<Emphasis Type="Italic">PIK3CA</Emphasis> alterations in primary (de novo) and secondary glioblastomas

We assessed alterations in the EGFR/PTEN/PI3K pathway in 107 primary (de novo) glioblastomas and 32 secondary glioblastomas that progressed from low-grade or anaplastic astrocytomas. SSCP followed by DNA sequencing in exons 9 and 20 of the PIK3CA gene revealed missense mutations in 5/107 (5%) primary and 1/32 (3%) secondary glioblastomas. Quantitative real-time PCR showed PIK3CA amplification (>3 copy numbers) in 14/107 (13%) primary and 3/32 (9%) secondary glioblastomas. Only one glioblastoma showed both PIK3CA mutation and amplification. Taken together with previously published data on EGFR amplification and PTEN mutations, at least one alteration in the EGFR, PTEN, or PIK3CA genes was detected in 63% of primary glioblastomas, which was significantly more frequent than in secondary glioblastomas (31%; P < 0.001). Furthermore, this signaling pathway was altered by either PTEN mutations or PIK3CA amplification in 10 of 12 (83%) malignant glioma cell lines analyzed. These results suggest that the EGFR/PTEN/PI3K pathway is frequently altered in glioblastomas and is a promising target for therapy, in particular for primary glioblastomas.     

Mutation analysis of the Ras pathway genes<Emphasis Type="Italic"> NRAS</Emphasis>,<Emphasis Type="Italic"> HRAS</Emphasis>,<Emphasis Type="Italic"> KRAS</Emphasis> and<Emphasis Type="Italic"> BRAF</Emphasis> in glioblastomas

Aberrant activation of Ras signaling is a common finding in human glioblastomas. To determine the contribution of Ras gene mutations to this aberration, we screened 94 glioblastomas for mutations in the three Ras family genes NRAS, KRAS and HRAS. All tumors were additionally analyzed for mutations in BRAF, which encodes a Ras-regulated serine/threonine kinase with oncogenic properties. Mutation analysis of the entire coding regions of NRAS and KRAS, as well as the known mutation hot-spot sites in HRAS, identified somatic point mutations in two glioblastomas, both affecting codon 12 of NRAS (c.35G>A, p.G12D). Three additional tumors carried BRAF mutations altering the known hot-spot codon 599 (c.1796T>A, p.V599E). None of these five glioblastomas showed amplification of the EGFR or PDGFRA genes, while three of the tumors, including two with NRAS and one with BRAF mutation, demonstrated PTEN missense mutations or loss of PTEN mRNA expression. Taken together, our data suggest activating mutations in NRAS or BRAF as a molecular alteration that contributes to aberrant Ras signaling in a small fraction of glioblastomas.     

<Emphasis Type="Italic">PTEN</Emphasis> methylation and expression in glioblastomas

The tumor suppressor gene PTEN on chromosome 10q23.3 regulates the Akt signaling pathway and modulates cell growth and apoptosis. The PTEN gene is mutated in 20–40% of glioblastomas. In this study, we assessed whether loss of PTEN expression is also caused epigenetically. Methylation-specific PCR revealed that CpG islands of the PTEN promoter were methylated in 27 of 77 (35%) glioblastomas and in 4 of 11 (36%) glioblastoma cell lines. Only two glioblastomas showed loss of PTEN immunoreactivity in the entire biopsy; both had a missense PTEN mutation and LOH at the PTEN locus, but lacked PTEN methylation. In biopsy specimens with focal loss of PTEN expression, DNA samples extracted from microdissected foci showed PTEN methylation only in areas with loss of PTEN expression. These results suggest that PTEN methylation occurs frequently in glioblastomas and may be associated with focal loss of PTEN expression. However, the correlation between PTEN methylation, PTEN mutations, LOH at the PTEN locus, and loss of PTEN protein expression was inconsistent. Possible reasons for discrepancies between gene status and protein expression include differences in the biological effect of specific PTEN mutations and the possibility that the processed PTEN pseudogene on 9p21 is expressed in glioblastomas and co-reacts with the PTEN antibody.     

