Impact of JQ1 treatment on seizures, hippocampal gene expression, and gliosis in a mouse model of temporal lobe epilepsy.

Epilepsy is a neurological disease characterised by recurrent seizures with complex aetiology. Temporal lobe epilepsy, the most common form in adults, can be acquired following brain insults including trauma, stroke, infection or sustained status epilepticus. The mechanisms that give rise to the formation and maintenance of hyperexcitable networks following acquired insults remain unknown, yet an extensive body of literature points towards persistent gene and epigenomic dysregulation as a potential mediator of this dysfunction. While much is known about the function of specific classes of epigenetic regulators (writers and erasers) in epilepsy, much less is known about the enzymes, which read the epigenome and modulate gene expression accordingly. Here, we explore the potential role for the epigenetic reader bromodomain and extra-terminal domain (BET) proteins in epilepsy. Using the intra-amygdala kainic acid model of temporal lobe epilepsy, we initially identified widespread dysregulation of important epigenetic regulators including EZH2 and REST as well as altered BRD4 expression in chronically epileptic mice. BRD4 activity was also notably affected by epilepsy-provoking insults as seen by elevated binding to and transcriptional regulation of the immediate early gene Fos. Despite influencing early aspects of epileptogenesis, blocking BET protein activity with JQ1 had no overt effects on epilepsy development in mice but did alter glial reactivity and influence gene expression patterns, promoting various neurotransmitter signalling mechanisms and inflammatory pathways in the hippocampus. Together, these results confirm that epigenetic reader activity is affected by epilepsy-provoking brain insults and that BET activity may exert cell-specific actions on inflammation in epilepsy.

Intratumoral histological and molecular heterogeneity in an adult diffuse glioma.

Adult-type diffuse gliomas are the most prevalent type of malignant adult brain tumors. Intratumoral heterogeneity can hinder accurate diagnosis and subsequent treatment. This case report documents a tumor with intratumoral heterogeneity, both histologically and by methylation analysis, located within the left cerebral hemisphere of a 29-year-old female. She presented after a witnessed generalized tonic clonic seizure at home. Two years prior she had a witnessed seizure; however, no brain imaging was done at the time. Magnetic resonance imaging (MRI), on this admission, showed a mass lesion in the left frontal operculum with poorly identified margins and right-sided midline shift. Sampling from the left temporal lobe showed an IDH-mutant, ATRX-mutant astrocytoma, which appeared grade 4 in the enhancing anterior portion and grade 2 in the left temporal lobe. Methylation analysis confirmed this heterogeneity. In summary, this is an excellent example of tumor heterogeneity both histologically and by molecular analysis. It is probable, given the clinical history of presentation 2 years prior, that this tumor originated as a low-grade glioma and subsequently evolved.

CPEB4-CLOCK crosstalk during temporal lobe epilepsy.

OBJECTIVE: Posttranscriptional mechanisms are increasingly recognized as important contributors to the formation of hyperexcitable networks in epilepsy. Messenger RNA (mRNA) polyadenylation is a key regulatory mechanism governing protein expression by enhancing mRNA stability and translation. Previous studies have shown large-scale changes in mRNA polyadenylation in the hippocampus of mice during epilepsy development. The cytoplasmic polyadenylation element-binding protein CPEB4 was found to drive epilepsy-induced poly(A) tail changes, and mice lacking CPEB4 develop a more severe seizure and epilepsy phenotype. The mechanisms controlling CPEB4 function and the downstream pathways that influence the recurrence of spontaneous seizures in epilepsy remain poorly understood. METHODS: Status epilepticus was induced in wild-type and CPEB4-deficient male mice via an intra-amygdala microinjection of kainic acid. CLOCK binding to the CPEB4 promoter was analyzed via chromatin immunoprecipitation assay and melatonin levels via high-performance liquid chromatography in plasma. RESULTS: Here, we show increased binding of CLOCK to recognition sites in the CPEB4 promoter region during status epilepticus in mice and increased Cpeb4 mRNA levels in N2A cells overexpressing CLOCK. Bioinformatic analysis of CPEB4-dependent genes undergoing changes in their poly(A) tail during epilepsy found that genes involved in the regulation of circadian rhythms are particularly enriched. Clock transcripts displayed a longer poly(A) tail length in the hippocampus of mice post-status epilepticus and during epilepsy. Moreover, CLOCK expression was increased in the hippocampus in mice post-status epilepticus and during epilepsy, and in resected hippocampus and cortex of patients with drug-resistant temporal lobe epilepsy. Furthermore, CPEB4 is required for CLOCK expression after status epilepticus, with lower levels in CPEB4-deficient compared to wild-type mice. Last, CPEB4-deficient mice showed altered circadian function, including altered melatonin blood levels and altered clustering of spontaneous seizures during the day. SIGNIFICANCE: Our results reveal a new positive transcriptional-translational feedback loop involving CPEB4 and CLOCK, which may contribute to the regulation of the sleep-wake cycle during epilepsy.

