Oligodendrocyte Progenitor Marker Research Articles

HGG-26. SINGLE-CELL RNA-SEQ OF PEDIATRIC HIGH-GRADE GLIOMAS IDENTIFIES COMMON ONCOGENIC PROCESSES AMONG DISTINCT TUMOR HISTOLOGIES

BackgroundPediatric high-grade glioma (PHGG) is a deadly childhood brain tumor that responds poorly to treatment. PHGG comprises two major subtypes: cortical tumors with wild-type H3K27 and diffuse midline gliomas (DMG) that occur in the midline and have characteristic H3K27M mutations. Cortical PHGG is heterogeneous with multiple molecular subtypes. In order to identify underlying commonalities in cortical PHGG that might lead to better treatment modalities, we performed molecular profiling, including single-cell RNA-Seq (scRNA-Seq), on PHGG samples from Children’s Hospital Colorado. MethodsNineteen cortical PHGG tumor samples, one DMG and one normal margin sample obtained at biopsy were disaggregated to isolate viable cells. Fifteen were glioblastomas (GBM), including five with epithelioid and/or giant cell features and five radiation-induced glioblastomas (RIG). There were also four non-GBM PHGG. We performed scRNA-Seq using 10X Genomics v.3 library preparation to enable capture of infiltrating immune cells. We also performed bulk RNA-Seq and DNA methylation profiling. ResultsAfter eliminating patient-specific and cell-cycle effects, RIG, epithelioid GBM, and other GBM each formed identifiable subgroups in bulk RNA-Seq and scRNA-Seq datasets. In the scRNA-Seq data, clusters with cells from multiple tumor samples included a PDGFRA-positive population expressing oligodendrocyte progenitor markers, astrocytic, mesenchymal and stemlike populations, macrophage/monocyte immune cells, and a smaller T-cell population. Analyses of DNA methylation data showed PDGFRA and CDK4 amplification and CDKN2A deletion are common alterations among PHGG. Inferred copy number variation analysis of the single-cell data confirmed that individual tumors include populations that both include and lack the molecular alterations identified in the methylation data. RNA velocity studies to define tumor cells of origin and further analyses of the immune cell populations are underway. ConclusionsSingle-cell analysis of PHGG confirms a large degree of tumor heterogeneity but also shows that PHGG have stemlike, mesenchymal and immune cell populations with common characteristics.

Low-Field Magnetic Stimulation Accelerates the Differentiation of Oligodendrocyte Precursor Cells via Non-canonical TGF-β Signaling Pathways.

Demyelination and oligodendrocyte loss are characteristic changes in demyelinating disorders. Low-field magnetic stimulation (LFMS) is a novel transcranial neuromodulation technology that has shown promising therapeutic potential for a variety of neuropsychiatric conditions. The cellular and molecular mechanisms of magnetic stimulation remain unclear. Previous studies mainly focused on the effects of magnetic stimulation on neuronal cells. Here we aimed to examine the effects of a gamma frequency LFMS on the glial progenitor cells. We used rat central glia-4 (CG4) cell line as an in vitro model. CG4 is a bipotential glial progenitor cell line that can differentiate into either oligodendrocyte or type 2-astrocyte. The cells cultured in a defined differentiation media were exposed to a 40-Hz LFMS 20min daily for five consecutive days. We found that LFMS transiently elevated the level of TGF-β1 in the culture media in the first 24h after the treatment. In correlation with the TGF-β1 levels, the percentage of cells possessing complex branches and expressing the late oligodendrocyte progenitor marker O4 was increased, indicating the accelerated differentiation of CG4 cells towards oligodendrocyte in LFMS-treated cultures. LFMS increased phosphorylation of Akt and Erk1/2 proteins, but not SMAD2/3. TGF-β1 receptor I specific inhibitor LY 364947 partially suppressed the effects of LFMS on differentiation and on levels of pAkt and pErk1/2, indicating that LFMS enhances the differentiation of oligodendrocyte progenitor cells via activation of non-canonical TGF-β-Akt and TGF-β-Erk1/2 pathways but not the canonical SMAD pathway. The data from this study reveal a novel mechanism of magnetic stimulation as a potential therapy for demyelination disorders.

