Peripheral Nervous System Myelin Research Articles

Laura Feltri: Of Schwann cells, matrix, and family.

Laura Feltri (1963–2023) has been a pioneer in the study of extracellular matrix in peripheral nervous system myelination.

Schwann cells are axo-protective after injury irrespective of myelination status in mouse Schwann cell-neuron cocultures.

Myelinating Schwann cell (SC)-dorsal root ganglion (DRG) neuron cocultures are an important technique for understanding cell-cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. Although methods using rat SCs and neurons or mouse DRG explants are commonplace, there are no established protocols for compartmentalised myelinating cocultures with dissociated mouse cells. There consequently is a need for a coculture protocol that allows separate genetic manipulation of mouse SCs or neurons, or use of cells from different transgenic animals to complement in vivo mouse experiments. However, inducing myelination of dissociated mouse SCs in culture is challenging. Here, we describe a new method to coculture dissociated mouse SCs and DRG neurons in microfluidic chambers and induce robust myelination. Cocultures can be axotomised to study injury and used for drug treatments, and cells can be lentivirally transduced for live imaging. We used this model to investigate axon degeneration after traumatic axotomy and find that SCs, irrespective of myelination status, are axo-protective. At later timepoints after injury, live imaging of cocultures shows that SCs break up, ingest and clear axonal debris.

Myelin protein zero - the structural foundation behind peripheral compact myelin.

Schwann cells are the myelinating glia of the peripheral nervous system (PNS). During PNS development, Schwann cell precursors differentiate into mature Schwann cells after the radial sorting of axons and form myelin around selected axonal segments. This multilayered, highly lipid-rich and insulative membrane structure significantly accelerates nerve impulse propagation, which is a pre-requisite for the efficient function of the PNS. During this differentiation process, protein zero (P0), a single-pass transmembrane protein, is expressed in high amounts. P0 is the most abundant PNS myelin protein, at up to 50% of total protein, and its accumulation in membranes eventually forms stable compact myelin. Failure to do so, like in the case of mutated or truncated protein, results in non-compacted myelin and demyelination. This subsequently causes incurable peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS), where CMT alone afflicts nearly 3 million people globally. To understand the compaction and maturation of PNS myelin, and the molecular etiology of disease, uncovering the structure-function relationships of P0 are of great importance. We demonstrate the molecular foundation of P0-mediated membrane stacking using a hybrid structural biology approach: on the extracellular side, immunoglobulin (Ig)-like domains form a zipper-like assembly of apposing protein monomers, whereas on the cytoplasmic side, the short, positively charged P0 tail (P0ct) glues membranes together. P0ct undergoes a substantial folding event during membrane binding, and we provide rationale to why membrane stacking is affected in diseases through functional in vitro mutagenesis studies. Additionally, we speculate on how post-translational modifications in P0ct help P0 locate to its target compartments, without inducing premature membrane stacking. Our results shed light on the essential role of P0 in during myelin maturation, and why other proteins fail to replace P0 when its function is compromised.

The TGR5 Agonist INT-777 Promotes Peripheral Nerve Regeneration by Activating cAMP-dependent Protein Kinase A in Schwann Cells.

Schwann cell (SC) myelination is a pivotal event in the normal physiological functioning of the peripheral nervous system (PNS), where myelination is finely controlled by a series of factors within SCs to ensure timely onset and correct myelin thickness for saltatory conduction. Among these, cyclic AMP (cAMP) is a promising factor for driving myelin gene expression in SCs. It has been shown that TGR5 activation is often associated with increased production of cAMP. Therefore, we speculated that the G-protein-coupled receptor (TGR5) might be involved in the PNS myelination. To test this hypothesis, sciatic nerve crush-injured mice were treated with INT-777, a specific agonist of TGR5, which significantly improved remyelination and functional recovery. Furthermore, rats that underwent sciatic nerve transection were treated with INT-777, which also promoted nerve regeneration and functional recovery. In primary SCs, the stimulatory effect of INT-777 on myelin gene expression was largely counteracted by H89, a potent inhibitor of cAMP-dependent protein kinase A (PKA). Additionally, INT-777 stimulated cell migration was blunted in the presence of H89. Overall, these data indicate that INT-777 is capable of promoting peripheral nerve regeneration and functional recovery after injury, and these benefits are likely due to the activation of the TGR5/cAMP/PKA axis. As such, INT-777, together with other TGR5 agonists, may hold great therapeutic potential for treating peripheral nerve injury.

The best of both worlds: mastering nerve regeneration combining biological and nanotechnological tools.

