Cyclin D1 is required for proliferation of olig2‐expressing progenitor cells in the injured cerebral cortex

Little is known about the molecular mechanisms driving proliferation of glial cells after an insult to the central nervous system (CNS). To test the hypothesis that the G1 regulator cyclin D1 is critical for injury‐induced cell division of glial cells, we applied an injury model that causes brain damage within a well‐defined region. For this, we injected the neurotoxin ibotenic acid into the prefrontal cortex of adult mice, which leads to a local nerve cell loss but does not affect the survival of glial cells. Here, we show that cyclin D1 immunoreativity increases drastically after neurotoxin injection. We find that the cyclin D1‐immunopositive (cyclin D1+) cell population within the lesioned area consists to a large extent of Olig2+ oligodendrocyte progenitor cells. Analysis of cyclin D1‐deficient mice demonstrates that the proliferation rate of Olig2+ cells diminishes upon loss of cyclin D1. Further, we show that cyclin‐dependent kinase (cdk) 4, but not cdk6 or cdk2, is essential for driving cell division of Olig2‐expressing cells in our injury model. These data suggest that distinct cell cycle proteins regulate proliferation of Olig2+ progenitor cells following a CNS insult.     

Olig2‐positive cells in glioneuronal tumors show both glial and neuronal characters: The implication of a common progenitor cell?

Glioneuronal tumors (GNTs) are rare neoplasms consisting of both glial and neuronal components. Among the GNTs, dysembryoplastic neuroepithelial tumors (DNTs), papillary glioneuronal tumors (PGNTs), and rosette‐forming glioneuronal tumors of the fourth ventricle (RGNTs) share the character of being mainly composed of small round Olig2‐positive tumor cells. Using immunohistochemistry and fluorescence in situ hybridization, we examined a series of 35 GNT cases (11 DNTs, 15 PGNTs and 9 RGNTs) on the characteristics of Olig2‐positive tumor cells. Histologically, Olig2‐positive cells showed small round forms in most GNTs; however, there were a small number of Olig2‐positive cells with neuronal morphology only in a PGNT case. These cells expressed both glial and neuronal markers by double immunostaining. With regard to labeling indices and intensity, only PGNT cells expressed neuronal markers, including α‐internexin and neurofilament. These findings also suggest that some Olig2‐positive PGNT cells may show neuronal differentiation. In GNTs, a considerable number of Olig2‐positive cells showed immunopositivity for cyclin D1 and/or platelet‐derived growth factor receptor alpha (PDGFRα), which are markers for oligodendrocyte progenitor cells. These immunostainings were particularly strong in DNTs. In RGNTs, Olig2‐positive cells formed “neurocytic rosettes”. Furthermore, they were also immunopositive for glial markers, including GFAP, PDGFRα and cyclin D1. These findings indicate the heterogeneous characteristics of Olig2‐positive cells in GNTs, and some of them also exhibited neuronal features. So it is possible that a part of Olig2‐positive GNT cells have characteristics similar to those of progenitor cells.     

Sema4D as an inhibitory regulator in oligodendrocyte development

The specific functions of intrinsic regulators of OL differentiation are poorly understood. Sema4D, originally found as a negative regulator of axon guidance, is mainly expressed by oligodendrocytes in the postnatal brain, and our previous study revealed that the lack of Sema4D induced an increase in the number of oligodendrocytes in the cerebral cortex, suggesting that Sema4D may function as an intrinsic regulator of oligodendrocyte development. In this study, we assessed the effects of Sema4D deficiency and of the exogenous addition of Sema4D on oligodendrocyte differentiation. Sema4D deficiency induced an increase in the number of oligodendrocytes in the cerebral cortex at postnatal day 14 and later, without increase in the number of oligodendrocyte progenitor cells. This increase was also observed in cultured oligodendrocytes obtained from Sema4D-deficient mice. Then we investigated whether Sema4D deficiency can increase the proliferation of the progenitor cells or influence the apoptosis. Apoptotic oligodendrocytes were markedly reduced in number in the developing cerebral cortex and in cultured oligodendrocytes obtained from Sema4D-deficient mice, although no significant change was found in proliferation of oligodendrocyte progenitor cells. Exogenous addition of Sema4D prevented the oligodendrocytes from this reduction of apoptosis, and further enhanced apoptosis in oligodendrocytes. Thus, Sema4D may act as an intrinsic inhibitory regulator of oligodendrocyte differentiation by promoting apoptosis.     