The Median Eminence, A New Oligodendrogenic Niche in the Adult Mouse Brain.

SummaryThe subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus are known as neurogenic niches. We show that the median eminence (ME) of the hypothalamus comprises BrdU+ newly proliferating cells co-expressing NG2 (oligodendrocyte progenitors) and RIP (pre-myelinating oligodendrocytes), suggesting their differentiation toward mature oligodendrocytes (OLs). ME cells can generate neurospheres (NS) in vitro, which differentiate mostly to OLs compared with SVZ-NS that typically generate neurons. Interestingly, this population of oligodendrocyte progenitors is increased in the ME from experimental autoimmune encephalomyelitis (EAE)-affected mice. Notably, the thrombospondin 1 (TSP1) expressed by astrocytes, acts as negative regulator of oligodendrogenesis in vitro and is downregulated in the ME of EAE mice. Importantly, transplanted ME-NS preferentially differentiate to MBP+ OLs compared with SVZ-NS in Shiverer mice. Hence, discovering the ME as a new site for myelin-producing cells has a great importance for advising future therapy for demyelinating diseases and spinal cord injury.

3D Bioprinting Pluripotent Stem Cell Derived Neural Tissues Using a Novel Fibrin Bioink Containing Drug Releasing Microspheres.

3D bioprinting combines cells with a supportive bioink to fabricate multiscale, multi-cellular structures that imitate native tissues. Here, we demonstrate how our novel fibrin-based bioink formulation combined with drug releasing microspheres can serve as a tool for bioprinting tissues using human induced pluripotent stem cell (hiPSC)-derived neural progenitor cells (NPCs). Microspheres, small spherical particles that generate controlled drug release, promote hiPSC differentiation into dopaminergic neurons when used to deliver small molecules like guggulsterone. We used the microfluidics based RX1 bioprinter to generate domes with a 1 cm diameter consisting of our novel fibrin-based bioink containing guggulsterone microspheres and hiPSC-derived NPCs. The resulting tissues exhibited over 90% cellular viability 1 day post printing that then increased to 95% 7 days post printing. The bioprinted tissues expressed the early neuronal marker, TUJ1 and the early midbrain marker, Forkhead Box A2 (FOXA2) after 15 days of culture. These bioprinted neural tissues expressed TUJ1 (15 ± 1.3%), the dopamine marker, tyrosine hydroxylase (TH) (8 ± 1%) and other glial markers such as glial fibrillary acidic protein (GFAP) (15 ± 4%) and oligodendrocyte progenitor marker (O4) (4 ± 1%) after 30 days. Also, quantitative polymerase chain reaction (qPCR) analysis showed these bioprinted tissues expressed TUJ1, NURR1 (gene expressed in midbrain dopaminergic neurons), LMX1B, TH, and PAX6 after 30 days. In conclusion, we have demonstrated that using a microsphere-laden bioink to bioprint hiPSC-derived NPCs can promote the differentiation of neural tissue.

The influence of TSA and VPA on the in vitro differentiation of bone marrow mesenchymal stem cells into neuronal lineage cells: Gene expression studies.

Epigenetic mechanisms regulate the transcription of genes, which can affect the differentiation of MSCs. The aim of the current work is to determine how the histone deacetylase inhibitors TSA and VPA affect the expression of neuronal lineage genes in a culture of rat MSCs (rMSCs). We analyzed the expression of early neuron marker gene (Tubb3), mature neuron markers genes (Vacht, Th, Htr2a) and the oligodendrocyte progenitor marker gene (GalC). Moreover, changes in the gene expression after three different periods of exposure to TSA and VPA were investigated for the first time. After six days of exposition to TSA and VPA, the expression of Tubb3 and GalC decreased, while the expression of Th increased. The highest increase of VAChT expression was observed after three days of TSA and VPA treatment. A decrease in Htr2a gene expression was observed after TSA treatment and an increase was observed after VPA treatment. We also observed that TSA and VPA inhibited cell proliferation and the formation of neurospheres in the rMSCs culture. The central findings of our study are that TSA and VPA affect the expression of neuronal lineage genes in an rMSCs culture. After exposure to TSA or VPA, the expression of early neuronal gene decreases but equally the expression of mature neuron genes increases. After TSA and VPA treatment ER of the oligodendrocyte progenitor marker decreased. TSA and VPA inhibit cell proliferation and the formation of neurospheres in rMSCs culture.

