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Epilepsy is a heterogenous group of disorders defined by recurrent seizure activity due to abnormal synchronized activity of neurons. A growing number of epilepsy cases are believed to be caused by genetic factors and copy number variants (CNV) contribute to up to 5% of epilepsy cases. However, CNVs in epilepsy are usually large deletions or duplications involving multiple neurodevelopmental genes. In patients who underwent seizure focus resection for treatment-resistant epilepsy, whole genome DNA methylation profiling identified 3 main clusters of which one showed strong association with receptor tyrosine kinase (RTK) genes. We identified focal copy number gains involving epidermal growth factor receptor (EGFR) and PDGFRA loci. The dysplastic neurons of cases with amplifications showed marked overexpression of EGFR and PDGFRA, while glial and endothelial cells were negative. Targeted sequencing of regulatory regions and DNA methylation analysis revealed that only enhancer regions of EGFR and gene promoter of PDGFRA were amplified, while coding regions did not show copy number abnormalities or somatic mutations. Somatic focal copy number gains of noncoding regulatory represent a previously unrecognized genetic driver in epilepsy and a mechanism of abnormal activation of RTK genes. Upregulated RTKs provide a potential avenue for therapy in seizure disorders.

The histone methyltransferase activity of PRC2 is central to the formation of H3K27me3-decorated facultative heterochromatin and gene silencing. In addition, PRC2 has been shown to automethylate its core subunits, EZH1/EZH2 and SUZ12. Here, we identify the lysine residues at which EZH1/EZH2 are automethylated with EZH2-K510 and EZH2-K514 being the major such sites in vivo. Automethylated EZH2/PRC2 exhibits a higher level of histone methyltransferase activity and is required for attaining proper cellular levels of H3K27me3. While occurring independently of PRC2 recruitment to chromatin, automethylation promotes PRC2 accessibility to the histone H3 tail. Intriguingly, EZH2 automethylation is significantly reduced in diffuse intrinsic pontine glioma (DIPG) cells that carry a lysine-to-methionine substitution in histone H3 (H3K27M), but not in cells that carry either EZH2 or EED mutants that abrogate PRC2 allosteric activation, indicating that H3K27M impairs the intrinsic activity of PRC2. Our study demonstrates a PRC2 self-regulatory mechanism through its EZH1/2-mediated automethylation activity.

SummaryDespite a prominent risk factor for Neurodevelopmental disorders (NDD), it remains unclear how Autism Susceptibility Candidate 2 (AUTS2) controls the neurodevelopmental program. Our studies investigated the role of AUTS2 in neuronal differentiation and discovered that AUTS2, together with WDR68 and SKI, forms a novel protein complex (AWS) specifically in neuronal progenitors and promotes neuronal differentiation through inhibiting BMP signaling. Genomic and biochemical analyses demonstrated that the AWS complex achieves this effect by recruiting the CUL4 E3 ubiquitin ligase complex to mediate poly-ubiquitination and subsequent proteasomal degradation of phosphorylated SMAD1/5/9. Furthermore, using primary cortical neurons, we observed aberrant BMP signaling and dysregulated expression of neuronal genes upon manipulating the AWS complex, indicating that the AWS-CUL4-BMP axis plays a role in regulating neuronal lineage specification in vivo. Thus, our findings uncover a sophisticated cellular signaling network mobilized by a prominent NDD risk factor, presenting multiple potential therapeutic targets for NDD.

SUMMARYThe heterogeneous complexes comprising the family of Polycomb Repressive Complex 1 (PRC1) are instrumental to establishing facultative heterochromatin that is repressive to transcription. Yet, two PRC1 species, PRC1.3 and PRC1.5, are known to comprise novel components, AUTS2, P300, and CK2 that convert this repressive function to that of transcription activation. Here, we report that patients harboring mutations in the HX repeat domain of AUTS2 exhibit defects in AUTS2 and P300 interaction as well as a developmental disorder reflective of Rubinstein-Taybi syndrome, which is mostly associated with a heterozygous pathogenic variant inCREBBP/EP300. As well, the absence of AUTS2 gives rise to a mis-regulation of a subset of developmental genes and curtails motor neuron differentiation from embryonic stem cells in the context of a well-defined system. Moreover, the transcription factor, Nuclear Respiratory Factor 1 (NRF1) exhibits a novel and integral role in this aspect of the neurodevelopmental process, being required for PRC1.3 recruitment to chromatin.

