Peter J. Gebicke-Haerter

Genome-wide Association Study of Alcohol Dependence

CONTEXT Alcohol dependence is a serious and common public health problem. It is well established that genetic factors play a major role in the development of this disorder. Identification of genes that contribute to alcohol dependence will improve our understanding of the mechanisms that underlie this disorder. OBJECTIVE To identify susceptibility genes for alcohol dependence through a genome-wide association study (GWAS) and a follow-up study in a population of German male inpatients with an early age at onset. DESIGN The GWAS tested 524,396 single-nucleotide polymorphisms (SNPs). All SNPs with P < 10(-4) were subjected to the follow-up study. In addition, nominally significant SNPs from genes that had also shown expression changes in rat brains after long-term alcohol consumption were selected for the follow-up step. SETTING Five university hospitals in southern and central Germany. PARTICIPANTS The GWAS included 487 male inpatients with alcohol dependence as defined by the DSM-IV and an age at onset younger than 28 years and 1358 population-based control individuals. The follow-up study included 1024 male inpatients and 996 age-matched male controls. All the participants were of German descent. MAIN OUTCOME MEASURES Significant association findings in the GWAS and follow-up study with the same alleles. RESULTS The GWAS produced 121 SNPs with nominal P < 10(-4). These, together with 19 additional SNPs from homologues of rat genes showing differential expression, were genotyped in the follow-up sample. Fifteen SNPs showed significant association with the same allele as in the GWAS. In the combined analysis, 2 closely linked intergenic SNPs met genome-wide significance (rs7590720, P = 9.72 x 10(-9); rs1344694, P = 1.69 x 10(-8)). They are located on chromosome region 2q35, which has been implicated in linkage studies for alcohol phenotypes. Nine SNPs were located in genes, including the CDH13 and ADH1C genes, that have been reported to be associated with alcohol dependence. CONCLUSIONS This is the first GWAS and follow-up study to identify a genome-wide significant association in alcohol dependence. Further independent studies are required to confirm these findings.

Microglial activation and amyloid-β clearance induced by exogenous heat-shock proteins

Alzheimers disease (AD) is characterized by the accumulation of fibrillar amyloid‐β (Aβ) peptides to form amyloid plaques. Understanding the balance of production and clearance of Aβ peptides is the key to elucidating amyloid plaque homeostasis. Microglia in the brain, associated with senile plaques, are likely to play a major role in maintaining this balance. Here, we show that heat‐shock proteins (HSPs), such as HSP90, HSP70, and HSP32, induce the production of interleukin 6 and tumor necrosis factor α and increase the phagocytosis and clearance of Aβ peptides. This suggests that microglial interaction with Aβ peptides is highly regulated by HSPs. The mechanism of microglial activation by exogenous HSPs involves the nuclear factor KB and p38 mitogen‐activated protein kinase pathways mediated by Toll‐like receptor 4 activation. In AD brains, levels of HSP90 were increased in both the cytosolic and membranous fractions, and HSP90 was colocalized with amyloid plaques. These observations suggest that HSP‐induced microglial activation may serve a neuroprotective role by facilitating Aβ clearance and cytokine production.

Expression of tumor necrosis factor alpha after focal cerebral ischaemia in the rat

Induction of tumor necrosis factor alpha was studied in the brain of rats after focal cerebral ischaemia by occlusion of the left middle cerebral artery. Using a specific antisense riboprobe for in situ hybridization histochemistry, cells positive for tumor necrosis factor alpha messenger RNA were detected within 30 min in the brain regions known to be necrotic within one to two days after onset of ischaemia. Their number increased over a time period of 1-8 h and then declined. Only a few tumor necrosis factor alpha messenger RNA positive cells could be detected four days after the onset of ischaemia. Reverse-transcription polymerase chain reaction experiments showed that maximal increase of tumor necrosis factor alpha messenger RNA level in the ischaemic brain hemisphere occurred 3 h after occlusion of the middle cerebral artery. Immunocytochemical experiments using an anti-tumor necrosis factor alpha antibody showed the presence of tumor necrosis factor alpha immunopositive cells as early as 30 min after occlusion of the middle cerebral artery in the same brain regions where tumor necrosis factor alpha messenger RNA positive cells were detected. Tumor necrosis factor alpha positive cells were highly abundant in the infarcted brain 8-24 h, but only few of them were detectable four days after the onset of ischaemia. Specificity of the anti-tumor necrosis factor alpha antibody and of the induction of tumor necrosis factor alpha protein was confirmed by western blot analysis. Tumor necrosis factor alpha messenger RNA- and protein-positive cells were also detected in the watershed zone and in some structures of the contralateral brain hemisphere. According to their morphology, tumor necrosis factor alpha-positive cells could be identified as microglial cells and macrophages at different states of activation. This assumption was further confirmed by double-labeling studies using the isolectin B4 from Griffonia simplicifolia, a specific microglial/macrophage cell marker. These results demonstrate that expression of tumor necrosis factor alpha is part of an intrinsic inflammatory reaction of the brain after ischaemia.

