Investigating the Function of Alternative Splicing in Synapse Formation

Synapse formation is essential for brain and neural circuit development, but the mechanisms of this process remain incompletely understood. Synaptogenesis in mouse hippocampi requires the postsynaptic adhesion G-protein-coupled-receptor (aGPCR) Latrophilin-3 (LPHN3), which is alternatively spliced into numerous different isoforms. Previous work indicates that some splice variants are not involved in synaptogenic activity, but not all variants have been characterized. Identifying which splice isoforms are necessary or extraneous for synaptogenesis will provide a basis for future research into potential mechanisms for synapse formation with a role for necessary exons. Functions for non-synaptogenic isoforms can also be explored. This project aims to determine whether splice variants that lack part of the GPCR autoproteolysis-inducing (GAIN) domain or the third intracellular loop are required for synapse formation. These domains, respectively, are hypothesized to contribute to the adhesion function of LPHN3 and known to be important for GPCR signaling through G-protein coupling. To test if these exons are necessary for synaptogenesis, LPHN3 isoform expression is genetically manipulated in primary hippocampal neuron cultures by either a CRISPR/Cas9 knock-out or a Cre/Lox rescue approach; the resulting synapse density is then calculated for both approaches. Here, we found that genetic manipulation of the two exons of interest using the CRISPR/Cas9 system resulted in loss of global LPHN3 protein levels, rendering synapse density uninterpretable and highlighting the limitations of using CRISPR/Cas9 to control splice isoform expression. The Cre/Lox rescue system resulted in overall increase in synapse density in all rescue constructs; this poor recapitulation of the endogenous system indicates a need for improved construct design. In demonstrating the challenges of controlling splice variant expression, this project offers insight into the strengths and limitations of various genetic manipulation strategies to study alternative splicing. This work also paves the way for future successes in characterizing the necessity of different LPHN3 splice isoforms in synaptogenesis.