Proteolipid Protein Research Articles

Brain and Serum Membrane Vesicle (Exosome) Profiles in Experimental Alcohol-Related Brain Degeneration: Forging the Path to Non-Invasive Liquid Biopsy Diagnostics

Background: Alcohol-related brain degeneration (ARBD) is associated with cognitive–motor impairments that can progress to disability and dementia. White matter (WM) is prominently targeted in ARBD due to chronic neurotoxic and degenerative effects on oligodendrocytes and myelin. Early detection and monitoring of WM pathology in ARBD could lead to therapeutic interventions. Objective: This study examines the potential utility of a non-invasive strategy for detecting WM ARBD using exosomes isolated from serum. Comparative analyses were made with paired tissue (Tx) and membrane vesicles (MVs) from the temporal lobe (TL). Methods: Long Evans rats were fed for 8 weeks with isocaloric liquid diets containing 37% or 0% caloric ethanol (n = 8/group). TL-Tx, TL-MVs, and serum exosomes (S-EVs) were used to examine ethanol’s effects on oligodendrocyte glycoprotein, astrocyte, and oxidative stress markers. Results: Ethanol significantly decreased the TL-Tx expression of platelet-derived growth factor receptor alpha (PDGFRA), 2′,3′-cyclic nucleotide 3′ phosphodiesterase (CNPase), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), glial fibrillary acidic protein (GFAP), and 8-OHdG, whereas in the TL-MVs, ethanol increased CNPase, PDGFRA, and 8-OHdG, but decreased MOG and GFAP concordantly with TL-Tx. Ethanol modulated the S-EV expression by reducing PLP, nestin, GFAP, and 4-hydroxynonenal (HNE). Conclusion: Chronic ethanol exposures differentially alter the expression of oligodendrocyte/myelin, astrocyte, and oxidative stress markers in the brain, brain MVs, and S-EVs. However, directionally concordant effects across all three compartments were limited. Future studies should advance these efforts by characterizing the relationship between ABRD and molecular pathological changes in brain WM-specific exosomes in serum.

Schwann cell-derived exosomes ameliorate peripheral neuropathy induced by ablation of dicer in Schwann cells.

MicroRNAs (miRNAs) in Schwann cells (SCs) mediate peripheral nerve function. Ablating Dicer, a key gene in miRNA biogenesis, in SCs causes peripheral neuropathy. Exosomes from healthy SCs (SC-Exo) ameliorate diabetic peripheral neuropathy in part via miRNAs. Thus, using transgenic mice with conditional and inducible ablation of Dicer in proteolipid protein (PLP) expressing SCs (PLP-cKO), we examined whether SC-Exo could reduce peripheral neuropathy in PLP-cKO mice. PLP-cKO mice at the age of 16 weeks (8 week post-Tamoxifen) were randomly treated with SC-Exo or saline weekly for 8 weeks. Age-and sex-matched wild-type (WT) littermates were used as controls. Peripheral neurological functions, sciatic nerve integrity, and myelination were analyzed. Quantitative RT-PCR and Western blot analyses were performed to examine miRNA and protein expression in sciatic nerve tissues, respectively. Compared to the WT mice, PLP-cKO mice exhibited a significant decrease in motor and sensory conduction velocities, thermal sensitivity, and motor coordination. PLP-cKO mice exhibited substantial demyelination and axonal damage of the sciatic nerve. Treatment of PLP-cKO mice with SC-Exo significantly ameliorated the peripheral neuropathy and sciatic nerve damage. PLP-cKO mice showed a substantial reduction in a set of Dicer-related miRNAs known to regulate myelination, axonal integrity, and inflammation such as miR-138, -146a and - 338 in the sciatic nerve. In addition, PLP-cKO mice exhibited significant reduction of myelin forming proteins, early growth response 2 (EGR2) and sex determining region Y-box10 (Sox10), but significantly increased myelination inhibitors, Notch1, c-Jun, and Sox2 and the axonal growth inhibitor phosphatase and tens in homolog (PTEN). However, SC-Exo treatment reversed the PLP-cKO altered miRNAs and proteins. This study demonstrates that exogenous SC-Exo ameliorate peripheral neuropathy induced by Dicer ablation in PLP expressing SCs. The therapeutic benefit may be mediated by the SC-Exo altered miRNAs and their targeted genes.

