Myelin Membrane Research Articles

New Insights into Risk Genes and Their Candidates in Multiple Sclerosis

Oligodendrocytes are central nervous system glial cells that wrap neuronal axons with their differentiated myelin membranes as biological insulators. There has recently been an emerging concept that multiple sclerosis could be triggered and promoted by various risk genes that appear likely to contribute to the degeneration of oligodendrocytes. Despite the known involvement of vitamin D, immunity, and inflammatory cytokines in disease progression, the common causes and key genetic mechanisms remain unknown. Herein, we focus on recently identified risk factors and risk genes in the background of multiple sclerosis and discuss their relationships.

Autophagic degradation of CNS myelin maintains axon integrity.

(Macro)autophagy is a major lysosome-dependent degradation mechanism which engulfs, removes and recycles unwanted cytoplasmic material, including damaged organelles and toxic protein aggregates. Although a few studies implicate autophagy in CNS demyelinating pathologies, its role, particularly in mature oligodendrocytes and CNS myelin, remains poorly studied. Here, using both pharmacological and genetic inhibition of the autophagic machinery, we provide evidence that autophagy is an essential mechanism for oligodendrocyte maturation in vitro. Our study reveals that two core myelin proteins, namely proteolipid protein (PLP) and myelin basic protein (MBP) are incorporated into autophagosomes in oligodendrocytes, resulting in their degradation. Furthermore, we ablated atg5, a core gene of the autophagic machinery, specifically in myelinating glial cells in vivo by tamoxifen administration (plp-Cre ERT2 ; atg5 f/f ) and showed that myelin maintenance is perturbed, leading to PLP accumulation. Significant morphological defects in myelin membrane such as decompaction accompanied with increased axonal degeneration are observed. As a result, the mice exhibit behavioral deficits. In summary, our data highlight that the maintenance of adult myelin homeostasis in the CNS requires the involvement of a fully functional autophagic machinery.

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.

CNS myelination requires VAMP2/3-mediated membrane expansion in oligodendrocytes

Myelin is required for rapid nerve signaling and is emerging as a key driver of CNS plasticity and disease. How myelin is built and remodeled remains a fundamental question of neurobiology. Central to myelination is the ability of oligodendrocytes to add vast amounts of new cell membrane, expanding their surface areas by many thousand-fold. However, how oligodendrocytes add new membrane to build or remodel myelin is not fully understood. Here, we show that CNS myelin membrane addition requires exocytosis mediated by the vesicular SNARE proteins VAMP2/3. Genetic inactivation of VAMP2/3 in myelinating oligodendrocytes caused severe hypomyelination and premature death without overt loss of oligodendrocytes. Through live imaging, we discovered that VAMP2/3-mediated exocytosis drives membrane expansion within myelin sheaths to initiate wrapping and power sheath elongation. In conjunction with membrane expansion, mass spectrometry of oligodendrocyte surface proteins revealed that VAMP2/3 incorporates axon-myelin adhesion proteins that are collectively required to form nodes of Ranvier. Together, our results demonstrate that VAMP2/3-mediated membrane expansion in oligodendrocytes is indispensable for myelin formation, uncovering a cellular pathway that could sculpt myelination patterns in response to activity-dependent signals or be therapeutically targeted to promote regeneration in disease.

Microscopic theory of spin-spin and spin-lattice relaxation of bound protons in cellular and myelin membranes-A lateral diffusion model (LDM).