Over the last decade, remarkable developments in nanotechnology have powered medical research, unveiling new approaches for the solution of public health issues such as the treatment of traumatic peripheral neuropathies. With an estimated incidence of 13 to 23 cases per 100,000 people per year in developed countries, traumatic nerve injuries constitute the most frequent type of peripheral nervous system (PNS) damage. Peripheral neuropathy results from accidents associated with everyday activities such as sports, work, recreation, and driving. Upon injury, the connection between the neuronal distal axon and soma is interrupted, which triggers the degeneration of the affected nerve with structural and functional loss, known as Wallerian degeneration. Patients suffer symptoms ranging from neuropathic pain to partial or even total sensory and motor limitations, depending on the degree of injury severity. The lack of nerve responsiveness to regeneration – in terms of axon regrowth and remyelination – and unsuccessful target organ reinnervation are the main obstacles hindering complete nerve recovery. Strategies commonly available for peripheral nerve lesion treatment are chosen according to the severity of the damage. Interventions include conventional approaches such as microsurgery to join nerve ends in the case of a transection, artificial and non-artificial nerve grafts, transplantation of conduits made of different biocompatible materials, polymer conduits coated with cells or growth factors that mimic a graft, and even therapies combining electrostimulation (Su et al., 2018) and physical exercise to promote nerve regeneration (Armada-da-Silva et al., 2013). Currently, the treatment of choice for the most severe transection injuries is microsurgery, although the functional outcome is often limited by inflammation, scar formation, and the pruning of sensory and motor axons. Less severe lesions in the PNS may undergo spontaneous regeneration. A cascade of cellular events associated with neuroinflammation (Figure 1A) takes place in the distal stump of the injured nerve and plays a critical role in regeneration, although a chronic inflammatory reaction may be an obstacle to the regenerative process. Among these cellular events, the activation and proliferation of Schwann cells (SCs) – due to their high plasticity – are key to nerve regeneration. After the injury, SCs lose contact with axons, acquire a dedifferentiated repair phenotype, and, together with resident macrophages, secrete a pool of cytokines in the early degeneration phase (Figure 1B). This event promotes the recruitment of hematogenous macrophages which contribute to the removal of axon and myelin debris, an essential step for peripheral nerve regeneration. Repair SCs generate Büngner bands to guide axonal regrowth and, finally, the regenerated axon triggers a switch through which repair SC recover their mature myelinating phenotype. In more severe lesions, the crosstalk between SCs and axons cannot be reestablished, and the regenerative process ultimately fails, causing cellular and axonal death. Unlike peripheral nerve lesions, traumatic injuries to the central nervous system (CNS) show poor regeneration ability. The low rate of myelin removal in the CNS generates a hostile environment for regeneration, as myelin and axonal debris contribute to the formation of the glial scar, which hampers axon regrowth and brings about loss of function.Figure 1: Summary of the relevant molecular events associated with the Wallerian degeneration process in the peripheral nervous system and therapeutic strategies for traumatic injuries.(A) Scheme of the in vivo degenerative-regenerative process. Sciatic nerve scheme showing a control nerve made up mainly of axons, connective tissue, myelinating Schwann cells (SC), and resident macrophages (RM). After the injury, the proximal stump of the nerve remains connected to the neuronal soma and preserves its structure; otherwise, the distal stump suffers a degenerative process after the lesion. SC lose their myelinating phenotype and proliferate, becoming repair Schwann cells (r-SC) which, together with RM, secrete cytokines and recruit hematogenous macrophages (HM). HM contributed to the removal of axon and myelin debris, while Büngner bands generated by r-SC guide axon regrowth to reinnervate the target site. At the same time, axons and SC reestablish the crosstalk and SC recovers their mature myelinating phenotype. (B) Temporal progression of cytokine secretion during degeneration. Within the first hours after injury, SC began to secrete pro-inflammatory cytokines [tumor necrosis factor (TNF) -α, interleukin (IL)-1β] which, together with factors secreted by fibroblasts, recruit HM which arrive at the site of injury 2–3 days post-injury. After the arrival of HM, the second stage of the inflammatory response begins, characterized by the second peak in IL-6 and a peak in IL-10, an anti-inflammatory cytokine, around 7 days post-injury. This event coincides with the peak in HM recruitment. Adapted from Rotshenker (2011). (C) Therapeutic strategies for traumatic peripheral nerve injuries. Conventional strategies (right) range from microsuture to nerve grafts and polymer conduits in more severe injuries where the nerve is completely transected. In less severe lesions, pharmacological therapy is commonly used for neuropathic pain treatment. In both cases, physical exercise and electrostimulation are used to improve regeneration and alleviate pain symptoms. Conventional strategies sometimes fail in restoring normal function and morphology of the nerve, which is why non-conventional strategies (left) such as cell therapy alone or combined with magnetic targeting have been developed over the last decades to improve cell recruitment to the injured nerve. The innovative hybrid therapeutic strategy appears as a promising tool to reach complete neuroregeneration after traumatic injuries. This technique has proven useful to improve and accelerate remyelination and distal latency recovery after injury in an animal model. AdMSC: Adipose-derived mesenchymal stem cell; GM-CSF: granulocyte-macrophage colony-stimulating factor; MNP: magnetic nanoparticles.The optimal temporal window for therapeutic interventions strongly depends on the severity of peripheral damage. The most severe cases, such as nerve transection, require the immediate restoration of nerve connection by microsurgery. If the gap between the two ends of the transected nerve is wide and does not allow suturing, acellular conduits are used to connect the two ends and guide the axonal path for efficient regeneration. Nevertheless, bioengineered conduits may recreate the extracellular matrix of the nerve and even be supplemented with trophic factors, but they still lack the critical cellular component. In cases of less severe transection or even severe non-transection injuries, the connective tissue remains intact, which bypasses the need for surgical intervention to allow the reinnervation of the target organ. On the other hand, non-surgical approaches such as medication and physical exercise can be a suitable option all along treatment (Armada-da-Silva et al., 2013), but electrical stimulation seems to have an optimum time window – between the first 21 days after damage – to yield good clinical results (Su et al., 2018). Even so, functional recovery remains a clinical challenge. PNS lesions thus exhibit a complex pathophysiology, and current surgical and pharmacological therapies are only partially effective. These reasons have led neuroregeneration research to develop and adopt hybrid therapeutic strategies that combine biological and nanotechnological methodologies (Figure 1C). Among biological strategies, adult multipotent mesenchymal stem cells (MSC) have received special attention and have been extensively