Identification of key regulatory genes involved in myelination after spinal cord injury by GSEA analysis

Multilayer dense myelin tissue provides insulating space and nutritional support for axons in healthy spinal cord tissue. Oligodendrocyte precursor cells (OPCs) are the main glial cells that complement myelin loss in the central nervous system and play an important role in the repair of spinal cord injury (SCI). However, the regulation of axonal remyelination after SCI is still insufficient. In this study, we focused on the changes in genes related to myelin repair after rat hemisection SCI by gene set enrichment analysis (GSEA). Key genes proteolipid protein 1 (Plp1), hexosaminidase subunit alpha (Hexa), and hexosaminidase subunit beta (Hexb) during remyelination after SCI were found. Through quantitative real-time polymerase chain reaction (qPCR) experiments, we confirmed that within 28 days after rat hemisection SCI, the mRNA expression of gene Plp1 gradually decreased, while the expressions of gene Hexa and Hexb gradually increased, which was consistent with RNA sequencing results. In vitro, we performed EdU proliferation assays on OPC cell line OLN-93 and primary rat OPCs. We found that interference of Plp1 promoted OPC proliferation, while interference of Hexa and Hexb inhibited OPC proliferation. In addition, we performed in vitro differentiation experiments on primary rat OPCs. By measuring myelin sheath branch outgrowth and the fluorescence intensity of the mature myelin sheath marker myelin basic protein (MBP), we found that interference of Hexa or Hexb promoted OPC differentiation and maturation, but interference of Plp1 inhibited this process. Finally, we injected Hexb siRNA in vivo and found that interfering Hexb could improve motor movements and myelin regeneration after SCI in rats. Our results provide new target genes that can selectively regulate the proliferation and differentiation of endogenous OPCs, providing new ideas for promoting remyelination and functional recovery after SCI.

Spatiotemporal Dysregulation of Neuron-Glia Related Genes and Pro-/Anti-Inflammatory miRNAs in the 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD), the leading cause of dementia, is a multifactorial disease influenced by aging, genetics, and environmental factors. miRNAs are crucial regulators of gene expression and play significant roles in AD onset and progression. This exploratory study analyzed the expression levels of 28 genes and 5 miRNAs (miR-124-3p, miR-125b-5p, miR-21-5p, miR-146a-5p, and miR-155-5p) related to AD pathology and neuroimmune responses using RT-qPCR. Analyses were conducted in the prefrontal cortex (PFC) and the hippocampus (HPC) of the 5xFAD mouse AD model at 6 and 9 months old. Data highlighted upregulated genes encoding for glial fibrillary acidic protein (Gfap), triggering receptor expressed on myeloid cells (Trem2) and cystatin F (Cst7), in the 5xFAD mice at both regions and ages highlighting their roles as critical disease players and potential biomarkers. Overexpression of genes encoding for CCAAT enhancer-binding protein alpha (Cebpa) and myelin proteolipid protein (Plp) in the PFC, as well as for BCL2 apoptosis regulator (Bcl2) and purinergic receptor P2Y12 (P2yr12) in the HPC, together with upregulated microRNA(miR)-146a-5p in the PFC, prevailed in 9-month-old animals. miR-155 positively correlated with miR-146a and miR-21 in the PFC, and miR-125b positively correlated with miR-155, miR-21, while miR-146a in the HPC. Correlations between genes and miRNAs were dynamic, varying by genotype, region, and age, suggesting an intricate, disease-modulated interaction between miRNAs and target pathways. These findings contribute to our understanding of miRNAs as therapeutic targets for AD, given their multifaceted effects on neurons and glial cells.

Knockdown of Rab9 Recovers Defective Morphological Differentiation Induced by Chemical ER Stress Inducer or PMD-Associated PLP1 Mutant Protein in FBD-102b Cells.

