OLIG2 Research Articles

Myelination deficiency in rats with genetically determined dopamine metabolism disorder

BACKGROUND: Attention deficit hyperactivity disorder is one of the most common neuropsychiatric disorders affecting children. The exact causes of attention deficit hyperactivity disorder are still not fully understood. Myelination deficiency in nerve fibers could be one of the possible factors in the pathogenesis of attention deficit hyperactivity disorder. Dopamine transporter knockout (DAT-KO) rats provide a good translational model for attention deficit hyperactivity disorder because they mimic behavioral symptoms of the disease: hyperactivity, cognitive impairment and compulsive behavior. This paper aims to determine abnormalities in the myelination process in the spinal cord of DAT-KO rats. Our findings can lead to a better understanding of the etiology and pathogenesis of attention deficit hyperactivity disorder. AIM: To determine the mRNA expression levels of myelination-related genes in the spinal cord of dopamine transporter knockout (DAT-KO) rats during their natural development. MATERIALS AND METHODS: The study was performed on 72 rat pups (DAT-KO, DAT-HET, DAT-WT) from 12 litters. Rat pups were born from the pairs of intercrossed DAT-HET x DAT-HET adult rats. Pups were decapitated on 7th, 14th and 21st day of their postnatal development. After that, mRNA levels of the main myelination-related genes were measured in the cervical and lumbar segments of the spinal cord using real-time PCR. RESULTS: The mRNA levels of the mag, olig2 and plp1 genes were significantly different in the cervical and lumbar spinal cords of DAT-KO rats compared to DAT-WT animals. A significant increase in the level of mag gene mRNA in DAT-HET and DAT-KO animals was observed on 14th and 21st days of postnatal development. Also, in these animals, the olig2 mRNA levels were increased on the 21st day of postnatal development. The plp1 mRNA level was increased on the 14th day, and reduced on the 21st day of postnatal development in DAT-KO animals. CONCLUSIONS: Myelination deficiency in nerve fibers in the spinal cord of DAT-KO rats is one of the effects of gene knockout. It is also of the possible causes of behavioral changes — hyperactivity, cognitive impairment and compulsive behavior.

Correlation between ABCB1 and OLIG2 polymorphisms and the severity and prognosis of patients with cerebral infarction.

This study investigated the relationship between ATP-binding cassette sub-family B member 1 (ABCB1) and OLIG2 single nucleotide polymorphism (SNP) and neurological injury severity and outcome in cerebral infarction (CI). The neurological injury severity of 298 CI patients was evaluated by the National Institutes of Health Stroke Scale. The prognosis of CI patients at 30 days after admission was evaluated by the modified Rankin Scale. And 322 healthy people were selected as the control group. The SNPs of the ABCB1 gene (rs1045642) and OLIG2 gene (rs1059004 and rs9653711) were detected by TaqMan probe PCR, and the distribution of SNPs genotype was analyzed. SNP rs9653711 was correlated with CI. Recessive models of rs1045642 and rs9653711 were correlated with CI. The genotypes of rs1045642 and rs9653711 and genetic models were associated with CI severity. rs1045642 had no correlation with CI prognosis, while rs9653711 had less correlation. The genotype distribution and recessive model were associated with CI prognosis. SNP rs1059004 was not associated with CI severity and prognosis. Alcohol consumption, hypertension, diabetes, hyperlipidemia, and high levels of homocysteine (HCY) were independent risk factors for CI, while hypertension, hyperlipidemia, and HCY were associated with poor prognosis of CI. ABCB1 rs1045642 and OLOG2 rs9653711 are associated with CI severity.

Biological functions of the Olig gene family in brain cancer and therapeutic targeting.

The Olig genes encode members of the basic helix-loop-helix (bHLH) family of transcription factors. Olig1, Olig2, and Olig3 are expressed in both the developing and mature central nervous system (CNS) and regulate cellular specification and differentiation. Over the past decade extensive studies have established functional roles of Olig1 and Olig2 in development as well as in cancer. Olig2 overexpression drives glioma proliferation and resistance to radiation and chemotherapy. In this review, we summarize the biological functions of the Olig family in brain cancer and how targeting Olig family genes may have therapeutic benefit.

