Toward the identification of peripheral epigenetic biomarkers of schizophrenia.

Schizophrenia (SZ) is a heritable, nonmendelian, neurodevelopmental disorder in which epigenetic dysregulation of the brain genome plays a fundamental role in mediating the clinical manifestations and course of the disease. The authors recently reported that two enzymes that belong to the dynamic DNA methylation/demethylation network-DNMT (DNA methyltransferase) and TET (ten-eleven translocase; 5-hydroxycytosine translocator)-are abnormally increased in corticolimbic structures of SZ postmortem brain, suggesting a causal relationship between clinical manifestations of SZ and changes in DNA methylation and in the expression of SZ candidate genes (e.g., brain-derived neurotrophic factor [BDNF], glucocorticoid receptor [GCR], glutamic acid decarboxylase 67 [GAD67], reelin). Because the clinical manifestations of SZ typically begin with a prodrome followed by a first episode in adolescence with subsequent deterioration, it is obvious that the natural history of this disease cannot be studied only in postmortem brain. Hence, the focus is currently shifting towards the feasibility of studying epigenetic molecular signatures of SZ in blood cells. Initial studies show a significant enrichment of epigenetic changes in lymphocytes in gene networks directly relevant to psychiatric disorders. Furthermore, the expression of DNA-methylating/demethylating enzymes and SZ candidate genes such as BDNF and GCR are altered in the same direction in both brain and blood lymphocytes. The coincidence of these changes in lymphocytes and brain supports the hypothesis that common environmental or genetic risk factors are operative in altering the epigenetic components involved in orchestrating transcription of specific genes in brain and peripheral tissues. The identification of DNA methylation signatures for SZ in peripheral blood cells of subjects with genetic and clinical high risk would clearly have potential for the diagnosis of SZ early in its course and would be invaluable for initiating early intervention and individualized treatment plans.

Clozapine and sulpiride but not haloperidol or olanzapine activate brain DNA demethylation.

Cortical GABAergic dysfunction, a hallmark of both schizophrenia (SZ) and bipolar (BP) disorder pathophysiologies may relate to the hypermethylation of GABAergic gene promoters (i.e., reelin and GAD67). Benefits elicited by a combination of atypical antipsychotics with valproate (VPA) (a histone deacetylase inhibitor that may also activate brain DNA demethylation) in SZ or BP disorder treatment prompted us to investigate whether the beneficial action of this association depends on induction of a putative DNA demethylase activity. To monitor this activity, we measured the ratio of 5-methyl cytosine to unmethylated cytosine in reelin and GAD67 promoters in the mouse frontal cortex and striatum. We compared normal mice with mice pretreated with l-methionine (5.2 mmol/kg s.c. twice a day for 7 days) to hypermethylate promoters, including reelin and GAD67. Clinically relevant doses of clozapine (CLZ) (3.8 to 15 micromol/kg twice a day s.c. for 3 days) and sulpiride (SULP) (12.5 to 50 micromol/kg twice a day for 3 days) but not clinically relevant doses of haloperidol (HAL) (1.3 to 4 micromol/kg twice a day s.c. for 3 days) or olanzapine (OLZ) (4 to 15 micromol/kg twice a day for 3 days) exhibited dose-related increases in the cortical and striatal demethylation of hypermethylated reelin and GAD67 promoters. These effects of CLZ and SULP were dramatically potentiated by a clinically relevant VPA dose (0.5 mmol/kg twice a day for 3 days). By activating a DNA demethylase, the association of CLZ or SULP with VPA may facilitate a chromatin remodeling that normalizes the GABAergic gene expression down-regulation detected in the telencephalic regions of SZ and BP patients.

One factor recognizes the liver-specific enhancers in alpha 1-antitrypsin and transthyretin genes.

Four different regulatory sites required for transcriptional stimulation by the enhancers of two unrelated liver-specific genes alpha 1-antitrypsin and transthyretin appear to bind the same nuclear protein that is found mainly in the liver. Such proteins may provide a basis for a coordinated, hepatocyte-specific control of gene transcription.

Low doses of prenatal ethanol exposure and maternal separation alter HPA axis function and ethanol consumption in adult male rats.