<Emphasis Type="Italic">CIC</Emphasis> and <Emphasis Type="Italic">FUBP1</Emphasis> mutations in oligodendrogliomas,oligoastrocytomas and astrocytomas

CIC and FUBP1 mutations have recently been detected in oligodendrogliomas but not in oligoastrocytomas. However, allelic losses in the regions on chromosomal arms 19q and 1p harboring CIC and FUBP1 are a common feature of both, oligodendrogliomas and oligoastrocytomas. To resolve this discrepancy, we analyzed CIC and FUBP1 mutations in a set of primary brain tumors including 18 oligodendrogliomas and 42 oligoastrocytomas. In addition, we analyzed 10 astrocytomas and 16 glioblastomas with allelic losses on 19q as well as a set of 12 medulloblastomas for CIC mutations. CIC mutations were found in 15/18 oligodendrogliomas, 14/42 oligoastrocytomas and 3/10 preselected astrocytomas. With the exception of a single case, all CIC mutations occurred in tumors with combined 1p/19q losses. In contrast to oligodendrogliomas where CIC mutations were always detected along with 1p/19q co-deletion, CIC mutations were only found in 52 % of the 1p/19q co-deleted oligoastrocytomas. FUBP1 mutations were detected in 7/61 tumors, all presenting with CIC mutations. FUBP1 mutations appear to cluster in the DNA binding domain spanning exons 5–14. CIC and FUBP1 mutations exclusively occurred in presence of either IDH1 or IDH2 mutations. Our data confirm CIC and FUBP1 mutations in oligodendrogliomas and demonstrate the presence of these mutations in oligoastrocytomas.     

Caspase-3 activation is required for reovirus-induced encephalitis <Emphasis Type="Italic">in vivo</Emphasis>

Reovirus infection of neonatal mice provides a classic experimental system for understanding the molecular pathogenesis of central nervous system (CNS) viral infection. CNS tissue injury, caused by many human neurotropic viruses, including herpes viruses and West Nile virus, is associated with caspase-dependent apoptotic neuronal cell death. We have previously shown that reovirus-induced CNS tissue injury results from apoptosis and is associated with activation of both death-receptor and mitochondrial apoptotic pathways culminating in the activation of the downstream effector caspase, caspase-3. In order to directly investigate the role of caspase-3 in virus-induced neuronal death and CNS tissue injury during encephalitis, we have compared the pathogenesis of reovirus CNS infection in mice lacking the caspase-3 gene (caspase-3 (−/−)) to syngeneic wild-type mice. Prior studies of antiapoptotic treatments for reovirus-infected mice have indicated that protection from reovirus-induced neuronal injury can occur without altering the viral titer in the brains of infected mice. We now show that reovirus infection of caspase-3 (−/−) mice was associated with dramatic reduction in severity of CNS tissue injury, decreased viral antigen and titer in the brain, and enhanced survival of infected mice. Following intracerebral inoculation, the authors also show that virus spread from the brain to the eyes in reovirus-infected caspase-3 (−/−) mice, indicating that viral spread was intact in these mice. Examination of brains of long-term survivors of reovirus infection among caspase-3 (−/−) mice showed that these mice eventually clear their CNS viral infection, and do not manifest residual or delayed CNS tissue injury.     

Lack of adrenoleukodystrophy protein enhances oligodendrocyte disturbance and microglia activation in mice with combined <Emphasis Type="Italic">Abcd1</Emphasis>/<Emphasis Type="Italic">Mag</Emphasis> deficiency