Diffuse intrinsic pontine glioma (DIPG) is a group of predominantly pediatric brain tumors with an average age of diagnosis of 6–7 years old, and a poor prognosis (median survival of ~1 year). Given the location of DIPG in the brainstem, surgical approaches are limited. Furthermore, the tumors have limited responsivity to traditional chemotherapy or radiotherapy, ergo new therapeutic options are needed. Recently, the drug ONC201 has emerged as a potential therapeutic option with outcomes sometimes surpassing progression-free and expected survival outcomes. However, the selectivity of its effect and mechanism in DIPG is still unclear. Here, we pursue a better understanding of ONC201 and its mechanism of action directly in DIPG patient-derived cell lines. First, we demonstrate that a range of DIPG cell lines are highly sensitive to ONC201 and compare this sensitivity to other patient-derived brain tumor and normal controls. Next, we directly show that the mitochondrial protease, ClpP is the primary target of ONC201 in DIPG. Given recent literature implicating the activation of ClpP by ONC201 and dysregulation of the metabolome in other tumors, we are currently examining these downstream effects in DIPG. Ultimately we hope to elucidate whether ClpP targeting can be used to better diagnose and improve therapeutic options in DIPG

Summary: Low-grade astrocytomas (LGAs) carry neomorphic mutations in isocitrate dehydrogenase (IDH) concurrently with P53 and ATRX loss. To model LGA formation, we introduced R132H IDH1, P53 shRNA, and ATRX shRNA into human neural stem cells (NSCs). These oncogenic hits blocked NSC differentiation, increased invasiveness in vivo, and led to a DNA methylation and transcriptional profile resembling IDH1 mutant human LGAs. The differentiation block was caused by transcriptional silencing of the transcription factor SOX2 secondary to disassociation of its promoter from a putative enhancer. This occurred because of reduced binding of the chromatin organizer CTCF to its DNA motifs and disrupted chromatin looping. Our human model of IDH mutant LGA formation implicates impaired NSC differentiation because of repression of SOX2 as an early driver of gliomagenesis. : In a human neural stem cell model of low-grade astrocytoma, Modrek et al. show that mutant IDH1 and loss of P53 and ATRX together block differentiation via disassociation of SOX2 from putative enhancers. This occurs because of disruption of chromatin looping secondary to hypermethylation at CTCF motifs. Keywords: low-grade glioma, astrocytoma, IDH, P53, ATRX, neural stem cells, SOX2, chromatin looping, CTCF, DNA methylation

IDH1/2 mutations are early drivers present in diverse human cancer types arising in various tissue sites. IDH1/2 mutation is known to induce a global hypermethylator phenotype. However, the effects on DNA methylation across IDH mutant cancers and functionally different genome regions, remain unknown. We analyzed DNA methylation data from IDH1/2 mutant acute myeloid leukemia, oligodendroglioma, astrocytoma, solid papillary breast carcinoma with reverse polarity, sinonasal undifferentiated carcinoma and cholangiocarcinoma, which clustered by their embryonal origin. Hypermethylated common probes affect predominantly gene bodies while promoters in IDH1/2 mutant cancers remain unmethylated. Enhancers showed global hypermethylation, however commonly hypomethylated enhancers were associated with tissue differentiation and cell fate determination. We demonstrate that some chromosomes, chromosomal arms and chromosomal regions are more affected by IDH1/2 mutations while others remain resistant to IDH1/2 mutation induced methylation changes. Therefore IDH1/2 mutations have different methylation effect on different parts of the genome, which may be regulated by different mechanisms.

Chromatin-bound LEDGF and HDGF2 proteins allow RNAPII to overcome the nucleosome-induced block to transcription., FACT (facilitates chromatin transcription) is a protein complex that allows RNA polymerase II (RNAPII) to overcome the nucleosome-induced barrier to transcription. While abundant in undifferentiated cells and many cancers, FACT is not abundant or is absent in most tissues. Therefore, we screened for additional proteins that might replace FACT upon differentiation. We identified two proteins, lens epithelium-derived growth factor (LEDGF) and hepatoma-derived growth factor 2 (HDGF2), each containing two high mobility group A (HMGA)–like AT-hooks and a methyl-lysine reading Pro-Trp-Trp-Pro (PWWP) domain that binds to H3K36me2 and H3K36me3.LEDGF and HDGF2 colocalize with H3K36me2/3 at genomic regions containing active genes. In myoblasts, LEDGF and HDGF2 are enriched on most active genes. Upon differentiation to myotubes, LEDGF levels decrease, while HDGF2 levels are maintained. Moreover, HDGF2 is required for their proper expression. HDGF2 knockout myoblasts exhibit an accumulation of paused RNAPII within the transcribed region of many HDGF2 target genes, indicating a defect in early elongation.

Although mTOR signaling is known as a broad regulator of cell growth and proliferation, in neurons it regulates synaptic transmission, which is thought to be a major mechanism through which altered mTOR signaling leads to neurological disease. Although previous studies have delineated postsynaptic roles for mTOR, whether it regulates presynaptic function is largely unknown. Moreover, the mTOR kinase operates in two complexes, mTORC1 and mTORC2, suggesting that mTOR’s role in synaptic transmission may be complex-specific. To better understand their roles in synaptic transmission, we genetically inactivated mTORC1 or mTORC2 in cultured mouse glutamatergic hippocampal neurons. Inactivation of either complex reduced neuron growth and evoked EPSCs (eEPSCs), however, the effects of mTORC1 on eEPSCs were postsynaptic and the effects of mTORC2 were presynaptic. Despite postsynaptic inhibition of evoked release, mTORC1 inactivation enhanced spontaneous vesicle fusion and replenishment, suggesting that mTORC1 and mTORC2 differentially modulate postsynaptic responsiveness and presynaptic release to optimize glutamatergic synaptic transmission.

