Oligodendrocyte Precursor Research Articles

Integrative Analyses of scRNA-seq, Bulk mRNA-seq, and DNA Methylation Profiling in Depressed Suicide Brain Tissues.

Suicidal behaviors have become a serious public health concern globally due to the economic and human cost of suicidal behavior to individuals, families, communities, and society. However, the underlying etiology and biological mechanism of suicidal behavior remains poorly understood. We collected different single omic data, including single-cell RNA sequencing (scRNA-seq), bulk mRNA-seq, DNA methylation microarrays from the cortex of Major Depressive Disorder (MDD) in suicide subjects' studies, as well as fluoxetine-treated rats brains. We matched subject IDs that overlapped between the transcriptome dataset and the methylation dataset. The differential expression genes and differentially methylated regions were calculated with a 2-group comparison analysis. Cross-omics analysis was performed to calculate the correlation between the methylated and transcript levels of differentially methylated CpG sites and mapped transcripts. Additionally, we performed a deconvolution analysis for bulk mRNA-seq and DNA methylation profiling with scRNA-seq as the reference profiles. Difference in cell type proportions among 7 cell types. Meanwhile, our analysis of single-cell sequence from the antidepressant-treated rats found that drug-specific differential expression genes were enriched into biological pathways, including ion channels and glutamatergic receptors. This study identified some important dysregulated genes influenced by DNA methylation in 2 brain regions of depression and suicide patients. Interestingly, we found that oligodendrocyte precursor cells (OPCs) have the most contributors for cell-type proportions related to differential expression genes and methylated sites in suicidal behavior.

Carnosine synthase deficiency aggravates neuroinflammation in multiple sclerosis

Multiple sclerosis (MS) pathology features autoimmune-driven neuroinflammation, demyelination, and failed remyelination. Carnosine is a histidine-containing dipeptide (HCD) with pluripotent homeostatic properties that is able to improve outcomes in an animal MS model (EAE) when supplied exogenously. To uncover if endogenous carnosine is involved in, and protects against, MS-related neuroinflammation, demyelination or remyelination failure, we here studied the HCD-synthesizing enzyme carnosine synthase (CARNS1) in human MS lesions and two preclinical mouse MS models (EAE, cuprizone). We demonstrate that due to its presence in oligodendrocytes, CARNS1 expression is diminished in demyelinated MS lesions and mouse models mimicking demyelination/inflammation, but returns upon remyelination. Carns1-KO mice that are devoid of endogenous HCDs display exaggerated neuroinflammation and clinical symptoms during EAE, which could be partially rescued by exogenous carnosine treatment. Worsening of the disease appears to be driven by a central, not peripheral immune-modulatory, mechanism possibly linked to impaired clearance of the reactive carbonyl acrolein in Carns1-KO mice. In contrast, CARNS1 is not required for normal oligodendrocyte precursor cell differentiation and (re)myelin to occur, and neither endogenous nor exogenous HCDs protect against cuprizone-induced demyelination. In conclusion, the loss of CARNS1 from demyelinated MS lesions can aggravate disease progression through weakening the endogenous protection against neuroinflammation.

Aerobic exercise attenuates abnormal myelination and oligodendrocyte differentiation in 3xTg-AD mice

Pathological features of Alzheimer's Disease (AD) include alterations in the structure and function of neurons as well as of myelin sheaths. Accumulated evidence shows that aerobic type of exercise can enhance neuroplasticity in mouse models of AD. However, whether and how aerobic exercise can affect myelin sheath repair and neuroprotection in the AD models remains unclear. In this study we tested the hypotheses that 1) myelin structural alterations in 3xTg-AD mice would be related to abnormalities in oligodendrocyte lineage cells, resulting in impaired learning and memory, and 2) a 6-month aerobic exercise intervention would have beneficial effects on such alterations. Two-month-old male 3xTg-AD mice were randomly assigned to a control (AC) or an exercise (AE) group, and age-matched male C57BL/6;129 mice were also randomly assigned to a normal control (NC) or an exercise (NE) group, with n = 12 in each group. Mice in the exercise groups were trained on a motor-drive treadmill, 60 min per day, 5 days per week for 6 months. Cognitive function was assessed at the end of the intervention period. Then, brain specimens were obtained for assessments of morphological and oligodendrocyte lineage cell changes. The results of electron microscopy showed that myelin ultrastructure demonstrated a higher percentage of loose and granulated myelin sheath around axons in the temporal lobe in the AC, as compared with the NC group, along with greater cognitive dysfunction at 8-months of age. These differences were accompanied by significantly greater myelin basic protein (MBP) expression and less neuron-glial antigen-2 (NG2) protein and mRNA levels in the AC, compared to the NC. However, there were no significant between-group differences in the G-ratio (the ratio of axon diameter to axon plus myelin sheath diameter) and 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase) protein and mRNA levels. The aerobic exercise ameliorated cognitive deterioration and appeared to keep components of myelin sheath and oligodendrocyte precursor cells stabilized, resulting in a decrease in the percentage of loose and granulated myelin sheath and MBP protein, and an increase in NG2 protein and mRNA levels in the AE group. Therefore, the 6-month exercise intervention demonstrated beneficial effects on myelin lesions, abnormal differentiation of oligodendrocytes and general brain function in the 3xTg-AD mice, providing further insights into the role of aerobic exercise in management of neurodegeneration in AD by maintaining intact myelination.

