Terminal Schwann Cells Research Articles

Differential evaluation of neuromuscular injuries to understand re-innervation at the neuromuscular junction

Peripheral nerve-crush injury is a well-established model of neuromuscular junction (NMJ) denervation and subsequent re-innervation. Functionally, the skeletal muscle follows a similar pattern as neural recovery, with immediate loss of force production that steadily improves in parallel with rates of re-innervation. On the other hand, traumatic injury to the muscle itself, specifically volumetric muscle loss (VML), results in an irrecoverable loss of muscle function. Recent work has indicated significant impairments to the NMJ following this injury that appear chronic in nature, alongside the lack of functional recovery. Thus, the goal of this study was to compare the effects of nerve and muscle injury on NMJ remodeling. Even numbers of adult male and female mice were used with three experimental groups: injury Naïve, nerve crush, and VML injury; and three terminal timepoints: 3-, 48-, and 112-days post-injury. Confirming the assumed recoverability of the two injury models, we found in vivo maximal torque was fully restored following nerve-crush injury but remained at a significant deficit following VML. Compared to injury Naïve and nerve-crush injury, we found VML results in aberrantly high trophic signaling (e.g., neuregulin-1) and numbers of supporting cells, including terminal Schwann cells and sub-synaptic nuclei. In some cases, sex differences were detected, including higher rates of innervation in females than males. Both nerve crush and VML injury display chronic changes to NMJ morphology, such as increased fragmentation and nerve sprouting, highlighting the potential of VML for modeling NMJ regeneration in adulthood, alongside the established nerve-injury models.

Mitochondrial disorders are associated with morphological neuromuscular junction defects

We aimed to evaluate whether inherited mitochondrial dysfunction is associated with neuromuscular junction remodeling in patients with mitochondrial disorders.Muscle biopsies from 15 patients with mitochondrial disorders and 10 control patients were analyzed through immunostaining for various neuromuscular junction components. The patient group, with a mean age of 49.9 years, exhibited various mitochondrial disorders including chronic progressive external ophthalmoplegia, Kearns-Sayre syndrome, and mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes. Patients with mitochondrial disorders had a high percentage of remodeled (p=0.0001), neoformed (p=0.0049) and dilated (p=0.016) endplates. There was a trend toward an increased proportion of neuromuscular junctions with terminal Schwann cell extension in these patients (p=0.052). No significant difference was found in myofiber diameter between the groups. The observed neuromuscular junction defects varied widely across different mitochondrial disorder phenotypes and were present even without accompanying muscle weakness or neuropathy.This suggest that mitochondrial disorders are associated with a primary NMJ remodeling independent of muscle structural damage. Pathomechanisms underpinning this remodeling of the neuromuscular junction, as well as clinical factors predictive of this remodeling, remain to be fully characterized.

Inducible deletion of endothelial cell Efnb2 delays capillary regeneration and attenuates myofibre reinnervation following myotoxin injury in mice.

Acute injury of skeletal muscle disrupts myofibres, microvessels and motor innervation. Myofibre regeneration is well characterized, however its relationship with the regeneration of microvessels and motor nerves is undefined. Endothelial cell (EC) ephrin-B2 (Efnb2) is required for angiogenesis during embryonic development and promotes neurovascular regeneration in the adult. We hypothesized that, following acute injury to skeletal muscle, loss of EC Efnb2 would impair microvascular regeneration and the recovery of neuromuscular junction (NMJ) integrity. Mice (aged 3-6months) were bred for EC-specific conditional knockout (CKO) of Efnb2 following tamoxifen injection with non-injected CKO mice as controls (CON). The gluteus maximus, tibialis anterior or extensor digitorum longus muscle was then injured with local injection of BaCl2. Intravascular staining with wheat germ agglutinin revealed diminished capillary area in the gluteus maximus of CKO vs. CON at 5days post-injury (dpi); both recovered to uninjured (0dpi) level by 10dpi. At 0dpi, tibialis anterior isometric force of CKO was less than CON. At 10dpi, isometric force was reduced by half in both groups. During intermittent contractions (75Hz, 330mss-1, 120s), isometric force fell during indirect (sciatic nerve) stimulation whereas force was maintained during direct (electrical field) stimulation of myofibres. Neuromuscular transmission failure correlated with perturbed presynaptic (terminal Schwann cells) and postsynaptic (nicotinic acetylcholine receptors) NMJ morphology in CKO. Resident satellite cell number on extensor digitorum longus myofibres did not differ between groups. Following acute injury of skeletal muscle, loss of Efnb2 in ECs delays capillary regeneration and attenuates recovery of NMJ structure and function. KEY POINTS: The relationship between microvascular regeneration and motor nerve regeneration following skeletal muscle injury is undefined. Expression of Efnb2 in endothelial cells (ECs) is essential to vascular development and promotes neurovascular regeneration in the adult. To test the hypothesis that EfnB2 in ECs is required for microvascular regeneration and myofibre reinnervation, we induced conditional knockout of Efnb2 in ECs of mice. Acute injury was then induced by BaCl2 injection into gluteus maximus, tibialis anterior or extensor digitorum longus (EDL) muscle. Capillary regeneration was reduced at 5days post-injury (dpi) in gluteus maximus of conditional knockout vs. controls; at 10dpi, neither differed from uninjured. Nerve stimulation revealed neuromuscular transmission failure in tibialis anterior with perturbed neuromuscular junction structure. Resident satellite cell number on EDL myofibres did not differ between groups. Conditional knockout of EC Efnb2 delays capillary regeneration and attenuates recovery of neuromuscular junction structure and function.