As the process that silences gene expression ensues during development, the stage is set for the activity of Polycomb-repressive complex 2 (PRC2) to maintain these repressed gene profiles. PRC2 catalyzes a specific histone posttranslational modification (hPTM) that fosters chromatin compaction. PRC2 also facilitates the inheritance of this hPTM through its self-contained “write and read” activities, key to preserving cellular identity during cell division. As these changes in gene expression occur without changes in DNA sequence and are inherited, the process is epigenetic in scope. Mutants of mammalian PRC2 or of its histone substrate contribute to the cancer process and other diseases, and research into these aberrant pathways is yielding viable candidates for therapeutic targeting. The effectiveness of PRC2 hinges on its being recruited to the proper chromatin sites; however, resolving the determinants to this process in the mammalian case was not straightforward and thus piqued the interest of many in the field. Here, we chronicle the latest advances toward exposing mammalian PRC2 and its high maintenance.

Emerging evidence from clinical and preclinical studies suggests that the imipridone ONC201 is well tolerated and may have some clinical impact in discrete diffuse intrinsic pontine glioma patients (DIPG). A primary goal of our work is to determine if DIPG are uniquely sensitive to ONC201 and if so, whether ONC201 itself can be used as a tool to illuminate novel vulnerabilities in DIPG. To accomplish this, we are utilizing a combination of patient-derived cell lines as well as mouse xenografts that dovetail with a variety of molecular, epigenetic and metabolomic tools. A central finding from our work is that ONC201 primarily activates the mitochondrial protease, ClpP in DIPG patient-derived cell lines, an effect consistent with recently described ONC201 mechanism of action in other tumors. We further demonstrate that activation of ClpP by ONC201 leads to a host of downstream effects in DIPG model systems including distinctive effects on the metabolome leading to direct alterations in the unique epigenetic signature of DIPG. By directly manipulating these metabolic and epigenetic factors we provide prospective mechanistic insight into how ONC201 as well as ClpP activity impacts DIPG growth and tumorigenicity. These preclinical research findings shed light on potential therapeutic vulnerabilities in DIPG as well as ways that these strategies may be combined to enhance their potential.

FACT (facilitates chromatin transcription) is a protein complex that allows RNAPII to overcome the nucleosome-induced barrier to transcription. While abundant in undifferentiated cells and many cancers, FACT is not abundant or is absent in most tissues. Therefore, we screened for additional proteins that might replace FACT upon differentiation. Here we report the identification of two such proteins, LEDGF and HDGF2, each containing two HMGA-like AT-hooks and a PWWP methyl-lysine reading domain known to bind to H3K36me2 and H3K36me3. LEDGF and HDGF2 localize with H3K36me2/3 at genomic regions containing active genes, usually adjacent to H3K27me3 domains. In myoblasts, where FACT expression is low, LEDGF and HDGF2 are enriched on most active genes. Upon differentiation to myotubes, LEDGF levels decrease across the genome while HDGF2 levels are maintained. Moreover, HDGF2 is recruited to the majority of myotube up-regulated genes and is required for their proper expression. HDGF2 knockout myoblasts exhibit an accumulation of paused RNAPII proximal to the first nucleosome within the transcribed region of many HDGF2 target genes, indicating a defect in early elongation. We propose that LEDGF and HDGF2 substitute for FACT in differentiated tissues and that their distribution on the genome helps maintain transcriptional programs unique to particular cell types. One Sentence Summary Chromatin bound LEDGF and HDGF2 proteins allow RNAPII to overcome the nucleosome-induced block to transcription.

Purpose: Diffuse intrinsic pontine glioma (DIPG) is the most aggressive primary pediatric brain tumor, with

H3K27M transiently recruits PRC2 to chromatin but persistently affects its activity, leading to an aberrant epigenome in DIPG., A methionine substitution at lysine-27 on histone H3 variants (H3K27M) characterizes ~80% of diffuse intrinsic pontine gliomas (DIPG) and inhibits polycomb repressive complex 2 (PRC2) in a dominant-negative fashion. Yet, the mechanisms for this inhibition and abnormal epigenomic landscape have not been resolved. Using quantitative proteomics, we discovered that robust PRC2 inhibition requires levels of H3K27M greatly exceeding those of PRC2, seen in DIPG. While PRC2 inhibition requires interaction with H3K27M, we found that this interaction on chromatin is transient, with PRC2 largely being released from H3K27M. Unexpectedly, inhibition persisted even after PRC2 dissociated from H3K27M-containing chromatin, suggesting a lasting impact on PRC2. Furthermore, allosterically activated PRC2 is particularly sensitive to H3K27M, leading to the failure to spread H3K27me from PRC2 recruitment sites and consequently abrogating PRC2’s ability to establish H3K27me2-3 repressive chromatin domains. In turn, levels of polycomb antagonists such as H3K36me2 are elevated, suggesting a more global, downstream effect on the epigenome. Together, these findings reveal the conditions required for H3K27M-mediated PRC2 inhibition and reconcile seemingly paradoxical effects of H3K27M on PRC2 recruitment and activity.