Breastfeeding promotes oligodendrocyte precursor cells division and myelination in the demyelinated corpus callosum

Demyelination alters the conduction of neuronal signals and hampers sensory-motor functions. Experimental and clinical evidence suggest that breastfeeding exerts a promyelinating impact on the maternal brain. The mechanism underlying this neuroprotective effect is not well-understood. In the present paper, we assessed the impact of rat lactation on lysolecithin-induced demyelination injury within the corpus callosum of lactating and non-lactating postpartum rats. We show that lactation enhanced the cell density of oligodendrocyte precursor cells (OPCs), but not that of activated microglia and astrocytes, within the demyelination lesion. Lactation also increased the expression of myelin markers involved in the initial stage of myelin recovery (Myelin-associated glycoprotein and 2′,3′-cyclic nucleotide 3′-phosphodiesterase) and reduced the demyelination injury. Altogether, these data suggest that lactation creates a conducive promyelinating environment through increased OPCs cell division, enhanced expression of select myelin proteins, and reduced number of non-myelinated axons.

Novel method of isolating nuclei of human oligodendrocyte precursor cells reveals substantial developmental changes in gene expression and H3K27ac histone modification.

Oligodendrocyte precursor cells (OPCs) generate differentiated mature oligodendrocytes (MOs) during development. In adult brain, OPCs replenish MOs in adaptive plasticity, neurodegenerative disorders, and after trauma. The ability of OPCs to differentiate to MOs decreases with age and is compromised in disease. Here we explored the cell specific and age-dependent differences in gene expression and H3K27ac histone mark in these two cell types. H3K27ac is indicative of active promoters and enhancers. We developed a novel flow-cytometry-based approach to isolate OPC and MO nuclei from human postmortem brain and profiled gene expression and H3K27ac in adult and infant OPCs and MOs genome-wide. In adult brain, we detected extensive H3K27ac differences between the two cell types with high concordance between gene expression and epigenetic changes. Notably, the expression of genes that distinguish MOs from OPCs appears to be under a strong regulatory control by the H3K27ac modification in MOs but not in OPCs. Comparison of gene expression and H3K27ac between infants and adults uncovered numerous developmental changes in each cell type, which were linked to several biological processes, including cell proliferation and glutamate signaling. A striking example was a subset of histone genes that were highly active in infant samples but fully lost activity in adult brain. Our findings demonstrate a considerable rearrangement of the H3K27ac landscape that occurs during the differentiation of OPCs to MOs and during postnatal development of these cell types, which aligned with changes in gene expression. The uncovered regulatory changes justify further in-depth epigenetic studies of OPCs and MOs in development and disease.

Single-cell brain organoid screening identifies developmental defects in autism

The development of the human brain involves unique processes (not observed in many other species) that can contribute to neurodevelopmental disorders1–4. Cerebral organoids enable the study of neurodevelopmental disorders in a human context. We have developed the CRISPR–human organoids–single-cell RNA sequencing (CHOOSE) system, which uses verified pairs of guide RNAs, inducible CRISPR–Cas9-based genetic disruption and single-cell transcriptomics for pooled loss-of-function screening in mosaic organoids. Here we show that perturbation of 36 high-risk autism spectrum disorder genes related to transcriptional regulation uncovers their effects on cell fate determination. We find that dorsal intermediate progenitors, ventral progenitors and upper-layer excitatory neurons are among the most vulnerable cell types. We construct a developmental gene regulatory network of cerebral organoids from single-cell transcriptomes and chromatin modalities and identify autism spectrum disorder-associated and perturbation-enriched regulatory modules. Perturbing members of the BRG1/BRM-associated factor (BAF) chromatin remodelling complex leads to enrichment of ventral telencephalon progenitors. Specifically, mutating the BAF subunit ARID1B affects the fate transition of progenitors to oligodendrocyte and interneuron precursor cells, a phenotype that we confirmed in patient-specific induced pluripotent stem cell-derived organoids. Our study paves the way for high-throughput phenotypic characterization of disease susceptibility genes in organoid models with cell state, molecular pathway and gene regulatory network readouts.

