Myelin-Specific microRNA-23a/b Cluster Deletion Inhibits Myelination in the Central Nervous System during Postnatal Growth and Aging.

Microribonucleic acids (miRNAs) comprising miR-23a/b clusters, specifically miR-23a and miR-27a, are recognized for their divergent roles in myelination within the central nervous system. However, cluster-specific miRNA functions remain controversial as miRNAs within the same cluster have been suggested to function complementarily. This study aims to clarify the role of miR-23a/b clusters in myelination using mice with a miR-23a/b cluster deletion (KO mice), specifically in myelin expressing proteolipid protein (PLP). Inducible conditional KO mice were generated by crossing miR-23a/b clusterflox/flox mice with PlpCre-ERT2 mice; the offspring were injected with tamoxifen at 10 days or 10 weeks of age to induce a myelin-specific miR-23a/b cluster deletion. Evaluation was performed at 10 weeks or 12 months of age and compared with control mice that were not treated with tamoxifen. KO mice exhibit impaired motor function and hypoplastic myelin sheaths in the brain and spinal cord at 10 weeks and 12 months of age. Simultaneously, significant decreases in myelin basic protein (MBP) and PLP expression occur in KO mice. The percentages of oligodendrocyte precursors and mature oligodendrocytes are consistent between the KO and control mice. However, the proportion of oligodendrocytes expressing MBP is significantly lower in KO mice. Moreover, changes in protein expression occur in KO mice, with increased leucine zipper-like transcriptional regulator 1 expression, decreased R-RAS expression, and decreased phosphorylation of extracellular signal-regulated kinases. These findings highlight the significant influence of miR-23a/b clusters on myelination during postnatal growth and aging.

[Repair effect of different doses of human umbilical cord mesenchymal stem cells on white matter injury in neonatal rats]. / 不同剂量人脐带间å è´¨å¹²ç»†èƒžç§»æ¤å¯¹æ–°ç”Ÿå¤§é¼ è„‘ç™½è´¨æŸä¼¤çš„ä¿®å¤ä½œç”¨.

OBJECTIVES: To compare the repair effects of different doses of human umbilical cord mesenchymal stem cells (hUC-MSCs) on white matter injury (WMI) in neonatal rats. METHODS: Two-day-old Sprague-Dawley neonatal rats were randomly divided into five groups: sham operation group, WMI group, and hUC-MSCs groups (low dose, medium dose, and high dose), with 24 rats in each group. Twenty-four hours after successful establishment of the neonatal rat white matter injury model, the WMI group was injected with sterile PBS via the lateral ventricle, while the hUC-MSCs groups received injections of hUC-MSCs at different doses. At 14 and 21 days post-modeling, hematoxylin and eosin staining was used to observe pathological changes in the tissues around the lateral ventricles. Real-time quantitative polymerase chain reaction was used to detect the quantitative expression of myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) mRNA in the brain tissue. Immunohistochemistry was employed to observe the expression levels of GFAP and neuron-specific nuclear protein (NeuN) in the tissues around the lateral ventricles. TUNEL staining was used to observe cell apoptosis in the tissues around the lateral ventricles. At 21 days post-modeling, the Morris water maze test was used to observe the spatial learning and memory capabilities of the neonatal rats. RESULTS: At 14 and 21 days post-modeling, numerous cells with nuclear shrinkage and rupture, as well as disordered arrangement of nerve fibers, were observed in the tissues around the lateral ventricles of the WMI group and the low dose group. Compared with the WMI group, the medium and high dose groups showed alleviated pathological changes; the arrangement of nerve fibers in the medium dose group was relatively more orderly compared with the high dose group. Compared with the WMI group, there was no significant difference in the expression levels of MBP and GFAP mRNA in the low dose group (P>0.05), while the expression levels of MBP mRNA increased and GFAP mRNA decreased in the medium and high dose groups. The expression level of MBP mRNA in the medium dose group was higher than that in the high dose group, and the expression level of GFAP mRNA in the medium dose group was lower than that in the high dose group (P<0.05). Compared with the WMI group, there was no significant difference in the protein expression of GFAP and NeuN in the low dose group (P>0.05), while the expression of NeuN protein increased and GFAP protein decreased in the medium and high dose groups. The expression of NeuN protein in the medium dose group was higher than that in the high dose group, and the expression of GFAP protein in the medium dose group was lower than that in the high dose group (P<0.05). Compared with the WMI group, there was no significant difference in the number of apoptotic cells in the low dose group (P>0.05), while the number of apoptotic cells in the medium and high dose groups was less than that in the WMI group, and the number of apoptotic cells in the medium dose group was less than that in the high dose group (P<0.05). Compared with the WMI group, there was no significant difference in the escape latency time in the low dose group (P>0.05); starting from the third day of the latency period, the escape latency time in the medium dose group was less than that in the WMI group (P<0.05). The medium and high dose groups crossed the platform more times than the WMI group (P<0.05). CONCLUSIONS: Low dose hUC-MSCs may yield unsatisfactory repair effects on WMI in neonatal rats, while medium and high doses of hUC-MSCs have significant repair effects, with the medium dose demonstrating superior efficacy.

