Myelin-associated glycoprotein alters the neuronal secretome and stimulates the release of TGFß and proteins that affect neural plasticity.

Myelin-associated glycoprotein (MAG) and Nogo inhibit neurite outgrowth by binding to receptors such as NgR1, PirB and LRP1, and they have also been shown to induce phosphorylation of Smad2, a key intermediate in the transforming growth factor ß (TGFß) signalling pathway. In this study, we determined that MAG and Nogo do not transactivate the TGFß receptor through their canonical receptors or discoidin domain receptor 1, which we identified as a novel receptor for MAG and Nogo. Instead, MAG and Nogo promoted Smad2 phosphorylation by stimulating secretion of TGFß. Proteomic analysis of the neuronal secretome revealed that MAG also regulated the secretion of proteins that affect central nervous system plasticity-inducing the secretion of S100A6, septin-7 and neurofascin 186, while inhibiting the secretion of frataxin, MAP6, syntenin-1 and GAP-43. This represents a novel function for MAG that has broad implications for the treatment for spinal cord injury.

Long-Term Disease Prevention with a Gene Therapy Targeting Oligodendrocytes in a Mouse Model of Adrenomyeloneuropathy.

Adrenomyeloneuropathy (AMN) is a late-onset axonopathy of spinal cord tracts caused by mutations of the ABCD1 gene that encodes adrenoleukodystrophy protein (ALDP), a peroxisomal transporter of very long-chain fatty acids (VLCFA). Disturbed metabolic interaction between oligodendrocytes (OL) and axons is suspected to play a major role in AMN axonopathy. To develop a vector targeting OL, the human ABCD1 gene driven by a short 0.3 kb part of the human myelin-associated glycoprotein (MAG) promoter was packaged into an adeno-associated viral serotype 9 (rAAV9). An intravenous injection of this vector on postnatal day 10 in Abcd1-/- mice, a model of AMN, allowed a near normal motor performance to persist for 24 months, while age-matched untreated mice developed major defects of balance and motricity. Three weeks postvector, 50-54% of spinal cord white matter OL was expressing human ALDP (hALDP) at the cervical level, and only 6-7% after 24 months. In addition, 29-32% of cervical spinal cord astrocytes at 3 weeks and 16-19% at 24 months also expressed ALDP. C26:0-lysoPC, a sensitive VLCFA marker of AMN, was lower by 41% and 50%, respectively, in the spinal cord and brain of vector-treated compared with untreated mice. In a nonhuman primate, the intrathecal injection of the rAAV9-MAG vector induced abundant ALDP expression at 3 weeks in spinal cord OL (43%, 29%, and 26% at cervical, thoracic, and lumbar levels) and cerebellum OL (35%). In addition, 33-41% of spinal cord astrocytes expressed hALDP, and 27% of cerebellar astrocytes. To our knowledge, OL targeting had not been obtained before in primates with other vectors or promoters. The current results thus provide a robust proof-of-concept not only for the gene therapy of AMN but also for other central nervous system diseases, where the targeting of OL with the rAAV9-MAG vector may be of interest.

Imperative role of glycosylation in human MOG-HLA interaction: molecular insights of MOG-Ab associated demyelination.

Myelin oligodendrocyte glycoprotein is a transmembrane protein found on the outer lamella of the myelin sheath. The autoimmune attack on the MOG leads to demyelination which differs from normal multiple sclerosis. MOG has three epitope regions MOG1-22, MOG35-55, and MOG92-106 in the extracellular region, and the crucial MOG35-55 epitope and Human Leukocyte Antigen (HLA) interaction is the initial step for autoantibody generation. To study the effective role of glycosylation in MOG-HLA interaction, we performed molecular dynamics simulations of the complex where HLA interacts with three MOG epitopes both in the absence and presence of glycan. The results projected that the epitope MOG1-22 is decisive for the HLA interaction in the absence of glycan and HLA interacts with the epitope MOG35-55 irrespective of glycan existence. The residues Arg9, Arg46, and Arg66 were found to interact strongly with HLA even in the presence of glycan. The glycan increased the flexibility of hMOG and enhanced the interaction of MOG with water molecules.