Norepinephrine modulates calcium dynamics in cortical oligodendrocyte precursor cells promoting proliferation during arousal in mice.

Oligodendrocytes, the myelinating cells of the central nervous system (CNS), are generated from oligodendrocyte precursor cells (OPCs) that express neurotransmitter receptors. However, the mechanisms that affect OPC activity in vivo and the physiological roles of neurotransmitter signaling in OPCs are unclear. In this study, we generated a transgenic mouse line that expresses membrane-anchored GCaMP6s in OPCs and used longitudinal two-photon microscopy to monitor OPC calcium (Ca2+) dynamics in the cerebral cortex. OPCs exhibit focal and transient Ca2+ increases within their processes that are enhanced during locomotion-induced increases in arousal. The Ca2+ transients occur independently of excitatory neuron activity, rapidly decline when OPCs differentiate and are inhibited by anesthesia, sedative agents or noradrenergic receptor antagonists. Conditional knockout of α1A adrenergic receptors in OPCs suppresses spontaneous and locomotion-induced Ca2+ increases and reduces OPC proliferation. Our results demonstrate that OPCs are directly modulated by norepinephrine in vivo to enhance Ca2+ dynamics and promote population homeostasis.

Transgenic expression of the HERV-W envelope protein leads to polarized glial cell populations and a neurodegenerative environment

The human endogenous retrovirus type W (HERV-W) has been identified and repeatedly confirmed as human-specific pathogenic entity affecting many cell types in multiple sclerosis (MS). Our recent contributions revealed the encoded envelope (ENV) protein to disturb myelin repair by interfering with oligodendroglial precursor differentiation and by polarizing microglial cells toward an axon-damage phenotype. Indirect proof of ENV's antiregenerative and degenerative activities has been gathered recently in clinical trials using a neutralizing anti-ENV therapeutic antibody. Yet direct proof of its mode of action can only be presented here based on transgenic ENV expression in mice. Upon demyelination, we observed myelin repair deficits, neurotoxic microglia and astroglia, and increased axon degeneration. Experimental autoimmune encephalomyelitis activity progressed faster in mutant mice equally accompanied by activated glial cells. This study therefore provides direct evidence on HERV-W ENV's contribution to the overall negative impact of this activated viral entity in MS.

Conditional Deletion of Foxg1 Delayed Myelination during Early Postnatal Brain Development.

FOXG1 (forkhead box G1) syndrome is a neurodevelopmental disorder caused by variants in the Foxg1 gene that affect brain structure and function. Individuals affected by FOXG1 syndrome frequently exhibit delayed myelination in neuroimaging studies, which may impair the rapid conduction of nerve impulses. To date, the specific effects of FOXG1 on oligodendrocyte lineage progression and myelination during early postnatal development remain unclear. Here, we investigated the effects of Foxg1 deficiency on myelin development in the mouse brain by conditional deletion of Foxg1 in neural progenitors using NestinCreER;Foxg1fl/fl mice and tamoxifen induction at postnatal day 0 (P0). We found that Foxg1 deficiency resulted in a transient delay in myelination, evidenced by decreased myelin formation within the first two weeks after birth, but ultimately recovered to the control levels by P30. We also found that Foxg1 deletion prevented the timely attenuation of platelet-derived growth factor receptor alpha (PDGFRα) signaling and reduced the cell cycle exit of oligodendrocyte precursor cells (OPCs), leading to their excessive proliferation and delayed maturation. Additionally, Foxg1 deletion increased the expression of Hes5, a myelin formation inhibitor, as well as Olig2 and Sox10, two promoters of OPC differentiation. Our results reveal the important role of Foxg1 in myelin development and provide new clues for further exploring the pathological mechanisms of FOXG1 syndrome.

