Jason Aoto - US grants

DESCRIPTION (provided by applicant): Increasing evidence suggests that abnormalities in synaptic transmission may be a key mechanism underlying some neuropsychiatric disorders. How might synaptic properties be specified an maintained? Synaptic cell adhesion molecules (SCAMs) are primary candidates that play a critical role regulating synapse function. Not surprisingly, genomic studies have found mutations in neurexin-3 (Nrx3) a prototypical presynaptic SCAM that is part of the neurexin/neuroligin complex. that are linked to Schizophrenia (SZ) and reward-seeking behavior. These disorders are associated with an enormous social and economic burden and share a common pathophysiological basis - dopamine system dysregulation due to abnormalities in synaptic transmission in the ventral subiculum (vSub) within the vSub-nucleus accumbens (NAc) shell circuit. Two distinct populations of subicular projection neurons serve as the major output of the hippocampus, receive input from CA1 and project to cortical and subcortical regions, including to two distinct medium spiny interneuron (MSN) cell- types in the NAc shell. A fundamental understanding how Nrx3 shapes cell-type specific synaptic properties at two subicular synapses involved in DA dysregulation - CA1 input to the vSub and subicular output to the NAc shell - is unexplored; thus, the dissection of cell-type specific pre- and post-synaptic functions of subicular neurons within this disease circuit may open new avenues for treatment strategies. To this end, we generated a Nrx3 mouse where splice site 4 (SS4), the splice site that dictates binding to nearly all neurexin ligands, is constitutively included (SS4+) but can be conditionally excluded (SS4-). We observed a selective reduction in AMPAR synaptic transmission, due to reduced surface AMPAR stability, in the dorsal subiculum of Nrx3SS4+ mice. Altered transsynaptic interactions in the Nrx3SS4 mouse may imitate disease-related Nrx3 mutations because most mutations affect surface exposed residues and alternative splicing - rarely resulting in complete protein loss. The overarching goal of this grant is to dissect presynaptic Nrx3SS4-dependent functions in distinct cell-types at the CA1-vSub synapse and the vSub-NAc shell synapse. The mentored aims will 1.) assess cell-type specific synaptic responses in vSub neurons that project to the NAc shell at the CA1-vSub synapse using stereotactic co-injection of retrograde virus and cre-recombinase in WT and Nrx3SS4 animals and 2.) assay vSub projections to two distinct populations of NAc shell MSNs in the striatal circuit by utilizing stereotactic injection of channelrhodopsin to selectively recruit ventral subicular fibers. For the R00 phase of this grant, will 1.) expand the study of synapse-specific striatal circuitry by dissecting the dorsal Sub-NAc core circuit using techniques acquired during the K99 phase and 2.) identify novel SCAMs by next-generation single-cell RNA sequencing of subicular neurons with the goal long-term of assessing their synaptic function in disease-relevant circuits. I anticipate that this proposal wil uncover new insights into cell-type specific synaptic properties in the subicular-striatal circuit and create a platform for future independent investigations into how other SCAMs function in subicular and striatal circuitry.

Neurexins (Nrxns) are a family of essential but poorly understood presynaptic cell-adhesion molecules that are frequently linked to neuropsychiatric and neurodevelopmental disorders such as autism spectrum disorders (ASDs), schizophrenia and intellectual disability (ID). Three evolutionarily conserved neurexin genes produce longer alpha and shorter beta neurexin mRNAs that undergo extensive alternative splicing. ?- and ?-Nrxns share common transmembrane and cytoplasmic sequences but differ in the length and complexity of their extracellular domains (ECDs; 9 ?-Nrxn domains compared to 1 ?-Nrxn domain). Individual ?-Nrxns are associated with distinct neuropsychiatric disorders and disease-relevant mutations are commonly located in genomic regions that code for ?-Nrxn-specific extracellular sequences, suggesting that individual alpha neurexin ECDs may control distinct aspects of synapse function. Despite their discovery over twenty years ago, the fundamental question regarding the essential role of individual ?-Nrxn ECDs at the synapse remains unresolved. As an important first step in understanding how ?-Nrxn-specific extracellular sequences function at the synapse, our laboratory has identified an Nrxn3? compound heterozygous patient with profound ID and epilepsy. One allele produces a non-functional protein and the second harbors a missense mutation in an extracellular sequence shared by all ?-Nrxns. Intriguingly, there are multiple ASD associated mutations in the equivalent region of Nrxn1? indicating that this region plays an important role at the synapse. Preliminary data from primary neurons and ex vivo acute slices revealed that expression of the missense Nrxn3? mutant produced striking morphological and functional phenotypes at excitatory and inhibitory synapses. Biochemically, the Nrxn3? missense mutation unexpectedly differentially modulated binding to two excitatory postsynaptic ligands. Based on our preliminary data, we hypothesize that extracellular sequences of individual alpha neurexins control distinct aspects of excitatory and inhibitory synapse function. Here, we will test our central hypothesis in three specific aims: 1. Determine the impact of Nrxn3? extracellular sequences on synaptic morphology and function in in vitro neuron cultures; 2. Biochemically assess how the Nrxn3? missense mutation affects transsynaptic binding; and 3. Manipulate Nrxn3? ECD in vivo and assess its impact on basal excitatory and inhibitory synaptic transmission and activity-dependent plasticity in ex vivo slices. To accomplish aims 1 and 3, we will use molecular replacement, shRNA-mediated knockdown of endogenous Nrxn3? and replacement with wild-type or mutant Nrxn3? to faithfully recapitulate the disease state, combined with immunocytochemistry, electrophysiology and electron microscopy. Aim 2 will use in vitro biochemical and structure/function approaches to measure binding affinities to known Nrxn ligands. These aims will provide first insight into the morphological, functional and biochemical properties of Nrxn3? extracellular sequences and how mutations in this region contribute to cognitive disease.