Small GTP-binding proteins of the Rab family regulate intracellular vesicle trafficking across many aspects of the transport system. Among these, Rab9 is recognized for its role in controlling the transport system not only around the trans-Golgi network but also around the late endosome. However, the specific functions across different cell types and tissues remain unclear. Here, for the first time, we report that Rab9 negatively regulates morphological changes in the FBD-102b cell line, an oligodendroglial precursor cell line undergoing morphological differentiation. The knockdown of Rab9 led to an increase in cell shape alterations characterized by widespread membrane extensions. These changes were accompanied by increased expression levels of oligodendroglial cell differentiation and myelination marker proteins. Notably, the knockdown of Rab9 was capable of recovering defective cell morphological changes induced by tunicamycin, an inducer of endoplasmic reticulum (ER) stress, which is one of the major causes of oligodendroglial cell diseases such as Pelizaeus-Merzbacher disease (PMD, currently known as hypomyelinating leukodystrophy type 1 [HLD1]). In addition, Rab9 knockdown recovered levels of ER stress marker proteins and differentiation markers. Similar results were obtained in the cases of dithiothreitol (DTT), another chemical ER stress inducer, as well as HLD1-associated proteolipid protein 1 (PLP1) mutant protein. These results indicate a unique role for Rab9 in oligodendroglial cell morphological changes, suggesting its potential as a therapeutic target for mitigating diseases such as HLD1 at the molecular and cellular levels.

Single-Nucleus Landscape of Glial Cells and Neurons in Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disease with a projected significant increase in incidence. Therefore, this study analyzed single-nucleus AD data to provide a theoretical basis for the clinical development and treatment of AD. We downloaded AD-related monocyte data from the Gene Expression Omnibus database, annotated cells, compared cell abundance between groups, and investigated glial and neuronal cell biological processes and pathways through functional enrichment analysis. Furthermore, we constructed a global regulatory network for AD based on cell communication and ecological analyses. Our findings revealed increased abundance of Capping Protein Regulator And Myosin 1 linker 1 (CARMIL1)+ astrocytes (AST), Immunoglobulin Superfamily Member 21 (IGSF21)+ microglia (MIC), SRY-Box Transcription Factor 6 (SOX6)+ inhibitory neurons (InNeu), and laminin alpha-2 chain (LAMA2)+ oligodendrocytes (OLI) cell subgroups in tissues of patients with AD, while prostaglandin D2 synthase (PTGDS)+ AST, Src Family Tyrosine Kinase (FYN)+ MIC, and Proteolipid Protein 1 (PLP1)+ InNeu subgroups specifically decreased. We found that the cell phenotype of patients with AD shifted from a simpler to a more complex state compared to the control group. Cell communication analysis revealed strong communication between MIC and NEU. Furthermore, AST, MIC, NEU, and OLI were involved in oxidative stress- and inflammation-related pathways, potentially contributing to disease development. This study provides a theoretical basis for further exploring the specific mechanisms underlying AD.

Comparative Neuroprotective Potential of Nanoformulated and Free Resveratrol Against Cuprizone-Induced Demyelination in Rats.

Demyelination is a frequent yet crippling neurological disease associated with multiple sclerosis (MS). The cuprizone (CZ) model, which causes demyelination through oxidative stress and neuroinflammation, is a popular tool used by researchers to examine this process. The polyphenol resveratrol (RESV) has become a promising neuroprotective agent in seeking for efficient therapies. In a rat model given CZ, we created and examined iron oxide nanoparticles (IONPs) loaded with RESV (IONP-RESV) to see how effective they were as a therapeutic agent against free RESV. According to molecular mechanisms, exposure to CZ resulted in a marked downregulation of myelin proteolipid protein (PLP) expression and an overexpression of the inflammatory markers tumor necrosis factor-α (TNF-α) and S100β, which are indicators of demyelination and neuroinflammation. It is remarkable that these CZ-induced alterations could be reversed by therapy with either RESV or IONP-RESV. Interestingly, IONP-RESV showed even stronger anti-inflammatory activity, as shown by a more noticeable downregulation of TNF-α and S100β expression. These results were confirmed by histopathological examination of the cerebral cortices. Our findings support the better neuroprotective benefits of RESV-loaded IONPs over free RESV in reducing demyelination and neuroinflammation brought on by CZ. Owing to their pro-remyelinating, anti-inflammatory, and antioxidant properties, RESV-loaded IONPs show promise as a neurotherapeutic intervention in the future for neurological diseases such as multiple sclerosis.