Deciphering salient features of biological tissue cellular microstructure in health and diseases is an ultimate goal of MRI. While most MRI approaches are based on studying MR properties of tissue "free" water indirectly affected by tissue microstructure, other approaches, such as magnetization transfer (MT), directly target signals from tissue-forming macromolecules. However, despite three-decades of successful applications, relationships between MT measurements and tissue microstructure remain elusive, hampering interpretation of experimental results. The goal of this paper is to develop microscopic theory connecting the structure of cellular and myelin membranes to their MR properties. Herein we introduce a lateral diffusion model (LDM) that explains the T2 (spin-spin) and T1 (spin-lattice) MRI relaxation properties of the macromolecular-bound protons by their dipole-dipole interaction modulated by the lateral diffusion of long lipid molecules forming cellular and myelin membranes. LDM predicts anisotropic T1 and T2 relaxation of membrane-bound protons. Moreover, their T2 relaxation cannot be described in terms of a standard R2 =1/T2 relaxation rate parameter, but rather by a relaxation rate function R2 (t) that depends on time t after RF excitation, having, in the main approximation, a logarithmic behavior: R2 (t) ∼ lnt. This anisotropic non-linear relaxation leads to an absorption lineshape that is different from Super-Lorentzian traditionally used in interpreting MT experiments. LDM-derived analytical equations connect the membrane-bound protons T1 and T2 relaxation with dynamic distances between protons in neighboring membrane-forming lipid molecules and their lateral diffusion. This sheds new light on relationships between MT parameters and microstructure of cellular and myelin membranes.

Implantable Electroceutical Approach Improves Myelination by Restoring Membrane Integrity in a Mouse Model of Peripheral Demyelinating Neuropathy.

Although many efforts are undertaken to treat peripheral demyelinating neuropathies based on biochemical interventions, unfortunately, there is no approved treatment yet. Furthermore, previous studies have not shown improvement of the myelin membrane at the biomolecular level. Here, an electroceutical treatment is introduced as a biophysical intervention to treat Charcot-Marie-Tooth (CMT) disease-the most prevalent peripheral demyelinating neuropathy worldwide-using a mouse model. The specific electrical stimulation (ES) condition (50mV mm-1 , 20Hz, 1h) for optimal myelination is found via an in vitro ES screening system, and its promyelinating effect is validated with ex vivo dorsal root ganglion model. Biomolecular investigation via time-of-flight secondary ion mass spectrometry shows that ES ameliorates distribution abnormalities of peripheral myelin protein 22 and cholesterol in the myelin membrane, revealing the restoration of myelin membrane integrity. ES intervention in vivo via flexible implantable electrodes shows not only gradual rehabilitation of mouse behavioral phenotypes (balance and endurance), but also restored myelin thickness, compactness, and membrane integrity. This study demonstrates, for the first time, that an electroceutical approach with the optimal ES condition has the potential to treat CMT disease and restore impaired myelin membrane integrity, shifting the paradigm toward practical interventions for peripheral demyelinating neuropathies.

Hyaluronic acid and its receptor CD44, acting through TMEM2, inhibit morphological differentiation in oligodendroglial cells

Hyaluronic acid is a main extracellular matrix component in the central nervous system (CNS), which provides structural support under physical and physiological conditions to maintain cellular homeostasis. However, hyaluronic acid and its degradation products are present within focal demyelinating lesions in multiple sclerosis (MS) patients and autoimmune encephalomyelitis (EAE) mouse models. Differentiated plasma membranes called myelin membranes are generated by oligodendrocytes (also called oligodendroglial cells), which are glial cells that wrap neuronal axons in the CNS. Despite these positive or negative relationships of hyaluronic acid with oligodendroglial cell differentiation and/or myelination, it remains unclear whether and how hyaluronic acid affects oligodendroglial cells. Here, we showed that hyaluronic acid and the cognate receptor CD44 are directly involved in inhibiting morphological differentiation in FBD-102b cells, which are differentiation models of oligodendroglial precursor cells, and primary oligodendroglial precursor cells. Their phenotype changes were supported by decreased oligodendroglial cell differentiation, myelin marker protein expression levels, and Akt kinase phosphorylation levels as a marker kinase. Furthermore, the effects of hyaluronic acid required transmembrane protein 2 (TMEM2), a cell surface hyaluronidase. These results suggest that hyaluronic acid and the CD44 receptor, acting through TMEM2, contribute to inhibiting morphological differentiation of oligodendroglial cells, providing a mechanism underlying cell physiological and possible pathological effects responsible for hyaluronic acid.