Regulation of neuronal repair and regeneration through inhibition of oligodendrocyte myelin glycoprotein (OMgp).

The inability of neural cells to regenerate themselves after an injury represents the major difference between neural cells and other cells of the body. Various factors are responsible for this, as the expression of myelin-derived inhibitors of axonal outgrowth such as neurite outgrowth inhibitor (Nogo), myelin-associated growth factor, and oligodendrocyte-myelin glycoprotein (OMgp) hinder the central nervous system (CNS) axons to recover properly and inhibit the neuron regeneration. The patient with spinal cord injury can even permanently lose their function due to the inability of axons to regenerate. However, their role in neural regeneration in vivo is not known completely. During the study, we found that once CNS gets injured, the axon growth inhibitor OMgp binds to the Nogo-66 Receptor 1 (NgR1) which in turn restricts the normal functioning of CNS. Considering the OMgp as the target protein, two flavonoid libraries (curcumin and piperine) were screened against it to get potential inhibitors. The effectiveness of the ligands was first screened by three-tier structure-based virtual screening by Glide, Schrödinger. Based on the docking score, the best-docked compounds were taken for absorption, distribution, metabolism, and excretion analysis and the top two complexes from each library were chosen for simulation studies. Flavonoid ligands showed a much better binding affinity when compared with already known inhibitors Riluzole and Minocycline. To date, no natural inhibitors are known for OMgp. Hence, this study can provide novel insight for upcoming research in this area. Communicated by Ramaswamy H. Sarma.

Myelin-associated glycoprotein activation triggers glutamate uptake by oligodendrocytes in vitro and contributes to ameliorate glutamate-mediated toxicity in vivo.

BACKGROUND: Myelin-associated glycoprotein (MAG) is a key molecule involved in the nurturing effect of myelin on ensheathed axons. MAG also inhibits axon outgrowth after injury. In preclinical stroke models, administration of a function-blocking anti-MAG monoclonal antibody (mAb) aimed to improve axon regeneration demonstrated reduced lesion volumes and a rapid clinical improvement, suggesting a mechanism of immediate neuroprotection rather than enhanced axon regeneration. In addition, it has been reported that antibody-mediated crosslinking of MAG can protect oligodendrocytes (OLs) against glutamate (Glu) overload by unknown mechanisms. PURPOSE: To unravel the molecular mechanisms underlying the protective effect of anti-MAG therapy with a focus on neuroprotection against Glu toxicity. RESULTS: MAG activation (via antibody crosslinking) triggered the clearance of extracellular Glu by its uptake into OLs via high affinity excitatory amino acid transporters. This resulted not only in protection of OLs but also nearby neurons. MAG activation led to a PKC-dependent activation of factor Nrf2 (nuclear-erythroid related factor-2) leading to antioxidant responses including increased mRNA expression of metabolic enzymes from the glutathione biosynthetic pathway and the regulatory chain of cystine/Glu antiporter system xc- increasing reduced glutathione (GSH), the main antioxidant in cells. The efficacy of early anti-MAG mAb administration was demonstrated in a preclinical model of excitotoxicity induced by intrastriatal Glu administration and extended to a model of Experimental Autoimmune Encephalitis showing axonal damage secondary to demyelination. CONCLUSIONS: MAG activation triggers Glu uptake into OLs under conditions of Glu overload and induces a robust protective antioxidant response.

Strategies to neutralize RhoA/ROCK pathway after spinal cord injury.

Regeneration is bungled following CNS injuries, including spinal cord injury (SCI). Inherent decay of permissive conditions restricts the regrowth of the mature CNS after an injury. Hypertrophic scarring, insignificant intrinsic axon-growth activity, and axon-growth inhibitory molecules such as myelin inhibitors and scar inhibitors constitute a significant hindrance to spinal cord repair. Besides these molecules, a combined absence of various mechanisms responsible for axonal regeneration is the main reason behind the dereliction of the adult CNS to regenerate. The neutralization of specific inhibitors/proteins by stymieing antibodies or encouraging enzymatic degradation results in improved axon regeneration. Previous efforts to induce regeneration after SCI have stimulated axonal development in or near lesion sites, but not beyond them. Several pathways are responsible for the axonal growth obstruction after a CNS injury, including SCI. Herein, we summarize the axonal, glial, and intrinsic factor which impedes the regeneration. We have also discussed the methods to stabilize microtubules and through this to maintain the proper cytoskeletal dynamics of growth cone as disorganized microtubules lead to the failure of axonal regeneration. Moreover, we primarily focus on diverse inhibitors of axonal growth and molecular approaches to counteract them and their downstream intracellular signaling through the RhoA/ROCK pathway.