P03.13.B RADIOTHERAPY-INDUCED COGNITIVE IMPAIRMENT IN CANCER PATIENTS: IS SENESCENCE INVOLVED?

Abstract BACKGROUND Patients with primary and metastatic brain tumors that survive may experience cognitive problems including progressive impairments in memory, attention, and executive function attributed to radiation-induced brain injury. Irradiation is known to bring tumor cells into senescence, a state of permanent proliferation arrest that is accompanied by metabolic changes and a secretory phenotype, which may cause inflammation. We hypothesize that senescence in the healthy brain, caused by whole brain radiotherapy (WBRT), plays an important role in cognitive problems. MATERIAL AND METHODS In this pilot experiment the effect of three different dosages (12, 30 and 60 Gy) of fractionated WBRT was assessed at different intervals after irradiation using male C57BL/6JRj mice. We performed senescence associated β-glucosidase (SA-β-gal) staining, cytokine ELISA assays, RT-qPCRs and immunohistochemistry to identify cellular senescence in the healthy brain. RESULTS Unexpectedly, no clear indications of induced cellular senescence in the brain could be observed by assessing SA-β-gal, cytokines and senescence markers on RNA and protein level. However, we found persistent DNA damage foci, microglial activation, a reduction in oligodendrocyte precursor cells and neurogenesis in the subvertricular zone. CONCLUSION No clear evidence for cellular senescence after WBRT was detected in the mouse brain, whereas profound effects were found on glia, neurogenesis and DNA damage. Difficulties in detecting cellular senescence might be explained by differences in presence of so-called markers for cellular senescence in the brain, or the rare nature of senescence in vivo. In future studies neurobiological changes will be assessed at later time points after WBRT, using a senescence reporter mouse and will be combined with behavioral studies.

P19.02.A FUNCTIONAL CONSEQUENCES OF IDH1 AND CIC MUTATIONS ON OLIGODENDROGLIOMA CELLS OF ORIGIN

Abstract BACKGROUND Oligodendrogliomas are primitive tumors of the central nervous system characterized by mutations in isocitrate dehydrogenase 1 (IDH1R132H) gene, codeletion of 1p and 19q chromosomic arms and mutations in Capicua (CIC) gene. Oligodendrocyte precursor cells (OPCs) are potential cells of origin for oligodendrogliomas and OPC-like tumor cells are responsible for tumor amplification at early stages of gliomagenesis. Although genetic alterations are well characterized for these tumors, little is known about the roles of IDH1 and CIC in oligodendroglioma initiation and development. Individually, IDH1 and CIC mutations can induce neoplastic characteristics but are not sufficient for tumoral development. We aim to understand how IDH1 and CIC mutations impact OPC development and whether these alterations are sufficient for tumoral initiation. METHODS We have generated an inducible mouse model allowing endogenous expression of IDH1R132H and inactivation of CIC in postnatal OPCs. We analyzed by immunofluorescence the proliferation and differentiation status of mutated oligodendroglial cells and other brain cells, at postnatal and adult stages. RESULTS We describe distinct effects of these mutations on oligodendroglial cell number and proliferation, at early and late time points. In addition, we found that IDH1R132H increases the proliferation of other brain cell types, suggesting paracrine effects of this mutation in the brain. We find no evidence of tumor in the brain of mice mutated for both IDH1 and CIC at late time points. CONCLUSION Our study describes for the first time the phenotypic impact of the combined expression of IDH1 and CIC mutations on oligodendroglial lineage cells and other brain cells, leading to a better understanding of oligodendroglioma development.

