Identification of the cysteine residue responsible for disulfide linkage of Na+ channel α and ß2 subunits.

Voltage-gated Na(+) channels in the brain are composed of a single pore-forming α subunit, one non-covalently linked ß subunit (ß1 or ß3), and one disulfide-linked ß subunit (ß2 or ß4). The final step in Na(+) channel biosynthesis in central neurons is concomitant α-ß2 disulfide linkage and insertion into the plasma membrane. Consistent with this, Scn2b (encoding ß2) null mice have reduced Na(+) channel cell surface expression in neurons, and action potential conduction is compromised. Here we generated a series of mutant ß2 cDNA constructs to investigate the cysteine residue(s) responsible for α-ß2 subunit covalent linkage. We demonstrate that a single cysteine-to-alanine substitution at extracellular residue Cys-26, located within the immunoglobulin (Ig) domain, abolishes the covalent linkage between α and ß2 subunits. Loss of α-ß2 covalent complex formation disrupts the targeting of ß2 to nodes of Ranvier in a myelinating co-culture system and to the axon initial segment in primary hippocampal neurons, suggesting that linkage with α is required for normal ß2 subcellular localization in vivo. WT ß2 subunits are resistant to live cell Triton X-100 detergent extraction from the hippocampal axon initial segment, whereas mutant ß2 subunits, which cannot form disulfide bonds with α, are removed by detergent. Taken together, our results demonstrate that α-ß2 covalent association via a single, extracellular disulfide bond is required for ß2 targeting to specialized neuronal subcellular domains and for ß2 association with the neuronal cytoskeleton within those domains.

Dicer loss in striatal neurons produces behavioral and neuroanatomical phenotypes in the absence of neurodegeneration.

MicroRNAs (miRNAs) are small noncoding RNAs that can act to repress target mRNAs by suppressing translation and/or reducing mRNA stability. Although it is clear that miRNAs and Dicer, an RNase III enzyme that is central to the production of mature miRNAs, have a role in the early development of neurons, their roles in the postmitotic neuron in vivo are largely unknown. To determine the roles of Dicer in neurons, we ablated Dicer in dopaminoceptive neurons. Mice that have lost Dicer in these cells display a range of phenotypes including ataxia, front and hind limb clasping, reduced brain size, and smaller neurons. Surprisingly, dopaminoceptive neurons without Dicer survive over the life of the animal. The lack of profound cell death contrasts with other mouse models in which Dicer has been ablated. These studies highlight the complicated nature of Dicer ablation in the brain and provide a useful mouse model for studying dopaminoceptive neuron function.

Conditional loss of Dicer disrupts cellular and tissue morphogenesis in the cortex and hippocampus.

To investigate the role of Dicer and microRNAs in the mammalian CNS, we used mice in which the second RNase III domain of Dicer was conditionally floxed. Conditional Dicer mice were bred with mice expressing an alpha-calmodulin kinase II Cre to selectively inactivate Dicer in excitatory forebrain neurons in vivo. Inactivation of Dicer results in an array of phenotypes including microcephaly, reduced dendritic branch elaboration, and large increases in dendritic spine length with no concomitant change in spine density. Microcephaly is likely caused by a 5.5-fold increase in early postnatal apoptosis in these animals as determined by active caspase-3 and TUNEL (terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling) staining in the cortex. Loss of Dicer function had no measurable effect on cortical lamination as determined by in situ hybridization, suggesting that microcephaly is not caused by defects in neuronal migration. Together, these results illustrate the in vivo significance of Dicer and miRNAs in the mammalian CNS and provide additional support for previous in vitro studies indicating that misregulation of this pathway may result in gross abnormalities in cell number and function that may contribute to a variety of neurological disorders.

Voltage-gated Na+ channel beta1 subunit-mediated neurite outgrowth requires Fyn kinase and contributes to postnatal CNS development in vivo.

Voltage-gated Na(+) channel beta1 subunits are multifunctional, participating in channel modulation and cell adhesion in vitro. We previously demonstrated that beta1 promotes neurite outgrowth of cultured cerebellar granule neurons (CGNs) via homophilic adhesion. Both lipid raft-associated kinases and nonraft fibroblast growth factor (FGF) receptors are implicated in cell adhesion molecule-mediated neurite extension. In the present study, we reveal that beta1-mediated neurite outgrowth is abrogated in Fyn and contactin (Cntn) null CGNs. beta1 protein levels are unchanged in Fyn null brains, whereas levels are significantly reduced in Cntn null brain lysates. FGF or EGF (epidermal growth factor) receptor kinase inhibitors have no effect on beta1-mediated neurite extension. These results suggest that beta1-mediated neurite outgrowth occurs through a lipid raft signaling mechanism that requires the presence of both fyn kinase and contactin. In vivo, Scn1b null mice show defective CGN axon extension and fasciculation indicating that beta1 plays a role in cerebellar microorganization. In addition, we find that axonal pathfinding and fasciculation are abnormal in corticospinal tracts of Scn1b null mice consistent with the suggestion that beta1 may have widespread effects on postnatal neuronal development. These data are the first to demonstrate a cell-adhesive role for beta1 in vivo. We conclude that voltage-gated Na(+) channel beta1 subunits signal via multiple pathways on multiple timescales and play important roles in the postnatal development of the CNS.

Sodium channel beta1 subunits promote neurite outgrowth in cerebellar granule neurons.

Many immunoglobulin superfamily members are integral in development through regulation of processes such as growth cone guidance, cell migration, and neurite outgrowth. We demonstrate that homophilic interactions between voltage-gated sodium channel beta1 subunits promote neurite extension in cerebellar granule neurons. Neurons isolated from wild-type or beta1(-/-) mice were plated on top of parental, mock-, or beta1-transfected fibroblasts. Wild-type neurons consistently showed increased neurite length when grown on beta1-transfected monolayers, whereas beta1(-/-) neurons showed no increase compared with control conditions. beta1-mediated neurite extension was mimicked using a soluble beta1 extracellular domain and was blocked by antibodies directed against the beta1 extracellular domain. Immunohistochemical analysis suggests that the beta1 and beta4 subunits, but not beta2 and beta3, are expressed in cerebellar Bergmann glia as well as granule neurons. These results suggest a novel role for beta1 during neuronal development and are the first demonstration of a functional role for sodium channel beta subunit-mediated cell adhesive interactions.