Long-standing Multifocal Motor Neuropathy Presenting With Delayed Clinical Features of Anti-Myelin-Associated Glycoprotein Neuropathy and Elevated Anti-Myelin-Associated Glycoprotein Antibody Titers.

ABSTRACT: Multifocal motor neuropathy with conduction block (MMN) and anti-myelin-associated glycoprotein (MAG) neuropathy are rare chronic acquired demyelinating neuropathies with distinct clinical and electrophysiological characteristics. These neuropathies are generally not known to coexist. This report describes a patient with long-standing MMN who subsequently developed clinical features of anti-MAG neuropathy. This suggests that subtypes of chronic inflammatory neuropathies may not be sharply defined. In addition, a presentation of MMN with anti-MAG titers may be a prognostic indicator of poor response to standard MMN treatment.

Iron Heterogeneity in Early Active Multiple Sclerosis Lesions.

OBJECTIVE: Multiple sclerosis (MS) is a heterogeneous inflammatory demyelinating disease. Iron distribution is altered in MS patients' brains, suggesting iron liberation within active lesions amplifies demyelination and neurodegeneration. Whether the amount and distribution of iron are similar or different among different MS immunopatterns is currently unknown. METHODS: We used synchrotron X-ray fluorescence imaging, histology, and immunohistochemistry to compare the iron quantity and distribution between immunopattern II and III early active MS lesions. We analyzed archival autopsy and biopsy tissue from 21 MS patients. RESULTS: Immunopattern II early active lesions contain 64% more iron (95% confidence interval [CI] = 17-127%, p = 0.004) than immunopattern III lesions, and 30% more iron than the surrounding periplaque white matter (95% CI = 3-64%, p = 0.03). Iron in immunopattern III lesions is 28% lower than in the periplaque white matter (95% CI = -40 to -14%, p < 0.001). When normalizing the iron content of early active lesions to that of surrounding periplaque white matter, the ratio is significantly higher in immunopattern II (p < 0.001). Microfocused X-ray fluorescence imaging shows that iron in immunopattern II lesions localizes to macrophages, whereas macrophages in immunopattern III lesions contain little iron. INTERPRETATION: Iron distribution and content are heterogeneous in early active MS lesions. Iron accumulates in macrophages in immunopattern II, but not immunopattern III lesions. This heterogeneity in the two most common MS immunopatterns may be explained by different macrophage polarization, origin, or different demyelination mechanisms, and paves the way for developing new or using existing iron-sensitive magnetic resonance imaging techniques to differentiate among immunopatterns in the general nonbiopsied MS patient population. ANN NEUROL 2021;89:498-510.

The Effect of Chronic Ethanol Exposure and Thiamine Deficiency on Myelin-related Genes in the Cortex and the Cerebellum.

