Differential cellular gene expression in ganglioglioma

PURPOSE: Gangliogliomas (GGs) are neuronal-glial tumors highly associated with epilepsy. We hypothesized that the expression of select gene families including neurotransmitter receptor subunits and growth factors would be distinct in neurons and astrocytes within GG compared with adjacent cortex and that these changes would yield insights into seizure onset and lesion formation. METHODS: Candidate gene expression was defined in single immunohistochemically labeled neurons and astrocytes microdissected from GG specimens compared with neurons and astrocytes microdissected from morphologically intact cortex adjacent to the GG or normal control cortex. RESULTS: Differential expression of 16 genes including glutamate transporter (EAAC1) and receptor (NMDA2C, mGluR5), growth factor (hepatocyte growth factor), and receptor (platelet derived growth factor receptor beta, fibroblast growth factor receptor 3) mRNAs was detected in GG neurons compared with control neurons. In astrocytes, altered expression of p75NGF, mGluR3, TGFbeta3 and Glt-1 mRNAs was detected. Nestin mRNA, a gene that exhibits enhanced expression in balloon cell cortical dysplasia, was increased in GG neurons. Because of the morphological similarities between GG and cortical dysplasia, we show that there is activation of the mTOR cascade in GG as evidenced by enhanced expression of phospho-p70S6kinase and phosphoribosomal S6 proteins. CONCLUSION: We find differential candidate gene expression in neurons and astrocytes in GG compared with adjacent cortex and show that there is activation of the mTOR pathway. These changes highlight pathways that may be pivotal for epileptogenesis and lesion growth.     

Astrocyte characterization in the multiple sclerosis glial scar

Introduction:  Dense astrocytic scarring in chronic multiple sclerosis (MS) plaques inhibits tissue repair. However, at the rim of a lesion the glial scar fails to form despite the presence of reactive astrocytes. Animal studies have shown that astrocyte antigen expression varies depending on astrocyte type and location. Characterization of human astrocytes in MS tissue may identify markers relevant to the glial scar.
Materials and methods:  Astrocyte antigenic phenotype was investigated in subventricular white matter by immunocytochemistry and Western blotting. Snap-frozen tissue from normal controls ( n  = 4), MS normal appearing white matter ( n  = 5) and lesions [acute ( n  = 7), subacute ( n  = 7) and chronic ( n  = 13)] was studied.
Results:  As expected, glial fibrillary acidic protein and vimentin expression was elevated in scar astrocytes. In addition there was increased expression of nestin, embryonic neural cell adhesion molecule, epidermal growth factor receptor, nerve growth factor and its receptor p75, and in a subpopulation of scar astrocytes, basic fibroblast growth factor.
Conclusion:  Changes in expression of proteins associated with development, growth factors and growth factor receptors are characteristic of the scar astrocyte phenotype in chronic MS lesions. These proteins may be of relevance to glial scar formation and tissue repair.     

Activation of mammalian target of rapamycin in cytomegalic neurons of human cortical dysplasia

OBJECTIVE: The cortex of patients with cortical dysplasia contains several abnormal cell types. Among the dysplastic cells, cytomegalic neurons are known to be electrically hyperactive and may contribute to epileptic activity. In this study, we sought to identify molecular markers of cytomegalic neurons in focal or hemispheric cortical dysplasia and to determine whether the activity of the mammalian target of rapamycin (mTOR) kinase is abnormally high in these cells. METHODS: Microarray analysis of gene expression in large dysplastic cells microdissected from cortical dysplasia surgical specimens was used to identify markers of cytomegalic neurons. Immunohistochemistry and immunofluorescence analysis of cortical sections was used to validate the microarray results and to probe the activity of mTOR in cytomegalic neurons using phospho-specific antibodies directed against known mTOR targets. RESULTS: We demonstrate that the neurofilament heavy chain is a reliable marker of cytomegalic neurons and that targets of the mTOR kinase, such as the ribosomal protein S6, eIF4G, and Akt, are hyperphosphorylated in these dysplastic neurons. INTERPRETATION: We conclude that mTOR kinase hyperactivation is a molecular mechanism underlying the development of cytomegalic neurons. This finding may lead to the development of novel therapeutic approaches for childhood epilepsy associated with cortical dysplasia.     