Pineoblastoma is a rare and highly aggressive brain cancer of childhood, histologically belonging to the spectrum of primitive neuroectodermal tumors. Patients with germline mutations in DICER1, a ribonuclease involved in microRNA processing, have increased risk of pineoblastoma, but genetic drivers of sporadic pineoblastoma remain unknown. Here, we analyzed pediatric and adult pineoblastoma samples (n = 23) using a combination of genome-wide DNA methylation profiling and whole-exome sequencing or whole-genome sequencing. Pediatric and adult pineoblastomas showed distinct methylation profiles, the latter clustering with lower-grade pineal tumors and normal pineal gland. Recurrent variants were found in genes involved in PKA- and NF-κB signaling, as well as in chromatin remodeling genes. We identified recurrent homozygous deletions of DROSHA, acting upstream of DICER1 in microRNA processing, and a novel microduplication involving chromosomal region 1q21 containing PDE4DIP (myomegalin), comprising the ancient DUF1220 protein domain. Expresion of PDE4DIP and DUF1220 proteins was present exclusively in pineoblastoma with PDE4DIP gain., Pineoblastoma is a highly aggressive and rare childhood brain cancer, and the genetic drivers of sporadic pineoblastoma are unknown. Here, the authors genomically interrogated pediatric and adult pineoblastomas and found novel variants including recurrent homozygous deletions of DROSHA.

The catalytic activity of PRC2 is central to maintain transcriptional repression by H3K27me3-decorated facultative heterochromatin in mammalian cells. To date, multiple factors have been reported to regulate PRC2 activity. Here, we demonstrate that PRC2 methylates itself on EZH1/2 and SUZ12 subunits, with EZH1/2-K514 being the major automethylation site in cells. The functional studies of automethylation on EZH2 indicate automethylation as a self-activating mechanism for PRC2 in the absence of stimulatory cofactors like AEBP2. Together, our study reveals PRC2 automethylation as a novel regulatory mechanism of PRC2 activity on chromatin.

BACKGROUND: Pineoblastoma is a rare and highly aggressive brain cancer of childhood, histologically belonging to the spectrum of primitive neuroectodermal tumors. Patients with germline mutations in DICER1, a ribonuclease involved in microRNA processing, have increased risk of pineoblastoma, but genetic drivers of sporadic pineoblastoma remain unknown. METHODS: We analyzed pediatric and adult pineoblastoma samples (n=23) using integrated genomic studies, including genome-wide DNA methylation profiling, whole-exome or whole-genome sequencing, and whole-transcriptome analysis. RESULTS: Pediatric and adult pineoblastomas showed distinct methylation profiles, the latter clustering with lower grade pineal tumors and normal pineal gland. Recurrent somatic mutations were found in genes involved in PKA- and NF-κB signaling, as well as in chromatin remodeling genes. We identified recurrent homozygous deletions of DROSHA, acting upstream of DICER1 in microRNA processing, and a novel microduplication involving chromosomal region 1q21 containing PDE4DIP (myomegalin), comprising the ancient DUF1220 protein domain. Expression of PDE4DIP and DUF1220 proteins was present exclusively in pineoblastoma with PDE4DIP gain. Whole-transcriptome analysis showed that homozygous loss of DROSHA led to distinct changes in RNA expression profile. Disruption of the DROSHA locus in human neural stem cells using the CRISPR/Cas9 system, led to decrease of the DROSHA protein, and massive loss of miRNAs. CONCLUSION: We identified recurrent homozygous deletions of DROSHA in pineoblastoma, suggesting that different mechanisms disrupting miRNA processing are involved in the pathogenesis of familial versus sporadic pineoblastoma. Furthermore, a novel microduplication of PDE4DIP leading to upregulation of DUF1220 protein suggests DUF1220 as a novel oncogenic driver in pineoblastoma.

© The Author(s) 2017. High-grade gliomas in children are different from those that arise in adults. Recent collaborative molecular analyses of these rare cancers have revealed previously unappreciated connections among chromatin regulation, developmental signaling, and tumorigenesis. As we begin to unravel the unique developmental origins and distinct biological drivers of this heterogeneous group of tumors, clinical trials need to keep pace. It is important to avoid therapeutic strategies developed purely using data obtained from studies on adult glioblastoma. This approach has resulted in repetitive trials and ineffective treatments being applied to these children, with limited improvement in clinical outcome. The authors of this perspective, comprising biology and clinical expertise in the disease, recently convened to discuss the most effective ways to translate the emerging molecular insights into patient benefit. This article reviews our current understanding of pediatric high-grade glioma and suggests approaches for innovative clinical management.

Multiple circuitries ensure that cells respond correctly to the environmental cues within defined cellular programs. There is increasing evidence suggesting that cellular memory for these adaptive processes can be passed on through cell divisions and generations. However, the mechanisms by which this epigenetic information is transferred remain elusive, largely because it requires that such memory survive through gross chromatin remodeling events during DNA replication, mitosis, meiosis, and developmental reprogramming. Elucidating the processes by which epigenetic information survives and is transmitted is a central challenge in biology. In this review, we consider recent advances in understanding mechanisms of epigenetic inheritance with a focus on histone segregation at the replication fork, and how an epigenetic memory may get passed through the paternal lineage.

