Cellular and molecular alterations to muscles and neuromuscular synapses in a mouse model of MEGF10-related myopathy.

Loss-of-function mutations in MEGF10 lead to a rare and understudied neuromuscular disorder known as MEGF10-related myopathy. There are no treatments for the progressive respiratory distress, motor impairment, and structural abnormalities in muscles caused by the loss of MEGF10 function. In this study, we deployed cellular and molecular assays to obtain additional insights about MEGF10-related myopathy in juvenile, young adult, and middle-aged Megf10 knockout (KO) mice. We found fewer muscle fibers in juvenile and adult Megf10 KO mice, supporting published studies that MEGF10 regulates myogenesis by affecting satellite cell differentiation. Interestingly, muscle fibers do not exhibit morphological hallmarks of atrophy in either young adult or middle-aged Megf10 KO mice. We next examined the neuromuscular junction (NMJ), in which MEGF10 has been shown to concentrate postnatally, using light and electron microscopy. We found early and progressive degenerative features at the NMJs of Megf10 KO mice that include increased postsynaptic fragmentation and presynaptic regions not apposed by postsynaptic nicotinic acetylcholine receptors. We also found perisynaptic Schwann cells intruding into the NMJ synaptic cleft. These findings strongly suggest that the NMJ is a site of postnatal pathology in MEGF10-related myopathy. In support of these cellular observations, RNA-seq analysis revealed genes and pathways associated with myogenesis, skeletal muscle health, and NMJ stability dysregulated in Megf10 KO mice compared to wild-type mice. Altogether, these data provide new and valuable cellular and molecular insights into MEGF10-related myopathy.

Origin, identity, and function of terminal Schwann cells.

The highly specialized nonmyelinating glial cells present at somatic peripheral nerve endings, known collectively as terminal Schwann cells (TSCs), play critical roles in the development, function and repair of their motor and sensory axon terminals and innervating tissue. Over the past decades, research efforts across various vertebrate species have revealed that while TSCs are a diverse group of cells, they share a number of features among them. In this review, we summarize the state-of-knowledge about each TSC type and explore the opportunities that TSCs provide to treat conditions that afflict peripheral axon terminals.

Cellular and molecular evidence that synaptic Schwann cells contribute to aging of mouse neuromuscular junctions.

Age-induced degeneration of the neuromuscular junction (NMJ) is associated with motor dysfunction and muscle atrophy. While the impact of aging on the NMJ presynapse and postsynapse is well-documented, little is known about the changes perisynaptic Schwann cells (PSCs), the synaptic glia of the NMJ, undergo during aging. Here, we examined PSCs in young, middle-aged, and old mice in three muscles with different susceptibility to aging. Using light and electron microscopy, we found that PSCs acquire age-associated cellular features either prior to or at the same time as the onset of NMJ degeneration. Notably, we found that aged PSCs fail to completely cap the NMJ even though they are more abundant in old compared with young mice. We also found that aging PSCs form processes that either intrude into the synaptic cleft or guide axonal sprouts to innervate other NMJs. We next profiled the transcriptome of PSCs and other Schwann cells (SCs) to identify mechanisms altered in aged PSCs. This analysis revealed that aged PSCs acquire a transcriptional pattern previously shown to promote phagocytosis that is absent in other SCs. It also showed that aged PSCs upregulate unique pro-inflammatory molecules compared to other aged SCs. Interestingly, neither synaptogenesis genes nor genes that are typically upregulated by repair SCs were induced in aged PSCs or other SCs. These findings provide insights into cellular and molecular mechanisms that could be targeted in PSCs to stave off the deleterious effects of aging on NMJs.

Kir4.1 is specifically expressed and active in non-myelinating Schwann cells.

Non-myelinating Schwann cells (NMSC) play important roles in peripheral nervous system formation and function. However, the molecular identity of these cells remains poorly defined. We provide evidence that Kir4.1, an inward-rectifying K+ channel encoded by the KCNJ10 gene, is specifically expressed and active in NMSC. Immunostaining revealed that Kir4.1 is present in terminal/perisynaptic SCs (TPSC), synaptic glia at neuromuscular junctions (NMJ), but not in myelinating SCs (MSC) of adult mice. To further examine the expression pattern of Kir4.1, we generated BAC transgenic Kir4.1-CreERT2 mice and crossed them to the tdTomato reporter line. Activation of CreERT2 with tamoxifen after the completion of myelination onset led to robust expression of tdTomato in NMSC, including Remak Schwann cells (RSC) along peripheral nerves and TPSC, but not in MSC. In contrast, activating CreERT2 before and during the onset of myelination led to tdTomato expression in NMSC and MSC. These observations suggest that immature SC express Kir4.1, and its expression is then downregulated selectively in myelin-forming SC. In support, we found that while activating CreERT2 induces tdTomato expression in immature SC, it fails to induce tdTomato in MSC associated with sensory axons in culture. NMSC derived from neonatal sciatic nerve were shown to express Kir4.1 and exhibit barium-sensitive inwardly rectifying macroscopic K+ currents. Thus, this study identified Kir4.1 as a potential modulator of immature SC and NMSC function. Additionally, it established a novel transgenic mouse line to introduce or delete genes in NMSC.