Adverse maternal behaviors during pregnancy and unfavorable postnatal experiences during development are associated with an increased risk of developing psychiatric disorders, as well as, a vulnerability to alcohol addiction in adulthood. Here, we examined the effects of combined ethanol exposure during late pregnancy and postnatal maternal separation (MS) on HPA responsiveness, anxiety behavior and preference for alcohol consumption in adult male rats. Animals exposed to both conditions revealed a decrease in blood levels of allopregnanolone accompanied by increased anxiety behavior. In addition, basal blood levels of corticosterone were markedly decreased in all experimental groups while increases in the foot-shock-induced corticosterone levels were more pronounced in MS animals. Finally, evaluating EtOH drinking behavior, MS animals exhibited a remarkable EtOH preference even at low doses (0.1-1%). Altogether, these data suggest that adverse conditions, alone or in combination, may alter anxiety-like states as well as modify behavior towards alcohol consumption.

Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and alpha 1-antitrypsin genes.

Transthyretin (TTR) and alpha 1-antitrypsin (alpha 1-AT) are expressed at high levels in the liver and also in at least one other cell type. We report here a detailed analysis of the proximal regulatory region of the TTR gene, which has uncovered two new DNA-binding factors that are present mainly (or only) in hepatocytes. One of these new factors, hepatocyte nuclear factor 3 (HNF-3), binds to two sites that are crucial in TTR expression as well as to two additional sites in the alpha 1-AT proximal enhancer region. The second new factor, HNF-4, binds to two sites in TTR that are required for gene activity. We had previously identified binding sites for another hepatocyte-enriched DNA-binding protein (C/EBP or a relative thereof), and additional promoter-proximal sites for that protein in both TTR and alpha 1-AT are also reported here. From these results it seems clear that cell-specific expression is not simply the result of a single cell-specific factor for each gene but the result of a combination of such factors. The variation and distribution of such factors among different cell types could be an important basis for the coordinate expression of the TTR and alpha 1-AT genes in the liver or the discordant transcriptional activation of these genes in a few other cell types. The identification of such cell-enriched factors is a necessary prelude to understanding the basis for cell specificity.

A cell-specific enhancer of the mouse alpha 1-antitrypsin gene has multiple functional regions and corresponding protein-binding sites.

We have previously described the isolation and characterization of genomic clones corresponding to the mouse alpha 1-antitrypsin gene (Krauter et al., DNA 5:29-36, 1986). In this report, we have analyzed the DNA sequences upstream of the RNA start site that direct hepatoma cell-specific expression of this gene when incorporated into recombinant plasmids. The 160 nucleotides 5' to the cap site direct low-level expression in hepatoma cells, and sequences between -520 and -160 bp upstream of the RNA start site functioned as a cell-specific enhancer of expression both with the alpha 1-antitrypsin promoter and when combined with a functional beta-globin promoter. Within the enhancer region, three binding sites for proteins present in hepatoma nuclear extracts were identified. The location of each site was positioned, using both methylation protection and methylation interference experiments. Each protein-binding site correlated with a functionally important region necessary for full enhancer activity. These experiments demonstrated a complex arrangement of regulatory elements comprising the alpha 1-antitrypsin enhancer. Significant qualitative differences exist between the findings presented here and the cis-acting elements operative in regulating expression of the human alpha 1-antitrypsin gene (Ciliberto et al., Cell 41:531-540, 1985; De Simone et al., EMBO J. 6:2759-2766, 1987).

The cell-specific enhancer of the mouse transthyretin (prealbumin) gene binds a common factor at one site and a liver-specific factor(s) at two other sites.

We previously defined two distinct cell-specific DNA elements controlling the transient expression of the transthyretin gene in Hep G2 (human hepatoma) cells: a proximal promoter region (-202 base pairs [bp] to the cap site), and a far-upstream cell-specific enhancer located between 1.6 and 2.15 kilobases (kb) 5' of the cap site (R. H. Costa, E. Lai, and J. E. Darnell, Jr., Mol. Cell. Biol. 6:4697-4708, 1986). In this report, we located the effective transthyretin enhancer element within a 100-bp region between 1.96 and 1.86 kb 5' to the mRNA cap site. In Hep G2 nuclear extracts, three protein-binding sites within this minimal enhancer element were identified by gel mobility and methylation protection experiments. Each binding site was required for full enhancer activity in Hep G2 transient expression assays. Competition experiments in protein-binding assays suggested that two of the three sites were recognized by a similar factor and that the protein interaction with the third site was different. The nuclear protein(s) which bound to the two homologous sites was found mainly or only in cells of hepatic origin, suggesting an involvement of this region in the cell-specific function of this enhancer. The nuclear protein(s) recognizing the third enhancer region was also found in HeLa and spleen cells.