BACKGROUND: Long-term alcohol consumption has been linked to structural and functional brain abnormalities. Furthermore, with persistent exposure to ethanol (EtOH), nutrient deficiencies often develop. Thiamine deficiency is a key contributor to alcohol-related brain damage and is suspected to contribute to white matter pathology. The expression of genes encoding myelin proteins in several cortical brain regions is altered with EtOH exposure. However, there is limited research regarding the impact of thiamine deficiency on myelin dysfunction. METHODS: A rat model was used to assess the impact of moderate chronic EtOH exposure (CET; 20% EtOH in drinking water for 1 or 6 months), pyrithiamine-induced thiamine deficiency treatment (PTD), both conditions combined (CET-PTD), or CET with thiamine injections (CET + T) on myelin-related gene expression (Olig1, Olig2, MBP, MAG, and MOG) in the frontal and parietal cortices and the cerebellum. RESULTS: The CET-PTD treatments caused the greatest suppression in myelin-related genes in the cortex. Specifically, the parietal cortex was the region that was most susceptible to PTD-CET-induced alterations in myelin-related genes. In addition, PTD treatment, with and without CET, caused minor fluctuations in the expression of several myelin-related genes in the frontal cortex. In contrast, CET alone and PTD alone suppressed several myelin-related genes in the cerebellum. Regardless of the region, there was significant recovery of myelin-related genes with extended abstinence and/or thiamine restoration. CONCLUSION: Moderate chronic EtOH alone had a minor effect on the suppression of myelin-related genes in the cortex; however, when combined with thiamine deficiency, the reduction was amplified. There was a suppression of myelin-related genes following long-term EtOH and thiamine deficiency in the cerebellum. However, the suppression in the myelin-related genes mostly occurred 24 h after EtOH removal or following thiamine restoration; within 3 weeks of abstinence or thiamine recovery, gene expression rebounded.

A Novel Siglec-4 Derived Spacer Improves the Functionality of CAR T Cells Against Membrane-Proximal Epitopes.

A domain that is often neglected in the assessment of chimeric antigen receptor (CAR) functionality is the extracellular spacer module. However, several studies have elucidated that membrane proximal epitopes are best targeted through CARs comprising long spacers, while short spacer CARs exhibit highest activity on distal epitopes. This finding can be explained by the requirement to have an optimal distance between the effector T cell and target cell. Commonly used long spacer domains are the CH2-CH3 domains of IgG molecules. However, CARs containing these spacers generally show inferior in vivo efficacy in mouse models compared to their observed in vitro activity, which is linked to unspecific Fcγ-Receptor binding and can be abolished by mutating the respective regions. Here, we first assessed a CAR therapy targeting membrane proximal CD20 using such a modified long IgG1 spacer. However, despite these mutations, this construct failed to unfold its observed in vitro cytotoxic potential in an in vivo model, while a shorter but less structured CD8α spacer CAR showed complete tumor clearance. Given the shortage of well-described long spacer domains with a favorable functionality profile, we designed a novel class of CAR spacers with similar attributes to IgG spacers but without unspecific off-target binding, derived from the Sialic acid-binding immunoglobulin-type lectins (Siglecs). Of five constructs tested, a Siglec-4 derived spacer showed highest cytotoxic potential and similar performance to a CD8α spacer in a CD20 specific CAR setting. In a pancreatic ductal adenocarcinoma model, a Siglec-4 spacer CAR targeting a membrane proximal (TSPAN8) epitope was efficiently engaged in vitro, while a membrane distal (CD66c) epitope did not activate the T cell. Transfer of the TSPAN8 specific Siglec-4 spacer CAR to an in vivo setting maintained the excellent tumor killing characteristics being indistinguishable from a TSPAN8 CD8α spacer CAR while outperforming an IgG4 long spacer CAR and, at the same time, showing an advantageous central memory CAR T cell phenotype with lower release of inflammatory cytokines. In summary, we developed a novel spacer that combines cytotoxic potential with an advantageous T cell and cytokine release phenotype, which make this an interesting candidate for future clinical applications.

Early evidence of delayed oligodendrocyte maturation in the mouse model of mucolipidosis type IV.