1,2-Dichloroethane induces cortex demyelination by depressing myelin basic protein via inhibiting aquaporin 4 in mice.

1,2-Dichloroethane (1,2-DCE) is a pervasive environmental pollutant, and overexposure to this hazardous material causes brain edema and demyelination in humans. We found that 1,2-DCE inhibits aquaporin 4 (AQP4) and is a primary pathogenic effector of 1,2-DCE-induced brain edema in animals. However, AQP4 down-regulation's link with cortex demyelination after 1,2-DCE exposure remains unclear. Thus, we exposed wild-type (WT) CD-1 mice and AQP4 knockout (AQP4-KO) mice to 0, 100, 350 and 700 mg/m3 1,2-DCE by inhalation for 28 days. We applied label-free proteomics and a cell co-culture system to elucidate the role of AQP4 inhibition in 1,2-DCE-induced demyelination. The results showed that 1,2-DCE down-regulated AQP4 in the WT mouse cortexes. Both 1,2-DCE exposure and AQP4 deletion induced neurotoxicity in mice, including increased brain water content, abnormal pathological vacuolations, and neurobehavioral damage. Tests for interaction of multiple regression analysis highlighted different effects of 1,2-DCE exposure level depending on the genotype, indicating the core role of AQP4 in regulation on 1,2-DCE-caused neurotoxicity. We used label-free quantitative proteomics to detect differentially expressed proteins associated with 1,2-DCE exposure and AQP4 inhibition, and identified down-regulation in myelin basic protein (MBP) and tyrosine-protein kinase Fyn (FYN) in a dose-dependent manner in WT mice but not in AQP4-KO mice. 1,2-DCE and AQP4 deletion separately resulted in demyelination, as detected by Luxol fast blue staining, and manifested as disordered nerve fibers and cavitation in the cortexes. Western blot and immunofluorescence confirmed the decreased AQP4 in the astrocytes and the down-regulated MBP in the oligodendrocytes by 1,2-DCE exposure and AQP4 inhibition, respectively. Finally, the co-culture results of SVG p12 and MO3.13 cells showed that 1,2-DCE-induced AQP4 down-regulation in the astrocytes was responsible for demyelination, by decreasing MBP in the oligodendrocytes. In conclusion, 1,2-DCE induced cortex demyelination by depressing MBP via AQP4 inhibition in the mice.

DNA Methylation Array Identifies Golli-MBP as a Biomarker for Disease Severity in Childhood Atopic Dermatitis.

In this study, we investigated the changes in global methylation status and its functional relevance in childhood atopic dermatitis (AD). Differences in epigenome-scale methylation events in peripheral blood associated with childhood AD were screened using DNA methylation arrays of 24 patients with AD compared with 24 control subjects. Of the 16,840 differentially methylated CpG regions between AD and control subjects, >97% CpG loci revealed hypomethylation in patients with childhood AD. Among the globally hypomethylated loci, we identified two CpG clusters within the golli-mbp locus of the MBP gene, which was functionally enriched by subnetwork enrichment analysis as an orchestrator among associated genes. The differential hypomethylation of the top-ranked cg24700313 cluster in the golli-mbp locus was validated by pyrosequencing in an independent cohort of 224 children with AD and 44 control subjects. DNA methylation was found to be negatively correlated with disease severity but showed no significant correlation with IgE levels after age adjustment. The multivariate correlation analysis represents a higher score in AD intensity with significantly increased IgE levels and decreased methylation levels in cg27400313. We concluded that methylation loss in the golli-mbp locus is an epigenetic factor associated with disease severity of childhood AD.

Effect of fluoxetine treatment on neurotoxicity induced by lysolecithin in male rats.