MicroRNA inhibition using antimiRs in acute human brain tissue sections.

Antisense inhibition of microRNAs is an emerging preclinical approach to pharmacoresistant epilepsy. A leading candidate is an "antimiR" targeting microRNA-134 (ant-134), but testing to date has used rodent models. Here, we develop an antimiR testing platform in human brain tissue sections. Brain specimens were obtained from patients undergoing resective surgery to treat pharmacoresistant epilepsy. Neocortical specimens were submerged in modified artificial cerebrospinal fluid (ACSF) and dissected for clinical neuropathological examination, and unused material was transferred for sectioning. Individual sections were incubated in oxygenated ACSF, containing either ant-134 or a nontargeting control antimiR, for 24 h at room temperature. RNA integrity was assessed using BioAnalyzer processing, and individual miRNA levels were measured using quantitative reverse transcriptase polymerase chain reaction. Specimens transported in ACSF could be used for neuropathological diagnosis and had good RNA integrity. Ant-134 mediated a dose-dependent knockdown of miR-134, with approximately 75% reduction of miR-134 at 1 µmol L-1 and 90% reduction at 3 µmol L-1 . These doses did not have off-target effects on expression of a selection of three other miRNAs. This is the first demonstration of ant-134 effects in live human brain tissues. The findings lend further support to the preclinical development of a therapy that targets miR-134 and offer a flexible platform for the preclinical testing of antimiRs, and other antisense oligonucleotide therapeutics, in human brain.

Melanoma brain metastases in Ireland: surgical and systemic considerations.

BACKGROUND: Cerebral metastases is a common complication in patients with melanoma. There is a paucity of information in the Republic of Ireland regarding the factors associated with melanoma brain metastases (MBM). METHODS: Patients diagnosed with melanoma brain metastases in Ireland were retrospectively identified in Beaumont Hospital between 1999 and 2018. Patient demographics; age at diagnosis of primary melanoma, age at detection of MBM, year of detection of MBM, anatomical location of primary melanoma, BRAF mutation analysis and the number of metastases were investigated. Follow-up data were also derived, including overall survival. RESULTS: There has being a 158% increase in the incidence of primary melanoma from 1999 compared to 2016. Over the same time period 128 patients with melanoma brain metastases were diagnosed. There was a significant male predominance (n = 77/128; 60%; p < 0.0001). BRAF mutation and leptomeningeal disease were independent prognostic factors in our cohort with a median survival 8 months and 0.5 months, respectively. CONCLUSIONS: Male predominance, leptomeningeal disease and BRAF mutation represent important considerations in this population group. The results of this study add to our knowledge concerning outcomes in melanoma brain metastases and may be useful in clinical planning and future treatments.

Desmoplastic myxoid tumor, SMARCB1-mutant: clinical, histopathological and molecular characterization of a pineal region tumor encountered in adolescents and adults.

Atypical teratoid/rhabdoid tumor (ATRT) is a highly malignant brain tumor predominantly occurring in infants. Mutations of the SMARCB1 gene are the characteristic genetic lesion. SMARCB1-mutant tumors in adolescents and adults are rare and may show uncommon histopathological and clinical features. Here we report seven SMARCB1-deficient intracranial tumors sharing distinct clinical, histopathological and molecular features. Median age of the four females and three males was 40 years (range 15-61 years). All tumors were located in the pineal region. Histopathologically, these tumors displayed spindled and epithelioid cells embedded in a desmoplastic stroma alternating with a variable extent of a loose myxoid matrix. All cases showed loss of nuclear SMARCB1/INI1 protein expression, expression of EMA and CD34 was frequent and the Ki67/MIB1 proliferation index was low in the majority of cases (median 3%). Three cases displayed heterozygous SMARCB1 deletions and two cases a homozygous SMARCB1 deletion. On sequencing, one tumor showed a 2 bp deletion in exon 4 (c.369_370del) and one a short duplication in exon 3 (c.237_276dup) both resulting in frameshift mutations. Most DNA methylation profiles were not classifiable using the Heidelberg Brain Tumor Classifier (version v11b4). By unsupervised t-SNE analysis and hierarchical clustering analysis, however, all tumors grouped closely together and showed similarities with ATRT-MYC. After a median observation period of 48 months, three patients were alive with stable disease, whereas one patient experienced tumor progression and three patients had succumbed to disease. In conclusion, our series represents an entity with distinct clinical, histopathological and molecular features showing epigenetic similarities with ATRT-MYC. We propose the designation desmoplastic myxoid tumor (DMT), SMARCB1-mutant, for these tumors.

