Beyond Wrapping: Canonical and Noncanonical Functions of Schwann Cells.

Schwann cells in the peripheral nervous system (PNS) are essential for the support and myelination of axons, ensuring fast and accurate communication between the central nervous system and the periphery. Schwann cells and related glia accompany innervating axons in virtually all tissues in the body, where they exhibit remarkable plasticity and the ability to modulate pathology in extraordinary, and sometimes surprising, ways. Here, we provide a brief overview of the various glial cell types in the PNS and describe the cornerstone cellular and molecular processes that enable Schwann cells to perform their canonical functions. We then dive into discussing exciting noncanonical functions of Schwann cells and related PNS glia, which include their role in organizing the PNS, in regulating synaptic activity and pain, in modulating immunity, in providing a pool of stem cells for different organs, and, finally, in influencing cancer.

Neuron-specific ablation of the Krabbe disease gene galactosylceramidase in mice results in neurodegeneration.

Krabbe disease is caused by a deficiency of the lysosomal galactosylceramidase (GALC) enzyme, which results in the accumulation of galactosylceramide (GalCer) and psychosine. In Krabbe disease, the brunt of demyelination and neurodegeneration is believed to result from the dysfunction of myelinating glia. Recent studies have shown that neuronal axons are both structurally and functionally compromised in Krabbe disease, even before demyelination, suggesting a possible neuron-autonomous role of GALC. Using a novel neuron-specific Galc knockout (CKO) model, we show that neuronal Galc deletion is sufficient to cause growth and motor coordination defects and inflammatory gliosis in mice. Furthermore, psychosine accumulates significantly in the nervous system of neuron-specific Galc-CKO. Confocal and electron microscopic analyses show profound neuro-axonal degeneration with a mild effect on myelin structure. Thus, we prove for the first time that neuronal GALC is essential to maintain and protect neuronal function independently of myelin and may directly contribute to the pathogenesis of Krabbe disease.

A new mouse model of Charcot-Marie-Tooth 2J neuropathy replicates human axonopathy and suggest alteration in axo-glia communication.

Myelin is essential for rapid nerve impulse propagation and axon protection. Accordingly, defects in myelination or myelin maintenance lead to secondary axonal damage and subsequent degeneration. Studies utilizing genetic (CNPase-, MAG-, and PLP-null mice) and naturally occurring neuropathy models suggest that myelinating glia also support axons independently from myelin. Myelin protein zero (MPZ or P0), which is expressed only by Schwann cells, is critical for myelin formation and maintenance in the peripheral nervous system. Many mutations in MPZ are associated with demyelinating neuropathies (Charcot-Marie-Tooth disease type 1B [CMT1B]). Surprisingly, the substitution of threonine by methionine at position 124 of P0 (P0T124M) causes axonal neuropathy (CMT2J) with little to no myelin damage. This disease provides an excellent paradigm to understand how myelinating glia support axons independently from myelin. To study this, we generated targeted knock-in MpzT124M mutant mice, a genetically authentic model of T124M-CMT2J neuropathy. Similar to patients, these mice develop axonopathy between 2 and 12 months of age, characterized by impaired motor performance, normal nerve conduction velocities but reduced compound motor action potential amplitudes, and axonal damage with only minor compact myelin modifications. Mechanistically, we detected metabolic changes that could lead to axonal degeneration, and prominent alterations in non-compact myelin domains such as paranodes, Schmidt-Lanterman incisures, and gap junctions, implicated in Schwann cell-axon communication and axonal metabolic support. Finally, we document perturbed mitochondrial size and distribution along MpzT124M axons suggesting altered axonal transport. Our data suggest that Schwann cells in P0T124M mutant mice cannot provide axons with sufficient trophic support, leading to reduced ATP biosynthesis and axonopathy. In conclusion, the MpzT124M mouse model faithfully reproduces the human neuropathy and represents a unique tool for identifying the molecular basis for glial support of axons.

Loss of YAP in Schwann cells improves HNPP pathophysiology.