Demyelination disorder is an unusual pathologic event, which occurs in the central nervous system (CNS). Multiple sclerosis (MS) is an inflammatory demyelinating disease that affects the CNS, and it is the leading cause of disability in young adults. Lysolecithin (LPC) is one of the best toxin-induced demyelination models. In this study, a suitable model is created, and the effect of fluoxetine treatment is examined on this model. In this case, it was assumed that daily fluoxetine treatment had increased the endogenous remyelination in the LPC model. This study was focused on investigating the influence of the fluoxetine dose of 5 or 10 mg/kg per day for 1 and 4 weeks on LPC-induced neurotoxicity in the corpus callosum region. It was performed as a demyelinating model in male Wistar rats. After 3 days, fluoxetine was injected intraperitoneally (5 or 10 mg/kg per day) for 1 and 4 weeks in each group. After completing the treatment course, the corpus callosum was removed to examine the gene expression and histological analysis was performed. The results of the histopathological study of hematoxylin and eosin staining of the corpus callosum showed that in 1 and 4-week treatment groups, fluoxetine has reduced the level of inflammation at the LPC injection site (5 and 10 mg/kg per day). Fluoxetine treatment in the luxol fast blue (LFB) staining of the corpus callosum has been led to an increase in myelination capacity in all doses and times. The results of the genetic study showed that the fluoxetine has significantly reduced the expression level of tumor necrosis factor-α, nuclear factor κß, and induced nitric oxide synthase in comparison with the untreated LPC group. Also, the fluoxetine treatment has enhanced the expression level of the forkhead box P3 (FOXP3) gene in comparison with the untreated group. Fluoxetine has increased the expression level of myelination and neurotrophic genes such as myelin basic protein (MBP), oligodendrocyte transcription factor 2 (OLIG2), and brain-derived neurotrophic factor (BDNF). The outcomes demonstrated that fluoxetine reduces inflammation and strengthens the endogenous myelination in the LPC-induced demyelination model; however, supplementary studies are required for specifying the details of its mechanisms.

CCAAT/Enhancer-Binding Protein ε27 Antagonism of GATA-1 Transcriptional Activity in the Eosinophil Is Mediated by a Unique N-Terminal Repression Domain, Is Independent of Sumoylation and Does Not Require DNA Binding.

CCAAT/enhancer binding protein epsilon (C/EBPε) is required for eosinophil differentiation, lineage-specific gene transcription, and expression of C/EBPε32 and shorter 27kD and 14kD isoforms is developmentally regulated during this process. We previously defined the 27kD isoform (C/EBPε27) as an antagonist of GATA-1 transactivation of the eosinophil's major basic protein-1 (MBP1) P2-promoter, showing C/EBPε27 and GATA-1 physically interact. In the current study, we used a Tat-C/EBPε27 fusion protein for cell/nuclear transduction of an eosinophil myelocyte cell line to demonstrate that C/EBPε27 is a potent repressor of MBP1 transcription. We performed structure-function analyses of C/EBPε27 mapping its repressor domains, comparing it to C/EBPε32 and C/EBPε14, using GATA-1 co-transactivation of the MBP1-P2 promoter. Results show C/EBPε27 repression of GATA-1 is mediated by its unique 68aa N-terminus combined with previously identified RDI domain. This repressor activity does not require, but is enhanced by, DNA binding via the basic region of C/EBPε27 but independent of sumoylation of the RDI core "VKEEP" sumoylation site. These findings identify the N-terminus of C/EBPε27 as the minimum repressor domain required for antagonism of GATA-1 in the eosinophil. C/EBPε27 repression of GATA-1 occurs via a combination of both C/EBPε27-GATA-1 protein-protein interaction and C/EBPε27 binding to a C/EBP site in the MBP1 promoter. The C/EBPε27 isoform may serve to titrate and/or turn off eosinophil granule protein genes like MBP1 during eosinophil differentiation, as these genes are ultimately silenced in the mature cell. Understanding the functionality of C/EBPε27 in eosinophil development may prove promising in developing therapeutics that reduce eosinophil proliferation in allergic diseases.

Anti-PD1 antibody enhances the anti-tumor efficacy of MUC1-MBP fusion protein vaccine via increasing Th1, Tc1 activity and decreasing the proportion of MDSC in the B16-MUC1 melanoma mouse model.

