Affinity of various benzodiazepine site ligands in mice with a point mutation in the GABA(A) receptor gamma2 subunit

The benzodiazepine binding site of GABA(A) receptors is located at the interface of the alpha and gamma subunits. Certain point mutations in these subunits have been demonstrated to dramatically reduce the affinity of benzodiazepine binding site ligands for these receptors. Recently, mice were generated with a phenylalanine (F) to isoleucine (I) substitution at position 77 in the gamma2 subunit of GABA(A) receptors. Here we tested the potency of 24 benzodiazepine binding site ligands from 16 different structural classes for inhibition of [(3)H]flunitrazepam binding to brain membranes of these gamma2F77I mice. Results indicate that the potency of the classical 1,4-benzodiazepines, of the 1,4-thienodiazepine clotiazepam, the 1,5-benzodiazepine clobazam, or the pyrazoloquinoline CGS 9896 is only 2-7-fold reduced by this gamma2F77I point mutation. The potency of the imidazopyrimidines Ru 32698, Ru 33203, and Ru 33356, of the imidazoquinoline Ru 31719, or the pyrazolopyridine CGS 20625 is reduced 10-20-fold, whereas the potency of some imidazobenzodiazepines, beta-carbolines, cyclopyrrolones, imidazopyridines, triazolopyridazines, or quinolines is 100-1000-fold reduced. Interestingly, the extent of potency reduction induced by the gamma2F77I point mutation varied within the structural classes of compounds. Results support and significantly extend previous observations indicating that the residue gamma2F77 is important for high affinity binding of some, but not all benzodiazepine site ligands.     

Non-association of dopamine D4 and D2 receptor genes with personality in healthy individuals

Recently, different research groups reported conflicting results with regard to an association of dopamine 4 receptor (DRD4) genotypes and the personality dimension of novelty seeking (NS). High scores for NS seemed to be associated with long alleles of a DRD4 polymorphism. Furthermore, an association between personality traits and the dopamine 2 (DRD2) receptor gene was reported. NS and persistence (PS) high scores seemed to be associated with alleles of DRD2. We examined 109 (78 female and 31 male) normal healthy individuals using Cloninger's Temperament and Character Inventory (TCI) in order to replicate these findings. We genotyped a 48 base pair variable number of tandem repeats (from two to eight repeats) polymorphism in the third exon of DRD4 and a Cys311Ser polymorphism in exon 7 of DRD2. We tested alleles and genotypes of DRD4 (allele 7 absent or present; genotype 4,4 versus 4,7), and Ser/Cys and Cys/Cys genotypes of DRD2 for associations with TCI values. NS and the alleles and genotypes of DRD4 did not show any association. In associating the genotypes of DRD2 with TCI scales (NS, harm avoidance, reward dependence and PS), we also found no association. Recent findings associating NS with DRD4 could not be replicated. With regard to DRD2, we tested a different polymorphism as published recently and could not find an association of TCI scales with the gene. The present results therefore do not provide evidence that the DRD2 and DRD4 receptor genes contribute a common and relevant effect to personality traits.     

[S]tert.-butylbicyclophosphorothionate and avermectin bind to different sites associated with the γ-aminobutyric acid-benzodiazepine receptor complex

Low concentrations of avermectin B₁a (AVM) stimulated the specific high affinity binding of [³⁵S]tert.-butylbicyclophosphorothionate ([³⁵S]TBPT) to membranes from rat cerebral cortex in the absence or presence of chloride or bromide ions. In contrast, TBPT either weakly stimulates or does not significantly influence the specific high affinity binding of [³H]AVM to the same membranes in the absence or presence of chloride ions, respectively. These results indicate that [³H]AVM and [³⁵S]TBPT bind to different but closely associated binding sites.     