Cyclooxygenase-2 expression in rat microglia is induced by adenosine A2a-receptors

We investigated the regulation of COX‐2 expression and activity by adenosine receptors in rat microglial cells. The selective adenosine A2a‐receptor agonist CGS21680 and the non‐selective adenosine A1‐ and A2‐receptor agonist 5′‐N‐ethylcarboxi‐amidoadenosine (NECA) induced an increase in COX‐2 mRNA levels and the synthesis of prostaglandin E2 (PGE2). The adenosine A1‐receptor agonist cyclopentyladenosine (CPA) was less potent, and the adenosine A3‐receptor‐specific agonist N6‐2‐(‐aminophenylo)ethyladenosine (APNEA) showed only marginal effects. Microglia expressed adenosine A1‐, A2a‐, and A3‐, but not A2b‐receptor mRNAs, whereas astroglial cells expressed adenosine A2b‐ but not A2a‐receptor mRNA. The adenosine A2a‐receptor selective antagonist (E)‐8‐(3,4‐dimethoxystyryl)‐1,3‐dipropyl‐7‐methylxanthine (KF17837) inhibited both CGS21680‐induced COX‐2 expression and PGE2 release. CGS21680‐increased PGE2 levels were inhibited by dexamethasone, by the nonsteroidal antiinflammatory drug meloxicam, and by the adenylyl cyclase inhibitor 9‐(tetrahydro‐2‐furanyl)‐9H‐purine‐6‐amine (SQ22536). CGS21680 and NECA both increased intracellular cAMP levels in microglial cells. Dibutyryl cAMP as well as forskolin induced the release of PGE2. The results strongly suggest that adenosine A2a‐receptor‐induced intracellular signaling events cause an up‐regulation of the COX‐2 gene and the release of PGE2. Apparently, the cAMP second messenger system plays a crucial role in COX‐2 gene regulation in rat microglial cells. The results are discussed with respect to neurodegenerative disorders of the CNS such as Alzheimers disease, in which activated microglia are critically involved and COX inhibitors may be of therapeutic benefit.

Sphingosine 1-phosphate induces chemotaxis of immature and modulates cytokine-release in mature human dendritic cells for emergence of Th2 immune responses

Sphingosine 1‐phosphate (S1P) is a potent extracellular lysolipid phosphoric acid mediator that is released after IgE‐stimulation of mast cells. Here we investigated the biological activity and intracellular signaling of S1P on human dendritic cells (DC), which are specialized antigen presenting cells with the ability to migrate into peripheral tissues and lymph nodes, as well as control the activation of naive T cells. We show that immature and mature DC express the mRNA for different S1P receptors, such as endothelial differentiation gene (EDG)‐1, EDG‐3, EDG‐5, and EDG‐6. In immature DC, S1P stimulated pertussis toxin‐sensitive Ca2+ increase actin‐polymerization and chemotaxis. These responses were lost by DC matured with lipopolysaccharide. In maturing DC, however, S1P inhibited the secretion of tumor necrosis factor α and interleukin (IL)‐12, whereas it enhanced secretion of IL‐10. As a consequence, mature DC exposed to S1P showed a reduced and increased capacity to generate allogeneic Th1 and Th2 responses, respectively. In summary, our study implicates that S1P might regulate the trafficking of DC and ultimately favor Th2 lymphocyte‐dominated immunity.

Expression and function of adenosine receptors in human dendritic cells

Dendritic cells (DCs) are specialized antigen‐presenting cells characterized by their ability to migrate into target sites, process antigens, and activate naive T cells. In this study, we analyzed the biological activity and intracellular signaling of adenosine by using reverse transcriptase‐polymerase chain reaction assays to investigate mRNA expression of A1,A2a and A3 adenosine receptors in immature and mature human DCs. Functional experiments on adenosine stimulation showed chemotaxis, intracellular calcium transients, and actin polymerization, but no activation of adenylate cyclase in immature DCs. Experiments with receptor isotype‐selective agonists and antagonists as well as pertussis toxin revealed that chemotaxis, calcium transients, and actin polymerization were mediated via Gi‐or G0‐protein‐coupled A1 and A3 receptors. Maturation of DCs induced by lipopolysaccharide (LPS) resulted in down‐regulation of A1 and A3 receptor mRNAs, although A2a receptor mRNA was still expressed. However, in LPS‐differentiated DCs, adenosine and an A2a receptor agonist stimulated adenylate cyclase activity, enhanced intracellular cAMP levels, and inhibited in‐terleukin 12 (IL‐12) production. These effects could be completely prevented by pretreatment with A2 receptor antagonist. These findings strongly suggest that adenosine has important but distinct biological effects in DCs activity as a chemotaxin for immature DCs and as a modulator of IL‐12 production in mature DCs. These effects can be explained by differential expression of adenosine receptor subtypes.