In previous studies, we have obtained a notable anti-tumor efficacy of the recombinant MUC1-MBP vaccine in the process of mouse B16-MUC1 melanoma treatment. However, the tumor cannot be eliminated completely. We found that the tumor inhibition rate decreased from 81.67% (five immunizations) to 43.67% (eight immunizations) after more than five immunizations, indicating persistent vaccine stimulation may activate immunosuppressive factors. In the present study, we revealed that programmed cell death 1 (PD1), an inhibitory molecule suppressing T cell function, expressed on splenic and tumor-infiltrating T cells were up-regulated by the vaccine. Therefore, to optimize the anti-tumor efficacy of the vaccine, we employed combination immunotherapy with MUC1-MBP vaccine and αPD1 (anti-PD1 antibody). Results showed that combination immunotherapy induced a more remarkable anti-tumor efficacy, the tumor clearance being increased to 80% from 20% which obtain by MUC1-MBP vaccine immunizations. To investigate the possible underlying mechanism, IFN-γ secretion and cytotoxic T lymphocyte (CTL) cytotoxicity were measured by enzyme-linked immunosorbent assay (ELISA) and xCELLigence real-time cell analyzer (RTCA) respectively. T cell subsets and immunosuppressive cells in the mouse spleen and tumor microenvironment were analyzed by FACS. Results showed that the proportion of splenic CD8+T cells and tumor infiltration was increased and the activity of CTL killing, T helper 1 (Th1), Type 1 CD8+T (Tc1) was enhanced, indicating that the anti-tumor efficacy enhanced by combination immunotherapy was mainly through boosting CD8+T cells mediated anti-tumor cellular immunity. Additionally, combination immunotherapy significantly decreased the splenic and tumor-infiltrating myeloid derived suppressor cells (MDSCs). These results demonstrated that combination immunotherapy with MUC1-MBP vaccine and αPD1 was capable to invoke a more potent anti-tumor immune response and provide a foundation for further research.

The Absence of Myelin Basic Protein Reduces Non-Amyloidogenic Processing of Amyloid Precursor Protein.

BACKGROUND: The accumulation of amyloid ß-protein (Aß) in the brain is a pathological feature of Alzheimer's disease (AD). Aß peptides originate from amyloid precursor protein (APP). APP can be proteolytically cleaved through amyloidogenic or non-amyloidogenic pathways. The molecular effects on APP metabolism/processing may be influenced by myelin and the breakdown of myelin basic protein (MBP) in AD patients and mouse models of AD pathology. METHODS: We directly tested whether MBP can alter influence APP processing in MBP-/- mice, known as Shiverer (shi/shi) mice, in which no functional MBP is produced due to gene breakage from the middle of MBP exon ll. RESULTS: A significant reduction of the cerebral sAPPα level in Shiverer (shi/shi) mice was found, although the levels of both total APP and sAPPß remain unchanged. The reduction of sAPPα was considered to be due to the changes in the expression levels of a disintegrin and metalloproteinase-9 (ADAM9) catalysis and non-amyloid genic processing of APP in the absence of MBP because it binds to ADAM9. MBP -/- mice exhibited increased Aß oligomer production. CONCLUSION: These findings suggest that in the absence of MBP, there is a marked reduction of nonamyloidogenic APP processing to sAPPα, and targeting myelin of oligodendrocytes may be a novel therapy for the prevention and treatment of AD.

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles attenuate experimental autoimmune encephalomyelitis via regulating pro and anti-inflammatory cytokines.

The therapeutic effects of mesenchymal stem cells-extracellular vesicles have been proved in many inflammatory animal models. In the current study, we aimed to investigate the effect of extracellular vesicles (EVs) derived from human umbilical cord-MSC (hUCSC-EV) on the clinical score and inflammatory/anti-inflammatory cytokines on the EAE mouse model. After induction of EAE in C57Bl/6 mice, they were treated intravenously with hUCSC-EV or vehicle. The clinical score and body weight of all mice was registered every day. On day 30, mice were sacrificed and splenocytes were isolated for cytokine assay by ELISA. Cytokine expression of pro-/anti-inflammatory cytokine by real-time PCR, leukocyte infiltration by hematoxylin and eosin (H&E) staining, and the percent of glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) positive cells by immunohistochemistry were assessed in the spinal cord. Our results showed that hUCSC-EV-treated mice have lower maximum mean clinical score (MMCS), pro-inflammatory cytokines, and inflammatory score in comparison to the control mice. We also showed that hUCSC-EV administration significantly improved body weight and increased the anti-inflammatory cytokines and the frequency of Treg cells in the spleen. There was no significant difference in the percent of GFAP and MBP positive cells in the spinal cord of experimental groups. Finally, we suggest that intravenous administration of hUCSC-EV alleviate induce-EAE by reducing the pro-inflammatory cytokines, such as IL-17a, TNF-α, and IFN-γ, and increasing the anti-inflammatory cytokines, IL-4 and IL-10, and also decrease the leukocyte infiltration in a model of MS. It seems that EVs from hUC-MSCs have the same therapeutic effects similar to EVs from other sources of MSCs, such as adipose or bone marrow MSCs.

Assessment of the Oral Delivery of a Myelin Basic Protein Gene Promoter with Antiapoptotic bcl-xL (pMBP-bcl-xL) DNA by Cyclic Peptide Nanotubes with Two Aspect Ratios and Its Biodistribution in the Brain and Spinal Cord.

