Evaluation of a novel antibody to define histone 3.3 G34R mutant brain tumours

Missense somatic mutations affecting histone H3.1 and H3.3 proteins are now accepted as the hallmark of paediatric diffuse intrinsic pontine gliomas (DIPG), non-brain stem paediatric high grade gliomas (pHGG) as well as a subset of adult glioblastoma multiforme (GBM). Different mutations give rise to one of three amino acid substitutions at two critical positions within the histone tails, K27M, G34R/V. Several studies have highlighted gene expression and epigenetic changes associated with histone H3 mutations; however their precise roles in tumourigenesis remain incompletely understood. Determining how such amino acid substitutions in a protein affect its properties can be challenging because of difficulties in detecting and tracking mutant proteins within cells and tissues. Here we describe a strategy for the generation of antibodies to discriminate G34R and G34V mutant histone H3 proteins from their wild-type counterparts. Antibodies were validated by western blotting and immunocytochemistry, using recombinant H3.3 proteins and paediatric GBM cell lines. The H3-G34R antibody demonstrated a high degree of selectivity towards its target sequence. Accordingly, immunostaining on a cohort of 22 formalin-fixed paraffin embedded tumours with a previously known H3.3 G34R mutation status, detected successfully the corresponding mutant protein in 11/11 G34R cases. Since there was a high concordance between genotype and immunohistochemical analysis of G34R mutant tumour samples, we analysed a series of tissue microarrays (TMAs) to assess the specificity of the antibody in a range of paediatric brain tumours, and noted immunoreactivity in 2/634 cases. Importantly, we describe the generation and validation of highly specific antibodies for G34 mutations. Overall our work adds to an extremely valuable portfolio of antibodies, not only for histopathologic detection of tumour-associated mutant histone sequences, but also facilitating the study of spatial/anatomical aspects of tumour formation and the identification of downstream targets and pathways in malignant glioma progression

Characterization of adenosine receptors in bovine chondrocytes and fibroblast-like synoviocytes exposed to low frequency low energy pulsed electromagnetic fields

SummaryObjectiveThe present study describes the presence and binding parameters of the A1, A2A, A2B and A3 adenosine receptors in bovine chondrocytes and fibroblast-like synoviocytes. The effect of low frequency low energy pulsed electromagnetic fields (PEMFs) on the adenosine receptor affinity and density was studied.MethodsSaturation, competition binding experiments and Western blotting assays in the absence and in the presence of PEMFs on the adenosine receptors in bovine chondrocytes or fibroblast-like synoviocytes were performed. Thermodynamic analysis of the A2A or A3 binding was studied to investigate the forces driving drug–receptor coupling. In the adenylyl cyclase and proliferation assays the potency of typical high-affinity A2A or A3 agonists in the absence and in the presence of PEMFs was evaluated.ResultsBovine chondrocytes and fibroblast-like synoviocytes expressed all adenosine receptors. PEMFs evoked an up-regulation of A2A and A3 receptors and thermodynamic parameters indicate that adenosine binding is enthalpy and entropy driven. In PEMF-treated cells the potency of typical A2A or A3 agonists on cyclic AMP assays was significantly increased when compared with the untreated cells. PEMFs potentiated the effect of A2A or A3 agonists on cell proliferation in both cell types.ConclusionsPEMFs mediate an up-regulation of A2A and A3 receptors related to an increase of their functional activities in bovine chondrocytes and fibroblast-like synoviocytes. No differences are present in adenosine affinity and in the drug–receptor interactions. Our data could be used as a trigger to future studies addressed to PEMFs and adenosine therapeutic intervention in inflammatory joint diseases

Pharmacological characterization of P2X1 and P2X3 purinergic receptors in bovine chondrocytes