Rapid nerve conduction in the peripheral nervous system (PNS) is facilitated by the multilamellar myelin sheath encasing many axons of peripheral nerves. Charcot-Marie-Tooth type 1A (CMT1A), and hereditary neuropathy with liability to pressure palsy (HNPP) are common demyelinating inherited peripheral neuropathies and are caused by mutations in the peripheral myelin protein 22 (PMP22) gene. Duplication of PMP22 leads to its overexpression and causes CMT1A, while its deletion results in PMP22 under expression and causes HNPP. Here, we investigated novel targets for modulating the protein level of PMP22 in HNPP. We found that genetic attenuation of the transcriptional coactivator Yap in Schwann cells reduces p-TAZ levels, increased TAZ activity, and increases PMP22 in peripheral nerves. Based on these findings, we ablated Yap alleles in Schwann cells of the Pmp22-haploinsufficient mouse model of HNPP and identified fewer tomacula on morphological assessment and improved nerve conduction in peripheral nerves. These findings suggest YAP modulation may be a new avenue for treatment of HNPP.

Raising cGMP restores proteasome function and myelination in mice with a proteotoxic neuropathy.

Agents that raise cyclic guanosine monophosphate (cGMP) by activating protein kinase G increase 26S proteasome activities, protein ubiquitination and degradation of misfolded proteins. Therefore, they may be useful in treating neurodegenerative and other diseases caused by an accumulation of misfolded proteins. Mutations in myelin protein zero (MPZ) cause the peripheral neuropathy Charcot-Marie-Tooth type 1B (CMT1B). In peripheral nerves of a mouse model of CMT1B, where the mutant MPZS63del is expressed, proteasome activities are reduced, mutant MPZS63del and polyubiquitinated proteins accumulate and the unfolded protein response (p-eif2α) is induced. In HEK293 cells, raising cGMP stimulated ubiquitination and degradation of MPZS63del, but not of wild-type MPZ. Treating S63del mice with the phosphodiesterase 5 inhibitor, sildenafil-to raise cGMP-increased proteasome activity in sciatic nerves and reduced the levels of polyubiquitinated proteins, the proteasome reporter ubG76V-GFP and p-elF2α. Furthermore, sildenafil treatment reduced the number of amyelinated axons, and increased myelin thickness and nerve conduction velocity in sciatic nerves. Thus, agents that raise cGMP, including those widely used in medicine, may be useful therapies for CMT1B and other proteotoxic diseases.

Heparanome-Mediated Rescue of Oligodendrocyte Progenitor Quiescence following Inflammatory Demyelination.

The proinflammatory cytokine IFN-γ, which is chronically elevated in multiple sclerosis, induces pathologic quiescence in human oligodendrocyte progenitor cells (OPCs) via upregulation of the transcription factor PRRX1. In this study using animals of both sexes, we investigated the role of heparan sulfate proteoglycans in the modulation of IFN-γ signaling following demyelination. We found that IFN-γ profoundly impaired OPC proliferation and recruitment following adult spinal cord demyelination. IFN-γ-induced quiescence was mediated by direct signaling in OPCs as conditional genetic ablation of IFNγR1 (Ifngr1) in adult NG2+ OPCs completely abrogated these inhibitory effects. Intriguingly, OPC-specific IFN-γ signaling contributed to failed oligodendrocyte differentiation, which was associated with hyperactive Wnt/Bmp target gene expression in OPCs. We found that PI-88, a heparan sulfate mimetic, directly antagonized IFN-γ to rescue human OPC proliferation and differentiation in vitro and blocked the IFN-γ-mediated inhibitory effects on OPC recruitment in vivo Importantly, heparanase modulation by PI-88 or OGT2155 in demyelinated lesions rescued IFN-γ-mediated axonal damage and demyelination. In addition to OPC-specific effects, IFN-γ-augmented lesions were characterized by increased size, reactive astrogliosis, and proinflammatory microglial/macrophage activation along with exacerbated axonal injury and cell death. Heparanase inhibitor treatment rescued many of the negative IFN-γ-induced sequelae suggesting a profound modulation of the lesion environment. Together, these results suggest that the modulation of the heparanome represents a rational approach to mitigate the negative effects of proinflammatory signaling and rescuing pathologic quiescence in the inflamed and demyelinated human brain.SIGNIFICANCE STATEMENT The failure of remyelination in multiple sclerosis contributes to neurologic dysfunction and neurodegeneration. The activation and proliferation of oligodendrocyte progenitor cells (OPCs) is a necessary step in the recruitment phase of remyelination. Here, we show that the proinflammatory cytokine interferon-γ directly acts on OPCs to induce pathologic quiescence and thereby limit recruitment following demyelination. Heparan sulfate is a highly structured sulfated carbohydrate polymer that is present on the cell surface and regulates several aspects of the signaling microenvironment. We find that pathologic interferon-γ can be blocked by modulation of the heparanome following demyelination using either a heparan mimetic or by treatment with heparanase inhibitor. These studies establish the potential for modulation of heparanome as a regenerative approach in demyelinating disease.