Behavioral and molecular neuroepigenetic alterations in prenatally stressed mice: relevance for the study of chromatin remodeling properties of antipsychotic drugs.

We have recently reported that mice born from dams stressed during pregnancy (PRS mice), in adulthood, have behavioral deficits reminiscent of behaviors observed in schizophrenia (SZ) and bipolar (BP) disorder patients. Furthermore, we have shown that the frontal cortex (FC) and hippocampus of adult PRS mice, like that of postmortem chronic SZ patients, are characterized by increases in DNA-methyltransferase 1 (DNMT1), ten-eleven methylcytosine dioxygenase 1 (TET1) and exhibit an enrichment of 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC) at neocortical GABAergic and glutamatergic gene promoters. Here, we show that the behavioral deficits and the increased 5MC and 5HMC at glutamic acid decarboxylase 67 (Gad1), reelin (Reln) and brain-derived neurotrophic factor (Bdnf) promoters and the reduced expression of the messenger RNAs (mRNAs) and proteins corresponding to these genes in FC of adult PRS mice is reversed by treatment with clozapine (5 mg kg(-1) twice a day for 5 days) but not by haloperidol (1 mg kg(-1) twice a day for 5 days). Interestingly, clozapine had no effect on either the behavior, promoter methylation or the expression of these mRNAs and proteins when administered to offspring of nonstressed pregnant mice. Clozapine, but not haloperidol, reduced the elevated levels of DNMT1 and TET1, as well as the elevated levels of DNMT1 binding to Gad1, Reln and Bdnf promoters in PRS mice suggesting that clozapine, unlike haloperidol, may limit DNA methylation by interfering with DNA methylation dynamics. We conclude that the PRS mouse model may be useful preclinically in screening for the potential efficacy of antipsychotic drugs acting on altered epigenetic mechanisms. Furthermore, PRS mice may be invaluable for understanding the etiopathogenesis of SZ and BP disorder and for predicting treatment responses at early stages of the illness allowing for early detection and remedial intervention.

Decrease in reelin and glutamic acid decarboxylase67 (GAD67) expression in schizophrenia and bipolar disorder: a postmortem brain study.

BACKGROUND: Reelin (RELN) is a glycoprotein secreted preferentially by cortical gamma-aminobutyric acid-ergic (GABAergic) interneurons (layers I and II) that binds to integrin receptors located on dendritic spines of pyramidal neurons or on GABAergic interneurons of layers III through V expressing the disabled-1 gene product (DAB1), a cytosolic adaptor protein that mediates RELN action. To replicate earlier findings that RELN and glutamic acid decarboxylase (GAD)(67), but not DAB1 expression, are down-regulated in schizophrenic brains, and to verify whether other psychiatric disorders express similar deficits, we analyzed, blind, an entirely new cohort of 60 postmortem brains, including equal numbers of patients matched for schizophrenia, unipolar depression, and bipolar disorder with nonpsychiatric subjects. METHODS: Reelin, GAD(65), GAD(67), DAB1, and neuron-specific-enolase messenger RNAs (mRNAs) and respective proteins were measured with quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) or Western blot analyses. Reelin-positive neurons were identified by immunohistochemistry using a monoclonal antibody. RESULTS: Prefrontal cortex and cerebellar expression of RELN mRNA, GAD(67) protein and mRNA, and prefrontal cortex RELN-positive cells was significantly decreased by 30% to 50% in patients with schizophrenia or bipolar disorder with psychosis, but not in those with unipolar depression without psychosis when compared with nonpsychiatric subjects. Group differences were absent for DAB1,GAD(65) and neuron-specific-enolase expression implying that RELN and GAD(67) down-regulations were unrelated to neuronal damage. Reelin and GAD(67) were also unrelated to postmortem intervals, dose, duration, or presence of antipsychotic medication. CONCLUSIONS: The selective down-regulation of RELN and GAD(67) in prefrontal cortex of patients with schizophrenia and bipolar disorder who have psychosis is consistent with the hypothesis that these parameters are vulnerability factors in psychosis; this plus the loss of the correlation between these 2 parameters that exists in nonpsychotic subjects support the hypothesis that these changes may be liability factors underlying psychosis.

