Single substitution in H3.3G34 alters DNMT3A recruitment to cause progressive neurodegeneration.

Germline histone H3.3 amino acid substitutions, including H3.3G34R/V, cause severe neurodevelopmental syndromes. To understand how these mutations impact brain development, we generated H3.3G34R/V/W knock-in mice and identified strikingly distinct developmental defects for each mutation. H3.3G34R-mutants exhibited progressive microcephaly and neurodegeneration, with abnormal accumulation of disease-associated microglia and concurrent neuronal depletion. G34R severely decreased H3K36me2 on the mutant H3.3 tail, impairing recruitment of DNA methyltransferase DNMT3A and its redistribution on chromatin. These changes were concurrent with sustained expression of complement and other innate immune genes possibly through loss of non-CG (CH) methylation and silencing of neuronal gene promoters through aberrant CG methylation. Complement expression in G34R brains may lead to neuroinflammation possibly accounting for progressive neurodegeneration. Our study reveals that H3.3G34-substitutions have differential impact on the epigenome, which underlie the diverse phenotypes observed, and uncovers potential roles for H3K36me2 and DNMT3A-dependent CH-methylation in modulating synaptic pruning and neuroinflammation in post-natal brains.

Histone H3.3G34-Mutant Interneuron Progenitors Co-opt PDGFRA for Gliomagenesis.

Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here we show that 50% of G34R/V tumors (n = 95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. Although considered gliomas, G34R/V tumors actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V mutations impair neuronal differentiation. The lineage of origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V tumors harbor dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell fate specification. G34R/V may become dispensable for tumor maintenance, whereas mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumors. G34R/V gliomas are neuronal malignancies where interneuron progenitors are stalled in differentiation by G34R/V mutations and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signaling.

p75NTR-dependent Rac1 activation requires receptor cleavage and activation of an NRAGE and NEDD9 signaling cascade.

The p75 neurotrophin receptor (p75NTR, also known as tumor necrosis factor receptor superfamily member 16) is implicated in diverse cellular events, but fundamental aspects of its signaling mechanisms remain unclear. To address this, we have established a novel bioassay to characterize signaling cascades activated by p75NTR. We show that in COS7 cells, p75NTR expression causes a large increase in cell surface area that relies on the activation of Rac1, and we demonstrate that the p75NTR-dependent COS7 phenotype is dependent on ADAM17- and c-secretase-dependent cleavage of p75NTR and generation of the p75NTR intracellular domain (p75NTRICD). We show that the p75NTR adaptor protein NRAGE (also known as MAGED1) acts downstream of the p75NTRICD in this cascade and, through a yeast two-hybrid screen, identify NEDD9, a Cas family adaptor protein, as a novel NRAGE-binding partner that mediates p75NTR-dependent Rac1 activation and cell spreading. Our results demonstrate a crucial role for p75NTR cleavage in small GTPase activation and define a novel Rac1 activation pathway involving the p75NTRICD, NRAGE andNEDD9.

Germline and somatic FGFR1 abnormalities in dysembryoplastic neuroepithelial tumors.

