Axon initial segments (AISs) and nodes of Ranvier are sites of action potential generation and propagation, respectively. Both domains are enriched in sodium channels complexed with adhesion molecules (neurofascin [NF] 186 and NrCAM) and cytoskeletal proteins (ankyrin G and βIV spectrin). We show that the AIS and peripheral nervous system (PNS) nodes both require ankyrin G but assemble by distinct mechanisms. The AIS is intrinsically specified; it forms independent of NF186, which is targeted to this site via intracellular interactions that require ankyrin G. In contrast, NF186 is targeted to the node, and independently cleared from the internode, by interactions of its ectodomain with myelinating Schwann cells. NF186 is critical for and initiates PNS node assembly by recruiting ankyrin G, which is required for the localization of sodium channels and the entire nodal complex. Thus, initial segments assemble from the inside out driven by the intrinsic accumulation of ankyrin G, whereas PNS nodes assemble from the outside in, specified by Schwann cells, which direct the NF186-dependent recruitment of ankyrin G.

Summary We have investigated the source(s) and targeting of components to PNS nodes of Ranvier. We show adhesion molecules are diffusible within the axon membrane and accumulate at forming nodes from local axonal sources. In contrast, ion channels and cytoskeletal components exhibit limited planar mobility and require transport to the node. We have characterized further targeting of NF186, an axonal adhesion molecule, which pioneers node formation. NF186 redistributes to nascent nodes from a mobile, surface pool. This initial accumulation, and clearance from the internode require extracellular interactions whereas subsequent targeting to mature nodes, i.e. those flanked by paranodal junctions, requires intracellular interactions. After incorporation into the node, NF186 is immobile, stable, and promotes node integrity. Thus nodes assemble from two sources: adhesion molecules, which initiate assembly, accumulate by diffusion trapping via interactions with Schwann cells whereas ion channels and cytoskeletal components accumulate via subsequent transport. In mature nodes, components turnover slowly and are replenished via transport.

The molecular mechanisms that govern the maturation of oligodendrocyte lineage cells remain unclear. Emerging studies have shown that N 6 -methyladenosine (m 6 A), the most common internal RNA modification of mammalian mRNA, plays a critical role in various developmental processes. Here, we demonstrate that oligodendrocyte lineage progression is accompanied by dynamic changes in m 6 A modification on numerous transcripts. In vivo conditional inactivation of an essential m 6 A writer component, METTL14, results in decreased oligodendrocyte numbers and CNS hypomyelination, although oligodendrocyte precursor cell (OPC) numbers are normal. In vitro Mettl14 ablation disrupts postmitotic oligodendrocyte maturation and has distinct effects on OPC and oligodendrocyte transcriptomes. Moreover, the loss of Mettl14 in oligodendrocyte lineage cells causes aberrant splicing of myriad RNA transcripts, including those that encode the essential paranodal component neurofascin 155 (NF155). Together, our findings indicate that dynamic RNA methylation plays an important regulatory role in oligodendrocyte development and CNS myelination.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, characterized by motor neuron death in the brain and spinal cord. Mutations in the Cu/Zn superoxide dismutase (SOD1) gene account for ~20% of all familial ALS forms, corresponding to 1%-2% of all ALS cases. One of the suggested mechanisms by which mutant SOD1 (mtSOD1) exerts its toxic effects involves intracellular accumulation of abnormal mtSOD1 aggregates, which trigger endoplasmic reticulum (ER) stress and activate its adaptive signal transduction pathways, including the unfolded protein response (UPR). PERK, an eIF2α kinase, is central to the UPR and is the most rapidly activated pathway in response to ER stress. Previous reports using mtSOD1 transgenic mice indicated that genetic or pharmacological enhancement of the UPR-PERK pathway may be effective in treating ALS. We investigated the response to PERK haploinsufficiency, and the response to deficiency of its downstream effectors GADD34 and CHOP, in five distinct lines of mtSOD1 mice. We demonstrate that, in contrast to a previously published study, PERK haploinsufficiency has no effect on disease in all mtSOD1 strains examined. We also show that deficiency of GADD34, which enhances the UPR by prolonging the phosphorylation of eIF2α does not ameliorate disease in these mtSOD1 mouse strains. Finally, we demonstrate that genetic ablation of CHOP transcription factor, which is known to be pro-apoptotic, does not ameliorate disease in mtSOD1 mice. Cumulatively, our studies reveal that neither genetic inhibition of the UPR via ablation of PERK, nor genetic UPR enhancement via ablation of GADD34, is beneficial for mtSOD1-induced motor neuron disease. Therefore, the PERK pathway is not a likely target for therapeutic intervention in ALS.

While connexin46 (Cx46) and connexin50 (Cx50) are crucial for maintaining lens transparency and growth, the contributions of a more recently identified lens fiber connexin, Cx23, are poorly understood. Therefore, we studied the consequences of absence of Cx23 in mouse lenses. Cx23-null mice were generated by homologous Cre recombination. Cx23 mRNA was abundantly expressed in wild type lenses, but not in Cx23-null lenses. The transparency and refractive properties of Cx23-null lenses were similar to wild type lenses when examined by darkfield microscopy. Neither the focusing ability nor the light scattering was altered in the Cx23-null lenses. While both Cx46 and Cx50 localized to appositional fiber cell membranes (as in wild type lenses), their levels were consistently (but not significantly) decreased in homozygous Cx23-null lenses. These results suggest that although Cx23 expression can influence the abundance of the co-expressed lens fiber connexins, heterozygous or homozygous expression of a Cx23-null allele does not alter lens transparency.