Human myelin proteolipid protein structure and lipid bilayer stacking

The myelin sheath is an essential, multilayered membrane structure that insulates axons, enabling the rapid transmission of nerve impulses. The tetraspan myelin proteolipid protein (PLP) is the most abundant protein of compact myelin in the central nervous system (CNS). The integral membrane protein PLP adheres myelin membranes together and enhances the compaction of myelin, having a fundamental role in myelin stability and axonal support. PLP is linked to severe CNS neuropathies, including inherited Pelizaeus-Merzbacher disease and spastic paraplegia type 2, as well as multiple sclerosis. Nevertheless, the structure, lipid interaction properties, and membrane organization mechanisms of PLP have remained unidentified. We expressed, purified, and structurally characterized human PLP and its shorter isoform DM20. Synchrotron radiation circular dichroism spectroscopy and small-angle X-ray and neutron scattering revealed a dimeric, α-helical conformation for both PLP and DM20 in detergent complexes, and pinpoint structural variations between the isoforms and their influence on protein function. In phosphatidylcholine membranes, reconstituted PLP and DM20 spontaneously induced formation of multilamellar myelin-like membrane assemblies. Cholesterol and sphingomyelin enhanced the membrane organization but were not crucial for membrane stacking. Electron cryomicroscopy, atomic force microscopy, and X-ray diffraction experiments for membrane-embedded PLP/DM20 illustrated effective membrane stacking and ordered organization of membrane assemblies with a repeat distance in line with CNS myelin. Our results shed light on the 3D structure of myelin PLP and DM20, their structure–function differences, as well as fundamental protein–lipid interplay in CNS compact myelin.

CRISPR/CasRx-Mediated RNA Knockdown Reveals That ACE2 Is Involved in the Regulation of Oligodendroglial Cell Morphological Differentiation.

Angiotensin-converting enzyme 2 (ACE2) plays a role in catalyzing angiotensin II conversion to angiotensin (1–7), which often counteracts the renin-angiotensin system. ACE2 is expressed not only in the cells of peripheral tissues such as the heart and kidney, but also in those of the central nervous system (CNS). Additionally, ACE2 acts as the receptor required for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), whose binding leads to endocytotic recycling and possible degradation of the ACE2 proteins themselves. One of the target cells for SARS-CoV-2 in the CNS is oligodendrocytes (oligodendroglial cells), which wrap neuronal axons with their differentiated plasma membranes called myelin membranes. Here, for the first time, we describe the role of ACE2 in FBD-102b cells, which are used as the differentiation models of oligodendroglial cells. Unexpectedly, RNA knockdown of ACE2 with CasRx-mediated gRNA or the cognate siRNA promoted oligodendroglial cell morphological differentiation with increased expression or phosphorylation levels of differentiation and/or myelin marker proteins, suggesting the negative role of ACE2 in morphological differentiation. Notably, ACE2′s intracellular region preferentially interacted with the active GTP-bound form of Ras. Thus, knockdown of ACE2 relatively increased GTP-bound Ras in an affinity-precipitation assay. Indeed, inhibition of Ras resulted in decreasing both morphological differentiation and expression or phosphorylation levels of marker proteins, confirming the positive role of Ras in differentiation. These results indicate the role of ACE2 itself as a negative regulator of oligodendroglial cell morphological differentiation, newly adding ACE2 to the list of regulators of oligodendroglial morphogenesis as well as of Ras-binding proteins. These findings might help us to understand why SARS-CoV-2 causes pathological effects in the CNS.

Hesperetin, a Citrus Flavonoid, Ameliorates Inflammatory Cytokine-Mediated Inhibition of Oligodendroglial Cell Morphological Differentiation.