Dysembryoplastic neuroepithelial tumor (DNET) is a benign brain tumor associated with intractable drug-resistant epilepsy. In order to identify underlying genetic alterations and molecular mechanisms, we examined three family members affected by multinodular DNETs as well as 100 sporadic tumors from 96 patients, which had been referred to us as DNETs. We performed whole-exome sequencing on 46 tumors and targeted sequencing for hotspot FGFR1 mutations and BRAF p.V600E was used on the remaining samples. FISH, copy number variation assays and Sanger sequencing were used to validate the findings. By whole-exome sequencing of the familial cases, we identified a novel germline FGFR1 mutation, p.R661P. Somatic activating FGFR1 mutations (p.N546K or p.K656E) were observed in the tumor samples and further evidence for functional relevance was obtained by in silico modeling. The FGFR1 p.K656E mutation was confirmed to be in cis with the germline p.R661P variant. In 43 sporadic cases, in which the diagnosis of DNET could be confirmed on central blinded neuropathology review, FGFR1 alterations were also frequent and mainly comprised intragenic tyrosine kinase FGFR1 duplication and multiple mutants in cis (25/43; 58.1 %) while BRAF p.V600E alterations were absent (0/43). In contrast, in 53 cases, in which the diagnosis of DNET was not confirmed, FGFR1 alterations were less common (10/53; 19 %; p < 0.0001) and hotspot BRAF p.V600E (12/53; 22.6 %) (p < 0.001) prevailed. We observed overexpression of phospho-ERK in FGFR1 p.R661P and p.N546K mutant expressing HEK293 cells as well as FGFR1 mutated tumor samples, supporting enhanced MAP kinase pathway activation under these conditions. In conclusion, constitutional and somatic FGFR1 alterations and MAP kinase pathway activation are key events in the pathogenesis of DNET. These findings point the way towards existing targeted therapies.

p75 neurotrophin receptor cleavage by α- and γ-secretases is required for neurotrophin-mediated proliferation of brain tumor-initiating cells.

Malignant gliomas are highly invasive, proliferative, and resistant to treatment. Previously, we have shown that p75 neurotrophin receptor (p75NTR) is a novel mediator of invasion of human glioma cells. However, the role of p75NTR in glioma proliferation is unknown. Here we used brain tumor-initiating cells (BTICs) and show that BTICs express neurotrophin receptors (p75NTR, TrkA, TrkB, and TrkC) and their ligands (NGF, brain-derived neurotrophic factor, and neurotrophin 3) and secrete NGF. Down-regulation of p75NTR significantly decreased proliferation of BTICs. Conversely, exogenouous NGF stimulated BTIC proliferation through α- and γ-secretase-mediated p75NTR cleavage and release of its intracellular domain (ICD). In contrast, overexpression of the p75NTR ICD induced proliferation. Interestingly, inhibition of Trk signaling blocked NGF-stimulated BTIC proliferation and p75NTR cleavage, indicating a role of Trk in p75NTR signaling. Further, blocking p75NTR cleavage attenuated Akt activation in BTICs, suggesting role of Akt in p75NTR-mediated proliferation. We also found that p75NTR, α-secretases, and the four subunits of the γ-secretase enzyme were elevated in glioblastoma multiformes patients. Importantly, the ICD of p75NTR was commonly found in malignant glioma patient specimens, suggesting that the receptor is activated and cleaved in patient tumors. These results suggest that p75NTR proteolysis is required for BTIC proliferation and is a novel potential clinical target.

Neurotrophins in the regulation of cellular survival and death.

The neurotrophins play crucial roles regulating survival and apoptosis in the developing and injured nervous system. The four neurotrophins exert profound and crucial survival effects on developing peripheral neurons, and their expression and action is intimately tied to successful innervation of peripheral targets. In the central nervous system, they are dispensable for neuronal survival during development but support neuronal survival after lesion or other forms of injury. Neurotrophins also regulate apoptosis of both peripheral and central neurons, and we now recognize that there are regulatory advantages to having the same molecules regulate life and death decisions. This chapter examines the biological contexts in which these events take place and highlights the specific ligands, receptors, and signaling mechanisms that allow them to occur.

Immune landscape of oncohistone-mutant gliomas reveals diverse myeloid populations and tumor-promoting function.