Ionotropic and metabotropic glutamate receptor protein expression in glioneuronal tumours from patients with intractable epilepsy

Glioneuronal tumours are an increasingly recognized cause of chronic pharmaco-resistant epilepsy. In the present study the immunocytochemical expression of various glutamate receptor (GluR) subtypes was investigated in 41 gangliogliomas (GG) and 16 dysembryoplastic neuroepithelial tumours (DNT) from patients with intractable epilepsy. Immunocytochemistry with antibodies specific for ionotropic NR1, NR2A/B (NMDA) GluR1, GluR2 (AMPA), GluR5-7 (kainate), and metabotropic mGluR1, mGluR2-3, mGluR5, mGluR7a subtypes demonstrated in both GG and DNT the presence of an highly differentiated neuronal population, containing subunits from each receptor class. More than 50% of tumours contained a high percentage of neuronal cells immunolabelled for NMDA, AMPA and kainate receptor subunits. A high percentage of neurones showed strong expression of NR2A-B, which co-localized with NR1. Group I mGluRs (mGluR1 and mGluR5) were highly represented in the neuronal component of the tumours. Immunolabelling for several GluRs was also present in the glial component. Increased expression of mGluR2-3, mGluR5 and GluR5-7 was observed in reactive astrocytes in the perilesional zone compared to normal cortex. The neurochemical profile of glioneuronal tumours, with high expression of specific GluR subtypes, supports the central role of glutamatergic transmission in the mechanisms underlying the intrinsic and high epileptogenicity of these lesions.     

Expression and cell distribution of group I and group II metabotropic glutamate receptor subtypes in taylor-type focal cortical dysplasia

PURPOSE: Focal cortical dysplasia (FCD) is known to be a major cause of intractable epilepsy. The cellular mechanism(s) underlying the epileptogenicity of FCD remain largely unknown. Because recent studies indicate that metabotropic glutamate receptor subtypes (mGluRs) play a role in epileptogenesis, we investigated the expression and cellular distribution pattern of mGluRs in FCD specimens. METHODS: Immunocytochemical expression of group I and group II mGluR subtypes was investigated in 15 specimens of human FCD obtained during epilepsy surgery. RESULTS: Strong mGluR1alpha and mGluR5 (group I mGluRs) immunoreactivity (IR) was observed in the majority of FCD specimens in dysplastic as well as in heterotopic neurons. mGluR1alpha was expressed in a subpopulation of neurons (mainly large dysplastic cells), whereas mGluR5 was represented in a higher percentage of dysplastic neuronal cells. Group II mGluRs (mGluR2/3) IR was observed less frequently than that in group I mGluRs and generally appeared in <10% of the dysplastic neurons. IR for all three mGluR subtypes was observed in balloon cells. mGluR2/3 appeared to be most frequently expressed in glial fibrillary acidic protein (GFAP)-positive balloon cells (glial type), and mGluR1alpha, in microtubule-associated protein (MAP)2-positive cells (neuronal type). mGluR5 was present in the majority of balloon cells. Occasionally glial mGluR1alpha IR was observed in bizarre glial cells with di- or multinuclei. Reactive astrocytes were intensively stained, mainly with mGluR5 and mGluR2/3. CONCLUSIONS: The cellular distribution of mGluR subtypes, with high expression of mGluR1alpha and mGluR5 in dysplastic neurons, suggests a possible contribution of group I mGluRs to the intrinsic and high epileptogenicity of dysplastic cortical regions.     

βAPP Gene Expression is Increased in the Rat Brain After Motor Neuron Axotomy

The response of the βAPP gene to neuronal injury was studied in the facial and hypoglossal nerve nuclei of the rat after corresponding nerve axotomy. Increased levels of βAPP 695, 714, 751 and 770 mRNAs were observed after either facial or hypoglossal nerve axotomy in the parent ipsilateral motor neurons. The increase was gradual, with maximal values 7 days after axotomy. βAPP mRNA expression returned to normal values 60 days after the lesion. Increased βAPP immunostaining was also detected in ipsilateral chromatolytic motor neurons. No change in βAPP immunoreactivity was observed in oligodendrocytes, another cell type expressing βAPP under normal conditions. A rapid increase in the expression of the GFAP gene was observed in reactive astrocytes surrounding chromatolytic neurons in the ipsilateral facial or hypoglossal nuclei. Thus, in contrast with other models of neuronal injury, where only the Kunitz protease inhibitor-containing βAPP mRNA isoforms are increased, all βAPP mRNAs are increased in the axotomy model. Furthermore, although βAPP expression has been shown to be increased in reactive astrocytes following neuronal injury, in the present study the increase was essentially found in the motor neurons reacting to axotomy.     