A methionine substitution at lysine-27 on histone H3 variants (H3K27M) is present in more than 80% of diffuse intrinsic pontine gliomas (DIPG) and inhibits polycomb repressive complex 2 (PRC2) in a dominant-negative fashion. We discovered that a number of parameters were involved in H3K27M-dependent inhibition of PRC2, including the ratio between H3K27M and PRC2 present in vivo, an enhanced affinity between H3K27M and the allosterically activated state of PRC2, and the displacement of PRC2 from chromatin containing H3K27M. Following these early events upon an introduction of H3K27M into wildtype cells, the steady-state chromatin manifested a global loss of H3K27 methylation but a gain of H3K36 methylaiton, an antagonistic histone modification associated with transcriptional elongation. These observations suggested that H3K36 methylation may function in displacing PRC2 from chromatin and the oncogenic transcription facilitated by H3K36 methylation could be a vulnerability of H3K27M DIPG. Indeed, ectopic expression of H3K36M in H3K27M DIPG cells depleted global H3K36 methylation and exerted an anti-proliferative effect, implicating a potential strategy in targeting the writer and reader proteins for H3K36 methylation against H3K27M DIPG. Citation Format: Jia-Ray Yu, Gary LeRoy, James Stafford, Chul-Hwan Lee, Danny Reinberg. Epigenetic and transcriptional alterations as potential vulnerabilities of diffuse intrinsic pontine glioma (DIPG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-104.

An issue of increasing theoretical and translational importance is to understand the conditions under which learned fear can be suppressed, or even eliminated. Basic research has pointed to extinction, in which an organism is exposed to a fearful stimulus (such as a context) in the absence of an expected aversive outcome (such as a shock). This extinction process results in the suppression of fear responses, but is generally thought to leave the original fearful memory intact. Here, we investigate the effects of extinction during periods of memory lability on behavioral responses and on expression of the immediate–early gene c-Fos within fear conditioning and extinction circuits. Our results show that long-term extinction is impaired when it occurs during time periods during which the memory should be most vulnerable to disruption (soon after conditioning or retrieval). These behavioral effects are correlated with hyperactivation of medial prefrontal cortex and amygdala subregions associated with fear expression rather than fear extinction. These findings demonstrate that behavioral experiences during periods of heightened fear prevent extinction and prolong the conditioned fear response.

The imipridone ONC201 is the first selective antagonist of DRD2 for clinical oncology. Several Phase 1, Phase 1/2 and Phase 2 studies in patients with advanced cancers have established the single agent recommended Phase 2 dose (RP2D) of 625mg ONC201 administered orally once a week in adults. ONC201 induces p53-independent apoptosis in newly diagnosed and recurrent high-grade glioma in vitro, ex vivo and in vivo. Furthermore, radiographic regressions in adult recurrent H3 K27M-mutant glioblastoma patients in response to single agent ONC201 have been reported. Based on this adult experience and complementary preclinical results demonstrating the increased susceptibility of H3 K27M-mutant gliomas to ONC201, we initiated the first Phase 1 pediatric clinical trial of ONC201 January 30, 2018. This trial will determine the safety and RP2D of ONC201 in pediatric post-radiation H3 K27M-mutant glioma patients as a single agent and in newly diagnosed diffuse intrinsic pontine glioma (DIPG) patients in combination with radiation ({"type":"clinical-trial","attrs":{"text":"NCT03416530","term_id":"NCT03416530"}}NCT03416530). Patients without known H3 K27M-mutation status by a CLIA-lab can enroll with commitment to post-term biopsy. This is a multicenter, open-label, 2 arm, dose-escalation and dose-expansion study. Ten children with H3 K27M-mutant gliomas ages 5–18 years have been treated post-radiation: 3 at dose level 1; 3 at dose level 2; 4 as part of the dose expansion cohort on dose level 2. Patients have received 2–12 doses (median= 5). The ONC201 has been tolerated very well. Grade III/IV events include: decreased neutrophil count grade III (n=1) spontaneously resolved without dose modification and elevated AST grade III (n=1) returned to grade II after holding 1 dose. Additional safety data as well as pharmacokinetics, pharmacodynamics, and progression-free survival results from this trial will be reported.

