Comparative modeling of GABA(A) receptors: limits,insights, future developments

GABA(A) receptors are chloride ion channels that mediate fast synaptic transmission and belong to a superfamily of pentameric ligand-gated ion channels. The recently published crystal structure of the acetylcholine binding protein can be used as a template for comparative modeling of the extracellular domain of GABA(A) receptors. In this commentary, difficulties with comparative modeling at low sequence identity are discussed, the degree of structural conservation to be expected within the superfamily is analyzed and numerical estimates of model uncertainties in functional regions are provided. Topography of the binding sites at subunit-interfaces is examined and possible targets for rational mutagenesis studies are suggested. Allosteric motions are considered and a mechanism for mediation of positive cooperativity at the benzodiazepine site is proposed.     

GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain

GABA(A) receptors are ligand-operated chloride channels assembled from five subunits in a heteropentameric manner. Using immunocytochemistry, we investigated the distribution of GABA(A) receptor subunits deriving from 13 different genes (alpha1-alpha6, beta1-beta3, gamma1-gamma3 and delta) in the adult rat brain. Subunit alpha1-, beta1-, beta2-, beta3- and gamma2-immunoreactivities were found throughout the brain, although differences in their distribution were observed. Subunit alpha2-, alpha3-, alpha4-, alpha5-, alpha6-, gamma1- and delta-immunoreactivities were more confined to certain brain areas. Thus, alpha2-subunit-immunoreactivity was preferentially located in forebrain areas and the cerebellum. Subunit alpha6-immunoreactivity was only present in granule cells of the cerebellum and the cochlear nucleus, and subunit gamma1-immunoreactivity was preferentially located in the central and medial amygdaloid nuclei, in pallidal areas, the substantia nigra pars reticulata and the inferior olive. The alpha5-subunit-immunoreactivity was strongest in Ammon's horn, the olfactory bulb and hypothalamus. In contrast, alpha4-subunit-immunoreactivity was detected in the thalamus, dentate gyrus, olfactory tubercle and basal ganglia. Subunit alpha3-immunoreactivity was observed in the glomerular and external plexiform layers of the olfactory bulb, in the inner layers of the cerebral cortex, the reticular thalamic nucleus, the zonal and superficial layers of the superior colliculus, the amygdala and cranial nerve nuclei. Only faint subunit gamma3-immunoreactivity was detected in most areas; it was darkest in midbrain and pontine nuclei. Subunit delta-immunoreactivity was frequently co-distributed with alpha4 subunit-immunoreactivity, e.g. in the thalamus, striatum, outer layers of the cortex and dentate molecular layer. Striking examples of complementary distribution of certain subunit-immunoreactivities were observed. Thus, subunit alpha2-, alpha4-, beta1-, beta3- and delta-immunoreactivities were considerably more concentrated in the neostriatum than in the pallidum and entopeduncular nucleus. In contrast, labeling for the alpha1-, beta2-, gamma1- and gamma2-subunits prevailed in the pallidum compared to the striatum. With the exception of the reticular thalamic nucleus, which was prominently stained for subunits alpha3, beta1, beta3 and gamma2, most thalamic nuclei were rich in alpha1-, alpha4-, beta2- and delta-immunoreactivities. Whereas the dorsal lateral geniculate nucleus was strongly immunoreactive for subunits alpha4, beta2 and delta, the ventral lateral geniculate nucleus was predominantly labeled for subunits alpha2, alpha3, beta1, beta3 and gamma2; subunit alpha1- and alpha5-immunoreactivities were about equally distributed in both areas. In most hypothalamic areas, immunoreactivities for subunits alpha1, alpha2, beta1, beta2 and beta3 were observed. In the supraoptic nucleus, staining of conspicuous dendritic networks with subunit alpha1, alpha2, beta2, and gamma2 antibodies was contrasted by perykarya labeled for alpha5-, beta1- and delta-immunoreactivities. Among all brain regions, the median emminence was most heavily labeled for subunit beta2-immunoreactivity. In most pontine and cranial nerve nuclei and in the medulla, only subunit alpha1-, beta2- and gamma2-immunoreactivities were strong, whereas the inferior olive was significantly labeled only for subunits beta1, gamma1 and gamma2. In this study, a highly heterogeneous distribution of 13 different GABA(A) receptor subunit-immunoreactivities was observed. This distribution and the apparently typical patterns of co-distribution of these GABA(A) receptor subunits support the assumption of multiple, differently assembled GABA(A) receptor subtypes and their heterogeneous distribution within the adult rat brain.     

