| Abstract |
The correct function of cortical microcircuits depends in part on GABAergic interneurons that provide the main source of inhibition. Impaired interneuronal function results in severe neurodevelopmental disorders such as schizophrenia, epilepsy and autism. A pivotal process in the development of cortical interneurons, is their migration from the ventral telencephalon, where they originate towards the developing cortex. It is a process that is determined by intrinsic properties of the interneurons and extracellular factors, which modify their leading processes in response to them through activation of intracellular pathways. Rac-proteins are intracellular mediators of numerous developmental processes such as cytoskeleton organization, vesicle trafficking, transcription, cell cycle progression in diverse cell types. In the present study we focused in the role of the ubiquitously expressed Rac1 and the neural-specific Rac3 in interneurons derived from the medial ganglionic eminence (MGE), a population comprising the majority of cortical interneurons. Previous work from our lab uncovered, using Cre/loxP technology, a cell autonomous and stage-specific requirement for Rac1 activity within proliferating interneuron precursors (Vidaki et al., 2012). Mutant cortical interneurons aggregate ventrally due to defects in the actin organization in the leading processes and a delay in cell cycle progression of their progenitors. As a consequence, only 50% of interneurons populate the cortex postnatally. Mice deficient for Rac3 show no obvious developmental defects, while the results of behavioral tests revealed a hyperactive behavior, hypereactivity to the presentation of new stimuli and some differences in motor coordination and motor learning(Corbetta et al., 2005; Corbetta et al., 2008). The fact that the ablation of Rac3 did not severely affect cortical development, even though it is a nervous system-specific GTPase and also that the ablation of Rac1 from the MGE progenitors resulted in a severe, yet partial reduction of MGE-derived interneurons has led to the assumption that the two GTPases might ’ . difficulty that arises in exploring the role of Racs, the approach followed in this work, was to examine interneurons with both Racs absent. To do so, Rac1/Rac3 double mutant mice were generated by crossing Rac1 conditional KO with Rac3 KO animals. In the first part of this work the phenotype of the Rac1/Rac3 double mutants was described. The double ablation of Rac1/Rac3 leads to an even more severe reduction of cortical interneurons in the postnatal cortex. 80% of the ΜGE-derived interneurons fail to migrate to the cortex due to a delay in the cell cycle exit (of the same extent as the one described in Rac1 single mutants) and cytoskeletal defects. In fact, Rac1/Rac3-deficient interneurons exhibit gross cytoskeletal abnormalities, due to impairments in actin-microtubule dynamics, including reduction of axon length, splitting of the leading process, abnormal growth cone formation and reduction of microtubule stability (Tivodar et al., 2015). Pharmacological treatment of the interneurons with taxol, a reagent that stabilizes microtubules, improved the described features described and partially rescued the phenotype of the cells. As migration of the Rac1/Rac3 double mutant interneurons was impaired, a process that requires constant cytoskeletal reorganization, in the second part of this work the migratory behavior of interneurons was analyzed by time lapse microscopy. Several aspects of their movement, like velocity, frequency of translocation, amplitude of translocation and others were found affected. The subcellular localization of the centrosome and Golgi complex, key elements of proper migration, were altered in the absence of Rac1/Rac3 and also actomyosin dynamics was impaired, explaining the deficits in their migratory behavior. Moreover, axon outgrowth, the initiation of this process and also axonal transport, as evidenced by lysosomal locomotion were significantly impacted. Lysosomal positioning was also found affected during the migration of the cells. RNA-sequencing revealed a number of genes with altered expression in MGE-derived interneurons upon ablation of Rac1 and Rac3. Among them the two-pore channel 2 (TPCN2 encoding TPC2), a member of the superfamily of voltage-gated ion channels, that is mainly located in late endosomes/lysosomes and mediates Ca2+ release (Marchant and Patel, 2015; Patel and Kilpatrick, 2018). The protein levels of TPC2 were found reduced and its subcellular localization was affected. Lastly, pharmacological inhibition of TPC2 in wild type interneurons severely affected their axon growth and the initiation of this process, similarly to the ablation of Rac1/3. Pharmacological inhibition of TPC2 also affected the migration as the velocity and the frequency of translocation were found decreased. Collectively, these data support the idea that reduction of TPC2 upon ablation of Rac1 and Rac3 in cortical interneurons could contribute to the phenotype observed regarding the initiation of axon outgrowth, axon extension and interneuron migration.
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