Murine muscle stem cell response to perturbations of the neuromuscular junction are attenuated with aging.

During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume and function impacting mobility and quality of life. Muscle loss is often associated with denervation and a loss of resident muscle stem cells (satellite cells or MuSCs); however, the relationship between MuSCs and innervation has not been established. Herein, we administered severe neuromuscular trauma to a transgenic murine model that permits MuSC lineage tracing. We show that a subset of MuSCs specifically engraft in a position proximal to the neuromuscular junction (NMJ), the synapse between myofibers and motor neurons, in healthy young adult muscles. In aging and in a mouse model of neuromuscular degeneration (Cu/Zn superoxide dismutase knockout - Sod1-/-), this localized engraftment behavior was reduced. Genetic rescue of motor neurons in Sod1-/- mice reestablished integrity of the NMJ in a manner akin to young muscle and partially restored MuSC ability to engraft into positions proximal to the NMJ. Using single cell RNA-sequencing of MuSCs isolated from aged muscle, we demonstrate that a subset of MuSCs are molecularly distinguishable from MuSCs responding to myofiber injury and share similarity to synaptic myonuclei. Collectively, these data reveal unique features of MuSCs that respond to synaptic perturbations caused by aging and other stressors.

Morphological remodeling during recovery of the neuromuscular junction from terminal Schwann cell ablation in adult mice.

Schwann cells (SCs) are integral to the formation and function of the peripheral nervous system (PNS). Exemplifying their importance, the loss or dysfunction of SCs is a feature of a myriad of diseases and conditions that compromise the PNS. Thus, it remains essential to understand the rules that govern the proliferation, differentiation and reconnection of Schwann cells with peripheral axons. Here, we examined the consequences of locally and acutely ablating terminal Schwann cells (tSCs) at the adult mouse neuromuscular junction (NMJ) by using mice expressing diphtheria toxin receptor (DTR) preferentially in tSCs compared to myelinating SCs followed by local application of diphtheria toxin (DTX). After DTX application, tSCs died but, importantly and contrary to expectations, their associated motor axons did not fully degenerate. Within 3 weeks, tSCs returned and reestablished coverage of the synapse with increased numbers. Furthermore, the post-synaptic muscle fibers displayed increased distinct clusters of acetylcholine receptors and axon terminals exhibited numerous terminal varicosities. The lack of degeneration of bare motor axon terminals and the morphological remodeling that occurs upon the return of tSCs to the NMJ may have wider implications for the mechanisms governing tSC occupancy of the adult NMJ and for conditions that adversely affect tSCs.

Exclusive vital labeling of myonuclei for studying myonuclear arrangement in mouse skeletal muscle tissue.

BACKGROUND: The arrangement of myonuclei in skeletal muscle tissue has long been used as a biomarker for muscle health, but there is a dearth of in vivo exploration of potential effects of myonuclear organization on the function and regeneration of skeletal muscle because traditional nuclear stains are performed on postmortem tissue. Therefore, we sought a transgenic method to produce a selective and persistent myonuclear label in whole muscles of living mice. METHODS: We bred together a mouse line with skeletal muscle fiber-selective expression of Cre recombinase and a second mouse line with a Cre-inducible fluorescently tagged histone protein to generate a mouse line that produces a myonuclear label suitable for vital imaging and histology of fixed tissue. We tested the effectiveness of this vital label in three conditions known to generate abnormal myonuclear positioning. First, we injured myofibers of young mice with cardiotoxin. Second, this nuclear label was bred into a murine model of Duchenne muscular dystrophy. Finally, we examined old mice from this line that have undergone the natural aging process. Welch's t test was used to compare wild type and transgenic mice. RESULTS: The resulting mouse line transgenically produces a vital red fluorescent label of myonuclei, which facilitates their in vivo imaging in skeletal muscle tissue. Transgenic fluorescent labeling of myonuclei has no significant effect on skeletal muscle function, as determined by twitch and tetanic force recordings. In each muscle examined, including those under damaged, dystrophic, and aged conditions, the labeled myonuclei exhibit morphology consistent with established literature, and reveal a specialized arrangement of subsynaptic myonuclei at the neuromuscular junction. CONCLUSIONS: Taken together, our results demonstrate that this mouse line provides a versatile tool to selectively visualize myonuclei within both living and fixed preparations of healthy, injured, diseased, and aged muscles.