Truncated isoform Vav3.1 is highly expressed in ovarian cancer stem cells and clinically relevant in predicting prognosis and platinum‐response

Vav3 is a key modulator of GTP‐hydrolases of the Rho/Rac family, which are crucially involved in cell proliferation. Vav3 is alternatively spliced in full‐length Vav3‐alpha and N‐terminal truncated Vav3.1 lacking its self‐regulatory domains. The aim of our study was to estimate the clinical impact of Vav3 and all other Vav family members in ovarian cancer. Purification of a stem‐cell like side‐population (SP) from ovarian cancer cell lines was performed by flow cytometry/FACS. Differences in gene expression between SP and NSP were assessed by Gene Array analysis and confirmed by RT‐PCR and immunoblot. In addition, Vav mRNA expression was determined in 150 epithelial ovarian cancers. Clinicopathological parameters, platinum‐sensitivity and survival were analyzed and associated with Vav expression. SP fractions of ovarian cancer cell lines exhibited marked overexpression of Vav3.1 (p < 0.001). Vav1 and Vav2 did not prove to be of clinicopathologic relevance in ovarian cancer. High Vav3.1 expression correlated with higher FIGO stage and residual disease. Furthermore, Vav3.1 overexpression was associated with poor progression‐free (HR = 2.820, p = 0.0001) and overall survival (HR = 2.842, p = 0.0001). Subgroup analyses revealed an impact of Vav3.1 on survival in Type‐II but not in Type‐I cancers. Notably, platinum‐refractory cancers showed marked overexpression of Vav3.1 compared to other subsets of platinum‐sensitivity (15.848 vs. 6.653, p = 0.0001). In conclusion, Vav3.1 is over‐expressed in stem‐cell like SP fractions and is clinically relevant in the pathophysiology of ovarian cancer. The N‐terminal truncated Vav3.1 may be decisively involved in mechanisms causing genuine multi‐drug resistance.     

Effects of Selegiline in a retroviral rat model for neurodegenerative disease

Upon inoculation into neonatal rats, murine leukemia virus (MuLV) NT40 causes a non-inflammatory degeneration of the central nervous system. While microglia cells appear to be the major target cells within the brain parenchyma for neurovirulent MuLV, degenerating neurons do not express retroviral gene products. In order to protect rats from neuronal damage we treated retrovirally infected rats once with monoamine oxidase (MAO) B inhibitor Selegiline which--under different conditions--exerts neuroprotective effects. Unexpectedly, when administered at 17 days post-infection (d.p.i.) a single intraperitoneal dose of Selegilin (1 mg/kg bodyweight) significantly shortened the incubation period for neurological disease. In contrast, Selegiline given in a lower dosage (0.05 mg/kg bodyweight) and/or at a different time point (13 d.p.i.) at the low (0.05 mg/kg bodyweight) and the high dose (1.0 mg/kg bodyweight) had no effect on the outcome of neurological disease. Animals treated with Selegiline (1.0 mg/kg bodyweight at 17 d.p.i.) contained higher amounts of viral loads in the CNS, higher numbers of brain cells expressing major histocompatibility complex class II molecules, and exhibited inhibition of MAO-B in comparison to untreated yet infected (control) animals. Supposedly, Selegiline activated the major target cell population of the CNS for MuLV-NT40, microglia, with the consequence of enhanced susceptibility for retroviral infection and triggered endogenous mechanism(s) involved in the pathogenesis of retroviral neurodegeneration.     

