Action potential conduction along myelinated axons depends on high densities of voltage-gated Na⁺ channels at the nodes of Ranvier. Flanking each node, paranodal junctions (paranodes) are formed between axons and Schwann cells in the peripheral nervous system (PNS) or oligodendrocytes in the CNS. Paranodal junctions contribute to both node assembly and maintenance. Despite their importance, the molecular mechanisms responsible for paranode assembly and maintenance remain poorly understood. βII spectrin is expressed in diverse cells and is an essential part of the submembranous cytoskeleton. Here, we show that Schwann cell βII spectrin is highly enriched at paranodes. To elucidate the roles of glial βII spectrin, we generated mutant mice lacking βII spectrin in myelinating glial cells by crossing mice with a floxed allele of Sptbn1 with Cnp-Cre mice, and analyzed both male and female mice. Juvenile (4 weeks) and middle-aged (60 weeks) mutant mice showed reduced grip strength and sciatic nerve conduction slowing, whereas no phenotype was observed between 8 and 24 weeks of age. Consistent with these findings, immunofluorescence microscopy revealed disorganized paranodes in the PNS and CNS of both postnatal day 13 and middle-aged mutant mice, but not in young adult mutant mice. Electron microscopy confirmed partial loss of transverse bands at the paranodal axoglial junction in the middle-aged mutant mice in both the PNS and CNS. These findings demonstrate that a spectrin-based cytoskeleton in myelinating glia contributes to formation and maintenance of paranodal junctions. SIGNIFICANCE STATEMENT Myelinating glia form paranodal axoglial junctions that flank both sides of the nodes of Ranvier. These junctions contribute to node formation and maintenance and are essential for proper nervous system function. We found that a submembranous spectrin cytoskeleton is highly enriched at paranodes in Schwann cells. Ablation of βII spectrin in myelinating glial cells disrupted the paranodal cell adhesion complex in both peripheral and CNSs, resulting in muscle weakness and sciatic nerve conduction slowing in juvenile and middle-aged mice. Our data show that a spectrin-based submembranous cytoskeleton in myelinating glia plays important roles in paranode formation and maintenance.

Nodes of Ranvier and associated paranodal and juxtaparanodal domains along myelinated axons are essential for normal function of the peripheral and central nervous systems. Disruption of these domains as well as increases in the reactive carbonyl species methylglyoxal are implicated as a pathophysiology common to a wide variety of neurological diseases. Here, using an ex vivo nerve exposure model, we show that increasing methylglyoxal produces paranodal disruption, evidenced by disorganized immunostaining of axoglial cell-adhesion proteins, in both sciatic and optic nerves from wild-type mice. Consistent with previous studies showing that increase of methylglyoxal can alter intracellular calcium homeostasis, we found upregulated activity of the calcium-activated protease calpain in sciatic nerves after methylglyoxal exposure. Methylglyoxal exposure altered clusters of proteins that are known as calpain substrates: ezrin in Schwann cell microvilli at the perinodal area and zonula occludens 1 in Schwann cell autotypic junctions at paranodes. Finally, treatment with the calpain inhibitor calpeptin ameliorated methylglyoxal-evoked ezrin loss and paranodal disruption in both sciatic and optic nerves. Our findings strongly suggest that elevated methylglyoxal levels and subsequent calpain activation contribute to the disruption of specialized axoglial domains along myelinated nerve fibers in neurological diseases.

Recent evidence suggests that alteration of axon initial segment (AIS) geometry (i.e., length or location along the axon) contributes to CNS dysfunction in neurological diseases. For example, AIS length is shorter in the prefrontal cortex of type 2 diabetic mice with cognitive impairment. To determine the key type 2 diabetes-related factor that produces AIS shortening we modified levels of insulin, glucose, or the reactive glucose metabolite methylglyoxal in cultures of dissociated cortices from male and female mice and quantified AIS geometry using immunofluorescent imaging of the AIS proteins AnkyrinG and βIV spectrin. Neither insulin nor glucose modification altered AIS length. Exposure to 100 but not 1 or 10 μmmethylglyoxal for 24 h resulted in accumulation of the methylglyoxal-derived advanced glycation end-product hydroimidazolone and produced reversible AIS shortening without cell death. Methylglyoxal-evoked AIS shortening occurred in both excitatory and putative inhibitory neuron populations and in the presence of tetrodotoxin (TTX). In single-cell recordings resting membrane potential was depolarized at 0.5–3 h and returned to normal at 24 h. In multielectrode array (MEA) recordings methylglyoxal produced an immediate ∼300% increase in spiking and bursting rates that returned to normal within 2 min, followed by a ∼20% reduction of network activity at 0.5–3 h and restoration of activity to baseline levels at 24 h. AIS length was unchanged at 0.5–3 h despite the presence of depolarization and network activity reduction. Nevertheless, these results suggest that methylglyoxal could be a key mediator of AIS shortening and disruptor of neuronal function during type 2 diabetes.

Recent evidence suggests that alteration of axon initial segment (AIS) geometry (i.e., length or position along the axon) contributes to CNS dysfunction in neurological diseases. For example, AIS length is shorter in the prefrontal cortex of type 2 diabetic mice with cognitive impairment. The key type 2 diabetes-related factor that alters AIS geometry is unknown. Here, we tested whether modifying the levels of insulin, glucose, or methylglyoxal, a reactive carbonyl species that is a metabolite of glucose, changes AIS geometry in mature cultures of dissociated postnatal mouse cortex using immunofluorescent imaging of the AIS proteins AnkyrinG and βIV spectrin. Neither insulin nor glucose modification appreciably altered AIS length. Elevation of methylglyoxal produced reversible AIS shortening without cell death. Multi-electrode array recordings revealed a biphasic effect of methylglyoxal on neuronal network activity: an immediate, transient ∼300% increase in spiking and bursting rates was followed by a ∼20% reduction from baseline at 3 h. AIS length was unchanged at 0.5 h or 3 h after adding methylglyoxal, whereas development of AIS shortening at 24 h was associated with restoration of spiking to baseline levels. Immunostaining for the excitatory neuron marker Ca ²⁺ /calmodulin-dependent protein kinase II alpha revealed AIS shortening in both excitatory and inhibitory neuron populations. This suggests that complex mechanisms maintain neuronal network operation after acute exposure to the disease metabolite methylglyoxal. Importantly, our results indicate that methylglyoxal could be a key mediator of AIS shortening during type 2 diabetes. Significance Statement: Small changes in the structure of the axon initial segment affect neuronal function and may be a key mediator of neurological complications in various disease states. However, the specific disease factors that mediate structural changes at the axon initial segment are relatively unknown. This is the first study to show that increase of methylglyoxal is sufficient to reduce axon initial segment length and modulate neuronal network function. Methylglyoxal is a disease factor implicated in a wide variety of conditions including type 2 diabetes, Alzheimer’s disease, and aging. Thus, these findings could significantly impact the understanding of neurological complications in several disease states and are of broad pathophysiological relevance.