Upregulation of Group I metabotropic glutamate receptors in neurons and astrocytes in the dorsal horn following spinal cord injury

Of the glutamate receptor types, the metabotropic glutamate receptors (mGluRs) are G proteins coupled and can initiate a number of intracellular pathways leading to hyperexcitability of spinal neurons. In this study, we tested the expression of mGluRs to determine which cell types might contribute to sustained neuronal hyperexcitability in the lumbar enlargement with postoperative day (POD) 7 (early), 14 (late), and 30 (chronic phase) following spinal cord injury (SCI) by unilateral hemisection at T13 in Sprague-Dawley rats. Expression was determined by confocal analyses of immunocytochemical reaction product of neurons (NeuN positive) and astrocytes (GFAP positive) in the dorsal horn on both sides of the L4 segment. Neurons were divided into two sizes: small (<20 microm) and large (>35 microm), for physiological reasons. We report a significant increase of mGluR(1) expression in large and small neurons of the dorsal horn on both sides of the cord in late and chronic phases when compared to control sham groups. Expression of mGluR(2/3) significantly increased in large neurons on the ipsilateral (hemisected) side in the late phase. Expression of mGluR(5) significantly increased in large neurons in early, late, and chronic phases. In addition, mGluR(1) and mGluR(5) expression after hemisection was significantly increased in astrocytes in early, late, and chronic phases; whereas mGluR(2/3) did not display any significant changes. In conclusion, our data demonstrate long-term changes in expression levels of Group I mGluRs (mGluR(1) and mGluR(5)) in both neurons and astrocytes in segments below a unilateral SCI. Thus, permanent alterations in dorsal horn receptor expression may play important roles in transmission of nociceptive responses in the spinal cord following SCI.     

mGluR5-dependent increases in immediate early gene expression in the rat striatum following acute administration of amphetamine

Metabotropic glutamate receptor 5 (mGluR5) is densely expressed in medium-sized spiny projection neurons of the rat striatum. Activation of mGluR5 increases intracellular Ca2+, resulting in Ca(2+)-dependent cellular responses. Acute administration of the psychostimulant amphetamine (AMPH) induces immediate early gene (IEG) expression in the striatum, which is considered an important molecular event for the development of striatal neuroplasticity related to the addictive properties of drugs of abuse. This study investigated the role of mGluR5 in the mediation of IEG expression in the rat striatum induced by a single dose of AMPH (4 mg/kg, i.p.) in vivo. We found that systemic administration of the mGluR5-selective antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP) at a dose of 10 mg/kg, i.p. reduced AMPH-stimulated c-fos mRNA levels in the dorsal (caudoputamen) and ventral (nucleus accumbens) striatum as revealed by quantitative in situ hybridization. Similar results were observed in the three areas of cerebral cortex (cingulate, sensory, and piriform cortex). In contrast to c-fos mRNAs, AMPH-stimulated mRNA expression of another IEG, zif/268, was not significantly altered by the blockade of mGluR5 with MPEP in the entire striatum and the three areas of cortex. Treatment with MPEP alone had no effect on basal levels of c-fos and zif/268 mRNAs in the striatal and cortical areas. These results indicate that an mGluR5-dependent mechanism selectively contributes to c-fos expression in the striatum and cortex in response to acute exposure to AMPH.     