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurometabolic disease associated with the accumulation of very long-chain fatty acids. Mutations in the ABCD1 gene encoding ALD protein (ALDP) cause this clinically heterogeneous disorder, ranging from adrenocortical insufficiency and neurodegeneration to severe cerebral inflammation and demyelination. ALDP-deficient mice replicate metabolic dysfunctions and develop late-onset axonopathy but lack histological signs of cerebral inflammation and demyelination. To test the hypothesis that subtle destabilization of myelin may initiate inflammatory demyelination in Abcd1 deficiency, we generated mice with the combined metabolic defect of X-ALD and the mild myelin abnormalities of myelin-associated glycoprotein (MAG) deficiency. A behavioural phenotype, impaired motor performance and tremor, developed in middle-aged Mag null mice, independent of Abcd1 genotype. Routine histology revealed no signs of inflammation or demyelination in the CNS, but immunohistochemical analyses of spinal cord neuropathology revealed microglia activation and axonal degeneration in Mag and Abcd1/Mag double-knockout (ko) and, less severe and of later onset, in Abcd1 mutants. While combined Abcd1/Mag deficiency showed an additive effect on microglia activation, axonal degeneration, quantified by accumulation of amyloid precursor protein (APP) in axonal spheroids, was not accelerated. Interestingly, abnormal APP reactivity was enhanced within compact myelin of Abcd1/Mag double-ko mice compared to single mutants already at 13 months. These results suggest that ALDP deficiency enhances metabolic distress in oligodendrocytes that are compromised a priori by destabilised myelin. Furthermore, the age at which this occurs precedes by far the onset of axonal degeneration in Abcd1-deficient mice, implying that oligodendrocyte/myelin disturbances may precede axonopathy in X-ALD.     

Association study of <Emphasis Type="Italic">interleukin 2</Emphasis> (<Emphasis Type="Italic">IL2</Emphasis>) and <Emphasis Type="Italic">IL4</Emphasis> with schizophrenia in a Japanese population

Interleukin 2 (IL-2) and IL-4 are pleiotropic cytokines regulating Th1/Th2 balance and have a regulatory activity in brain function. Thus these cytokines have been implicated in the pathophysiology of schizophrenia. The latest studies provided controversial results regarding the genetic associations of these cytokines. The functional polymorphisms, IL2-330T/G and IL4-590C/T, were associated with schizophrenia in a German population, although contradictory findings were also reported in a Korean population. To ascertain whether IL2 and IL4 contribute to vulnerability to schizophrenia, we conducted a moderate-scale case-control (536 patients and 510 controls) association study for seven polymorphisms in Japanese subjects. There were no significant associations of these genes with schizophrenia using either single marker or haplotype analyses. The present study suggests that IL2 and IL4 do not contribute to vulnerability to schizophrenia in the Japanese population.     

The <Emphasis Type="Italic">GABRB3</Emphasis> Polymorphism and its Association with Schizophrenia

The aim of this study was to explore whether schizophrenia occurrence is associated with polymorphisms in the 5′ regulatory region of GABRB3 (gamma-aminobutyric acid type A receptor beta 3, subunit gene). The study included 324 patients with schizophrenia and 327 unaffected participants; all individuals were northern Han Chinese. Genotype and haplotype frequency distributions were compared for the 2 groups by means of PCR amplification and direct sequencing of the promoter region of GABRB3. The genotype distribution among control participants was in accordance with the Hardy-Weinberg equilibrium. Five common single-nucleotide polymorphism (SNP) sites were detected in the 5′ promoter region of GABRB3: rs4243768, rs7171660, rs4363842, rs4906902, and rs8179184. Only rs8179184 and rs4906902 differed significantly in frequency between controls and cases (P?<?0.05); this difference remained significant when only women in each group were compared. The 2 SNP sites showed linkage disequilibrium, resulting in 2 haplotypes: T-G and C-A. The frequency of C-A was significantly higher among patients with schizophrenia than among controls. Our findings suggest that rs4906902 and rs8179184 in the 5′ promoter region of GABRB3 are associated with schizophrenia. The C-A haplotype may entail an increased risk of schizophrenia, and the onset of schizophrenia may be gender-specific.     