Both adenosine A1- and A2-receptors are required to stimulate microglial proliferation.

The neuromodulator adenosine is one of the major endogenous inhibitors of overactive excitatory neurotransmission. Adenosine receptors have been identified on neuronal but also on glial surfaces, indicating a role of glial cells in mediation of adenosine effects. Microglia, the immunocompetent cells of the brain, typically respond with proliferation, migration and production of inflammatory substances to viral or bacterial stimuli or to cell damage and degeneration. Since adenosine is released in large amounts in conditions of, for example, hypoxic or ischemic stress, it might be involved in the activation process of microglia. Proliferation of microglia was determined by incorporation of [3H]thymidine into microglial DNA after stimulation with adenosine A1- and A2-receptor agonists. N6-Cyclopentyl adenosine (CPA) and CGS-21680, a specific adenosine A2-receptor agonist had no effect on microglial proliferation. However, combinations of CPA and CGS-21680 as well as the mixed agonist, N6-ethyl-carboxamido adenosine (NECA) increased incorporation of radiolabel above controls. The effect of NECA was inhibited by the adenosine A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). From these results, it is concluded that proliferation of microglia can be increased only by simultaneous stimulation of both adenosine A1- and A2-receptors. Targeted interference with the activation of A1-adenosine receptors by specific drugs appears to be sufficient to reduce microglial activation. The findings may have implications for the treatment of neurodegenerative diseases in which microglial activation is supposed to play a causative role.

Lipopolysaccharide induces expression of tumour necrosis factor alpha in rat brain: inhibition by methylprednisolone and by rolipram

1 We have investigated the effects of the phosphodiesterase (PDE) type IV inhibitor rolipram and of the glucocorticoid methylprednisolone on the induction of tumour necrosis factor alpha (TNF‐α) mRNA and protein in brains of rats after peripheral administration of lipopolysaccharide (LPS). 2 After intravenous administration of LPS, a similar time‐dependent induction of both TNF‐α mRNA and protein was observed in rat brain. Peak mRNA and protein levels were found 7 h after administration of LPS. 3 In situ hybridization experiments with a specific antisense TNF‐α riboprobe suggested that the cells responsible for TNF‐α production in the brain were microglia. 4 Intraperitoneal administration of methylprednisolone inhibited the induction of TNF‐α protein in a dose‐dependent manner. A maximal inhibition of TNF‐α protein production by 42.9±10.2% was observed at a dose regimen consisting of two injections of each 30 mg kg−1 methylprednisolone. 5 Intraperitoneal administration of rolipram also inhibited the induction of TNF‐α protein in a dose‐dependent manner. The maximal inhibition of TNF‐α protein production was 96.1±12.2% and was observed at a dose regimen of three separate injections of each 3 mg kg−1 rolipram. 6 In situ hybridization experiments showed that the level of TNF‐α mRNA induced in rat brain by LPS challenge was reduced by intraperitoneal administration of methylprednisolone (2×15 mg kg−1) and of rolipram (3×3 mg kg−1). 7 We suggest that peripheral administration of LPS induces a time‐dependent expression of TNF‐α in rat brain, presumably in microglial cells, and that methylprednisolone and rolipram inhibit LPS‐induced expression of TNF‐α in these cells via a decrease of TNF‐α mRNA stability and/or TNF‐α gene transcription.

Characterization and possible function of adenosine 5'-triphosphate receptors in activated rat microglia.