Secreted Phosphoprotein-1 Mediates Muscle Resident Schwann Cell Dynamics and NMJ Regeneration Post Nerve Injury

The maintenance of neuromuscular junctions (NMJs) is crucial for combating degenerative neuromuscular diseases. Maintenance of the NMJ involves turnover and regeneration with these processes dependant on the activation of Schwann cells by specific molecular cues within the muscular environment. Secreted phosphoprotein-1 (SPP1), known as osteopontin, is a chemokine that is rapidly upregulated following nerve injury. Its precise influence on Schwann cell-mediated nerve regeneration, however, remains to be elucidated. Our recent single-cell RNA sequencing data has uncovered a previously unrecognized interaction between myelinating Schwann cells and terminal Schwann cells (tSCs), mediated by SPP1 signaling. We propose that this interaction, orchestrated by SPP1, is crucial for tSC proliferation and ultimately, successful reinnervation of muscle fibers post-injury. To investigate the effect of nerve injury on SPP1 signaling gene expression within skeletal muscle, we conducted sciatic nerve crush experiments in two-month-old C57BL/6 mice. We compared gene expression profiles associated with denervation and SPP1 signaling in the gastrocnemius muscles of uninjured controls to those at 7, 14, and 28 days post-injury (DPI). We found significant transient increases in Spp1 and its receptor genes Cd44 and Itgav, peaking at 7 DPI and returning to baseline by 14 DPI. To further elucidate the role of SPP1 in muscle reinnervation and its impact on tSC responses following nerve injury, we conducted peroneal nerve injuries in S100-GFP transgenic mice and administered either an SPP1 neutralizing antibody (Spp1-nAb) or a saline solution intramuscularly. Direct muscle stimulation force testing revealed comparable evoked forces between both groups, however nerve-evoked muscle forces were significantly diminished by 43% in the Spp1-nAb-treated mice. The Spp1-nAb treated mice also demonstrated a 38% reduction in nerve-to-muscle force ratios compared to the saline group. Histological assessments post-injury showed that mice given Spp1-nAb at 7 DPI had markedly reduced nerve terminal and synaptic areas compared to saline-treated controls. Similarly, a greater percentage of muscle fibers remained denervated with Spp1-nAb treatment (67% vs 33% in saline), accompanied by a reduction in NMJ synaptic area. An analysis of tSCs further indicated a decreased count per NMJ and a reduction in overall tSC area upon SPP1 signaling inhibition. Our comprehensive analysis establishes the essential function of Spp1 in muscle reinnervation, as evidenced by its role in enhancing tSC numbers at the NMJ. These findings provide significant contributions to our understanding of the molecular mechanisms essential for NMJ repair and maintenance, with a particular focus on the role of tSCs in this process. This work was supported by the National institutes of Health (NIH) under the awards R01(AG050676) (SVB), P01 (AG051442) (SVB), P30 (AR069620)(SVB), T32 (AG000114)(SDG), and the American Physiological Society Porter Fellowship (SDG). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neuromuscular Junction Remodeling: the Role of Sub-Synaptic Nuclei, Terminal Schwann Cells, and Trophic Signaling Following Neuromusculoskeletal Injuries