Although radiotherapy is the mainstay of treatment for children with diffuse intrinsic pontine gliomas (DIPG), fewer than 10% of patients survive past two years from diagnosis despite 30 years of clinical trials. ONC201 is an oral, selective DRD2 antagonist that induces p53-independent apoptosis and has been reported to produce a durable response in an adult with a H3 K27M mutant recurrent glioblastoma. In April 2017, a 33 month old female presented with headaches, right 6(th) nerve palsy and MRI findings consistent with a DIPG. A biopsy revealed an H3.3 K27M mutant glioblastoma. A patient-derived DIPG tumorsphere cell line was created from the biopsy sample and in vitro studies revealed a potent reduction in cell viability following 5 days of treatment with ONC201, with an IC50 of approximately 600nM. She received involved field irradiation (54Gy). Starting one month following irradiation, single agent, oral ONC201 125mg (11mg/kg) was given weekly on a compassionate use protocol. An MRI, 4 months later revealed stable disease. Clinically, her 6(th) nerve palsy and left hemiparesis have improved. She is otherwise asymptomatic. Pharmacokinetic studies are being analyzed. She has had no side effects detected. Based on our in vitro and clinical findings as well as other experience in vitro and in adults, ONC201 is currently being investigated with a phase 1 trial in pediatric patients with H3 K27M mutant gliomas.

An issue of increasing theoretical interest in the study of learning is to compare the processes that follow an initial learning experience (such as learning an association between a context and a shock; memory consolidation processes) with those that follow retrieval of that learning experience (such as exposure to the context in the absence of shock; memory reconsolidation and extinction processes). Much of what is known about these processes comes from separate experiments examining one process or the other; there have been few attempts to compare these processes directly in a single experiment. A challenge in between-experiment comparisons of consolidation and reconsolidation deficits is that they frequently involve comparisons between groups that are not matched on factors that may influence the size and persistence of these deficits (e.g., prior learning experience, memory expression prior to deficit). The following experiments examined the size and persistence of these deficits after matching both the amount of experience with a context and the levels of performance in that context prior to delivery of the protein synthesis inhibitor anisomycin. We found that systemic or intrahippocampal administration of anisomycin caused a deficit in groups receiving context conditioning (consolidation groups) or reactivation (reconsolidation groups) immediately prior to the injections. With systemic injections, the deficit was larger and more persistent in consolidation groups; with intrahippocampal injections, the initial deficit was statistically identical, yet was more persistent in the consolidation group. These experiments showed that when experiences and performance are matched prior to anisomycin injections, consolidation deficits are generally larger and more persistent compared to reconsolidation deficits.

History A 24-year-old Egyptian man with a history of hemophilia A presented with enlargement of the left thigh and lower leg by multiple masses (Fig 1). The patient had a 2-year history of progressive pain and swelling despite treatment with cryoprecipitate and bed rest. Leg weakness prevented him from bearing weight for any extended period of time, and ultimately the leg became nearly functionless. The patient traveled from Egypt to the United States for treatment. Physical examination confirmed severe enlargement and deformity of the middle to distal thigh, knee, and proximal calf secondary to numerous pliable masses. There was bluish discoloration of the skin. Laboratory findings were as follows: hemoglobin, 9.2 mg/dL; hematocrit, 29.6%; prothrombin time, 13.3 seconds; partial thromboplastin time, 84.7 seconds; and factor VIII assay, less than 2%.

INTRODUCTION Histone H3 K27M mutations distinguish a subgroup of midline gliomas in children and young adults with devastating prognosis for which there are no effective medical therapies. In a recent phase 2 trial in adult recurrent glioblastoma with ONC201, a small molecule selective antagonist of the dopamine receptor D2 (DRD2) and D3 (DRD3), a 22 year old female with recurrent H3 K27M mutant glioblastoma had tumor regression of 92% that has persisted >15 months. Furthermore, antiseizure medication was discontinued and no ONC201 side effects were observed. Based on this exceptional response, we tested whether H3 K27M mutant gliomas were selectively sensitive to ONC201-induced apoptosis.

Noninvasive functional imaging holds great promise for serving as a translational bridge between human and animal models of various neurological and psychiatric disorders. However, despite a depth of knowledge of the cellular and molecular underpinnings of atypical processes in mouse models, little is known about the large-scale functional architecture measured by functional brain imaging, limiting translation to human conditions. Here, we provide a robust processing pipeline to generate high-resolution, whole-brain resting-state functional connectivity MRI (rs-fcMRI) images in the mouse. Using a mesoscale structural connectome (i.e., an anterograde tracer mapping of axonal projections across the mouse CNS), we show that rs-fcMRI in the mouse has strong structural underpinnings, validating our procedures. We next directly show that large-scale network properties previously identified in primates are present in rodents, although they differ in several ways. Last, we examine the existence of the so-called default mode network (DMN)--a distributed functional brain system identified in primates as being highly important for social cognition and overall brain function and atypically functionally connected across a multitude of disorders. We show the presence of a potential DMN in the mouse brain both structurally and functionally. Together, these studies confirm the presence of basic network properties and functional networks of high translational importance in structural and functional systems in the mouse brain. This work clears the way for an important bridge measurement between human and rodent models, enabling us to make stronger conclusions about how regionally specific cellular and molecular manipulations in mice relate back to humans.