DNA-methyltransferase1 (DNMT1) binding to CpG rich GABAergic and BDNF promoters is increased in the brain of schizophrenia and bipolar disorder patients.

The down regulation of glutamic acid decarboxylase67 (GAD1), reelin (RELN), and BDNF expression in brain of schizophrenia (SZ) and bipolar (BP) disorder patients is associated with overexpression of DNA methyltransferase1 (DNMT1) and ten-eleven translocase methylcytosine dioxygenase1 (TET1). DNMT1 and TET1 belong to families of enzymes that methylate and hydroxymethylate cytosines located proximal to and within cytosine phosphodiester guanine (CpG) islands of many gene promoters, respectively. Altered promoter methylation may be one mechanism underlying the down-regulation of GABAergic and glutamatergic gene expression. However, recent reports suggest that both DNMT1 and TET1 directly bind to unmethylated CpG rich promoters through their respective Zinc Finger (ZF-CXXC) domains. We report here, that the binding of DNMT1 to GABAergic (GAD1, RELN) and glutamatergic (BDNF-IX) promoters is increased in SZ and BP disorder patients and this increase does not necessarily correlate with enrichment in promoter methylation. The increased DNMT1 binding to these promoter regions is detected in the cortex but not in the cerebellum of SZ and BP disorder patients, suggesting a brain region and neuron specific dependent mechanism. Increased binding of DNMT1 positively correlates with increased expression of DNMT1 and with increased binding of MBD2. In contrast, the binding of TET1 to RELN, GAD1 and BDNF-IX promoters failed to change. These data are consistent with the hypothesis that the down-regulation of specific GABAergic and glutamatergic genes in SZ and BP disorder patients may be mediated, at least in part, by a brain region specific and neuronal-activity dependent DNMT1 action that is likely independent of its DNA methylation activity.

Structure of the rat gene encoding the mitochondrial benzodiazepine receptor.

The gene encoding the rat mitochondrial benzodiazepine receptor (MBR) was cloned and characterized. Hybridization of a previously cloned cDNA for MBR to genomic Southern blots indicated that the gene was probably present at one copy per haploid genome. Rapid amplification of cDNA ends with rat adrenal RNA was used to obtain 47 nt of additional sequence upstream from our previously cloned MBR cDNA proving to be a crucial step in cloning the first exon of this gene. The MBR gene is comprised of four exons spanning approx. 10 kb. The first intron, contained within a 8-kb stretch of this gene, is located within the 5'-untranslated sequence, whereas the remaining two introns are much shorter (641 and 854 bp) and interrupt the coding sequence. The third intron contains sequences homologous to rodent B1 repetitive elements and a novel sequence closely resembling part of a repetitive element belonging to the Alu family in humans. The transcription start point was mapped by S1 nuclease protection assays suggesting that the first exon is just 56 bp in length. The sequence upstream from this region contains three GC boxes but lacks other known consensus recognition sites for sequence-specific transcription factors.

Developmental and mature expression of full-length and truncated TrkB receptors in the rat forebrain.