Effect of Young�s modulus on neural differentiation of human induced pluripotent stem cell derived neural stem cells.

Event Abstract Back to Event Effect of Young’s modulus on neural differentiation of human induced pluripotent stem cell derived neural stem cells. Matthew C. Mosley1, 2, Hyun Ju Lim1, 2, Jing Chen1, 2, Shenglan Li1, 2, Ying Liu1, 2 and Laura Smith Callahan1, 2, 3 1 University of Texas Medical School at Houston, Neurosurgery, United States 2 University of Texas Health Science Center at Houston, Center for Stem Cells & Regenerative Medicine, United States 3 University of Texas Medical School at Houston, Nanomedicine & Biomedical Engineering, United States Introduction: The Young’s modulus of culture substrates has been shown to have a significant effect on the lineage choice and differentiation state of stem cells[1],[2]. Typically, these studies have examined a mechanical range over several orders of magnitude using discrete samples to identify optimal mechanical regions for the differentiation of specific lineages. However, a more detailed examination of the region of interest for a specific lineage, such as neurons, could further improve the optimization of biomaterial mechanics for specific applications. In this study the optimal range for neural differentiation of human induced pluripotent stem cell (hiPSC) derived neural stem cells (NSC) will be examined using poly ethylene glycol dimethacrylate (PEGDM) hydrogels containing a continuous gradient in Young’s Modulus. Material and Methods: 50 mm × 10 mm × 1 mm hydrogel gradients were fabricated by dispensing 10% and 55% 10 kDa PEGDM solutions containing 0.1% Matrigel through two syringe pumps running in inverse linear ramping profiles ranging from 0 mL/h to 52 mL/h, respectively, over 75s into a custom mold, followed by photopolymerization with 2.3 mJ/cm2 for 7 min. After swelling in PBS, one 9.6-mm disc was punched out every 10 mm along each gradient. Discs were then evaluated for swelling ratio, mesh size and mechanical properties. For cellular experiments, the discs were placed in the wells of 48-well plates, seeded with ND2.0 hiPSC derived NCS and cultured with serum-free neural differentiation media for 14 days with medium exchanges every other day. Cell-seeded constructs were stained for neuron-specific β3-tubulin (TUJ1) and length of neurite extension was measured. Cultured cells on constructs were harvested for analyses of gene expression. Statistical significance was determined using one-way or two-way ANOVA in conjunction with the paired t-test with a p-value of < 0.05. Results and Discussion: The Young’s modulus of gradient samples ranged from 0.5 kPA to 3.5 kPA. Gene expression of TUJ1 (immature neuron marker) in cells cultures cultured on hydrogels decreases with increasing hydrogel Young’s modulus (Figure 1). The gene expression of PAX6 (neural stem cell marker), MAP2 (mature neuron marker), GFAP (astrocyte marker) and Oligo2 (oligodendrocyte progenitor marker) were not affected by Young’s modulus changes in this range and oligodendrocyte marker, PDGFRa, was not detected over the time course. Maximal neurite extension was observed in cells cultured on hydrogels with a Young’s modulus of 0.91 kPa (Figure 2). Conclusion: Hydrogel Young’s modulus effects hiPSC derived NSC gene expression of TUJ1 and neurite extension, but does not have a significant effect on the gene expression of other tested neural lineage markers. Modulation of matrix mechanics for neural differentiation is an important matrix property requiring futher optimization and study to fully understand its complex effect on cellular differentiation. Funding from Mission Connect a TIRR Foundation program and William Stamps Farish Fund supported this work.

Neural Placode Tissue Derived From Myelomeningocele Repair Serves as a Viable Source of Oligodendrocyte Progenitor Cells.