Association study of polymorphisms in synaptic vesicle-associated genes, <Emphasis Type="Italic">SYN2</Emphasis> and <Emphasis Type="Italic">CPLX2</Emphasis>, with schizophrenia

Background  

The occurrence of aberrant functional connectivity in the neuronal circuit is one of the integrative theories of the etiology of schizophrenia. Previous studies have reported that the protein and mRNA levels of the synapsin 2 (SYN2) and complexin 2 (CPLX2) genes were decreased in patients with schizophrenia. Synapsin 2 and complexin 2 are involved in synaptogenesis and the modulation of neurotransmitter release. This report presents a study of the association of polymorphisms of SYN2 and CPLX2 with schizophrenia in the Korean population.     

Poliovirus type 1 infection of murine <Emphasis Type="Italic">PRNP</Emphasis>-knockout neuronal cells

Transfection of the prion protein gene (Prnp) into prion-deficient mouse cells was shown to reduce the replication of coxsackievirus B3, an enterovirus. Because mice can be susceptible to poliovirus infection by parenteral routes, the authors tested the susceptibility to poliovirus-1 (PV-1) of a panel of murine neuronal cell lines differing in their ability to express Prnp. The investigated cell lines (prionless HpL3.4 cells, HpL3.4 cells transfected with a Prnp vector, HpL3.4 cells transfected with a void vector, wild-type Hw3.5 Prnp ^+/+ cells) expressed the murine homologue (Tage4) of human poliovirus receptor (CD155/hPVR). PV-1 infection of Prnp ^−/− HpL3.4 cells resulted in the production of high viral titers, though viral antigens could be detected in only 0.5% to 2% of cells. Wild-type Prnp ^+/+ cells and prionless cells transfected with the Prnp gene were not permissive to PV-1. Results of viral titration and immunofluorescence were confirmed by conventional polymerase chain reaction (PCR) and quantitative real-time PCR. Exposure to PV-1 had no influence on the gene expression profile of Prnp ^+/+ cells. In contrast, PV-1 infection was associated with up regulation of several genes in permissive Prnp ^−/− cell cultures: type I interferon (IFN) genes, IFN-related developmental regulator 1 (IFNRD1), tumor necrosis factor superfamily member 13b (TNFSF13b), interleukin (IL) − 7, granulocyte/macrophage colony-stimulating factors (CSFs), hepatocyte growth factor (HGF), vascular endothelial growth factor-A, transforming growth factors beta1 and beta3 (TGFb1, TGFb3), as well as a variety of bone morphogenetic proteins endowed with neuroprotective activity. Distinction of permissive from nonpermissive neuronal cells on the basis of Prnp expression suggests that prion-deficient mice could represent an extraordinarily sensitive animal model for poliovirus infection.     

Association of <Emphasis Type="Italic">CILP</Emphasis>, <Emphasis Type="Italic">COL9A2</Emphasis> and <Emphasis Type="Italic">MMP3</Emphasis> Gene Polymorphisms with Lumbar Disc Degeneration in an Indian Population

Lumbar disc degeneration (LDD) is a multifactorial disorder caused by genetic and environmental factors. Polymorphisms in several structural and inflammatory genes like collagens, aggrecan, matrix metalloproteinases are associated with the risk of disc degeneration. In this study, we analyzed the role of a few important single nucleotide polymorphisms in cartilage intermediate layer protein (CILP), collagen 9A2 (COL9A2) and matrix metalloproteinase 3 (MMP3) genes in LDD from an Indian population. Two hundred patients with LDD and 200 healthy controls were recruited for the study. Genotyping was performed by allelic discrimination assay. The rs2073711 polymorphism (CILP gene - GG genotype) was associated with reduced risk of LDD in the Indian population (OR?=?0.43, p?=?0.016). The rs591058 polymorphism (MMP3 gene - TT genotype) is found to be associated with lower risk among women (OR?=?0.34, p?=?0.041). No significant association was found between COL9A2 polymorphism rs7533552 and the risk of LDD. We conclude that the CILP gene polymorphism (rs2073711) is associated with a lower risk of LDD, the MMP3 (rs591058) gene polymorphism is associated with LDD among women, and the TT genotype confers a lower risk of LDD.     