Cyclo-(D-Trp-Tyr) peptide nanotubes (PNTs) were reported to be potential carriers for oral gene delivery in our previous study; however, the effect of the aspect ratio (AR) of these PNTs on gene delivery in vivo could affect penetration or interception in biological environments. The aim of this study was to assess the feasibility of cyclo-(D-Trp-Tyr) PNTs with two ARs as carriers for oral pMBP-bcl-xL-hRluc delivery to the spinal cord to treat spinal cord injury (SCI). We evaluated the biodistribution of oligodendrocyte (OLG)-specific myelin basic protein gene promoter-driven antiapoptotic DNA (pMBP-bcl-xL) to the brain and spinal cord delivered with cyclo-(D-Trp-Tyr) PNTs with large (L) and small (S) PNTs with two ARs. After complex formation, the length, width, and AR of the L-PNTs/DNA were 77.86 ± 3.30, 6.51 ± 0.28, and 13.75 ± 7.29 µm, respectively, and the length and width of the S-PNTs/DNA were 1.17 ± 0.52 and 0.17 ± 0.05 µm, respectively, giving an AR of 7.12 ± 3.17 as detected by scanning electron microscopy. Each of these three parameters exhibited significant differences (p < 0.05) between L-PNTs/DNA and S-PNTs/DNA. However, there were no significant differences (p > 0.05) between the L-PNTs and S-PNTs for either their DNA encapsulation efficiency (29.72 ± 14.19 and 34.31 ± 16.78%, respectively) or loading efficiency (5.15 ± 2.58 and 5.95 ± 2.91%). The results of the in vitro analysis showed that the S-PNT/DNA complexes had a significantly higher DNA release rate and DNA permeation in the duodenum than the L-PNT/DNA complexes. Using Cy5 and TM-rhodamine to individually and chemically conjugate the PNTs with plasmid DNA, we observed, using laser confocal microscopy, that the PNTs and DNA colocalized in complexes. We further confirmed the complexation between DNA and the PNTs using fluorescence resonance energy transfer (FRET). Data from an in vivo imaging system (IVIS) showed that there was no significant difference (p > 0.05) in PNT distribution between L-PNTs/DNA and S-PNTs/DNA within 4 h. However, the S-PNT/DNA group had a significantly higher DNA distribution (p < 0.05) in several organs, including the ilium, heart, lungs, spleen, kidneys, testes, brain, and spinal cord. Finally, we determined the bcl-xL protein expression levels in the brain and spinal cord regions for the L-PNT/DNA and S-PNT/DNA complex formulations. These results suggested that either L-PNTs or S-PNTs may be used as potential carriers for oral gene delivery to treat SCI.

Modulation of vigabatrin induced cerebellar injury: the role of caspase-3 and RIPK1/RIPK3-regulated cell death pathways.

Vigabatrin is the drug of choice in resistant epilepsy and infantile spasms. Ataxia, tremors, and abnormal gait have been frequently reported following its use indicating cerebellar involvement. This study aimed, for the first time, to investigate the involvement of necroptosis and apoptosis in the VG-induced cerebellar cell loss and the possible protective role of combined omega-3 and vitamin B12 supplementation. Fifty Sprague-Dawley adult male rats (160-200 g) were divided into equal five groups: the control group received normal saline, VG200 and VG400 groups received VG (200 mg or 400 mg/kg, respectively), VG200 + OB and VG400 + OB groups received combined VG (200 mg or 400 mg/kg, respectively), vitamin B12 (1 mg/kg), and omega-3 (1 g/kg). All medications were given daily by gavage for four weeks. Histopathological changes were examined in H&E and luxol fast blue (LFB) stained sections. Immunohistochemical staining for caspase-3 and receptor-interacting serine/threonine-protein kinase-1 (RIPK1) as well as quantitative real-time polymerase chain reaction (qRT-PCR) for myelin basic protein (MBP), caspase-3, and receptor-interacting serine/threonine-protein kinase-3 (RIPK3) genes were performed. VG caused a decrease in the granular layer thickness and Purkinje cell number, vacuolations, demyelination, suppression of MBP gene expression, and induction of caspases-3, RIPK1, and RIPK3 in a dose-related manner. Combined supplementation with B12 and omega-3 improved the cerebellar histology, increased MBP, and decreased apoptotic and necroptotic markers. In conclusion, VG-induced neuronal cell loss is dose-dependent and related to both apoptosis and necroptosis. This could either be ameliorated (in low-dose VG) or reduced (in high-dose VG) by combined supplementation with B12 and omega-3.

Murine Esophagus Expresses Glial-Derived Central Nervous System Antigens.

