Neural/glial Antigen 2 Research Articles

Distribution of type VI collagen in association with osteoblast lineages in the groove of Ranvier during rat postnatal development

In the groove of Ranvier (GOR), osteoblast lineages form bone bark, which develops into endosteal cortical bone. This ossification process is thought to be regulated by the microenvironment in the GOR. Type VI collagen (Col VI), an extracellular matrix (ECM) protein found in the periosteum/perichondrium, mediates osteoblast differentiation via the cell-surface receptor neural/glial antigen 2 (NG2) chondroitin sulfate proteoglycan. In order to clarify the function of Col VI during osteoblast differentiation in the GOR, in the present study, we examined the distribution of Col VI and osteoblast lineages expressing NG2 in the rat tibia proximal end during postnatal growing periods by immunohistochemistry. Our data revealed that Col VI accumulated in the ECM of the GOR middle layer and that Col VI accumulation was reduced and disappeared in the inner and middle lower regions. Runt-related transcription factor 2-immunoreactive pre-osteoblasts expressed NG2 in Col VI-immunopositive areas. However, Osterix-immunoreactive mature osteoblasts were only found in the Col VI-immunonegative area. These findings indicate that Col VI provided a characteristic microenvironment in the GOR and that NG2-Col VI interactions may regulate the differentiation of osteoblast lineages prior to terminal maturation.

Vascular Notch proteins and Notch signaling in the peri-implantation mouse uterus.

BackgroundAngiogenesis is essential for uterine decidualization, the progesterone-mediated transformation of the uterus allowing embryo implantation and initiation of pregnancy. In the current study, we define the vasculature, expression of Notch proteins and Notch ligands, and Notch activity in both endothelial cells and vascular-associated mural cells of blood vessels in the pre-implantation endometrium and post-implantation decidua of the mouse uterus.MethodsWe used immunofluorescence to determine the expression of Notch in endothelial cells and mural cells by co-staining for the endothelial cell marker, CD31, the pan-mural cell marker, platelet-derived growth factor receptor beta (PDGFR-β), the pericyte markers, neural/glial antigen 2 (NG2) and desmin, or the smooth muscle cell marker, alpha smooth muscle actin (SMA). A fluorescein isothiocyanate-labeled dextran tracer, was used to identify functional peri-implantation vasculature. CBF:H2B-Venus Notch reporter transgenic mice were used to determine Notch activity.ResultsNotch signaling is observed in endothelial cells and pericytes in the peri-implantation uterus. Prior to implantation, Notch1, Notch2 and Notch4 and Notch ligand, Delta-like 4 (Dll4) are expressed in capillary endothelial cells, while Notch3 is expressed in the pericytes. Jagged1 is expressed in both capillary endothelial cells and pericytes. After implantation, Notch1, Notch4 and Dll4 are expressed in endothelial cells of newly formed decidual capillaries. Jagged1 is expressed in endothelial cells of spiral arteries and a subset of decidual pericytes. Notch proteins are not expressed in lymphatic vessels or macrophages in the peri-implantation uterus.ConclusionsWe show Notch activity and distinct expression patterns for Notch proteins and ligands, suggesting unique roles for Notch1, Notch4, Dll4, and Jag1 during decidual angiogenesis and early placentation. These data set the stage for loss-of-function and gain-of-function studies that will determine the cell-type specific requirements for Notch proteins in decidual angiogenesis and placentation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13221-015-0034-y) contains supplementary material, which is available to authorized users.

Differentiation of oligodendrocyte precursors is impaired in the prefrontal cortex in schizophrenia

The pathophysiology of schizophrenia involves disturbances of information processing across brain regions, possibly reflecting, at least in part, a disruption in the underlying axonal connectivity. This disruption is thought to be a consequence of the pathology of myelin ensheathment, the integrity of which is tightly regulated by oligodendrocytes. In order to gain insight into the possible neurobiological mechanisms of myelin deficit, we determined the messenger RNA (mRNA) expression profile of laser captured cells that were immunoreactive for 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), a marker for oligodendrocyte progenitor cells (OPCs) in addition to differentiating and myelinating oligodendrocytes, in the white matter of the prefrontal cortex in schizophrenia subjects. Our findings pointed to the hypothesis that OPC differentiation might be impaired in schizophrenia. To address this hypothesis, we quantified cells that were immunoreactive for neural/glial antigen 2 (NG2), a selective marker for OPCs, and those that were immunoreactive for oligodendrocyte transcription factor 2 (OLIG2), an oligodendrocyte lineage marker that is expressed by OPCs and maturing oligodendrocytes. We found that the density of NG2-immunoreactive cells was unaltered, but the density of OLIG2-immunoreactive cells was significantly decreased in subjects with schizophrenia, consistent with the notion that OPC differentiation impairment may contribute to oligodendrocyte disturbances and thereby myelin deficits in schizophrenia.