Mucolipidosis type IV (MLIV) is a lysosomal disease caused by mutations in the MCOLN1 gene that encodes the endolysosomal transient receptor potential channel mucolipin-1, or TRPML1. MLIV results in developmental delay, motor and cognitive impairments, and vision loss. Brain abnormalities include thinning and malformation of the corpus callosum, white-matter abnormalities, accumulation of undegraded intracellular 'storage' material and cerebellar atrophy in older patients. Identification of the early events in the MLIV course is key to understanding the disease and deploying therapies. The Mcoln1-/- mouse model reproduces all major aspects of the human disease. We have previously reported hypomyelination in the MLIV mouse brain. Here, we investigated the onset of hypomyelination and compared oligodendrocyte maturation between the cortex/forebrain and cerebellum. We found significant delays in expression of mature oligodendrocyte markers Mag, Mbp and Mobp in the Mcoln1-/- cortex, manifesting as early as 10 days after birth and persisting later in life. Such delays were less pronounced in the cerebellum. Despite our previous finding of diminished accumulation of the ferritin-bound iron in the Mcoln1-/- brain, we report no significant changes in expression of the cytosolic iron reporters, suggesting that iron-handling deficits in MLIV occur in the lysosomes and do not involve broad iron deficiency. These data demonstrate very early deficits of oligodendrocyte maturation and critical regional differences in myelination between the forebrain and cerebellum in the mouse model of MLIV. Furthermore, they establish quantitative readouts of the MLIV impact on early brain development, useful to gauge efficacy in pre-clinical trials.

Modeling beta-sheet peptide-protein interactions: Rosetta FlexPepDock in CAPRI rounds 38-45.

Peptide-protein docking is challenging due to the considerable conformational freedom of the peptide. CAPRI rounds 38-45 included two peptide-protein interactions, both characterized by a peptide forming an additional beta strand of a beta sheet in the receptor. Using the Rosetta FlexPepDock peptide docking protocol we generated top-performing, high-accuracy models for targets 134 and 135, involving an interaction between a peptide derived from L-MAG with DLC8. In addition, we were able to generate the only medium-accuracy models for a particularly challenging target, T121. In contrast to the classical peptide-mediated interaction, in which receptor side chains contact both peptide backbone and side chains, beta-sheet complementation involves a major contribution to binding by hydrogen bonds between main chain atoms. To establish how binding affinity and specificity are established in this special class of peptide-protein interactions, we extracted PeptiDBeta, a benchmark of solved structures of different protein domains that are bound by peptides via beta-sheet complementation, and tested our protocol for global peptide-docking PIPER-FlexPepDock on this dataset. We find that the beta-strand part of the peptide is sufficient to generate approximate and even high resolution models of many interactions, but inclusion of adjacent motif residues often provides additional information necessary to achieve high resolution model quality.

Myelin-associated glycoprotein inhibits neurite outgrowth through inactivation of the small GTPase Rap1.

Rap1 is a small GTPase that has been implicated in dendritic development and plasticity. In this study, we investigated the role of Rap1 in axonal growth and its activation in response to neurotrophins and myelin-associated inhibitors. We report that Rap1 is activated by brain-derived neurotrophic factor and that this activation can be blocked by myelin-associated glycoprotein (MAG) or central nervous system myelin, which also induced increases in Rap1GAP1 levels. In addition, we demonstrate that adenoviral overexpression of Rap1 enhances neurite outgrowth in the presence of MAG and myelin, while inhibition of Rap1 activity through overexpression of Rap1GAP1 blocks neurite outgrowth. These findings suggest that Rap1GAP1 negatively regulates neurite outgrowth, making it a potential therapeutic target to promote axonal regeneration.

Membrane Progesterone Receptors (mPRs/PAQRs) Differently Regulate Migration, Proliferation, and Differentiation in Rat Schwann Cells.

Several studies in the last decade demonstrated that progestogens play an important role in the biology of Schwann cells, the main neuroglial cells of the peripheral nervous system. Since a recent study showed that the S42 rat Schwann cell line expressed membrane progesterone receptors (mPRs), members of the PAQR family, in this study, we examined mPR expression in a more physiological model, primary rat Schwann cells. We demonstrated that primary rat Schwann cells show a different pattern of mPR expression compared to the previously studied model; mPRα (PAQR7) and ß (PAQR8) isoforms were the major mPR members identified, with different sub-cellular localizations. Activation of the nuclear progesterone receptor (PR) with the specific agonist R5020 upregulated mPR expression, while activation of mPRs with the specific agonist Org OD 02-0 changed their sub-cellular localization. An in-depth analysis revealed additional effects of mPR activation, such as AKT activation, reduced expression of the myelin-associated glycoprotein (MAG), morphological changes, altered expression of several Schwann cell differentiation markers, and increased Schwann cell migration and proliferation. In conclusion, we identified mPRα and mPRß in primary rat Schwann cells, and our findings suggest a putative role for mPRs in the regulation of Schwann cell migration, proliferation, and differentiation. Therefore, mPRs are a potential pharmacological target for Schwann cell-related disorders and neurodegenerative diseases, especially those in which Schwann cell-mediated axon remyelination is desirable.