CNS lymphoma, the Irish experience: A retrospective review of neuropathologically confirmed cases over 10 years.

BACKGROUND: Central nervous system (CNS) lymphoma is rare, representing 2% of all brain tumors. The commonest subtype is diffuse large B-cell lymphoma (DLBCL), with primary T-cell lymphomas (PCNSTL) accounting for ~ 2%. OBJECTIVE: To determine the frequency and describe the key features of CNS lymphoma over a 10-year period in an Irish population. MATERIALS AND METHODS: This retrospective review was carried out using the neuropathology database in Beaumont hospital, the largest of two national neurosurgical centers, to identify all cases of CNS lymphoma from 2007 to 2017. Clinical, radiological, morphological, immunophenotype, and molecular information was recorded where available. RESULTS: We identified 149 cases of CNS lymphoma from 2007 to 2017, which equated to a cumulative incidence rate of 0.4/100,000 persons. Median age at diagnosis was 66 years, and 46% were male. 86% were classified as DLBCL (n = 128), 10% immunodeficiency-associated CNS lymphoma (n = 15), 3% PCNSTL (n = 4), and 1% (n = 2) cases of intravascular large B-cell lymphoma. Location in declining frequency was as follows: supratentorial (n = 125), infratentorial (n = 22), spinal (n = 1), and orbital (n = 1). CONCLUSION: This is the first study in an Irish population to determine a cumulative incidence rate of CNS lymphoma, which is in line with larger international population-based registries. No significant trends in incidence rate have been observed from 2007 to 2017. DLBCL is the commonest subtype encountered. Rare variants including PCNSTL can pose a significant diagnostic challenge, and in this setting, molecular studies can be useful to confirm diagnoses.

IgG4 hypophysitis - a rare and underdiagnosed cause of pituitary gland and stalk mass-like thickening.

Our aim is to present a typical case of IgG4-related hypophysitis, which will offer insight into the aetiology and pathogenesis of this relatively newly described disease. IgG4 Related Disease is a protean systemic condition that mimics inflammatory, infectious, and malignant processes. Biopsy of affected organs will show a typical histopathological pattern.

Reactive gliosis mimicking tumor recurrence - a case series documenting MRI abnormalities and neuropathological correlates.

The aim of this study is to identify, in our center, all cases of foreign-body reactions to hemostatic agents or other prostheses resulting in a radiological suspicion of tumor recurrence. We interrogated our internal database to identify all such cases and systematically evaluated the MRI brain scans of patients: (i) at the time of initial tumor diagnosis, (ii) postoperatively, (iii) and at the time of suspected tumor recurrence. In addition, we reviewed each patient's operative notes and reviewed the histology of all cases following a second surgical intervention. In total, we identified 8 patients, 7 of whom had a WHO grade II glioma at initial surgery. We did not identify any distinguishing radiological abnormalities from the initial diagnostic brain scan to the suspected recurrence, and histologically all cases were characterized by extensive gliosis; with both macrophages and reactive astrocytes present throughout. The cause of gliosis was identified as being relating to hemostatic agents in 4 cases; in the other 4 cases, the foreign-body reaction was presumed to be caused be materials used in a craniotomy or cranioplasty. This study highlights the difficulty in radiologically diagnosing a foreign-body reaction and also identifies that such a gliotic reaction may occur as a consequence of exogenous materials used in a craniotomy or cranioplasty.
.

The DNA copy number landscape of a collision tumor.

Intracranial collision tumors are composed of two histologically distinct but merging components, and are rare. Their genetic profile has rarely been described. Comparative genome hybridization of a combined meningioma and oligodendroglioma demonstrated deletion of chromosome 22q and of 19q in both tumors. Somatic deletion of chromosome 22q and 19q is associated with development of an intracranial collision tumor.
.

Immunoproteasome deficiency results in age-dependent development of epilepsy.

