1910570
Phospholipase C Beta 1: a Candidate Signature Gene for Proneural Subtype High-Grade Glioma
Phospholipase C beta 1 (PLCβ1) expresses in gliomas and cultured glial cells, but its expression is barely detectable in normal glial cells. We analyzed data from Gene Expression Omnibus (GEO-GDSxxx), The Cancer Genome Atlas (TCGA), and the Repository for Molecular Brain Neoplasia Data (REMBRANDT) to explore the potential role of PLCβ1 as a biomarker in high-grade glioma (HGG). PLCβ1 expression is significantly higher in grade III gliomas than that in grade IV gliomas from GDS1815 (n = 24 vs. 76), GDS1962 (n = 19 vs. 81), and GDS1975 (n = 26 vs. 59). In GDS1815, PLCβ1 expression correlates with several known proneural (PN) signature genes; its expression from PN subtype (n = 15) is significantly higher than that from mesenchymal (Mes) subtype (n = 33) HGG. In GDS1962, PLCβ1 expression is the highest in nontumor brain tissue (n = 23) and is significantly higher than its expression in grade II gliomas [astrocytomas (n = 7) and oligodendrogliomas (n = 37)]. A Kaplan-Meier survival curve from a REMBRANDT cohort demonstrates that glioma patients with intermediate PLCβ1 expression (n = 103) survived significantly longer than PLCβ1 downregulated (2X) groups (n = 226). From TCGA data, PLCβ1 RNA-Seq signal inversely correlates with the pathological grades, and PLCβ1 expression in PN (n = 8) is of significantly higher levels than that in Mes (n = 8) subtypes of glioblastoma. The top 50 % of PLCβ1 expression subgroup (n = 294) of gliomas (grades II to IV merged) survived significantly longer than the low 50 percentile of the PLCβ1 expression subgroup (n = 293). p values are less than 0.05 for all these analyses. We conclude that PLCβ1 is a candidate signature gene for PN subtype HGG, and its expression inversely correlates with glioma pathological grade and is a potential prognostic factor.
33610094
Population-based study on incidence, survival rates, and genetic alterations of low-grade diffuse astrocytomas and oligodendrogliomas
We carried out a population-based study on low-grade diffuse gliomas in the Canton of Zurich, Switzerland (population 1.16 million). From 1980 to 1994, 987 astrocytic and oligodendroglial tumors were diagnosed, of which 122 (12.4%) were low-grade (WHO grade II). The incidence rates adjusted to the World Standard Population, per million population per year, were 2.28 for low-grade diffuse astrocytomas, 0.89 for oligoastrocytomas, and 2.45 for oligodendrogliomas. The survival rate (mean follow-up 7.5±4.8 years) was highest for patients with oligodendroglioma (78% at 5 years, 51% at 10 years), followed by those with oligoastrocytoma (70% at 5 years, 49% at 10 years) and fibrillary astrocytoma (65% at 5 years, 31% at 10 years). Survival of patients with gemistocytic astrocytoma was poor, with survival rates of 16% at 5 years and 0% at 10 years. Younger patients (<50 years) survived significantly longer than older patients (>50 years; P=0.013). DNA sequencing, performed in 84% of cases, revealed that TP53 mutations were most frequent in gemistocytic astrocytomas (88%), followed by fibrillary astrocytomas (53%) and oligoastrocytomas (44%), but were infrequent (13%) in oligodendrogliomas. The presence of TP53 mutations was associated with shorter survival of patients with low-grade diffuse gliomas (log-rank test; P=0.047), but when each histological type was analyzed separately, an association was observed only for oligoastrocytoma (P=0.05). Loss on 1p and 19q were assessed by quantitative microsatellite analysis in 67% of cases. These alterations were frequent in oligodendrogliomas (1p, 57%; 19q, 69%), less common in oligoastrocytomas (1p, 27%; 19q, 45%), rare in fibrillary astrocytomas (1p, 7%; 19q, 7%), and absent in gemistocytic astrocytomas. None of these alterations were predictive of survival. These results establish the frequency of key genetic alterations in low-grade diffuse gliomas at a population-based level. Multivariate Cox’s regression analysis indicates that only age and histological type, but not genetic alterations, are significant predictive factors.
3750884
MGMT testing—the challenges for biomarker-based glioma treatment
Many patients with malignant gliomas do not respond to alkylating agent chemotherapy. Alkylator resistance of glioma cells is mainly mediated by the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Epigenetic silencing of the MGMT gene by promoter methylation in glioma cells compromises this DNA repair mechanism and increases chemosensitivity. MGMT promoter methylation is, therefore, a strong prognostic biomarker in paediatric and adult patients with glioblastoma treated with temozolomide. Notably, elderly patients (>65–70 years) with glioblastoma whose tumours lack MGMT promoter methylation derive minimal benefit from such chemotherapy. Thus, MGMT promoter methylation status has become a frequently requested laboratory test in neuro-oncology. This Review presents current data on the prognostic and predictive relevance of MGMT testing, discusses clinical trials that have used MGMT status to select participants, evaluates known issues concerning the molecular testing procedure, and addresses the necessity for molecular-context-dependent interpretation of MGMT test results. Whether MGMT promoter methylation testing should be offered to all individuals with glioblastoma, or only to elderly patients and those in clinical trials, is also discussed. Justifications for withholding alkylating agent chemotherapy in patients with MGMT-unmethylated glioblastomas outside clinical trials, and the potential role for MGMT testing in other gliomas, are also discussed.
42512447
Extent of resection and radiotherapy in GBM: A 1973 to 2007 surveillance, epidemiology and end results analysis of 21,783 patients.
Surgery, radiation and chemotherapy are the standard of care for GBM patients, however, the impact of extent of resection (EOR) and radiotherapy (RT) on patient survival across age groups has not been established. Therefore, we present the current largest study on EOR and RT in GBM over the past three decades. Using the population based Surveillance, Epidemiology and End Results (SEER) registry, we identified a total of 21,783 GBM patients (1973-2007). Survival analysis based on EOR and RT was performed by means of factor analysis, Kaplan-Meier survival and Cox proportional hazard's ratio. Age, RT and EOR were highly prognostic (p<0.00001). Combined gross total resection (GTR) and RT showed the longest median survival (11 months) compared to subtotal resection (STR) and RT (9 months). Survival times after monotherapy with RT, GTR and STR were 5, 3 and 2 months, respectively. Patients without therapy showed a median survival of 1 month. RT and GTR demonstrated highest median survival. Interestingly, survival advantage of GTR versus STR amounted to only 1-2 months. Monotherapy (GTR, STR or RT) showed a significantly lower survival rate compared to combination therapies. RT alone yielded significantly better survival compared to any resective approach. Relative to overall age-specific median survival, elderly patients still reasonably benefit from RT alone. However, across all age groups multimodality treatment with surgery and RT continues to provide the largest survival benefit compared to either treatment alone and, thus, should be pursued whenever feasible.
8636959
DNA-microarray analysis of brain cancer: molecular classification for therapy
Primary brain tumours are among the most lethal of all cancers, largely as a result of their lack of responsiveness to current therapy. Numerous new therapies hold great promise for the treatment of patients with brain cancer, but the main challenge is to determine which treatment is most likely to benefit an individual patient. DNA-microarray-based technologies, which allow simultaneous analysis of expression of thousands of genes, have already begun to uncover previously unrecognized patient subsets that differ in their survival. Here, we review the progress made so far in using DNA microarrays to optimize brain cancer therapy.
2996477
IDH Mutation and Neuroglial Developmental Features Define Clinically Distinct Subclasses of Lower Grade Diffuse Astrocytic Glioma
Purpose: Diffuse gliomas represent the most prevalent class of primary brain tumor. Despite significant recent advances in the understanding of glioblastoma [World Health Organization (WHO) IV], its most malignant subtype, lower grade (WHO II and III) glioma variants remain comparatively understudied, especially in light of their notable clinical heterogeneity. Accordingly, we sought to identify and characterize clinically relevant molecular subclasses of lower grade diffuse astrocytic gliomas. Experimental Design: We conducted multidimensional molecular profiling, including global transcriptional analysis, on 101 lower grade diffuse astrocytic gliomas collected at our own institution and validated our findings using publically available gene expression and copy number data from large independent patient cohorts. Results: We found that IDH mutational status delineated molecularly and clinically distinct glioma subsets, with IDH mutant (IDH mt) tumors exhibiting TP53 mutations, platelet—derived growth factor receptor (PDGFR)A overexpression, and prolonged survival, and IDH wild-type (IDH wt) tumors exhibiting EGFR amplification, PTEN loss, and unfavorable disease outcome. Furthermore, global expression profiling revealed three robust molecular subclasses within lower grade diffuse astrocytic gliomas, two of which were predominantly IDH mt and one almost entirely IDH wt. IDH mt subclasses were distinguished from each other on the basis of TP53 mutations, DNA copy number abnormalities, and links to distinct stages of neurogenesis in the subventricular zone. This latter finding implicates discrete pools of neuroglial progenitors as cells of origin for the different subclasses of IDH mt tumors. Conclusion: We have elucidated molecularly distinct subclasses of lower grade diffuse astrocytic glioma that dictate clinical behavior and show fundamental associations with both IDH mutational status and neuroglial developmental stage. Clin Cancer Res; 18(9); 2490–501. ©2012 AACR.