Nerve sprouting capacity in a pharmacologically induced mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by loss-of-function mutations in the survival of motoneuron gene 1 (SMN1). SMA is characterized by motoneuron death, skeletal muscle denervation and atrophy. Disease severity inversely correlates with copy number of a second gene (SMN2), which harbors a splicing defect that causes the production of inadequate levels of functional SMN protein. Small molecules that modify SMN2 splicing towards increased production of functional SMN significantly ameliorate SMA phenotypes in mouse models of severe SMA. At suboptimal doses, splicing modifiers, such as SMN-C1, have served to generate mice that model milder SMA, referred to as pharmacological SMA mice, which survive into early adulthood. Nerve sprouting at endplates, known as terminal sprouting, is key to normal muscle fiber reinnervation following nerve injury and its promotion might mitigate neuromuscular symptoms in mild SMA. Sprouting has been difficult to study in severe SMA mice due to their short lifespan. Here, we show that pharmacological SMA mice are capable of terminal sprouting following reinnervation that is largely SMN-C1 dose-independent, but that they display a reinnervation delay that is critically SMN-C1 dose-dependent. Data also suggest that SMN-C1 can induce by itself a limited terminal sprouting response in SMA and wild-type normally-innervated endplates.

Polyethylene glycol solutions rapidly restore and maintain axonal continuity, neuromuscular structures, and behaviors lost after sciatic nerve transections in female rats.

Complete severance of major peripheral mixed sensory-motor nerve proximally in a mammalian limb produces immediate loss of action potential conduction and voluntary behaviors mediated by the severed distal axonal segments. These severed distal segments undergo Wallerian degeneration within days. Denervated muscles atrophy within weeks. Slowly regenerating (∼1 mm/day) outgrowths from surviving proximal stumps that often nonspecifically reinnervate denervated targets produce poor, if any, restoration of lost voluntary behaviors. In contrast, in this study using completely transected female rat sciatic axons as a model system, we provide extensive morphometric, immunohistochemical, electrophysiological, and behavioral data to show that these adverse outcomes are avoided by microsuturing closely apposed axonal cut ends (neurorrhaphy) and applying a sequence of well-specified solutions, one of which contains polyethylene glycol (PEG). This "PEG-fusion" procedure within minutes reestablishes axoplasmic and axolemmal continuity and signaling by nonspecifically fusing (connecting) closely apposed open ends of severed motor and/or sensory axons at the lesion site. These PEG-fused axons continue to conduct action potentials and generate muscle action potentials and muscle twitches for months and do not undergo Wallerian degeneration. Continuously innervated muscle fibers undergo much less atrophy compared with denervated muscle fibers. Dramatic behavioral recovery to near-unoperated levels occurs within days to weeks, almost certainly by activating many central nervous system and peripheral nervous system synaptic and other plasticities, some perhaps to a greater extent than most neuroscientists would expect. Negative control transections in which neurorrhaphy and all solutions except the PEG-containing solution are applied produce none of these remarkably fortuitous outcomes observed for PEG-fusion.

Effects of extracellular calcium and surgical techniques on restoration of axonal continuity by polyethylene glycol fusion following complete cut or crush severance of rat sciatic nerves.

Complete crush or cut severance of sciatic nerve axons in rats and other mammals produces immediate loss of axonal continuity. Loss of locomotor functions subserved by those axons is restored only after months, if ever, by outgrowths regenerating at ∼1 mm/day from the proximal stumps of severed axonal segments. The distal stump of a severed axon typically begins to degenerate in 1-3 days. We recently developed a polyethylene glycol (PEG) fusion technology, consisting of sequential exposure of severed axonal ends to hypotonic Ca(2+) -free saline, methylene blue, PEG in distilled water, and finally Ca(2+) -containing isotonic saline. This study examines factors that affect the PEG fusion restoration of axonal continuity within minutes, as measured by conduction of action potentials and diffusion of an intracellular fluorescent dye across the lesion site of rat sciatic nerves completely cut or crush severed in the midthigh. Also examined are factors that affect the longer-term PEG fusion restoration of lost behavioral functions within days to weeks, as measured by the sciatic functional index. We report that exposure of cut-severed axonal ends to Ca(2+) -containing saline prior to PEG fusion and stretch/tension of proximal or distal axonal segments of cut-severed axons decrease PEG fusion success. Conversely, trimming cut-severed ends in Ca(2+) -free saline just prior to PEG fusion increases PEG fusion success. PEG fusion prevents or retards the Wallerian degeneration of cut-severed axons, as assessed by measures of axon diameter and G ratio. PEG fusion may produce a paradigm shift in the treatment of peripheral nerve injuries. © 2016 Wiley Periodicals, Inc.