1N, altered the
distribution of actin and 4.1N. In contrast, the KCC2-C568A mutant, which shows a reduced binding affinity to 4.1N, did not affect the cytoskeleton. Thus, we suggest that the interaction between KCC2 and 4.1N plays a key role in the induction of the developmental defects observed in the transgenic embryos. As KCC2-FL and KCC2-ΔNTD had an effect on migration of neural crest cells, we assessed whether ectopic expression could also affect neuronal migration in vitro. C17.2 selleck products cells were transfected with control, KCC2-FL, KCC2-ΔNTD and KCC2-C568A plasmids. After 48 h, a scratch was made through the cell layer and the cells were incubated in serum-reduced medium for 18 h to allow migration in the wound area. In control cultures, the wound area was invaded by a moderate number of cells (Fig. 9A). KCC2-FL (Fig. 9B) and KCC2-ΔNTD (Fig. 9C) transfections significantly reduced the number of migrating cells (73 and 72% of control; P = 0.016 and P = 0.011, respectively). Transfection with KCC2-C568A (Fig. 9D) did not affect the number of cells in the wound area (96% of control; P = 0.627). Thus, KCC2-FL and KCC2-ΔNTD perturbed migration of neuronal cells in vitro, similar to the effect on neural crest migration in vivo. Our work shows that ectopic expression of KCC2 in mouse embryos leads to disturbances in the actin cytoskeleton, which in turn interferes
with neuronal differentiation and migration. The results are consistent with a structural role for KCC2 during early neuronal development that is not dependent VE-822 cell line on the ion transport function of KCC2. In several parts of the central nervous system, such as the spinal cord (Delpy et al., 2008) and brainstem
(Balakrishnan et al., 2003; Blaesse et al., 2006), KCC2 is expressed before the onset of functional Cl− extrusion. Moreover, the levels Cell Penetrating Peptide of KCC2 expression in the auditory brainstem do not change at the periods of the hyperpolarizing EGABA shift (Balakrishnan et al., 2003; Vale et al., 2005). It has been suggested that the early expressed protein is inactive and requires regulation of its localization, state of phosphorylation, or oligomerization for functional activation (Vale et al., 2005; Blaesse et al., 2006; Lee et al., 2007; Hartmann et al., 2009). KCC2 shows a high level of expression in the proximity of excitatory synapses and within dendritic spines (Gulyas et al., 2001) and, more recently, is has been shown that KCC2 promotes the development of spines through interaction with the cytoskeleton-associated protein 4.1N (Li et al., 2007). Thus, KCC2 has a morphogenic role that is independent of its ion transport function. This morphogenic role may explain the early presence of KCC2 prior to the hyperpolarizing EGABA shift. The present results show that KCC2 is already endogenously expressed at E9.5 in neuronal cells of mouse embryos. This is earlier than previously shown time points for KCC2 expression (Li et al.