Cdk5-dependent Mst3 phosphorylation and activity regulate neuronal migration through RhoA inhibition.

The radial migration of newborn neurons is critical for the lamination of the cerebral cortex. Proper neuronal migration requires precise and rapid reorganization of the actin and microtubule cytoskeleton. However, the underlying signaling mechanisms controlling cytoskeletal reorganization are not well understood. Here, we show that Mst3, a serine/threonine kinase highly expressed in the developing mouse brain, is essential for radial neuronal migration and final neuronal positioning in the developing mouse neocortex. Mst3 silencing by in utero electroporation perturbed the multipolar-to-bipolar transition of migrating neurons and significantly retards radial migration. Although the kinase activity of Mst3 is essential for its functions in neuronal morphogenesis and migration, it is regulated via its phosphorylation at Ser79 by a serine/threonine kinase, cyclin-dependent kinase 5 (Cdk5). Our results show that Mst3 regulates neuronal migration through modulating the activity of RhoA, a Rho-GTPase critical for actin cytoskeletal reorganization. Mst3 phosphorylates RhoA at Ser26, thereby negatively regulating the GTPase activity of RhoA. Importantly, RhoA knockdown successfully rescues neuronal migration defect in Mst3-knockdown cortices. Our findings collectively suggest that Cdk5-Mst3 signaling regulates neuronal migration via RhoA-dependent actin dynamics.

Cdk5-mediated phosphorylation of Axin directs axon formation during cerebral cortex development.

Axon formation is critical for the establishment of connections between neurons, which is a prerequisite for the development of neural circuitry. Kinases such as cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase-3ß (GSK-3ß), have been implicated to regulate axon outgrowth. Nonetheless, the in vivo roles of these kinases in axon development and the underlying signaling mechanisms remain essentially unknown. We report here that Cdk5 is important for axon formation in mouse cerebral cortex through regulating the functions of axis inhibitor (Axin), a scaffold protein of the canonical Wnt pathway. Knockdown of Axin in utero abolishes the formation and projection of axons. Importantly, Axin is phosphorylated by Cdk5, and this phosphorylation facilitates the interaction of Axin with GSK-3ß, resulting in inhibition of GSK-3ß activity and dephosphorylation of its substrate collapsin response mediator protein-2 (CRMP-2), a microtubule-associated protein. Specifically, both phosphorylation of Axin and its interaction with GSK-3ß are critically required for axon formation in mouse cortex development. Together, our findings reveal a new regulatory mechanism of axon formation through Cdk5-dependent phosphorylation of Axin.

Increased Axin expression enhances adult hippocampal neurogenesis and exerts an antidepressant effect.

Major depressive disorders are emerging health problems that affect millions of people worldwide. However, treatment options and targets for drug development are limited. Impaired adult hippocampal neurogenesis is emerging as a key contributor to the pathology of major depressive disorders. We previously demonstrated that increasing the expression of the multifunctional scaffold protein Axis inhibition protein (Axin) by administration of the small molecule XAV939 enhances embryonic neurogenesis and affects social interaction behaviors. This prompted us to examine whether increasing Axin protein level can enhance adult hippocampal neurogenesis and thus contribute to mood regulation. Here, we report that stabilizing Axin increases adult hippocampal neurogenesis and exerts an antidepressant effect. Specifically, treating adult mice with XAV939 increased the amplification of adult neural progenitor cells and neuron production in the hippocampus under both normal and chronic stress conditions. Furthermore, XAV939 injection in mice ameliorated depression-like behaviors induced by chronic restraint stress. Thus, our study demonstrates that Axin/XAV939 plays an important role in adult hippocampal neurogenesis and provides a potential therapeutic approach for mood-related disorders.

Overproduction of Neurons Is Correlated with Enhanced Cortical Ensembles and Increased Perceptual Discrimination.

Brains vary greatly in neuronal number and density, even across individuals within the same species, yet it remains unclear whether such variation leads to differences in brain function or behavior. By imaging cortical activity of a mouse model in which neuronal production is moderately enhanced in utero, we find that animals with more cortical neurons also develop enhanced functional correlations and more distinct neuronal ensembles in primary visual cortex. These mice also have sharper orientation discrimination in their visual behavior. These results unveil a correlation between neuronal ensembles and behavior and suggest that neuronal number is linked to functional modularity and perceptual discrimination of visual cortex. By experimentally linking differences in neuronal number and behavior, our findings could help explain how evolutionary and developmental variability of individual and species brain size may lead to perceptual and cognitive differences.

Cloning and characterization of a novel splice variant of human U2AF1L3 gene.