26054757
Histomolecular classification of adult diffuse gliomas: the diagnostic value of immunohistochemical markers.
Adult gliomas are most often infiltrative. The World Health Organization (WHO) has classed them into three major groups according to the presomptive cell of origin: astrocytoma, oligodendroglioma and mixed oligoastrocytoma. Depending on the presence or absence of a small number of signs of anaplasia (mitosis, nuclear atypia, cell density, microvascular proliferation and necrosis) the WHO distinguishes grade II (LGG), III (anaplastic), and IV (glioblastomas, GBM). Mutation in the isocitrate deshydrogenase I and II (IDH1 and 2) genes distinguishes grade II, III and secondary GBM from primary GBM. Moreover two additional genetic alterations are recorded in grade II and III gliomas: TP53 mutations that characterize astrocytomas and 1p19q codeletion (as the result of t(1;19)(q10;p10) translocation) recorded in oligodendrogliomas. Mixed gliomas, the most non-reproducible category, share with astrocytomas and oligodendrogliomas the same genetic alterations. Interestingly TP53 mutation (p53+) and 1p19q codeletion (1p19q+) are mutually exclusive and involve IDH mutated (IDH+) glial precursor cells. According to IDH, TP53, and 1p19q status, four major subtypes of LGG are recorded: IDH+/p53-/1p19q-, IDH+/p53+/1p19q-, IDH+/p53-/1p19q+ and triple negative, this last subgroup having the worst prognosis. Interestingly, p53 expression and internexin alpha (INA) expression can replace to some extent TP53 mutation and 1p19 codeletion, respectively. Moreover the antibody directed against the IDH1R132H isoform is highly specific. Because this mutation is the most frequent it is sufficient to assess IDH status in more than 80% of grade II and III gliomas. Taken together these three immunohistochemical markers are contribute greatly to the classification of gliomas and should be tested routinely as diagnostic markers. Finally, although GBM are genetically heterogeneous, the vast majority display EGFR amplification, often associated with EGFR expression, which can be helpful for diagnosis in certain cases.
5801583
Whole-genome sequencing identifies genetic alterations in pediatric low-grade gliomas
The most common pediatric brain tumors are low-grade gliomas (LGGs). We used whole-genome sequencing to identify multiple new genetic alterations involving BRAF, RAF1, FGFR1, MYB, MYBL1 and genes with histone-related functions, including H3F3A and ATRX, in 39 LGGs and low-grade glioneuronal tumors (LGGNTs). Only a single non-silent somatic alteration was detected in 24 of 39 (62%) tumors. Intragenic duplications of the portion of FGFR1 encoding the tyrosine kinase domain (TKD) and rearrangements of MYB were recurrent and mutually exclusive in 53% of grade II diffuse LGGs. Transplantation of Trp53-null neonatal astrocytes expressing FGFR1 with the duplication involving the TKD into the brains of nude mice generated high-grade astrocytomas with short latency and 100% penetrance. FGFR1 with the duplication induced FGFR1 autophosphorylation and upregulation of the MAPK/ERK and PI3K pathways, which could be blocked by specific inhibitors. Focusing on the therapeutically challenging diffuse LGGs, our study of 151 tumors has discovered genetic alterations and potential therapeutic targets across the entire range of pediatric LGGs and LGGNTs.
21692235
Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1.
The Cancer Genome Atlas Network recently cataloged recurrent genomic abnormalities in glioblastoma multiforme (GBM). We describe a robust gene expression-based molecular classification of GBM into Proneural, Neural, Classical, and Mesenchymal subtypes and integrate multidimensional genomic data to establish patterns of somatic mutations and DNA copy number. Aberrations and gene expression of EGFR, NF1, and PDGFRA/IDH1 each define the Classical, Mesenchymal, and Proneural subtypes, respectively. Gene signatures of normal brain cell types show a strong relationship between subtypes and different neural lineages. Additionally, response to aggressive therapy differs by subtype, with the greatest benefit in the Classical subtype and no benefit in the Proneural subtype. We provide a framework that unifies transcriptomic and genomic dimensions for GBM molecular stratification with important implications for future studies.
3954095
Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis.
Previously undescribed prognostic subclasses of high-grade astrocytoma are identified and discovered to resemble stages in neurogenesis. One tumor class displaying neuronal lineage markers shows longer survival, while two tumor classes enriched for neural stem cell markers display equally short survival. Poor prognosis subclasses exhibit markers either of proliferation or of angiogenesis and mesenchyme. Upon recurrence, tumors frequently shift toward the mesenchymal subclass. Chromosomal locations of genes distinguishing tumor subclass parallel DNA copy number differences between subclasses. Functional relevance of tumor subtype molecular signatures is suggested by the ability of cell line signatures to predict neurosphere growth. A robust two-gene prognostic model utilizing PTEN and DLL3 expression suggests that Akt and Notch signaling are hallmarks of poor prognosis versus better prognosis gliomas, respectively.
174047
Gene Expression Profiling of Gliomas Strongly Predicts Survival
In current clinical practice, histology-based grading of diffuse infiltrative gliomas is the best predictor of patient survival time. Yet histology provides little insight into the underlying biology of gliomas and is limited in its ability to identify and guide new molecularly targeted therapies. We have performed large-scale gene expression analysis using the Affymetrix HG U133 oligonucleotide arrays on 85 diffuse infiltrating gliomas of all histologic types to assess whether a gene expression-based, histology-independent classifier is predictive of survival and to determine whether gene expression signatures provide insight into the biology of gliomas. We found that gene expression-based grouping of tumors is a more powerful survival predictor than histologic grade or age. The poor prognosis samples could be grouped into three different poor prognosis groups, each with distinct molecular signatures. We further describe a list of 44 genes whose expression patterns reliably classify gliomas into previously unrecognized biological and prognostic groups: these genes are outstanding candidates for use in histology-independent classification of high-grade gliomas. The ability of the large scale and 44 gene set expression signatures to group tumors into strong survival groups was validated with an additional external and independent data set from another institution composed of 50 additional gliomas. This demonstrates that large-scale gene expression analysis and subset analysis of gliomas reveals unrecognized heterogeneity of tumors and is efficient at selecting prognosis-related gene expression differences which are able to be applied across institutions.
16776393
Molecular classification of gliomas based on whole genome gene expression: a systematic report of 225 samples from the Chinese Glioma Cooperative Group.
Defining glioma subtypes based on objective genetic and molecular signatures may allow for a more rational, patient-specific approach to molecularly targeted therapy. However, prior studies attempting to classify glioma subtypes have given conflicting results. We aim to complement and validate the existing molecular classification system on a large number of samples from an East Asian population. A total of 225 samples from Chinese patients was selected for whole genome gene expression profiling. Consensus clustering was applied. Three major groups of gliomas were identified (referred to as G1, G2, and G3). The G1 subgroup correlates with a good clinical outcome, young age, and extremely high frequency of IDH1 mutations. Relative to the G1 subgroup, the G3 subgroup is correlated with a poorer clinical outcome, older age, and a very low rate of mutations in the IDH1 gene. Correlations of the G2 subgroup with respect to clinical outcome, age, and IDH1 mutation fall between the G1 and G3 subgroups. In addition, the G2 subtype was associated with a higher percentage of loss of 1p/19q when compared with G1 and G3 subtypes. Furthermore, our classification scheme was validated on 2 independent datasets derived from the cancer genome atlas (TCGA) and Rembrandt. With use of the TCGA classification system, proneural, neural, and mesenchymal, but not classical subtype, associated gene signatures were clearly defined. In summary, our results reveal that 3 main subtypes stably exist in Chinese patients with glioma. Our classification scheme may reflect the clinical and genetic alterations more clearly. Classical subtype-associated gene signature was not found in our dataset.
305627
Differential distribution of phospholipase C beta isoforms and diaglycerol kinase-beta in rodents cerebella corroborates the division of unipolar brush cells into two major subtypes
Sublineage diversification of specific neural cell classes occurs in complex as well as simply organized regions of the central and peripheral nervous systems; the significance of the phenomenon, however, remains insufficiently understood. The unipolar brush cells (UBCs) are glutamatergic cerebellar interneurons that occur at high density in vestibulocerebellum. As they are classified into subsets that differ in chemical phenotypes, intrinsic properties, and lobular distribution, they represent a valuable neuronal model to study subclass diversification. In this study, we show that cerebellar UBCs of adult rats and mice form two subclasses—type I and type II UBCs—defined by somatodendritic expression of calretinin (CR), mGluR1α, phospholipases PLCβ1 and PLCβ4, and diacylglycerol kinase-beta (DGKβ). We demonstrate that PLCβ1 is associated only with the CR+ type I UBCs, while PLCβ4 and DGKβ are exclusively present in mGluR1α+ type II UBCs. Notably, all PLCβ4+ UBCs, representing about 2/3 of entire UBC population, also express mGluR1α. Furthermore, our data show that the sum of CR+ type I UBCs and mGluR1α+ type II UBCs accounts for the entire UBC class identified with Tbr2 immunolabeling. The two UBC subtypes also show a very different albeit somehow overlapping topographical distribution as illustrated by detailed cerebellar maps in this study. Our data not only complement and extend the previous knowledge on the diversity and subclass specificity of the chemical phenotypes within the UBC population, but also provide a new angle to the understanding of the signaling networks in type I and type II UBCs.