Dynamic changes in myelin aberrations and oligodendrocyte generation in chronic amyloidosis in mice and men

Myelin loss is frequently observed in human Alzheimer's disease (AD) and may constitute to AD‐related cognitive decline. A potential source to repair myelin defects are the oligodendrocyte progenitor cells (OPCs) present in an adult brain. However, until now, little is known about the reaction of these cells toward amyloid plaque deposition neither in human AD patients nor in the appropriate mouse models. Therefore, we analyzed cells of the oligodendrocyte lineage in a mouse model with chronic plaque deposition (APPPS1 mice) and samples from human patients. In APPPS1 mice defects in myelin integrity and myelin amount were prevalent at 6 months of age but normalized to control levels in 9‐month‐old mice. Concomitantly, we observed an increase in the proliferation and differentiation of OPCs in the APPPS1 mice at this specific time window (6–8 months) implying that improvements in myelin aberrations may result from repair mechanisms mediated by OPCs. However, while we observed a higher number of cells of the oligodendrocyte lineage (Olig2+ cells) in APPPS1 mice, OLIG2+ cells were decreased in number in postmortem human AD cortex. Our data demonstrate that oligodendrocyte progenitors specifically react to amyloid plaque deposition in an AD‐related mouse model as well as in human AD pathology, although with distinct outcomes. Strikingly, possible repair mechanisms from newly generated oligodendrocytes are evident in APPPS1 mice, whereas a similar reaction of oligodendrocyte progenitors seems to be strongly limited in final stages of human AD pathology. © 2012 Wiley Periodicals, Inc.     

Radixin expression in microglia after cortical stroke lesion

Stroke induces extensive tissue remodeling, resulting in the activation of several cell types in the brain as well as recruitment of blood‐borne leucocytes. Radixin is part of a cytoskeleton linker protein family with the ability to connect transmembrane proteins to the actin cytoskeleton, promoting cell functions involving a dynamic cytoskeleton such as morphological changes, cell division and migration which are common events of different cell types after stroke. In the healthy adult brain radixin is expressed in Olig2⁺ cells throughout the brain and in neural progenitor cells in the subventricular zone. In the current study, we detected a 2.5 fold increase in the number of radixin positive cells in the peri‐infarct cortex two weeks after the induction of cortical stroke by photothrombosis. Similarly, the number of Olig2⁺ cells increased in the peri‐infarct area after stroke; however, the number of radixin⁺/Olig2⁺ cells was unchanged. Neural progenitor cells maintained radixin expression on their route to the infarct. More surprising however, was the expression of radixin in activated microglia in the peri‐infarct cortex. Seventy percent of Iba1⁺ cells expressed radixin after stroke, a population which was not present in the control brain. Furthermore, activation of radixin was predominantly detected in the peri‐infarct region of oligodendrocyte progenitors and microglia. The specific location of radixin⁺ cells in the peri‐infarct region and in microglia suggests a role for radixin in microglial activation after stroke.     

PARP-1 deletion promotes subventricular zone neural stem cells toward a glial fate