Calcineurin Activity Is Increased in Charcot-Marie-Tooth 1B Demyelinating Neuropathy.

Schwann cells produce a considerable amount of lipids and proteins to form myelin in the PNS. For this reason, the quality control of myelin proteins is crucial to ensure proper myelin synthesis. Deletion of serine 63 from P0 (P0S63del) protein in myelin forming Schwann cells causes Charcot-Marie-Tooth type 1B neuropathy in humans and mice. Misfolded P0S63del accumulates in the ER of Schwann cells where it elicits the unfolded protein response (UPR). PERK is the UPR transducer that attenuates global translation and reduces ER stress by phosphorylating the translation initiation factor eIF2alpha. Paradoxically, Perk ablation in P0S63del Schwann cells (S63del/PerkSCKO ) reduced the level of P-eIF2alpha, leaving UPR markers upregulated, yet unexpectedly improved S63del myelin defects in vivo We therefore investigated the hypothesis that PERK may interfere with signals outside of the UPR and specifically with calcineurin/NFATc4 pro-myelinating pathway. Using mouse genetics including females and males in our experimental setting, we show that PERK and calcineurin interact in P0S63del nerves and that calcineurin activity and NFATc4 nuclear localization are increased in S63del Schwann cells, without altering EGR2/KROX20 expression. Moreover, genetic manipulation of the calcineurin subunits appears to be either protective or toxic in S63del in a context-dependent manner, suggesting that Schwann cells are highly sensitive to alterations of calcineurin activity.SIGNIFICANCE STATEMENT Our work shows a novel activity and function for calcineurin in Schwann cells in the context of ER stress. Schwann cells expressing the S63del mutation in P0 protein induce the unfolded protein response and upregulate calcineurin activity. Calcineurin interacts with the ER stress transducer PERK, but the relationship between the UPR and calcineurin in Schwann cells is unclear. Here we propose a protective role for calcineurin in S63del neuropathy, although Schwann cells appear to be very sensitive to its regulation. The paper uncovers a new important role for calcineurin in a demyelinating diseases.

Laminin α2 controls mouse and human stem cell behaviour during midbrain dopaminergic neuron development.

Development of the central nervous system requires coordination of the proliferation and differentiation of neural stem cells. Here, we show that laminin alpha 2 (lm-α2) is a component of the midbrain dopaminergic neuron (mDA) progenitor niche in the ventral midbrain (VM) and identify a concentration-dependent role for laminin α2ß1γ1 (lm211) in regulating mDA progenitor proliferation and survival via a distinct set of receptors. At high concentrations, lm211-rich environments maintain mDA progenitors in a proliferative state via integrins α6ß1 and α7ß1, whereas low concentrations of lm211 support mDA lineage survival via dystroglycan receptors. We confirmed our findings in vivo, demonstrating that the VM was smaller in the absence of lm-α2, with increased apoptosis; furthermore, the progenitor pool was depleted through premature differentiation, resulting in fewer mDA neurons. Examination of mDA neuron subtype composition showed a reduction in later-born mDA neurons of the ventral tegmental area, which control a range of cognitive behaviours. Our results identify a novel role for laminin in neural development and provide a possible mechanism for autism-like behaviours and the brainstem hypoplasia seen in some individuals with mutations of LAMA2.

Peripheral glia diversity.

Recent years have seen an evolving appreciation for the role of glial cells in the nervous system. As we move away from the typical neurocentric view of neuroscience, the complexity and variability of central nervous system glia is emerging, far beyond the three main subtypes: astrocytes, oligodendrocytes, and microglia. Yet the diversity of the glia found in the peripheral nervous system remains rarely discussed. In this review, we discuss the developmental origin, morphology, and function of the different populations of glia found in the peripheral nervous system, including: myelinating Schwann cells, Remak Schwann cells, repair Schwann cells, satellite glia, boundary cap-derived glia, perineurial glia, terminal Schwann cells, glia found in the skin, olfactory ensheathing cells, and enteric glia. The morphological and functional heterogeneity of glia found in the periphery reflects the diverse roles the nervous system performs throughout the body. Further, it highlights a complexity that should be appreciated and considered when it comes to a complete understanding of the peripheral nervous system in health and disease.