7384152
Cellular neurochemical characterization and subcellular localization of phospholipase C β1 in rat brain
The present study describes a complete and detailed neuroanatomical distribution map of the phospholipase C beta1 (PLCβ1) isoform along the adult rat neuraxis, and defines the phenotype of cells expressing PLCβ1, along with its subcellular localization in cortical neurons as assessed by double-immunofluorescence staining and confocal laser scanning. Immunohistochemical labeling revealed a considerable morphological heterogeneity among PLCβ1-positive cells in the cortex, even though there was a marked predominance of pyramidal morphologies. As an exception to the general non-matching distribution of GFAP and PLCβ1, a high degree of co-expression was observed in radial glia-like processes of the spinal cord white matter. In the somatosensory cortex, the proportion of GABAergic neurons co-stained with PLCβ1 was similar (around 2/3) in layers I, II-III, IV and VI, and considerably lower in layer V (around 2/5). Double immunofluorescence against PLCβ1 and nuclear speckle markers SC-35 and NeuN/Fox3 in isolated nuclei from the rat cortex showed a high overlap of both markers with PLCβ1 within the nuclear matrix. In contrast, there was no apparent co-localization with markers of the nuclear envelope and lamina. Finally, to assess whether the subcellular expression pattern of PLCβ1 involved specifically one of the two splice variants of PLCβ1, we carried out Western blot experiments in cortical subcellular fractions. Notably, PLCβ1a/1b ratios were statistically higher in the cytoplasm than in the nuclear and plasma membrane fractions. These results provide a deeper knowledge of the cellular distribution of the PLCβ1 isoform in different cell subtypes of the rat brain, and of its presence in the neuronal nuclear compartment.
27058985
Predominant expression of phospholipase Cβ1 in telencephalic principal neurons and cerebellar interneurons, and its close association with related signaling molecules in somatodendritic neuronal elements
Upon activation of receptors coupled to the Gq subclass of G proteins, phospholipase C (PLC)β hydrolyses membrane phospholipid to yield a pair of second messengers, inositol 1,4,5‐trisphosphate and 1,2‐diacylglycerol. Of four PLCβ isoforms, PLCβ1 is transcribed predominantly in the telencephalon and its gene inactivation in mice impairs metabotropic glutamate receptor‐ and muscarinic acetylcholine receptor‐dependent hippocampal oscillations, endocannabinoid production in the hippocampus and barrel formation in the somatosensory cortex. Here we examined cellular and subcellular distributions of PLCβ1 in adult mouse brains. In the telencephalon, high levels of PLCβ1 were observed in principal neurons, including pyramidal cells in the cortex and hippocampus, granule cells and mossy cells in the dentate gyrus, and medium spiny neurons in the caudate‐putamen, whereas most interneurons had low levels of or were negative for PLCβ1 and, instead, expressed PLCβ4. By immunofluorescence, tiny clusters of PLCβ1 were distributed in somatodendritic compartments of principal neurons and positioned close to those of metabotropic glutamate receptor 5, muscarinic acetylcholine receptor M1 and diacylglycerol lipase‐α, respectively. Immunoelectron microscopy revealed that PLCβ1 was often associated with the smooth endoplasmic reticulum, cell membrane or postsynaptic density. In particular, it was highly accumulated at the perisynapse of dendritic spines forming asymmetrical synapses. In the cerebellum, PLCβ1 was generally low but was enriched in axons and dendrites of basket cells. These results suggest that PLCβ1 is the key effector in telencephalic principal neurons and cerebellar interneurons. Furthermore, the well‐orchestrated molecular arrangement appears to be the anatomical basis for the specificity, efficiency and convergence of the neuronal phosphoinositide signaling system.
31059621
Brain phospholipase C isozymes: differential mRNA localizations by in situ hybridization.
mRNAs for isozymes of phospholipase C (PLC) were localized in rat brain by in situ hybridization with oligonucleotide probes for PLC isozymes I, II, and III of Rhee's group [Suh, P.-G., Ryu, S. H., Moon, K. H., Suh, H. W. & Rhee, S. G. (1988) Proc. Natl. Acad. Sci. USA 85, 5419-5423 and (1988) Cell 54, 161-169], and isozyme I of Bennett and Crooke [Bennett, C. F., Balcarek, J. M., Varrichio, A. & Crooke, S. T. (1988) Nature (London) 334, 268-270], which we designate PLC-A. The isozymes displayed different localizations. PLC-A mRNA was highest in the mitral cell layer of the olfactory bulb, choroid plexus, hippocampus and dentate gyrus, magnocellular hypothalamic nuclei, rostral raphe nuclei, and cerebellar Purkinje cells. PLC-I was highest in the internal granular cell layer of the olfactory bulb, cerebral cortex, caudate, nucleus of the lateral olfactory tract, reticular nucleus of thalamus, hippocampus and dentate gyrus, and granule cell layer of the cerebellum. PLC-II had a more widespread distribution, with relatively high levels in the internal granular layer of the olfactory bulb, hippocampus and dentate gyrus, and cerebellar Purkinje and granule cells. PLC-III label was low throughout the brain. These distributions suggest selective coupling of individual PLC isozymes with particular postsynaptic receptors. PLC-A may be preferentially associated with 5-hydroxytryptamine 1C receptors, vasopressin V1 receptors, and a subtype of glutamate receptors. PLC-I may be linked to muscarinic m1 and m3 receptors as well as other receptors. The distribution of PLC-II mRNA resembles that of src protooncogene, with which it displays sequence homology.
1152249
Phospholipase C-beta1 expression correlates with neuronal differentiation and synaptic plasticity in rat somatosensory cortex.
Receptor-mediated signal transduction is thought to play an important role in neuronal differentiation and the modification of synaptic connections during brain development. The intracellular signalling molecule phospholipase C-beta1 (PLC-beta1), which is activated via specific neurotransmitter receptors, has recently been implicated in activity-dependent plasticity in the cat visual cortex. PLC-beta1 has been shown to be concentrated in an intermediate compartment-like organelle, the botrysome, which is present in 5-week-old, but not adult, cat cortical neurons. We have characterized the spatial and temporal regulation of PLC-beta1 expression in the developing rat cerebral cortex. PLC-beta1-positive botrysome-like organelles are observed during early postnatal cortical development, but not at postnatal day 14 or later stages. In the postnatal somatosensory cortex, there is also striking spatial variation in diffuse neuropilar immunoreactivity of layer IV and above, in a pattern corresponding to the thalamocortical recipient zones known as barrels. This expression pattern is specific to the developing barrel field and is most distinct at postnatal days 4-7, when cellular components of barrels are capable of activity-dependent modification. During later stages of cortical maturation, stained botrysomes disappear, expression of PLC-beta1 is down-regulated and only diffuse immunoreactivity remains in dendritic processes. Our results are consistent with a role for PLC-beta1 in activity-dependent, receptor-mediated neuronal plasticity during development of the somatosensory cortex.
34572527
Phospholipase C isozymes in neurons and glial cells in culture: an immunocytochemical and immunochemical study
Expression of 3 brain isozymes of phospholipase (PLC-beta, PLC-gamma and PLC-delta) was investigated in relation to cell types found in rat CNS cultures. Immunoreactivity of cultured neurons, astrocytes and oligodendrocytes was demonstrated for all of the 3 isozymes by immunocytochemical staining and immunoblotting, with some differences in reaction intensity. Immunoblotting revealed that the level of expression was neurons greater than oligodendrocytes greater than astrocytes for PLC-beta and PLC-gamma, and astrocytes greater than oligodendrocytes greater than neurons for PLC-delta.
24926954
Expression of phosphoinositide‐specific phospholipase C isoenzymes in cultured astrocytes
Signal transduction from plasma membrane to cell nucleus is a complex process depending on various components including lipid signaling molecules, in particular phosphoinositides and their related enzymes, which act at cell periphery and/or plasma membrane as well as at nuclear level. As far as the nervous system may concern the inositol lipid cycle has been hypothesized to be involved in numerous neural as well as glial functions. In this context, however, a precise panel of glial PLC isoforms has not been determined yet. In the present experiments we investigated astrocytic PLC isoforms in astrocytes obtained from foetal primary cultures of rat brain and from an established cultured (C6) rat astrocytoma cell line, two well known cell models for experimental studies on glia. Identification of PLC isoforms was achieved by using a combination of RT‐PCR and immunocytochemistry experiments. While in both cell models the most represented PI‐PLC isoforms were β4, γ1, δ4, and ε, isoforms PI‐PLC β2 and δ3 were not detected. Moreover, in primary astrocyte cultures PI‐PLC δ3 resulted well expressed in C6 cells but was absent in astrocytes. Immunocytochemistry performed with antibodies against specific PLC isoforms substantially confirmed this pattern of expression both in astrocytes and C6 glioma cells. In particular while some isoenzymes (namely isoforms β3 and β4) resulted mainly nuclear, others (isoforms δ4 and ε) were preferentially localized at cytoplasmic and plasma membrane level. J. Cell. Biochem. 100: 952–959, 2007. © 2006 Wiley‐Liss, Inc.