SummaryObjectiveThe aim of the present study is that of characterizing, for the first time in a quantitative way, from a biochemical, physico chemical and functional point of view P2X1 and P2X3 purinergic receptors in bovine chondrocytes. The affinity and the potency of typical purinergic ligands were studied through competition binding experiments and their role in modulating chondrocyte actvities was investigated by analyzing nitric oxide (NO) and prostaglandin E2 (PGE2) release.MethodsSaturation, competition binding experiments, western blotting and immunohistochemistry assays on the P2X1 and P2X3 purinergic receptors in bovine chondrocytes were performed. Thermodynamic analysis of the P2X1 and P2X3 purinergic binding was studied to investigate the forces driving drug-receptor coupling. In the functional assays (NO and PGE2 release) the potency of purinergic agonists and antagonists was evaluated.ResultsBovine chondrocytes expressed P2X1 and P2X3 purinergic receptors and thermodynamic parameters indicated that purinergic binding is enthalpy- and entropy-driven for agonists and totally entropy-driven for antagonists. Typical purinergic agonists such as adenosine 5′-triphosphate (ATP) and α,β-methyleneATP were able to increase NO and PGE2 release. A purinergic antagonist, A317491, was able to block the stimulatory effect on functional experiments mediated by the agonists.ConclusionsThese data demonstrate for the first time the presence of functional P2X1 and P2X3 purinergic receptors in bovine chondrocytes. Agonists and antagonists are thermodynamically discriminated and are able to modulate functional responses such as NO and PGE2 release. These results suggest the potential role of novel purinergic antagonists in the treatment of pathophysiological diseases linked to the inflammation and involved in articular cartilage resorption

Adenosine and lymphocyte regulation

Adenosine is a potent extracellular messenger that is produced in high concentrations under metabolically unfavourable conditions. Tissue hypoxia, consequent to a compromised cellular energy status, is followed by the enhanced breakdown of ATP leading to the release of adenosine. Through the interaction with A2 and A3 membrane receptors, adenosine is devoted to the restoration of tissue homeostasis, acting as a retaliatory metabolite. Several aspects of the immune response have to be taken into consideration and even though in general it is very important to dampen inflammation, in some circumstances, such as the case of cancer, it is also necessary to increase the activity of immune cells against pathogens. Therefore, adenosine receptors that are defined as ‘sensors–of metabolic changes in the local tissue environment may be very important targets for modulation of immune responses and drugs devoted to regulating the adenosinergic system are promising in different clinical situations

Pathophysiological Role and Medicinal Chemistry of A2A Adenosine Receptor Antagonists in Alzheimer's Disease

The A(2A) adenosine receptor is a protein belonging to a family of four GPCR adenosine receptors. It is involved in the regulation of several pathophysiological conditions in both the central nervous system and periphery. In the brain, its localization at pre- and postsynaptic level in striatum, cortex, hippocampus and its effects on glutamate release, microglia and astrocyte activation account for a crucial role in neurodegenerative diseases, including Alzheimer's disease (AD). This ailment is considered the main form of dementia and is expected to exponentially increase in coming years. The pathological tracts of AD include amyloid peptide-beta extracellular accumulation and tau hyperphosphorylation, causing neuronal cell death, cognitive deficit, and memory loss. Interestingly, in vitro and in vivo studies have demonstrated that A(2A) adenosine receptor antagonists may counteract each of these clinical signs, representing an important new strategy to fight a disease for which unfortunately only symptomatic drugs are available. This review offers a brief overview of the biological effects mediated by A(2A) adenosine receptors in AD animal and human studies and reports the state of the art of A(2A) adenosine receptor antagonists currently in clinical trials. As an original approach, it focuses on the crucial role of pharmacokinetics and ability to pass the blood-brain barrier in the discovery of new agents for treating CNS disorders. Considering that A(2A) receptor antagonist istradefylline is already commercially available for Parkinson's disease treatment, if the proof of concept of these ligands in AD is confirmed and reinforced, it will be easier to offer a new hope for AD patients

Molecular profiling of a rare rosette-forming glioneuronal tumor arising in the spinal cord