Schwann cells ER-associated degradation contributes to myelin maintenance in adult nerves and limits demyelination in CMT1B mice.

In the peripheral nervous system (PNS) myelinating Schwann cells synthesize large amounts of myelin protein zero (P0) glycoprotein, an abundant component of peripheral nerve myelin. In humans, mutations in P0 cause the demyelinating Charcot-Marie-Tooth 1B (CMT1B) neuropathy, one of the most diffused genetic disorders of the PNS. We previously showed that several mutations, such as the deletion of serine 63 (P0-S63del), result in misfolding and accumulation of P0 in the endoplasmic reticulum (ER), with activation of the unfolded protein response (UPR). In addition, we observed that S63del mouse nerves display the upregulation of many ER-associated degradation (ERAD) genes, suggesting a possible involvement of this pathway in the clearance of the mutant P0. In ERAD in fact, misfolded proteins are dislocated from the ER and targeted for proteasomal degradation. Taking advantage of inducible cells that express the ER retained P0, here we show that the P0-S63del glycoprotein is degraded via ERAD. Moreover, we provide strong evidence that the Schwann cell-specific ablation of the ERAD factor Derlin-2 in S63del nerves exacerbates both the myelin defects and the UPR in vivo, unveiling a protective role for ERAD in CMT1B neuropathy. We also found that lack of Derlin-2 affects adult myelin maintenance in normal nerves, without compromising their development, pinpointing ERAD as a previously unrecognized player in preserving Schwann cells homeostasis in adulthood. Finally, we provide evidence that treatment of S63del peripheral nerve cultures with N-Acetyl-D-Glucosamine (GlcNAc), known to enhance protein quality control pathways in C.elegans, ameliorates S63del nerve myelination ex vivo. Overall, our study suggests that potentiating adaptive ER quality control pathways might represent an appealing strategy to treat both conformational and age-related PNS disorders.

Deletion of Calcineurin in Schwann Cells Does Not Affect Developmental Myelination, But Reduces Autophagy and Delays Myelin Clearance after Peripheral Nerve Injury.

In the PNS, myelination occurs postnatally when Schwann cells (SCs) contact axons. Axonal factors, such as Neuregulin-1 Type III, trigger promyelinating signals that upregulate myelin genes. Neuregulin-1 Type III has been proposed to activate calcineurin signaling in immature SCs to initiate differentiation and myelination. However, little is known about the role of calcineurin in promyelinating SCs after birth. By creating a SC conditional KO of calcineurin B (CnBscko), we assessed the effects of CnB ablation on peripheral myelination after birth in both male and female mice. Surprisingly, CnBscko mice have minimal myelination defects, no alteration of myelin thickness, and normal KROX20 expression. In contrast, we did find a unique role for calcineurin in SCs after nerve injury. Following nerve crush, CnBscko mice have slower degeneration of myelin compared with WT mice. Furthermore, absence of CnB in primary SCs delays clearance of myelin debris. SCs clear myelin via autophagy and recent literature has demonstrated that calcineurin can regulate autophagy via dephosphorylation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy. We demonstrate that loss of CnB reduces autophagic flux in primary SCs, indicating a possible mechanism for impaired myelin clearance. In addition, ablation of CnB impairs TFEB translocation to the nucleus 3 d after crush, suggesting that calcineurin may regulate autophagy in SCs via TFEB activation. Together, our data indicate that calcineurin is not essential for myelination but has a novel role in myelin clearance after injury.SIGNIFICANCE STATEMENT Our data offer a novel mechanism for activation of autophagy after peripheral nerve injury. Efficient clearance of myelin after injury by Schwann cells is important for axonal regrowth and remyelination, which is one reason why the PNS is significantly better at recovery compared with the CNS. Improved understanding of myelin clearance allows for the identification of pathways that are potentially accessible to increase myelin clearance and improve remyelination and recovery. Finally, this paper clarifies the role of calcineurin in Schwann cells and myelination.

Phosphorylation of eIF2α Promotes Schwann Cell Differentiation and Myelination in CMT1B Mice with Activated UPR.