40837611
Expression of phosphoinositide‐specific phospholipase C isoenzymes in cultured astrocytes activated after stimulation with lipopolysaccharide
Signal transduction pathways, involved in cell cycle and activities, depend on various components including lipid signalling molecules, such as phosphoinositides and related enzymes. Many evidences support the hypothesis that inositol lipid cycle is involved in astrocytes activation during neurodegeneration. Previous studies investigated the pattern of expression of phosphoinositide‐specific phospholipase C (PI‐PLC) family isoforms in astrocytes, individuating in cultured neonatal rat astrocytes, supposed to be quiescent cells, the absence of some isoforms, accordingly to their well known tissue specificity. The same study was conducted in cultured rat astrocytoma C6 cells and designed a different pattern of expression of PI‐PLCs in the neoplastic counterpart, accordingly to literature suggesting a PI signalling involvement in tumour progression. It is not clear the role of PI‐PLC isoforms in inflammation; recent data demonstrate they are involved in cytokines production, with special regard to IL‐6. PI‐PLCs expression in LPS treated neonatal rat astrocytes performed by using RT‐PCR, observed at 3, 6, 18 and 24 h intervals, expressed: PI‐PLC beta1, beta4 and gamma1 in all intervals analysed; PI‐PLC delta1 at 6, 18 and 24 h; PI‐PLC delta3 at 6 h after treatment. PI‐PLC beta3, delta4 and epsilon, present in untreated astrocytes, were not detected after LPS treatment. Immunocytochemical analysis, performed to visualize the sub‐cellular distribution of the expressed isoforms, demonstrated different patterns of localisation at different times of exposure. These observations suggest that PI‐PLCs expression and distribution may play a role in ongoing inflammation process of CNS. J. Cell. Biochem. 109: 1006–1012, 2010. © 2010 Wiley‐Liss, Inc.
6776087
Localization of two forms of phospholipase C-beta1, a and b, in C6Bu-1 cells.
Phospholipase C-beta1 (PLC-beta1), one of the PLC-beta isozymes, exists as two immunologically distinguishable polypeptides of 150 (PLC-beta1a) and 140 kDa (PLC-beta1b) which are encoded in two distinct transcripts and generated by alternative splicing of a single gene. In this study, the subcellular localization of the two phospholipases C-beta1 proteins was examined in rat C6Bu-1 glioma cells using immunological techniques. Immunoblot analysis revealed that the two forms of PLC-beta1 were detectable in both cytosolic and nuclear fractions. PLC-beta1a appeared to be located preferentially in the cytosol, whereas PLC-beta1b was found predominantly in the nuclei of C6Bu-1 cells. Immunocytochemical experiments confirmed the differential localization of the two PLC-beta1 species in C6Bu-1 cells. These results suggest that the two PLC-beta1 proteins may have different physiological roles in the cell.
1836154
Comprehensive genomic characterization defines human glioblastoma genes and core pathways
Human cancer cells typically harbour multiple chromosomal aberrations, nucleotide substitutions and epigenetic modifications that drive malignant transformation. The Cancer Genome Atlas (TCGA) pilot project aims to assess the value of large-scale multi-dimensional analysis of these molecular characteristics in human cancer and to provide the data rapidly to the research community. Here we report the interim integrative analysis of DNA copy number, gene expression and DNA methylation aberrations in 206 glioblastomas—the most common type of adult brain cancer—and nucleotide sequence aberrations in 91 of the 206 glioblastomas. This analysis provides new insights into the roles of ERBB2, NF1 and TP53, uncovers frequent mutations of the phosphatidylinositol-3-OH kinase regulatory subunit gene PIK3R1, and provides a network view of the pathways altered in the development of glioblastoma. Furthermore, integration of mutation, DNA methylation and clinical treatment data reveals a link between MGMT promoter methylation and a hypermutator phenotype consequent to mismatch repair deficiency in treated glioblastomas, an observation with potential clinical implications. Together, these findings establish the feasibility and power of TCGA, demonstrating that it can rapidly expand knowledge of the molecular basis of cancer.
14050561
Reversing HOXA9 oncogene activation by PI3K inhibition: epigenetic mechanism and prognostic significance in human glioblastoma.
HOXA genes encode critical transcriptional regulators of embryonic development that have been implicated in cancer. In this study, we documented functional relevance and mechanism of activation of HOXA9 in glioblastoma (GBM), the most common malignant brain tumor. Expression of HOXA genes was investigated using reverse transcription-PCR in primary gliomas and glioblastoma cell lines and was validated in two sets of expression array data. In a subset of GBM, HOXA genes are aberrently activated within confined chromosomal domains. Transcriptional activation of the HOXA cluster was reversible by a phosphoinostide 3-kinase (PI3K) inhibitor through an epigenetic mechanism involving histone H3K27 trimethylation. Functional studies of HOXA9 showed its capacity to decrease apoptosis and increase cellular proliferation along with tumor necrosis factor-related apoptosis-including ligand resistance. Notably, aberrant expression of HOXA9 was independently predictive of shorter overall and progression-free survival in two GBM patient sets and improved survival prediction by MGMT promoter methylation. Thus, HOXA9 activation is a novel, independent, and negative prognostic marker in GBM that is reversible through a PI3K-associated epigenetic mechanism. Our findings suggest a transcriptional pathway through which PI3K activates oncogenic HOXA expression with implications for mTOR or PI3K targeted therapies.
17152532
Neuronal and glioma-derived stem cell factor induces angiogenesis within the brain.
Stem cell factor (SCF) is overexpressed by neurons following brain injury as well as by glioma cells; however, its role in gliomagenesis remains unclear. Here, we demonstrate that SCF directly activates brain microvascular endothelial cells (ECs) in vitro and induces a potent angiogenic response in vivo. Primary human gliomas express SCF in a grade-dependent manner and induce normal neurons to express SCF in brain regions infiltrated by glioma cells, areas that colocalize with prominent angiogenesis. Downregulation of SCF inhibits tumor-mediated angiogenesis and glioma growth in vivo, whereas overexpression of SCF is associated with shorter survival in patients with malignant gliomas. Thus, the SCF/c-Kit pathway plays an important role in tumor- and normal host cell-induced angiogenesis within the brain.
21430110
Overexpression of the EGFR/FKBP12/HIF-2alpha pathway identified in childhood astrocytomas by angiogenesis gene profiling.
Intense angiogenesis proliferation, a histopathological hallmark distinguishing malignant from benign astrocytoma, is vital for tumor progression. Thus, identifying and targeting specific pathways that promote malignant astrocytoma-induced angiogenesis could have substantial therapeutic benefit. Expression profiling of 13 childhood astrocytomas to determine the expression pattern of 133 angiogenesis-related genes revealed that 44 (33%) genes were differentially expressed (17 were overexpressed, and 27 were underexpressed) between malignant high-grade astrocytomas (HGAs) and benign low-grade astrocytomas. Hierarchical clustering and principal components analysis using only the 133 angiogenesis-related genes distinguished HGA from low-grade astrocytoma in 100% of the samples analyzed, as did unsupervised analyses using the entire set of 9198 expressed genes represented on the array, indicating that the angiogenesis-related genes were reliable markers of pathological grade. A striking new finding was significant overexpression of hypoxia-inducible transcription factor (HIF)-2alpha as well as high-level expression of FK506-binding protein (FKBP) 12 by HGA. Furthermore, 9 of 21 (43%) genes overexpressed by HGA were HIF/FKBP-associated genes. This group included the epidermal growth factor receptor (EGFR), which promotes HIF synthesis, as well as insulin-like growth factor-binding protein 2 (IGFBP2), a target gene of HIF activity. Differential protein expression of HIF-2alpha was validated in an independent group of 16 astrocytomas (P = 0.02). We conclude that the EGFR/FKBP12/HIF-2alpha pathway is important in childhood HGA and represents a potential new therapeutic target.
120386719
Distance-Weighted Discrimination
High-dimension low–sample size statistical analysis is becoming increasingly important in a wide range of applied contexts. In such situations, the popular support vector machine suffers from "data piling" at the margin, which can diminish generalizability. This leads naturally to the development of distance-weighted discrimination, which is based on second-order cone programming, a modern computationally intensive optimization method.
29689803
The epidemiology of glioma in adults: a "state of the science" review.
