We have identified novel functions for IKKα in enhancing the differentiation of human NPCs. Elevated IKKα indirectly lowers the level of REST/NRSF repressor, which is a global inhibitor of neurogenesis 
. The ability of IKKα to enhance neuronal differentiation is further exemplified by the induction of neuron-enriched miRNAs such as miR-124a and -7, and proteins including MeCP2, PSD95, and BDNF, which are involved in neurite outgrowth, neuronal maturation, and synaptic plasticity. Thus, increasing the level and/or the activity of IKKα may be a useful strategy to promote neuronal differentiation in vitro
and potentially in vivo
. Our results also highlight a direct link between IKKα and MeCP2, which could be instrumental in regulating MeCP2-dependent gene expression and neurodevelopment.
Elevation of IKKα inhibits self-renewal and accelerates the differentiation of MESC2.10 NPCs, and reduction of REST expression may play a role. As a repressor of neuronal genes, REST promotes the proliferation of NSCs as well as neuroblastoma cell lines, whereas reduction of REST induces neuronal differentiation 
. We propose that the effect of IKKα on REST expression is indirect, since elevated IKKα does not lower the REST promoter activity. However, REST promoters have several NF-κB binding sites 
and the regulation of NF-κB activity by IKKα may influence REST levels under certain physiological conditions. We have not been successful in establishing a link between IKKα/NF-κB and REST transcription, however.
A critical step in the initiation of NSC differentiation is the induction of miR-124, which is repressed by REST 
. miR-124 is enriched in the brain and is recognized as the “micromanager of neurogenesis” in vivo
. Indeed, miR-124 promotes the direct conversion of human fibroblasts into functional neurons, where it instructs chromatin remodeling and promotes brain-specific alternative splicing of mRNAs essential for neuronal differentiation 
. Thus, the reduced levels of REST and reciprocal elevation of miR-124 in IKKα+ cells will likely cause global changes in gene expression that inhibit proliferation and engage the differentiation programming (). In addition, miR-124 plays an important role in synaptic plasticity and memory formation in post-mitotic neurons in Aplysia 
. In vivo
studies indicate that IKKα is involved in hippocampal-dependent memory reconsolidation 
. It will be interesting to examine whether elevated expression of IKKα induces miR-124 and enhances memory formation and learning, possibly by affecting neurogenesis in the adult hippocampus.
IKKα accumulates in the nuclei of differentiating NPCs (, and ), and nuclear transfer of IKKα is implicated in the phosphorylation of histone-3 (Ser10), which leads to enhanced expression of various genes 
. Our transcriptome analysis (mRNA–seq) of differentiating control and IKKα+ NPCs reveals significant changes in the expression of several hundred mRNAs in IKKα+ cells; some of these encode proteins involved in neurodevelopment and the splicing of neuron-specific mRNAs (A. Khoshnan et al., unpublished data). Characterization of some of these genes may shed further light on the mechanism of how IKKα accelerates neuronal differentiation and regulates complex epigenetic changes such as neurite outgrowth. It is intriguing that miR-7, which is implicated in neuronal homeostasis and neurite outgrowth 
, is selectively induced in differentiating IKKα+ NPCs. miR-7 also protects dopaminergic neurons against oxidative stress, where it reduces the expression of α-synuclein and leads to enhanced survival 
. We have previously shown that IKKα protects MESC2.10 neurons against oxidative stress-induced neuronal death and preserves the integrity of neuron-enriched huntingtin protein, which has neuroprotective properties 
. Thus, in addition to promoting neurite outgrowth, IKKα-induced miR-7 may also contribute to the resiliency of neurons under adverse environmental conditions.
The ability of IKKα to regulate MeCP2 levels and activity is another novel aspect of this study. These interactions were characterized in the context of BDNF expression, which is induced by elevated IKKα and suppressed when MeCP2 levels are knocked down (). BDNF plays a critical role in neuronal differentiation and survival, miRNA processing, and synaptic plasticity 
. The MeCP2-dependent induction of BDNF may therefore be important in these processes, which has implications for neurological and psychiatric disorders. While earlier studies supported an inhibitory role for MeCP2, recent findings are consistent with a positive effect of MeCP2 on BDNF expression 
. Moreover, in animal models where MeCP2 is inactive or deleted, BDNF levels are significantly reduced 
. Our data are also consistent with a positive effect of elevated MeCP2 on BDNF and highlight the involvement of IKKα.
Recent studies propose that MeCP2 may function both as a repressor and activator of the same target genes, depending on its association with other proteins. For example, MeCP2-dependent recruitment of HDAC2 or CREB to the glial-derived neurotrophic factor promoter can inhibit or promote gene expression, respectively 
. We find that IKKα associates with MeCP2 and both are recruited to the BDNF exon-IV promoter, which may be crucial for the induction of BDNF. Thus, similar to CREB, binding of IKKα to MeCP2 may enhance MeCP2-dependent gene expression. Moreover, maximal BDNF expression in IKKα+ neurons coincides with elevated levels of MeCP2 (). We posit that changes in the homeostasis of MeCP2 may dictate whether it acts as repressor or activator of gene expression. At steady state, MeCP2 may simply function as a chromatin organizer and control the noise in global gene expression 
. On the other hand, when MeCP2 levels are elevated, it may facilitate selective gene expression by associating by other regulatory proteins such as IKKα and CREB. It is relevant that elevation of MeCP2 in transgenic mice induces the expression of ~2200 genes including CREB 
. Moreover, the levels of MeCP2 and its phosphorylation at Ser421 are increased by exogenous factors such as amphetamine, cocaine, and the anti-depressant fluoxetine 
. These findings support the dynamic nature of MeCP2 expression in neurons and how fluctuations in its levels and/or its phosphorylation may dictate various functions. Exogenous stimuli including growth factors and cytokines also regulate IKKα activity 
. The elevation of MeCP2 in IKKα+ neurons and the phosphorylation of MeCP2 by IKKα raise the possibility that environmental activation of IKKα may affect MeCP2 homeostasis and activity. Further characterization of IKKα-MeCP2 interactions may shed light on the complex nature of MeCP2 activities in neurons.