The presence, characteristics, and potential clinical relevance of neural progenitor populations within the neural placodes of myelomeningocele patients remain to be studied. Neural stem cells are known to reside adjacent to ependyma-lined surfaces along the central nervous system axis. Given such neuroanatomic correlation and regenerative capacity in fetal development, we assessed myelomeningocele-derived neural placode tissue as a potentially novel source of neural stem and progenitor cells. Nonfunctional neural placode tissue was harvested from infants during the surgical repair of myelomeningocele and subsequently further analyzed by in vitro studies, flow cytometry, and immunofluorescence. To assess lineage potential, neural placode-derived neurospheres were subjected to differential media conditions. Through assessment of platelet-derived growth factor receptor α (PDGFRα) and CD15 cell marker expression, Sox2+Olig2+ putative oligodendrocyte progenitor cells were successfully isolated. PDGFRαCD15 cell populations demonstrated the highest rate of self-renewal capacity and multipotency of cell progeny. Immunofluorescence of neural placode-derived neurospheres demonstrated preferential expression of the oligodendrocyte progenitor marker, CNPase, whereas differentiation to neurons and astrocytes was also noted, albeit to a limited degree. Neural placode tissue contains multipotent progenitors that are preferentially biased toward oligodendrocyte progenitor cell differentiation and presents a novel source of such cells for use in the treatment of a variety of pediatric and adult neurological disease, including spinal cord injury, multiple sclerosis, and metabolic leukoencephalopathies.

Evidence that the central canal lining of the spinal cord contributes to oligodendrogenesis during postnatal development and adulthood in intact rats

Two waves of oligodendrogenesis in the ventricular zone of the spinal cord (SC-VZ) during rat development, which take place between embryonic days 14 and 18 (E14-E18) and E20-E21, have been described. In the VZ of the brain, unlike the SC-VZ, a third wave of oligodendrogenesis occurs during the first weeks of postnatal development. Using immunofluorescence staining of intact rat SC tissue, we noticed the presence of small numbers of Olig2(+) /Sox-10(+) cells inside the lining of the central canal (CC) during postnatal development and adulthood. Olig2(+) /Sox-10(+) cells appeared inside the lining of the CC shortly after birth, and their number reached a maximum of approximately 0.65 ± 0.14 cell/40-μm section during the second postnatal week. After the latter development, the number of Olig2(+) /Sox-10(+) cells decreased to 0.21 ± 0.07 (P36) and 0.18 ± 0.1 cell/section (P120). At P21, Olig2(+) /Sox-10(+) cells inside the CC lining started to express other oligodendroglial markers such as CNPase, RIP, and APC. Olig2(+) /Sox-10(+) cells usually did not proliferate inside the CC lining and were only rarely found to be immunoreactive against oligodendrocyte progenitor markers such as NG2 or PDGFRα. Using 5-bromo-2-deoxyuridine administration at P2, P11, P22, or P120-P125, we revealed that these cells arose in the CC lining during postnatal development and adulthood. Our findings confirmed that the CC lining is the source of a small number of cells with an oligodendroglial phenotype during postnatal development and adulthood in the SC of intact rats.

Myelin Gene Regulatory Factor Is Required for Maintenance of Myelin and Mature Oligodendrocyte Identity in the Adult CNS

Although the transcription factors required for the generation of oligodendrocytes and CNS myelination during development have been relatively well established, it is not known whether continued expression of the same factors is required for the maintenance of myelin in the adult. Here, we use an inducible conditional knock-out strategy to investigate whether continued oligodendrocyte expression of the recently identified transcription factor myelin gene regulatory factor (MRF) is required to maintain the integrity of myelin in the adult CNS. Genetic ablation of MRF in mature oligodendrocytes within the adult CNS resulted in a delayed but severe CNS demyelination, with clinical symptoms beginning at 5 weeks and peaking at 8 weeks after ablation of MRF. This demyelination was accompanied by microglial/macrophage infiltration and axonal damage. Transcripts for myelin genes, such as proteolipid protein, MAG, MBP, and myelin oligodendrocyte glycoprotein, were rapidly downregulated after ablation of MRF, indicating an ongoing requirement for MRF in the expression of these genes. Subsequently, a proportion of the recombined oligodendrocytes undergo apoptosis over a period of weeks. Surviving oligodendrocytes gradually lose the expression of mature markers such as CC1 antigen and their association with myelin, without reexpressing oligodendrocyte progenitor markers or reentering the cell cycle. These results demonstrate that ongoing expression of MRF within the adult CNS is critical to maintain mature oligodendrocyte identity and the integrity of CNS myelin.

