Postnatal regulation of dendritic spine formation and refinement in cortical pyramidal neurons is critical for excitatory/inhibitory balance in neocortical networks. Recent studies have identified a selective spine pruning mechanism in the mouse prefrontal cortex (PFC) mediated by class 3 Semaphorins and the L1-CAM cell adhesion molecules Neuron-glia related CAM (NrCAM), Close Homolog of L1 (CHL1), and L1. L1-CAMs bind Ankyrin B (AnkB), an actin-spectrin adaptor encoded by Ankyrin2 (ANK2), a high confidence gene for autism spectrum disorder (ASD). In a new inducible mouse model (Nex1Cre-ERT2: Ank2flox: RCE), Ank2 deletion in early postnatal pyramidal neurons increased spine density on apical dendrites in PFC layer 2/3 of homozygous and heterozygous Ank2-deficient mice. In contrast, Ank2 deletion in adulthood had no effect on spine density. Sema3F-induced spine pruning was impaired in cortical neuron cultures from AnkB-null mice and was rescued by re-expression of the 220 kDa AnkB isoform but not 440 kDa AnkB. AnkB bound to NrCAM at a cytoplasmic domain motif (FIGQY1231), and mutation to FIGQH inhibited binding, impairing Sema3F-induced spine pruning in neuronal cultures. Identification of a novel function for AnkB in dendritic spine regulation provides insight into cortical circuit development, as well as potential molecular deficiencies in ASD.
The L1 cell adhesion molecule NrCAM (Neuron-glia related cell adhesion molecule) functions as a co-receptor for secreted class 3 Semaphorins to prune subpopulations of dendritic spines on apical dendrites of pyramidal neurons in the developing mouse neocortex. The developing spine cytoskeleton is enriched in actin filaments but a small number of microtubules have been shown to enter the spine apparently trafficking vesicles to the membrane. Doublecortin-like kinase 1 (DCLK1) is a member of the Doublecortin (DCX) family of microtubule-binding proteins with serine/threonine kinase activity. To determine if DCLK1 plays a role in spine remodeling, we generated a tamoxifen-inducible mouse line (Nex1Cre-ERT2: DCLK1flox/flox : RCE) to delete microtubule binding isoforms of DCLK1 from pyramidal neurons during postnatal stages of spine development. Homozygous DCLK1 conditional mutant mice exhibited decreased spine density on apical dendrites of pyramidal neurons in the prefrontal cortex (layer 2/3). Mature mushroom spines were selectively decreased upon DCLK1 deletion but dendritic arborization was unaltered. Mutagenesis and binding studies revealed that DCLK1 bound NrCAM at the conserved FIGQY1231 motif in the NrCAM cytoplasmic domain, a known interaction site for the actin-spectrin adaptor Ankyrin. These findings demonstrate that DCLK1 facilitates spine growth and maturation on cortical pyramidal neurons in the mouse prefrontal cortex potentially through microtubule and NrCAM interactions.
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