Myelin Protein Zero (MPZ/P0) is the most abundant glycoprotein of peripheral nerve myelin. P0 is synthesized by myelinating Schwann cells, processed in the endoplasmic reticulum (ER) and delivered to myelin via the secretory pathway. The mutant P0S63del (deletion of serine 63 in the extracellular domain of P0), that causes Charcot-Marie-Tooth type 1B (CMT1B) neuropathy in humans and a similar demyelinating neuropathy in transgenic mice, is instead retained the ER where it activates an unfolded protein response. Under ER-stress conditions, protein kinase R-like endoplasmic reticulum kinase (PERK) phosphorylates eukaryotic initiation factor 2α (eIF2α) to attenuate global translation, thus reducing the misfolded protein overload in the ER. Genetic and pharmacological inactivation of Gadd34 (damage-inducible protein 34), a subunit of the PP1 phosphatase complex that promotes the dephosphorylation of eIF2α, prolonged eIF2α phosphorylation and improved motor, neurophysiological, and morphologic deficits in S63del mice. However, PERK ablation in S63del Schwann cells ameliorated, rather than worsened, S63del neuropathy despite reduced levels of phosphorylated eIF2α. These contradictory findings prompted us to genetically explore the role of eIF2α phosphorylation in P0S63del-CMT1B neuropathy through the generation of mice in which eIF2α cannot be phosphorylated specifically in Schwann cells. Morphologic and electrophysiological analysis of male and female S63del mice showed a worsening of the neuropathy in the absence of eIF2α phosphorylation. However, we did not detect significant changes in ER stress levels, but rather a dramatic increase of the MEK/ERK/c-Jun pathway accompanied by a reduction in expression of myelin genes and a delay in Schwann cell differentiation. Our results support the hypothesis that eIF2α phosphorylation is protective in CMT1B and unveil a possible cross talk between eIF2α and the MEK/ERK pathway in neuropathic nerves.SIGNIFICANCE STATEMENT In the P0S63del (deletion of serine 63 in the extracellular domain of P0) mouse model of Charcot-Marie-Tooth type 1B (CMT1B), the genetic and pharmacological inhibition of Gadd34 (damage-inducible protein 34) prolonged eukaryotic initiation factor 2α (eIF2α) phosphorylation, leading to a proteostatic rebalance that significantly ameliorated the neuropathy. Yet, ablation of protein kinase R-like endoplasmic reticulum kinase (PERK) also ameliorated the S63del neuropathy, despite reduced levels of eIF2α phosphorylation (P-eIF2α). In this study, we provide genetic evidence that eIF2α phosphorylation has a protective role in CMT1B Schwann cells by limiting ERK/c-Jun hyperactivation. Our data support the targeting of the P-eIF2α/Gadd34 complex as a therapeutic avenue in CMT1B and also suggest that PERK may hamper myelination via mechanisms outside its role in the unfolded protein response.

Alveolar progenitor cells in the mammary gland are dependent on the ß4 integrin.

Understanding how progenitor cell function is regulated in the mammary gland is an important developmental problem that has significant implications for breast cancer. Although it had been assumed that the expression the α6ß4 integrin (ß4) is restricted to the basal lineage, we report that alveolar progenitor cells in the mouse mammary gland also express this integrin based on analysis of single cell RNA-Seq data. Subsequent experiments using a mouse mammary epithelial cell line (NMuMG) confirmed this finding and revealed that ß4 is essential for maintaining progenitor function as assessed by serial passage mammosphere assays. These data were substantiated by analyzing the alveolar progenitor population isolated from nulliparous mouse mammary glands. Based on the finding that the alveolar progenitor cells express Whey Acidic Protein (WAP), WAP-Cre mice were crossed with itgß4flox/flox mice to generate conditional knock-out of ß4 in alveolar progenitor cells. These itgß4flox/floxWAP-Cre+ mice exhibited significant defects in alveologenesis and milk production during pregnancy compared to itgß4flox/floxWAP-Cre- mice, establishing a novel role for the ß4 integrin in alveolar progenitor function and alveologenesis.