Naturally occurring variations of Polycomb repressive complex 1 (PRC1) comprise a core assembly of Polycomb group proteins and additional factors that include, surprisingly, autism susceptibility candidate 2 (AUTS2). Although AUTS2 is often disrupted in patients with neuronal disorders, the mechanism underlying the pathogenesis is unclear. We investigated the role of AUTS2 as part of a previously identified PRC1 complex (PRC1–AUTS2), and in the context of neurodevelopment. In contrast to the canonical role of PRC1 in gene repression, PRC1–AUTS2 activates transcription. Biochemical studies demonstrate that the CK2 component of PRC1–AUTS2 neutralizes PRC1 repressive activity, whereas AUTS2-mediated recruitment of P300 leads to gene activation. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) demonstrated that AUTS2 regulates neuronal gene expression through promoter association. Conditional targeting of Auts2 in the mouse central nervous system (CNS) leads to various developmental defects. These findings reveal a natural means of subverting PRC1 activity, linking key epigenetic modulators with neuronal functions and diseases. Polycomb group proteins are known to maintain gene repression during development; however, when autism susceptibility candidate 2 (AUTS2) associates with some Polycomb group complexes, these complexes have an unexpected gene activation role, offering new insight into the role of AUTS2 in neurological disorders. Polycomb group proteins, which maintain gene repression during development, comprise two main complexes (PRC1 and PRC2), with distinct enzymatic activities. Some PRC1 complexes associate with autism susceptibility candidate 2 (AUTS2), the gene for which is often disrupted in neuronal disorders. Here, Danny Reinberg and colleagues find that AUTS2 confers an unexpected transcriptional activation function on PRC1, and the PRC1–AUTS2 complex regulates neuronal genes. Deletion of the Auts2 locus in the mouse central nervous system leads to developmental defects. AUTS2 may have a key role in modulating PRC1 activity during normal brain development.

We conducted a series of experiments to investigate the neural basis of the immediate extinction deficit, the lack of extinction when the interval between fear memory acquisition and extinction is short. In experiment 1, rats were given extinction training composed of 15 conditioned stimuli (CSs) either 15 min (immediate extinction: I-EXT) or 24 h (delayed extinction: D-EXT) after five tone–shock pairings. In the retention test performed 48 h after conditioning, I-EXT group exhibited significantly higher freezing than D-EXT group. In experiment 2, functional activation in the medial prefrontal cortex (mPFC) was detected using c-fos immunoreactivity. The number of Fos-positive neurons in the mPFC was significantly lower in I-EXT group than in D-EXT group. In experiment 3, rats received immediate extinction with microstimulation of the infralimbic region (IL) of the mPFC, either contingently paired or unpaired with the CS. In a subsequent retention test, the paired stimulation group exhibited decreased freezing relative to the unpaired stimulation group. Together, our results suggest that the immediate extinction deficit may be linked to the lack of neuronal activity in the IL.

Background A key finding from recent studies of epigenetic mechanisms of memory is that increasing histone acetylation after a learning experience enhances memory consolidation. This has been demonstrated in several preparations, but little is known about whether excitatory and inhibitory memories are equally sensitive to drugs that promote histone acetylation and how transcriptional changes in the hippocampal-medial prefrontal cortex network contribute to these drug effects. Methods We compare the long-term behavioral consequences of systemic, intrahippocampal and intra–medial prefrontal cortex administration of the histone deacetylase inhibitor sodium butyrate (NaB) after contextual fear conditioning and extinction 1 and/or 14 days later in male c57BL/6J mice ( n = 302). Levels of histone acetylation and expression of the product of the immediate-early gene c-Fos were assessed by immunohistochemistry following infusion of NaB into the hippocampus ( n = 26). Results Across a variety of conditions, the effects of NaB on extinction were larger and more persistent compared to the effects on initial memory formation. NaB administered following weak extinction induced behavioral extinction, infralimbic histone acetylation and c-Fos expression consistent with strong extinction. No similar effect was seen in the prelimbic cortex. The involvement of the infralimbic cortex was confirmed as infusions of NaB into the infralimbic, but not prelimbic cortex, induced extinction enhancements. Conclusions These studies show that the memory modulating ability of drugs that enhance acetylation is sensitive to a variety of behavioral and molecular conditions. We further identify transcriptional changes in the hippocampal-infralimbic circuit associated with extinction enhancements induced by the histone deacetylase inhibitor NaB.

Many studies of learning have demonstrated that conditioned behavior can be eliminated when previously established relations between stimuli are severed. This extinction process has been extremely important for the development of learning theories and, more recently, for delineating the neurobiological mechanisms that underlie memory. A key finding from behavioral studies of extinction is that extinction eliminates behavior without eliminating the original memory; extinguished behavior often returns with time or with a return to the context in which the original learning occurred. This persistence of the original memory after extinction creates a challenge for clinical applications that use extinction as part of a treatment intervention. Consequently, a goal of recent neurobiological research on extinction is to identify potential pharmacological targets that may result in persistent extinction. Drugs that promote epigenetic changes are particularly promising because they can result in a long-term molecular signal that, combined with the appropriate behavioral treatment, can cause persistent changes in behavior induced by extinction. We will review evidence demonstrating extinction enhancements by drugs that target epigenetic mechanisms and will describe some of the challenges that epigenetic approaches face in promoting persistent suppression of memories.