Peripheral nerve crush injury is a well-established model of neuromuscular junction (NMJ) denervation and subsequent re-innervation. Functionally, the muscle tissue follows a similar pattern as neural recovery, with immediate loss of force production that steadily improves in parallel with rates of re-innervation. On the other hand, traumatic injury to the muscle tissue itself, specifically volumetric muscle loss (VML), results in an irrecoverable loss of muscle function. Recent work has indicated significant impairments to the NMJ following VML that appear chronic in nature, alongside functional recovery. Thus, the goal of this study was to compare the effects of nerve and muscle injury on NMJ remodeling, with the hypothesis that VML will result in chronic alterations to the NMJ, including a higher degree of cellular localization to the NMJ. Even numbers of adult male and female mice were used with three experimental groups: injury naive, nerve crush, and VML-injury; and three terminal timepoints: 3-, 48-, and 112-days post injury. Data were analyzed by three-way ANOVA with Tukey’s HSD post-hoc. Confirming the assumed recoverability of the two injury models, we found in vivo maximal torque was fully restored following nerve crush injury (Tukey’s HSD post hoc p=0.730), but remained at a significant deficit following VML (~41% lower than injury naïve; Tukey’s HSD post hoc p<0.001). Compared to nerve crush and injury naïve, we found VML results in aberrantly high trophic signaling and numbers of supporting cells. Protein expression of neuregulin-1 and connective tissue growth factor were both highest in the VML group (~1.2-fold and 12-fold increase compared to injury naïve, respectively; Main effect of group, p<0.001). While nerve crush injury elicited a transient increase in terminal Schwann cell numbers (~38% higher than injury naïve at 48dpi; Tukey’s HSD post hoc p<0.001), VML resulted in a chronic increase (~53% and 27% higher than injury naïve at 48 and 112dpi, respectively; Tukey’s HSD post hoc p<0.001). Moreover, nerve crush injury did not alter the number of sub-synaptic nuclei per NMJ, while VML resulted in significant increases at both 48- and 112-days post injury (~27% and 24% higher than injury naïve, respectively; Tukey’s HSD post hoc p=0.002). In some cases, sex differences were detected, including higher rates of innervation in females than males (Main effect of sex p=0.003), particularly following VML-injury (Tukey’s HSD post hoc p=0.004). The findings herein bear a striking resemblance to NMJs found in dystrophic muscle, which are thought to be the result of continuous cycles of muscle fiber degeneration and regeneration. Thus, the NMJ changes observed following VML may be indicative of a larger issue, such as chronic muscle fiber remodeling and pervasive fibrotic accumulation. R01-AR078903 (JAC & SMG); K02-AG081488 (SMG). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sensory Schwann cells set perceptual thresholds for touch and selectively regulate mechanical nociception

Previous work identified nociceptive Schwann cells that can initiate pain. Consistent with the existence of inherently mechanosensitive sensory Schwann cells, we found that in mice, the mechanosensory function of almost all nociceptors, including those signaling fast pain, were dependent on sensory Schwann cells. In polymodal nociceptors, sensory Schwann cells signal mechanical, but not cold or heat pain. Terminal Schwann cells also surround mechanoreceptor nerve-endings within the Meissner’s corpuscle and in hair follicle lanceolate endings that both signal vibrotactile touch. Within Meissner´s corpuscles, two molecularly and functionally distinct sensory Schwann cells positive for Sox10 and Sox2 differentially modulate rapidly adapting mechanoreceptor function. Using optogenetics we show that Meissner’s corpuscle Schwann cells are necessary for the perception of low threshold vibrotactile stimuli. These results show that sensory Schwann cells within diverse glio-neural mechanosensory end-organs are sensors for mechanical pain as well as necessary for touch perception.