studied due to their potential application in the field of regenerative and reconstructive medicine. MSC are adult stem cells originally described in the bone marrow but later found in different adult tissues. MSC regenerative ability exceeds that of other stem cells as a result of advantages such as self-renewal rates, high plasticity, and low immunogenicity and tumorigenicity. Most relevant, the use of MSC avoids ethical concerns usually associated with embryonic stem cells (Bacakova et al., 2018). Among the most popular MSCs, adipose-derived MSCs (AdMSCs) emerged as an interesting cell population for regenerative therapies, as they rapidly migrate to and integrate into the target tissue after systemic administration. AdMSCs mediate regenerative processes through mechanisms such as transdifferentiation, the induction of angiogenesis, and the secretion of growth factors and extracellular vesicles (LeBlanc and Uchida, 2019). Also, AdMSC has been proven to be more active in the autocrine production of trophic and immunomodulatory factors than other cell types. AdMSC has been thoroughly studied for their potential therapeutic application in a variety of pathologies with diverse etiology. Safety and absence of adverse effects have been demonstrated after AdMSC transplantation for the treatment of acute respiratory syndrome (Zheng et al., 2014) and secondary progressive multiple sclerosis (Fernández et al., 2018). In addition, good clinical results and an improvement in joint function have been observed in osteoarthritis treatment (Lu et al., 2019). It is noteworthy that the success of cell transplantation treatment depends on factors such as the number of transplanted cells, the administration route, and the number of cells that arrive and are retained at the target site. In this scenario, the incorporation of nanotechnological tools appears as a promising strategy to enhance treatment outcomes. Magnetic nanoparticles (MNP), particularly iron oxide ones in the maghemite and magnetite phase (γ-Fe2O3 and Fe3O4), are the most frequently proposed nano-developments in medical research due to their outstanding properties. Their great response to magnetic fields allows remote and non-invasive manipulation, enabling cells loaded with MNP to be magnetically targeted to a specific tissue or organ. One of the key properties of iron oxide MNP which makes them excellent candidates for biomedical use is their high biocompatibility. Indeed, iron plays a fundamental role in numerous cellular processes such as oxygen storage and transport, electron transport and energy metabolism, and antioxidant and prooxidant reactions. The regulation of iron metabolism and homeostasis are complex processes, and an imbalance caused by excessive exogenous iron administration may trigger toxicity. Furthermore, proper iron uptake is essential for SC maturation in PNS myelination and remyelination along with development and after injury (Santiago González et al., 2019), as iron is an indispensable cofactor for several enzymes involved in the production and maintenance of the myelin sheath. Even though the use of MNP for different applications is widely disseminated, their toxicological profile has not been well defined yet and remains controversial. When MNP internalization by cells is required, nano-cytotoxicity must be carefully considered for MNP validation as a biomedical tool. Moreover, MNP cytotoxicity is dose-dependent, and the generation of reactive oxygen species is chiefly responsible for cell damage. For these reasons, the evaluation of labile iron levels is of great interest for the use of MNP in regenerative medicine applications. In addition, special attention should be given to the analysis of the MNP degradation process. MNP degradation depends on cell factors such as type, metabolism, and proliferation rate, and inherent MNP factors such as size, coating, and shape. Iron oxide MNP degradation produces free iron ions which either are incorporated into hemoglobin or join the native iron pool of the body and are degraded by normal iron-recycling pathways. However, excessive iron doses could affect cell morphology, signaling processes, and differentiation potential. Nevertheless, MNP use in controlled conditions allows their efficient internalization by cells without affecting cell functions or differentiation capacity. As mentioned above, a crucial step in PNS regeneration is the successful delivery of cells to the target tissue and their retention at the lesion site. To overcome these obstacles, our group has improved cell therapy outcomes through the implementation of magnetic targeting of AdMSC loaded with responsive MNP (Soto et al., 2021). Magnetic targeting is an encouraging method for the treatment of CNS and PNS lesions, as it optimizes the rates at which systemically transplanted cells arrive and remain at the site of injury. This brings about an increased concentration of trophic factors secreted by AdMSC, which improves cell immunomodulatory effects. Moreover, magnetic targeting allows the in vivo remote manipulation of transplanted cells using small magnets. This approach avoids invasive methods of cell transplantation or an extra surgical procedure that could per se promote an inflammatory reaction and the exacerbation of neuropathic pain. This strategy, including AdMSC magnetic targeting, also allows cell transplantation in the acute phase after lesion. Given that the therapeutic time window for traumatic lesions in the PNS is short and represents a critical point in neuroregeneration processes, acute phase transplantation may contribute to more satisfactory results. Good results using magnetic targeting have been reported for the treatment of CNS lesions (Grayston et al., 2022). Also, employing spinal cord injury models, different authors have demonstrated that MSC labeled with MNP respond to the applied magnetic field and, more relevant, improve motor function (Vaněček et al., 2012). Furthermore, some studies have reported the use of magnetic targeting to deliver growth factors to the injured sciatic nerve (Marcus et al., 2018), while others have documented the use of MSC in the treatment of peripheral nerve injuries (Fernandes et al., 2018). Recently, in a rat model of sciatic nerve lesion, our group demonstrated the efficacy of AdMSC magnetic targeting to speed up nerve regeneration from a morphological and functional point of view. The proposed strategy uses a hybrid material consisting of AdMSC loaded with 10 nm iron oxide MNP in their endosomes and submitted to the force produced by a permanent external Neodymium Iron Boron magnet, which warrants animal comfort and full mobility during treatment (Soto et al., 2021). Therefore, to the best of our knowledge and despite the substantial evidence provided in previous studies, a hybrid approach combining the two strategies for in vivo sciatic nerve regeneration has so far only been reported by our group. Hybrid materials have proven useful, and the combination of different properties improves the therapeutic outcomes for numerous CNS and PNS injuries. Beyond the encouraging results, several challenges must be met prior to the translation of magnetic targeting to clinical applications. Some of them are optimal conditions for cell magnetic targeting – including thorough studies of the bio-nano interphase (MNP and biological environment interaction) – and the interaction between magnetically loaded cells and the magnetic fields applied once cells are intravenously administered. Another pressing need is the evaluation of MNP degradation and clearance in patients receiving magnetically loaded cells. Further experiments are necessary to fully elucidate this process, which depends on the metabolism and proliferation rate of the cell type involved. C-Editors: Zhao M, Liu WJ, Wang Lu; T-Editor: Jia Y