Monoclonal antibody specific for <Emphasis Type="Italic">IDH1</Emphasis> R132H mutation

IDH1 R132H mutations occur in approximately 70% of astrocytomas and oligodendroglial tumors. We developed a mouse monoclonal antibody targeting the IDH1 R132H mutation. Here, we show the high specificity and sensitivity of this antibody on Western blots and tissue sections from formalin fixed paraffin embedded tumor specimens. This antibody is highly useful for tumor classification, in detecting single infiltrating tumor cells and for the characterization of the cellular role of mutant IDH1 protein.     

Coexistence of <Emphasis Type="Italic">CLCN1</Emphasis> and <Emphasis Type="Italic">SCN4A</Emphasis> mutations in one family suffering from myotonia

Non-dystrophic myotonias are characterized by clinical overlap making it challenging to establish genotype-phenotype correlations. We report clinical and electrophysiological findings in a girl and her father concomitantly harbouring single heterozygous mutations in SCN4A and CLCN1 genes. Functional characterization of N1297S hNav1.4 mutant was performed by patch clamp. The patients displayed a mild phenotype, mostly resembling a sodium channel myotonia. The CLCN1 c.501C>G (p.F167L) mutation has been already described in recessive pedigrees, whereas the SCN4A c.3890A>G (p.N1297S) variation is novel. Patch clamp experiments showed impairment of fast and slow inactivation of the mutated Nav1.4 sodium channel. The present findings suggest that analysis of both SCN4A and CLCN1 genes should be considered in myotonic patients with atypical clinical and neurophysiological features.     

Two four-marker haplotypes on 7q36.1 region indicate that the potassium channel gene <Emphasis Type="Italic">HERG1</Emphasis> (<Emphasis Type="Italic">KCNH2</Emphasis>, <Emphasis Type="Italic">Kv11.1</Emphasis>) is related to schizophrenia: a case control study

Background  

The pathobiology of schizophrenia is still unclear. Its current treatment mainly depends on antipsychotic drugs. A leading adverse effect of these medications is the acquired long QT syndrome, which results from the blockade of cardiac HERG1 channels (human ether-a-go-go-related gene potassium channels 1) by antipsychotic agents. The HERG1 channel is encoded by HERG1 (KCNH2, Kv11.1) gene and is most highly expressed in heart and brain. Genetic variations in HERG1 predispose to acquired long QT syndrome. We hypothesized that the blockade of HERG1 channels by antipsychotics might also be significant for their therapeutic mode of action, indicating a novel mechanism in the pathogenesis of schizophrenia.     

Regulation of the immediate-early genes <Emphasis Type="Italic">arc</Emphasis> and <Emphasis Type="Italic">zif268</Emphasis> in a mouse operant model of cocaine seeking reinstatement

Reinstatement of extinguished operant responding for drug is an appropriate model of relapse to drug abuse. Due to the difficulty of implementing in mice the procedure of instrumental intravenous self-administration, mechanisms of reinstatement have so far been studied almost exclusively in rats. A mouse model of reinstatement of cocaine seeking has recently been characterized (Soria et al. 2008). The aim of the present study was to assess regional brain activation, as measured by induction of the immediate early genes (IEG) arc and zif268, during priming- or cue-elicited reinstatement of cocaine seeking using this new mouse model and the in situ hybridization technique. We have demonstrated that cue-elicited reinstatement of cocaine seeking was associated with induction of the IEG in the medial prefrontal cortex (prelimbic and infralimbic) and basolateral amygdala. Priming-induced reinstatement produced a more widespread up-regulation of those genes in forebrain regions including medial prefrontal, orbitofrontal and motor cortex, dorsal striatum and basolateral amygdala. These patterns of IEG expression are in agreement with previous results obtained in rats and thus indicate that the new mouse model of reinstatement is functionally equivalent to rat models. That comparability adds to the usefulness of the mouse model as a tool for addressing neurobiological mechanisms of addiction.