Pre-mRNA splicing allows individual genes to produce multiple protein isoforms with diverse functions. Recognition of functional splice sites in pre-mRNAs is very important in this splicing process and requires some protein auxiliary factors such as U2 small nuclear ribonucleoprotein auxiliary factor small subunit (U2AF35, encoded by U2AF1). By its RNA binding domains, U2AF35 interacts with U2AF65 to bind 3' splice site of pre-mRNA and initiates splicing. Another protein, which is named as U2AF1-like3 (U2AF1L3), shows high similarity with U2AF35 and may have related function in pre-mRNA splicing. Here, we report a splice variant of U2AF1L3, which is 767 bp in length and has an open reading frame (ORF) coding a predicted 181 amino acids protein. Reverse transcription-PCR (RT-PCR) shows that this isoform has different expression pattern with U2AF1L3 and is highly expressed in heart, brain and lung.

Axin Regulates Dendritic Spine Morphogenesis through Cdc42-Dependent Signaling.

During development, scaffold proteins serve as important platforms for orchestrating signaling complexes to transduce extracellular stimuli into intracellular responses that regulate dendritic spine morphology and function. Axin ("axis inhibitor") is a key scaffold protein in canonical Wnt signaling that interacts with specific synaptic proteins. However, the cellular functions of these protein-protein interactions in dendritic spine morphology and synaptic regulation are unclear. Here, we report that Axin protein is enriched in synaptic fractions, colocalizes with the postsynaptic marker PSD-95 in cultured hippocampal neurons, and interacts with a signaling protein Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in synaptosomal fractions. Axin depletion by shRNA in cultured neurons or intact hippocampal CA1 regions significantly reduced dendritic spine density. Intriguingly, the defective dendritic spine morphogenesis in Axin-knockdown neurons could be restored by overexpression of the small Rho-GTPase Cdc42, whose activity is regulated by CaMKII. Moreover, pharmacological stabilization of Axin resulted in increased dendritic spine number and spontaneous neurotransmission, while Axin stabilization in hippocampal neurons reduced the elimination of dendritic spines. Taken together, our findings suggest that Axin promotes dendritic spine stabilization through Cdc42-dependent cytoskeletal reorganization.

Cloning and characterization of a splice variant of human Bardet-Biedl syndrome 4 gene (BBS4).

Bardet-Biedl syndrome (BBS) is a heterogeneous multisystemic disorder characterized primarily by five cardinal features of retinal degeneration, obesity, polydactyly, hypogenitalism and mental retardation. To date, six distinct BBS loci that have been identified on different chromosomes. BBS4 gene is mapped to 15q22.2-23, which when mutated can cause BBS4. Its protein shows strong homology to O-linked N-acetylglucosamine (O-GlcNAc) transferase. Here we report a splice variant of BBS4, which is 2556 bp in length and has an open reading frame coding a predicted 527 amino-acids protein. RT-PCR shows that the cDNA is widely expressed while it has higher expression levels in pancreas, liver and prostate.

Overproduction of upper-layer neurons in the neocortex leads to autism-like features in mice.

The functional integrity of the neocortex depends upon proper numbers of excitatory and inhibitory neurons; however, the consequences of dysregulated neuronal production during the development of the neocortex are unclear. As excess cortical neurons are linked to the neurodevelopmental disorder autism, we investigated whether the overproduction of neurons leads to neocortical malformation and malfunction in mice. We experimentally increased the number of pyramidal neurons in the upper neocortical layers by using the small molecule XAV939 to expand the intermediate progenitor population. The resultant overpopulation of neurons perturbs development of dendrites and spines of excitatory neurons and alters the laminar distribution of interneurons. Furthermore, these phenotypic changes are accompanied by dysregulated excitatory and inhibitory synaptic connection and balance. Importantly, these mice exhibit behavioral abnormalities resembling those of human autism. Thus, our findings collectively suggest a causal relationship between neuronal overproduction and autism-like features, providing developmental insights into the etiology of autism.

Axin directs the amplification and differentiation of intermediate progenitors in the developing cerebral cortex.

The expansion of the mammalian cerebral cortex is safeguarded by a concerted balance between amplification and neuronal differentiation of intermediate progenitors (IPs). Nonetheless, the molecular controls governing these processes remain unclear. We found that the scaffold protein Axin is a critical regulator that determines the IP population size and ultimately the number of neurons during neurogenesis in the developing cerebral cortex. The increase of the IP pool is mediated by the interaction between Axin and GSK-3 in the cytoplasmic compartments of the progenitors. Importantly, as development proceeds, Axin becomes enriched in the nucleus to trigger neuronal differentiation via ß-catenin activation. The nuclear localization of Axin and hence the switch of IPs from proliferative to differentiative status are strictly controlled by the Cdk5-dependent phosphorylation of Axin at Thr485. Our results demonstrate an important Axin-dependent regulatory mechanism in neurogenesis, providing potential insights into the evolutionary expansion of the cerebral cortex.