Targeted inactivation of the Septin2 and Septin9 genes in myelinating Schwann cells of mice.

The formation of axon-enwrapping myelin sheaths by oligodendrocytes in the central nervous system involves the assembly of a scaffolding septin filament comprised of the subunits SEPTIN2, SEPTIN4, SEPTIN7 and SEPTIN8. Conversely, in the peripheral nervous system (PNS), myelin is synthesized by a different cell type termed Schwann cells, and it remained unknown if septins also assemble as a multimer in PNS myelin. According to prior proteome analysis, PNS myelin comprises the subunits SEPTIN2, SEPTIN7, SEPTIN8, SEPTIN9, and SEPTIN11, which localize to the paranodal and abaxonal myelin subcompartments. Here, we use the Cre/loxP-system to delete the Septin9-gene specifically in Schwann cells, causing a markedly reduced abundance of SEPTIN9 in sciatic nerves, implying that Schwann cells are the main cell type expressing SEPTIN9 in the nerve. However, Septin9-deficiency in Schwann cells did not affect the abundance or localization of other septin subunits. In contrast, when deleting the Septin2-gene in Schwann cells the abundance of all relevant septin subunits was markedly reduced, including SEPTIN9. Notably, we did not find evidence that deleting Septin2 or Septin9 in Schwann cells impairs myelin biogenesis, nerve conduction velocity or motor/sensory capabilities, at least at the assessed timepoints. Our data thus show that SEPTIN2 but not SEPTIN9 is required for the formation or stabilization of a septin multimer in PNS myelin in vivo; however, its functional relevance remains to be established.

Transgenic Schwann cells overexpressing POU6F1 promote sciatic nerve regeneration within acellular nerve allografts