Efficient generation of a self-organizing neuromuscular junction model from human pluripotent stem cells

The complex neuromuscular network that controls body movements is the target of severe diseases that result in paralysis and death. Here, we report the development of a robust and efficient self-organizing neuromuscular junction (soNMJ) model from human pluripotent stem cells that can be maintained long-term in simple adherent conditions. The timely application of specific patterning signals instructs the simultaneous development and differentiation of position-specific brachial spinal neurons, skeletal muscles, and terminal Schwann cells. High-content imaging reveals self-organized bundles of aligned muscle fibers surrounded by innervating motor neurons that form functional neuromuscular junctions. Optogenetic activation and pharmacological interventions show that the spinal neurons actively instruct the synchronous skeletal muscle contraction. The generation of a soNMJ model from spinal muscular atrophy patient-specific iPSCs reveals that the number of NMJs and muscle contraction is severely affected, resembling the patient’s pathology. In the future, the soNMJ model could be used for high-throughput studies in disease modeling and drug development. Thus, this model will allow us to address unmet needs in the neuromuscular disease field.

Using the Neuromuscular Junction Cellular Response to Predict Functional Recovery after Nerve Grafting in a Mouse Experimental Model.

Functional recovery following peripheral nerve injury worsens with increasing time of denervation before repair. Denervated muscle undergoes progressive atrophy that limits the extent to which motor endplates can be reinnervated. The aims of this study were to assess nerve injuries reconstructed at different time points and to identify various neural and muscle-based markers to predict functional outcome, including an in-depth look at the neuromuscular junction (NMJ). Adult wild-type C57BL/6J mice underwent surgery on the sciatic nerve and were divided into 5 groups: (1) nerve cut and repaired, (2) acute (nerve cut and immediately repaired with a 1-cm autograft), (3) subacute (nerve grafted 2 weeks after injury), (4) delayed (nerve grafted 4 weeks after injury), (5) nerve cut and capped. Functional recovery was measured by treadmill and electrodiagnostic tests. Nerves were harvested for histologic evaluation, and leg muscles, for histologic evaluation and NMJ immunofluorescent staining of motor endplate innervation and terminal Schwann cells (tSCs). The delayed graft group performed worst in nearly all parameters. The subacute graft group shared more similarities with the acute group, especially the tSC response (subacute, 48%; acute, 51%) and motor endplate innervation pattern (subacute, 75%; acute, 72%). The only parameters to elucidate differences were muscle weight and motor endplate fragmentation. Traditional axon count failed to capture differences among the 3 groups. The tSC activity and NMJ innervation pattern can be used as predictive markers of functional recovery that capture differences among acute, subacute, and delayed nerve injuries. The tSC activity at the NMJ displayed by immunohistochemical methods can accurately reflect target muscle innervation over time. This can be used as a predictor of reinnervation when timing of nerve reconstruction is delayed, which can be a critically needed tool in clinical scenarios.

Neuromuscular junction denervation and terminal Schwann cell loss in the hTDP-43 overexpression mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with complex aetiology. Despite evidence of neuromuscular junction (NMJ) denervation and 'dying-back' pathology in models of SOD1-dependent ALS, evidence in other genetic forms of ALS is limited by a lack of suitable animal models. TDP-43, a key mediator protein in ALS, is overexpressed in neurons in Thy1-hTDP-43WT mice. We therefore aimed to comprehensively analyse NMJ pathology in this model of ALS. Expression of TDP-43 was assessed via western blotting. Immunohistochemistry techniques, alongside NMJ-morph quantification, were used to analyse motor neuron number, NMJ denervation status and terminal Schwann cell morphology. We present a time course of progressive, region-specific motor neuron pathology in Thy1-hTDP-43WT mice. Thy1-driven hTDP-43 expression increased steadily, correlating with developing hindlimb motor weakness and associated motor neuron loss in the spinal cord with a median survival of 21 days. Pronounced NMJ denervation was observed in hindlimb muscles, mild denervation in cranial muscles but no evidence of denervation in either forelimb or trunk muscles. NMJ pathology was restricted to motor nerve terminals, with denervation following the same time course as motor neuron loss. Terminal Schwann cells were lost from NMJs in hindlimb muscles, directly correlating with denervation status. Thy1-hTDP-43WT mice represent a severe model of ALS, with NMJ pathology/denervation of distal muscles and motor neuron loss, as observed in ALS patients. This model therefore provides an ideal platform to investigate mechanisms of dying-back pathology, as well as NMJ-targeting disease-modifying therapies in ALS.

Pan-Neurofascin autoimmune nodopathy - a life-threatening, but reversible neuropathy.

