Intricate molecular interactions between neurons and glial cells underlie the creation of unique domains that are essential for saltatory conduction of action potentials by myelinated axons. Previously, the cell surface adhesion molecule Neurofascin (Nfasc) has been shown to have a dual-role in the establishment of axonal domains from both the glial and neuronal interface. While the neuron-specific isoform of Neurofascin (NF186) is indispensable for clustering of voltage-gated sodium channels at nodes of Ranvier; the glial-specific isoform of Neurofascin (NF155) is required for myelinating glial cells to organize the paranodal domain. Although many studies have addressed the individual roles of NF155 and NF186 in assembling paranodes and nodes, respectively; critical questions about their roles in the maintenance and long-term health of the myelinated axons remain, which we aimed to address in these studies. Here using spatiotemporal ablation of Neurofascin in neurons alone or together with myelinating glia, we report that loss of NF186 individually from postnatal mice leads to progressive nodal destabilization and axonal degeneration. While individual ablation of paranodal NF155 does not disrupt nodes of Ranvier; loss of NF186 combined with NF155 causes more accelerated nodal destabilization than loss of NF186 alone, providing strong evidence regarding a supporting role for paranodes in nodal maintenance. In both cases of NF186 loss, myelinating axons show ultrastructural changes and degeneration. Our studies reveal that long-term maintenance of nodes and ultimately the health of axons is correlated with the stability of NF186 within the nodal complex and the presence of auxiliary paranodes.

The mechanisms that govern node of Ranvier organization, stability, and long-term maintenance remain to be fully elucidated. One of the molecular components of the node is the cytoskeletal scaffolding protein, ankyrin G (AnkG), which interacts with multiple members of the nodal complex. The role of AnkG in nodal organization and maintenance is still not clearly defined as to whether AnkG functions as an initial nodal organizer or whether it functions as a nodal stabilizer after the nodal complex has been assembled. Using a mouse model system, we report here that perinatal and juvenile neuronal ablation of AnkG has differential consequences on nodal stability. Early loss of AnkG creates immature nodes with abnormal morphology, which undergo accelerated destabilization within a month, resulting in rapid voltage-gated sodium (Na V ) channel and ␤IV spectrin loss with reduced effects on neurofascin 186. On the other hand, late ablation of AnkG from established nodal complexes leads to slow but progressive nodal destabilization over 10 months, primarily affecting ␤IV spectrin, followed by Na V channels, with modest impact on neurofascin 186. We also show that ankyrin R and ␤I spectrin are not sufficient to prevent nodal disorganization after AnkG ablation. Additionally, nodal disorganization in both early and late AnkG mutants is accompanied by axonal pathology and neurological dysfunction. Together, our results suggest that AnkG plays an indispensable role in the maturation and long-term stabilization of the newly assembled nodal complex, and that loss of AnkG after nodal stabilization does not lead to rapid nodal disassembly but to loss of specific nodal components in a time-dependent manner.

The roles of myelin in maintaining axonal integrity and action potential (AP) propagation are well established, but its role in synapse maintenance and neurotransmission remains largely understudied. Here, we investigated how Purkinje axon myelination regulates synaptic transmission in the Purkinje to deep cerebellar nuclei (DCN) synapses using the Long Evans Shaker (LES) rat, which lacks compact myelin and thus displays severe locomotion deficits. DCN neurons fired spontaneous action potentials (APs), whose frequencies were dependent on the extent of myelin. In the LES cerebellum with severe myelin deficiency, DCN neurons were hyper-excitable, exhibiting spontaneous AP firing at a much higher frequency compared to those from wild type (LE) and heterozygote (LEHet) rats. The hyper-excitability in LES DCN neurons resulted from reduced inhibitory GABAergic inputs from Purkinje cells to DCN neurons. Corresponding with functional alterations including failures of AP propagation, electron microscopic analysis revealed anatomically fewer active zones at the presynaptic terminals of Purkinje cells in both LEHet and LES rats. Taken together, these studies suggest that proper axonal myelination critically regulates presynaptic terminal structure and function and directly impacts synaptic transmission in the Purkinje cell-DCN cell synapse in the cerebellum.

Disorganization of nodes of Ranvier is associated with motor and sensory dysfunctions. Mechanisms that allow nodal recovery during pathological processes remain poorly understood. A highly enriched nodal cytoskeletal protein βIV spectrin anchors and stabilizes the nodal complex to actin cytoskeleton. Loss of murine βIV spectrin allows the initial nodal organization, but causes gradual nodal destabilization. Mutations in human βIV spectrin cause auditory neuropathy and impairment in motor coordination. Similar phenotypes are caused by nodal disruption due to demyelination. Here we report on the precise timelines of nodal disorganization and reorganization by following disassembly and reassembly of key nodal proteins in βIV spectrin mice of both sexes before and after βIV spectrin re-expression at specifically chosen developmental time points. We show that the timeline of nodal restoration has different outcomes in the PNS and CNS with respect to nodal reassembly and functional restoration. In the PNS, restoration of nodes occurs within 1 month regardless of the time of βIV spectrin re-expression. In contrast, the CNS nodal reorganization and functional restoration occurs within a critical time window; after that, nodal reorganization diminishes, leading to less efficient motor recovery. We demonstrate that timely restoration of nodes can improve both the functional properties and the ultrastructure of myelinated fibers affected by long-term nodal disorganization. Our studies, which indicate a critical timeline for nodal restoration together with overall motor performance and prolonged life span, further support the idea that nodal restoration is more beneficial if initiated before any axonal damage, which is critically relevant to demyelinating disorders. Nodes of Ranvier are integral to efficient and rapid signal transmission along myelinated fibers. Various demyelinating disorders are characterized by destabilization of the nodal molecular complex, accompanied by severe reduction in nerve conduction and the onset of motor and sensory dysfunctions. This study is the first to report reassembly of destabilized nodes with sequential improvement in overall motor performance. Our study reveals that nodal restoration is achievable before any axonal damage, and that long-term nodal destabilization causes irreversible axonal structural changes that prevent functional restoration. Our studies provide significant insights into timely restoration of nodal domains as a potential therapeutic approach in treatment of demyelinating disorders.

Mutations in human tuberous sclerosis complex (TSC) genes TSC1 and TSC2 are the leading causes of developmental brain abnormalities and large tumors in other tissues. Murine Tsc1/2 have been shown to negatively regulate the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in most tissues, and this pathway has been shown to be essential for proper oligodendrocytes/Schwann cell differentiation and myelination. Here, we report that ablation of Tsc1 gene specifically in oligodendrocytes/Schwann cells activates mTORC1 signaling resulting in severe motor disabilities, weight loss, and early postnatal death. The mutant mice of either sex showed reduced myelination, disrupted paranodal domains in myelinated axons, and disorganized unmyelinated Remak bundles. mRNA and protein expression analyses revealed strong reduction in the RNA–binding protein Quaking (Qk) and the 155 kDa glial Neurofascin (NfascNF155). Re-introduction of exogenous Qk gene in Tsc1 mutant oligodendrocytes restored NfascNF155 protein levels indicating that Qk is required for the stabilization of NfascNF155 mRNA. Interestingly, injection of Rapamycin, a pharmacological mTORC1 inhibitor, to pregnant mothers increased the lifespan of the mutant offspring, restored myelination as well as the levels of Qk and NfascNF155, and consequently the organization of the paranodal domains. Together our studies show a critical role of mTORC1 signaling in the differentiation of myelinating glial cells and proper organization of axonal domains and provide insights into TSC-associated myelinated axon abnormalities.