Mechanisms of demyelination and neurodegeneration in globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD), also known as Krabbe disease, is a lysosomal storage disorder causing extensive demyelination in the central and peripheral nervous systems. GLD is caused by loss-of-function mutations in the lysosomal hydrolase, galactosylceramidase (GALC), which catabolizes the myelin sphingolipid galactosylceramide. The pathophysiology of GLD is complex and reflects the expression of GALC in a number of glial and neural cell types in both the central and peripheral nervous systems (CNS and PNS), as well as leukocytes and kidney in the periphery. Over the years, GLD has garnered a wide range of scientific and medical interests, especially as a model system to study gene therapy and novel preclinical therapeutic approaches to treat the spontaneous murine model for GLD. Here, we review recent findings in the field of Krabbe disease, with particular emphasis on novel aspects of GALC physiology, GLD pathophysiology, and therapeutic strategies.

Enhanced axonal neuregulin-1 type-III signaling ameliorates neurophysiology and hypomyelination in a Charcot-Marie-Tooth type 1B mouse model.

Charcot-Marie-Tooth (CMT) neuropathies are a group of genetic disorders that affect the peripheral nervous system with heterogeneous pathogenesis and no available treatment. Axonal neuregulin 1 type III (Nrg1TIII) drives peripheral nerve myelination by activating downstream signaling pathways such as PI3K/Akt and MAPK/Erk that converge on master transcriptional regulators of myelin genes, such as Krox20. We reasoned that modulating Nrg1TIII activity may constitute a general therapeutic strategy to treat CMTs that are characterized by reduced levels of myelination. Here we show that genetic overexpression of Nrg1TIII ameliorates neurophysiological and morphological parameters in a mouse model of demyelinating CMT1B, without exacerbating the toxic gain-of-function that underlies the neuropathy. Intriguingly, the mechanism appears not to be related to Krox20 or myelin gene upregulation, but rather to a beneficial rebalancing in the stoichiometry of myelin lipids and proteins. Finally, we provide proof of principle that stimulating Nrg1TIII signaling, by pharmacological suppression of the Nrg1TIII inhibitor tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17), also ameliorates the neuropathy. Thus, modulation of Nrg1TIII by TACE/ADAM17 inhibition may represent a general treatment for hypomyelinating neuropathies.

Neuregulin 1 type III improves peripheral nerve myelination in a mouse model of congenital hypomyelinating neuropathy.

Myelin sheath thickness is precisely regulated and essential for rapid propagation of action potentials along myelinated axons. In the peripheral nervous system, extrinsic signals from the axonal protein neuregulin 1 (NRG1) type III regulate Schwann cell fate and myelination. Here we ask if modulating NRG1 type III levels in neurons would restore myelination in a model of congenital hypomyelinating neuropathy (CHN). Using a mouse model of CHN, we improved the myelination defects by early overexpression of NRG1 type III. Surprisingly, the improvement was independent from the upregulation of Egr2 or essential myelin genes. Rather, we observed the activation of MAPK/ERK and other myelin genes such as peripheral myelin protein 2 and oligodendrocyte myelin glycoprotein. We also confirmed that the permanent activation of MAPK/ERK in Schwann cells has detrimental effects on myelination. Our findings demonstrate that the modulation of axon-to-glial NRG1 type III signaling has beneficial effects and improves myelination defects during development in a model of CHN.

The Hippo pathway: Horizons for innovative treatments of peripheral nerve diseases.

Initially identified in Drosophila, the Hippo signaling pathway regulates how cells respond to their environment by controlling proliferation, migration and differentiation. Many recent studies have focused on characterizing Hippo pathway function and regulation in mammalian cells. Here, we present a brief overview of the major components of the Hippo pathway, as well as their regulation and function. We comprehensively review the studies that have contributed to our understanding of the Hippo pathway in the function of the peripheral nervous system and in peripheral nerve diseases. Finally, we discuss innovative approaches that aim to modulate Hippo pathway components in diseases of the peripheral nervous system.

Sustained Expression of Negative Regulators of Myelination Protects Schwann Cells from Dysmyelination in a Charcot-Marie-Tooth 1B Mouse Model.