Anti-MAG IgM: differences in antibody tests and correlation with clinical findings.

OBJECTIVES: Anti-myelin-associated glycoprotein (MAG) antibody is associated with clinically heterogeneous polyneuropathies. Our purpose was to compare neuropathy phenotypes identified by different anti-MAG tests' results. METHODS: Cohort study: Sera from 40 neuropathy anti-MAG EIA positive patients were tested for anti-MAG by Western blot (WB), for anti-peripheral nerve myelin (PNM) on monkey nerve by immunofluorescence assay (IFA), and for anti-HNK1 on rat CNS slices by IFA. Anti-sulfatide antibodies, for comparison, were also tested by EIA. RESULTS: Among 40 anti-MAG EIA positive sera, 85% also had anti-PNM IFA reactivity and 67.5% bind HNK1 on rat CNS. Anti-HNK1 positive patients had the classical predominantly distal acquired demyelinating symmetric (DADS) neuropathy with a benign course, while anti-PNM positive but anti-HNK1 negative patients had predominantly axonal neuropathy with a high frequency of anti-sulfatide reactivity and the worst long-term prognosis. Anti-MAG EIA positive patients without anti-PNM or anti-HNK1 IFA reactivity had a CIDP-like polyneuropathy. CONCLUSION: Different methods to test for anti-MAG antibodies identify different clinical and electrophysiological findings, as well as long-term outcome. HNK1 reactivity is the strongest marker of DADS.

A comparative study for the intermediate states of myelin oligodendrocyte glycoprotein in the absence and presence of glycan - A computational approach.

Myelin Oligodendrocyte glycoprotein (MOG) is found to play an important role in providing structural integrity to myelin sheath at the same time it acts as an auto-antigen which might lead to Multiple Sclerosis (MS). What causes this specific property of being an auto-antigen is still not known. Here we present molecular dynamics simulation studies of unfolding and folding of the protein MOG in both the absence and presence of N-glycan in order to understand the role of glycosylation in the stability and flexibility of the protein. The main results from these studies show that the glycosylation increases the stability of the protein MOG and inhibits the complete unfolding of MOG in the SMD. From the folding studies using TMD, it was observed that the glycan helps the protein to attain the near-native folded conformation. However, it was also observed from the direct TMD studies that the pathway of protein folding was enhanced by the trace-back of intermediate states in the presence of glycan.

In-silico and Molecular Analysis of Reticulon 4asNovel therapeutic option for axonal regeneration.

OBJECTIVES: To find interactions of the ligand with axonali nhibitors, and to check the optimum interactions of existing drugs as inhibitors for the protein that hinders the growth of injured neurons. METHODS: The study was conducted at Kongju National University, Korea from May 2016 to March 2017. It consisted of two parts. Molecular analysis and bioinformatics analysis. The study comprised a family of six with Charcot-Marie-Tooth phenotypes, recommended by a neurologist for molecular analysis on the clinical symptoms to find the mutations responsible for the disease. Blood samples were collected from each family member and total deoxyribonucleic acid was extracted and it was analysed for Reticulon 4 gene by sequencing the coding and intronic regions. However, a missense mutation was found on exon 2 of the gene in the proband and the whole family was subsequently analysed. Bioinformatics analysis and docking studies were carried out to investigate the potential behaviour of Reticulon 4 as therapeutic agent. Sequencing analysis was performed to find the pathoegenic variant responsible for Charcot-Marie-Tooth type 1. RESULTS: After checking pathogenicity of the mutation, Reticulon 4 gene was found to be not involved in Charcot- Marie-Tooth disease type 1. CONCLUSIONS: Reticulon 4 gene was not found to be involved in causing Charcot-Marie-Tooth disease type 1.