Impacts of dual active-ingredient bed nets on the behavioural responses of pyrethroid resistant Anopheles gambiae determined by room-scale infrared video tracking

BackgroundThe success of insecticide treated bed nets (ITNs) for malaria vector control in Africa relies on the behaviour of various species of Anopheles. Previous research has described mosquito behavioural alterations resulting from widespread ITN coverage, which could result in a decrease in net efficacy. Here, behaviours were compared including timings of net contact, willingness to refeed and longevity post-exposure to two next-generation nets, PermaNet® 3.0 (P3 net) and Interceptor® G2 (IG2 net) in comparison with a standard pyrethroid-only net (Olyset Net™ (OL net)) and an untreated net.MethodsSusceptible and resistant Anopheles gambiae mosquitoes were exposed to the nets with a human volunteer host in a room-scale assay. Mosquito movements were tracked for 2 h using an infrared video system, collecting flight trajectory, spatial position and net contact data. Post-assay, mosquitoes were monitored for a range of sublethal insecticide effects.ResultsMosquito net contact was focused predominantly on the roof for all four bed nets. A steep decay in activity was observed for both susceptible strains when P3 net and OL net were present and with IG2 net for one of the two susceptible strains. Total mosquito activity was higher around untreated nets than ITNs. There was no difference in total activity, the number, or duration, of net contact, between any mosquito strain, with similar behaviours recorded in susceptible and resistant strains at all ITNs. OL net, P3 net and IG2 net all killed over 90% of susceptible mosquitoes 24 h after exposure, but this effect was not seen with resistant mosquitoes where mortality ranged from 16 to 72%. All treated nets reduced the willingness of resistant strains to re-feed when offered blood 1-h post-exposure, with a more pronounced effect seen with P3 net and OL net than IG2 net.ConclusionThese are the first results to provide an in-depth description of the behaviour of susceptible and resistant Anopheles gambiae strains around next-generation bed nets using a room-scale tracking system to capture multiple behaviours. These results indicate that there is no major difference in behavioural responses between mosquito strains of differing pyrethroid susceptibility when exposed to these new ITNs under the experimental conditions used.

Nose to brain delivery of ibudilast micelles for treatment of multiple sclerosis in an experimental autoimmune encephalomyelitis animal model

Multiple sclerosis is a chronic inflammatory disease of the central nervous system ultimate to neurodegeneration and demyelination. Ibudilast is a phosphodiesterase inhibitor, effective on the function of glial cells and lymphocytes, and inhibits the release of TNF-α by inflammatory cells. Dysregulation of glia is one of the most important pathological causes of MS. Therefore, ibudilast as a glial attenuator can be a useful treatment. The objective of the present study was to investigate the effect of nasal spray of polydopamine coated micelles of surfactin, a biosurfactant, loaded with ibudilast on its brain targeted delivery and effectiveness in remylination and neuroprotection in animal model of MS. In animal studies the micelles were administrated intranasally in different doses of 10, 25 and 50 mg/kg/day in C57/BL6 mice immunized by experimental autoimmune encephalomyelitis (EAE) model. The results of Luxol fast blue staining indicated increment in myelin fiber percent more significantly (p < 0.05) in the groups treated with the polydopamine coated micelles (PDAM) compared to nasal spray of free drug or oral administration. These formulations also increased expression of Mbp, Olig2 and Mog genes in the corpus callosum. These results suggest a positive outcome of polydopamine coated micelles loaded with ibudilast in active MS as an anti-inflammatory and neuroprotective agent.

The preventive effect of Zingiber officinale essential oil on demyelination of corpus callosum in a cuprizone rat model of multiple sclerosis.

Multiple sclerosis (MS) is the most prevalent neurological disability among young adults. Anti-inflammatory drugs have shown to be effective in MS. The anti-inflammatory and antioxidative properties of Zingiber officinale (ginger) have been shown and proven in many phytotherapy studies. This study aimed to evaluate effects of ginger essential oil on preventing myelin degradation in a rat model of MS. In this study, we divided 49 rats into 7 groups; 4 control and 3 experimental groups that received 3 different dose of ginger essential oil (50, 100, and 150 mg/kg/day) for treatment of cuprizone-induced demyelinated rats. Basket test and transmission electron microscopy were performed in this study. Olig2 and Mbp genes and proteins were respectively evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Histologically, cuprizone created demyelination in the corpus callosum fibers. Remyelination of fibers was seen in the group treated with the medium dose of ginger essence, by toluidine blue staining. transmission electron microscopy (TEM) revealed increased thickness of the myelin of fibers in all 3 treated groups (p<0.05). Feeding by the medium dose of ginger essence significantly increased the levels of Mbp and Olig2 genes (p<0.05). ELISA test showed that 100 mg/kg/day of ginger caused a significant difference between experimental and the cuprizone-induced groups (p<0.05). Our findings suggested that administration of ginger essential oil prevented demyelination and improved remyelination of rats` corpus callusom and can be used as an effective substance in the prevention of MS.

