From Synapse to Behavior: Dissecting the Role of Presynaptic Rac1 in the Regulation of Synaptic Vesicles and Memory

Abstract

Rac1 is a small Rho GTPase that functions as a molecular switch, cycling between active GTP-bound and inactive GDP-bound states in response to extracellular signals. In neurons, Rac1 is important for its role in actin cytoskeleton organization, a process fundamental for neuronal migration, morphogenesis, and synaptic plasticity. Its significance is particularly evident in postsynaptic compartments, where Rac1 and its downstream effector kinases and actin regulatory WAVE1 complex are known to influence spine morphology and neurotransmission. Consistent with Rac1’s important roles in synaptic signaling, alterations to Rac1 activity or its signaling pathways are associated with a variety of neurological disorders, including autism spectrum disorder (ASD) and schizophrenia. In the hippocampus, the Rac1-cofilin signaling regulates F-actin dynamics and thereby affects both long-term potentiation and depression, key processes in modulating synaptic strength and thereby influencing learning and memory. While actin cytoskeleton remodeling is believed to be a key contributor to Rac1’s involvement in learning and memory, the specific molecular mechanisms translating actin remodeling to altered synaptic efficacy remain to be explored. Moreover, the majority of the literature on Rac1 has focused on its functions at the postsynaptic sites, while actin remodeling also governs presynaptic functions. Our previous study showed that presynaptic Rac1 is activated by action potentials and negatively regulates the replenishment rate of synaptic vesicles of hippocampal neurons and thus modulates short-term presynaptic plasticity. This finding underscores the importance of understanding presynaptic Rac1 in answering the long-standing question of how presynaptic signaling and short-term presynaptic plasticity may influence learning and memory. Hence, this dissertation explores the behavioral impacts and regulatory mechanisms of Rac1 at presynaptic terminals. To investigate the behavioral impacts of synaptic Rac1 activity, we expressed a Rac1 inhibitor construct fused with a presynaptic or postsynaptic marker in C57BL/6J mice and performed memory performance tasks. We observed that presynaptic Rac1 inhibition impairs spatial working memory without affecting other types of memory, such as spatial or fear long-term memory. Conversely, postsynaptic Rac1 inhibition did not significantly impact spatial working memory but did impair remote fear memory, indicating a site-specific function of synaptic Rac1 in its effects on learning and memory. These experiments highlighted the synaptic compartmentalization of Rac1's functions in learning and memory and challenged traditional views of hippocampal roles in memory by suggesting that working memory and long-term memory processes may be more dissociated than previously believed. Next, to dissect the mechanisms by which presynaptic Rac1 inhibition selectively regulates working memory, we examined the effects of presynaptic Rac1 inhibition on synaptic ultrastructure using electron microscopy (EM) analysis of primary neuron cultures expressing the presynaptic Rac1 inhibitor construct. We found no significant changes in the bouton size, number of synaptic vesicles per bouton, and spine density compared to the control. However, inhibition of presynaptic Rac1 led to an increase in the diameter of synaptic vesicles, which could be attributed to disruptions in synaptic vesicle endocytosis. Furthermore, vesicles were observed to be distributed further from the active zone, suggesting potential alterations in vesicle trafficking or dynamics. Additionally, a slight increase in spine area hinted at transsynaptic effects resulting from the altered presynaptic functions. Overall, the EM analysis showed how presynaptic Rac1 inhibition modulates synaptic vesicle morphology and distribution, implying Rac1's potential involvement in presynaptic endocytosis and transsynaptic communication. Lastly, we leveraged in vivo proximity-dependent biotin identification (BioID) coupled with advanced mass spectrometry to map the functional proteomic landscape of presynaptic Rac1. In mice, we expressed a presynaptic Rac1 BioID construct, which is a fusion protein composed of the presynaptic marker synaptotagmin1, the biotin ligase UltraID, and Rac1 in either its constitutively active (CA) or dominant negative (DN) form. We identified 149 proteins that were biotinylated, suggesting their proximity to Rac1 at presynaptic terminals. This set was highly enriched for proteins localized at presynaptic sites and involved in GTPase regulation, actin filament organization, and kinase activity. Notably, 19 proteins significantly enriched in the CA Rac1 proteome likely represent the specific portion of proteins interacting with Rac1 at presynaptic terminals in an activity-dependent manner. Additionally, we identified 42 peptides corresponding to 22 proteins that were also significantly enriched in the CA proteome. Interestingly, analysis of this set suggested that Rac1's presynaptic roles may extend through phosphorylation-mediated mechanisms, affecting synaptic vesicle dynamics and GTPase activity. This proteomic study revealed a complex network of Rac1 interactions at presynaptic terminals, highlighting the kinase-mediated modulation of synaptic function, and suggesting potential pathways through which Rac1 influences neuronal signaling and plasticity. In summary, this dissertation demonstrated the function of presynaptic Rac1 in selectively influencing spatial working memory, possibly through phosphorylation processes that affect synaptic vesicle dynamics. Our study combined behavioral analyses with electron microscopy and proteomics to explore Rac1's interactions with synaptic proteins and its impact on synaptic structure and function. The findings indicated intricate mechanisms underlying working memory and long-term memory, mediated through site-specific functions of hippocampal Rac1. The future directions of this research include investigation into Rac1's precise role in memory by examining synaptic plasticity and the broader implications of presynaptic Rac1 inhibition on memory formation and cognitive processes.