Deletions in the MAL gene result in loss of Mal protein, defining the rare inherited AnWj-negative blood group phenotype

The genetic background of the high prevalence red blood cell antigen AnWj has remained unresolved since its identification in 1972, despite reported associations with both CD44 and Smyd1 histone methyltransferase. Development of anti-AnWj, which may be clinically significant, is usually due to transient suppression of antigen expression, but a small number of individuals with persistent, autosomally-recessive inherited AnWj-negative phenotype have been reported. Whole exome sequencing of individuals with the rare inherited AnWj-negative phenotype revealed no shared mutations in CD44H or SMYD1, but instead we discovered homozygosity for the same large exonic deletion in MAL, which was confirmed in additional unrelated AnWj-negative individuals. MAL encodes an integral multi-pass membrane proteolipid, Myelin and Lymphocyte protein (Mal), which has been reported to have essential roles in cell transport and membrane stability. AnWj-positive individuals were shown to express full-length Mal on their red cell membranes, which was not present on the membranes of AnWj-negative individuals, whether of an inherited or suppression background. Furthermore, binding of anti-AnWj was able to inhibit binding of anti-Mal to AnWj-positive red cells, demonstrating the antibodies bind to the same molecule. Over-expression of Mal in an erythroid cell-line resulted in expression of AnWj antigen, regardless of the presence or absence of CD44, demonstrating that Mal is both necessary and sufficient for AnWj expression. Our data resolve the genetic background of the inherited AnWj-negative phenotype, forming the basis of a new blood group system, further reducing the number of remaining unsolved blood group antigens.

Targeting the PAC1 receptor mitigates degradation of myelin and synaptic markers and diminishes locomotor deficits in the cuprizone demyelination model.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system with a strong neuroinflammatory component. Current treatments principally target the immune system but fail to preserve long-term myelin health and do not prevent neurological decline. Studies over the past two decades have shown that the structurally related neuropeptides VIP and PACAP (vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide, respectively) exhibit pronounced anti-inflammatory activities and reduce clinical symptoms in MS disease models, largely via actions on their bivalent VIP receptor type 1 and 2. Here, using the cuprizone demyelination model, we demonstrate that PACAP and VIP, and strikingly the PACAP-selective receptor PAC1 agonist maxadilan, prevented locomotor deficits in the horizontal ladder and open field tests. Moreover, only PACAP and maxadilan were able to prevent myelin deterioration, as assessed by a reduction in the expression of the myelin markers proteolipid protein 1, oligodendrocyte transcription factor 2, quaking-7 (APC) and Luxol Fast Blue staining. Furthermore, PACAP and maxadilan (but not VIP), prevented striatal synaptic loss and diminished astrocyte and microglial activation in the corpus callosum of cuprizone-fed mice. Invitro, PACAP or maxadilan prevented lipopolysaccharide (LPS)-induced polarisation of primary astrocytes at 12-24 h, an effect that was not seen with maxadilan in LPS-stimulated microglia. Taken together, our data demonstrates for the first time that PAC1 agonists provide distinctive protective effects against white matter deterioration, neuroinflammation and consequent locomotor dysfunctions in the cuprizone model. The results indicate that targeting the PAC1 receptor may provide a path to treat myelin-related diseases in humans.

Myelination by signaling through Arf guanine nucleotide exchange factor.

During myelination, large quantities of proteins are synthesized and transported from the endoplasmic reticulum (ER)-trans-Golgi network (TGN) to their appropriate locations within the intracellular region and/or plasma membrane. It is widely believed that oligodendrocytes uptake neuronal signals from neurons to regulate the endocytosis- and exocytosis-mediated intracellular trafficking of major myelin proteins such as myelin-associated glycoprotein (MAG) and proteolipid protein 1 (PLP1). The small GTPases of the adenosine diphosphate (ADP) ribosylation factor (Arf) family constitute a large group of signal transduction molecules that act as regulators for intracellular signaling, vesicle sorting, or membrane trafficking in cells. Studies on mice deficient in Schwann cell-specific Arfs-related genes have revealed abnormal myelination formation in peripheral nerves, indicating that Arfs-mediated signaling transduction is required for myelination in Schwann cells. However, the complex roles in these events remain poorly understood. This review aims to provide an update on signal transduction, focusing on Arf and its activator ArfGEF (guanine nucleotide exchange factor for Arf) in oligodendrocytes and Schwann cells. Future studies are expected to provide important information regarding the cellular and physiological processes underlying the myelination of oligodendrocytes and Schwann cells and their function in modulating neural activity.