Effect of 6-hydroxydopamine (6-OHDA) on proliferation of glial cells in the rat cortex and striatum: evidence for de-differentiation of resident astrocytes

Reactive astrogliosis is the universal response to any brain insult. It is characterized by cellular hypertrophy, up-regulation of the astrocyte marker glial fibrillary acidic protein (GFAP), and proliferation. The source of these proliferating cells is under intense debate. Progenitor cells derived from the subventricular zone (SVZ), cells positive for chondroitin sulfate proteoglycan (NG2(+)), and de-differentiated astrocytes have been proposed as the origin of proliferating cells following injury. We have analyzed the effect of intraventricular-applied 6-hydroxydopamine (6-OHDA) on the proliferation and morphology of astrocytes in rat cortex and striatum by means of immunohistochemistry and confocal laser microscopy. At 4 days post-lesion, GFAP expression increased markedly. A subpopulation of the GFAP(+) cells co-expressed Ki-67, indicating that these cells were proliferating. To investigate whether these cells (1) arose from migrating SVZ progenitor cells, (2) derived from NG2(+) progenitor cells, or (3) de-differentiated from resident astrocytes, we studied the expression of the migration marker doublecortin (Dcx), the oligodendrocyte progenitor marker NG2, and the progenitor markers Nestin and Pax6. The proliferating Ki-67(+) cells co-expressed Nestin and Pax6, whereas no co-expression of Ki-67 with NG2 or the migration marker Dcx was observed. Thus, resident astrocytes de-differentiate, in response to the intraventricular application of 6-OHDA, to a phenotype resembling radial glia cells, which represent transient astrocyte precursors during development. An understanding of the mechanisms of the de-differentiation of mature astrocytes might be useful for designing new approaches to cell therapy in neurodegenerative diseases such as Parkinson's disease.

Environmental enrichment stimulates progenitor cell proliferation in the amygdala

Enriched environments enhance hippocampal neurogenesis, synaptic efficacy, and learning and memory functions. Recent studies have demonstrated that enriched environments can restore learning behavior and long-term memory after significant brain atrophy and neural loss. Emotional and anxiety-related behaviors were also improved by enriched stimuli, but the effect of enriched environments on the amygdala, one of the major emotion-related structures in the central nervous system, remains largely unknown. In this study, we have focused on the effects of an enriched environment on cell proliferation and differentiation in the murine amygdala. The enriched environment increased bromodeoxyuridine (BrdU)-positive (newborn) cell numbers in the amygdala, almost all of which, immediately after a 1-week period of enrichment, expressed the oligodendrocyte progenitor marker Olig2. Furthermore, enriched stimuli significantly suppressed cell death in the amygdala. Some of the BrdU-positive cells in mice exposed to the enriched environment, but none in animals housed in the standard environment, later differentiated into astrocytes. Our findings, taken together with previous behavioral studies, suggest that progenitor proliferation and differentiation in the amygdala may contribute to the beneficial aspects of environmental enrichment such as anxiolytic effects.

A GLI1-p53 inhibitory loop controls neural stem cell and tumour cell numbers

How cell numbers are determined is not understood. Hedgehog-Gli activity is involved in precursor cell proliferation and stem cell self-renewal, and its deregulation sustains the growth of many human tumours. However, it is not known whether GLI1, the final mediator of Hh signals, controls stem cell numbers, and how its activity is restricted to curtail tumourigenesis. Here we have altered the levels of GLI1 and p53, the major tumour suppressor, in multiple systems. We show that GLI1 expression in Nestin+ neural progenitors increases precursor and clonogenic stem cell numbers in vivo and in vitro. In contrast, p53 inhibits GLI1-driven neural stem cell self-renewal, tumour growth and proliferation. Mechanistically, p53 inhibits the activity, nuclear localisation and levels of GLI1 and in turn, GLI1 represses p53, establishing an inhibitory loop. We also find that p53 regulates the phosphorylation of a novel N' truncated putative activator isoform of GLI1 in human cells. The balance of GLI1 and p53 functions, thus, determines cell numbers, and prevalence of p53 restricts GLI1-driven stem cell expansion and tumourigenesis.