Histone H3-mutant gliomas are deadly brain tumors characterized by a dysregulated epigenome and stalled differentiation. In contrast to the extensive datasets available on tumor cells, limited information exists on their tumor microenvironment (TME), particularly the immune infiltrate. Here, we characterize the immune TME of H3.3K27M and G34R/V-mutant gliomas, and multiple H3.3K27M mouse models, using transcriptomic, proteomic and spatial single-cell approaches. Resolution of immune lineages indicates high infiltration of H3-mutant gliomas with diverse myeloid populations, high-level expression of immune checkpoint markers, and scarce lymphoid cells, findings uniformly reproduced in all H3.3K27M mouse models tested. We show these myeloid populations communicate with H3-mutant cells, mediating immunosuppression and sustaining tumor formation and maintenance. Dual inhibition of myeloid cells and immune checkpoint pathways show significant therapeutic benefits in pre-clinical syngeneic mouse models. Our findings provide a valuable characterization of the TME of oncohistone-mutant gliomas, and insight into the means for modulating the myeloid infiltrate for the benefit of patients.

Targeting the IGF-Axis in Cultured Pediatric High-Grade Glioma Cells Inhibits Cell Cycle Progression and Survival.

Pediatric high-grade gliomas (pHGG) accounts for approximately 8-12% of primary brain tumors in children. Prognosis is poor, with a median survival of 9-15 months. Insulin-like growth factor 1-receptor (IGF-1R) gene amplifications have been identified in high-grade gliomas and may contribute to its highly aggressive phenotype, but the effect of IGF inhibitors on pHGG is yet to be determined. In the present study, we analyzed the response of patient-derived pediatric high-grade glioma cells to a novel IGF-1R inhibitor, the IGF-Trap. Using immunohistochemistry, we found that IGF-1R was localized to both the nucleus and cell membrane in different pHGG patient-derived xenograft (PDX) lines under basal conditions. In response to ligand binding, nuclear levels of the receptor increased, and this was associated with the transcriptional upregulation of both the receptor and cyclin D1, suggesting that IGF-1R could regulate its own expression and cell cycle progression in these cells. Insulin-like growth factor-1 (IGF-1) increased the proliferation of the pHGG cells DIPG13 and SGJ2, and this could be blocked by the addition of the IGF-Trap. The IGF-Trap reduced the colony formation of these cells in an optimal growth medium and impeded the ability of IGF-1 to rescue DIPG13 cells from starvation-induced apoptosis. Collectively, these results implicate the IGF-1 axis in the regulation of cell cycle progression, cellular proliferation, and cell survival in pHGG, and identify the IGF-axis as a target and the IGF-Trap as a potential inhibitor of this axis in pHGG.

Potential neuroprotective effects of continuous topiramate therapy in the developing brain.

Because antiepileptic drug therapy is usually given chronically with resulting concerns about long-term neurotoxicity, and because short-term topiramate (TPM) therapy has been reported to be neuroprotective against the effects of acute hypoxia, we investigated the long-term effects of continuous TPM therapy during early stages of development. Four groups of rat pups were studied: two sham manipulated normoxia groups and two acute hypoxia groups (at postnatal day [P] 10 down to 4% O(2)), each injected intraperitoneally daily with either vehicle or TPM (30 mg/kg) from P0 to P21. TPM therapy prevented hypoxia-induced long-term (P81) memory impairment (Morris water maze) as well as aggressivity (handling test). The hypoxia group receiving TPM also showed a trend toward reduced CA1 hippocampal cell loss. The aforementioned TPM therapy had no long-term deleterious effects on memory, hyperactivity, or CA1 cell counts in the TPM normoxia group as compared with normal controls.

Glioblastoma cell populations with distinct oncogenic programs release podoplanin as procoagulant extracellular vesicles.