Cystatin C expression in ischemic white matter lesions

Objectives –  To study the involvement of cystatin C in the progression of ischemic white matter lesions (WMLs).
Materials and methods –  Cystatin C levels in the cerebrospinal fluid (CSF) of patients with cerebrovascular disease, and also in primary and established human neural cell cultures were investigated. For pathologic analysis, cystatin C immunoreactivity was investigated in the white matter of patients with severe WMLs, mild WMLs or controls.
Results –  Cystatin C levels in the CSF of patients with Fazekas WML grade 3 [14 with hypertension; W/HT(+) and nine without hypertension; W/HT(−)] were lower than those in 38 patients with grade 0–1 ( P  = 0.0022 and P  < 0.0001 respectively). Immunohistochemical study showed that the cystatin C immunoreactivity was found in astrocytes, and the number of astrocytes in the white matter in the severe WML group was decreased when compared with that in controls ( P  = 0.0027) and in the mild WML group ( P  = 0.0024). In human neural cell cultures, treatments with thrombin, matrix metalloproteinases and interleukin 1β increased the expression of cystatin C mRNA in human astrocytes and hybrid neurons, but an enzyme-linked immunosorbent assay revealed that only thrombin significantly increased the production and secretion of cystatin C in astrocytes.
Conclusions –  These results suggest that low levels of CSF cystatin C in ischemic WMLs might be due to the decreased number of astrocytes that secrete cystatin C in response to the stimuli of proteases and inflammatory cytokines.     

Effect of Aging on Gene Expression of Metabotropic Glutamate Receptor 5 and Glutamate Transporter-1 in the Prefrontal Cortex of Schizophrenics

Background : The involvement of the glutamatergic system in the patho-physiology of schizophrenia is attracting interest because an antagonist of N-methyl-D-aspartate (NMDA) glutamate receptor has been demonstrated to induce schizophrenic-like symptoms in normal subjects. The results of previous studies have also suggested that glutamatergic function is reduced in the prefrontal cortex (PFC) of diagnosed schizophrenics. In order to provide additional evidence for this, we investigated whether expression of metabotropic glutamate receptor 5 (mGluR5) and glutamate transporter-1 (GLT-1) in the PFC is altered in schizophrenia.
Methods : We compared the expression of mGluRS and GLT-1 by in situ hybridization in Brodmann area 9 (B9) and 10 (B10) of the prefrontal cortex in 5 normal individuals and 5 schizophrenics that were younger than in previous studies, and examined the relationship between age and mRNA expression.
Results : There were no significant differences in either mGluR5 or GLT-1 mRNA expression between the schizophrenics and controls, however, there was a significant correlation to increase mGluR5 mRNA levels in the schizophrenics in both layer III and layer V of B9 with age, a finding not observed in the controls.
Conclusion : The results suggest that the brains of schizophrenics may be vulnerable to aging and that the glutamatergic dysfunctions previously reported in schizophrenics may be partly explained by the aging process.     

Ventral Mesencephalon Astrocytes Are More Efficient Than Those of Other Regions in Inducing Dopaminergic Neurons Through Higher Expression Level of TGF-β3

Being supportive cells for neurons in the central nervous system, astrocytes have recently found to be associated with neurogenesis. Ventral mesencephalon (VM) astrocytes were also detected being instructive for VM dopaminergic (DA) neurogenesis, but the underling mechanisms are still unclear. This research is to figure out whether VM astrocytes are more efficient than those from other brain regions in inducing VM DA neurons from their precursors and whether transforming growth factor-βs (TGF-βs) are the underlying molecules. We found that, compared with astrocytes preparations from striatum and hippocampus, VM astrocytes preparations displayed markedly higher efficacy in inducing DA neurogenesis. Besides, they also expressed higher level of TGF-β3 than those of two other regions. When TGF-β3 gene expression in astrocytes preparations was inhibited by its antisense oligonucleotide, the induction of DA neurons decreased to a similar level among these three astrocytes preparations. Thus, our experiment indicates that VM astrocytes preparations which contained highly purified astrocytes are more efficient in inducing DA neurogenesis than those from other regions. Furthermore, it also suggests that the regional differences are regulated by different expression levels of TGF-β3 in those astrocytes preparations from different derivations.     