Identification of critical factors involved in oligodendroglial fate specification from endogenous neural stem cells is relevant to the development of therapeutic interventions that aim to promote remyelination. Here we report a novel role of the DNA repair protein poly-ADP-ribose polymerase-1 (PARP-1) in regulating the neural stem cell profile in the postnatal mouse forebrain subventricular zone (SVZ). We observed increased expression of Sox2 and Sox10 in the SVZ of postnatal day 11 (P11) PARP-1 knockout mice. This increase corresponded to increased Olig2 expression in Sox2-positive cells of the PARP-1 knockout mouse SVZ and decreased Map2abc expression compared with Sox2/Olig2 and Sox2/Map2abc expression in wild-type mice. We noted enhanced expression of proliferating oligodendrocyte progenitor cells (OPCs) at the expense of proliferating neuroblasts in the SVZ of PARP-1 knockout mice, by using Olig1/Ki67/DCX, NG2/Ki67/DCX, and PDGFR/BrdU/TuJ1 immunofluorescence labeling. In addition, the percentage of BrdU/Olig2 double-labeled cells increased in the SVZ and corpus callosum of PARP-1 knockout mice compared with wild-type mice. We also observed a decrease in DCX-positive cells without a decrease in the overall SVZ area in PARP-1 knockout mice, further indicating a switch from neuroblast to OPC fate. PARP-1 knockout mice displayed thinning of MBP expression in the corpus callosum and external capsule, suggesting that the enhanced OPC proliferation in the SVZ might compensate for deficiency in myelination. Together, our results show that PARP-1 deletion promotes SVZ neural stem cells toward a glial fate and suggest that future studies target PARP-1 as a potential therapeutic strategy for demyelinating diseases.     

TGF‐β pro‐oligodendrogenic effects on adult SVZ progenitor cultures and its interaction with the Notch signaling pathway

Adult neural progenitor cells (NPCs) are capable of differentiating into neurons, astrocytes, and oligodendrocytes throughout life. Notch and transforming growth factor β1 (TGF‐β) signaling pathways play critical roles in controlling these cell fate decisions. TGF‐β has been previously shown to exert pro‐neurogenic effects on hippocampal and subventricular zone (SVZ) NPCs in vitro and to interact with Notch in different cellular types. Therefore, the aim of our work was to study the effect of TGF‐β on adult rat brain SVZ NPC glial commitment and its interaction with Notch signaling. Initial cell characterization revealed a large proportion of Olig2+, Nestin+, and glial fibrillary acidic protein (GFAP+) cells, a low percentage of platelet‐derived growth factor receptor α (PDGFRα+) or NG2+ cells, and <1% Tuj1+ cells. Immunocytochemical analyses showed a significant increase in the percentage of PDGFRα+, NG2+, and GFAP+ cells upon four‐day TGF‐β treatment, which demonstrates the pro‐gliogenic effect of this growth factor on adult brain SVZ NPCs. Real‐time polymerase chain reaction analyses showed that TGF‐β induced the expression of Notch ligand Jagged1 and downstream gene Hes1. Notch signaling inhibition in cultures treated with TGF‐β produced a decrease in the proportion of PDGFRα+ cells, while TGF‐β receptor II (TβRII) inhibition also rendered a decrease in the proportion of PDGFRα+ cells, concomitantly with a decrease in Jagged1 levels. These findings demonstrate the participation of Notch signaling in TGF‐β effects and illustrate the impact of TGF‐β on glial cell fate decisions of adult brain SVZ NPCs, as well as on oligodendroglial progenitor cell proliferation and maturation.     

Diversity of glial cell components in pilocytic astrocytoma

To characterize the cellular density and proliferative activity of GFAP‐negative cells in pilocytic astrocytoma (PA), surgically excised tissues of PAs (n = 37) and diffuse astrocytomas (DAs) (n = 11) were examined morphologically and immunohistochemically using antibodies against GFAP, Olig2, Iba1 and Ki‐67 (MIB‐1). In PA, Olig2 immunoreactivity was significantly expressed in protoplasmic astrocytes in microcystic, loose areas and cells in oligodendroglioma‐like areas. Iba1‐positive, activated microglia/macrophages were also commonly observed in microcystic areas. In compact areas, a prominent reaction for GFAP was observed, but for Olig2 and Iba1 to a lesser degree. On semiquantitative analysis, the number of Olig2‐positive cells was significantly higher in PAs (mean labeling index (LI) ± standard deviation (SD): 46.8 ± 15.4%) than in DAs (13.3 ± 7.8%) (P < 0.001). Many Iba1‐positive, microglia/macrophages were observed in PAs (19.9 ± 6.5%), similarly to DAs (20.9 ± 9.9%). Re‐immunostaining of PA demonstrated that most Ki‐67‐positive, proliferating cells expressed Olig2, whereas GFAP or Iba1 expression in Ki‐67‐positive cells was less frequent (14.7 ± 13.7%, and 8.8 ± 13.6%) in a double immunostaining study. Conversely, the percentage of Olig2‐positive, proliferating cells in total Olig2‐positive cells (7.2 ± 3.9%) was higher than that of Iba1‐positive, proliferating cells in total Iba1‐positive cells (0.9 ± 0.6%). In conclusion, the present study found that PA consisted of numerous GFAP‐negative cells, including Olig2‐positive cells with high proliferation. Semiquantitative analysis of Olig2 immunohistochemistry in microcystic areas might therefore be useful for the differential diagnosis of PA and DA.     