Autoimmune nodopathies are immune-mediated neuropathies associated with antibodies targeting the peripheral node of Ranvier. Recently, antibodies against all neurofascin-isoforms (pan-neurofascin) have been linked to a clinical phenotype distinct from previously described autoimmune nodopathies. Here, we aim at highlighting the molecular background and the red flags for diagnostic assessment and provide treatment and surveillance approaches for this new disease. Neurofascin-isoforms are located at different compartments of the node of Ranvier: Neurofascin-186 at the axonal nodal gap, and Neurofascin-155 at the terminal Schwann cell loops at the paranode. Pan-neurofascin antibodies recognize a common epitope on both isoforms and can access the node of Ranvier directly. Depending on their subclass profile, antibodies can induce direct structural disorganization and complement activation. Affected patients present with acute and immobilizing sensorimotor neuropathy, with cranial nerve involvement and long-term respiratory insufficiency. Early antibody-depleting therapy is crucial to avoid axonal damage, and remission is possible despite extended disease and high mortality. The antibody titer and serum neurofilament light chain levels can serve as biomarkers for diagnosis and therapy monitoring. Pan-neurofascin-associated autoimmune nodopathies has unique molecular and clinical features. Testing should be considered in severe and prolonged Guillain-Barré-like phenotype.

A Call for Discovery and Therapeutic Development for Cutaneous Neurofibromas

A Call for Discovery and Therapeutic Development for Cutaneous Neurofibromas

Cover Image

Immunostaining of lymphoid enhancer-binding factor 1 (LEF1, Green), a downstream mediator of the Wnt/β-catenin signaling pathway, in the postnatal back skin of mice. In addition to the dermal papilla (DP), LEF1 is expressed throughout the hair cycle in the terminal Schwann cells (TSCs), forming a ring-like structure surrounding the isthmus of the hair follicle; From: Expression dynamics of lymphoid enhancer-binding factor 1 in terminal Schwann cells, dermal papilla, and interfollicular epidermis; H Song, XB Zhao, QS Chu, J Zhang, L Gao, XH Liao; DevDyn 252:4, https://doi.org/10.1002/dvdy.562.

Response of terminal Schwann cells following volumetric muscle loss injury

An often-overlooked component of traumatic skeletal muscle injuries is the impact on the nervous system and resultant innervation of the affected muscles. Recent work in a rodent model of volumetric muscle loss (VML) injury demonstrated a progressive, secondary loss of neuromuscular junction (NMJ) innervation, supporting a role of NMJ dysregulation in chronic functional deficits. Terminal Schwann cells (tSCs) are known to be vital for the maintenance of NMJ structure and function, in addition to guiding repair and regeneration after injury. However, the tSC response to a traumatic muscle injury such as VML is not known. Thus, a study was conducted to investigate the effect of VML on tSC morphological characteristics and neurotrophic signaling proteins in adult male Lewis rats that underwent VML injury to the tibialis anterior muscle using a temporal design with outcome assessments at 3, 7, 14, 21, and 48 days post-injury. The following salient observations were made; first, although there is a loss of innervation over time, the number of tSCs per NMJ increases, significantly so at 48 days post-injury compared to control. The degree of NMJ fragmentation was positively correlated with tSC number after injury. Moreover, neurotrophic factors such as NRG1 and BDNF are elevated after injury through at least 48 days. These results were unanticipated and in contrast to neurodegenerative disease models, in which there is a reduction in tSC number that precedes denervation. However, we found that while there are more tSCs per NMJ after injury, they cover a significantly smaller percent of the post-synaptic endplate area compared to control. These findings support a sustained increase in neurotrophic activity and tSC number after VML, which is a maladaptive response occurring in parallel to other aspects of the VML injury, such as over-accumulation of collagen and aberrant inflammatory signaling.

Lack of Sod1 induces disruption of innervation accompanied by Schwann cell proliferation in skeletal muscle

