PPM1D mutations are oncogenic drivers of de novo diffuse midline glioma formation.

The role of PPM1D mutations in de novo gliomagenesis has not been systematically explored. Here we analyze whole genome sequences of 170 pediatric high-grade gliomas and find that truncating mutations in PPM1D that increase the stability of its phosphatase are clonal driver events in 11% of Diffuse Midline Gliomas (DMGs) and are enriched in primary pontine tumors. Through the development of DMG mouse models, we show that PPM1D mutations potentiate gliomagenesis and that PPM1D phosphatase activity is required for in vivo oncogenesis. Finally, we apply integrative phosphoproteomic and functional genomics assays and find that oncogenic effects of PPM1D truncation converge on regulators of cell cycle, DNA damage response, and p53 pathways, revealing therapeutic vulnerabilities including MDM2 inhibition.

Potential role of NGF, BDNF, and their receptors in oligodendrocytes differentiation from neural stem cell: An in vitro study.

Neural stem cells (NSCs) or neuronal progenitor cells are cells capable of differentiating into oligodendrocytes, myelin-forming cells that have the potential of remyelination. Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are two neurotrophic factors that have been studied to stimulate NSC differentiation thus playing a role in multiple sclerosis pathogenesis and several other demyelinating disorders. While several studies have demonstrated the proliferative and protective capabilities of these neurotrophic factors, their cellular and molecular functions are still not well understood. Thus, in the present study, we focus on understanding the role of these neurotrophins (BDNF and NGF) in oligodendrogenesis from NSCs. Both neurotrophic factors have been shown to promote NSC proliferation and NSC differentiation particularly into oligodendroglial lineage in a dose-dependent fashion. Further, to establish the role of these neurotrophins in NSC differentiation, we have employed pharmacological inhibitors for TrkA and TrkB receptors in NSCs. The use of these inhibitors suppressed NSC differentiation into oligodendrocytes along with the downregulation of phosphorylated ERK suggesting active involvement of ERK in the functioning of these neurotrophins. The morphometric analysis also revealed the important role of both neurotrophins in oligodendrocytes development. These findings highlight the importance of neurotrophic factors in stimulating NSC differentiation and may pave a role for future studies to develop neurotrophic factor replacement therapies to achieve remyelination.

Gamma-Radiation-Induced Endoplasmic Reticulum Stress and Downregulation of WFS1, Nectin 3, and Sostdc1 Gene Expression in Mice Hippocampus.

INTRODUCTION: Neurogenesis mainly occurs in the hippocampus that is sensitive to radiation. More histological changes are reported at higher doses of radiation, while low dose radiation causes cognitive dysfunction in adult mammals. In the present study, we tried to correlate the Endoplasmic Reticulum (ER) stress-mediated hippocampus dysfunction after whole-body gamma radiation of mice. METHODS: Mice were exposed to a series of gamma radiations, followed by isolation of hippocampus. To elucidate the gene expression profile, qPCR was performed for ER stress markers CHOP, BiP, and hippocampal specific genes WFS1, Nectin 3, and Sostdc 1 on the isolated hippocampus. Expression of CHOP and ERK½ were analyzed by western blot on exposure to gamma radiation. RESULTS: qPCR results showed a significant increase in the expression of ER stress-specific genes CHOP, BiP, and decrease in hippocampal specific genes WFS1, Nectin3, and Sostdc1. Western blot study suggests a significant increase in ER stress proteins like CHOP and ERK½ expression. CONCLUSION: Exposure to gamma radiation significantly increased the expression of ER-stress genes, suggesting that ER stress plays a major role in inducing radiation mediated dysfunction of the hippocampus. Also, significant downregulation of WFS1, Nectin3, and Sostdc1 genes suggests radiation mediated effect of hippocampal CA 1, CA 2, and CA 3 regions. A further significant increase of ERK½ shows involvement of the ERK pathway in mediating radiation-induced ER stress dysfunction in mice hippocampus. The present findings may lead to the identification of ER stress as a new marker to study radiation-induced neurodegenerative disorder.