Olig2-expressing progenitor cells preferentially differentiate into oligodendrocytes in cuprizone-induced demyelinated lesions

Many oligodendrocyte progenitor cells (OPCs) are found in acute or chronic demyelinated area, but not all of them differentiate efficiently into mature oligodendrocytes in the demyelinated central nervous system (CNS). Recent studies have shown that the basic helix-loop-helix transcription factor Olig2, which stimulates OPCs to differentiate into oligodendrocyte, is strongly up-regulated in many pathological conditions including acute or chronic demyelinating lesions in the adult CNS. Despite their potential role in the treatment of demyelinating diseases, the long-term fate of these up-regulated Olig2 cells has not been identified due to the lack of stable labeling methods. To trace their fate we have used double-transgenic mice, in which we were able to label Olig2-positive cells conditionally with green fluorescent protein (GFP). Demyelination was induced in these mice by feeding cuprizone, a copper chelator. After 6 weeks of cuprizone exposure, GFP-positive (GFP +) cells were processed for a second labeling with antibodies to major neural cell markers APC (mature oligodendrocyte marker), GFAP (astrocyte marker), NeuN (neuron marker), Iba1 (microglia marker) and NG2 proteoglycan (oligodendrocyte progenitor marker). More than half of the GFP + cells in the external capsule showed co-localization with NG2 proteoglycan. While the percentages of NG2-positive (NG2 +) and APC-positive (APC +) oligodendrocyte lineage cells in cuprizone-treated mice were significantly higher than those in the normal diet group, no significant difference was observed for GFAP-positive (GFAP +) astrocytic lineage cells. Our data therefore provide direct evidence that proliferation and differentiation of local and/or recruited Olig2 progenitors contribute to remyelination in demyelinated lesions.

Disease-Responsive Neural Precursor Cells are Present in Multiple Sclerosis Lesions

Spontaneous tissue repair occurs in multiple sclerosis (MS), but the origin of remyelinating cells remains obscure. Here we explore the hypothesis that endogenous neural precursors are involved in MS disease processes. We studied postmortem brain and spinal cord samples from MS patients using immunocytochemical techniques. We show that cells co-positive for nestin and musashi-1 are not merely present in lesions, but found in markedly increased numbers (up to fivefold). Small numbers of nestin-positive cells show direct evidence of proliferation, co-staining for Ki67; some also coexpress glial fibrillary acidic protein or oligodendrocyte progenitor markers (NG-2 or PDGF-alpha receptor), or the early neuronal marker doublecortin, consistent with transition from neural precursors. These findings suggest that endogenous neural precursors react to disease processes in MS.

Up-regulation of NG2 proteoglycan and interferon-induced transmembrane proteins 1 and 3 in mouse astrocytoma: A membrane proteomics approach

Although brain tumors are classified as if their lineage were well understood, the relationship between the molecular events that specify neural cell lineage and brain tumors remains enigmatic. Traditionally, cell surface membrane antigens have served as biomarkers that distinguish brain tumor origin and malignancy. In this study, membrane proteins were identified from a terminally differentiated mouse astrocyte (AC) and CT-2A astrocytoma (CT-2A) cell line using liquid-chromatography coupled with tandem mass spectrometry (LC-MS/MS). A total of 321 and 297 protein groups with at least one unique peptide were identified in the AC and CT-2A cells. Using a label-free quantitative MS approach, 25 plasma membrane proteins in CT-2A were found significantly up- or down-regulated compared with those in AC. Three of the up-regulated proteins, chondroitin sulfate proteoglycan-4 (Cspg4), interferon-induced transmembrane protein-2 (IFITM2) and -3 (IFITM3) were further validated by semi-quantitative RT-PCR analysis. In addition, a third member of the IFITM family, interferon-induced transmembrane protein-1 (IFITM1) was also analyzed. Expression of Cspg4, IFITM1 and IFITM3 was significantly greater in the CT-2A cells than that in the AC cells. Interestingly, Cspg4, also known as neuronal/glial 2 (NG2) proteoglycan in human, is an oligodendrocyte progenitor marker. Therefore, our data suggest that the CT-2A tumor may be derived from NG2 glia rather than from fully differentiated astrocytes. Moreover, the CT-2A cells also express a series of interferon-induced signature proteins that may be specific to this tumor. These data highlight the utility of LC-MS/MS for the identification of brain tumor membrane biomarkers.