Valproic acid effects on human adipose-derived stem cell differentiation into oligodendrocytes and improved remyelination in a mouse model of Multiple Sclerosis.

Valproic acid (VPA), a neuroprotective agent and inhibitor of GSK3-β, along with human Adipose-Derived Stem Cells (hADSCs) have been proposed to be potential therapeutic agents for neurodegenerative disorders. In the present study, we have assessed the effects of VPA alone or in combination with hADSCs on oligodendrocyte differentiation, remyelination, and functional recovery in a mouse model of Multiple Sclerosis (MS). These MS-model mice were randomly divided into cuprizone, sham, VPA, hADSC, and VPA/hADSC groups, with 10 mice considered a control group (healthy mice). The hanging wire test was used to measure motor behavior. To estimate the level of myelination, we performed toluidine blue staining and immunofluorescent staining for OLIG2 and MOG-positive cells. Real-time PCR was used to evaluate the expression of β-catenin, human and mouse Mbp, Mog, and Olig2 genes. Remyelination and motor function improved in mice receiving VPA, hADSCs, and especially VPA/hADSCs compared to the Cup and Sham groups (P < 0.01). Additionally, the number of MOG and OLIG2 positive cells significantly increased in the VPA/hADSCs group compared to the Cup and Sham groups (P < 0.01). The expression of β-catenin, myelin and the other oligodendrocyte-specific genes was significantly higher in the VPA recipient groups. Valproic acid can enhance the differentiation of stem cells into oligodendrocytes, making it a potential candidate for MS treatment.

Expression of myelin transcription factor 1 and lamin B receptor mediate neural progenitor fate transition in the zebrafish spinal cord pMN domain

The pMN domain is a restricted domain in the ventral spinal cord, defined by the expression of the olig2 gene. Though it is known that the pMN progenitor cells can sequentially generate motor neurons and oligodendrocytes, the lineages of these progenitors are controversial and how their progeny are generated is not well understood. Using single-cell RNA sequencing, here, we identified a previously unknown heterogeneity among pMN progenitors with distinct fates and molecular signatures in zebrafish. Notably, we characterized two distinct motor neuron lineages using bioinformatic analysis. We then went on to investigate specific molecular programs that regulate neural progenitor fate transition. We validated experimentally that expression of the transcription factor myt1 (myelin transcription factor 1) and inner nuclear membrane integral proteins lbr (lamin B receptor) were critical for the development of motor neurons and neural progenitor maintenance, respectively. We anticipate that the transcriptome features and molecular programs identified in zebrafish pMN progenitors will not only provide an in-depth understanding of previous findings regarding the lineage analysis of oligodendrocyte progenitor cells and motor neurons but will also help in further understanding of the molecular programming involved in neural progenitor fate transition.

Evaluation of Edaravone effects on the differentiation of human adipose derived stem cells into oligodendrocyte cells in multiple sclerosis disease in rats