Replication,immunogenicity, and protective properties of live-attenuated simian immunodeficiency viruses expressing interleukin-4 or interferon-gamma

Nef deletion mutants of SIV-expressing interleukin-4 (SIV-IL4) or interferon-gamma (SIV-IFN) were constructed to study the effect of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) on viral load, immunogenicity, and protective properties. Four rhesus monkeys were infected with SIV-IL4 and four were infected with SIV-IFN. During the acute phase of infection, the cell-associated viral load, but not the plasma viral RNA load, was approximately 10-fold lower in SIV-IFN-infected macaques than in SIV-IL4-infected rhesus monkeys. The viral load declined to hardly detectable levels 4 months postinfection in all animals. SIV antibody titers and the affinity of these antibodies were higher in SIV-IL4-infected macaques than in SIV-IFN-infected animals, consistent with a stimulation of T helper cell type 2 immune responses by IL-4. At peak viremia, there was a trend to higher interleukin-12 and perforin mRNA levels of the lymph nodes in the SIV-IFN-infected macaques than in the SIV-IL4-infected monkeys. Deletion of the viral IFN gene, but not the viral IL-4 gene, after the development of antiviral immune responses suggests a repressive effect of IFN, but not IL-4, on virus spread in vivo. A trend to higher set point viral RNA levels in SIV-IL4-infected monkeys in comparison to monkeys infected with the parental nef deletion mutant and similar viral RNA levels during the acute phase of infection suggest that IL-4 expression leads to a slight reduction in the control of virus replication by host immune responses. However, SIV-IL4 and SIV-IFN induced protection against a homologous challenge virus. Subsequent challenge with an SIV-HIV-1 hybrid virus (SHIV) also revealed protection in the absence of neutralizing antibodies.     

Postnatal development of proteins irreversibly labeled by [3H]flunitrazepam

The irreversible labeling by [³H]flunitrazepam of three proteins with apparent molecular weights of 51,000 (P₅₁), 55,000 (P₅₅) and 59,000 (P₅₉) was investigated using hippocampal membranes isolated from rats at various timepoints after birth. The present results indicate that [³H]flunitrazepam predominantly labels P₅₅ and P₅₉ in the early days after birth whereas labeling of P₅₁ starts to increase significantly in the second postnatal week. A possible association of P₅₅ and P₅₉ with type 2 and of P₅₁ with type 1 benzo diazepine receptors is suggested.     

GABAA receptor subunits in the rat hippocampus II: Altered distribution in kainic acid-induced temporal lobe epilepsy

Intraperitoneal injection of kainic acid in the rat represents a widely used animal model of human temporal lobe epilepsy. Injection of kainic acid induces acute limbic seizures which are accompanied by seizure-induced brain damage and late spontaneous recurrent seizures. There is considerable evidence for an altered transmission of GABA in human temporal lobe epilepsy and in the kainic acid model. We therefore investigated by immunocytochemistry the distribution of 13 GABA receptor subunits in the hippocampus of rats 12 h, 24 h, and two, seven and 30 days after injection of kainic acid. Within the molecular layer of the dentate gyrus, decreases in α₂- and δ- and slight increases in α₁-, β₂- and β₃-immunoreactivities were observed at early intervals (12 to 24 h) after kainic acid injection. These changes were succeeded by marked increases in α₁-, α₂-, α₄-, α₅-, β₁-, β₃-, γ₂- and δ-immunoreactivities in the same area after seven to 30 days. Within the hippocampus proper, changes in expression of GABA_A receptor subunits were demarcated by considerable neurodegeneration of CA1 and CA3 pyramidal neurons. All subunits present within dendritic areas of CA1 and CA3 were affected. These were α₁, α₂, α₅, β₁–β₃, γ₂ and α₄ (present only in CA1). Decreases in these subunits were followed by increased expression of α₂-, α₅-, β₃-, γ₂- and δ-subunits in the hippocampus proper notably in CA3 at later intervals (up to 30 days). α₁-, β₂-, γ₂- and δ-subunits were found in presumed GABA containing interneurons throughout the hippocampus. Their immunoreactivity was augmented after two to seven days. Some α₄-, γ₃- and δ-immunoreactivity was also found in astrocytes 48 h after kainic acid injection.Our data indicate an impairment of GABA-mediated neurotransmission due to a lasting loss of GABA_A receptor containing cells after kainic acid-induced seizures. The seizure-induced loss in GABA_A receptors within the hippocampus may in part be compensated by increased expression of GABA_A receptor subunits within the molecular layer of the dentate gyrus and in pyramidal cells.     