Proliferating cells in the adolescent rat amygdala: Characterization and response to stress

The amygdala is a heterogeneous group of nuclei that plays a role in emotional and social learning. As such, there has been increased interest in its development in adolescent animals, a period in which emotional/social learning increases dramatically. While many mechanisms of amygdala development have been studied, the role of cell proliferation during adolescence has received less attention. Using bromodeoxyuridine (BrdU) injections in adolescent and adult rats, we previously found an almost fivefold increase in BrdU-positive cells in the amygdala of adolescents compared to adults. Approximately one third of BrdU-labeled cells in the amygdala contained the putative neural marker doublecortin (DCX), suggesting a potential for neurogenesis. To further investigate this possibility in adolescents, we examined the proliferative dynamics of DCX/BrdU-labeled cells. Surprisingly, DCX/BrdU-positive cells were found to comprise a stable subpopulation of BrdU-containing cells across survivals up to 56days, and there was no evidence of neural maturation by 28days after BrdU injection. Additionally, we found that approximately 50% of BrdU+ cells within the adolescent amygdala contain neural-glial antigen (NG2) and are therefore presumptive oligodendrocyte precursors (OPCs). We next characterized the response to a short-lived stressor (3-day repeated variable stress, RVS). The total BrdU-labeled cell number decreased by ∼30% by 13days following RVS (10days post-BrdU injection) as assessed by stereologic counting methods, but the DCX/BrdU-labeled subpopulation was relatively resistant to RVS effects. In contrast, NG2/BrdU-labeled cells were strongly influenced by RVS. We conclude that typical neurogenesis is not a feature of the adolescent amygdala. These findings point to several possibilities, including the possibility that DCX/BrdU cells are late-developing neural precursors, or a unique subtype of NG2 cell that is relatively resistant to stress. In contrast, many proliferating OPCs are significantly impacted by a short-lived stressor, suggesting consequences for myelination in the developing amygdala.

How do culture media influence in vitro perivascular cell behavior?

Perivascular cells are multilineage cells located around the vessel wall and important for wall stabilization. In this study, we evaluated a stem cell media and a perivascular cell-specific media for the culture of primary perivascular cells regarding their cell morphology, doubling time, stem cell properties, and expression of cell type-specific markers. When the two cell culture media were compared to each other, perivascular cells cultured in the stem cell medium had a more elongated morphology and a faster doubling rate and cells cultured in the pericyte medium had a more typical morphology, with several filopodia, and a slower doubling rate. To evaluate stem cell properties, perivascular cells, CD146(-) cells, and mesenchymal stem cells (MSCs) were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. It was seen that perivascular cells, as well as CD146(-) cells and MSCs, cultured in stem cell medium showed greater differentiation than cells cultured in pericyte-specific medium. The expression of pericyte-specific markers CD146, neural/glial antigen 2 (NG2), platelet-derived growth factor receptor-β (PDGFR-β), myosin, and α-smooth muscle actin (α-SMA) could be found in both pericyte cultures, as well as to varying amounts in CD146(-) cells, MSCs, and endothelial cells. The here presented work shows that perivascular cells can adapt to their in vitro environment and cell culture conditions influence cell functionality, such as doubling rate or differentiation behavior. Pericyte-specific markers were shown to be expressed also from cells other than perivascular cells. We can further conclude that CD146(+) perivascular cells are inhomogeneous cell population probably containing stem cell subpopulations, which are located perivascular around capillaries.

4,4'-Diisothiocyanostilbene-2,2'-disulfonic Acid (DIDS) Ameliorates Ischemia-Hypoxia-Induced White Matter Damage in Neonatal Rats through Inhibition of the Voltage-Gated Chloride Channel ClC-2.