Oligodendrocytes (oligodendroglial cells) are glial cells that wrap neuronal axons with their differentiated plasma membranes called myelin membranes. In the pathogenesis of inflammatory cytokine-related oligodendroglial cell and myelin diseases such as multiple sclerosis (MS), typical inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) are thought to contribute to the degeneration and/or progression of the degeneration of oligodendroglial cells and, in turn, the degeneration of naked neuronal cells in the central nervous system (CNS) tissues. Despite the known involvement of these inflammatory cytokines in disease progression, it has remained unclear whether and how TNFα or IL-6 affects the oligodendroglial cells themselves or indirectly. Here we show that TNFα or IL-6 directly inhibits morphological differentiation in FBD-102b cells, which are differentiation models of oligodendroglial cells. Their phenotype changes were supported by the decreased expression levels of oligodendroglial cell differentiation and myelin marker proteins. In addition, TNFα or IL-6 decreased phosphorylation levels of Akt kinase, whose upregulation has been associated with promoting oligodendroglial cell differentiation. Hesperetin, a flavonoid mainly contained in citrus fruit, is known to have neuroprotective effects. Hesperetin might also be able to resolve pre-illness conditions, including the irregulated secretion of cytokines, through diet. Notably, the addition of hesperetin into cells recovered TNFα- or IL-6-induced inhibition of differentiation, as supported by increased levels of marker protein expression and phosphorylation of Akt kinase. These results suggest that TNFα or IL-6 itself contributes to the inhibitory effects on the morphological differentiation of oligodendroglial cells, possibly providing information not only on their underlying pathological effects but also on flavonoids with potential therapeutic effects at the molecular and cellular levels.

The Extracellular Matrix Proteins Tenascin-C and Tenascin-R Retard Oligodendrocyte Precursor Maturation and Myelin Regeneration in a Cuprizone-Induced Long-Term Demyelination Animal Model.

Oligodendrocytes are the myelinating cells of the central nervous system. The physiological importance of oligodendrocytes is highlighted by diseases such as multiple sclerosis, in which the myelin sheaths are degraded and the axonal signal transmission is compromised. In a healthy brain, spontaneous remyelination is rare, and newly formed myelin sheaths are thinner and shorter than the former ones. The myelination process requires the migration, proliferation, and differentiation of oligodendrocyte precursor cells (OPCs) and is influenced by proteins of the extracellular matrix (ECM), which consists of a network of glycoproteins and proteoglycans. In particular, the glycoprotein tenascin-C (Tnc) has an inhibitory effect on the differentiation of OPCs and the remyelination efficiency of oligodendrocytes. The structurally similar tenascin-R (Tnr) exerts an inhibitory influence on the formation of myelin membranes in vitro. When Tnc knockout oligodendrocytes were applied to an in vitro myelination assay using artificial fibers, a higher number of sheaths per single cell were obtained compared to the wild-type control. This effect was enhanced by adding brain-derived neurotrophic factor (BDNF) to the culture system. Tnr−/− oligodendrocytes behaved differently in that the number of formed sheaths per single cell was decreased, indicating that Tnr supports the differentiation of OPCs. In order to study the functions of tenascin proteins in vivo Tnc−/− and Tnr−/− mice were exposed to Cuprizone-induced demyelination for a period of 10 weeks. Both Tnc−/− and Tnr−/− mouse knockout lines displayed a significant increase in the regenerating myelin sheath thickness after Cuprizone treatment. Furthermore, in the absence of either tenascin, the number of OPCs was increased. These results suggest that the fine-tuning of myelin regeneration is regulated by the major tenascin proteins of the CNS.

Tenascins Interfere With Remyelination in an Ex Vivo Cerebellar Explant Model of Demyelination

Oligodendrocytes form myelin membranes and thereby secure the insulation of axons and the rapid conduction of action potentials. Diseases such as multiple sclerosis highlight the importance of this glial cell population for brain function. In the adult brain, efficient remyelination following the damage to oligodendrocytes is compromised. Myelination is characterized by proliferation, migration, and proper integration of oligodendrocyte precursor cells (OPCs). These processes are among others controlled by proteins of the extracellular matrix (ECM). As a prominent representative ECM molecule, tenascin-C (Tnc) exerts an inhibitory effect on the migration and differentiation of OPCs. The structurally similar paralogue tenascin-R (Tnr) is known to promote the differentiation of oligodendrocytes. The model of lysolecithin-induced demyelination of cerebellar slice cultures represents an important tool for the analysis of the remyelination process. Ex vivo cerebellar explant cultures of Tnc −/− and Tnr −/− mouse lines displayed enhanced remyelination by forming thicker myelin membranes upon exposure to lysolecithin. The inhibitory effect of tenascins on remyelination could be confirmed when demyelinated wildtype control cultures were exposed to purified Tnc or Tnr protein. In that approach, the remyelination efficiency decreased in a dose-dependent manner with increasing concentrations of ECM molecules added. In order to examine potential roles in a complex in vivo environment, we successfully established cuprizone-based acute demyelination to analyze the remyelination behavior after cuprizone withdrawal in SV129, Tnc −/− , and Tnr −/− mice. In addition, we documented by immunohistochemistry in the cuprizone model the expression of chondroitin sulfate proteoglycans that are inhibitory for the differentiation of OPCs. In conclusion, inhibitory properties of Tnc and Tnr for myelin membrane formation could be demonstrated by using an ex vivo approach.