Vascular anomalies, including local and peripheral thrombosis, are a hallmark of glioblastoma (GBM) and an aftermath of deregulation of the cancer cell genome and epigenome. Although the molecular effectors of these changes are poorly understood, the upregulation of podoplanin (PDPN) by cancer cells has recently been linked to an increased risk for venous thromboembolism (VTE) in GBM patients. Therefore, regulation of this platelet-activating protein by transforming events in cancer cells is of considerable interest. We used single-cell and bulk transcriptome data mining, as well as cellular and xenograft models in mice, to analyze the nature of cells expressing PDPN, as well as their impact on the activation of the coagulation system and platelets. We report that PDPN is expressed by distinct (mesenchymal) GBM cell subpopulations and downregulated by oncogenic mutations of EGFR and IDH1 genes, along with changes in chromatin modifications (enhancer of zeste homolog 2) and DNA methylation. Glioma cells exteriorize their PDPN and/or tissue factor (TF) as cargo of exosome-like extracellular vesicles (EVs) shed from cells in vitro and in vivo. Injection of glioma-derived podoplanin carrying extracelluar vesicles (PDPN-EVs) activates platelets, whereas tissue factor carrying extracellular vesicles (TF-EVs) activate the clotting cascade. Similarly, an increase in platelet activation (platelet factor 4) or coagulation (D-dimer) markers occurs in mice harboring the corresponding glioma xenografts expressing PDPN or TF, respectively. Coexpression of PDPN and TF by GBM cells cooperatively affects tumor microthrombosis. Thus, in GBM, distinct cellular subsets drive multiple facets of cancer-associated thrombosis and may represent targets for phenotype- and cell type-based diagnosis and antithrombotic intervention.

Gap junctional intercellular communication in hypoxia-ischemia-induced neuronal injury.

Brain hypoxia-ischemia is a relatively common and serious problem in neonates and in adults. Its consequences include long-term histological and behavioral changes and reduction in seizure threshold. Gap junction intercellular communication is pivotal in the spread of hypoxia-ischemia related injury and in mediating its long-term effects. This review provides a comprehensive and critical review of hypoxia-ischemia and hypoxia in the brain and the potential role of gap junctions in the spread of the neuronal injury induced by these insults. It also presents the effects of hypoxia-ischemia and of hypoxia on the state of gap junctions in vitro and in vivo. Understanding the mechanisms involved in gap junction-mediated neuronal injury due to hypoxia will lead to the development of novel therapeutic strategies.

H3K27M induces defective chromatin spread of PRC2-mediated repressive H3K27me2/me3 and is essential for glioma tumorigenesis.

Lys-27-Met mutations in histone 3 genes (H3K27M) characterize a subgroup of deadly gliomas and decrease genome-wide H3K27 trimethylation. Here we use primary H3K27M tumor lines and isogenic CRISPR-edited controls to assess H3K27M effects in vitro and in vivo. We find that whereas H3K27me3 and H3K27me2 are normally deposited by PRC2 across broad regions, their deposition is severely reduced in H3.3K27M cells. H3K27me3 is unable to spread from large unmethylated CpG islands, while H3K27me2 can be deposited outside these PRC2 high-affinity sites but to levels corresponding to H3K27me3 deposition in wild-type cells. Our findings indicate that PRC2 recruitment and propagation on chromatin are seemingly unaffected by K27M, which mostly impairs spread of the repressive marks it catalyzes, especially H3K27me3. Genome-wide loss of H3K27me3 and me2 deposition has limited transcriptomic consequences, preferentially affecting lowly-expressed genes regulating neurogenesis. Removal of H3K27M restores H3K27me2/me3 spread, impairs cell proliferation, and completely abolishes their capacity to form tumors in mice.

Mutant H3 histones drive human pre-leukemic hematopoietic stem cell expansion and promote leukemic aggressiveness.

Our ability to manage acute myeloid leukemia (AML) is limited by our incomplete understanding of the epigenetic disruption central to leukemogenesis, including improper histone methylation. Here we examine 16 histone H3 genes in 434 primary AML samples and identify Q69H, A26P, R2Q, R8H and K27M/I mutations (1.6%), with higher incidence in secondary AML (9%). These mutations occur in pre-leukemic hematopoietic stem cells (HSCs) and exist in the major leukemic clones in patients. They increase the frequency of functional HSCs, alter differentiation, and amplify leukemic aggressiveness. These effects are dependent on the specific mutation. H3K27 mutation increases the expression of genes involved in erythrocyte and myeloid differentiation with altered H3K27 tri-methylation and K27 acetylation. The functional impact of histone mutations is independent of RUNX1 mutation, although they at times co-occur. This study establishes that H3 mutations are drivers of human pre-cancerous stem cell expansion and important early events in leukemogenesis.