Immunohistochemical and neurochemical evidence for GABAA receptor heterogeneity between the hypothalamus and cortex

This study examined both the function of the GABA_A receptor complex and the expression of its α1, α2 and α3 subunits within the hypothalamus as compared to that of the cerebral cortex. A large number of different GABA_A receptor subunit combinations potentially exist in various brain regions which, presumably, would intimate differing receptor structure and function. Here, we present evidence that the average functional characteristics of GABA_A receptors within the rat hypothalamus are considerably different from those of the cerebral cortex. We assessed two neurochemical measures of GABA_A receptor function: namely, chloride-facilitation of [³H]flunitrazepam binding and GABA-mediated ³⁶chloride uptake. [³H]Flunitrazepam binding in the rat cortex was facilitated by increasing concentrations (12.5–500 mM) of chloride, and this facilitation was responsive to 15 min restraint. Yet, hypothalamic [³H]flunitrazepam binding was not responsive to increasing chloride-concentration in either the basal or restraint conditions. Also, maximal facilitation of GABA-mediated ³⁶chloride uptake was significantly blunted in the hypothalamus relative to cortex (7.4 ± 0.9 versus 35.8 ± 1.5 nmoles/mg protein, respectively). While in vitro addition of 10 μM diazepam shifted GABA-mediated ³⁶chloride uptake curves of the cortex to the left, diazepam addition appeared to be without effect in the hypothalamus. However, the blunted maximal facilitation of GABA on hypothalamic ³⁶chloride uptake made accurate determination of the EC₅₀ for the diazepam-potentiation difficult. In addition to these functional disparities between the regions, differences in subunit expression were also apparent. Distributions of α1, α2 and α3 subunit immunoreactivities within cingulate, parietal and temporal cortices and 8 major hypothalamic regions were assessed. Staining of the α1 subunit was prevalent throughout the hypothalamus and cortex, and dense in both regions. However, the α2 and α3 subunits, while of intermediate density in cortex, were of low density or absent (α3) in the hypothalamus. The α2-immunoreactivity was restricted to cell bodies of the arcuate nucleus, dorsomedial nucleus and overlying dorsal area and to neuropil staining of the median eminence. Thus, functional responsiveness of the GABA_A receptor differs in the hypothalamus relative to the cortex and this would seem related to the presence of different receptor α subunits in homogenate preparations of the two regions.     

Binge alcohol drinking is associated with GABAA alpha2-regulated Toll-like receptor 4 (TLR4) expression in the central amygdala

Binge drinking (blood-alcohol levels ≥ 0.08 g% in a 2-h period), is a significant public health burden in need of improved treatment. Gene therapy may offer beneficial alternatives to current psychosocial and pharmacotherapeutic interventions, but identification of the target genes is a clinical challenge. We report that a GABA(A) α2 siRNA vector (pHSVsiLA2) infused into the central nucleus of the amygdala (CeA) of alcohol-preferring (P) rats caused profound and selective reduction of binge drinking associated with inhibition of α2 expression, decreased GABA(A) receptor density, and inhibition of Toll-like receptor 4 (TLR4). CeA infusion of a TLR4 siRNA vector (pHSVsiLTLR4a) also inhibited binge drinking, but neither vector functioned when infused into the ventral pallidum. Binge drinking was inhibited by a GABA(A) α1 siRNA vector (pHSVsiLA1) infused into the ventral pallidum, unrelated to TLR4. The vectors did not alter sucrose intake and a scrambled siRNA vector was negative. The data indicate that GABA(A) α2-regulated TLR4 expression in the CeA contributes to binge drinking and may be a key early neuroadaptation in excessive drinking.     