CNSC-03. LINKING CELL FATE DETERMINATION IN THE EMBRYONIC FOREBRAIN TO PEDIATRIC HIGH-GRADE GLIOMA

Abstract BACKGROUND Pediatric high-grade gliomas are treatment resistant, usually fatal cancers encompassing diffuse midline glioma, K27-altered (DMG), and diffuse hemispheric glioma, G34R/V (DHG). Most patients harbour canonical and non-canonical mutations in histone variants H3.1 and H3.3, leading to global epigenetic dysregulation and stalled differentiation states as oligodendroglial progenitor cells (OPC) in DMG and GABAergic interneuron precursors in DHG. DLX2 is a homeobox transcription factor that directly represses OPC cell fate and promotes GABAergic interneuron cell fate and migration during early neurogenesis. Methods & Results Our lab has shown DLX2 promoter occupancy of Gad1/Gad2, and early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required for OPC differentiation in vivo. Co-expression of DLX2 with these target sequences reduces reporter gene expression in vitro, and the Dlx1/Dlx2 double knockout (DKO) mouse showed increases in Olig2, Nkx2.2, and Plp-1 expression in vivo. Transient over-expression of a Dlx2-GFP construct into murine DMG cells resulted in significant increased expression of Gad1/2 isoforms and decreased Olig2 and Nkx2.2, global restoration of H3K27me3 and loss of H3K27 acetylation, as well as a reduction in migration, invasion, and colony formation in vitro. To validate in human pHGG, we first probed an RNA-seq database containing 62 patient-derived pHGG cell lines which demonstrated an inverse correlation between DLX2 expression and OLIG1/2. Cell fate changes in DHG cell lines were evaluated by knockout of DLX2 using CRISPR/Cas9. Concurrently, OLIG1/2-expressing DMG cell lines underwent stable transfection of a DLX2-mCherry overexpression construct in conjunction with a SOX2-BFP overexpression construct, confirmed by qPCR and immunoblot. Analysis of histone H3 marks, K27M, and OPC signature genes will be compared to OPC genes from Dlx1/Dlx2 DKO mouse ganglionic eminences. CONCLUSIONS Manipulation of DLX2 in pHGG provides a model system for assessing the contribution of a homeobox factor to cell fate decisions regulating differentiation and tumorigenicity.

Ido2 Deficiency Exacerbates Motor Impairment and Reduces Aryl Hydrocarbon Receptor Activity through Decreased Kynurenine in a Chronic Demyelinating Mouse Model.

Demyelinating diseases including multiple sclerosis (MS) are chronic inflammatory diseases of the central nervous system. Indoleamine 2,3-dioxygenase 2 (Ido2) is a recently identified as catalytic enzyme involved in the rate-limiting step of the tryptophan-kynurenine pathway that influences susceptibility to inflammatory diseases. However, the pathological role of Ido2 in demyelination remains unclear. In this study, we investigated whether Ido2 deficiency influences the pathogenesis of proteolipid protein transgenic (Plp tg) mice, an animal model of chronic demyelination. Ido2 deficiency exacerbates impairments of motor function in the locomotor activity test, wire hanging test, and rotarod test. Ido2 deficiency caused severe demyelination associated with CD68-positive microglial activation in Plp tg mice. In the cerebellum of Plp tg mice, Ido2 deficiency significantly increased the expression of Tnfα. Ido2 deficiency reduced tryptophan metabolite kynurenine (KYN) levels and subsequent aryl hydrocarbon receptor (AhR) activity, which play an important role in anti-inflammatory response. These results suggest that Ido2 has an important role in preventing demyelination through AhR. Taken together, Ido2 could be a potential therapeutic target for demyelinating diseases.

