Rho family GTPases, Rac and Cdc42, control the localization of neonatal dentate granule cells during brain development

Abstract

The hippocampus is generally considered as a brain center for learning and memory. We have recently established an electroporation‐mediated gene transfer method to investigate the development of neonatal dentate granule cells in vivo. Using this new technique, we introduced knockdown vectors against Rac1 small GTPase into precursors for dentate granule cells at postnatal day 0. After 21 days, Rac1‐deficient cells were frequently mispositioned between the granule cell layer (GCL) and hilus. About 60% of these mislocalized cells expressed a dentate granule cell marker, Prox1. Both the dendritic spine density and the ratio of mature spine were reduced when Rac1 was silenced. Notably, the deficient cells have immature thin processes during migrating in the early neonatal period. Knockdown of another Rac isoform, Rac3, also resulted in mislocalization of neonatally born dentate granule cells. In addition, knockdown of Cdc42, another Rho family protein, also caused mislocalization of the cell, although the effects were moderate compared to Rac1 and 3. Despite the ectopic localization, Rac3‐ or Cdc42‐disrupted mispositioned cells expressed Prox1. These results indicate that Rho signaling pathways differentially regulate the proper localization and differentiation of dentate granule cells.

The synaptic scaffolding protein CNKSR2 interacts with CYTH2 to mediate hippocampal granule cell development

“…In the control (shCont) treatment, labeled cells exhibited a polarized shape with dendrites projecting to the molecular layer (Fig. 6A), a finding consistent with previous reports (25,30). In contrast, however, cells transduced with shCNKSR2 and shCYTH2 vectors showed features consistent with a loss of neuronal polarity and abnormal neurite morphology (Fig.…”

“…This method is highly effective for the analysis of hippocampal cell differentiation and maturation. We previously took this approach to clarify the functions of the small GTPases, Rac1, Rac3, and Cdc42, and their roles in the localization and differentiation of dentate granule cells (30).…”

“…CYTH2 is characterized as a GEF for ARF small GTPases (35) to activate Rac signaling, while ARHGAP39, a GAP for Rac and Cdc42, serves as a negative regulator for Rac (36). It is noteworthy that Rac and Cdc42 are essential to hippocampal development (30), and their functional deficits are implicated in neurodevelopmental disorders (37)(38)(39)(40)(41)(42). This, CYTH2 and ARHGAP39 both stabilize CNKSR2 expression yet play distinct roles in dentate granule cell development.…”

“…Immunostaining was performed with free-floating sections (30). To quantify GFP-labeled cells in the dentate gyrus, we usually prepared serial brain sections containing dentate gyrus (100 µm-thickness), picked one slice from every three slices (six slices in total), stained for GFP, and quantified.…”

The Rac3 GTPase in Neuronal Development, Neurodevelopmental Disorders, and Cancer

“…Electroporation-mediated transfer of knockdown vectors for Rac1 and Rac3 in newborn mice has recently been used to address the development of neonatal dentate granule cells in vivo [47]. Knockdown of either Rac3 or Rac1 results in frequently mispositioned neonatally born dentate granule cells, with partially different effects of the two GTPases on the differentiation of these cells, revealing the independent role of the two GTPases in regulating the proper localization and differentiation of dentate granule cells.…”