Neuroprotective effect of quercetin against radiation-induced endoplasmic reticulum stress in neurons.

The endoplasmic reticulum (ER) plays an important role in the regulation and maintenance of cellular homeostasis. However, unresolved ER stress leads to deleterious effects by inducing the accumulation of unfolded proteins in the cell. Here we have demonstrated the protective aspects of quercetin against radiation-induced ER stress and against inflammation in primary cultured dorsal root ganglion (DRG) neurons. The mature DRG neurons were pretreated with different concentrations of quercetin (5-100 µM) for 24 hours before 2 Gy gamma radiation exposure and then subjected to a cytotoxicity assay, quantitative real-time polymerase chain reaction and Western blot analysis. The results showed that quercetin decreased the expression of BiP and C/EBP-homologous protein, the ER stress marker genes along with downregulation of tumor necrosis factor-α, JNK in irradiated DRG neurons. Furthermore, quercetin pretreatment significantly increased the cytoskeletal protein Tuj1 and the neurotrophin brain-derived neurotrophic factor in the neuron. These results indicate that quercetin plays a neuroprotective role against radiation-mediated ER stress and inflammatory responses.

Role of astrocytic MeCP2 in regulation of CNS myelination by affecting oligodendrocyte and neuronal physiology and axo-glial interactions.

Astrocytes perform several critical functions such as promoting neuronal maturation, neuronal survival, maintaining and supporting neurons and oligodendrocytes. Astrocytes participate in the formation of nodes of Ranvier. Recently, studies emphasizing on the role of astrocytes in regulating myelination by secreting pro-myelinating factors like growth factors, neurotrophins and ECM proteins, have been investigated by many researchers. Methyl-CpG-Binding Protein 2 (MeCP2), an epigenetic protein, binds to CpG islands in the genome and induces multiple gene regulatory functions by conforming changes in the chromatin structure and resulting in cell-specific gene expression. MeCP2 deficient astrocytes have been linked with abnormal neuronal function including decreased dendritic arborization and decreased dendritic outgrowth. However, role of astrocytic MeCP2 in central nervous system myelination is largely not known. The data from the current study indicate altered mRNA levels (Lif, Cntf, Pdgfa, Cxcl10) of astrocyte-secreted factors involved in myelination. Bdnf and Ngf mRNA levels were also altered in MeCP2 knockdown astrocytes. Moreover, the secreted BDNF levels were significantly altered whereas there were no significant changes in NGF secretion. We also observed that astrocytic MeCP2 affects the morphology, physiology and survival of oligodendrocytes and neurons-two of the key players in myelination. Further, we report that some of the axo-glial interaction genes, namely Caspr, Notch1, Nf155 and Nrg1 are under the regulation of astrocytic MeCP2 along with key myelin genes and proteins.

ER stress and genomic instability induced by gamma radiation in mice primary cultured glial cells.

Ionizing radiation induces various pathophysiological conditions by altering central nervous system (CNS) homeostasis, leading to neurodegenerative diseases. However, the potential effect of ionizing radiation response on cellular physiology in glial cells is unclear. In the present study, micronucleus test, comet assay, and RT-PCR were performed to investigate the potential effect of gamma radiation in cultured oligodendrocytes and astrocytes with respect to genomic instability, Endoplasmic Reticulum (ER) stress, and inflammation. Further, we studied the effect of alteration in ER stress specific gene expression in cortex post whole body radiation in mice. Results showed that exposure of gamma radiation of 2Gy in-vitro cultured astrocytes and oligodendrocytes and 7Gy in-vivo induced ER stress and Inflammation along with profuse DNA damage and Chromosomal abnormality. Additionally, we observed downregulation of myelin basic protein levels in cultured oligodendrocytes exposed to radiation. The present data suggests that ER stress and pro inflammatory cytokines serve as the major players in inducing glial cell dysfunction post gamma irradiation along with induction of genomic instability. Taken together, these results indicate that ER stress, DNA damage, and inflammatory pathways may be critical events leading to glial cell dysfunction and subsequent cell death following exposure to ionizing radiation.