Zebrafish Models to Study NMDA Receptors in Early Nervous System Development and Disease (2022)

Zoodsma, J. (2022). Zebrafish Models to Study NMDA Receptors in Early Nervous System Development and Disease [Doctoral thesis, State University of New York at Stony Brook]. https://www.proquest.com/openv...


N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that play critical roles in nervous system function and neuronal development. NMDARs are obligate heterotetramers, typically comprised of a combination of GluN1 and GluN2(AD) subunits. Highlighting the developmental importance of these receptors, diseaseassociated mutations in each of these subunits result in various neurodevelopmental disorders including epilepsy, schizophrenia, and autism spectrum disorders (ASD). How myriad missense mutations across an array of proteins from the same receptor all converge to neurodevelopmental dysfunction is poorly understood. Here, we establish the relevancy of the zebrafish model to study developmental functions of NMDARs in vivo. We generated CRISPR-mediated lesions in the genes encoding the primary NMDAR subunits GluN1, GluN2A, GluN2B, GluN2C and GluN2D, disrupting their iv function. Surprisingly, we found that zebrafish lacking all NMDAR function survive longer than their murine counterparts, offering a unique opportunity to interrogate NMDAR roles in neural development. Through an array of larval zebrafish behavior assays, we found that zebrafish lacking all NMDARs had robust behavioral alterations, including abnormal responses to light and acoustic stimuli, and diminished prey capture abilities. Not all these behaviors mirrored acute pharmacological inhibition, suggesting additional developmental roles for these receptors. Zebrafish lacking any of the GluN2(A-D) subunits survive into adulthood, affording an opportunity to assay subunitspecific developmental effects. We found that zebrafish lacking GluN2B displayed social deficits and a reduction of inhibitory neurons in the subpallium. GluN2B is encoded by the high-confidence ASD-associated gene grin2B, and this social deficit reflects a central feature of ASD. While removing individual subunits is a robust way to interrogate developmental roles of these proteins, most disease-associated mutations are missense mutations not resulting in complete loss of function. Because of this, we have begun to generate zebrafish containing human missense mutations in the endogenous zebrafish genes using prime editing. Future studies will interrogate the developmental effects of these missense mutations in an in vivo environment. Together, these findings highlight the unique opportunity to study, in zebrafish, the roles of NMDARs in development and disease etiology and afford a system for future examination of the developmental effects of disease-associated missense mutations.