Human NG2 Research Articles

Cover Image, Volume 71, Issue 6

Cover Illustration: Human glial chimeric mouse cortex engrafted with fetal human glial progenitor cells (human NG2, red), visualized 1 year after neonatal transplant. A fraction of the donor cells were tagged before transplant with a lentiviral vector expressing membranebound EGFP (green), thereby visualizing in adulthood the morphological complexity, domain structure and tiling of cortical hGPCs in vivo. (See Osorio, MJ, et al. https://doi.org/10.1002/glia.24291)

Protein Kinase CK2 Regulates Nerve/Glial Antigen (NG)2-Mediated Angiogenic Activity of Human Pericytes.

Protein kinase CK2 is a crucial regulator of endothelial cell proliferation, migration and sprouting during angiogenesis. However, it is still unknown whether this kinase additionally affects the angiogenic activity of other vessel-associated cells. In this study, we investigated the effect of CK2 inhibition on primary human pericytes. We found that CK2 inhibition reduces the expression of nerve/glial antigen (NG)2, a crucial factor which is involved in angiogenic processes. Reporter gene assays revealed a 114 bp transcriptional active region of the human NG2 promoter, whose activity was decreased after CK2 inhibition. Functional analyses demonstrated that the pharmacological inhibition of CK2 by CX-4945 suppresses pericyte proliferation, migration, spheroid sprouting and the stabilization of endothelial tubes. Moreover, aortic rings of NG2−/− mice showed a significantly reduced vascular sprouting when compared to rings of NG2+/+ mice, indicating that NG2 is an important regulator of the angiogenic activity of pericytes. In vivo, implanted Matrigel plugs containing CX-4945-treated pericytes exhibited a lower microvessel density when compared to controls. These findings demonstrate that CK2 regulates the angiogenic activity of pericytes through NG2 gene expression. Hence, the inhibition of CK2 represents a promising anti-angiogenic strategy, because it does not only target endothelial cells, but also vessel-associated pericytes.

CSIG-14. PROTEIN KINASE CK2 REGULATES THE EXPRESSION OF NERVE/GLIAL ANTIGEN (NG)2 IN GLIOBLASTOMA

Abstract Nerve/glial antigen (NG)2 enhances cell proliferation, migration as well as chemoresistance and is associated with a poor clinical outcome in glioblastoma. Since regulatory mechanisms of NG2 expression are still largely unknown, we herein investigated the impact of protein kinase CK2 inhibition on NG2 expression in human glioblastoma cells. CK2 was inhibited in NG2-positive human glioblastoma cell lines (A1207, U87) and primary human glioblastoma cells by pharmacological treatment (CX-4945, TBB) or siRNA. NG2 expression was analyzed by flow cytometry, Western Blot and qRT-PCR. Cytotoxicity and viability were assessed by WST-1, LDH and BrdU assays. Scratch, sprouting and BrdU assays as well as growth curves were performed to examine cell migration and proliferation. Truncated fragments of the human NG2 promotor were generated and their transcriptional activity was assessed using reporter gene assays. The effect of CK2 inhibition on tumor growth was investigated in xenografts within the flanks of NOD/SCID mice. Finally, effects of CK2 inhibition were analyzed in primary human glioblastoma cells. We found that inhibition of CK2 significantly reduces NG2 protein levels in A1207 (19%±6.2; Mean±SD), U87 (35%±11.3) and primary human glioblastoma cells (41%) when compared to controls. Further analyses revealed that this is due to a decreased NG2 mRNA level. Of note, we identified a 200 base pair fragment, including a binding site for the CK2-dependent transcription factor SP-1. Functional assays showed no cytotoxic effects of the decreased NG2 expression after CK2 inhibition, whereas cell migration and proliferation were markedly reduced. Moreover, we found that CX-4945 treatment decreases tumor growth, which is associated with a diminished NG2 expression (56%±13.0) when compared to controls. In conclusion, we identified CK2 as a novel regulator of NG2 gene expression in human glioblastoma cells. Hence, pharmacological inhibition of CK2 may represent a novel strategy in the therapy of NG2-positive glioblastoma.