Abstract 30 Cord Blood Derived Macrophages Promote Remyelination

Abstract Introduction Autoimmune diseases of the central nervous system, like multiple sclerosis (MS), result from autoreactive immune cells attacking myelin. Although numerous therapies targeting the immune system are effective for slowing the progression of MS, there are no therapies to promote remyelination. Therefore, we developed DUOC-01, a first-generation macrophage cell-therapy product manufactured from banked cord blood to promote remyelination. Using mouse models of demyelination, we determined that DUOC-01 blocked progression of disease and enhanced remyelination. Objectives Although preclinical data using DUOC-01 are promising, shortfalls in manufacturing and an unknown mechanism of action limits their use for MS. Here, we examined the effects of DUOC-01 on oligodendrocyte precursor cells (OPCs) to develop a second generation product that promotes the maturation of OPCs. Methods We first developed an ex vivo system, using organotypic brain slices treated with lysophosphatidylcholine (LPC), to measure remyelination by imaging the co-localization of myelin basic protein (MBP), a marker for myelin, and neurofilament, a marker for neuronal axons. Next we developed a primary OPC differentiation assay and measured maturation through markers of proliferation and differentiation. Using these assays, we set out to determine the ability of DUOC-01 and a second generation product to promote remyelination. Results In culture, our brain slices maintained well-preserved cytoarchitecture and myelinated axons, indicating a health three-dimensional system. Treating the slices with LPC caused massive demyelination of neuron processes three days post exposure. After washing out LPC, we treated cultures with vehicle or DUOC-01 and determined that DUOC-01 promoted remyelination when compared to controls. Using primary OPC cultures, we determined that DUOC-01 skewed OPC cultures to a more mature phenotype, demonstrated by the increased expression of the mature oligodendrocyte marker MBP. When treating OPC cultures with DUOC-01 conditioned media or separating DUOC-01 from OPCs using a transwell, OPC cultures still upregulated expression of MBP, indicating that bioactivity was due to factors released by DUOC-01. Discussion Overall, our data demonstrated that DUOC-01 promoted remyelination, and the factors released by DUOC-01 drive the maturation of oligodendrocytes. Thus, these remyelination factors released by DUOC-01 have the potential to work as a cell-free therapeutic for treating MS and other diverse neurological demyelinating diseases.

The role and potential therapeutic targets of astrocytes in central nervous system demyelinating diseases.

Astrocytes play vital roles in the central nervous system, contributing significantly to both its normal functioning and pathological conditions. While their involvement in various diseases is increasingly recognized, their exact role in demyelinating lesions remains uncertain. Astrocytes have the potential to influence demyelination positively or negatively. They can produce and release inflammatory molecules that modulate the activation and movement of other immune cells. Moreover, they can aid in the clearance of myelin debris through phagocytosis and facilitate the recruitment and differentiation of oligodendrocyte precursor cells, thereby promoting axonal remyelination. However, excessive or prolonged astrocyte phagocytosis can exacerbate demyelination and lead to neurological impairments. This review provides an overview of the involvement of astrocytes in various demyelinating diseases, emphasizing the underlying mechanisms that contribute to demyelination. Additionally, we discuss the interactions between oligodendrocytes, oligodendrocyte precursor cells and astrocytes as therapeutic options to support myelin regeneration. Furthermore, we explore the role of astrocytes in repairing synaptic dysfunction, which is also a crucial pathological process in these disorders.

Oligodendrocyte precursor cells stop sensory axons regenerating into the spinal cord

Primary somatosensory axons stop regenerating as they re-enter the spinal cord, resulting in incurable sensory loss. What arrests them has remained unclear. We previously showed that axons stop by forming synaptic contacts with unknown non-neuronal cells. Here, we identified these cells in adult mice as oligodendrocyte precursor cells (OPCs). We also found that only a few axons stop regenerating by forming dystrophic endings, exclusively at the CNS:peripheral nervous system (PNS) borderline where OPCs are absent. Most axons stop in contact with a dense network of OPC processes. Live imaging, immuno-electron microscopy (immuno-EM), and OPC-dorsal root ganglia (DRG) co-culture additionally suggest that axons are rapidly immobilized by forming synapses with OPCs. Genetic OPC ablation enables many axons to continue regenerating deep into the spinal cord. We propose that sensory axons stop regenerating by encountering OPCs that induce presynaptic differentiation. Our findings identify OPCs as a major regenerative barrier that prevents intraspinal restoration of sensory circuits following spinal root injury.

Myelination-independent functions of oligodendrocyte precursor cells in health and disease.

Oligodendrocyte precursor cells (OPCs) are a population of tissue-resident glial cells found throughout the CNS, constituting approximately 5% of all CNS cells and persisting from development to adulthood and aging. The canonical role of OPCs is to give rise to myelinating oligodendrocytes. However, additional functions of OPCs beyond this traditional role as precursors have been suggested for a long time. In this Perspective, we provide an overview of the multiple myelination-independent functions that have been described for OPCs in the context of neuron development, angiogenesis, inflammatory response, axon regeneration and their recently discovered roles in neural circuit remodeling.