The neurotrophins brain-derived neurotrophic factor (BDNF) and NT-4/5 exert their trophic effects on the nervous system via signaling through trkB receptors. These receptors occur as splice variants of the trkB gene that encodes a full-length receptor containing the signal transducing tyrosine kinase domain as well as truncated forms lacking this domain. Because the importance of the trkB isoforms for development and maturation of the nervous system is unknown, we have examined the expression of trkB receptor isoforms during development of the rat forebrain using 1) a sensitive ribonuclease protection assay to distinguish full-length and truncated trkB transcripts, 2) western blot analysis to characterize developmental changes in trkB proteins, and 3) immunohistochemistry to determine the cellular localization of trkB receptors. In the rat forebrain, adult mRNA levels for full-length trkB are reached by birth, whereas truncated trkB message does not peak until postnatal days 10-15. Western blot analysis indicates that full-length trkB protein is the major form during early development, whereas truncated trkB protein predominates in all forebrain regions of late postnatal and adult rats. These data also suggest that the glycosylation state of these receptors changes during postnatal maturation. TrkB immunoreactivity is present predominately within neurons, where it is localized to axons, cell soma, and dendrites. Strong dendritic immunostaining is particularly evident in certain neuronal populations, such as pyramidal neurons in the hippocampus and in layer V of the neocortex. The dendritic localization of trkB receptors supports the hypothesis that dendrites, as well as axons, are important sites for neurotrophin actions in the central nervous system.

Pharmacological characterization of regulation of phosphoinositide metabolism by recombinant 5-HT2 receptors of the rat.

Transfection of 5-HT2 receptor cDNA in 293 cells induced the expression of a protein binding domain, exhibiting the classical 5-HT2 receptor transduction mechanism. Both [3H]DOB and [3H]spiperone high affinity binding sites were present in membranes of sense but not of antisense, 5-HT2 receptor cDNA transfected cells. Addition of 1 microM 5-HT induced a time-dependent increase of phosphoinositide (PI) metabolism in sense but not in antisense, 5-HT2 receptor cDNA transfected cells. Graded concentrations of 5-HT and of different serotonergic agonists showed different potencies (DOI greater than 5-HT greater than quipazine greater than DOM greater than alpha-methyl-5-HT greater than 8-OH-DPAT greater than 2-methyl-5-HT greater than CGS-12066B) in stimulating turnover of PI in cells transfected with cDNA encoding for 5-HT2 receptors of the rat. The ability of different antagonists to inhibit 5-HT-stimulated turnover of PI bore a direct relationship with their potency to inhibit 5-HT2 receptor binding in cells transfected with 5-HT2 receptor cDNA (spiperone greater than ketanserin greater than ritanserin greater than mianserin greater than haloperidol). Preincubation of transfected 293 cells with pertussis toxin failed to modify either 5-HT- or DOI-induced activation of metabolism of PI. Pretreatment of transfected 293 cells with DOI (100 nM) for 2 hr or more, significantly reduced activation of turnover of PI elicited by graded doses of 5-HT. When the transfected 293 cells were exposed to DOI (100 nM) for 12 hr and the challenge was performed after a 2-hr wash-out period, the desensitization of the response to 5-HT was virtually abolished.(ABSTRACT TRUNCATED AT 250 WORDS)

GABAA receptors and benzodiazepines: a role for dendritic resident subunit mRNAs.

This review is designed to describe the evolution of the seminal observation made simultaneously in 1975 by Dr. W. Haefely's laboratory (Hoffman La Roche, Basel, Switzerland) and in the Laboratory of Preclinical Pharmacology (NIH, St. Elizabeths Hospital, Washington DC), that benzodiazepine action was mediated by a modulation of GABA action at GABA(A) receptors. In fact, our suggestion was that the benzodiazepine receptor was "a receptor on a receptor" and that this receptor was GABA(A). Needless to say, this suggestion created opposition, but we did not abandon the original idea, in fact, as shown in this review, there is now universal agreement with our hypothesis on the mode of action of benzodiazepines. Hence, this review deals with the allosteric modulation of GABA(A) receptors by benzodiazepines, the role of GABA(A) receptors and benzodiazepine structure diversities in this modulation, and describes the results of our attempts to establish a benzodiazepine (imidazenil) devoid of tolerance, withdrawal symptoms, and changes in the expression of GABA(A) receptor subunits during tolerance. It also deals with the idea that the synthesis of GABA(A) receptor subunits triggered by tolerance resides in dendrites and spines where mRNAs and the apparatus for this translation is located. New analytic procedures may foster progress in the understanding of tolerance to and withdrawal from benzodiazepines.

A GABAergic cortical deficit dominates schizophrenia pathophysiology.