Objective. Acellular nerve allograft (ANA) is an effective surgical approach used to bridge the sciatic nerve gap. The molecular regulators of post-surgical recovery are not well-known. Here, we explored the effect of transgenic Schwann cells (SCs) overexpressing POU domain class 6, transcription factor 1 (POU6F1) on sciatic nerve regeneration within ANAs. We explored the functions of POU6F1 in nerve regeneration by using a cell model of H2O2-induced SCs injury and transplanting SCs overexpressing POU6F1 into ANA to repair sciatic nerve gaps. Approach. Using RNA-seq, Protein–Protein Interaction network analysis, gene ontology enrichment, and Kyoto Encyclopedia of Genes and Genomes pathway analysis, we identified a highly and differentially expressed transcription factor, POU6F1, following ANA treatment of sciatic nerve gap. Expressing a high degree of connectivity, POU6F1 was predicted to play a role in peripheral nervous system myelination. Main results. To test the role of POU6F1 in nerve regeneration after ANA, we infected SCs with adeno-associated virus—POU6F1, demonstrating that POU6F1 overexpression promotes proliferation, anti-apoptosis, and migration of SCs in vitro. We also found that POU6F1 significantly upregulated JNK1/2 and c-Jun phosphorylation and that selective JNK1/2 inhibition attenuated the effects of POU6F1 on proliferation, survival, migration, and JNK1/2 and c-Jun phosphorylation. The direct interaction of POU6F1 and activated JNK1/2 was subsequently confirmed by co-immunoprecipitation. In rat sciatic nerve injury model with a 10 mm gap, we confirmed the pattern of POU6F1 upregulation and co-localization with transplanted SCs. ANAs loaded with POU6F1-overexpressing SCs demonstrated the enhanced survival of transplanted SCs, axonal regeneration, myelination, and functional motor recovery compared to the ANA group loaded by SCs-only in line with in vitro findings. Significance. This study identifies POU6F1 as a novel regulator of post-injury sciatic nerve repair, acting through JNK/c-Jun signaling in SCs to optimize therapeutic outcomes in the ANA surgical approach.

Coordinated Regulation of Myelination by Growth Factor and Amino-acid Signaling Pathways.

Myelin-forming oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS) are essential for structural and functional homeostasis of nervous tissue. Albeit with certain similarities, the regulation of CNS and PNS myelination is executed differently. Recent advances highlight the coordinated regulation of oligodendrocyte myelination by amino-acid sensing and growth factor signaling pathways. In this review, we discuss novel insights into the understanding of differential regulation of oligodendrocyte and Schwann cell biology in CNS and PNS myelination, with particular focus on the roles of growth factor-stimulated RHEB-mTORC1 and GATOR2-mediated amino-acid sensing/signaling pathways. We also discuss recent progress on the metabolic regulation of oligodendrocytes and Schwann cells and the impact of their dysfunction on neuronal function and disease.

Imbalance of NRG1-ERBB2/3 signalling underlies altered myelination in Charcot-Marie-Tooth disease 4H.

Charcot-Marie-Tooth (CMT) disease is one of the most common inherited neurological disorders, affecting either axons from the motor and/or sensory neurons or Schwann cells of the peripheral nervous system (PNS) and caused by more than 100 genes. We previously identified mutations in FGD4 as responsible for CMT4H, an autosomal recessive demyelinating form of CMT disease. FGD4 encodes FRABIN, a GDP/GTP nucleotide exchange factor, particularly for the small GTPase Cdc42. Remarkably, nerves from patients with CMT4H display excessive redundant myelin figures called outfoldings that arise from focal hypermyelination, suggesting that FRABIN could play a role in the control of PNS myelination. To gain insights into the role of FGD4/FRABIN in Schwann cell myelination, we generated a knockout mouse model (Fgd4SC-/-), with conditional ablation of Fgd4 in Schwann cells. We show that the specific deletion of FRABIN in Schwann cells leads to aberrant myelination in vitro, in dorsal root ganglia neuron/Schwann cell co-cultures, as well as in vivo, in distal sciatic nerves from Fgd4SC-/- mice. We observed that those myelination defects are related to an upregulation of some interactors of the NRG1 type III/ERBB2/3 signalling pathway, which is known to ensure a proper level of myelination in the PNS. Based on a yeast two-hybrid screen, we identified SNX3 as a new partner of FRABIN, which is involved in the regulation of endocytic trafficking. Interestingly, we showed that the loss of FRABIN impairs endocytic trafficking, which may contribute to the defective NRG1 type III/ERBB2/3 signalling and myelination. Using RNA-Seq, in vitro, we identified new potential effectors of the deregulated pathways, such as ERBIN, RAB11FIP2 and MAF, thereby providing cues to understand how FRABIN contributes to proper ERBB2 trafficking or even myelin membrane addition through cholesterol synthesis. Finally, we showed that the re-establishment of proper levels of the NRG1 type III/ERBB2/3 pathway using niacin treatment reduces myelin outfoldings in nerves of CMT4H mice. Overall, our work reveals a new role of FRABIN in the regulation of NRG1 type III/ERBB2/3 NRG1signalling and myelination and opens future therapeutic strategies based on the modulation of the NRG1 type III/ERBB2/3 pathway to reduce CMT4H pathology and more generally other demyelinating types of CMT disease.

[Introduction to Myelin Research].

Myelin is a multilamellar membrane structure formed by oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). It has been recognized as an insulator that is essential for the rapid and efficient propagation of action potentials by saltatory conduction. However, recently many studies have shown that myelin and myelin-forming cells interact with axons and regulate the nervous system far more actively than previously thought. For example, myelination changes axons dynamically and divides them into four distinct functional domains: node of Ranvier, paranode, juxtaparanode, and internode. Voltage-gated Na+ channels are clustered at the node, while K+ channels are at the juxtaparanode, and segregation of these channels by paranodal axoglial junction is necessary for proper axonal function. My research experience began at the neurology ward of the Niigata University Medical Hospital, where I saw a patient with peripheral neuropathy of unknown etiology more than 37 years ago. In the patient's serum, we found an autoantibody against a glycolipid enriched in the PNS. Since then, I have been interested in myelin because of its beautiful structure and unique roles in the nervous system. In this review, our recent studies related to CNS and PNS myelin are presented.