The neuromuscular junction (NMJ) is an excitatory synapse that constitutes the primary site of communication between the nervous system and skeletal muscle and therefore any degenerative changes at the NMJ have the potential to impair muscle function. The primary cellular components of the NMJ are the motor neuron, the muscle fiber and terminal Schwann cells (tSC) that surround the NMJ. The importance of redox homeostasis for the maintenance of NMJ integrity is supported by work from our group showing degenerative changes at NMJs in mice lacking copper zinc superoxide dismutase (CuZnSOD; Sod1 -/- ) as early as 4 months of age. Prior work from others suggests that tSCs contribute trophic support for the NMJ and facilitate motor unit remodeling, but changes in tSC structure and function under conditions of elevated oxidative stress have not been thoroughly explored. Using in vitro assays, we previously showed that primary Schwann cells treated with a sublethal dose of the reactive oxygen species inducing agent Paraquat (PQ) resulted in increased Schwann cell number, ki67+ staining, and gene expression of trophic factors and the critical NMJ organizing protein Agrin. Furthermore, differentiated C2C12 myotubes treated with conditioned media from PQ treated Schwann cell cultures showed increased number of acetylcholine receptor (AChR) plaques. The goal of the current study was to characterize NMJs in young Sod1 -/- mice (2-months) to determine the early effects of elevated oxidative stress on NMJ structure in vivo with a specific focus on tSCs. We hypothesized that loss of Sod1 would lead to aberrant tSC and NMJ structure associated with decreased muscle function. We assessed maximum isometric force, NMJ morphology, and number of Schwann cells in gastrocnemius muscles of WT (n = 3) and Sod1 -/- (n = 3) mice. In Sod1 -/- mice, muscle force was reduced by 26% ( p = 2e-4) with nerve stimulation compared to direct muscle stimulation, and overlap between motor nerve terminals and AChRs was reduced by nearly 60% compared to WT controls. Post-synaptic AChR area was increased by 37% in Sod1 -/- mice, and tSC area and numbers per NMJ were increased by 60% and 155%, respectively in Sod1 -/- mice. Finally, Schwann cells proximal to NMJs showed increased Ki67+ labeling in Sod1 -/- mice compared to WT mice. This analysis demonstrates that early changes in both pre- and post-synaptic NMJ components and reduced neurotransmission due to loss of Sod1 are associated with marked increases in tSC proliferation. Our data implicate a protective role of muscle-resident Schwann cells in preserving NMJ integrity in response to alterations in redox homeostasis that may be mediated in part through activation and proliferation of these cells. These findings indicate that further investigation of the dynamics between Schwann cells and NMJ function is important to increase understanding of neuromuscular degenerativeconditions that involve altered redox homeostasis such as sarcopenia. Supported by the University of Michigan Medical School Office of Research, the American Physiological Society, and NIH AR069620. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Direct electrical stimulation impacts on neuromuscular junction morphology on both stimulated and unstimulated contralateral soleus.

There is increasing evidence of crosstalk between organs. The neuromuscular junction (NMJ) is a peripheral chemical synapse whose function and morphology are sensitive to acetylcholine (ACh) release and muscle depolarization. In an attempt to improve our understanding of NMJ plasticity and muscle crosstalk, the effects of unilateral direct electrical stimulation of a hindlimb muscle on the NMJ were investigated in rats exposed long-term post-synaptic neuromuscular blockade. Sprague Dawley rats were subjected to post-synaptic blockade of neuromuscular transmission by systemic administration of α-cobrotoxin and mechanically ventilated for up to 8days and compared with untreated sham operated controls and animals exposed to unilateral chronic electrical stimulation 12h/day for 5 or 8days. NMJs produced axonal and glial sprouts (growth of processes that extend beyond the confines of the synapse defined by high-density aggregates of acetylcholine receptors [AChRs]) in response to post-synaptic neuromuscular blockade, but less than reported after peripheral denervation or pre-synaptic blockade. Direct electrical soleus muscle stimulation reduced the terminal Schwann cell (tSC) and axonal sprouting in both stimulated and non-stimulated contralateral soleus. Eight days chronic stimulation reduced (P<0.001) the number of tSC sprouts on stimulated and non-stimulated soleus from 6.7±0.5 and 6.9±0.5 sprouts per NMJ, respectively, compared with 10.3±0.9 tSC per NMJ (P<0.001) in non-stimulated soleus from rats immobilized for 8days. A similar reduction of axonal sprouts (P<0.001) was observed in stimulated and non-stimulated contralateral soleus in response to chronic electrical stimulation. RNAseq-based gene expression analyses confirmed a restoring effect on both stimulated and unstimulated contralateral muscle. The cross-over effect was paralleled by increased cytokine/chemokine levels in stimulated and contralateral unstimulated muscle as well as in plasma. Motor axon terminals and terminal Schwann cells at NMJs of rats subjected to post-synaptic neuromuscular blockade exhibited sprouting responses. These axonal and glial responses were likely dampened by a muscle-derived myokines released in an activity-dependent manner with both local and systemic effects.