Two adhesive systems cooperatively regulate axon ensheathment and myelin growth in the CNS.

Central nervous system myelin is a multilayered membrane produced by oligodendrocytes to increase neural processing speed and efficiency, but the molecular mechanisms underlying axonal selection and myelin wrapping are unknown. Here, using combined morphological and molecular analyses in mice and zebrafish, we show that adhesion molecules of the paranodal and the internodal segment work synergistically using overlapping functions to regulate axonal interaction and myelin wrapping. In the absence of these adhesive systems, axonal recognition by myelin is impaired with myelin growing on top of previously myelinated fibers, around neuronal cell bodies and above nodes of Ranvier. In addition, myelin wrapping is disturbed with the leading edge moving away from the axon and in between previously formed layers. These data show how two adhesive systems function together to guide axonal ensheathment and myelin wrapping, and provide a mechanistic understanding of how the spatial organization of myelin is achieved.

Coordinated internodal and paranodal adhesion controls accurate myelination by oligodendrocytes.

Oligodendrocyte-axon contact is mediated by several cell adhesion molecules (CAMs) that are positioned at distinct sites along the myelin unit, yet their role during myelination remains unclear. Cadm4 and its axonal receptors, Cadm2 and Cadm3, as well as myelin-associated glycoprotein (MAG), are enriched at the internodes below the compact myelin, whereas NF155, which binds the axonal Caspr/contactin complex, is located at the paranodal junction that is formed between the axon and the terminal loops of the myelin sheath. Here we report that Cadm4-, MAG-, and Caspr-mediated adhesion cooperate during myelin membrane ensheathment. Genetic deletion of either Cadm4 and MAG or Cadm4 and Caspr resulted in the formation of multimyelinated axons due to overgrowth of the myelin away from the axon and the forming paranodal junction. Consequently, these mice displayed paranodal loops either above or underneath compact myelin. Our results demonstrate that accurate placement of the myelin sheath by oligodendrocytes requires the coordinated action of internodal and paranodal CAMs.

FTY720-Mitoxy reduces toxicity associated with MSA-like α-synuclein and oxidative stress by increasing trophic factor expression and myelin protein in OLN-93 oligodendroglia cell cultures.

Multiple system atrophy (MSA) is a fatal demyelinating disorder lacking any disease-modifying therapies. MSA pathology stems from aggregated α-synuclein (aSyn) accumulation in glial cytosolic inclusions of oligodendroglial cell (OLGs), the myelinating cells of brain. In MSA brains and in MSA animal models with aSyn accumulation in OLGs, aberrant expression of brain-derived neurotrophic factor (BDNF) and glial-cell-line-derived neurotrophic factor (GDNF) occur. Nerve growth factor (NGF) expression can also be altered in neurodegenerative diseases. It is unclear if oxidative stress impacts the viability of aSyn-accumulating OLG cells. Here, we show that OLN-93 cells stably expressing human wild type aSyn or the MSA-associated-aSyn-mutants G51D or A53E, are more vulnerable to oxidative stress. In dose response studies we found that OLN-93 cells treated 48 h with 160 nM FTY720 or our new non-immunosuppressive FTY720-C2 or FTY720-Mitoxy derivatives sustained normal viability. Also, FTY720, FTY720-C2, and FTY720-Mitoxy all stimulated NGF expression at 24 h. However only FTY720-Mitoxy also increased BDNF and GDNF mRNA at 24 h, an effect paralleled by increases in histone 3 acetylation and ERK1/2 phosphorylation. Myelin associated glycoprotein (MAG) levels were also increased in OLN-93 cells after 48 h treatment with FTY720-Mitoxy. FTY720, FTY720-C2, and FTY720-Mitoxy all prevented oxidative-stress-associated-cell-death of OLN-93 cells that lack any aSyn expression. However, only FTY720-Mitoxy protected MSA-like aSyn-expressing-OLN-93-cells against oxidative-cell-death. These data identify potent protective effects for FTY720-Mitoxy with regard to trophic factors as well as MAG expression by OLG cells. Testing of FTY720-Mitoxy in mice is thus a judicious next step for neuropharmacological preclinical development.