Comprehensive Glycomic and Proteomic Analysis of Mouse Striatum and Lateral Hypothalamus Following Repeated Exposures to Cocaine or Methamphetamine

Substance use disorder is a major concern, with few therapeutic options. Heparan sulfate (HS) and chondroitin sulfate (CS) interact with a plethora of growth factors and their receptors and have profound effects on cellular signaling. Thus, targeting these dynamic interactions might represent a potential novel therapeutic modality. In the present study, we performed mass spectrometry–based glycomic and proteomic analysis to understand the effects of cocaine and methamphetamine (METH) on HS, CS, and the proteome of two brain regions critically involved in drug addiction: the lateral hypothalamus and the striatum. We observed that cocaine and METH significantly alter HS and CS abundances as well as sulfate contents and composition. In particular, repeated METH or cocaine treatments reduced CS 4-O-sulfation and increased CS 6-O-sulfation. Since C4S and C6S exercise differential effects on axon growth, regeneration, and plasticity, these changes likely contribute to drug-induced neural plasticity in these brain regions. Notably, we observed that restoring these alterations by increasing CS 4-0 levels in the lateral hypothalamus by adeno-associated virus delivery of an shRNA to arylsulfatase B (N-acetylgalactosamine-4-sulfatase) ameliorated anxiety and prevented the expression of preference for cocaine in a novelty induced conditioned place preference test during cocaine withdrawal. Finally, proteomics analyses revealed a number of aberrant proteins in METH- and cocaine-treated versus saline-treated mice, including myelin proteolipid protein, calcium/calmodulin-dependent protein kinase type II subunit alpha, synapsin-2, tenascin-R, calnexin, annexin A7, hepatoma-derived growth factor, neurocan, and CSPG5, and oxidative phosphorylation among the top perturbed pathway. Taken together, these data support the role of HS, CS, and associated proteins in stimulants abuse and suggest that manipulation of HSPGs can represent a novel therapeutic strategy.

ACKR3 Antagonism Enhances the Repair of Demyelinated Lesions Through Both Immunomodulatory and Remyelinating Effects

Addressing inflammation, demyelination, and associated neurodegeneration in inflammatory demyelinating diseases like multiple sclerosis (MS) remains challenging. ACT-1004-1239, a first-in-class and potent ACKR3 antagonist, currently undergoing clinical development, showed promise in preclinical MS models, reducing neuroinflammation and demyelination. However, its effectiveness in treating established disease and impact on remyelination after the occurrence of demyelinated lesions remain unexplored. This study assessed the therapeutic effect of ACT-1004-1239 in two demyelinating disease models. In the proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) model, ACT-1004-1239 administered upon the detection of the first signs of paralysis, resulted in a dose-dependent reduction in EAE disease severity, concomitant with diminished immune cell infiltrates in the CNS and reduced demyelination. Notably, efficacy correlated with elevated plasma concentrations of CXCL11 and CXCL12, two pharmacodynamic biomarkers of ACKR3 antagonism. Combining ACT-1004-1239 with siponimod, an approved immunomodulatory treatment for MS, synergistically reduced EAE severity. In the cuprizone-induced demyelination model, ACT-1004-1239 administered after 5 weeks of cuprizone exposure, significantly accelerated remyelination, already quantifiable one week after cuprizone withdrawal. Additionally, ACT-1004-1239 penetrated the CNS, elevating brain CXCL12 concentrations. These results demonstrate that ACKR3 antagonism significantly reduces the severity of experimental demyelinating diseases, even when treatment is initiated therapeutically, after the occurrence of lesions. It confirms the dual mode of action of ACT-1004-1239, exhibiting both immunomodulatory effects by reducing neuroinflammation and promyelinating effects by accelerating myelin repair. The results further strengthen the rationale for evaluating ACT-1004-1239 in clinical trials for patients with demyelinating diseases.

Whole exome sequencing identified a homozygous novel variant in DOP1A gene in the Pakistan family with neurodevelopmental disabilities: case report and literature review.