Interferon-gamma inhibits cell cycle exit in differentiating oligodendrocyte progenitor cells

The developmental processes of the oligodendrocyte progenitor cell (OPC) lineage that are targeted by interferon-gamma (IFN-gamma) were studied in primary rat OPC cultures. Under conditions of thyroid hormone-mediated oligodendrocyte differentiation, IFN-gamma produced a dose-dependent apoptotic response in OPCs. The lowest dose tested (15 ng/ml or 75 U/ml) was nonapoptotic, but activated detectable STAT1 DNA-binding. At this dose, IFN-gamma reduced the percentage of mature O1+ cells and increased the percentage of immature A2B5+ OPCs. This was observed without significant change in total cell number and cytotoxicity, and was accompanied by an increase in BrdU-labeled A2B5+ and O4+ cells. FACS analysis confirmed a lack of apoptotic sub-G1 cells and revealed a greater percentage of S- and G2/M-phase OPCs with IFN-gamma treatment. Dual immunostaining with Ki-67 and Olig2 showed a smaller percentage of Olig2+ cells in G0 phase in IFN-gamma-treated OPCs, indicating loss of G1 control. Instead, increased levels and phosphorylation of the checkpoint protein p34cdc2 by IFN- suggested increased partial arrest in G2. IFN-gamma not only sustained expression of PCNA and the G1-S regulators retinoblastoma protein, cyclin D1, cyclin E, and cdk2, but also decreased p27 levels. In addition to changes in cell proliferation and differentiation, IFN-gamma attenuated myelin basic protein (MBP) expression significantly, which was associated with decreased expression of both MBP and Sox10 RNAs. These findings indicate that IFN-gamma not only maintains cell cycle activity that could predispose OPCs to apoptosis, but also overrides G1-G0 signals leading to thyroid hormone-mediated terminal differentiation and myelin gene expression.     

Glial alterations from early to late stages in a model of Alzheimer's disease: Evidence of autophagy involvement in Aβ internalization

Alzheimer's disease (AD) is a progressive neurodegenerative disease without effective therapy. Brain amyloid deposits are classical histopathological hallmarks that generate an inflammatory reaction affecting neuronal and glial function. The identification of early cell responses and of brain areas involved could help to design new successful treatments. Hence, we studied early alterations of hippocampal glia and their progression during the neuropathology in PDAPP‐J20 transgenic mice, AD model, at 3, 9, and 15 months (m) of age. At 3 m, before deposits formation, microglial Iba1+ cells from transgenic mice already exhibited signs of activation and larger soma size in the hilus, alterations appearing later on stratum radiatum. Iba1 immunohistochemistry revealed increased cell density and immunoreactive area in PDAPP mice from 9 m onward selectively in the hilus, in coincidence with prominent amyloid Congo red + deposition. At pre‐plaque stages, GFAP+ astroglia showed density alterations while, at an advanced age, the presence of deposits was associated with important glial volume changes and apparently being intimately involved in amyloid degradation. Astrocytes around plaques were strongly labeled for LC3 until 15 m in Tg mice, suggestive of increased autophagic flux. Moreover, β‐Amyloid fibrils internalization by astrocytes in in vitro conditions was dependent on autophagy. Co‐localization of Iba1 with ubiquitin or p62 was exclusively found in microglia contacting deposits from 9 m onward, suggesting torpid autophagy. Our work characterizes glial changes at early stages of the disease in PDAPP‐J20 mice, focusing on the hilus as an especially susceptible hippocampal subfield, and provides evidence that glial autophagy could play a role in amyloid processing at advanced stages. © 2015 Wiley Periodicals, Inc.     