Growth factor pre-treatment differentially regulates phosphoinositide turnover downstream of lysophospholipid receptor and metabotropic glutamate receptors in cultured rat cerebrocortical astrocytes

Reactive gliosis is an aspect of neural plasticity and growth factor (GF) stimulation of astrocytes in vitro is widely regarded as a model system to study astrocyte plasticity. Astrocytes express receptors for several ligands including lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P), agonists for the G-protein-coupled lysophospholipid receptors (lpRs). Activation of lpRs by LPA or S1P leads to multiple pharmacological effects including the influx of calcium, phosphoinositide (PI) hydrolysis, phosphorylation of extracellular receptor regulated kinase (ERK), release of arachidonic acid, and induces mitogenesis. Treatment of astrocytes in vitro with a growth factor cocktail (containing epidermal growth factor [EGF], basic fibroblast growth factor [bFGF] and insulin) led to a marked attenuation of lpR-induced PI hydrolysis. In contrast, under identical conditions, GF treatment led to marked potentiation of PI hydrolysis downstream of activation of another abundantly expressed G-protein coupled receptor, mGluR5. Quantitative gene expression analysis of GF-treated or control astrocytes by TaqMan RT-PCR indicated that GF treatment did not change gene expression of lpa1 and s1p1, but increased gene expression of s1p5 which is expressed at very low levels in basal conditions. These results suggest that GF differentially affected PLC activation downstream of mGluR5 versus lpR activation and that the changes in mRNA levels of lpRs do not account for marked attenuation of agonist-induced phosphoinositide turnover.     

Laser microdissection of immunolabeled astrocytes allows quantification of astrocytic gene expression

Astrocytes represent the major glial cell population within the central nervous system. In order to elucidate the function of astrocytes under physiological conditions and during the course of neurological disease, astrocytic gene expression profiling is necessary. However, since astrocytes form an intimately connected network with neurons and other cell types in the brain, gene expression analysis of astrocytes with a sufficient degree of cellular specificity is difficult. Here we are presenting a rapid and, thus, RNA preserving immunostaining protocol for the detection of astrocytes in rodent brain. This protocol can readily be combined with laser microdissection (Leica AS LMD platform) and quantitative RT-PCR (qPCR). Employing this method, we studied changes in glial fibrillary acidic protein (GFAP) expression in astrocytes of mouse entorhinal cortex following entorhinal cortex lesion. Using laser microdissection, astrocytes (n = 60) were collected in the tissue surrounding the lesion, the entorhinal cortex contralateral to the lesion, and in unlesioned control animals. Changes in GFAP mRNA were quantified using qPCR. GFAP mRNA levels were 82-fold higher in astrocytes of lesioned animals at the site of the lesion compared to GFAP mRNA levels in entorhinal cortex astrocytes of control mice. GFAP mRNA levels were only slightly elevated at the contralateral side (lesioned animals). This optimized protocol for immunolabeling and laser microdissection of astrocytes followed by qPCR allows quantification of astrocytic gene expression levels with a high degree of cellular specificity. It may similarly be employed in different settings where other cell types need to be identified and microdissected for gene expression profiling.     

Impaired preprodynorphin, but not preproenkephalin, mRNA induction in the striatum of mGluR1 mutant mice in response to acute administration of the full dopamine D(1) agonist SKF-82958

Metabotropic glutamate receptor 1 (mGluR1) is highly expressed in striatonigral projection neurons of rat striatum. To define the role of mGluR1 in the regulation of striatal gene expression, the responsiveness of the three neuropeptide gene expression to a single injection of the dopamine D(1) agonist SKF-82958 was compared between mGluR1 mutant and wild-type control mice. We found that acute injection of SKF-82958 increased preprodynorphin (PPD), substance P (SP), and preproenkephalin (PPE) mRNAs in the dorsal and ventral striatum of mutant and wild-type mice in a dose-dependent manner (0.125, 0.5, and 2 mg/kg, i.p.) as revealed by quantitative in situ hybridization. However, the induction of PPD mRNA in both the dorsal and ventral striatum of mGluR1 minus sign/minus sign mice was significantly less than that of wild-type +/+ mice in response to the two higher doses of SKF-82958. In contrast to PPD, SP and PPE in the dorsal and ventral striatum of mGluR1 mutant mice were elevated to a similar level as that of wild-type mice. There were no differences in basal levels and distribution patterns of all three mRNAs between the two genotypes of mice treated with saline. These results indicate that mGluR1 selectively participates in striatonigral PPD induction in response to D(1) receptor stimulation.