Changes in sphingomyelinases, ceramide, Bax, Bcl(2), and caspase-3 during and after experimental status epilepticus.

Status epilepticus (SE) induces a number of events leading to programmed cell death (PCD). The aim of our work is to study the time sequence of activation of different factors in experimental SE (intraperitoneal kainic acid (KA) model). We studied ceramide, a known mediator of apoptosis in multiple models, sphingomyelinases (SMases), enzymes that break down sphingomyelin and increase ceramide thus leading to apoptosis in many models, Bcl(2), Bax, and caspase-3. SE induced a sustained ceramide increase starting 2h after kainic acid injection followed by an increase in Bax protein at 6 and 12h, and the appearance of caspase-3-activated fragment (caspase-3a) immunostaining and TUNEL positivity at 12h. Status epilepticus also induced an increase in acidic and neutral sphingomyelinases that preceded (acidic sphingomyelinase) and parallelled (acidic and neutral sphingomyelinase) the increases in ceramide. These data suggest that, in this model, Bax is activated early in the process and that its increase is sustained till 12h after kainic acid injection which is the time of first appearance of caspase-3 activation and TUNEL positivity, and that SMases contribute to increases in ceramide levels during and after status epilepticus.

Programmed cell death in the lithium pilocarpine model: evidence for NMDA receptor and ceramide-mediated mechanisms.

Ceramide is known to induce programmed cell death (PCD) in neural and non-neural tissues and to increase after kainic acid (KA) status epilepticus (SE). Ceramide increases have been shown to depend on NMDA receptor activation in the KA model, but these changes have not been studied in the lithium pilocarpine (LiPC) model. Thus, the purpose of this study was to determine if hippocampal ceramide levels increase after LiPC induced SE and if NMDA receptor blockade prevents PCD and any such ceramide increases. We found that LiPC induced SE resulted in ceramide increases and DNA fragmentation in the hippocampus of adult, P21, and P7 rats. The administration of MK-801, the NMDA receptor antagonist, in adults, 15min prior to pilocarpine, prevented ceramide increases, and DNA fragmentation.

Long-term effects of acute and of chronic hypoxia on behavior and on hippocampal histology in the developing brain.

Ten-day-old rat pups (P10) subjected to acute hypoxia (down to 4% O2) had as adults increased aggression (handling test), memory impairment (water maze test), and decreased CA1 cell counts. Pups subjected to chronic hypoxia (10% O2 from P0 to P21) had increased aggression, hyperactivity (open-field test), and decreased CA1 cell counts. Chronic hypoxia with superimposed acute hypoxia resulted in consequences that were not different from those of chronic hypoxia.

Differential expression of hippocampal connexins after acute hypoxia in the developing brain.

Acute hypoxia at postnatal day (P) 10 is an accepted model of human neonatal hypoxia which results, among other consequences, in increased hippocampal excitability. Hypoxic-ischemic injury, which mimics stroke, has been shown to result in changes in connexins (Cxs), however, changes in Cxs have not been studied in the P10 hypoxia model. The aim of this study was to investigate changes in the hippocampal expression of three different connexins at consecutive developmental stages after acute hypoxia at P10 (10min and 30min after reoxygenation, P11, P14, P17, P29, and P45) as compared to sham manipulated pups. After acute hypoxia at P10, Cx30 protein levels were increased at 30min after reoxygenation, at P11 and at P14, and then returned to control levels. Cx36 protein levels transiently decreased at P11 after acute hypoxia then returned to control levels. Cx43 protein levels did not change at any of the time points. Although changes in mRNA expression were observed during development for Cx30 only, acute hypoxia did not result in changes in mRNA expression of all these Cxs when compared to age matched controls suggesting that acute hypoxia induced posttranslational changes in protein expression.