Gliomas are the most common primary intracranial tumor, representing 81% of malignant brain tumors. Although relatively rare, they cause significant mortality and morbidity. Glioblastoma, the most common glioma histology (∼45% of all gliomas), has a 5-year relative survival of ∼5%. A small portion of these tumors are caused by Mendelian disorders, including neurofibromatosis, tuberous sclerosis, and Li-Fraumeni syndrome. Genomic analyses of glioma have also produced new evidence about risk and prognosis. Recently discovered biomarkers that indicate improved survival include O⁶-methylguanine-DNA methyltransferase methylation, isocitrate dehydrogenase mutation, and a glioma cytosine-phosphate-guanine island methylator phenotype. Genome-wide association studies have identified heritable risk alleles within 7 genes that are associated with increased risk of glioma. Many risk factors have been examined as potential contributors to glioma risk. Most significantly, these include an increase in risk by exposure to ionizing radiation and a decrease in risk by history of allergies or atopic disease(s). The potential influence of occupational exposures and cellular phones has also been examined, with inconclusive results. We provide a “state of the science” review of current research into causes and risk factors for gliomas in adults.
10628465
Prognostic impact of the isocitrate dehydrogenase 1 single‐nucleotide polymorphism rs11554137 in malignant gliomas
The IDH1 gene, which encodes isocitrate dehydrogenase 1, is frequently mutated in gliomas and acute myeloid leukemia. The single‐nucleotide polymorphism (SNP) (reference SNP no. rs11554137:C>T) located on IDH1 codon 105 has been associated with a poor outcome in patients with acute myeloid leukemia but has not been investigated in patients with gliomas.
24065690
Moving toward molecular classification of diffuse gliomas in adults
Diffuse gliomas are a heterogenous group of neoplasms traditionally classified as grades II to IV based on histologic features, and with prognosis determined mainly by histologic grade and pretreatment clinical factors. Our understanding of the molecular basis of glioma initiation, tumor progression, and treatment failure is rapidly evolving. A molecular profile of diffuse gliomas is emerging. Studies evaluating gene expression and DNA methylation profile have found multiple glioma subtypes and an association between subtype and survival. The recent discovery of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) mutations in glioma has provided reproducible prognostic biomarkers and novel therapeutic targets. Glioblastomas that exhibit CpG island hypermethylator phenotype, proneural gene expression, or IDH1 mutation identify a subset of patients with markedly improved prognosis. Accumulated evidence supports the stratification of both low-grade and anaplastic diffuse gliomas into prognostic groups using 1p/19q codeletion and IDH mutation status. A classification scheme incorporating clinical, pathologic, and molecular information may facilitate improved prognostication for patients treated in the clinic, the development of more effective clinical trials, and rational testing of targeted therapeutics.
1696137
Isoform-level gene signature improves prognostic stratification and accurately classifies glioblastoma subtypes
Molecular stratification of tumors is essential for developing personalized therapies. Although patient stratification strategies have been successful; computational methods to accurately translate the gene-signature from high-throughput platform to a clinically adaptable low-dimensional platform are currently lacking. Here, we describe PIGExClass (platform-independent isoform-level gene-expression based classification-system), a novel computational approach to derive and then transfer gene-signatures from one analytical platform to another. We applied PIGExClass to design a reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) based molecular-subtyping assay for glioblastoma multiforme (GBM), the most aggressive primary brain tumors. Unsupervised clustering of TCGA (the Cancer Genome Altas Consortium) GBM samples, based on isoform-level gene-expression profiles, recaptured the four known molecular subgroups but switched the subtype for 19% of the samples, resulting in significant (P = 0.0103) survival differences among the refined subgroups. PIGExClass derived four-class classifier, which requires only 121 transcript-variants, assigns GBM patients’ molecular subtype with 92% accuracy. This classifier was translated to an RT-qPCR assay and validated in an independent cohort of 206 GBM samples. Our results demonstrate the efficacy of PIGExClass in the design of clinically adaptable molecular subtyping assay and have implications for developing robust diagnostic assays for cancer patient stratification.
37521483
Clinical impact of molecular biomarkers in gliomas
The World Health Organization (WHO) classification system for glial tumors provides guidance as to the predicted course of the disease and choice of treatment. However, histologically identical tumors may have a very different outcome and response to treatment. Molecular markers that carry both diagnostic and prognostic information add valuable tools by redefining tumor subtypes within each WHO category. Therefore, molecular biomarkers have become an integral part of tumor assessment in modern neuro-oncology and biomarker status now guides clinical decisions in some subtypes of gliomas, including anaplastic oligodendroglioma and glioblastoma in the elderly. This review discusses the prognostic and predictive impact of molecular markers that have undergone extensive study in recent years. The clinical relevance of contemporary molecular classification of gliomas using the routine assessment of IDH mutations, promoter methylation of MGMT, chromosomal deletion of 1p/19q, mutations of EGFR and ATRX genes, and BRAF fusion or point mutation is highlighted. The potential of molecular biomarker-based classification to guide future therapeutic approach is discussed and accentuated.
26766038
Key determinants of short-term and long-term glioblastoma survival: A 14-year retrospective study of patients from the Hermelin Brain Tumor Center at Henry Ford Hospital
OBJECTIVE
Glioblastoma (GBM) is a heterogeneous neoplasm with a small percentage of long-term survivors. Despite aggressive surgical resection and advances in radiotherapy and chemotherapy, the median survival for patients with GBM is 12-14 months. Factors associated with a favorable prognosis include young age, high performance status, gross resection >98%, non-eloquent tumor location and O6-methylguanine methyltransferase (MGMT) promoter methylation. We retrospectively analyzed the relationship of clinical, epidemiologic, genetic and molecular characteristics with survival in patients with GBM.


METHODS
This retrospective analysis of overall survival looked at the outcomes of 480 patients diagnosed with GBM over 14 years at a single institution. Multivariate analysis was performed examining multiple patient characteristics.


RESULTS
Median survival time improved from 11.8 months in patients diagnosed from 1995 to 1999 to 15.9 months in those diagnosed from 2005 to 2008. Factors associated with survivor groups were age, KPS, tumor resection, treatment received and early progression. 18 cancer-related genes were upregulated in short-term survivors and five genes were downregulated in short-term survivors.


CONCLUSIONS
Epidemiologic, clinical, and molecular characteristics all contribute to GBM prognosis. Identifying factors associated with survival is important for treatment strategies as well as research for novel therapeutics and technologies. This study demonstrated improved survival for patients over time as well as significant differences among survivor groups.
6517763
Molecular characterization of long‐term survivors of glioblastoma using genome‐ and transcriptome‐wide profiling
The prognosis of glioblastoma, the most malignant type of glioma, is still poor, with only a minority of patients showing long‐term survival of more than three years after diagnosis. To elucidate the molecular aberrations in glioblastomas of long‐term survivors, we performed genome‐ and/or transcriptome‐wide molecular profiling of glioblastoma samples from 94 patients, including 28 long‐term survivors with >36 months overall survival (OS), 20 short‐term survivors with <12 months OS and 46 patients with intermediate OS. Integrative bioinformatic analyses were used to characterize molecular aberrations in the distinct survival groups considering established molecular markers such as isocitrate dehydrogenase 1 or 2 (IDH1/2) mutations, and O6‐methylguanine DNA methyltransferase (MGMT) promoter methylation. Patients with long‐term survival were younger and more often had IDH1/2‐mutant and MGMT‐methylated tumors. Gene expression profiling revealed over‐representation of a distinct (proneural‐like) expression signature in long‐term survivors that was linked to IDH1/2 mutation. However, IDH1/2‐wildtype glioblastomas from long‐term survivors did not show distinct gene expression profiles and included proneural, classical and mesenchymal glioblastoma subtypes. Genomic imbalances also differed between IDH1/2‐mutant and IDH1/2‐wildtype tumors, but not between survival groups of IDH1/2‐wildtype patients. Thus, our data support an important role for MGMT promoter methylation and IDH1/2 mutation in glioblastoma long‐term survival and corroborate the association of IDH1/2 mutation with distinct genomic and transcriptional profiles. Importantly, however, IDH1/2‐wildtype glioblastomas in our cohort of long‐term survivors lacked distinctive DNA copy number changes and gene expression signatures, indicating that other factors might have been responsible for long survival in this particular subgroup of patients.© 2014 UICC
2175440
Patterns of expression for the mRNA corresponding to the four isoforms of phospholipase Cβ in mouse brain
Ligand binding to neurotransmitter and hormone receptors which couple to the Gq subclass of GTP‐binding protein leads to the activation of phospholipase Cβ (PLCβ) which hydrolyses phosphatidyl‐inositol 4,5‐bisphosphate, yielding a pair of second messengers, diacylglycerol and inositol 1,4,5‐trisphosphate (IP3). The expression of PLCβ1–4 mRNAs was comparatively examined by in situ hybridization in the mouse brain. In adults, PLCβ1 mRNA was expressed predominantly in the telencephalon, including the cerebral cortex, hippocampus, amygdala, lateral septum and olfactory bulb, with little expression in most thalamic nuclei. PLCβ2 mRNA was distributed in the white matter, suggesting its expression in non‐neuronal cells, most likely oligodendrocytes. PLCβ3 mRNA was specifically expressed in cerebellar Purkinje cells. The highest levels of PLCβ4 mRNA were detected in Purkinje cells. High levels of PLCβ4 mRNA were also found in the thalamus and medial septum, whereas weak signals were detected in most telencephalic regions, thus showing an expression pattern almost reciprocal to that of PLCβ1 mRNA. During development, such characteristic regional expression of PLCβ1 and PLCβ4 were observed starting in late foetal stages, while specific expression of PLCβ2 and PLCβ3 appeared in early postnatal stages. We conclude that despite the existence of four PLCβ isoforms, only one or two of them is expressed in individual neurons and glial cells. The distinct expression of PLCβs provides a molecular basis for analysing the nature of the specific signal transduction pathway leading to the production of diacylglycerol and IP3 in distinct cell types and in different regions of the brain.