White matter integrity in mice\xa0requires continuous myelin synthesis at the inner tongue

Myelin, the electrically insulating sheath on axons, undergoes dynamic changes over time. However, it is composed of proteins with long lifetimes. This raises the question how such a stable structure is renewed. Here, we study the integrity of myelinated tracts after experimentally preventing the formation of new myelin in the CNS of adult mice, using an inducible Mbp null allele. Oligodendrocytes survive recombination, continue to express myelin genes, but they fail to maintain compacted myelin sheaths. Using 3D electron microscopy and mass spectrometry imaging we visualize myelin-like membranes failing to incorporate adaxonally, most prominently at juxta-paranodes. Myelinoid body formation indicates degradation of existing myelin at the abaxonal side and the inner tongue of the sheath. Thinning of compact myelin and shortening of internodes result in the loss of about 50% of myelin and axonal pathology within 20 weeks post recombination. In summary, our data suggest that functional axon-myelin units require the continuous incorporation of new myelin membranes.

Cellular and Molecular Gradients in the Ventral Horns With Increasing Distance From the Injury Site After Spinal Cord Contusion.

To identify cellular and molecular gradients following spinal cord injury (SCI), a rat contusion model of severe SCI was used to investigate the expression of NG2 and molecules that identify astrocytes and axons of the ventral horns (VH) at different distances on 7 and 30 days post-injury (dpi). A gradient of expression of NG2+/Olig2+ cells was determined, with the highest concentrations focused close to the injury site. A decrease in NG2 mean intensity correlates with a decrease in the number of NG2+ cells more distally. Immunoelectron microscopy subsequently revealed the presence of NG2 in connection with the membrane and within the cytoplasm of NG2+ glial cells and in large amounts within myelin membranes. Analysis of the astrocyte marker GFAP showed increased expression local to injury site from 7 dpi, this increase in expression spread more distally from the injury site by 30 dpi. Paradoxically, astrocyte perisynaptic processes marker GLT-1 was only increased in expression in areas remote from the epicenter, which was traced both at 7 and 30 dpi. Confocal microscopy showed a significant decrease in the number of 5-HT+ axons at a distance from the epicenter in the caudal direction, which is consistent with a decrease in β3-tubulin in these areas. The results indicate significant cellular and molecular reactions not only in the area of the gray matter damage but also in adjacent and remote areas, which is important for assessing the possibility of long-distance axonal growth.

Purified myelin lipids display a critical mixing point at low surface pressure

Lipids extracted from Purified Myelin Membranes (LPMM) were spread as monomolecular films at the air/aqueous interface. The films were visualized by Brewster Angle Microscopy (BAM) at different lateral pressures (π) and ionic environments. Coexistence of Liquid-Expanded (LE) and cholesterol-enriched (CE) rounded domains persisted up to π ≈ 5 mN/m but the monolayers became homogeneous at higher surface pressures. Before mixing, the domains distorted to non-rounded domains. We experimentally measured the line tension (λ) for the lipid monolayers at the domain borders by a shape relaxation technique using non-homogeneous electric fields. Regardless of the subphase conditions, the obtained line tensions are of the order of pN and tended to decrease as lateral pressure increased toward the mixing point. From the mean square displacement of nested trapped domains, we also calculated the dipole density difference between phases (μ). A non-linear drop was detected in this parameter as the mixing point is approached. Here we quantitively evaluated the π-dependance of both parameters with proper power laws in the vicinity of the critical mixing surface pressure, and the exponents showed to be consistent with a critical phenomenon in the two-dimensional Ising universality class. This idea of bidimensionality was found to be compatible only for simplified lipidic systems, while for whole myelin monolayers, that means including proteins, no critical mixing point was detected.Finally, the line tension values were related with the thickness differences between phases (Δt) near the critical point.