Additional support for linkage of schizophrenia and bipolar disorder to chromosome 3q29.

After publishing a genome scan and follow-up fine mapping, suggesting schizophrenia and bipolar disorder linkage to chromosome 3q29, we now genotyped 11 additional SNPs (single nucleotide polymorphisms), in order to narrow down a potential candidate region. Linkage was performed using the GENEHUNTER program version 2.1r3. A NPL score Z(all) of 3.891 (p=0.000156) was observed with SNP rs225. In short, we found significant linkage scores most telomeric on chromosome 3q29, spanning 3.46 Mbp (7 SNPs).     

Sequestration of nNOS in neurofilamentous aggregate bearing neurons in vitro leads to enhanced NMDA-mediated calcium influx

The significance of copper/zinc superoxide dismutase (SOD1) and neuronal nitric oxide synthase (nNOS) co-localization to neurofilamentous (NF) aggregates in amyotrophic lateral sclerosis (ALS) is unknown. In this study, we have used dissociated motor neurons from either C57BL/6 or mice that over-express the human low molecular weight neurofilament protein (hNFL+/+) to examine the relationship between NF aggregate formation, SOD1 and nNOS co-localization, and the regulation of NMDA-mediated calcium influx in vitro. The intracellular distribution of NF aggregates, SOD1 and nNOS was examined by confocal microscopy and NMDA-induced alterations in intracellular calcium levels using either Oregon green fluorescence or FURA-2 photometric imaging. Cell death was assessed using an antibody to activated caspase-3. C57 Bl/6 motor neurons expressed nNOS in a punctate manner, whereas SOD1 was distributed homogeneously throughout the cytosol. In contrast, hNFL+/+ motor neurons demonstrated co-localization of SOD1 and nNOS by day 9 post-plating, preceding the formation of NF aggregates. Both proteins co-localized to NF aggregates once formed. With NMDA stimulation, aggregate-bearing hNFL+/+ motor neurons demonstrated significant increases in intracellular calcium, whereas only a minimal alteration in intracellular calcium was observed in C57 Bl/6 neurons. Following stimulation with 100 microM NMDA, 75.5+/-5.5% of hNFL+/+ neurons became apoptotic, whereas only 16.3+/-5.3% of C57 Bl/6 were. These observations suggest that the presence of NF aggregates results in a failure of regulation of NMDA-mediated calcium influx, and that this occurs due to the sequestration of nNOS to the NF aggregate, preventing its down-regulation of the NMDA receptor.     

Possible linkage of schizophrenia and bipolar affective disorder to chromosome 3q29: a follow-up

The present linkage study is a follow-up within the chromosome 3q29 region in schizophrenia and bipolar affective disorder families, based on our recently published genome scan, resulting in evidence for linkage of both disorders to this region (marker D3S1265: NPL [non parametric lod] score Z_all=3.74, P=0.003). Using the same family sample (five pedigrees with schizophrenic index patients and three pedigrees with index bipolar disorder patients N=86; 50 of them were available for genotyping), genotyping of eight additional markers close to D3S1265 was done. Five of those new markers (three centromeric and two telomeric of D3S1265) spanning 4.14 cM (centiMorgan) could be used for statistical analyses (“new markers”). Moreover, marker D3S1265, genotyped within the published genome scan, was used for additional calculations. Linkage analysis was performed using the GENEHUNTER program version 2.1r3. Within newly genotyped markers the highest NPL score Z_all observed was 1.93296 with the telomeric SNP (single nucleotide polymorphism) rs1835669, corresponding to P=0.032166. Statistical analysis including D3S1265, located in between the newly genotyped markers, resulted in a peak NPL score Z_all=4.00179 with marker D3S1265, that is P=0.000128. Doing subset analyses of the bipolar disorder and schizophrenia families separately with new markers and D3S1265, linkage signals arose substantially from bipolar disorder families, with contribution from schizophrenia families, too. The results of our follow-up study support our previous linkage finding of schizophrenia and bipolar affective disorder to chromosome 3q29.