Chronic cerebral hypoperfusion is believed to cause white matter lesions (WMLs), leading to cognitive impairment. Previous studies have shown that inflammation and apoptosis of oligodendrocytes (OLs) are involved in the pathogenesis of WMLs, but effective treatments have not been studied. In this study, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a chloride (Cl−) channel blocker, was injected into chronic cerebral ischemia-hypoxia rat models at different time points. Our results showed that DIDS significantly reduced the elevated mRNA levels and protein expression of chloride channel 2 (ClC-2) in neonatal rats induced by ischemia-hypoxia. Meanwhile, DIDS application significantly decreased the concentrations of reactive oxygen species (ROS); and the mRNA levels of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha TNF-α in neonatal rats with hypoxic-ischemic damage. Myelin staining was weaker in neonatal rats with hypoxic-ischemic damage compared to normal controls in corpus callosum and other white matter, which was ameliorated by DIDS. Furthermore, the elevated number of caspase-3 and neural/glial antigen 2 (NG-2) double-labeled positive cells was attenuated by DIDS after ischemia anoxic injury. Administration of DIDS soon after injury alleviated damage to OLs much more effectively in white matter. In conclusion, our study suggests that early application of DIDS after ischemia-hypoxia injury may partially protect developing OLs.

Genetic mapping of ASIC4 and contrasting phenotype to ASIC1a in modulating innate fear and anxiety.

Although ASIC4 is a member of the acid-sensing ion channel (ASIC) family, we have limited knowledge of its expression and physiological function in vivo. To trace the expression of this ion channel, we generated the ASIC4-knockout/CreERT(2)-knockin (Asic4(Cre) (ERT) (2)) mouse line. After tamoxifen induction in the Asic4(Cre) (ERT)(2)::CAG-STOP(floxed)-Td-tomato double transgenic mice, we mapped the expression of ASIC4 at the cellular level in the central nervous system (CNS). ASIC4 was expressed in many brain regions, including the olfactory bulb, cerebral cortex, striatum, hippocampus, amygdala, thalamus, hypothalamus, brain stem, cerebellum, spinal cord and pituitary gland. Colocalisation studies further revealed that ASIC4 was expressed mainly in three types of cells in the CNS: (i) calretinin (CR)-positive and/or vasoactive intestine peptide (VIP)-positive interneurons; (ii) neural/glial antigen 2 (NG2)-positive glia, also known as oligodendrocyte precursor cells; and (iii) cerebellar granule cells. To probe the possible role of ASIC4, we hypothesised that ASIC4 could modulate the membrane expression of ASIC1a and thus ASIC1a signaling in vivo. We conducted behavioral phenotyping of Asic4(Cre) (ERT)(2) mice by screening many of the known behavioral phenotypes found in Asic1a knockouts and found ASIC4 not involved in shock-evoked fear learning and memory, seizure termination or psychostimulant-induced locomotion/rewarding effects. In contrast, ASIC4 might play an important role in modulating the innate fear response to predator odor and anxious state because ASIC4-mutant mice showed increased freezing response to 2,4,5-trimethylthiazoline and elevated anxiety-like behavior in both the open-field and elevated-plus maze. ASIC4 may modulate fear and anxiety by counteracting ASIC1a activity in the brain.

Functional regeneration of the brain: white matter matters.

Functional regeneration of the brain: white matter matters.

Characterization of Endoneurial Fibroblast-like Cells from Human and Rat Peripheral Nerves

Endoneurial fibroblast-like cells (EFLCs) are one of the cell populations present in the peripheral nervous system. The role and immunophenotypic characteristics of EFLCs are not well known and led us to perform a histological and cytological study of EFLCs in normal human and rat peripheral nerves. We found that all EFLCs express CD34, neural/glial antigen 2 (NG2), and prolyl-4-hydrolase-beta. In addition, half of the EFLCs in normal peripheral nerves express platelet-derived growth factor receptor-β (PDGFR-β) and some also express the intermediate filament nestin in vivo (at a lower level than Schwann cells, which express high levels of nestin). Using cell cultures of purified EFLCs, we characterized subpopulations of EFLCs expressing PDGFR-β alone or PDGFR-β and nestin. Experimental nerve lesions in rat resulted in an increase in nestin-positive EFLCs, which returned to normal levels after 8 days. This suggests that some EFLCs could have a different proliferative and/or regenerative potential than others, and these EFLCs may play a role in the initial phase of nerve repair. These "activated" EFLCs share some immunophenotypic similarities with pericytes and Interstitial cells of Cajal, which have progenitor cell potentials. This raises the questions as to whether a proportion of EFLCs have a possible role as endoneurial progenitor cells.