Glial Cell Lineages in the Rat Cerebral Cortex

I have traced the fates of glial cell progenitors in the rat cerebral cortex marked with a recombinant retrovirus throughout most of the period of corticogenesis, from embryonic (E) day 14 to postnatal (P) day 14. Discrete clusters of clonally related glia were examined in serially cut sections, and their phenotypes identified using reliable light and electron microscopic criteria. Analysis of a large number of clones marked with retrovirus at various stages of embryonic life contained, with very few exceptions, either all astrocytes or all oligodendrocytes. This observation suggests that the ventricular zone contains separate progenitor cells for the two glial cell types. Oligodendrocyte clones were rarely seen in the cortices injected with retrovirus at the early stages of corticogenesis (E14-E16), suggesting that there is a very small number of oligodendrocyte progenitors in the ventricular zone at these early stages. Their frequency increased significantly at later embryonic ages. At these later stages, ventricular zone cells also give rise to progenitor cells that make up the subventricular zone in early postnatal life. Injections of retrovirus in this proliferative zone shortly after birth resulted in the generation of labeled astrocyte and oligodendrocyte clones in the cortical gray and white matter, with the astrocyte clones being in the majority. Injections at increasingly later stages resulted in the presence, predominantly in the white matter of both hemispheres and in the corpus callosum, of progressively more oligodendrocyte clones and fewer astrocyte clones. Injections at P14 generated only oligodendrocyte clones in the white matter of both hemispheres. A small number of clusters (<10%) generated after subventricular zone injections contained both astrocytes and oligodendrocytes, suggesting that single subventricular zone cells can differentiate into both glial cell types.     

Oligodendrocytes in mouse corpus callosum are coupled via gap junction channels formed by connexin47 and connexin32

According to previously published ultrastructural studies, oligodendrocytes in white matter exhibit gap junctions with astrocytes, but not among each other, while in vitro oligodendrocytes form functional gap junctions. We have studied functional coupling among oligodendrocytes in acute slices of postnatal mouse corpus callosum. By whole‐cell patch clamp we dialyzed oligodendrocytes with biocytin, a gap junction‐permeable tracer. On average 61 cells were positive for biocytin detected by labeling with streptavidin‐Cy3. About 77% of the coupled cells stained positively for the oligodendrocyte marker protein CNPase, 9% for the astrocyte marker GFAP and 14% were negative for both CNPase and GFAP. In the latter population, the majority expressed Olig2 and some NG2, markers for oligodendrocyte precursors. Oligodendrocytes are known to express Cx47, Cx32 and Cx29, astrocytes Cx43 and Cx30. In Cx47‐deficient mice, the number of coupled cells was reduced by 80%. Deletion of Cx32 or Cx29 alone did not significantly reduce the number of coupled cells, but coupling was absent in Cx32/Cx47‐double‐deficient mice. Cx47‐ablation completely abolished coupling of oligodendrocytes to astrocytes. In Cx43‐deficient animals, oligodendrocyte‐astrocyte coupling was still present, but coupling to oligodendrocyte precursors was not observed. In Cx43/Cx30‐double deficient mice, oligodendrocyte‐to‐astrocyte coupling was almost absent. Uncoupled oligodendrocytes showed a higher input resistance. We conclude that oligodendrocytes in white matter form a functional syncytium predominantly among each other dependent on Cx47 and Cx32 expression, while astrocytic connexins expression can promote the size of this network. © 2010 Wiley‐Liss, Inc.     