Amyloid‐beta 1‐40 is associated with alterations in NG2+ pericyte population ex vivo and in vitro

SummaryThe population of brain pericytes, a cell type important for vessel stability and blood brain barrier function, has recently been shown altered in patients with Alzheimer's disease (AD). The underlying reason for this alteration is not fully understood, but progressive accumulation of the AD characteristic peptide amyloid‐beta (Aβ) has been suggested as a potential culprit. In the current study, we show reduced number of hippocampal NG2+ pericytes and an association between NG2+ pericyte numbers and Aβ1‐40 levels in AD patients. We further demonstrate, using in vitro studies, an aggregation‐dependent impact of Aβ1‐40 on human NG2+ pericytes. Fibril‐EP Aβ1‐40 exposure reduced pericyte viability and proliferation and increased caspase 3/7 activity. Monomer Aβ1‐40 had quite the opposite effect: increased pericyte viability and proliferation and reduced caspase 3/7 activity. Oligomer‐EP Aβ1‐40 had no impact on either of the cellular events. Our findings add to the growing number of studies suggesting a significant impact on pericytes in the brains of AD patients and suggest different aggregation forms of Aβ1‐40 as potential key regulators of the brain pericyte population size.

NG2 cells, a new trail for Alzheimer’s disease mechanisms?

BackgroundNeuron Glial 2 (NG2) cells are glial cells known to serve as oligodendrocyte progenitors as well as modulators of the neuronal network. Altered NG2 cell morphology and up-regulation as well as increased shedding of the proteoglycan NG2 expressed on the cell surface have been described in rodent models of brain injury. Here we describe alterations in the human NG2 cell population in response to pathological changes characteristic of Alzheimer’s disease (AD).ResultsImmunohistological stainings of postmortem brain specimens from clinically diagnosed and postmortem verified AD patients and non-demented controls revealed reduced NG2 immunoreactivity as well as large numbers of NG2 positive astrocytes in individuals with high amyloid beta plaque load. Since fibrillar amyloid beta (Aβ)1-42 is the major component of AD-related senile plaques, we exposed human NG2 cells to oligomer- and fibril enriched preparations of Aβ1-42. We found that both oligomeric and fibrillar Aβ1-42 induced changes in NG2 cell morphology. Further, in vitro exposure to fibrillar Aβ1-42 decreased the NG2 concentrations in both cell lysates and supernatants. Interestingly, we also found significantly decreased levels of soluble NG2 in the cerebrospinal fluid (CSF) from clinically diagnosed AD patients compared to non-demented individuals. Additionally, the CSF NG2 levels were found to significantly correlate with the core AD biomarkers Aß1-42, T-tau and P-tau.ConclusionOur results demonstrate major alterations in the NG2 cell population in relation to AD pathology which highlights the NG2 cell population as a new attractive research target in the search for cellular mechanisms associated with AD pathogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/2051-5960-1-7) contains supplementary material, which is available to authorized users.

Human Ng2+ adipose stem cells loaded in vivo on a new crosslinked hyaluronic acid‐lys scaffold fabricate a skeletal muscle tissue

Mesenchymal stem cell (MSC) therapy holds promise for treating diseases and tissue repair. Regeneration of skeletal muscle tissue that is lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. Human Adipose stem cells (ASCs) have been reported to regenerate muscle fibers and reconstitute the pericytic cell pool after myogenic differentiation in vitro. Our aim was to evaluate the differentiation potential of constructs made from a new cross-linked hyaluronic acid (XHA) scaffold on which different sorted subpopulations of ASCs were loaded. Thirty days after engraftment in mice, we found that NG2(+) ASCs underwent a complete myogenic differentiation, fabricating a human skeletal muscle tissue, while NG2(-) ASCs merely formed a human adipose tissue. Myogenic differentiation was confirmed by the expression of MyoD, MF20, laminin, and lamin A/C by immunofluorescence and/or RT-PCR. In contrast, adipose differentiation was confirmed by the expression of adiponectin, Glut-4, and PPAR-γ. Both tissues formed expressed Class I HLA, confirming their human origin and excluding any contamination by murine cells. In conclusion, our study provides novel evidence that NG2(+) ASCs loaded on XHA scaffolds are able to fabricate a human skeletal muscle tissue in vivo without the need of a myogenic pre-differentiation step in vitro. We emphasize the translational significance of our findings for human skeletal muscle regeneration.

