
synaptic transmission. Heterozygosity for the GEF1-defective TRIOK1431M allele causes reduced brain size
and behavioral defects, consistent with our hypothesis that selective alterations in TRIO GEF1 activity
compromise normal neuronal development and synaptic function. We will use quantitative histopathology and
electron microscopy in mice bearing the K1431M and R1078Q variants to reveal how altered TRIO GEF1 activity
impacts axon, dendritic arbor, and synapse development. Whole-cell electrophysiology and optogenetic
manipulation will enable us to identify the consequences of changes in TRIO GEF1 activity on neuronal
excitability, synaptic function, and circuit connectivity.