Multiple sclerosis (MS) has been considered to specifically affect the central nervous system (CNS) for a long time. As autonomic dysfunction including dysphagia can occur as accompanying phenomena in patients, the enteric nervous system has been attracting increasing attention over the past years. The aim of this study was to identify glial and myelin markers as potential target structures for autoimmune processes in the esophagus. RT-PCR analysis revealed glial fibrillary acidic protein (GFAP), proteolipid protein (PLP), and myelin basic protein (MBP) expression, but an absence of myelin oligodendrocyte glycoprotein (MOG) in the murine esophagus. Selected immunohistochemistry for GFAP, PLP, and MBP including transgenic mice with cell-type specific expression of PLP and GFAP supported these results by detection of (1) GFAP, PLP, and MBP in Schwann cells in skeletal muscle and esophagus; (2) GFAP, PLP, but no MBP in perisynaptic Schwann cells of skeletal and esophageal motor endplates; (3) GFAP and PLP, but no MBP in glial cells surrounding esophageal myenteric neurons; and (4) PLP, but no GFAP and MBP in enteric glial cells forming a network in the esophagus. Our results pave the way for further investigations regarding the involvement of esophageal glial cells in the pathogenesis of dysphagia in MS.

Hericium erinaceus mycelium and its small bioactive compounds promote oligodendrocyte maturation with an increase in myelin basic protein.

Oligodendrocytes (OLs), myelin-producing glia in the central nervous system (CNS), produce a myelin extension that enwraps axons to facilitate action potential propagation. An effective approach to induce oligodendrogenesis and myelination is important to foster CNS development and promote myelin repair in neurological diseases. Hericium (H.) erinaceus, an edible and culinary-medicinal mushroom, has been characterized as having neuroprotective activities. However, its effect on OL differentiation has not yet been uncovered. In this study using oligodendrocyte precursor cell (OPC) cultures and an ex vivo cerebellar slice system, we found that the extract from H. erinaceus mycelium (HEM) not only promoted the differentiation of OPCs to OLs in the differentiation medium, but also increased the level of myelin basic protein (MBP) on neuronal fibers. Moreover, daily oral administration of HEM into neonatal rat pups for 7 days enhanced MBP expression and OLs in the corpus callosum of the postnatal rat brain. The effect of HEM-derived bioactive compounds, the diterpenoid xylosides erinacine A (HeA) and HeC and a sesterterpene with 5 isoprene units called HeS, were further evaluated. The results showed that HeA and HeS more potently stimulated MBP expression in OLs and increased the number of OLs. Moreover, overlap between MBP immunoreactivity and neuronal fibers in cultured cerebellar tissue slices was significantly increased in the presence of HeA and HeS. In summary, our findings indicate that HEM extract and its ingredients HeA and HeS display promising functional effects and promote OL maturation, providing insights into their potential for myelination in neurodevelopmental disorders.

Loss of Quaking RNA binding protein disrupts the expression of genes associated with astrocyte maturation in mouse brain.

Quaking RNA binding protein (QKI) is essential for oligodendrocyte development as myelination requires myelin basic protein mRNA regulation and localization by the cytoplasmic isoforms (e.g., QKI-6). QKI-6 is also highly expressed in astrocytes, which were recently demonstrated to have regulated mRNA localization. Here, we define the targets of QKI in the mouse brain via CLIPseq and we show that QKI-6 binds 3'UTRs of a subset of astrocytic mRNAs. Binding is also enriched near stop codons, mediated partially by QKI-binding motifs (QBMs), yet spreads to adjacent sequences. Using a viral approach for mosaic, astrocyte-specific gene mutation with simultaneous translating RNA sequencing (CRISPR-TRAPseq), we profile ribosome associated mRNA from QKI-null astrocytes in the mouse brain. This demonstrates a role for QKI in stabilizing CLIP-defined direct targets in astrocytes in vivo and further shows that QKI mutation disrupts the transcriptional changes for a discrete subset of genes associated with astrocyte maturation.

Myelin-specific T cells in animals with Japanese macaque encephalomyelitis.

OBJECTIVE: To determine whether animals with Japanese macaque encephalomyelitis (JME), a spontaneous demyelinating disease similar to multiple sclerosis (MS), harbor myelin-specific T cells in their central nervous system (CNS) and periphery. METHODS: Mononuclear cells (MNCs) from CNS lesions, cervical lymph nodes (LNs) and peripheral blood of Japanese macaques (JMs) with JME, and cervical LN and blood MNCs from healthy controls or animals with non-JME conditions were analyzed for the presence of myelin-specific T cells and changes in interleukin 17 (IL-17) and interferon gamma (IFNγ) expression. RESULTS: Demyelinating JME lesions contained CD4+ T cells and CD8+ T cells specific to myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), and/or proteolipid protein (PLP). CD8+ T-cell responses were absent in JME peripheral blood, and in age- and sex-matched controls. However, CD4+ Th1 and Th17 responses were detected in JME peripheral blood versus controls. Cervical LN MNCs from eight of nine JME animals had CD3+ T cells specific for MOG, MBP, and PLP that were not detected in controls. Mapping myelin epitopes revealed a heterogeneity in responses among JME animals. Comparison of myelin antigen sequences with those of JM rhadinovirus (JMRV), which is found in JME lesions, identified six viral open reading frames (ORFs) with similarities to myelin antigen sequences. Overlapping peptides to these JMRV ORFs did not induce IFNγ responses. INTERPRETATIONS: JME possesses an immune-mediated component that involves both CD4+ and CD8+ T cells specific for myelin antigens. JME may shed new light on inflammatory demyelinating disease pathogenesis linked to gamma-herpesvirus infection.