Prognostic factors in patients with advanced hepatocellular carcinoma treated with sorafenib

Background Sorafenib is the new reference standard for patients with advanced hepatocellular carcinoma (HCC). Aim To identify prognostic factors in sorafenib‐treated HCC patients and to evaluate outcomes with respect to liver function. Methods In this retrospective study, 148 HCC patients received sorafenib 400 mg b.d. across 11 Austrian institutions. Seventy‐eight HCC patients who received best supportive care (BSC) in the pre‐sorafenib era served as a control. Results In sorafenib‐treated patients, low baseline α‐fetoprotein, low Child–Pugh (CP) score, compensated cirrhosis, and low baseline aspartate aminotransferase (AST) were associated with significantly longer overall survival (OS) on univariate analysis. CP score and baseline AST remained independent prognostic factors on multivariate analysis. In patients with Barcelona Clinic liver Cancer (BCLC) stage B or C HCC (sorafenib: n = 139; BSC: n = 39), CP‐A patients had a median OS of 11.3 (sorafenib [n = 76]) vs. 6.4 (BSC [n = 17]) months (P = 0.010), and CP‐B patients had a median OS of 5.5 (sorafenib [n = 55]) vs. 1.9 (BSC [n = 22]) months (P = 0.021). In the sorafenib group, median OS according to baseline AST was 11.8 (<100 U/L [n = 58]) vs. 3.9 (≥100 U/L [n = 15]) months for CP‐A patients (P = 0.127), and 6.5 (<100 U/L [n = 33]) vs. 2.1 (≥100 U/L [n = 21]) months for CP‐B patients (P = 0.011). There was no survival difference between sorafenib and BSC in patients with BCLC stage D HCC (1.5 vs. 1.4 months; P = 0.116). Conclusions Sorafenib was associated with improved survival in both CP‐A and CP‐B patients. In CP‐B patients, baseline AST may be helpful in determining which patients are most likely to benefit from sorafenib.     

An updated unified pharmacophore model of the benzodiazepine binding site on gamma-aminobutyric acid(a) receptors: correlation with comparative models

A successful unified pharmacophore/receptor model which has guided the synthesis of subtype selective compounds is reviewed in light of recent developments both in ligand synthesis and structural studies of the binding site itself. The evaluation of experimental data in combination with a comparative model of the alpha1beta2gamma2 GABA(A) receptor leads to an orientation of the pharmacophore model within the Bz BS. Results not only are important for the rational design of selective ligands, but also for the identification and evaluation of possible roles which specific residues may have within the benzodiazepine binding pocket.     

Distribution of the major gamma-aminobutyric acid(A) receptor subunits in the basal ganglia and associated limbic brain areas of the adult rat

Within the basal ganglia, gamma-aminobutyric acid (GABA) exerts a fundamental role as neurotransmitter of local circuit and projection neurons. Its fast hyperpolarizing action is mediated through GABA(A) receptors. These ligand-gated chloride channels are assembled from five subunits, which derive from multiple genes. Using immunocytochemistry, we investigated the distribution of 12 major GABA(A) receptor subunits (alpha1-5, beta1-3, gamma1-3, and delta) in the basal ganglia and associated limbic brain areas of the rat. Immunoreactivity for an additional subunit (subunit alpha6) was not observed. The striatum, the nucleus accumbens, and the olfactory tubercle displayed strong, diffuse staining for the subunits alpha2, alpha4, beta3, and delta presumably located on dendrites of the principal medium spiny neurons. Subunit alpha1-, beta2-, and gamma2-immunoreactivities were apparently mostly restricted to interneurons of these areas. In contrast, the globus pallidus, the entopeduncular nucleus, the ventral pallidum, the subthalamic nucleus, and the substantia nigra pars reticulata revealed dense networks of presumable dendrites of resident projection neurons, which were darkly labeled for subunit alpha1-, beta2-, and gamma2-immunoreactivities. The globus pallidus, ventral pallidum, entopeduncular nucleus, and substantia nigra pars reticulata, all areas receiving innervations from the striatum, displayed strong subunit gamma1-immunoreactivity compared to other brain areas. In the substantia nigra pars compacta and in the ventral tegmental area, numerous presumptive dopaminergic neurons were labeled for subunits alpha3, gamma3, and/or delta. This highly heterogeneous distribution of individual GABA(A) receptor subunits suggests the existence of differently assembled, and presumably also functionally different, GABA(A) receptors within individual nuclei of the basal ganglia and associated limbic brain areas.