Rosette-forming glioneuronal tumor (RGNT) of the IV ventricle is a rare and recently recognized brain tumor entity. It is histologically composed by two distinct features: a glial component, resembling pilocytic astrocytoma, and a component forming neurocytic rosettes and/or perivascular rosettes. Herein, we describe a 33-year-old man with RGNT arising in the spinal cord. Following an immunohistochemistry validation, we further performed an extensive genomic analysis, using array-CGH (aCGH), whole exome and cancer-related hotspot sequencing, in order to better understand its underlying biology. We observed the loss of 1p and gain of 1q, as well as gain of the whole chromosomes 7, 9 and 16. Local amplifications in 9q34.2 and 19p13.3 (encompassing the gene SBNO2) were identified. Moreover, we observed focal gains/losses in several chromosomes. Additionally, on chromosome 7, we identified the presence of the KIAA1549:BRAF gene fusion, which was further validated by RT-PCR and FISH. Across all mutational analyses, we detected and validated the somatic mutations of the genes MLL2, CNNM3, PCDHGC4 and SCN1A. Our comprehensive molecular profiling of this RGNT suggests that MAPK pathway and methylome changes, driven by KIAA1549:BRAF fusion and MLL2 mutation, respectively, could be associated with the development of this rare tumor entity.Conselho Nacional de Desenvolvimento Científico e Tecnológico [475358/2011-2] to RMR (www.cnpq.br); Fundação de Amparo a Pesquisa do Estado de São Paulo [2012/19590-0] to RMR and [2011/08523-7 and 2012/08287-4] to LTB (www.fapesp.br); the Foundation for Science and Technology (FCT) [PTDC/SAU-ONC/115513/2009] to RMR; and the National Cancer Institute [P30CA046934] to MG

A computational approach to identify whole genome homozygosity mapping across multiple SNP mapping experiments

The recent development of microarray platforms, capable to genotype more than thousands of single nucleotide polymorphisms (SNPs) in individuals, had provided an opportunity to rapidly identify susceptibility loci for complex phenotypes. High density SNP mapping arrays have been widely applied to association studies, to copy number (CN) analysis in cancers and recently to investigate the role of homozygosity extended regions in individuals. Long stretches of CN neutral and homozygous SNPs, defined as runs of homozygosity (ROHs) can be found either in a single individual or shared across samples. The identification of ROHs among affected individuals of the same family or among unrelated ones with same disease, can underline loci potentially implicated in the genetic basis of the disease under study. Therefore the identification of ROHs in affected individuals or pathological datasets gives a chance to identify disease associated loci and new causative mutations. In order to identify ROHs pattern across Affymetrix SNP mapping datasets, we developed a computational strategy including several computational steps: 1) loss of heterozygosity analysis by dChip2007 software; 2) a within-subject step allowing the identification of ROHs in a single sample; 3) an across-subject step extracting the ROH fingerprint of the dataset and 4) the identification of a common ROHs pattern based on frequency across the dataset under study, varying the number of individuals carrying common ROHs; 5) the annotation step allowing the association of genes to selected ROHs. In order to obtain an effective ROHs visualization, we use dChip software for the entire samples dataset. We assess our strategy to two SNP mapping datasets including 100K leukemia and 250K congenital recessive diseases. The procedure allowed the identification of a unique genetic ROH fingerprint of clinical datasets potentially important to discover new diseases associated loci suitable for further investigations