An agonist of CXCR4 induces a rapid recovery from the neurotoxic effects of Vipera ammodytes and Vipera aspis venoms.

People bitten by Alpine vipers are usually treated with antivenom antisera to prevent the noxious consequences caused by the injected venom. However, this treatment suffers from a number of drawbacks and additional therapies are necessary. The venoms of Vipera ammodytes and of Vipera aspis are neurotoxic and cause muscle paralysis by inducing neurodegeneration of motor axon terminals because they contain a presynaptic acting sPLA2 neurotoxin. We have recently found that any type of damage to motor axons is followed by the expression and activation of the intercellular signaling axis consisting of the CXCR4 receptor present on the membrane of the axon stump and of its ligand, the chemokine CXCL12 released by activated terminal Schwann cells. We show here that also V. ammodytes and V. aspis venoms cause the expression of the CXCL12-CXCR4 axis. We also show that a small molecule agonist of CXCR4, dubbed NUCC-390, induces a rapid regeneration of the motor axon terminal with functional recovery of the neuromuscular junction. These findings qualify NUCC-390 as a promising novel therapeutics capable of improving the recovery from the paralysis caused by the snakebite of the two neurotoxic Alpine vipers.

Cellular interplay in skeletal muscle regeneration and wasting: insights from animal models.

Skeletal muscle wasting, whether related to physiological ageing, muscle disuse or to an underlying chronic disease, is a key determinant to quality of life and mortality. However, cellular basis responsible for increased catabolism in myocytes often remains unclear. Although myocytes represent the vast majority of skeletal muscle cellular population, they are surrounded by numerous cells with various functions. Animal models, mostly rodents, can help to decipher the mechanisms behind this highly dynamic process, by allowing access to every muscle as well as time-course studies. Satellite cells (SCs) play a crucial role in muscle regeneration, within a niche also composed of fibroblasts and vascular and immune cells. Their proliferation and differentiation is altered in several models of muscle wasting such as cancer, chronic kidney disease or chronic obstructive pulmonary disease (COPD). Fibro-adipogenic progenitor cells are also responsible for functional muscle growth and repair and are associated in disease to muscle fibrosis such as in chronic kidney disease. Other cells have recently proven to have direct myogenic potential, such as pericytes. Outside their role in angiogenesis, endothelial cells and pericytes also participate to healthy muscle homoeostasis by promoting SC pool maintenance (so-called myogenesis-angiogenesis coupling). Their role in chronic diseases muscle wasting has been less studied. Immune cells are pivotal for muscle repair after injury: Macrophages undergo a transition from the M1 to the M2 state along with the transition between the inflammatory and resolutive phase of muscle repair. T regulatory lymphocytes promote and regulate this transition and are also able to activate SC proliferation and differentiation. Neural cells such as terminal Schwann cells, motor neurons and kranocytes are notably implicated in age-related sarcopenia. Last, newly identified cells in skeletal muscle, such as telocytes or interstitial tenocytes could play a role in tissular homoeostasis. We also put a special focus on cellular alterations occurring in COPD, a chronic and highly prevalent respiratory disease mainly linked to tobacco smoke exposure, where muscle wasting is strongly associated with increased mortality, and discuss the pros and cons of animal models versus human studies in this context. Finally, we discuss resident cells metabolism and present future promising leads for research, including the use of muscle organoids.

Three-dimensional correlative light and focused ion beam scanning electron microscopy reveals the distribution and ultrastructure of lanceolate nerve endings surrounding terminal hair follicles in human scalp skin.