Deconvolution of the MBP-Bri2 Interaction by a Yeast Two Hybrid System and Synergy of the AlphaFold2 and High Ambiguity Driven Protein-Protein Docking

Myelin basic protein (MBP) is one of the key proteins in the development of multiple sclerosis (MS). However, very few intracellular MBP partners have been identified up to now. In order to find proteins interacting with MBP in the brain, an expression library from the human brain was screened using a yeast two-hybrid system. Here we showed that MBP interacts with the C-terminal 24-residue peptide of Integral transmembrane protein II associated with familial British and Danish dementia (ITM2B/Bri2 or Bri2). This peptide (Bri23R) was one residue longer than the known Bri23 peptide, which is cleaved from the C-terminus of Bri2 during its maturation in the Golgi and has physiological activity as a modulator of amyloid precursor protein processing. Since the spatial structures for both MBP and Bri2 were not known, we used computational methods of structural biology including an artificial intelligence system AlphaFold2 and high ambiguity driven protein-protein docking (HADDOCK 2.1) to gain a mechanistic explanation of the found protein-protein interaction and elucidate a possible structure of the complex of MBP with Bri23R peptide. As expected, MBP was mostly unstructured, although it has well-defined α-helical regions, while Bri23R forms a stable β-hairpin. Simulation of the interaction between MBP and Bri23R in two different environments, as parts of the two-hybrid system fusion proteins and in the form of single polypeptides, showed that MBP twists around Bri23R. The observed interaction results in the adjustment of the size of the internal space between MBP α-helices to the size of the β-hairpin of Bri23R. Since Bri23 is known to inhibit aggregation of amyloid oligomers, and the association of MBP to the inner leaflet of the membrane bilayer shares features with amyloid fibril formation, Bri23 may serve as a peptide chaperon for MBP, thus participating in myelin membrane assembly.

Effects of Lid Domain Structural Changes on the Interactions between Peripheral Myelin Protein 2 and a Lipid Bilayer.

Peripheral myelin protein 2 (P2) plays an important role in the stacking of the myelin membrane and lipid transport. Here we investigate the interactions between P2 and a model myelin membrane using molecular dynamics simulations, focusing on the effect of the L27D mutation and conformational changes in the α2-helix in the lid domain of P2. The L27D mutation weakens the binding of the lid domain of P2 on the membrane. The α2-helix is either folded or unfolded on the membrane. Compared with the α2-helix structure in water, the membrane stabilizes the structure of the α2-helix, whereas the unfolding of the α2-helix reduces the binding affinity of P2 on the membrane. These findings reveal the energetics of the mutant and the structural changes of P2 on the interactions between the protein and the lipid bilayer and help us to understand the microscopic mechanism of the formation of the myelin sheath structure and some neurological disorders.

Molecular characterization of myelin basic protein a (mbpa) gene from red-bellied pacu (Piaractus brachypomus)

