Molecular mechanisms of semaphorin 3A-Neuropilin1 / Plexin-A4 signaling in layer 5 pyramidal neurons of the mouse cortex during development
Creators: name: Abushalbaq, Oday M. H.
Publisher: name: No Publisher Supplied
Year: 2022

description: The proper establishment of neuronal circuits is of ultimate importance to the functionality of an organism as a whole. Neuronal circuit development is a complex process that largely revolves around the formation of the correct neuronal morphologies through directional growth and maturation of neurites, i.e., axons and dendrites. This is achieved through multiple developmental stages such as neuronal polarization, neurite outgrowth, guidance, synaptogenesis, and neurite pruning. The formation of abnormal neuronal connections due to aberrant dendritic morphologies, in particular, has been shown to contribute to a variety of neurological disorders such as autism spectrum disorders (ASDs), Schizophrenia, and epilepsy, just to name a few. The neurodevelopmental processes of neurite outgrowth and guidance are largely dependent on extracellular guidance cues. These cues mediate their effects through the regulation of cytoskeletal components, actin and microtubules, leading to morphological changes in neurites. A number of guidance cue families such as Slits, Netrins, Ephrins and Semaphorins have been shown to act as attractants or repellents for neurites during development. The semaphorins, a large family of secreted and membrane-bound guidance cues, were initially identified as repellents in axon guidance events. They have also been described in other crucial cellular processes that contribute to the formation of neuronal connections including, but not limited to, dendritic morphogenesis and synaptic development. Of particular interest, the class 3 secreted semaphorin 3A (Sema3A), signaling through its receptor complex Neuropilin-1 (Nrp1) / PlexinA4 (PlxnA4), has been shown to promote dendritic morphogenesis in cortical neuron development in vitro and in vivo. However, the underlying intracellular mechanisms employed by Sema3A-Nrp1/PlxnA4 signaling to control dendritic elaboration remain elusive. Furthermore, it is not clear whether dendritic development (positive effect) mediated by Sema3A-Nrp1/PlxnA4 employs the same or similar intracellular signaling pathways as those proposed for axon guidance (negative effect). Therefore, I asked these main questions in my thesis work: 1) Is the LVS motif in the H/RBD domain of the Plexin-A4 receptor required for Sema3A-mediated dendritic elaboration in vivo and in vitro? 2) Does Sema3A-mediated dendritic elaboration involve transcriptional and/or translational regulation, as seen in some Sema3A-mediated axon guidance events, in vitro? And 3) What are the potential novel downstream effectors in Sema3A-mediated dendritic elaboration in cortical pyramidal neuron development?To address the above questions, first I characterized the role of the LVS motif in the H/RBD cytoplasmic domain of the PlexinA4 receptor in layer 5 cortical neuron dendrite development. I showed that the LVS motif is required for basal dendritic branching and growth in vivo and in vitro following Sema3A treatment. Upon Sema3A binding to the Nrp1/PlxnA4 holoreceptor complex, the small Rnd1 GTPase is able to bind the PlxnA4 receptor at the LVS motif. In addition, siRNA knockdown of Rnd1 showed its requirement for Sema3A-induced dendritic elaboration. Next, I showed that the activity of the small RhoA GTPase is downregulated in wild type primary cortical neurons following Sema3A treatment, but not in PlxnA4LVS-GGA/LVS-GGA mutant neurons. The inactivation of RhoA following Sema3A treatment is supported by consequential inhibition of ROCK, which is downstream of RhoA signaling. Moreover, I also showed that the overexpression of the small GTPase Rac1 in PlxnA4LVS-GGA/LVS-GGA mutant neurons is able to rescue the dendritic phenotypes observed in vitro. Taken together, my findings suggest that Sema3A-Nrp1/PlxnA4-LVS interaction with the Rnd1 GTPase to decrease the activity of RhoA and ROCK downstream signaling leads to the permissive growth and branching of dendrites in developing mouse cortical neurons. To address the question of whether Sema3A-mediated dendritic elaboration also requires transcriptional and/or translational regulation, I took a pharmacological approach. Using transcriptional and translational inhibitors, I showed that primary cortical neurons no longer respond to Sema3A-induced dendritic branching and growth. I dissected the temporal windows at which each of these two mechanisms are employed during Sema3A-induced dendritic elaboration in vitro. Specifically, I showed that gene transcription is required as early as 30 minutes following Sema3A treatment up to 3 hours. I also found that protein translation is required starting at ~1 hour following Sema3A signaling up to 8 hours. Collectively, my findings suggest that gene transcription and protein translation are employed during the initial stages of Sema3A-induced dendritic branching in mouse cortical neurons in vitro. Finally, to determine the novel gene expression profile changes following Sema3A signaling in developing cortical neurons, I performed RNA sequencing (in collaboration with the Genomics Center, Rutgers NJ Medical School) in primary cortical neurons taken from the RiboTag mouse (where the Rpl22 protein is HA-tagged and floxed) crossed with the Etv1-CreERT2 driver line. Indeed, many genes are differentially regulated following Sema3A treatment as summarized in this thesis. Future experiments are required to verify which specific genes are involved in Sema3A-mediated dendritic elaboration versus axon guidance events. Taken together, the results in my thesis research provided novel insights to the intracellular mechanisms underlying Sema3A-Nrp1/PlxnA4-mediated dendritic morphogenesis during cortical development in vivo and in vitro. I also showed that both gene transcription and protein translation are required for Sema3A signaling in vitro and have identified a substantial number of potential candidates that are differentially regulated. Overall, my findings provided new knowledge that leads to a better understanding of the molecular mechanisms controlling neuronal connectivity in the developing mammalian neocortex.