Hereditary neurodevelopmental disorders (NDDs) are prevalent in poorly prognostic pediatric diseases, but the pathogenesis of NDDs is still unclear. Irregular myelination could be one of the possible causes of NDDs. Here, whole exome sequencing was carried out for a consanguineous Pakistani family with NDDs to identify disease-associated variants. The co-segregation of candidate variants in the family was validated using Sanger sequencing. The potential impact of the gene on NDDs has been supported by conservation analysis, protein prediction, and expression analysis. A novel homozygous variant DOP1A(NM_001385863.1):c.2561A>G was identified. It was concluded that the missense variant might affect the protein-protein binding sites of the critical MEC interaction region of DOP1A, and DOP1A-MON2 may cause stability deficits in Golgi-endosome protein traffic. Proteolipid protein (PLP) and myelin-associate glycoprotein (MAG) could be targets of the DOP1A-MON2 Golgi-endosome traffic complex, especially during the fetal stage and the early developmental stages. This further supports the perspective that disorganized myelinogenesis due to congenital DOP1A deficiency might cause neurodevelopmental disorders (NDDs). Our case study revealed the potential pathway of myelinogenesis-relevant NDDs and identified DOP1A as a potential NDDs-relevant gene in humans.

Buyang huanwu decoction promotes remyelination via miR-760-3p/GPR17 axis after intracerebral hemorrhage

Ethnopharmacological relevanceThe repairment of myelin sheaths is crucial for mitigating neurological impairments of intracerebral hemorrhage (ICH). However, the current research on remyelination processes in ICH remains limited. A representative traditional Chinese medicine, Buyang Huanwu decoction (BYHWD), shows a promising therapeutic strategy for ICH treatment. Aim of the studyTo investigate the pro-remyelination effects of BYHWD on ICH and explore the underlying mechanisms. Materials and methodsThe collagenase-induced mice ICH model was created for investigation. BYHWD's protective effects were assessed by behavioral tests and histological staining. Transmission electron microscopy was used for displaying the structure of myelin sheaths. The remyelination and oligodendrocyte differentiation were evaluated by the expressions of myelin proteolipid protein (PLP), myelin basic protein (MBP), MBP/TAU, Olig2/CC1, and PDGFRα/proliferating cell nuclear antigen (PCNA) through RT-qPCR and immunofluorescence. Transcriptomics integrated with disease database analysis and experiments in vivo and in vitro revealed the microRNA-related underlying mechanisms. ResultsHere, we reported that BYHWD promoted the neurological function of ICH mice and improved remyelination by increasing PLP, MBP, and TAU, as well as restoring myelin structure. Besides, we showed that BYHWD promoted remyelination by boosting the differentiation of PDGFRα+ oligodendrocyte precursor cells into olig2+/CC1+ oligodendrocytes. Additionally, we demonstrated that the remyelination effects of BYHWD worked by inhibiting G protein-coupled receptor 17 (GPR17). miRNA sequencing integrated with miRNA database prediction screened potential miRNAs targeting GPR17. By applying immunofluorescence, RNA in situ hybridization and dual luciferase reporter gene assay, we confirmed that BYHWD suppressed GPR17 and improved remyelination by increasing miR-760-3p. ConclusionsBYHWD improves remyelination and neurological function in ICH mice by targeting miR-760-3p to inhibit GPR17. This study may shed light on the orchestration of remyelination mechanisms after ICH, thus providing novel insights for developing innovative prescriptions with brain-protective properties.

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.

Palmitoylation of proteolipid protein M6 promotes tricellular junction assembly in epithelia of Drosophila.

Tricellular junctions (TCJs) seal epithelial cell vertices and are essential for tissue integrity and physiology, but how TCJs are assembled and maintained is poorly understood. In Drosophila, the transmembrane proteins Anakonda (Aka, also known as Bark), Gliotactin (Gli) and M6 organize occluding TCJs. Aka and M6 localize in an interdependent manner to vertices and act jointly to localize Gli, but how these proteins interact to assemble TCJs was not previously known. Here, we show that the proteolipid protein M6 physically interacts with Aka and with itself, and that M6 is palmitoylated on conserved juxta-membrane cysteine residues. This modification promotes vertex localization of M6 and binding to Aka, but not to itself, and becomes essential when TCJ protein levels are reduced. Abolishing M6 palmitoylation leads to delayed localization of M6 and Aka but does not affect the rate of TCJ growth or mobility of M6 or Aka. Our findings suggest that palmitoylation-dependent recruitment of Aka by M6 promotes initiation of TCJ assembly, whereas subsequent TCJ growth relies on different mechanisms that are independent of M6 palmitoylation.