Inhibition of Rho Family Functions by Lovastatin Promotes Myelin Repair in Ameliorating Experimental Autoimmune Encephalomyelitis

Impaired remyelination is critical to neuroinflammation in multiple sclerosis (MS), which causes chronic and relapsing neurological impairments. Recent studies revealed that immunomodulatory activity of statins in an experimental autoimmune encephalomyelitis (EAE) model of MS are via depletion of isoprenoids (farnesyl-pyrophosphate and geranylgeranyl-pyrophosphate) rather than cholesterol in immune cells. In addition, we previously documented that lovastatin impedes demyelination and promotes myelin repair in treated EAE animals. To this end, we revealed the underlying mechanism of lovastatin-induced myelin repair in EAE using in vitro and in vivo approaches. Survival, proliferation (chondroitin sulfate proteoglycan-NG2(+) and late oligodendrocyte progenitor marker(+)), and terminal-differentiation (myelin basic protein(+)) of OPs was significantly increased in association with induction of a promyelinating milieu by lovastatin in mixed glial cultures stimulated with proinflammatory cytokines. Lovastatin-induced effects were reversed by cotreatment with mevalonolactone or geranylgeranyl-pyrophosphate, but not by farnesyl-pyrophosphate or cholesterol, suggesting that depletion of geranygeranyl-pyrophosphate is more critical than farnesyl-pyrophosphate in glial cells. These effects of lovastatin were mimicked by inhibitors of geranylgeranyl-transferase (geranylgeranyl transferase inhibitor-298) and downstream effectors {i.e., Rho-family functions (C3-exoenzyme) and Rho kinase [Y27632 (N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride)]} but not by an inhibitor of farnesyl-transferase (farnesyl transferase inhibitor-277). Moreover, activities of Rho/Ras family GTPases were reduced by lovastatin in glial cells. Corresponding with these findings, EAE animals exhibiting demyelination (on peak clinical day; clinical scores >/=3.0) when treated with lovastatin and aforementioned agents validated these in vitro findings. Together, these data provide unprecedented evidence that-like immune cells-geranylgeranyl-pyrophosphate depletion and thus inhibition of Rho family functions in glial cells by lovastatin promotes myelin repair in ameliorating EAE.

Postnatal Neurogenesis and Gliogenesis in the Olfactory Bulb from NG2-Expressing Progenitors of the Subventricular Zone

We used a 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP)-enhanced green fluorescent protein (EGFP) transgenic mouse to study postnatal subventricular zone (SVZ) progenitor fate, with a focus on the olfactory bulb (OB). The postnatal OB of the CNP-EGFP mouse contained EGFP+ interneurons and oligodendrocytes. In the anterior SVZ, the majority of EGFP+ progenitors were NG2+. These NG2+/EGFP+ progenitors expressed the OB interneuron marker Er81, the neuroblast markers doublecortin (DC) and Distalless-related homeobox (DLX), or the oligodendrocyte progenitor marker Nkx2.2. In the rostral migratory stream (RMS), EGFP+ cells displayed a migrating phenotype. A fraction of these cells were either NG2-/Er81+/DC+/DLX+ or NG2+/Nkx2.2+. DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) injection into the lateral ventricle (LV) of early postnatal mice demonstrated that NG2+/EGFP+ progenitors migrate from the SVZ through the RMS into the OB. Moreover, fluorescence-activated cell-sorting-purified NG2+/CNP-EGFP+ or NG2+/beta-actin-enhanced yellow fluorescent protein-positive (EYFP+) progenitors transplanted into the early postnatal LV displayed extensive rostral and caudal migration. EYFP+ or EGFP+ graft-derived cells within the RMS were DLX+/Er81+ or Nkx2.2+, migrated to the OB, and differentiated to interneurons and oligodendrocytes. In the subcortical white matter (SCWM), grafted cells differentiated to either oligodendrocytes or astrocytes. Transplantation of NG2+/EYFP+ progenitors selectively purified from the SVZ showed that these cells were migratory and generated glia and neurons in the OB, hippocampus, and striatum. In contrast, cortical, OB, or cerebellar NG2+ cells had a very limited migratory potential and gave rise to glia in the SCWM and striatum. Our findings indicate region-specific differences between NG2+ progenitor cells and show that NG2+ cells can migrate throughout the RMS and contribute to both gliogenesis and neurogenesis in the postnatal OB.