Schwann cell differentiation and myelination in the PNS are the result of fine-tuning of positive and negative transcriptional regulators. As myelination starts, negative regulators are downregulated, whereas positive ones are upregulated. Fully differentiated Schwann cells maintain an extraordinary plasticity and can transdifferentiate into "repair" Schwann cells after nerve injury. Reactivation of negative regulators of myelination is essential to generate repair Schwann cells. Negative regulators have also been implicated in demyelinating neuropathies, although their role in disease remains elusive. Here, we used a mouse model of Charcot-Marie-Tooth neuropathy type 1B (CMT1B), the P0S63del mouse characterized by ER stress and the activation of the unfolded protein response, to show that adult Schwann cells are in a partial differentiation state because they overexpress transcription factors that are normally expressed only before myelination. We provide evidence that two of these factors, Sox2 and Id2, act as negative regulators of myelination in vivo However, their sustained expression in neuropathy is protective because ablation of Sox2 or/and Id2 from S63del mice of both sexes results in worsening of the dysmyelinating phenotype. This is accompanied by increased levels of mutant P0 expression and exacerbation of ER stress, suggesting that limited differentiation may represent a novel adaptive mechanism through which Schwann cells counter the toxic effect of a mutant terminal differentiation protein.SIGNIFICANCE STATEMENT In many neuropathies, Schwann cells express high levels of early differentiation genes, but the significance of these altered expression remained unclear. Because many of these factors may act as negative regulators of myelination, it was suggested that their misexpression could contribute to dysmyelination. Here, we show that the transcription factors Sox2 and Id2 act as negative regulators of myelination in vivo, but that their sustained expression in Charcot-Marie-Tooth type 1B (CMT1B) represents an adaptive response activated by the Schwann cells to reduce mutant protein toxicity and prevent demyelination.

Muscarinic Receptor M3R Signaling Prevents Efficient Remyelination by Human and Mouse Oligodendrocyte Progenitor Cells.

Muscarinic receptor antagonists act as potent inducers of oligodendrocyte differentiation and accelerate remyelination. However, the use of muscarinic antagonists in the clinic is limited by poor understanding of the operant receptor subtype, and questions regarding possible species differences between rodents and humans. Based on high selective expression in human oligodendrocyte progenitor cells (OPCs), we hypothesized that M3R is the functionally relevant receptor. Lentiviral M3R knockdown in human primary CD140a/PDGFαR+ OPCs resulted in enhanced differentiation in vitro and substantially reduced the calcium response following muscarinic agonist treatment. Importantly, following transplantation in hypomyelinating shiverer/rag2 mice, M3R knockdown improved remyelination by human OPCs. Furthermore, conditional M3R ablation in adult NG2-expressing OPCs increased oligodendrocyte differentiation and led to improved spontaneous remyelination in mice. Together, we demonstrate that M3R receptor mediates muscarinic signaling in human OPCs that act to delay differentiation and remyelination, suggesting that M3 receptors are viable targets for human demyelinating disease.SIGNIFICANCE STATEMENT The identification of drug targets aimed at improving remyelination in patients with demyelination disease is a key step in development of effective regenerative therapies to treat diseases, such as multiple sclerosis. Muscarinic receptor antagonists have been identified as effective potentiators of remyelination, but the receptor subtypes that mediate these receptors are unclear. In this study, we show that genetic M3R ablation in both mouse and human cells results in improved remyelination and is mediated by acceleration of oligodendrocyte commitment from oligodendrocyte progenitor cells. Therefore, M3R represents an attractive target for induced remyelination in human disease.

Impairment of protein degradation and proteasome function in hereditary neuropathies.

In several neurodegenerative diseases in which misfolded proteins accumulate there is impairment of the ubiquitin proteasome system (UPS). We tested if a similar disruption of proteostasis occurs in hereditary peripheral neuropathies. In sciatic nerves from mouse models of two human neuropathies, Myelin Protein Zero mutation (S63del) and increased copy number (P0 overexpression), polyubiquitinated proteins accumulated, and the overall rates of protein degradation were decreased. 26S proteasomes affinity-purified from sciatic nerves of S63del mice were defective in degradation of peptides and a ubiquitinated protein, unlike proteasomes from P0 overexpression, which appeared normal. Nevertheless, cellular levels of 26S proteasomes were increased in both, through the proteolytic-activation of the transcription factor Nrf1, as occurs in response to proteasome inhibitors. In S63del, increased amounts of the deubiquitinating enzymes USP14, UCH37, and USP5 were associated with proteasomes, the first time this has been reported in a human disease model. Inhibitors of USP14 increased the rate of protein degradation in S63del sciatic nerves and unexpectedly increased the phosphorylation of eIF2α by Perk. Thus, proteasome content, composition and activity are altered in these diseases and USP14 inhibitors have therapeutic potential in S63del neuropathy.