The effects of auditory enrichment on zebrafish behavior and physiology

Environmental enrichment is widely used to improve welfare and behavioral performance of animal species. It ensures housing of laboratory animals in environments with space and complexity that enable the expression of their normal behavioral repertoire. Auditory enrichment by exposure to classical music decreases abnormal behaviors and endocrine stress responses in humans, non-humans primates, and rodents. However, little is known about the role of auditory enrichment in laboratory zebrafish. Given the growing importance of zebrafish for neuroscience research, such studies become critical. To examine whether auditory enrichment by classical music can affect fish behavior and physiology, we exposed adult zebrafish to 2 h of Vivaldi’s music (65–75 dB) twice daily, for 15 days. Overall, zebrafish exposed to such auditory stimuli were less anxious in the novel tank test and less active, calmer in the light-dark test, also affecting zebrafish physiological (immune) biomarkers, decreasing peripheral levels of pro-inflammatory cytokines and increasing the activity of some CNS genes, without overt effects on whole-body cortisol levels. In summary, we report that twice-daily exposure to continuous musical sounds may provide benefits over the ongoing 50–55 dB background noise of equipment in the laboratory setting. Overall, our results support utilizing auditory enrichment in laboratory zebrafish to reduce stress and improve welfare in this experimental aquatic organism

Angiocentric glioma-associated seizures: The possible role of EATT2, pyruvate carboxylase and glutamine synthetase

Purpose: Our purpose was to better understand the pathogenesis of seizures associated with angiocentric glioma. Angiocentric glioma is an indolent and rare low-grade glioma. Its typical clinical presentation is with epileptic seizures. The pathogenesis of tumor-associated seizures is poorly understood. Among the possible pathomechanisms, the increased neurotoxic concentrations of the glutamate has been proposed. Glutamate transporters, pyruvate carboxylase and glutamine synthetase are involved in maintaining the physiological concentration of glutamate in the inter synaptic spaces. Methods: We evaluated the immunohistochemical expression of EAAT2 (the most important glutamate transporter), pyruvate carboxylase and glutamine synthetase in 17 angiocentric gliomas. Results: EAAT2 was never expressed (0%) in the neoplastic cells in none of the cases studied. Pyruvate carboxylase was expressed in the cytoplasm of the neoplastic cells in 16/17 cases (94 %). Glutamine synthetase was expressed in the cytoplasm of the neoplastic cells in 15/17 cases (88 %). Conclusion: The net result of this enzymatic expression, in particular considering the loss of EAAT2, could be an increased glutamate concentration in the synaptic clef, which might increase local network excitability initially involving intratumoral neurons. The observation that the angiocentric glioma-associated epilepsy might be at least in part related to EAAT2 deficiency opens up interesting therapeutic perspectives

Pediatric high-grade gliomas and the WHO CNS Tumor Classification - Perspectives of pediatric neuro-oncologists and neuropathologists in light of recent updates

Background: The WHO Classification of Tumors of the Central Nervous System has undergone major restructuring. Molecularly defined diagnostic criteria were introduced in 2016 (revised 4th edition) and expanded in 2021 (5th edition) to incorporate further essential diagnostic molecular parameters. We investigated potential differences between specialists in perception of these molecularly defined subtypes for pediatric high-grade gliomas (pedHGG). Methods: We designed a 22-question survey studying the impact of the revised 4th edition of the WHO classification on pedHGG. Data were collected and statistically analyzed to examine the spectrum of viewpoints and possible differences between neuro-oncologists and neuropathologists. Results: 465 participants from 53 countries were included; 187 pediatric neuro-oncologists (40%), 160 neuropathologists (34%), and 118 additional experts (26%). Neuro-oncologists reported issues with the introduction of molecularly defined tumor types, as well as the abolishment or renaming of established tumor entities, while neuropathologists did not to the same extent. Both groups indicated less relevant or insufficient diagnostic definitions were available in 2016. Reported issues were classified and assessed in the 2021 WHO classification and a substantial improvement was perceived. However, issues of high clinical relevance remain to be addressed, including the definition of clinical phenotypes for diffuse intrinsic pontine glioma and gliomatosis cerebri. Conclusions: Within the WHO classification of pediatric brain tumors, such as pedHGG, rapid changes in molecular characterization have been introduced. This study highlights the ongoing need for cross talk between pathologist and oncologist to advance the classification of pedHGG subtypes and ensure biological relevance and clinical impact