4365900
Phospholipase C isozymes selectively couple to specific neurotransmitter receptors
A variety of extracellular signals are transduced across the cell membrane by the enzyme phosphoinositide-specific phospholipase C-β (PLC-β) coupled with guanine-nucleotide-binding G proteins. There are four isoenzymes of PLC-β, β1–β4, but their functions in vivo are not known. Here we investigate the role of PLC-β1 and PLC-β4 in the brain by generating null mutations in mice: we found that PLCβ1−/− mice developed epilepsy and PLCβ4−/− mice showed ataxia. We determined the molecular basis of these phenotypes and show that PLC-β1 is involved in signal transduction in the cerebral cortex and hippocampus by coupling predominantly to the muscarinic acetylcholine receptor, whereas PLC-β4 works through the metabotropic glutamate receptor in the cerebellum, illustrating how PLC-β isoenzymes are used to generate different functions in the brain.
24506431
Transmembrane signaling through phospholipase C‐β in the developing human prefrontal cortex
To investigate changes in muscarinic receptor‐stimulated phospholipase C‐β (PLC‐β) activity during brain development, we examined the functional coupling of each of the three major protein components of the phosphoinositide system (M1, M3, and M5 muscarinic receptor subtypes; Gq/11 proteins; PLC‐β1–4 isoforms) in membrane preparations from post‐mortem human prefrontal cerebral cortex collected at several stages of prenatal and postnatal development. In human prenatal brain membranes, PLC was found to be present and could be activated by calcium, but the ability of guanosine‐5′‐o‐3 thiotriphosphate (GTPγS) or carbachol (in the presence of GTPγS) to modulate prenatal PLC‐β was significantly weaker than that associated with postnatal PLC‐β. Western blot analysis revealed that the levels of Gαq/11 did not change significantly during development. In contrast, dramatically higher levels of expression of PLC‐β1–4 isoforms and of M1, M3, and M5 muscarinic receptors were detected in the child vs. the fetal brain, a finding that might underlie the observed increased activity of PLC. Thus, inositol phosphate production may be more efficiently regulated by altering the amount of effectors (PLC‐β1–4) and receptors (M1,3,5 subtypes) than by altering the level of Gαq/11 subunits. These results demonstrate that different PLC isoforms are expressed in the prefrontal cortex of the developing human brain in an age‐specific manner, suggesting specific roles not only in synaptic transmission but also in the differentiation and maturation of neurons in the developing brain. © 2006 Wiley‐Liss, Inc.
15316798
Primary glioblastoma with oligodendroglial differentiation has better clinical outcome but no difference in common biological markers compared with other types of glioblastoma.
BACKGROUND
Glioblastoma multiforme with an oligodendroglial component (GBMO) has been recognized in the World Health Organization classification-however, the diagnostic criteria, molecular biology, and clinical outcome of primary GBMO remain unclear. Our aim was to investigate whether primary GBMO is a distinct clinicopathological subgroup of GBM and to determine the relative frequency of prognostic markers such as loss of heterozygosity (LOH) on 1p and/or 19q, O(6)-methylguanine-DNA methyltransferase (MGMT) promoter methylation, and isocitrate dehydrogenase 1 (IDH1) mutation.


METHODS
We examined 288 cases of primary GBM and assessed the molecular markers in 57 GBMO and 50 cases of other primary GBM, correlating the data with clinical parameters and outcome.


RESULTS
GBMO comprised 21.5% of our GBM specimens and showed significantly longer survival compared with our other GBM (12 mo vs 5.8 mo, P = .006); there was also a strong correlation with younger age at diagnosis (56.4 y vs 60.6 y, P = .005). Singular LOH of 19q (P = .04) conferred a 1.9-fold increased hazard of shorter survival. There was no difference in the frequencies of 1p or 19q deletion, MGMT promoter methylation, or IDH1 mutation (P = .8, P = 1.0, P = 1.0, respectively).


CONCLUSIONS
Primary GBMO is a subgroup of GBM associated with longer survival and a younger age group but shows no difference in the frequency of LOH of 1p/19q, MGMT, and IDH1 mutation compared with other primary GBM.
15078127
Phospholipase C beta 1 deficiency is associated with early-onset epileptic encephalopathy.
The epileptic encephalopathies of infancy and childhood are a collection of epilepsy disorders characterized by refractory, severe seizures and poor neurological outcome, in which the mechanism of disease is poorly understood. We report the clinical presentation and evolution of epileptic encephalopathy in a patient, associated with a loss-of-function mutation in the phospholipase C-β 1 gene. We ascertained a consanguineous family containing a male infant who presented with early-onset epileptic encephalopathy for detailed clinical phenotyping and molecular genetic investigation. In addition, a cohort of 12 consanguineous families of children with infantile spasms were analysed for linkage to the phospholipase C-β 1 gene locus. The male infant presented with tonic seizures in early infancy and subsequently developed infantile spasms. Over time, he developed drug-resistant epilepsy associated with severe neurological regression and failure to thrive. Molecular genetic investigation revealed a homozygous loss-of-function 0.5-Mb deletion, encompassing the promoter element and exons 1, 2 and 3 of phospholipase C-β 1 in the index case. Linkage to the phospholipase C-β 1 locus was excluded in the 12 other consanguineous families, consistent with genetic heterogeneity in this disorder. Although phospholipase C-β 1 deficiency has not previously been reported in humans, the Plcb1 homozygote knockout mouse displays early-onset severe tonic seizures and growth retardation, thus recapitulating the human phenotype. Phospholipase C-β 1 has important functions in both hippocampal muscarinic acetylcholine receptor signalling and in cortical development. Thus, the discovery of a phospholipase C-β 1 mutation allows us to propose a novel potential underlying mechanism in early-onset epileptic encephalopathy.
5802365
Severe infantile epileptic encephalopathy due to mutations in PLCB1: expansion of the genotypic and phenotypic disease spectrum
Homozygous deletions of chromosome 20p12.3, disrupting the promoter region and first three coding exons of the phospholipase C β1 gene (PLCB1), have previously been described in two reports of early infantile epileptic encephalopathy (EIEE). Both children were born to consanguineous parents, one presented with infantile spasms, the other with migrating partial seizures of infancy. We describe an infant presenting with severe intractable epilepsy (without a specific EIEE electroclinical syndrome diagnosis) and neurodevelopmental delay associated with compound heterozygous mutations in PLCB1. A case note review and molecular genetic investigations were performed for a child, approximately 10 months of age, admitted to Johns Hopkins University Hospital for developmental delay and new‐onset seizures. The patient presented at 6 months of age with developmental delay, followed by the onset of intractable, focal, and generalized seizures associated with developmental regression from 10 months of age. Presently, at 2 years of age, the child has severe motor and cognitive delays. Diagnostic microarray revealed a heterozygous 476kb deletion of 20p12.3 (encompassing PLCB1), which was also detected in the mother. The genomic breakpoints for the heterozygous deletion were determined. In order to investigate the presence of a second PLCB1 mutation, direct Sanger sequencing of the coding region and flanking intronic regions was undertaken, revealing a novel heterozygous intron 1 splice site variant (c.99+1G>A) in both the index individual and the father. Advances in molecular genetic testing have greatly improved diagnostic rates in EIEE, and this report further confirms the important role of microarray investigation in this group of disorders. PLCB1‐EIEE is now reported in a number of different EIEE phenotypes and our report provides further evidence for phenotypic pleiotropy encountered in early infantile epilepsy syndromes.