Comprehensive Analysis of Age-related Changes in Lipid Metabolism and Myelin Sheath Formation in Sciatic Nerves.

To investigate the molecular changes related to myelin formation and lipid metabolism in the sciatic nerve in Sprague Dawley (SD) rats during aging. Thirty-six healthy male SD rats were divided into five groups according to age: 1 week, 1 month, 6 months, 12 months, and 24 months. Sciatic nerves were collected from 1-month-old and 24-month-old SD rats (n = 3) to perform next-generation sequencing (NGS) and bioinformatics analysis. Specimens from each group were harvested and analyzed by qPCR, Western blotting, and transmission electron microscopy (TEM). Protein-protein interaction (PPI) networks of differentially expressed mRNAs (DEmRNAs) related to myelin and lipid metabolism were constructed. DEmRNAs in subnetworks were verified using qPCR. A total of 4580 DEmRNAs were found during aging. The top enriched GO biological processes were primarily clustered in cholesterol and lipid metabolism, including the cholesterol biosynthetic process (RF = 3.16), sterol biosynthetic process (RF = 3.03), cholesterol metabolic process (RF = 2.15), sterol metabolic process (RF = 2.11), fatty acid biosynthetic process (RF = 2.09), and lipid biosynthetic process (RF = 1.79). The mRNA levels of MBP, PMP22, and MPZ were downregulated during aging, while the protein expression of MBP showed an increasing trend. The TEM results showed thin myelin sheaths and an increased number of unmyelinated axons in the 1-week-old rats, and the sheaths became thickened with degenerated axons appearing in older animals. Forty PPI subnetworks related to lipid metabolism were constructed, including one primary subnetwork and two smaller subnetworks. The hub genes were mTOR in sub-network 1, Akt1 in sub-network 2, and SIRT1 in sub-network 3. No gene expression was found consistent with the sequencing results, while in the downregulated genes, AKT1, CEBPA, LIPE, LRP5, PHB, and Rara were significantly downregulated in 24-month-old rats. Lipid metabolism might play an important role in maintaining the structure and physiological function in sciatic nerves during aging and could be candidates for nerve aging research.

Developmental expression of genes involved in progesterone synthesis, metabolism and action during the post-natal cerebellar myelination.

Progesterone is involved in dendritogenesis, synaptogenesis and maturation of cerebellar Purkinge cells, major sites of steroid synthesis in the brain. To study a possible time-relationship between myelination, neurosteroidogenesis and steroid receptors during development of the postnatal mouse cerebellum, we determined at postnatal days 5 (P5),18 (P18) and 35 (P35) the expression of myelin basic protein (MBP), components of the steroidogenic pathway, levels of endogenous steroids and progesterone's classical and non-classical receptors. In parallel with myelin increased expression during development, P18 and P35 mice showed higher levels of cerebellar progesterone and its reduced derivatives, higher expression of steroidogenic acute regulatory protein (StAR) mRNA, cholesterol side chain cleavage enzyme (P450scc) and 5α-reductase mRNA vs. P5 mice. Other steroids such as corticosterone and its reduced derivatives and 3ß-androstanodiol (ADIOL) showed a peak increase at P18 compared to P5. Progesterone membrane receptors and binding proteins (PGRMC1, mPRα, mPRß, mPRγ, and Sigma1 receptors) mRNAs levels increased during development while that of classical progesterone receptors (PR) remained invariable. PRKO mice showed similar MBP levels than wild type. Thus, these data suggests that progesterone and its neuroactive metabolites may play a role in postnatal cerebellar myelination.

In silico study of the association of the HLA-A*31:01 allele (human leucocyte antigen allele 31:01) with neuroantigenic epitopes of PLP (proteolipid protein), MBP (myelin basic protein) and MOG proteins (myelin oligodendrocyte glycoprotein) for studying the multiple sclerosis disease pathogenesis.