BackgroundMyelin basic protein (MBP) is one of the most important structural components of the myelin sheaths in both central and peripheral nervous systems. MBP has several functions including organization of the myelin membranes, reorganization of the cytoskeleton during the myelination process, and interaction with the SH3 domain in signaling pathways. Likewise, MBP has been proposed as a marker of demyelination in traumatic brain injury and chemical exposure. MethodsThe aim of this study was to molecularly characterize the myelin basic protein a (mbpa) gene from the Colombian native fish, red-bellied pacu, Piaractus brachypomus. Bioinformatic tools were used to identify the phylogenetic relationships, physicochemical characteristics, exons, intrinsically disordered regions, and conserved domains of the protein. Gene expression was assessed by qPCR in three models corresponding to sublethal chlorpyrifos exposure, acute brain injury, and anesthesia experiments. Resultsmbpa complete open reading frame was identified with 414 nucleotides distributed in 7 exons that encode 137 amino acids. MBPa was recognized as belonging to the myelin basic protein family, closely related with orthologous proteins, and two intrinsically disordered regions were established within the sequence. Gene expression of mbpa was upregulated in the optic chiasm of the chlorpyrifos exposed fish in contrast to the control group. ConclusionsThe physicochemical computed features agree with the biological functions of MBP, and basal gene expression was according to the anatomical distribution in the tissues analyzed. This study is the first molecular characterization of mbpa from the native species Piaractus brachypomus.

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.

Oral Administration of Probiotic Enterococcus durans to Ameliorate Experimental Autoimmune Encephalomyelitis in Mice.

Probiotics, including lactobacilli, have immunomodulatory activities with promising effects on inflammatory diseases. In this study, we evaluate the effect of Enterococcus durans (Edu) and three various strains of lactobacilli (Lacto-mix), including L. rhamnosus, L. casei, and L. plantarum, to prevent Experimental Autoimmune Encephalomyelitis (EAE) features. C57BL/6 female mice were inoculated with Myelin Oigodendrocyte Glycoprotein (MOG35-55) in CFA (complete Freund's adjuvant) to induce EAE. Five groups (n=6 in each group) of animals received saline or probiotics by oral gavage with 200 μL of lactobacilli (1.5×108 CFU/mL) for 2 weeks before the immunization and during the test for one month. Histopathological studies showed an increase in infiltration of inflammatory cells and destruction of the myelin membrane in the EAE group but a decrease in inflammatory cells in the probiotic-treated animals. Pro-inflammatory cytokines (Interleukin [IL]-17 and Interferon [IFN]-γ) concentration in the supernatant of the brain and spinal cord tissues showed a significant increase in the EAE compared with the normal saline group (P<0.01). While in the spinal cord tissue, there was a decrease in IL-17 in those animals treated with the Lactomix and Edu + Lacto-mix (P<0.01) and a significant decrease in IFN-γ in those animals that received Edu (P<0.05). Western blot analysis of matrix metalloproteinase-9 and myelin basic protein showed a decrease and increase in treatment and EAE groups, respectively, compared to the normal control group. Our data suggest that probiotic Enterococcus durans and Lacto-mix prevents EAE, but further studies are needed to clarify the exact mechanisms and their application in preclinical and clinical trials. Dysfunction of the blood-brain barrier, migration of inflammatory cells into the Central Nervous System (CNS), and an increase in the pro-inflammatory factors, are the hallmarks in the pathogenesis of Multiple Sclerosis (MS) and Experimental Autoimmune Encephalomyelitis (EAE).The optimal effects of probiotic strains may involve the simultaneous use of more than one strain.Probiotic Enterococcus durans and Lacto-mix have a preventive effect against EAE. Multiple Sclerosis (MS) is a myelin-degenerating autoimmune disease in the central nervous system. Experimental Autoimmune Encephalomyelitis (EAE), due to its similar clinical and pathologic features to MS, is widely used in many model studies of this disease. The microbiome refers to a genomic set of germs (bacteria, arches, fungi, and viruses), a commensal flora that lives in the intestine and niche of humans and other mammals. The microbiome affects the host's physiological system, especially the balance between health and disease. Additionally, the importance of the microbiome is evident in regulating the intestine-brain axis, or the coordination of the digestive and the central nervous system. In this regard, probiotics, including lactobacilli, have antioxidant and anti-inflammatory properties in vitro and in vivo. Probiotic strains have a wide range of health-improvement effects, and a combination of strains with specific properties provides a broader range of antimicrobial spectrum and stronger anti-inflammatory effects. Considering the critical role of probiotics in the immune system, this study aimed to investigate the possible role of Enterococcus durans alone or in combination with Lactobacillus mixture (L. rhamnosus, L. casei, and L. plantarum) on the EAE animal model of MS.