26688909
Towards a knowledge-based Human Protein Atlas
1. Anonymous. Nat. Biotechnol. 28, 987 (2010). 2. Plosila, W.H. Econ. Dev. Q. 18, 113–126 (2004). 3. Stayn, S. BNA Med. Law Pol. Rep. 5, 718–725 (2006). 4. Lomax, G. & Stayn, S. BNA Med. Law Pol. Rep. 7, 695–698 (2008). 5. Levine, A.D. Public Adm. Rev. 68, 681–694 (2008). 6. Levine, A.D. Nat. Biotechnol. 24, 865–866 (2006). 7. McCormick, J.B., Owen-Smith, J. & Scott, C.T. Cell Stem Cell 4, 107–110 (2009). 8. Fossett, J.W., Ouellette, A.R., Philpott, S., Magnus, D. & Mcgee, G. Hastings Cent. Rep. 37, 24–35 (2007). 9. Mintrom, M. Publius 39, 606–631 (2009). 10. Scott, C.T., McCormick, J.B. & Owen-Smith, J. Nat. Biotechnol. 27, 696–697 (2009). 11. Takahashi, K. & Yamanaka, S. Cell 126, 663–676 (2006). Foundation and the Georgia Research Alliance, and Georgia Tech. They thank J. Walsh at Georgia Tech for helpful comments on an earlier version of this manuscript. They also appreciate the assistance they received with data collection from officials in various state stem cell agencies. A.D.L. would also like to thank A. Jakimo, whose comment at a meeting of the Interstate Alliance on Stem Cell Research inspired collection of these data. stem cell programs, as well as similar state programs supporting other areas of science, is uncertain. The analysis here suggests that state stem cell funding programs are sufficiently large and established that simply ending the programs, at least in the absence of substantial investment in the field by other funding sources, could have deleterious effects. Such action would fail to capitalize on the initial efforts of scientists who have been drawn to the field of stem cell research by state programs and leave many stem cell scientists suddenly searching for funding to continue their research. Large-scale state funding for basic research is a relatively new phenomenon, and many questions remain about the impact of these programs on the development of scientific fields and the careers of scientists. The influence of state funding programs on the distribution of research publications, the acquisition of future external funding, the creation of new companies and the translation of basic research into medical practice, for instance, are important unanswered questions. Similarly, comparing state funding programs with federal funding programs as well as foundations could offer new insight into the relative priorities of different funding bodies and the extent to which their funding portfolios overlap or are distinct. We hope the analysis presented here and the public release of the underlying database will inspire additional analysis of state science funding programs generally and state-funded stem cell science in particular.
20275510
Two forms of phospholipase C-beta 1 generated by alternative splicing.
Phospholipase C-beta 1 (PLC-beta 1) exists as two immunologically indistinguishable polypeptides of 150 and 140 kDa and is encoded in rat brain by two distinct transcripts of 5.4 and 7.2 kilobases (kb). cDNA corresponding to the entire 5.4-kb transcript as reported previously reveals an open reading frame that is capable of coding a 1216-amino acid polypeptide (Suh, P. G., Ryu, S. H., Moon, K. H., Suh, H. W., and Rhee, S. G. (1988) Cell 54, 161-169). We have now isolated cDNAs corresponding to the entire 7.2-kb transcript from a rat brain cDNA library. The 7.2-kb transcript differs from the previously reported 5.4-kb transcript by possessing both an additional 118 nucleotides located near the end of the coding sequence and a 1738-nucleotide extension of the 3'-flanking region. The presence of the 118-nucleotide insert in the cumulative 7.2-kb sequence gives rise to an open reading frame that is capable of coding a 1173-amino acid polypeptide (PLC-beta 1b), the carboxyl-terminal sequence of which differs from that of the 1216-amino acid polypeptide (PLC-beta 1a) derived from the 5.4-kb transcript. Antibodies were raised against synthetic peptides corresponding to the carboxyl-terminal portions of PLC-beta 1a and PLC-beta 1b. Immunoblot analysis with these isozyme-specific antibodies revealed that both PLC-beta 1a and PLC-beta 1b are expressed in rat brain and C6Bu-1 glioma cells and that PLC-beta 1a and PLC-beta 1b correspond to the previously identified 150- and 140-kDa PLC-beta 1 enzymes, respectively. Analysis of PLC-beta 1 genomic DNA indicates that PLC-beta 1a and PLC-beta 1b are derived from a single gene by alternative RNA splicing.
38884836
Nuclear but not cytoplasmic phospholipase C beta 1 inhibits differentiation of erythroleukemia cells.
A body of evidence has shown the existence of a nuclear phosphoinositide cycle in different cell types. The cycle is endowed with kinases as well as phosphatases and phospholipase C (PLC). Among the PLC isozymes, the beta family is characterized by a long COOH-terminal tail that contains a cluster of lysine residues responsible for nuclear localization. Indeed, PLC beta 1 is the major isoform that has been detected in the nucleus of several cells. This isoform is activated by insulin-like growth factor I, and when this isoform is lacking, as a result of gene ablation, the onset of DNA synthesis induced by this hormone is abolished. On the contrary, PLC beta 1 is down-regulated during the erythroid differentiation of Friend erythroleukemia cells. A key question is how PLC beta 1 signaling at the nucleus fits into the erythroid differentiation program of Friend erythroleukemia cells, and whether PLC beta 1 signaling activity is directly responsible for the maintenance of the undifferentiated state of erythroleukemia cells. Here we present evidence that nuclear PLC beta 1 but not the isoform located at the plasma membrane is directly involved in maintaining the undifferentiated state of Friend erythroleukemia cells. Indeed, when wild-type PLC beta 1 is overexpressed in these cells, differentiation in response to DMSO is inhibited in that the expression of beta-globin is almost completely abolished, whereas when a mutant lacking the ability to localize to the nucleus is expressed, the cells differentiate, and the expression of beta-globin is the same as in wild-type cells.
23004095
Nuclear diacylglycerol lipase‐α in rat brain cortical neurons: evidence of 2‐arachidonoylglycerol production in concert with phospholipase C‐β activity
In this report, we describe the localization of diacylglycerol lipase‐α (DAGLα) in nuclei from adult cortical neurons, as assessed by double‐immunofluorescence staining of rat brain cortical sections and purified intact nuclei and by western blot analysis of subnuclear fractions. Double‐labeling assays using the anti‐DAGLα antibody and NeuN combined with Hoechst staining showed that only nuclei of neuronal origin were DAGLα positive. At high resolution, DAGLα‐signal displayed a punctate pattern in nuclear subdomains poor in Hoechst's chromatin and lamin B1 staining. In contrast, SC‐35‐ and NeuN‐signals (markers of the nuclear speckles) showed a high overlap with DAGLα within specific subdomains of the nuclear matrix. Among the members of the phospholipase C‐β (PLCβ) family, PLCβ1, PLCβ2, and PLCβ4 exhibited the same distribution with respect to chromatin, lamin B1, SC‐35, and NeuN as that described for DAGLα. Furthermore, by quantifying the basal levels of 2‐arachidonoylglycerol (2‐AG) by liquid chromatography and mass spectrometry (LC‐MS), and by characterizing the pharmacology of its accumulation, we describe the presence of a mechanism for 2‐AG production, and its PLCβ/DAGLα‐dependent biosynthesis in isolated nuclei. These results extend our knowledge about subcellular distribution of neuronal DAGLα, providing biochemical grounds to hypothesize a role for 2‐AG locally produced within the neuronal nucleus.
8212565
Nuclear PI-PLCβ1: an appraisal on targets and pathology.
Lipid signalling molecules are essential components of the processes that allow one extracellular signal to be transferred inside the nucleus, where specific lipid second messengers elicit reactions capable of regulating gene transcription, DNA replication or repair and DNA cleavage, eventually resulting in cell growth, differentiation, apoptosis or many other cell functions. Nuclear inositides are independently regulated, suggesting that the nucleus constitutes a functionally distinct compartment of inositol lipids metabolism. Indeed, nuclear inositol lipids themselves can modulate nuclear processes, such as transcription and pre-mRNA splicing, growth, proliferation, cell cycle regulation and differentiation. Nuclear PI-PLCβ1 is a key molecule for nuclear inositide signalling, where it plays a role in cell cycle progression, proliferation and differentiation. Here we review the targets and possible involvement of nuclear PI-PLCβ1 in human physiology and pathology.
35666035
Nuclear phospholipase C beta1, regulation of the cell cycle and progression of acute myeloid leukemia.
A large number of observations have hinted at the fact that location impinges on function of some of the main players of nuclear inositol lipid cycle. PLC beta1 is a well-known example, given that it has been shown that only the enzyme located in the nucleus targets the cyclin D3/cdk4 complex, playing, in turn, a key role in the control of normal progression through the G1 phase of the cell cycle. The PLC beta1 gene, which is constituted of 36 small exons and large introns, maps on the short arm of human chromosome 20 (20pl2, nearby markers D20S917 and D20S177) with the specific probe (PAC clone HS881E24) spanning from exon 19 to 32 of the gene itself. The chromosome band 20pl2 has been shown to be rearranged in human diseases such as solid tumors without a more accurate definition of the alteration, maybe because of the absence of candidate genes or specific probes. Moreover, non-specific alterations in chromosome 20 have been found in patients affected by MDS and acute myeloid leukemia AML. MDS is an adult hematological disease that evolves into AML in about 30% of the cases. The availability of a highly specific probe gave an opportunity to perform in patients affected with MDS/AML, associated with normal karyotype, painting and FISH analysis aimed to check the PLC beta1 gene, given that this signaling molecule is a key player in the control of some checkpoints of the normal progression through the cell cycle. FISH analysis disclosed in a small group of MDS/AML patients with normal karyotype the monoallelic deletion of the PLC beta1 gene. In contrast, PLC beta4, another gene coding for a signaling molecule, located on 20pl2.3 at a distance as far as less than 1 Mb from PLC beta1, is unaffected in MDS patients with the deletion of PLC beta1 gene, hinting at an interstitial deletion. The MDS patients, bearing the deletion, rapidly evolved to AML, whilst the normal karyotype MDS patients, showing non-deletion of PLC beta1 gene, are still alive at least 24 months after the diagnosis. The immunocytochemical analysis using an anti PLC beta1 monoclonal antibody showed that all the AML/MDS patients who were normal at FISH analysis also had normal staining of the nucleus, which is a preferential site for PLC beta1. In contrast, the monoallelic deletion gave rise to a dramatic decrease of the nuclear staining suggesting a decreased expression of the nuclear PLC beta1. The reported data strengthen the contention of a key role played by PLC beta1 in the nucleus, suggest a possible involvement of PLC beta1 in the progression of MDS to AML and pave the way for a larger investigation aimed at identifying a possible high risk group among MDS patients with a normal karyotype.