The immunoproteasome is a central protease complex required for optimal antigen presentation. Immunoproteasome activity is also associated with facilitating the degradation of misfolded and oxidized proteins, which prevents cellular stress. While extensively studied during diseases with increasing evidence suggesting a role for the immunoproteasome during pathological conditions including neurodegenerative diseases, this enzyme complex is believed to be mainly not expressed in the healthy brain. In this study, we show an age-dependent increase in polyubiquitination in the brains of wild-type mice, accompanied by an induction of immunoproteasomes, which was most prominent in neurons and microglia. In contrast, mice completely lacking immunoproteasomes (triple-knockout mice), displayed a strong increase in polyubiquitinated proteins already in the young brain and developed spontaneous epileptic seizures, beginning at the age of 6 months. Injections of kainic acid led to high epilepsy-related mortality of aged triple-knockout mice, confirming increased pathological hyperexcitability states. Notably, the expression of the immunoproteasome was reduced in the brains of patients suffering from epilepsy. In addition, the aged triple-knockout mice showed increased anxiety, tau hyperphosphorylation and degeneration of Purkinje cell population with the resulting ataxic symptoms and locomotion alterations. Collectively, our study suggests a critical role for the immunoproteasome in the maintenance of a healthy brain during ageing.

RET overexpression leads to increased brain metastatic competency in luminal breast cancer.

BACKGROUND: Breast cancer brain metastasis is a rising occurrence, necessitating a better understanding of the mechanisms involved for effective management. Breast cancer brain metastases diverge notably from the primary tumor, with gains in kinase and concomitant losses of steroid signaling observed. In this study, we explored the role of the kinase receptor RET in promoting breast cancer brain metastases and provide a rationale for targeting this receptor. METHODS: RET expression was characterized in a cohort of patients with primary and brain metastatic tumors. RET functionality was assessed using pharmacological inhibition and gene silencing in patient-derived brain metastatic tumor explants and in vivo models, organoid models, and brain organotypic cultures. RNA sequencing was used to uncover novel brain metastatic relevant RET mechanisms of action. RESULTS: A statistically significant enrichment of RET in brain metastases was observed in estrogen receptor-positive breast cancer, where it played a role in promoting cancer cell adhesion, survival, and outgrowth in the brain. In vivo, RET overexpression enhanced brain metastatic competency in patient-derived models. At a mechanistic level, RET overexpression was found to enhance the activation of gene programs involved in cell adhesion, requiring EGFR cooperation to deliver a pro-brain metastatic phenotype. CONCLUSION: Our results illustrate, for the first time, the role of RET in regulating colonization and outgrowth of breast cancer brain metastasis and provide data to support the use of RET inhibitors in the management strategy for patients with breast cancer brain metastases.

Case report: BRAF-inhibitor therapy in BRAF-mutated primary CNS tumours including one case of BRAF-mutated Rosai-Dorfman disease.

BRAF V600E oncogene mutations have been reported in multiple central nervous system (CNS) tumor types, and emerging evidence supports the use of targeted therapy in BRAF-mutated gliomas. BRAF oncogene mutations have been recently identified in Rosai-Dorfman disease (RDD)-a rare non-Langerhans cell histiocytosis. This series describes three patients from two neurosurgical centers in Ireland with BRAF V600E-mutated CNS tumors. The study participants include a 19-year-old male patient with ganglioglioma with anaplastic features, a 21-year-old male patient with CNS involvement of RDD, and a 28-year-old female patient with ganglioglioma with anaplastic features. Two patients received radiation with concurrent temozolomide before BRAF-targeted therapy. This case series describes clinical and radiological responses to BRAF-targeted therapy in BRAF V600E-mutated gliomas across multiple tumor grades and is only the second published report of response to targeted therapy in BRAF-mutated RDD. The durability of disease control with BRAF-targeted therapy was generally superior to that achieved with chemoradiation; one patient has experienced ongoing disease control for 5 years. The reported case of treatment response in BRAF-mutated RDD supports the strategy of genotyping and utilization of targeted therapy in this rare disease. The optimal sequencing of BRAF-targeted therapy in BRAF-mutated gliomas/glioneuronal tumors remains unclear, and further prospective studies are required to guide the use of genome-matched therapy in this patient population.

Microvascular stabilization via blood-brain barrier regulation prevents seizure activity.

