Using a mouse model system, we report here that perinatal and juvenile neuronal ablation of AnkG has differential consequences on nodal stability. AnkG creates immature nodes with abnormal morphology, which undergo accelerated destabilization within a month, resulting in rapid voltage-gated sodium (NaV) 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 MSDC-0160 over 10 months, primarily affecting IV spectrin, followed by NaV 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 MSDC-0160 of AnkG after nodal stabilization does not lead to rapid nodal disassembly but to loss of specific nodal components in a time-dependent manner. SIGNIFICANCE STATEMENT Nodes of Ranvier are the myelin-free gaps along myelinated axons that allow fast communication between neurons and their target cells by propagating action potentials in a saltatory manner. The cytoskeletal scaffolding protein ankyrin G (AnkG) has been thought to play an important role in node formation; however, its precise role in nodal assembly, stability, and maintenance is still not clear. By using spatiotemporal ablation of AnkG, we report its differential role in nodal maturation MSDC-0160 and stabilization. We show that early AnkG-deficient nodes fail to mature and undergo rapid destabilization. In contrast, nodes that assemble with AnkG are much more stable and undergo gradual disintegration with sequential loss of nodal components in the absence of AnkG. conditional knock-out mouse line (RRID:MGI:5538267) for AnkG ablation was provided by Vann Bennett (Duke University, Durham, NC; Paez-Gonzalez et al., 2011). The Single-neuron Labeling with Inducible Cre-mediated Knock-out ((derivative of transgenic mouse line (RRID:IMSR_JAX:019099) was obtained from the The Jackson Laboratory (stock #019099). Mouse lines used in these studies were on a mixed and genetic background. To obtain and animals were mated, which caused ubiquitous recombination of the allele and loss of one AnkG copy in strain to obtain females to get females were bred MMP2 to at 4C for 30 min. Supernatant was collected as a final lysate, heated for 5 min at 37C (to detect AnkG or pan-NaV channels) or boiled (for all other proteins), loaded onto SDS-PAGE gel, transferred to nitrocellulose, and probed with primary antibodies as previously described (Thaxton et al., 2011). Afterward, membranes were incubated in HRP- or IR-conjugated secondary antibodies followed by detection of the probes. Protein concentration in samples was estimated using a Pierce BCA protein assay kit (Thermo Scientific). Transmission electron microscopy. Animals were anesthetized and transcardially perfused with normal saline followed by 2.5% glutaraldehyde/4% PFA (dissolved in 0.16 m NaH2PO4/0.11 m NaOH buffer, to a final of pH 7.2C7.4) EM fixative for 30 min. All solutions were freshly prepared on the day of perfusion. After perfusion, entire mouse carcasses were postfixed for 2 weeks in the same EM fixative. SNs and SCs then were dissected out and incubated overnight in 0.1 m sodium cacodylate buffer followed by incubation in a 2% OsO4 solution and gradient ethanol dehydration. Samples were incubated in propylene oxide, left in 100% PolyBed resin with constant agitation for 36 h and embedded in flat molds at 55C for 36 h. After embedding, the molds were processed and imaged on a JEOL 1230 electron microscope at the University of Texas Health Science Center, San Antonio, Electron Microscopy Laboratory, as previously described (Green et al., 2013). nerve conduction measurements. All nerve conduction studies were performed under isoflurane anesthesia, and mouse external body temperature was constantly maintained between 33C and 34C with a warming lamp. Subdermal stainless steel needle electrodes were used for nerve stimulation with 0.02 ms impulses using the Nicolet Teca Synergy portable neurological.