Peripheral neuropathy in severe iron deficiency anemia: A case report

Anemia is a global health problem that need medical attention. Iron deficiency anemia (IDA), in particular is the most common nutritional deficiency anemia prevalent in India. Iron essential for effective myelination of peripheral nervous system.1 Nerve conduction studies are one of the neurophysiological investigations that can quantitatively detect peripheral nerve dysfunction.2 In this case report, we present findings of nerve conduction studies performed on a case of severe iron deficiency anemia and the associated peripheral neurodeficit observed in the case study. Nerve conduction study (NCS) was performed in bilateral median and ulnar motor and sensory nerves. CMAP (compound muscle action potential) and SNAP(sensory nerve action potential) and motor and sensory NCV (nerve conduction velocity) was decreased and the distal latency was prolonged in both ulnar and median nerves. These observations indicate a sensorimotor demyelinating and axonal peripheral neuropathy in the patient due to iron deficiency.

Ninj2 regulates Schwann cells development by interfering laminin-integrin signaling.

Rationale: Myelin sheath is an important structure to maintain normal functions of the nerves. Nerve Injury-Induced Protein 2 (Ninj2) was found upregulated in Schwann cells (SC) upon injury. However, whether and how Ninj2 plays a role in myelination remain unknown. Methods: In this study, we use transmission electron microscope imaging, immunofluorescent imaging, and behavioral tests to show the effects of Ninj2 on myelination and remyelination in peripheral nervous system (PNS) of SC-specific Ninj2 knockout mice (Dhhcre/+;Ninj2fl/fl ). For mechanism studies, we use RNA-Seq analysis to show the Ninj2-related pathways, and co-immunoprecipitation/mass-spectrometry to identify the Ninj2-interacting proteins in SCs. Furthermore, we evaluate the effect of integrin inhibitor GRGDSP during remyelination. Results: Ninj2 negatively regulates SC development. Ninj2-deficient mice exhibit precocious myelination phenotype, as well as the accelerated remyelination process after sciatic nerve injury. Loss of Ninj2 promotes myelination by promoting SC proliferation to augment its population. Mechanistically, Ninj2 interacted with ITGB1 on SC membrane, which inhibits laminin-integrin signaling. Removal of Ninj2 induces the activity of laminin-integrin signaling, resulting in the improved myelination in the Dhhcre/+;Ninj2fl/fl mice. Inhibition of laminin-integrin signaling by integrin inhibitor GRGDSP sufficiently delays the remyelination process in the Dhhcre/+;Ninj2fl/fl mice with sciatic nerve injury. Conclusion: Our study found Ninj2 as a negative regulator in the network controlling myelination in the PNS.

An electroceutical approach enhances myelination via upregulation of lipid biosynthesis in the dorsal root ganglion

As the myelin sheath is crucial for neuronal saltatory conduction, loss of myelin in the peripheral nervous system (PNS) leads to demyelinating neuropathies causing muscular atrophy, numbness, foot deformities and paralysis. Unfortunately, few interventions are available for such neuropathies, because previous pharmaceuticals have shown severe side effects and failed in clinical trials. Therefore, exploring new strategies to enhance PNS myelination is critical to provide solution for such intractable diseases. This study aimed to investigate the effectiveness of electrical stimulation (ES) to enhance myelination in the mouse dorsal root ganglion (DRG)—an ex vivo model of the PNS. Mouse embryonic DRGs were extracted at E13 and seeded onto Matrigel-coated surfaces. After sufficient growth and differentiation, screening was carried out by applying ES in the 1–100 Hz range at the beginning of the myelination process. DRG myelination was evaluated via immunostaining at the intermediate (19 days in vitro (DIV)) and mature (30 DIV) stages. Further biochemical analyses were carried out by utilizing ribonucleic acid sequencing, quantitative polymerase chain reaction and biochemical assays at both intermediate and mature myelination stages. Imaging of DRG myelin lipids was carried out via time-of-flight secondary ion mass spectrometry (ToF-SIMS). With screening ES conditions, optimal condition was identified at 20 Hz, which enhanced the percentage of myelinated neurons and average myelin length not only at intermediate (129% and 61%) but also at mature (72% and 17%) myelination stages. Further biochemical analyses elucidated that ES promoted lipid biosynthesis in the DRG. ToF-SIMS imaging showed higher abundance of the structural lipids, cholesterol and sphingomyelin, in the myelin membrane. Therefore, promotion of lipid biosynthesis and higher abundance of myelin lipids led to ES-mediated myelination enhancement. Given that myelin lipid deficiency is culpable for most demyelinating PNS neuropathies, the results might pave a new way to treat such diseases via electroceuticals.