Lanceolate nerve endings (LNEs) surrounding hair follicles (HFs) play an important role in detecting hair deflection. Complexes of the LNEs form a palisade-like structure along the longitudinal axis of hair roots in which axons are sandwiched between two processes of terminal Schwann cells (tSCs) at the isthmus of HFs. The structure and molecular mechanism of LNEs in animal sinus hair, pelage, and human vellus hairs have been investigated. Despite the high density of HFs in human scalp skin, the LNEs in human terminal HFs have not been investigated. In this study, we aimed to reveal the distribution and ultrastructure of LNEs in terminal HFs of human scalp skin. Using light-sheet microscopy and immunostaining, the LNEs were observed at one terminal HF but not at the other terminal HFs in the same follicular unit. The ultrastructure of the LNEs of terminal HFs in human scalp skin was characterized using correlated light and electron microscopy (CLEM). Confocal laser microscopy and transmission electron microscopy of serial transverse sections of HFs revealed that LNEs were aligned adjacent to the basal lamina outside the outer root sheath (ORS), at the isthmus of terminal HFs, and adjacent to CD200-positive ORS cells in the upper bulge region. Moreover, axons with abundant mitochondria were sandwiched between tSCs. Three-dimensional CLEM, specifically confocal laser microscopy and focused ion beam scanning electron microscopy, of stained serial transverse sections revealed that LNEs were wrapped with type I and type II tSCs, with the processes protruding from the space between the Schwann cells. Moreover, the ultrastructures of LNEs at miniaturized HFs were similar to those of LNEs at terminal HFs. Preembedding immunoelectron microscopy revealed that Piezo-type mechanosensitive ion channel component 2 (Piezo2), a gated ion channel, was in axons and tSCs and adjacent to the cell membrane of axons and tSCs, suggesting that LNEs function as mechanosensors. The number of LNEs increased as the diameter of the ORS decreased, suggesting that LNEs dynamically adapt to the HF environment as terminal HFs miniaturize into vellus-like hair. These findings will provide insights for investigations of mechanosensory organs, aging, and re-innervation during wound healing.

Expression dynamics of lymphoid enhancer-binding factor 1 in terminal Schwann cells, dermal papilla, and interfollicular epidermis.

Transcription factor lymphoid enhancer-binding factor 1 (LEF1) is a downstream mediator of the Wnt/β-catenin signaling pathway. It is expressed in dermal papilla and surrounding cells in the hair follicle, promoting cell proliferation, and differentiation. Here, we report that LEF1 is also expressed all through the hair cycle in the terminal Schwann cells (TSCs), a component of the lanceolate complex located at the isthmus. The timing of LEF1 appearance at the isthmus coincides with that of hair follicle innervation. LEF1 is not found at the isthmus in the aberrant hair follicles in nude mice. Instead, LEF1 in TSCs is found in the de novo hair follicles reconstituted on nude mice by stem cells chamber graft assay. Cutaneous denervation experiment demonstrates that the LEF1 expression in TSCs is independent of nerve endings. At last, LEF1 expression in the interfollicular epidermis during the early stage of skin development is significantly suppressed in transgenic mice with T-cell factor 3 (TCF3) overexpression. We reveal the expression dynamics of LEF1 in skin during development and hair cycle. LEF1 expression in TSCs indicates that the LEF1/Wnt signal might help to establish a niche at the isthmus region for the lanceolate complex, the bulge stem cells and other neighboring cells.

Terminal Schwann Cells Are Essential for Neuromuscular Junction Function and Recovery after Nerve Injury.

Terminal Schwann cells (tSCs), nonmyelinating glial cells at the neuromuscular junction (NMJ), are integral to NMJ development, function, remodeling, and response to injury. It is essential to understand their requirement for NMJ function. In this study, the authors assessed consequences of immune-mediated tSC ablation in adult S100 -GFP mice of both sexes in homeostasis and after nerve injury. The authors examined NMJ morphology and function in the extensor digitorum longus muscle during homeostasis at post-tSC ablation days 3, 14, and 42 and after peroneal nerve transection and immediate repair at 3 and 6 weeks after nerve injury and tSC ablation (postinjury and ablation). tSC ablation resulted in significant decreases ( P < 0.05) in tSC numbers per NMJ and end plate fragmentation. NMJ innervation and EDL tetanic force were significantly decreased at post-tSC ablation day 14 ( P < 0.05) and tSCs reestablished their NMJ coverage at post-tSC ablation day 42. After nerve injury, motor end plate fragmentation increased ( P < 0.01) with tSC ablation compared with injured control mice. NMJ reinnervation and extensor digitorum longus tetanic force were significantly reduced ( P < 0.001), even at 6 weeks postinjury and ablation, compared with control mice. These results add to the understanding that tSCs, with their proregenerative potential, help maintain NMJ integrity in homeostasis and are necessary for NMJ reinnervation after peripheral nerve injury. Terminal Schwann cells are integral for efficient NMJ recovery after nerve injury. This cell population may provide a novel therapeutic target to improve outcomes for patients with nerve injuries; additional investigation is warranted.