Several lines of evidence support the role of an epigenetic-induced GABAergic cortical dysfunction in schizophrenia psychopathology, which is probably dependent on an increase in the expression of DNA-methyltransferase-1 occurring selectively in GABAergic neurons. The key enzyme regulating GABA synthesis, termed glutamic acid decarboxylase 67 (GAD67) and the important neurodevelopmental protein called reelin are coexpressed in GABAergic neurons. Upon release, GABA and reelin bind to postsynaptic receptors located in dendrites, somata, or the axon initial segment of pyramidal neurons. Because GAD67 and reelin are downregulated in schizophrenia, it is suggested that schizophrenics may express GABAergic deficit-related alterations of pyramidal neuron function. A reduction of dendritic spines is a finding reported in the prefrontal cortex of schizophrenia patients. Because dendritic spines are innervated by glutamatergic axon terminals, very probably this reduction of dendritic spine expression is translated into a functional deficit of glutamatergic transmission. Plastic modifications of neuronal circuits are probably dependent on GABAergic transmitter tone, and it is likely that GABAergic dysfunction is at the root of synaptic plasticity deficits in schizophrenia. Thus, a possible avenue for the treatment of schizophrenia would be to address this GABAergic functional deficit using positive allosteric modulators of the action of GABA at GABAA receptors. Benzodiazepines (BZ) such as diazepam are effective in treating positive and negative symptoms of schizophrenia, but because they positively modulate GABAA receptors expressing alpha1 subunits, these BZs cause sedation and tolerance. In contrast, imidazenil, a full allosteric modulator of GABAA receptors expressing alpha5 subunits may reduce psychotic symptomatology without producing sedation. Hence, imidazenil should be appropriately studied as a prospective candidate for a pharmacological intervention in schizophrenia.

Exposure of neuronal cultures to K+ depolarization or to N-methyl-D-aspartate increases the transcription of genes encoding the alpha 1 and alpha 5 GABAA receptor subunits.

The transcription rates of the alpha 1, alpha 5, and alpha 6 gamma-aminobutyric acidA receptor subunit genes were analyzed in cultures maintained in low KCl (12.5 mM), in low KCl treated with NMDA (10 microM), and in high KCl (25 mM). alpha 1 and alpha 5 transcription rates were significantly increased in response to NMDA or high KCl treatment, while alpha 6 and cyclophilin transcription rates were not changed by either condition. These data suggest that following NMDA or high KCl treatment of granule cells, changes in alpha 1 and alpha 5 mRNA content are a consequence of a specific transcriptional rate increase of the corresponding subunit genes.

Regional distribution in the rat central nervous system of a mRNA encoding a portion of the cardiac sodium/calcium exchanger isolated from cerebellar granule neurons.

The cardiac Na+/Ca2+ exchanger is a bidirectional electrogenic ion transporter that exchanges three Na+ ions for each Ca2+ ion and plays a critical role in returning sarcolemma Ca2+ concentrations to their resting levels. Because of the importance that the Na+/Ca2+ exchanger may play in maintaining neuronal Ca2+ homeostasis in the central nervous system, we subcloned a 456 bp portion of the Na+/Ca2+ exchanger cDNA from RNA isolated from primary cultures of rat cerebellar granule neurons using the polymerase chain reaction (PCR). This cDNA fragment was sequenced and shown to share 91.4% sequence identity with the human and 88% sequence identity with the canine cardiac Na+/Ca2+ exchangers. The PCR amplification product was used to analyze the distribution of this portion of the Na+/Ca2+ exchanger mRNA in various regions of the CNS by both Northern blotting and in situ hybridization histochemistry. The Northern analysis showed that the rank order of abundance of this mRNA was: hippocampus > cortex > cerebellum > hypothalamus > midbrain > striatum. The in situ hybridization data indicated that the corresponding mRNA containing this portion of the exchanger was present in numerous brain regions including multiple cortical layers, the hippocampus, septal nuclei, various thalamic nuclei, cerebellum, hypothalamus, olfactory bulb, brainstem, in various regions of the thoracic spinal cord and to a lesser extent in the striatum. The differential distribution of the mRNA as revealed by the in situ hybridization pattern suggests that either additional molecular variants exist or that different Na+/Ca2+ exchange mechanisms may be operative in those cell types that contain low amounts of this fragment of the exchanger mRNA.