Adhesion G protein-coupled receptor Gpr126/Adgrg6 is essential for placental development.

Mutations in the G protein–coupled receptor GPR126/ADGRG6 cause human diseases, including defective peripheral nervous system (PNS) myelination. To study GPR126 function, we generated new genetic mice and zebrafish models. Murine Gpr126 is expressed in developing heart endocardium, and global Gpr126 inactivation is embryonically lethal, with mutants having thin-walled ventricles but unaffected heart patterning or maturation. Endocardial-specific Gpr126 deletion does not affect heart development or function, and transgenic endocardial GPR126 expression fails to rescue lethality in Gpr126-null mice. Zebrafish gpr126 mutants display unaffected heart development. Gpr126 is also expressed in placental trophoblast giant cells. Gpr126-null mice with a heterozygous placenta survive but exhibit GPR126-defective PNS phenotype. In contrast, Gpr126-null embryos with homozygous mutant placenta die but are rescued by placental GPR126 expression. Gpr126-deficient placentas display down-regulation of preeclampsia markers Mmp9, Cts7, and Cts8. We propose that the placenta-heart axis accounts for heart abnormalities secondary to placental defects in Gpr126 mutants.

Biochemical and Structural Characteristics, Gene Regulation, Physiological, Pathological and Clinical Features of Lipocalin-Type Prostaglandin D2 Synthase as a Multifunctional Lipocalin.

Lipocalin-type prostaglandin (PG) D2 synthase (L-PGDS) catalyzes the isomerization of PGH2, a common precursor of the two series of PGs, to produce PGD2. PGD2 stimulates three distinct types of G protein-coupled receptors: (1) D type of prostanoid (DP) receptors involved in the regulation of sleep, pain, food intake, and others; (2) chemoattractant receptor-homologous molecule expressed on T helper type 2 cells (CRTH2) receptors, in myelination of peripheral nervous system, adipocyte differentiation, inhibition of hair follicle neogenesis, and others; and (3) F type of prostanoid (FP) receptors, in dexamethasone-induced cardioprotection. L-PGDS is the same protein as β-trace, a major protein in human cerebrospinal fluid (CSF). L-PGDS exists in the central nervous system and male genital organs of various mammals, and human heart; and is secreted into the CSF, seminal plasma, and plasma, respectively. L-PGDS binds retinoic acids and retinal with high affinities (Kd < 100 nM) and diverse small lipophilic substances, such as thyroids, gangliosides, bilirubin and biliverdin, heme, NAD(P)H, and PGD2, acting as an extracellular carrier of these substances. L-PGDS also binds amyloid β peptides, prevents their fibril formation, and disaggregates amyloid β fibrils, acting as a major amyloid β chaperone in human CSF. Here, I summarize the recent progress of the research on PGD2 and L-PGDS, in terms of its “molecular properties,” “cell culture studies,” “animal experiments,” and “clinical studies,” all of which should help to understand the pathophysiological role of L-PGDS and inspire the future research of this multifunctional lipocalin.

Unraveling the etiology of myelin disorders: the P2 case in Charcot–Marie–Tooth disease

There are several examples of single mutations that lead to a well-defined disease through a well-known mechanism. In other cases, a collection of mutations of the same protein produces a pathology with different degrees of severity. The accompanying work by Uusitalo etal. studies several mutants of the fatty acid binding protein P2 of the peripheral nervous system myelin. They conserve the native tertiary structure but a remarkable difference in the capacity to interact with lipids. This could be a clue to unravel the complex way in which these mutations affect myelin structure and function in a variant of Charcot-Marie-Tooth disease. Comment on: https://doi.org/10.1111/febs.16079.

Human myelin protein P2: from crystallography to time-lapse membrane imaging and neuropathy-associated variants.

Peripheral myelin protein 2 (P2) is a fatty acid-binding protein expressed in vertebrate peripheral nervous system myelin, as well as in human astrocytes. Suggested functions of P2 include membrane stacking and lipid transport. Mutations in the PMP2 gene, encoding P2, are associated with Charcot-Marie-Tooth disease (CMT). Recent studies have revealed three novel PMP2 mutations in CMT patients. To shed light on the structure and function of these P2 variants, we used X-ray and neutron crystallography, small-angle X-ray scattering, circular dichroism spectroscopy, computer simulations and lipid binding assays. The crystal and solution structures of the I50del, M114T and V115A variants of P2 showed minor differences to the wild-type protein, whereas their thermal stability was reduced. Vesicle aggregation assays revealed no change in membrane stacking characteristics, while the variants showed altered fatty acid binding. Time-lapse imaging of lipid bilayers indicated formation of double-membrane structures induced by P2, which could be related to its function in stacking of two myelin membrane surfaces in vivo. In order to better understand the links between structure, dynamics and function, the crystal structure of perdeuterated P2 was refined from room temperature data using neutrons and X-rays, and the results were compared to simulations and cryocooled crystal structures. Our data indicate similar properties for all known human P2 CMT variants; while crystal structures are nearly identical, thermal stability and function of CMT variants are impaired. Our data provide new insights into the structure-function relationships and dynamics of P2 in health and disease.