AimsAmong all the treatments for Multiple Sclerosis, stem cell transplantation, such as ADSCs, has attracted a great deal of scientific attention. On the other hand, Edaravone, as an antioxidant component, in combination with stem cells, could increase the survival and differentiation potential of stem cells. Main methods42 rats were divided into: Control, Cuprizone (CPZ), Sham, Edaravone (Ed), hADSCs, and Ed/hADSCs groups. Following induction of cuprizone, induced MS model, behavioral tests were designed to evaluate motor function during. Luxal fast blue staining was done to measure the level of demyelination and remyelination. Immunofluorescent staining was used to evaluate the amount of MBP, OLIG2, and MOG proteins. The mRNA levels of human MBP, MOG, and OLIG2 and rat Mbp, Mog, and Olig2 were determined via RT-PCR. Key findingsFlow cytometry analysis exhibited that the extracted cells were positive for CD73 (93.8 ± 3%) and CD105 (91.6 ± 3%), yet negative for CD45 (2.06 ± 0.5%). Behavioral tests, unveiled a significant improvement in the Ed (P < 0.001), hADSCs (P < 0.001), and Ed/hADSCs (P < 0.001) groups compared to the others. In the Ed/hADSCs group, the myelin density was significantly higher than that in the Ed treated and hADSCs treated groups (P < 0.01). Edaravone and hADSCs increased the expression of Mbp, Mog, and Olig2 genes in the cuprizone rat models. Moreover, significant differences were seen between the Ed treated and hADSCs treated groups and the Ed/hADSCs group (P < 0.05 for Mbp and Olig2 and P < 0.01 for Mog). SignificanceEdaravone in combination with hADSCs reduced demyelination and increased oligodendrogenesis in the cuprizone rat models.

Glioblastoma multiforme: a multi-omics analysis of driver genes and tumour heterogeneity.

Glioblastoma (GBM) is the most aggressive and common brain cancer in adults with the lowest life expectancy. The current neuro-oncology practice has incorporated genes involved in key molecular events that drive GBM tumorigenesis as biomarkers to guide diagnosis and design treatment. This study summarizes findings describing the significant heterogeneity of GBM at the transcriptional and genomic levels, emphasizing 18 driver genes with clinical relevance. A pattern was identified fitting the stem cell model for GBM ontogenesis, with an upregulation profile for MGMT and downregulation for ATRX, H3F3A, TP53 and EGFR in the mesenchymal subtype. We also detected overexpression of EGFR, NES, VIM and TP53 in the classical subtype and of MKi67 and OLIG2 genes in the proneural subtype. Furthermore, we found a combination of the four biomarkers EGFR, NES, OLIG2 and VIM with a remarkable differential expression pattern which confers them a strong potential to determine the GBM molecular subtype. A unique distribution of somatic mutations was found for the young and adult population, particularly for genes related to DNA repair and chromatin remodelling, highlighting ATRX, MGMT and IDH1. Our results also revealed that highly lesioned genes undergo differential regulation with particular biological pathways for young patients. This multi-omic analysis will help delineate future strategies related to the use of these molecular markers for clinical decision-making in the medical routine.

Differentiation of human dental pulp stem cells into functional motor neuron: In vitro and ex vivo study

There are several therapeutic options for spinal cord injury (SCI), among these strategies stem cell therapy is a potential treatment. The stem cells based therapies have been investigating in acute phase of clinical trials for promoting spinal repair in humans through replacement of functional neuronal and glial cells. The aim of this study was to evaluate the differentiation of Human Dental Pulp Stem Cells (hDPSCs) into functional motor neuron like cells (MNLCs) and promote neuroregeneration by stimulating local neurogenesis in the adult spinal cord slice culture. The immunocytochemistry analysis demonstrated that hDPSCs were positive for mesenchymal stem cell markers (CD73, CD90 and CD105) and negative for the hematopoietic markers (CD34 and CD45). hDPSCs were induced to neurospheres (via implementing B27, EGF, and bFGF) and then neural stem cells (NSC). The NSC differentiated into MNLCs in two steps: first by Shh and RA and ; then with GDNF and BDNF administration. The NS and the NSC were assessed for Oct4, nestin, Nanog, Sox2 expression while the MNLCs were evaluated by ISLET1, Olig2, and HB9 genes. Our results showed that hDPSC can be differentiated into motor neuron phenotype with expression of the motor neuron genes. The functionality of MNLCs was demonstrated by FM1-43, intracellular calcium ion shift and co- culture with C2C12. We co-cultivated hDPSCs with adult rat spinal slices in vitro. Immunostaining and hoechst assay showed that hDPSCs were able to migrate, proliferate and integrate in both the anterolateral zone and the edges of the spinal slices.

Impact of the Olig Family on Neurodevelopmental Disorders.