Abstract 1016: Asymmetry-defective oligodendrocyte progenitors are glioma precursors

Abstract Mammalian oligodendrocyte progenitors expressing the chondroitine sulfate proteoglycan NG2 are the largest dividing population in the postnatal, mammalian brain. NG2+ oligodendrocyte progenitors self-renew and generate mature oligodendrocytes. The mechanism by which oligodendrocyte progenitors fulfill their dual function has been unclear. NG2+ oligodendrocyte progenitors are also a cellular origin of oligodendrogliomas, a chemo-sensitive but frequently progressive and lethal type of brain tumor. It is however poorly understood how the normal progenitors turn into tumor cells. We proposed to test whether oligodendrocyte progenitor cells undergo asymmetric cell divisions to generate daughter cells of distinct fate, by asymmetrically segregating cell fate determinants. We further speculated that alterations in asymmetric cell division might predispose cells for or even cause transformation by disrupting the normal balance of self-renewal and differentiation. By analyzing murine oligodendrocyte progenitors in the pair assay, which was developed to visualize asymmetric cell divisions, we discovered that the proteoglycan NG2 segregates asymmetrically, suggestive of asymmetric cell division. By determining the fate of the NG2+ and NG2- daughters, we found that the NG2+ progeny undergoes EGF-dependent self-renewal. In contrast, the NG2- progeny differentiates. By analyzing self-renewal of NG2 deficient oligodendrocyte progenitors from genetically engineered NG2 knock-out mice, we determined that NG2 is required for asymmetric segregation of NG2 and EGF-dependent self-renewal. In a transgenic mouse model for oligodendrogliomas, increased symmetric oligodendrocyte progenitor divisions coincide with abnormal self-renewal rather then differentiation at premalignant stages and leads to neoplastic transformation. Lastly, our analyses of human brain and brain tumor tissue-derived cells showed that asymmetric cell divisions are prevalent in non-neoplastic NG2+ cells and reduced in NG2+ cells from low-grade oligodendrogliomas. Mining of existing brain tumor databases for expression level changes revealed that candidate regulators of asymmetric cell division are frequently mis-expressed in low grade brain oligodendrogliomas. Taken together, we unraveled loss of asymmetric division in premalignant, aberrantly self-renewing differentiation-defective murine oligodendrocyte progenitors and human NG2+ oligodendroglioma cells, thereby explaining initial steps by which oligodendrocyte progenitors transform into oligodendroglioma cells. Our data suggest that asymmetric cell divisions maintain homeostasis in the postnatal oligodendrocyte lineage. In contrast, loss or disruption of asymmetric cell division might be causal to the neoplastic transformation of oligodendrocyte progenitor cells and provide novel points of disruption to which specific glioma therapies can be targeted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1016. doi:1538-7445.AM2012-1016

Asymmetry-Defective Oligodendrocyte Progenitors Are Glioma Precursors

Postnatal oligodendrocyte progenitor cells (OPC) self-renew, generate mature oligodendrocytes, and area cellular origin of oligodendrogliomas. We show that the proteoglycan NG2 segregates asymmetrically during mitosis to generate OPC cells of distinct fate. NG2 is required for asymmetric segregation of EGFR to the NG2(+) progeny, which consequently activates EGFR and undergoes EGF-dependent proliferation and self-renewal. In contrast, the NG2(-) progeny differentiates. In a mouse model, decreased NG2 asymmetry coincides with premalignant, abnormal self-renewal rather than differentiation and with tumor-initiating potential. Asymmetric division of human NG2(+) cells is prevalent in non-neoplastic tissue but is decreased in oligodendrogliomas. Regulators of asymmetric cell division are misexpressed in low-grade oligodendrogliomas. Our results identify loss of asymmetric division associated with the neoplastic transformation of OPC.

NG2 immunoreactivity on human brain endothelial cells.

In this study, we evaluated the expression of NG2 on human brain endothelial cells derived from temporal lobe tissue resected as a treatment for intractable epilepsy. Using dissociated cell cultures, we found expression of NG2 on both proliferating and non-proliferating cells, at the mRNA level by reverse transcription-polymerase chain reaction analyses, and at the protein level by immunocytochemistry and immunoblotting. We further observed that human cerebral microvessels in nonmalignant CNS tissues immunoreacted with NG2. NG2 protein was detected using both a rabbit antibody raised against the rodent NG2 and a monoclonal antibody raised against the human NG2 (9.2.27). Our findings further define the range of resident cells of the CNS that can express NG2 and indicate that expression of NG2 by endothelial cells is not restricted to proliferating CNS endothelial cells or to endothelial cells found in brain tumors.