Behavioral and pharmacological approaches to strengthen the long-term consequences of a learning experience often focus on ways of enhancing memory formation. This approach has been successful at strengthening new memories that form as a result of an initial learning experience (e.g., McGaugh, 2004). It also has been successful at strengthening processes involved in extinction, resulting in the rapid and persistent loss of a previously established conditioned response(e.g., Walker, Ressler, Lu, & Davis, 2002). Extinction has been a particularly interesting process to strengthen because behavioral enhancements in extinction may occur by facilitating the development of some new learning process during extinction, or by impairing some process associated with the neural representation of the original memory (see Lattal, Radulovic, & Lukowiak, 2006; Myers & Davis, 2007). Several recent studies have characterized some of the behavioral and molecular conditions that may favor memory erasure (or unlearning) as a mechanism for extinction. These findings are provocative, but there are many reasons to be cautious about interpreting any extinction effect as being caused by memory erasure. Although extinction is commonly described as a learning process that involves new memory formation, theories of extinction have indeed sometimes appealed to erasure mechanisms to explain aspects of extinction (e.g., Estes 1950; reviewed in Delamater, 2004; Rescorla, 2004). This idea that extinction may sometimes erase the original memory is explored in the experiments reported by Norrholm, Vervliet, Jovanovic, Boshoven, Myers, Davis, & Rothbaum (2008). Their approach is particularly noteworthy because they take a commonly used behavioral paradigm with rodents and extend it to humans. This enables them to ask questions about similar mechanisms between humans and rodents, and, more important for studies of extinction, this preparation allows Norrholm et al (2008) to examine how the behavioral response corresponds to subjects’ self-reported knowledge of the stimulus contingencies (revealed through an expectancy measure). Although the expectancy measure is not without complications, it gives the subjects two possible ways to answer the question, “Do you remember the initial contingencies?” Norrholm et al (2008) show that the answer to this question may be “no” when conditioned responses are examined, but it may be “yes” when expectancy measures are used. The designs of Norrholm et al’s extinction experiments are based on a key assumption that underlies many neurobiological accounts of memory, which is that a behavioral experience instigates a cascade of cellular and molecular events that consolidate an experience into a relatively stable, permanent memory (reviewed in McGaugh, 2000). This consolidation process can be impaired or enhanced by any number of pharmacological and behavioral interventions, provided those interventions occur in close proximity to the learning experience. One of these interventions may potentially include an extinction trial – if extinction occurs soon after acquisition (immediate extinction), the extinction memory may override or compete with the initial memory for consolidation, resulting in a permanently weakened initial memory (e.g., Myers, Ressler, & Davis, 2006). Norrholm et al (2008) demonstrate that although immediate extinction may weaken spontaneous recovery of a conditioned fear response, it does not prevent recovery of expectancy ratings. This difference emphasizes the importance of asking questions about extinction and memory erasure in multiple ways. Because a number of recent studies have suggested that extinction may involve unlearning mechanisms, it is important to give careful thought to the kind of evidence needed to demonstrate memory erasure. This needs to be considered in the context of extinction, as well as in the context of reconsolidation, where pharmacological interventions after a brief extinction trial may permanently erase (or enhance, depending on the intervention) the original memory. All of the studies that demonstrate memory erasure make compelling cases based on behavioral and molecular results, but they are faced with the same inherent challenge. That is, what are the behavioral and molecular mechanisms underlying memory formation and storage? Until we have a full understanding of the external and internal circumstances that cause memory formation, we will be hard pressed to conclude that any manipulation has retroactively eliminated that memory.

The heme coordination chemistry and spectroscopic properties of Rhodobacter capsulatus cytochrome c' (RCCP) have been compared to data from Alcaligenes xylosoxidans (AXCP), with the aim of understanding the basis for their different reactivities with nitric oxide (NO). Whereas ferrous AXCP reacts with NO to form a predominantly five-coordinate heme-nitrosyl complex via a six-coordinate intermediate, RCCP forms an equilibrium mixture of six-coordinate and five-coordinate heme-nitrosyl species in approximately equal proportions. Ferrous RCCP and AXCP both exhibit high Fe-His stretching frequencies (227 and 231 cm(-)(1), respectively), suggesting that factors other than the Fe-His bond strength account for their differences in heme-nitrosyl coordination number. Resonance Raman spectra of ferrous-nitrosyl RCCP confirm the presence of both five-coordinate and six-coordinate heme-NO complexes. The six-coordinate heme-nitrosyl of RCCP exhibits a fairly typical Fe-NO stretching frequency (569 cm(-)(1)), in contrast to the relatively high value (579 cm(-)(1)) of the AXCP six-coordinate heme-nitrosyl intermediate. It is proposed that NO experiences greater steric hindrance in binding to the distal face of AXCP, as compared to RCCP, leading to a more distorted Fe-N-O geometry and an elevated Fe-NO stretching frequency. Evidence that RCCP has a more accessible distal coordination site than in AXCP stems from the fact that ferric RCCP readily forms a heme complex with exogenous imidazole, whereas AXCP does not. A model is proposed in which distal heme-face accessibility, rather than the proximal Fe-His bond strength, determines the heme-nitrosyl coordination number in cytochromes c'.