1 Purinoceptor agonist‐induced currents in untreated (proliferating) and lipopolysaccharide (LPS; 100 ng ml−1)‐treated (non‐proliferating) rat microglial cells in culture were recorded by the whole‐cell patch‐clamp technique. These cells have two preferred resting membrane potentials, one at − 35 mV and another one at − 70 mV. 2 Most experiments were carried out in non‐proliferating cells. ATP, ATP‐γ‐S and α,β‐MeATP (1–1000 μm in all cases) evoked an inward current at a holding potential of − 70 mV, followed, in some experiments, by an outward current. At − 70 mV 2‐methylthio ATP (1–1000 μm) evoked an inward current, whereas at − 35 mV it produced an outward current only. 3 When K+ was replaced in the pipette solution by an equimolar concentration of Cs+ (150 mm), the main outward component of the ATP‐γ‐S (10 μm) induced response disappeared. Instead, an inward current was obtained. Replacement of K+ by Cs+ did not affect the inward current evoked by 2‐methylthio ATP (300 μm). 4‐Aminopyridine (1–10 mm), however, almost abolished this current and unmasked a smaller outward current. 4 The rank order of agonist potency was 2‐methylthio ATP >ATP>α,β‐MeATP. Adenosine and UTP were inactive. Suramin (300 μm) and reactive blue 2 (50 μm) antagonized the effect of 2‐methylthio ATP (300 μm). 5 I–V relations were determined by delivering fast voltage ramps before and during the application of 2‐methylthio ATP (300 μm). In the presence of extra‐ (1 mm) and intracellular (150 mm) Cs+, the 2‐methylthio ATP‐evoked current crossed the zero current level near 0 mV. When both Cs+ (1 mm) and 4‐aminopyridine (1 mm) were present in the bath medium, the intersection of the 2‐methylthio ATP current with the zero current level was near − 75 mV. 6 2‐Methylthio ATP (1–1000 μm) induced the same inward current both in proliferating and non‐proliferating microglia. However, the depolarizing response to 2‐methylthio ATP (300 μm) was larger and longer‐lasting in the proliferating cells. When the free Ca2+ concentration in the pipettes was increased from the standard 0.01 to 1 μm, the amplitude and duration of this depolarization was increased in non‐proliferating cells. 4‐Aminopyridine (1 mm) enhanced the duration, but not the amplitude of responses. 7 ATP and its structural analogues stimulate microglial purinoceptors of the P2Y‐type. This leads to the opening of non‐selective cationic channels and potassium channels. Depending on the resting membrane potential, depolarization or hyperpolarization prevails. Although the inward current produced by 2‐methylthio ATP is of similar amplitude in proliferating and non‐proliferating microglia, the resulting depolarization is smaller in the latter cell type because of the presence of voltage‐sensitive, outwardly rectifying potassium channels.

Voltage‐dependent potassium channels in activated rat microglia.

1. Voltage‐dependent currents of untreated (proliferating) and lipopolysaccharide (LPS)‐treated rat microglial cells in culture were recorded using the whole‐cell patch‐clamp technique. 2. Membrane potentials showed prominent peaks at ‐35 mV and ‐70 mV. Membrane potentials of LPS‐treated cells alternated between the two values. This may be due to a negative slope region of the I‐V relation resulting in two zero current potentials. 3. From a holding potential of ‐70 mV, hyperpolarizing steps evoked an inwardly rectifying current both in proliferating and in LPS‐treated cells, while depolarizing steps below ‐50 mV evoked an outwardly rectifying current only in LPS‐treated microglia. The currents were K+ selective, as indicated by their reversal potential of approximately 0 mV in symmetric K+ concentrations (150 mM both intra‐ and extracellularly) and the reversal potential of the outward tail currents of approximately ‐90 mV at a normal extracellular K+ concentration (4.5 mM). 4. The activation of the outward current could be fitted by Hodgkin‐Huxley‐type n4 kinetics. The time constant of activation depended on voltage. 5. The inactivation of the inward and outward currents could be fitted by a single exponential. The time constant of the inward current inactivation was dependent on voltage, whereas the time constant of the outward current inactivation was virtually independent of voltage, except near the threshold of activation. Recovery of the outward from inactivation was slow and could be fitted by two exponentials. Responses to depolarizing steps were stable at 0.125 Hz, but greatly decreased from the first to the second pulse at 1 Hz. 6. The inactivation of the inward, but not of the outward, current disappeared in a low Na(+)‐containing medium (5 mM). The inward current was selectively inhibited by extracellular Cs+ and Ba2+. The outward current was selectively inhibited by Cd2+, 4‐aminopyridine and charybdotoxin. Replacement of intracellular K+ by an equimolar concentration of Cs+, and the extracellular application of tetraethylammonium and quinine inhibited both currents. 7. An increase of extracellular Ca2+ from 2 to 20 mM resulted in outwardly rectifying K+ channels activating at more positive potentials. Omission of Ca2+ from the extracellular medium had the opposite effect. When the intracellular free Ca2+ was increased from 0.01 to 1 microM, the outward current amplitudes were depressed. The Ca2+ ionophore A23187 had a similar effect. 8. LPS‐treated microglial cells possess inwardly and outwardly rectifying K+ channels. The physiological and pharmacological characteristics of these two channel populations are markedly different.(ABSTRACT TRUNCATED AT 400 WORDS)