The Olig genes encode members of the basic helix-loop-helix (bHLH) family of transcription factors. Olig1, Olig2, and Olig3 are expressed in both the developing and mature central nervous system (CNS) and strictly regulate cellular specification and differentiation. Extensive studies have established functional roles of Olig1 and Olig2 in directing neuronal and glial formation during different stages in development. Recently, Olig2 overexpression was implicated in neurodevelopmental disorders down syndrome (DS) and autism spectrum disorder (ASD) but its influence on cognitive and intellectual defects remains unknown. In this review, we summarize the biological functions of the Olig family and how it uniquely promotes cellular diversity in the CNS. This is followed up with a discussion on how abnormal Olig2 expression impacts brain development and function in DS and ASD. Collectively, the studies described here emphasize vital features of the Olig members and their distinctive potential roles in neurodevelopmental disease states.

Association Between OLIG2 Gene SNP rs1059004 and Negative Self-Schema Constructing Trait Factors Underlying Susceptibility to Depression.

Recent evidence has indicated that the disruption of oligodendrocytes may be involved in the pathogenesis of depression. Genetic factors are likely to affect trait factors, such as characteristics, rather than state factors, such as depressive symptoms. Previously, a negative self-schema had been proposed as the major characteristic of constructing trait factors underlying susceptibility to depression. Thus, the association between a negative self-schema and the functional single nucleotide polymorphism (SNP) rs1059004 in the OLIG2 gene, which influences OLIG2 gene expression, white matter integrity, and cerebral blood flow, was evaluated. A total of 546 healthy subjects were subjected to genotype and psychological evaluation using the Beck Depression Inventory-II (BDI-II) and the Brief Core Schema Scale (BCSS). The rs1059004 SNP was found to be associated with the self-schema subscales of the BCSS and scores on the BDI-II in an allele dose-dependent manner, and to have a predictive impact on depressive symptoms via a negative-self schema. The results suggest the involvement of a genetic factor regulating oligodendrocyte function in generating a negative-self schema as a trait factor underlying susceptibility to depression.

Common Myelin Regulatory Factor Gene Variants Predisposing to Excellence in Sports.

In all sport disciplines, excellent coordination of movements is crucial for achieving mastery. The ability to learn new motor skills quickly and effectively is dependent on efficient myelination which varies between individuals. It has been suggested that these differences may play a role in athletic performance. The process of myelination is under transcriptional control by Myelin Regulatory Factor (MYRF) as well as other transcription factors (SOX10 and OLIG2). We analyze a panel of 28 single nucleotide polymorphisms (SNPs) located within the frequencies of common variants of MYRF, SOX10 and OLIG2 genes in professional athletes compared to non-athletes. No significant differences were detected after correction for multiple testing by false discovery rate (FDR) for any of the models tested. However, some deviations from the expected distribution was found for seven SNPs (rs174528, rs139884, rs149435516 and rs2238001, rs7943728, rs61747222, and rs198459). The MYRF alleles rs7943728 and rs61747222 showed a correlation with the level of sport achievement among the athletes. Even though the athletes did not differ from the non-athlete controls in the distribution of most SNPs analyzed, some interesting differences of several variants were noted. Presented results indicate that genetic variants of MYRF and SOX10 could be genetic factors weakly predisposing for successful athletic performance.

TBIO-08. BASE-RESOLUTION METHYLOMES OF GLIOMAS BEARING HISTONE H3.3 MUTATIONS REVEAL A G34 MUTANT-SPECIFIC SIGNATURE SHARED WITH BONE TUMORS

BACKGROUNDTwo recurrent mutations, K27M and G34R/V, in H3F3A, encoding non-canonical histone H3.3, are reported in pediatric and young adult gliomas, whereas G34W mutation was prevalent in bone tumors. In contrast to K27 mutation, it remains elusive how G34 mutations affect the epigenome. Here we performed whole-genome bisulfite sequencing of four G34R-mutated gliomas and the G34V-mutated glioma cell line KNS-42. Similarly, we analyzed seven and three gliomas harboring K27M and no mutations in H3F3A, respectively. These data were compared with those on bone tumors.RESULTSG34R-mutated gliomas exhibited lower global methylation levels, similar CpG island (CGI) methylation levels, and compromised hypermethylation of telomere-proximal CGIs compared with those bearing K27M and no mutations. Hypermethylated regions specific to G34R-mutated gliomas were enriched for CGIs, including those of OLIG1, OLIG2, and canonical histone genes in the HIST1 cluster. These CGIs were hypermethylated in osteosarcomas with, but not without, the G34W mutation. In KNS-42 cells, CGIs with G34V-mutated histone H3.3 exhibited higher methylation levels than those with wild-type histone H3.3. This effect was also observed in the G34R-mutated glioma samples.CONCLUSIONSGliomas bearing G34R/V mutations display characteristic methylomic alterations, some of which are shared by osteosarcomas with the G34W mutation. Deposition of G34 variants may lead to elevated methylation of otherwise hypomethylated, histone H3.3-bearing CGIs.