Blood-brain barrier (BBB) dysfunction is associated with worse epilepsy outcomes however the underlying molecular mechanisms of BBB dysfunction remain to be elucidated. Tight junction proteins are important regulators of BBB integrity and in particular, the tight junction protein claudin-5 is the most enriched in brain endothelial cells and regulates size-selectivity at the BBB. Additionally, disruption of claudin-5 expression has been implicated in numerous disorders including schizophrenia, depression and traumatic brain injury, yet its role in epilepsy has not been fully deciphered. Here we report that claudin-5 protein levels are significantly diminished in surgically resected brain tissue from patients with treatment-resistant epilepsy. Concomitantly, dynamic contrast-enhanced MRI in these patients showed widespread BBB disruption. We show that targeted disruption of claudin-5 in the hippocampus or genetic heterozygosity of claudin-5 in mice exacerbates kainic acid-induced seizures and BBB disruption. Additionally, inducible knockdown of claudin-5 in mice leads to spontaneous recurrent seizures, severe neuroinflammation, and mortality. Finally, we identify that RepSox, a regulator of claudin-5 expression, can prevent seizure activity in experimental epilepsy. Altogether, we propose that BBB stabilizing drugs could represent a new generation of agents to prevent seizure activity in epilepsy patients.

Progressive dysphagia in limb-girdle muscular dystrophy type 2B.

Dysphagia has not been reported in genetically confirmed limb-girdle muscular dystrophy type 2B (LGMD2B). A 40-year-old woman reported exercise-induced calf pain at age 34, followed by progressive lower and upper limb weakness. At age 38, progressive dysphagia for solids, and subsequently liquids, ensued. Endoscopic and videofluoroscopic-radiological findings indicated a myopathic swallowing disorder. Molecular genetic analysis confirmed two dysferlin gene mutations consistent with a compound heterozygote state. Progressive dysphagia should be considered as part of the expanding dysferlinopathy phenotype.

Temporal fossa intra-extracranial dumbbell schwannoma.

Intra-extracranial schwannomas arising unrelated to major cranial nerves are exceedingly rare neoplasms. We report the case of a 23-year-old male who presented with a 9 month history of progressive temporal swelling which was excised and found histologically to be a schwannoma. A succinct review of the relevant literature is presented.

Comparative analysis of the AIB1 interactome in breast cancer reveals MTA2 as a repressive partner which silences E-Cadherin to promote EMT and associates with a pro-metastatic phenotype.

Steroid regulated cancer cells use nuclear receptors and associated regulatory proteins to orchestrate transcriptional networks to drive disease progression. In primary breast cancer, the coactivator AIB1 promotes estrogen receptor (ER) transcriptional activity to enhance cell proliferation. The function of the coactivator in ER+ metastasis however is not established. Here we describe AIB1 as a survival factor, regulator of pro-metastatic transcriptional pathways and a promising actionable target. Genomic alterations and functional expression of AIB1 associated with reduced disease-free survival in patients and enhanced metastatic capacity in novel CDX and PDX ex-vivo models of ER+ metastatic disease. Comparative analysis of the AIB1 interactome with complementary RNAseq characterized AIB1 as a transcriptional repressor. Specifically, we report that AIB1 interacts with MTA2 to form a repressive complex, inhibiting CDH1 (encoding E-cadherin) to promote EMT and drive progression. We further report that pharmacological and genetic inhibition of AIB1 demonstrates significant anti-proliferative activity in patient-derived models establishing AIB1 as a viable strategy to target endocrine resistant metastasis. This work defines a novel role for AIB1 in the regulation of EMT through transcriptional repression in advanced cancer cells with a considerable implication for prognosis and therapeutic interventions.

Acute multiple sclerosis lesion pathology does not predict subsequent clinical course-a biopsy study.

BACKGROUND: Knowledge of the clinical outcome in tumefactive demyelination remains limited. AIMS: This study aims to characterise the natural history of biopsy-proven, pathogen-free, cerebral demyelination in an adult Irish population. METHODS: We identified all patients with biopsy-proven demyelination in a single neuropathology centre between 1999 and 2017. A baseline, and at least one follow-up MRI scan was available in each instance (mean of 3 scans per patient), together with both the presenting and most recent clinical details including disability level and disease-modifying drugs. RESULTS: In 21 patients, white matter biopsies showed the following: macrophages with myelin debris, myelin-axonal dissociation, reactive astrocytes and occasional lymphocytes. During a mean follow-up time of 8 years (± 4.4), 17 patients developed MS, confirmed both clinically and on MRI, using the 2010 McDonald criteria: 11 relapsing remitting (RR) MS, four secondary progressive and two primary progressive MS. Four patients had a monophasic illness with lesion regression, without clinical or radiological evidence of any further disease activity on follow-up. The patients with progressive MS had significantly higher levels of physical disability than either the RRMS or monophasic patients. CONCLUSION: Uniform white matter subacute demyelination is associated with a diverse clinical course ranging from a monophasic illness to progressive MS, suggesting that extraneous factors distinct from the basic pathology significantly influence the clinical course in MS.