9407951
ErbB1-4-dependent EGF/neuregulin signals and their cross talk in the central nervous system: pathological implications in schizophrenia and Parkinson's disease
Ligands for ErbB1-4 receptor tyrosine kinases, such as epidermal growth factor (EGF) and neuregulins, regulate brain development and function. Thus, abnormalities in their signaling are implicated in the etiology or pathology of schizophrenia and Parkinson's disease. Among the ErbB receptors, ErbB1, and ErbB4 are expressed in dopamine and GABA neurons, while ErbB1, 2, and/or 3 are mainly present in oligodendrocytes, astrocytes, and their precursors. Thus, deficits in ErbB signaling might contribute to the neurological and psychiatric diseases stemming from these cell types. By incorporating the latest cancer molecular biology as well as our recent progress, we discuss signal cross talk between the ErbB1-4 subunits and their neurobiological functions in each cell type. The potential contribution of virus-derived cytokines (virokines) that mimic EGF and neuregulin-1 in brain diseases are also discussed.
35725431
PLC and PI3K/Akt/mTOR signalling in disease and cancer.
Cancer cell metabolism is deregulated, and signalling pathways can be involved. For instance, PI3K/Akt/mTOR is associated with normal proliferation and differentiation, and its alteration is detectable in cancer cells, that exploit the normal mechanisms to overcome apoptosis. On the other hand, also the family of Phospholipase C (PLC) enzymes play a critical role in cell growth, and any change concerning these enzymes or their downstream targets can be associated with neoplastic transformation. Here, we review the role of PLC and PI3K/Akt/mTOR signal transduction pathways in pathophysiology.
22125374
PI‐PLCβ1b affects Akt activation, cyclin E expression, and caspase cleavage, promoting cell survival in pro‐B‐lymphoblastic cells exposed to oxidative stress
The phosphoinositide‐dependent signal transduction pathway has been implicated in the control of a variety of biologic processes, such as the regulation of cellular metabolism and homeostasis, cell proliferation and differentiation, and apoptosis. One of the key players in the regulation of inositol lipid signaling is the phospholipase Cβ1 (PI‐PLCβ1), that hydrolyzes phosphatidylinositol 4,5‐bisphosphate [PtIns(4,5)P2], giving rise to the second messengers inositol triphosphate and diacylglicerol. PI‐PLC/1 has been associated with the regulation of several cellular functions, some of which have not yet been fully understood. In particular, it has been reported that PI‐PLCβ1 protects murine fibroblasts from oxidative stress‐induced cell death. The mediators of oxidative stress, reactive oxygen species (ROS), have been shown to regulate major epigenetic processes, causing the silencing of tumor suppressors and enhancing the proliferation of leukemic cells under oxidative stress. Investigation of the interplay between ROS, PI‐PLCβ1, and their signaling mediators in leukemia might therefore reveal innovative targets of pharmacological therapy in the treatment for leukemia. In this work, we demonstrate that in pro‐B‐lymphoblastic cells (Ba/F3), treated with H2O2, PI‐PLCβ1b conferred resistance to cell death, promoting cell cycle progression and cell proliferation and influencing the expression of cyclin A and E. Interestingly, we found that, expression of PI‐PLCβ1b affects the activity of caspase‐3, caspase‐7, and of several protein kinases induced by oxidative stress. In particular, PI‐PLCβ1b expression completely abolished the phosphorylation of Erk1/2 MAP kinases, down‐regulated phosphatase and tensin homolog (PTEN), and up‐regulated the phosphorylation of Akt, thereby sustaining cellular proliferation.—Piazzi, M., Blalock, W. L., Bavelloni, A., Faenza, I., Raffini, M., Tagliavini, F., Manzoli, L. Cocco, L. PI‐PLCβ1b affects Akt activation, cyclin E expression, and caspase cleavage, promoting cell survival in pro‐B‐lymphoblastic cells exposed to oxidative stress. FASEB J. 29, 1383‐1394 (2015). www.fasebj.org
18779079
Presenilin-Dependent ErbB4 Nuclear Signaling Regulates the Timing of Astrogenesis in the Developing Brain
Embryonic multipotent neural precursors are exposed to extracellular signals instructing them to adopt different fates, neuronal or glial. However, the mechanisms by which precursors integrate these signals to make timely fate choices remained undefined. Here we show that direct nuclear signaling by a receptor tyrosine kinase inhibits the responses of precursors to astrocyte differentiation factors while maintaining their neurogenic potential. Upon neuregulin-induced activation and presenilin-dependent cleavage of ErbB4, the receptor's intracellular domain forms a complex with TAB2 and the corepressor N-CoR. This complex undergoes nuclear translocation and binds promoters of astrocytic genes, repressing their expression. Consistent with this observation, astrogenesis occurs precociously in ErbB4 knockout mice. Our studies define how presenilin-dependent nuclear signaling by a receptor tyrosine kinase directly regulates gene transcription and cell fate. This pathway could be of importance for neural stem cell biology and for understanding the pathogenesis of Alzheimer's disease.
207245644

 In situ hybridization reveals developmental regulation of ErbB1-4 mRNA expression in mouse midbrain: Implication of ErbB receptors for dopaminergic neurons
Although epidermal growth factor (EGF) and neuregulin-1 are neurotrophic factors for mesencephalic dopaminergic neurons and implicated in schizophrenia, the cellular localization and developmental regulation of their receptors (ErbB1-4) remain to be characterized. Here we investigated the distributions of mRNA for ErbB1-4 in the midbrain of the developing mouse with in situ hybridization and immunohistochemistry. The expression of ErbB1 and ErbB2 mRNAs was relatively high at the perinatal stage and frequently colocalized with mRNA for S100beta and Olig2, markers for immature astrocytes or oligodendrocyte precursors. Modest signal for ErbB1 mRNA was also detected in a subset of dopaminergic neurons. ErbB3 mRNA was detectable at postnatal day 10, peaked at postnatal day 18, and colocalized with 2',3'-cyclic nucleotide 3'-phosphodiesterase, a marker for oligodendrocytes. In contrast, ErbB4 mRNA was exclusively localized in neurons throughout development. Almost all of ErbB4 mRNA-expressing cells (94%-96%) were positive for tyrosine hydroxylase in the substantia nigra pars compacta but 66%-78% in the ventral tegmental area and substantia nigra pars lateralis. Conversely, 92%-99% of tyrosine hydroxylase-positive cells expressed ErbB4 mRNA. The robust and restricted expression of ErbB4 mRNA in the midbrain dopaminergic neurons suggests that ErbB4 ligands, neuregulin-1 and other EGF-related molecules, contribute to development or maintenance of this neuronal population.
13778198
Selective Expression of ErbB4 in Interneurons, But Not Pyramidal Cells, of the Rodent Hippocampus
NRG1 and ERBB4 have emerged as some of the most reproducible schizophrenia risk genes. Moreover, the Neuregulin (NRG)/ErbB4 signaling pathway has been implicated in dendritic spine morphogenesis, glutamatergic synaptic plasticity, and neural network control. However, despite much attention this pathway and its effects on pyramidal cells have received recently, the presence of ErbB4 in these cells is still controversial. As knowledge of the precise locus of receptor expression is crucial to delineating the mechanisms by which NRG signaling elicits its diverse physiological effects, we have undertaken a thorough analysis of ErbB4 distribution in the CA1 area of the rodent hippocampus using newly generated rabbit monoclonal antibodies and ErbB4-mutant mice as negative controls. We detected ErbB4 immunoreactivity in GABAergic interneurons but not in pyramidal neurons, a finding that was further corroborated by the lack of ErbB4 mRNA in electrophysiologically identified pyramidal neurons as determined by single-cell reverse transcription–PCR. Contrary to some previous reports, we also did not detect processed ErbB4 fragments or nuclear ErbB4 immunoreactivity. Ultrastructural analysis in CA1 interneurons using immunoelectron microscopy revealed abundant ErbB4 expression in the somatodendritic compartment in which it accumulates at, and adjacent to, glutamatergic postsynaptic sites. In contrast, we found no evidence for presynaptic expression in cultured GAD67-positive hippocampal interneurons and in CA1 basket cell terminals. Our findings identify ErbB4-expressing interneurons, but not pyramidal neurons, as a primary target of NRG signaling in the hippocampus and, furthermore, implicate ErbB4 as a selective marker for glutamatergic synapses on inhibitory interneurons.