Cytochemical and cytological properties of perineuronal oligodendrocytes in the mouse cortex

Neuronal cell bodies are associated with glial cells collectively referred to as perineuronal satellite cells. One such satellite cell is the perineuronal oligodendrocyte, which is unmyelinating oligodendrocytes attaching to large neurons in various neural regions. However, little is known about their cellular characteristics and function. In this study, we identified perineuronal oligodendrocytes as 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase‐positive cells attaching to neuronal perikarya immunostained for microtubule‐associated protein 2, and examined their cytochemical and cytological properties in the mouse cerebral cortex. 2′,3′‐Cyclic nucleotide 3′‐phosphodiesterase‐positive perineuronal oligodendrocytes were immunonegative to representative glial markers for astrocytes (brain‐type lipid binding protein and glial fibrillary acidic protein), microglia (Iba‐1) and NG2⁺ glia. However, almost all perineuronal oligodendrocytes expressed glia‐specific or glia‐enriched metabolic enzymes, i.e. the creatine synthetic enzyme S‐adenosylmethionine:guanidinoacetate N‐methyltransferase and l ‐serine biosynthetic enzyme 3‐phosphoglycerate dehydrogenase. As to molecules participating in the glutamate–glutamine cycle, none of the perineuronal oligodendrocytes expressed the plasmalemmal glutamate transporters GLAST and GLT‐1, although nearly half of the perineuronal oligodendrocytes were immunopositive for glutamine synthetase. Cytologically, perineuronal oligodendrocytes were mainly distributed in deep cortical layers (layers IV–VI), and attached directly and tightly to neuronal cell bodies, making a long concave impression to the contacting neurons. Interestingly, they attached more to glutamatergic principal neurons than to GABAergic interneurons, and this became evident at postnatal day 14, when the cerebral cortex develops and maturates. These cytochemical and cytological properties suggest that perineuronal oligodendrocytes are so differentiated as to fulfill metabolic support to the associating principal cortical neurons, rather than to regulate their synaptic transmission.     

Dividing Olig2-expressing progenitor cells derived from ES cells

G-Olig2 is a knock-in ES cell line with GFP inserted into the Olig2 gene so that ES cell-derived neural cells that express Olig2 also express GFP. This tool allows visualization of the subset of cells that differentiate along the Olig2-expressing pathway. By manipulating culture conditions, it is possible to induce Olig2 expression in rapidly dividing cells. These cells have many of the features of glial progenitor cells but, unlike other glial progenitors, are able to divide rapidly for at least 1 month while still expressing Olig2. Even after 1-month expansion, the cells differentiate readily into astrocyte-like and oligodendrocyte-like cells when switched to serum-containing medium. Cellular memory is the property whereby cells remain specified to a particular lineage or pathway while undergoing division. ES cell-derived neural cells show cellular memory for a glial progenitor phenotype and thus provide a new and tractable model for this basic feature of neural development.     

Development and differentiation of glial precursor cells in the rat cerebellum

The development and differentiation of bipotential glial precursor cells has been studied extensively in tissue culture, but little is known about the distribution and fate of these cells within intact animals. To analyze the development of glial progenitor cells in the developing rat cerebellum, we utilized immunofluorescent, immunocytochemical, and autoradiographic techniques. Glial progenitor cells were identified with antibodies against the NG2 chondroitin-sulfate proteoglycan, a cell-surface antigen of 02A progenitor cells in vitro, and the distribution of this marker antigen was compared to that of marker antigens that identify immature astrocytes, mature astrocytes, oligodendrocyte precursors, and mature oligodendrocytes. Cells expressing the NG2 antigen appeared in the cerebellum during the last 3-4 days of embryonic life. Over the first 10 days of postnatal life, the NG2-labeled cells incorporated ³H-thymidine into their nuclei and their total number increased. At all ages examined, the NG2-labeled cells did not contain either vimentin-like or glial fibrillary acidic protein (GFAP)-like immunoreactivity, suggesting that they do not develop along an astrocytic pathway. NG2-labeled cells of embryonic animals expressed G_D3 ganglioside antigens, a property of oligodendrocyte precursors, whereas NG2-positive cells of postnatal animals did not express G_D3 immunoreactivity. Nevertheless, the NG2-labeled cells of the nascent white matter expressed oligodendrocyte-specific marker antigens. Cells lyingoutside of the white matter continued to express the NG2 antigen. In adult animals, the NG2-labeled cells incorporated ³H-thymidine. Glial cells isolated from adult animals and grown in tissue culture express the NG2 antigen and display the phenotypic plasticity characteristic of 02A progenitor cells. These findings demonstrate that a population of glial progenitor cells is extensive within both young and adult animals.