Periodontal Ligament Stem Cells Possess the Characteristics of Pericytes

Periodontal ligament (PDL) contributes to maintaining homeostasis in periodontal tissues by supplying stem/progenitor cells. It has long been suggested that PDL stem cells/progenitors are located around blood vessels. Recently mesenchymal stem cells (MSCs) have been isolated and cultured from PDL in vitro, although the location of the stem cells in PDL is unclear. The purpose of this study is to test the characteristics of human PDL stem cells (PDLSCs) and examine their similarity to related vascular cell types, such as pericytes and endothelial cells. PDLSCs were obtained from healthy extracted teeth using the collagenase/dispase enzyme digestion method. MSC and pericyte characteristics of PDLSCs were examined by cell surface marker expression using flow cytometry. The expression of pericyte markers was tested using immunohistochemistry. Pericyte-like functions of PDLSCs were examined in co-culture of PDLSCs and umbilical vein endothelial cells on a gel matrix. Cultured PDLSCs were positive for both MSC markers and pericyte markers, including cluster of differentiation 146 (CD146), neural/glial antigen 2 (NG2), and CD140b. When pericyte marker expression was explored in rat periodontal tissue sections, CD146- and NG2-positive signals were observed in the perivascular area of the PDL. Further, when the cells were cultured with human umbilical cord endothelial cells under conditions for forming capillary-like structures in vitro, PDLSCs localized adjacent to endothelial cells and contributed to the stability of the capillary-like structure. PDLSCs possess pericyte-like characteristics and may localize as pericytes in the PDL. These data provide useful information for stem cell biology in periodontal research and stem cell-based periodontal therapy.

WIN55,212-2 protects oligodendrocyte precursor cells in stroke penumbra following permanent focal cerebral ischemia in rats

To explore whether the synthetic cannabinoid receptor agonist WIN55,212-2 could protect oligodendrocyte precursor cells (OPCs) in stroke penumbra, thereby providing neuroprotection following permanent focal cerebral ischemia in rats. Adult male SD rats were subjected to permanent middle cerebral artery occlusion (p-MCAO). The animals were administered WIN55,212-2 at 2 h, and sacrificed at 24 h after the ischemic insult. The infarct volumes and brain swelling were assessed. The expression of cannabinoid receptor type 1 (CB1) in the stroke penumbra was examined using Western blot assay. The pathological changes and proliferation of neural glial antigen 2-positive OPCs (NG2(+) cells) in the stroke penumbra were studied using immunohistochemistry staining. p-MCAO significantly increased the expression of CB1 within the stroke penumbra with the highest level appearing at 2 h following the ischemic insult. Administration of WIN55,212-2 (9 mg/kg, iv) significantly attenuated the brain swelling, and reduced the infarct volume as well as the number of tau-immunoreactive NG2(+) cells (tau-1(+)/NG2(+) cells) in the stroke penumbra. Moreover, WIN55,212-2 significantly promoted the proliferation of NG2(+) cells in the stroke penumbra and in the ipsilateral subventricular zone at 24 h following the ischemic insult. Administration of the selective CB1 antagonist rimonabant (1 mg/kg, iv) partially blocked the effects caused by WIN55,212-2. Tau-1 is expressed in NG2(+) cells following permanent focal cerebral ischemic injury. Treatment with WIN55,212-2 reduces the number of tau-1(+)/NG2(+) cells and promotes NG2(+) cell proliferation in the stroke penumbra, which are mediated partially via CB1 and may contribute to its neuroprotective effects.

Human bone marrow mesenchymal cells express NG2: possible increase in discriminative ability of flow cytometry during mesenchymal stromal cell identification

Mesenchymal stromal cells (MSC) exhibit non-specific hematopoietic cell and/or stromal cell markers (e.g. CD73, CD105 and CD166) that have been used to identify MSC by flow cytometry. Because a neural glial antigen, NG2 (a progenitor cell marker in the central nervous system), is expressed by several tissue cells originating in the mesenchyme but not hematopoietic cells, it might be useful for isolating and identifying MSC. We investigated NG2 expression on culture-expanded MSC by flow cytometry. Human bone marrow (BM) samples taken from 12 donors were cultured for MSC to be used in up to nine serial passages. Using flow cytometry, the neural glial antigen NG2 and commonly used MSC markers CD73, CD105 and CD166, were analyzed on the surface of culture-expanded MSC. The multipotential differentiation of the MSC was examined by adipogenic and osteogenic induction. The percentage of cells positive for NG2 was similar to the percentages of cells positive for CD73, CD105 and CD166 in all passages of BM samples. The mean fluorescent intensities of NG2 did not change with culture passage. The MSC was successfully differentiated into adipogenic and osteogenic lines. The cells showed no karyotypic abnormalities. NG2 seems to be a promising marker for investigating the biology of MSC.