GABAA receptor beta 2 and beta 3 subunits mRNA in the hippocampal formation of aged human brain with Alzheimer-related neuropathology.

Our work on the role of glutamate in Alzheimer's disease (AD)-related neuronal vulnerability and death provided significant insight into the potential contribution of the gamma-aminobutyric acid (GABA) neurotransmitter system as it participates in countering the neurotoxic effects of excessive glutamate receptor stimulation. Our previous studies demonstrate that beta2/3 GABAA receptor subunit immunoreactivity is relatively well preserved in hippocampi with AD pathology. To further elucidate the molecular basis for this observation, we employed in situ hybridization histochemistry to examine the levels of beta2 and beta3 receptor subunit mRNAs in the hippocampus of 19 elderly subjects presenting with a broad range of pathologic severity (i.e., Braak stage I-VI). Semi-quantitative analysis with film autoradiograms revealed that beta2 mRNA signal was highest in the granule cell layer, CA2 and CA1 subfields, while beta3 mRNA hybridization was highest in the granule cell layer, followed by CA2>/=CA3>/=CA1 regions. No significant difference in beta2 mRNA expression was detected among the pathologically mild, moderate or severe groups. In contrast, levels of beta3 mRNA in the pathologically severe group was significantly decreased compared to the mild group within all subregions examined except CA4. Our data suggest that alterations in the expression of GABAA receptor subunits in the AD hippocampus differ between specific receptor subunits with the amount of beta2 mRNA being relatively well-preserved, while beta3 mRNA levels were decreased.

GABA and NMDA in the prevention of apoptotic-like cell death in vitro.

We have shown recently that cerebellar granule neurons die in the absence of depolarizing concentrations of KCl through an apoptosis-like process. To study the contributions of inhibitory (gamma-aminobutyric acid; GABA) and excitatory (glutamate) neurotransmitters in the prevention of apoptotic-like cell death in cultures grown in the presence of reduced concentrations of KCl (12.5 mM), we treated these cultures either acutely or chronically with GABA, bicuculline methiodide, a GABAA receptor antagonist, N-methyl-D-aspartate (NMDA) and/or the NMDA receptor antagonist, MK-801. Cell viability was measured with fluorescein diacetate/propidium iodide (FDA/PI) and trypan blue exclusion tests. In addition, DNA fragmentation was assessed quantitatively using an in situ terminal deoxynucleotidyl transferase assay. Our results demonstrate that treatment of cerebellar granule cell cultures maintained in 12.5 mM KCl with the glutamate receptor agonist NMDA and/or bicuculline protects against cell death and reduces DNA fragmentation. In contrast, GABA potentiated cerebellar granule cell apoptosis mediated by KCl deprivation. These data indicate that signal transduction pathways activated following NMDA receptor stimulation mimic the anti-apoptotic action of high potassium in primary cultures of cerebellar granule neurons. Also, our data support an inhibitory (hyperpolarizing) role for GABA in these cultures. Collectively, the results suggest that the neurotrophic actions of NMDA on granule cells maintained in low KCl and GABA on granule cells cultured in high KCl are due to the necessity for maintaining appropriate intraneuronal calcium concentrations.

Nonselective inhibition by antisense oligonucleotides of cytosine arabinoside action.

Antisense oligonucleotides can be used in cell cultures to inhibit biosynthesis of neurotransmitter receptors. Hence, they operate as highly specific pharmacological antagonists. In obtaining a pure neuronal primary culture the suppression of non-neuronal cell proliferation is required; usually 1-beta-D-arabinofuranosylcytosine (AraC) is used. We report that in primary cultures of rat cerebellar cells, oligonucleotides, targeted to: (1) glutamate receptor, (2) the seven transmembrane spanning region of receptors coupled to GTP binding proteins, and (3) beta-adrenergic receptor kinase, nonspecifically inhibit the cell incorporation of 3H-AraC and curtail its antiproliferative action. This nonspecific action might occur at the level of the mechanism of action of AraC and should be taken into account when antisense probes as pharmacological antagonists are used.