BMAL1 loss in oligodendroglia contributes to abnormal myelination and sleep

Myelination depends on the maintenance of oligodendrocytes that arise from oligodendrocyte precursor cells (OPCs). We show that OPC-specific proliferation, morphology, and BMAL1 are time-of-day dependent. Knockout of Bmal1 in mouse OPCs during development disrupts the expression of genes associated with circadian rhythms, proliferation, density, morphology, and migration, leading to changes in OPC dynamics in a spatiotemporal manner. Furthermore, these deficits translate into thinner myelin, dysregulated cognitive and motor functions, and sleep fragmentation. OPC-specific Bmal1 loss in adulthood does not alter OPC density at baseline but impairs the remyelination of a demyelinated lesion driven by changes in OPC morphology and migration. Lastly, we show that sleep fragmentation is associated with increased prevalence of the demyelinating disorder multiple sclerosis (MS), suggesting a link between MS and sleep that requires further investigation. These findings have broad mechanistic and therapeutic implications for brain disorders that include both myelin and sleep phenotypes.

Characterization of tumor microenvironment in glioblastoma multiforme identifies ITGB2 as a key immune and stromal related regulator in glial cell types

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor characterized by inter and intra-tumor heterogeneity and complex tumor microenvironment. To uncover the molecular targets in this milieu, we systematically identified immune and stromal interactions at the glial cell type level that leverages on RNA-sequencing data of GBM patients from The Cancer Genome Atlas. The perturbed genes between the high vs low immune and stromal scored patients were subjected to weighted gene co-expression network analysis to identify the glial cell type specific networks in immune and stromal infiltrated patients. The intramodular connectivity analysis identified the highly connected genes in each module. Combining it with univariable and multivariable prognostic analysis revealed common vital gene ITGB2, between the immune and stromal infiltrated patients enriched in microglia and newly formed oligodendrocytes. We found following unique hub genes in immune infiltrated patients; COL6A3 (microglia), ITGAM (oligodendrocyte precursor cells), TNFSF9 (microglia), and in stromal infiltrated patients, SERPINE1 (microglia) and THBS1 (newly formed oligodendrocytes, oligodendrocyte precursor cells). To validate these hub genes, we used external GBM patient single cell RNA-sequencing dataset and this identified ITGB2 to be significantly enriched in microglia, newly formed oligodendrocytes, T-cells, macrophages and adipocyte cell types in both immune and stromal datasets. The tumor infiltration analysis of ITGB2 showed that it is correlated with myeloid dendritic cells, macrophages, monocytes, neutrophils, B-cells, fibroblasts and adipocytes. Overall, the systematic screening of tumor microenvironment components at glial cell types uncovered ITGB2 as a potential target in primary GBM.

Necrostatin-1s Suppresses RIPK1-driven Necroptosis and Inflammation in Periventricular Leukomalacia Neonatal Mice.

Periventricular leukomalacia (PVL), a predominant form of brain injury in preterm survivors, is characterized by hypomyelination and microgliosis and is also the major cause of long-term neurobehavioral abnormalities in premature infants. Receptor-interacting protein kinase 1 (RIPK1) plays a pivotal role in mediating cell death and inflammatory signaling cascade. However, very little is known about the potential effect of RIPK1 in PVL and the underlying mechanism. Herein, we found that the expression level of RIPK1 was drastically increased in the brain of PVL neonatal mice models, and treatment of PVL neonatal mice with Necrostatin-1s (Nec-1s), an inhibitor of RIPK1, greatly ameliorated cerebral ischemic injury, exhibiting an increase of body weights, reduction of cerebral infarct size, neuronal loss, and occurrence of necrosis-like cells, and significantly improved the long-term abnormal neurobehaviors of PVL. In addition, Nec-1s significantly suppressed hypomyelination and promoted the differentiation of oligodendrocyte precursor cells (OPCs), as demonstrated by the increased expression levels of MBP and Olig2, the decreased expression level of GPR17, a significant increase in the number of CC-1-positive cells, and suppression of myelin ultrastructure impairment. Moreover, the mechanism of neuroprotective effects of Nec-1s against PVL is closely associated with its suppression of the RIPK1-mediated necrosis signaling molecules, RIPK1, PIPK3, and MLKL. More importantly, inhibition of RIPK1 could reduce microglial inflammatory injury by triggering the M1 to M2 microglial phenotype, appreciably decreasing the levels of M1 marker CD86 and increasing the levels of M2 markers Arg1 or CD206 in PVL mice. Taken together, inhibition of RIPK1 markedly ameliorates the brain injury and long-term neurobehavioral abnormalities of PVL mice through the reduction of neural cell necroptosis and reversing neuroinflammation.