Base-resolution methylomes of gliomas bearing histone H3.3 mutations reveal a G34 mutant-specific signature shared with bone tumors

Two recurrent mutations, K27M and G34R/V, in H3F3A, encoding non-canonical histone H3.3, are reported in pediatric and young adult gliomas, whereas G34W mutation is prevalent in bone tumors. In contrast to K27M mutation, it remains elusive how G34 mutations affect the epigenome. Here we performed whole-genome bisulfite sequencing of four G34R-mutated gliomas and the G34V-mutated glioma cell line KNS-42 for comparison with gliomas harboring K27M and no mutations in H3F3A and with G34W-mutated bone tumors. G34R-mutated gliomas exhibited lower global methylation levels, similar CpG island (CGI) methylation levels, and compromised hypermethylation of telomere-proximal CGIs, compared to the other two glioma subgroups. Hypermethylated regions specific to G34R-mutated gliomas were enriched for CGIs, including those of OLIG1, OLIG2, and canonical histone genes in the HIST1 cluster. They were notably hypermethylated in osteosarcomas with, but not without, G34W mutation. Independent component analysis revealed that G34 mutation-specific components shared a significant similarity between glioma and osteosarcoma, suggesting that G34 mutations exert characteristic methylomic effects regardless of the tumor tissue-of-origin. CRISPR/Cas9-mediated disruption of G34V-allele in KNS-42 cells led to demethylation of a subset of CGIs hypermethylated in G34R-mutated gliomas. These findings will provide a basis for elucidating epigenomic roles of G34 oncohistone in tumorigenesis.

Unbiased Identification of Novel Transcription Factors in Striatal Compartmentation and Striosome Maturation

Striatal function is dependent on neuronal compartmentation into striosomes and matrix, which are selectively affected in disease. Striatal projection neurons are GABAergic medium spiny neurons (MSNs), subtyped by selective expression of receptors, neuropeptides, and other gene families. Neurogenesis of the striosome and matrix occurs in separate waves, but the factors regulating compartmentation and neuronal differentiation following migration are largely unidentified. We performed RNA- and ATAC-seq on sorted striosome and matrix cells at postnatal day 3 using the Nr4a1-EGFP striosome reporter mouse. With a focus on the striosome, we validated the localization and/or role of Irx1, Foxf2, Olig2 and Stat1/2 in the developing striosome and the in vivo enhancer function of a striosome-specific open chromatin region 4.4 Kb downstream of the Olig2 gene. These data provide novel tools to dissect and manipulate the networks regulating MSN compartmentation and differentiation, including in human iPSC-derived striatal neurons for disease modeling and drug discovery.

Ethnicity-Dependent Effects of Schizophrenia Risk Variants of the OLIG2 Gene on OLIG2 Transcription and White Matter Integrity.

Previous studies have indicated associations between several OLIG2 gene single-nucleotide polymorphisms (SNPs) and susceptibility to schizophrenia among Caucasians. Consistent with these findings, postmortem brain and diffusion tensor imaging studies have indicated that the schizophrenia-risk-associated allele (A) in the OLIG2 SNP rs1059004 predicts lower OLIG2 gene expression in the dorsolateral prefrontal cortex (DLPFC) of schizophrenia patients and reduced white matter (WM) integrity of the corona radiata in normal brains among Caucasians. In an effort to replicate the association between this variant and WM integrity among healthy Japanese, we found that the number of A alleles was positively correlated with WM integrity in some fiber tracts, including the right posterior limb of the internal capsule, and with mean blood flow in a widespread area, including the inferior frontal operculum, orbital area, and triangular gyrus. Because the A allele affected WM integrity in opposite directions in Japanese and Caucasians, we investigated a possible association between the OLIG2 gene SNPs and the expression level of OLIG2 transcripts in postmortem DLPFCs. We evaluated rs1059004 and additional SNPs in the 5' upstream and 3' downstream regions of rs1059004 to cover the broader region of the OLIG2 gene. The 2 SNPs (rs1059004 and rs9653711) had opposite effects on OLIG2 gene expression in the DLPFC in Japanese and Caucasians. These findings suggest ethnicity-dependent opposite effects of OLIG2 gene SNPs on WM integrity and OLIG2 gene expression in the brain, which may partially explain the failures in replicating associations between genetic variants and psychiatric phenotypes among ethnicities.