Differential Contribution of Cadm1-Cadm3 Cell Adhesion Molecules to Peripheral Myelinated Axons.

Cell adhesion proteins of the Cadm (SynCAM/Necl) family regulate myelination and the organization of myelinated axons. In the peripheral nervous system (PNS), intercellular contact between Schwann cells and their underlying axons is believed to be mediated by binding of glial Cadm4 to axonal Cadm3 or Cadm2. Nevertheless, given that distinct neurons express different combinations of the Cadm proteins, the identity of the functional axonal ligand for Cadm4 remains to be determined. Here, we took a genetic approach to compare the phenotype of Cadm4 null mice, which exhibit abnormal distribution of Caspr and Kv1 potassium channels, with mice lacking different combinations of Cadm1-Cadm3 genes. We show that in contrast to mice lacking the single Cadm1, Cadm2, or Cadm3 genes, genetic ablation of all three phenocopies the abnormalities detected in the absence of Cadm4. Similar defects were observed in double mutant mice lacking Cadm3 and Cadm2 (i.e., Cadm3-/-/Cadm2-/-) or Cadm3 and Cadm1 (i.e., Cadm3-/-/Cadm1-/-), but not in mice lacking Cadm1 and Cadm2 (i.e., Cadm1-/-/Cadm2-/-). Furthermore, axonal organization abnormalities were also detected in Cadm3 null mice that were heterozygous for the two other axonal Cadms. Our results identify Cadm3 as the main axonal ligand for glial Cadm4, and reveal that its absence could be compensated by the combined action of Cadm2 and Cadm1.SIGNIFICANCE STATEMENT Myelination by Schwann cells enables fast conduction of action potentials along motor and sensory axons. In these nerves, Schwann cell-axon contact is mediated by cell adhesion molecules of the Cadm family. Cadm4 in Schwann cells regulates axonal ensheathment and myelin wrapping, as well as the organization of the axonal membrane, but the identity of its axonal ligands is not clear. Here, we reveal that Cadm mediated axon-glia interactions depend on a hierarchical adhesion code that involves multiple family members. Our results provide important insights into the molecular mechanisms of axon-glia communication, and the function of Cadm proteins in PNS myelin.

Guillain-barre syndrome associated by covid-19 infection

Background: Coronavirus disease 2019 (COVID-19) typically causes breathing issues that can range from flu symptoms to extreme pneumonia but can also impair extra respiratory systems and cause multisystemic failure, including neurological complications. Case Presentation: A 55-year-old male with acute progressive symmetrical ascending quadriparesis complaints was admitted to the hospital. Twelve days prior to hospitalization, the patient with taste disruption, myalgia, fever, and polymerase chain reaction with reverse transcription (RT-PCR) was confirmed to have been positive for COVID-19 infection. The neurophysiological findings were consistent with the diagnosis of Guillain-Barre syndrome (GBS). Conclusion: COVID-19 activates inflammatory cells and creates a number of inflammatory cytokines and eventually produces immune-mediated processes. Both cell and humoral-dependent pathways of GBS pathogenesis are believed to be related. peripheral nervous system myelin, axons and, in some cases, both immune-mediated attacks are believed to be the cause of molecular expression. COVID-19 is believed to induce antibody formation against particular gangliosides. Further study is needed to understand the role of GBS caused by infection with COVID-19.

New evidence for secondary axonal degeneration in demyelinating neuropathies

Development of peripheral nervous system (PNS) myelin involves a coordinated series of events between growing axons and the Schwann cell (SC) progenitors that will eventually ensheath them. Myelin sheaths have evolved out of necessity to maintain rapid impulse propagation while accounting for body space constraints. However, myelinating SCs perform additional critical functions that are required to preserve axonal integrity including mitigating energy consumption by establishing the nodal architecture, regulating axon caliber by organizing axonal cytoskeleton networks, providing trophic and potentially metabolic support, possibly supplying genetic translation materials and protecting axons from toxic insults. The intermediate steps between the loss of these functions and the initiation of axon degeneration are unknown but the importance of these processes provides insightful clues. Prevalent demyelinating diseases of the PNS include the inherited neuropathies Charcot-Marie-Tooth Disease, Type 1 (CMT1) and Hereditary Neuropathy with Liability to Pressure Palsies (HNPP) and the inflammatory diseases Acute Inflammatory Demyelinating Polyneuropathy (AIDP) and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP). Secondary axon degeneration is a common feature of demyelinating neuropathies and this process is often correlated with clinical deficits and long-lasting disability in patients. There is abundant electrophysiological and histological evidence for secondary axon degeneration in patients and rodent models of PNS demyelinating diseases. Fully understanding the involvement of secondary axon degeneration in these diseases is essential for expanding our knowledge of disease pathogenesis and prognosis, which will be essential for developing novel therapeutic strategies.