Marginal Zinc Deficiency during Gestation and Lactation in Rats Affects Oligodendrogenesis, Motor Performance, and Behavior in the Offspring

BackgroundOligodendrocytes are responsible for myelin production in the central nervous system (CNS). Hypomyelination may slow saltatory nerve signal conduction and affect motor performance and behavior in adults. Gestational marginal zinc deficiency in rats significantly decreases proliferation of neural stem cells (NSCs) in the offspring brain. ObjectivesGiven that NSCs are precursors of oligodendrocytes, this study investigated if marginal zinc deficiency during early development in rats affects oligodendrogenesis in the offspring’s CNS. MethodsRat dams were fed an adequate (25 μg zinc/g diet) (C) or a marginal zinc diet (MZD) (10 μg zinc/g diet), from gestation day zero until postnatal day (P) 20, and subsequently all offspring was fed the control diet until P60. Oligodendrogenesis was evaluated in the offspring at P2, P5, P10, P20, and P60, by measuring parameters of oligodendrocyte progenitor cells (OPCs) proliferation, differentiation, maturation, and of myelination. ResultsThe expression of 1) proteins that regulate OPC proliferation (Shh, Sox10, Olig2); 2) OPC markers (NG2, PDGFRα); 3) myelin proteins (MBP, MAG, MOG, PLP) were lower in the brain cortex from MZD than C offspring at various stages in development. The amount of myelin after zinc replenishment continued to be low in the MZD young adult at P60. Accordingly, parameters of motor performance and behavior [grip strength, rotarod, elevated T-maze (ETM), and open-field tests] were impaired in the MZD offspring at P60. ConclusionsResults support the concept that maternal and early postnatal exposure to MZD affects oligodendrogenesis causing long-lasting effects on myelination and on motor performance in the young adult offspring.

Hypomyelination, hypodontia and craniofacial abnormalities in a Polr3b mouse model of leukodystrophy.

RNA polymerase III (Pol III)-related hypomyelinating leukodystrophy (POLR3-HLD), also known as 4H leukodystrophy, is a severe neurodegenerative disease characterized by the cardinal features of hypomyelination, hypodontia and hypogonadotropic hypogonadism. POLR3-HLD is caused by biallelic pathogenic variants in genes encoding Pol III subunits. While approximately half of all patients carry mutations in POLR3B encoding the RNA polymerase III subunit B, there is no in vivo model of leukodystrophy based on mutation of this Pol III subunit. Here, we determined the impact of POLR3BΔ10 (Δ10) on Pol III in human cells and developed and characterized an inducible/conditional mouse model of leukodystrophy using the orthologous Δ10 mutation in mice. The molecular mechanism of Pol III dysfunction was determined in human cells by affinity purification-mass spectrometry and western blot. Postnatal induction with tamoxifen induced expression of the orthologous Δ10 hypomorph in triple transgenic Pdgfrα-Cre/ERT; R26-Stopfl-EYFP; Polr3bfl mice. CNS and non-CNS features were characterized using a variety of techniques including microCT, ex vivo MRI, immunofluorescence, immunohistochemistry, spectral confocal reflectance microscopy and western blot. Lineage tracing and time series analysis of oligodendrocyte subpopulation dynamics based on co-labelling with lineage-specific and/or proliferation markers were performed. Proteomics suggested that Δ10 causes a Pol III assembly defect, while western blots demonstrated reduced POLR3BΔ10 expression in the cytoplasm and nucleus in human cells. In mice, postnatal Pdgfrα-dependent expression of the orthologous murine mutant protein resulted in recessive phenotypes including severe hypomyelination leading to ataxia, tremor, seizures and limited survival, as well as hypodontia and craniofacial abnormalities. Hypomyelination was confirmed and characterized using classic methods to quantify myelin components such as myelin basic protein and lipids, results which agreed with those produced using modern methods to quantify myelin based on the physical properties of myelin membranes. Lineage tracing uncovered the underlying mechanism for the hypomyelinating phenotype: defective oligodendrocyte precursor proliferation and differentiation resulted in a failure to produce an adequate number of mature oligodendrocytes during postnatal myelinogenesis. In summary, we characterized the Polr3bΔ10 mutation and developed an animal model that recapitulates features of POLR3-HLD caused by POLR3B mutations, shedding light on disease pathogenesis, and opening the door to the development of therapeutic interventions.