The main pathologic hallmark of multiple sclerosis is a demyelinating plaque that contains a prominent immunologic response dominated by T cells of the immune system. PLP (proteolipid protein), MPB (myelin basic protein), and Myelin oligodendrocyte glycoprotein (MOG) proteins are important autoantigens for the demyelinating of CNS in multiple sclerosis. There is good evidence indicating that T CD8+ cells and MHC class I molecules play an important role in this disease. The HLA-A*31:01 allele of MHC class I is a member of HLA-A3 superfamily and there is no clear report concerning the relationship of this allele with MS. Feeling this gap, we studied the possible association of the HLA-A*31:01 with MS by prediction of neuroantigenic epitopes of human MBP, PLP, and MOG proteins of myelin sheath using in silico methods. PLP did not show any neuroantigenic epitope, but the two epitopes of MBP and seven epitopes of MOG for HLA-A*31:01 were determined via bioinformatics servers. In silico study of the nine epitope showed that MOG195-204 (LIICYNWLHR) peptide of the membrane-associated/cytoplasmic part of human MOG has suitable binding affinity to the HLA-A*31:01 allele as a potential neuroantigenic epitope. Further investigations of this peptide revealed that the binding of C-terminal residue of this peptide has a more significant effect on binding to this allele than the N-terminal part of the peptide. Altogether, this combination of "LIICYNWLHR/A*31:01 allele "may play an important role in MS pathogenesis and this complex is suggested for further studies such as T cell receptor.Communicated by Ramaswamy H. Sarma.

Mannan-MOG35-55 Reverses Experimental Autoimmune Encephalomyelitis, Inducing a Peripheral Type 2 Myeloid Response, Reducing CNS Inflammation, and Preserving Axons in Spinal Cord Lesions.

CNS autoantigens conjugated to oxidized mannan (OM) induce antigen-specific T cell tolerance and protect mice against autoimmune encephalomyelitis (EAE). To investigate whether OM-peptides treat EAE initiated by human MHC class II molecules, we administered OM-conjugated murine myelin oligodendrocyte glycoprotein peptide 35-55 (OM-MOG) to humanized HLA-DR2b transgenic mice (DR2b.Ab°), which are susceptible to MOG-EAE. OM-MOG protected DR2b.Ab° mice against MOG-EAE by both prophylactic and therapeutic applications. OM-MOG reversed clinical symptoms, reduced spinal cord inflammation, demyelination, and neuronal damage in DR2b.Ab° mice, while preserving axons within lesions and inducing the expression of genes associated with myelin (Mbp) and neuron (Snap25) recovery in B6 mice. OM-MOG-induced tolerance was peptide-specific, not affecting PLP178-191-induced EAE or polyclonal T cell proliferation responses. OM-MOG-induced immune tolerance involved rapid induction of PD-L1- and IL-10-producing myeloid cells, increased expression of Chi3l3 (Ym1) in secondary lymphoid organs and characteristics of anergy in MOG-specific CD4+ T cells. The results show that OM-MOG treats MOG-EAE in a peptide-specific manner, across mouse/human MHC class II barriers, through induction of a peripheral type 2 myeloid cell response and T cell anergy, and suggest that OM-peptides might be useful for suppressing antigen-specific CD4+ T cell responses in the context of human autoimmune CNS demyelination.

In Vitro Differentiation of Human Placenta-Derived Multipotent Cells into Schwann-Like Cells.

Human placenta-derived multipotent stem cells (PDMCs) resembling embryonic stem cells can differentiate into three germ layer cells, including ectodermal lineage cells, such as neurons, astrocytes, and oligodendrocytes. The favorable characteristics of noninvasive cell harvesting include fewer ethical, religious, and legal considerations as well as accessible and limitless supply. Thus, PDMCs are attractive for cell-based therapy. The Schwann cell (SC) is the most common cell type used for tissue engineering such as nerve regeneration. However, the differentiation potential of human PDMCs into SCs has not been demonstrated until now. In this study, we evaluated the potential of PDMCs to differentiate into SC-like cells in a differentiation medium. After induction, PDMCs not only exhibited typical SC spindle-shaped morphology but also expressed SC markers, including S100, GFAP, p75, MBP, and Sox 10, as revealed by immunocytochemistry. Moreover, a reverse transcription-quantitative polymerase chain reaction analysis revealed the elevated gene expression of S100, GFAP, p75, MBP, Sox-10, and Krox-20 after SC induction. A neuroblastoma cell line, SH-SY5Y, was cultured in the conditioned medium (CM) collected from PDMC-differentiated SCs. The growth rate of the SH-SY5Y increased in the CM, indicating the function of PDMC-induced SCs. In conclusion, human PDMCs can be differentiated into SC-like cells and thus are an attractive alternative to SCs for cell-based therapy in the future.