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.

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title: Localization of two forms of phospholipase C-beta1, a and b, in C6Bu-1 cells.
abstract: 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.

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abstract: 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.

title: Overexpression of the EGFR/FKBP12/HIF-2alpha pathway identified in childhood astrocytomas by angiogenesis gene profiling.
abstract: 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.

title: Distance-Weighted Discrimination
abstract: 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.

title: The epidemiology of glioma in adults: a "state of the science" review.
abstract: 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.

title: Prognostic impact of the isocitrate dehydrogenase 1 single‐nucleotide polymorphism rs11554137 in malignant gliomas
abstract: 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.

title: Moving toward molecular classification of diffuse gliomas in adults
abstract: 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.

title: Isoform-level gene signature improves prognostic stratification and accurately classifies glioblastoma subtypes
abstract: 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.

title: Clinical impact of molecular biomarkers in gliomas
abstract: 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.

title: 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
abstract: 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.



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.



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.



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.

title: Molecular characterization of long‐term survivors of glioblastoma using genome‐ and transcriptome‐wide profiling
abstract: 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.

title: Patterns of expression for the mRNA corresponding to the four isoforms of phospholipase Cβ in mouse brain
abstract: 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.

title: Phospholipase C isozymes selectively couple to specific neurotransmitter receptors
abstract: 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.

title: Transmembrane signaling through phospholipase C‐β in the developing human prefrontal cortex
abstract: 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.

title: Primary glioblastoma with oligodendroglial differentiation has better clinical outcome but no difference in common biological markers compared with other types of glioblastoma.
abstract: 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.



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.



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).



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.

title: Phospholipase C beta 1 deficiency is associated with early-onset epileptic encephalopathy.
abstract: 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.

title: Severe infantile epileptic encephalopathy due to mutations in PLCB1: expansion of the genotypic and phenotypic disease spectrum
abstract: 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.

title: Towards a knowledge-based Human Protein Atlas
abstract: 1.  (2010). 2. Plosila, W.H. Econ. Dev. Q. 18, 113–126 (2004). 3. Stayn, S. BNA Med.  (2006). 4. Lomax, G. & Stayn, S. BNA Med.  (2008). 5. Levine, A.D. Public Adm. Rev. 68, 681–694 (2008). 6. Levine,  (2006). 7. McCormick, J.B., Owen-Smith, J. & Scott,  (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,  (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.

title: Two forms of phospholipase C-beta 1 generated by alternative splicing.
abstract: 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.

title: Nuclear but not cytoplasmic phospholipase C beta 1 inhibits differentiation of erythroleukemia cells.
abstract: 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.

title: Nuclear diacylglycerol lipase‐α in rat brain cortical neurons: evidence of 2‐arachidonoylglycerol production in concert with phospholipase C‐β activity
abstract: 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.

title: Nuclear PI-PLCβ1: an appraisal on targets and pathology.
abstract: 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.

title: Nuclear phospholipase C beta1, regulation of the cell cycle and progression of acute myeloid leukemia.
abstract: 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.

title: ErbB1-4-dependent EGF/neuregulin signals and their cross talk in the central nervous system: pathological implications in schizophrenia and Parkinson's disease
abstract: 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.

title: PLC and PI3K/Akt/mTOR signalling in disease and cancer.
abstract: 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.

title: PI‐PLCβ1b affects Akt activation, cyclin E expression, and caspase cleavage, promoting cell survival in pro‐B‐lymphoblastic cells exposed to oxidative stress
abstract: 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

title: Presenilin-Dependent ErbB4 Nuclear Signaling Regulates the Timing of Astrogenesis in the Developing Brain
abstract: 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.

title: 
 In situ hybridization reveals developmental regulation of ErbB1-4 mRNA expression in mouse midbrain: Implication of ErbB receptors for dopaminergic neurons
abstract: 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.

title: Selective Expression of ErbB4 in Interneurons, But Not Pyramidal Cells, of the Rodent Hippocampus
abstract: 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.