Muscle-building supplement β-hydroxy β-methylbutyrate stimulates the maturation of oligodendroglial progenitor cells to oligodendrocytes.

Oligodendrocytes are the myelinating cells in the CNS and multiple sclerosis (MS) is a demyelinating disorder that is characterized by progressive loss of myelin. Although oligodendroglial progenitor cells (OPCs) should be differentiated into oligodendrocytes, for multiple reasons, OPCs fail to differentiate into oligodendrocytes in MS. Therefore, increasing the maturation of OPCs to oligodendrocytes may be of therapeutic benefit for MS. The β-hydroxy β-methylbutyrate (HMB) is a muscle-building supplement in humans and this study underlines the importance of HMB in stimulating the maturation of OPCs to oligodendrocytes. HMB treatment upregulated the expression of different maturation markers including PLP, MBP, and MOG in cultured OPCs. Double-label immunofluorescence followed by immunoblot analyses confirmed the upregulation of OPC maturation by HMB. While investigating mechanisms, we found that HMB increased the maturation of OPCs isolated from peroxisome proliferator-activated receptor β-/- (PPARβ-/-) mice, but not PPARα-/- mice. Similarly, GW6471 (an antagonist of PPARα), but not GSK0660 (an antagonist of PPARβ), inhibited HMB-induced maturation of OPCs. GW9662, a specific inhibitor of PPARγ, also could not inhibit HMB-mediated stimulation of OPC maturation. Furthermore, PPARα agonist GW7647, but neither PPARβ agonist GW0742 nor PPARγ agonist GW1929, alone increased the maturation of OPCs. Finally, HMB treatment of OPCs led to the recruitment of PPARα, but neither PPARβ nor PPARγ, to the PLP gene promoter. These results suggest that HMB stimulates the maturation of OPCs via PPARα and that HMB may have therapeutic prospects in remyelination.

Abstract WP320: Microemboli Model of Vascular Cognitive Impairment/Dementia (VCID) Presents With Long-Term Brain Tissue Hypoxia: Relevance to Endothelin-1 (ET-1)

Diabetes doubles the risk of VCID but the underlying reasons remain unclear. Given that diabetes mediates early endothelial dysfunction and activates the ET system, post-mortem brain ET-1 levels correlate with tissue hypoxia and disease severity in patients with dementia, and microemboli (ME) are more common in diabetic individuals, the goals of the current study were to investigate 1) the relationship between the brain ET system, hypoxia and neuroinflammation in a clinically relevant model of VCID, and 2) the impact of improving endothelial function on the ET system. At 10 weeks after the onset of diabetes, control and diabetic rats received cholesterol crystal ME (40-70 μm) injections and were monitored for 16 weeks. Another cohort received isosorbide mononitrate (ISMN, 75 mg/kg/day) and cilostazol (CZL, 60 mg/kg/day). Behavioral tests included novel object recognition (NOR) and Y-maze. After termination, brain tissue levels of ET-1/ETA/ETB, hypoxia markers myelin-associated glycoprotein (MAG), proteolipid protein-1 (PLP-1) and hypoxia-inducible factor-1α (HIF-1α), and microglia activation were analyzed by ELISA and immunoblotting. Diabetic animals had baseline deficits in certain cognitive domains that progressively worsened and treatment with (ISMN/CZL) prevented decline in cognitive function. HIF-1α levels were higher and MAG levels were lower in the ME cohorts indicating tissue hypoxia. MAG/PLP-1 ratio was even lower in diabetic ME animals (Table 1). ISMN/CZL treatment reduced HIF1α levels in the brain. While there was no dramatic change in plasma and brain tissue ET-1 levels, brain ET-1 levels correlated with HIF-1α (r 2 =0.53, p=0.0073) and MAG/PLP (r 2 =0.45, p=0.0167) in this model of VCID. The causal relationship between ET-1 and brain hypoxia, as well as neuroinflammation, needs further investigation. Strategies to improve vascular function by modulation of the ET system blockade can be a preventive and therapeutic strategy for VCID.