The evolutionary demand for rapid nerve impulse conduction led to the process of myelinationdependent organization of axons into distinct molecular domains. These domains include the node of Ranvier flanked by highly specialized paranodal domains where myelin loops and axolemma orchestrate the axoglial septate junctions. These junctions are formed by interactions between a glial isoform of neurofascin (Nfasc NF155 ) and axonal Caspr and Cont. Here we report the generation of myelinating glia-specific Nfasc NF155 null mouse mutants. These mice exhibit severe ataxia, motor paresis, and death before the third postnatal week. In the absence of glial Nfasc NF155 , paranodal axoglial junctions fail to form, axonal domains fail to segregate, and myelinated axons undergo degeneration. Electrophysiological measurements of peripheral nerves from Nfasc NF155 mutants revealed dramatic reductions in nerve conduction velocities. By using inducible PLP-CreER recombinase to ablate Nfasc NF155 in adult myelinating glia, we demonstrate that paranodal axoglial junctions disorganize gradually as the levels of Nfasc NF155 protein at the paranodes begin to drop. This coincides with the loss of the paranodal region and concomitant disorganization of the axonal domains. Our results provide the first direct evidence that the maintenance of axonal domains requires the fence function of the paranodal axoglial junctions. Together, our studies establish a central role for paranodal axoglial junctions in both the organization and the maintenance of axonal domains in myelinated axons. The anatomical organization of myelinated axons into distinct molecular domains is the basis for rapid propagation of action potentials in a saltatory manner (Hartline and Colman, 2007). Although the signal transduction mechanisms that underlie the axonal organization into specific domains (i.e., the node, the paranode, the juxtaparanode, and the internode) are poorly understood, considerable progress has been made in identifying key molecular components within these axonal domains. The paranodal region is unique in its organization and ultrastructural characteristics and contains specialized axoglial junctions referred to as the paranodal axoglial septate junctions, which resemble the ladder-like invertebrate septate junctions (Einheber et al., 1997;Pedraza et al., 2001; Banerjee et al., 2006a, b). Three major paranodal proteins have been identified: Caspr or paranodin (Einheber et al., 1997;Menegoz et al., 1997;Peles et al., 1997;Bhat et al., 2001), and a GPI-anchored neural cell adhesion molecule Contactin (Cont;Berglund et al., 1999;Boyle et al., 2001) on the axonal side, and the 155-kDa neurofascin isoform (Nfasc NF155 ) on the glial side (Tait et al., 2000;Charles et al., 2002). Although Nfasc NF155 is the only known glial paranodal protein, many proteins expressed in myelinating glia are required at some level in the formation and/or stability of the paranodal junctions (Coetzee et al., 1996;Griffiths et al., 1998;Ishibashi et al., 2002; LappeSie...

SUMMARY Calcitonin gene-related peptide (CGRP) is a classic molecular marker of peptidergic primary somatosensory neurons. Despite years of research, it is unknown if these neurons are required to sense pain or other sensory stimuli. Here, we found that genetic ablation of CGRPα-expressing sensory neurons reduced sensitivity to noxious heat, capsaicin and itch (histamine and chloroquine) and impaired thermoregulation but did not impair mechanosensation or β-alanine itch—stimuli associated with nonpeptidergic sensory neurons. Unexpectedly, ablation enhanced behavioral responses to cold stimuli and cold mimetics without altering peripheral nerve responses to cooling. Mechanistically, ablation reduced tonic and evoked activity in postsynaptic spinal neurons associated with TRPV1/heat, while profoundly increasing tonic and evoked activity in spinal neurons associated with TRPM8/cold. Our data reveal that CGRPα sensory neurons encode heat and itch and tonically cross-inhibit cold-responsive spinal neurons. Disruption of this crosstalk unmasks cold hypersensitivity, with mechanistic implications for neuropathic pain and temperature perception.

Accumulation of voltage gated sodium (Nav) channels at nodes of Ranvier is paramount for action potential propagation along myelinated fibers, yet the mechanisms governing nodal development, organization and stabilization remain unresolved. Here, we report that genetic ablation of the neuron-specific isoform of Neurofascin (NfascNF186) in vivo results in nodal disorganization, including loss of Nav channel and ankyrin-G (AnkG) enrichment at nodes in the peripheral (PNS) and central (CNS) nervous systems. Interestingly, the presence of paranodal domains failed to rescue nodal organization in the PNS and the CNS. Most importantly, using ultrastructural analysis, we demonstrate that the paranodal domains invade the nodal space in NfascNF186 mutant axons and occlude node formation. Our results suggest that NfascNF186-dependent assembly of the nodal complex acts as a molecular boundary to restrict the movement of flanking paranodal domains into the nodal area, thereby facilitating the stereotypic axonal domain organization and saltatory conduction along myelinated axons.

The formation of paranodal axo-glial junctions is critical for the rapid and efficient propagation of nerve impulses. Genetic ablation of genes encoding the critical paranodal proteins Caspr, contactin (Cont), and the myelinating glia-specific isoform of Neurofascin (Nfasc NF155 ) results in the disruption of the paranodal axo-glial junctions, loss of ion channel segregation, and impaired nerve conduction, but the mechanisms regulating their interactions remain elusive. Here, we report that loss of immunoglobulin (Ig) domains 5 and 6 in Nfasc NF155 in mice phenocopies complete ablation of Nfasc NF155. The mutant mice lack paranodal septate junctions, resulting in the diffusion of Caspr and Cont from the paranodes, and redistribution of the juxtaparanodal potassium channels toward the nodes. Although critical for Nfasc NF155 function, we find that Ig5-6 are dispensable for nodal Nfasc NF186 function. Moreover, in vitro binding assays using Ig5-6 deletion constructs reveal their importance for the association of Nfasc NF155 with Cont. These findings provide the first molecular evidence demonstrating domain-specific requirements controlling the association of the paranodal tripartite complex in vivo. Our studies further emphasize that in vivo structure/function analysis is necessary to define the unique protein-protein interactions that differentially regulate the functions of Neurofascins during axonal domain organization.

Angelman syndrome (AS) is a debilitating neurodevelopmental disorder caused by loss of function of the maternally inherited allele. It is currently unclear how the consequences of this genetic insult unfold to impair neurodevelopment. We reasoned that by elucidating the basis of microcephaly in AS, a highly penetrant syndromic feature with early postnatal onset, we would gain new insights into the mechanisms by which maternal loss derails neurotypical brain growth and function. Detailed anatomical analysis of both male and female maternal -null mice reveals that microcephaly in the AS mouse model is primarily driven by deficits in the growth of white matter tracts, which by adulthood are characterized by densely packed axons of disproportionately small caliber. Our results implicate impaired axon growth in the pathogenesis of AS and identify noninvasive structural neuroimaging as a potentially valuable tool for gauging therapeutic efficacy in the disorder. People who maternally inherit a deletion or nonfunctional copy of the gene develop Angelman syndrome (AS), a severe neurodevelopmental disorder. To better understand how loss of maternal function derails brain development, we analyzed brain structure in a maternal knock-out mouse model of AS. We report that the volume of white matter (WM) is disproportionately reduced in AS mice, indicating that deficits in WM development are a major factor underlying impaired brain growth and microcephaly in the disorder. Notably, we find that axons within the WM pathways of AS model mice are abnormally small in caliber. This defect is associated with slowed nerve conduction, which could contribute to behavioral deficits in AS, including motor dysfunction.

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