Effects of progesterone on the cell number of gliomaspheres derived from human glioblastoma cell lines

AimsThe malignancy of the Glioblastomas (GBM), the most frequent and aggressive brain tumors, have been associated with the presence of glioma stem cells (GSCs) which can form gliomaspheres (GS) in vitro. Progesterone (P) increases the proliferation, migration, and invasion of GBM cell lines through the interaction with its intracellular receptor (PR). However, it is unknown if the PR is expressed and the possible effects of P in the formation/differentiation of GS. Main methodsGS were grown from U251 and U87 cell lines by selective culture with serum-free neural stem cell medium. GSCs were identified by the detection of Sox2, Ki67, Nestin, CD133, and CD15 by immunofluorescence. Additionally, the relative expression of PROM1, NES, SOX2, OLIG2, EZH2, BMI1 and PR genes was evaluated by RT-qPCR. The GS were treated with P, and the number of cells was quantified. By RT-PCR the βIII-TUB and GFAP differentiation genes were evaluated. Key findingsGS were maintained until passage four. The expression of all GSCs markers was significantly higher in GS as compared with the basal culture of U251 and U87 cells. We demonstrated for the first time that PR is expressed in GS and this expression was higher as compared with the U251 and U87 cells in basal conditions. Also, we observed that P increased the number of cells derived primary gliomaspheres (GS1) from the U251 line, as well as the expression of the neuronal differentiation marker βIII-TUB. SignificanceThese results suggest the participation of P in the growth of GSCs.

Temporal and partial inhibition of GLI1 in neural stem cells (NSCs) results in the early maturation of NSC derived oligodendrocytes in vitro

BackgroundOligodendrocytes are a type of glial cells that synthesize the myelin sheath around the axons and are critical for the nerve conduction in the CNS. Oligodendrocyte death and defects are the leading causes of several myelin disorders such as multiple sclerosis, progressive multifocal leukoencephalopathy, periventricular leukomalacia, and several leukodystrophies. Temporal activation of the Sonic Hedgehog (SHH) pathway is critical for the generation of oligodendrocyte progenitors, and their differentiation and maturation in the brain and spinal cord during embryonic development in mammals.MethodsOur protocol utilized adherent cultures of human induced pluripotent stem cells (iPSC) and human embryonic stem cells (hESCs) with a green fluorescent protein (GFP) reporter knocked into one allele of the OLIG2 gene locus, dual SMAD inhibition, and transient partial inhibition of glioma-associated oncogene 1 (GLI1) by the small molecule GANT61 during the formation of the SOX2/PAX6-positive neural stem cells (NSCs). The SHH pathway was later restimulated by a Smoothened agonist purmorphamine to induce the generation of OLIG2 glial precursors. One hundred ninety-two individual oligodendrocyte precursor cells (OPCs) from GANT61 and control group were analyzed by single-cell RNA sequencing (RNA-Seq).ResultsWe demonstrate here that transient and partial inhibition of the SHH pathway transcription factor GLI1 in NSCs by a small molecule inhibitor GANT61 was found to generate OPCs that were more migratory and could differentiate earlier toward myelin-producing oligodendrocytes. Single-cell transcriptomic analysis (RNA-Seq) showed that GANT61-NSC-derived oligodendrocyte precursor cells (OPCs) had differential activation of some of the genes in the cytoskeleton rearrangement pathways that are involved in OPC motility and induction of maturation. At the protein level, this was also associated with higher levels of myelin-specific genes in the GANT61 group compared to controls. GANT61-NSC-derived OPCs were functional and could generate compact myelin in vitro and in vivo after transplantation in myelin-deficient shiverer mice.ConclusionsThis is a small molecule-based in vitro protocol that leads to the faster generation of functional oligodendrocytes. The development of protocols that lead to efficient and faster differentiation of oligodendrocytes from progenitors provides important advances toward the development of autologous neural stem cell-based therapies using human iPSCs.