Electroconvulsive seizures induce proliferation of NG2-expressing glial cells in adult rat amygdala

BackgroundVolumetric changes and glial pathology have been reported in the amygdala in patients with major depressive disorder. Here we report an analysis of glial cell proliferation in response to electroconvulsive seizures (ECS), clinically used for the treatment of severe depression. MethodsMale Wistar rats were subjected to five ECS-treatments and then injected with bromodeoxyuridine (BrdU) to detect cell proliferation in the amygdala. The animals were transcardially perfused either 12 hours or 3 weeks after the last BrdU injection. Tissue sections were double-stained for BrdU and the cell-type markers NG2, OX-42, RIP, S-100β, Doublecortin, or NeuN. ResultsElectroconvulsive seizures dramatically increased the proliferation of amygdala cells expressing the oligodendrocyte progenitor marker NG2. Bromodeoxyuridine-labeled NG2-expressing cells were still present after 3 weeks of survival, and a small proportion of the proliferating cells had differentiated into mature oligodendrocytes. ConclusionsMajor depression has been associated with a reduction of glial cells. Our results show that ECS, an antidepressant treatment, significantly increases the number of NG2+ glial cells and mature oligodendrocytes in the adult rat amygdala.

Expression of the myelin and oligodendrocyte progenitor marker sulfatide in neurons and astrocytes of adult rat brain.

Sulfatide is a myelin component of the central (CNS) and peripheral nervous system (PNS) and is used extensively to identify oligodendrocyte progenitor cells. We have explored sulfatide expression in CNS gray matter (cerebellum, cerebral cortex, and hippocampus) and the PNS in adult rats using an anti-sulfatide antibody (Sulph I) and confocal microscopy. Biochemical analyses revealed two Sulph I antigens, sulfatide and seminolipid; sulfatide was present at about five times higher concentration, and the affinity of Sulph I for sulfatide was 2.5 times higher than that for seminolipid. Thus sulfatide was considered the dominant antigen. We found Sulph I immunostaining, in addition to that in myelinated areas in subpopulations of astrocytes and neurons. Astrocyte Sulph I staining was localized to the cell bodies and in some cases also to the processes. In the cerebellum, some Sulph I-positive astrocytes corresponded to Golgi epithelial cell bodies. We also found Sulph I staining in neuronal cell bodies, which in some neurons was clearly localized to the cytoplasm and in others to the nuclear membrane. Sulph I immunostaining in the PNS was located in the myelin sheath and paranodal end segments. These results demonstrate the expression of sulfatide in cell types other than oligodendrocytes and Schwann cells, showing that sulfatide is not a selective marker for adult oligodendrocyte progenitor cells. Moreover, these findings show that sulfatide is localized also to intracellular compartments and indicate that other roles of sulfatide in astrocytes and neurons, compared to myelin, might be considered.

Dynamic expression of basic helix-loop-helix Olig family members: implication of Olig2 in neuron and oligodendrocyte differentiation and identification of a new member, Olig3

Basic helix-loop-helix (bHLH) transcription factors have been shown to be essential for specification of various cell types. Here, we describe a novel bHLH family consisting of three members, two of which (Olig1, Olig2) are expressed in a nervous tissue-specific manner, whereas the third, Olig3 is found mainly in non-neural tissues. Olig1 and Olig2, which recently have been implicated in oligodendrogenesis, are expressed in the region of the ventral ventricular zone of late embryonic spinal cord where oligodendrocyte progenitors appear. In the embryonic brain, the Olig2 expression domain is broader than that of Olig1 and does not overlap with an oligodendrocyte progenitor marker, CNP. Furthermore, Olig2 is expressed in most cells in the ventral half of the early embryonic spinal cord, which do not yet express an early neuronal marker TuJ1. These results indicate that Olig2 expression is not limited to the oligodendrocyte lineage but includes immature neuronal progenitors and multipotential neuron/glia progenitors as well as embryonic olfactory neurons.