Sundari Rallapalli

The differential role of α1- and α5-containing GABAA receptors in mediating diazepam effects on spontaneous locomotor activity and water-maze learning and memory in rats

The clinical use of benzodiazepines (BZs) is hampered by sedation and cognitive deterioration. Although genetic and pharmacological studies suggest that alpha1- and alpha5-containing GABA(A) receptors mediate and/or modulate these effects, their molecular substrate is not fully elucidated. By the use of two selective ligands: the alpha1-subunit affinity-selective antagonist beta-CCt, and the alpha5-subunit affinity- and efficacy-selective antagonist XLi093, we examined the mechanisms of behavioural effects of diazepam in the tests of spontaneous locomotor activity and water-maze acquisition and recall, the two paradigms indicative of sedative- and cognition-impairing effects of BZs, respectively. The locomotor-activity decreasing propensity of diazepam (significant at 1.5 and 5 mg/kg) was antagonized by beta-CCt (5 and 15 mg/kg), while it tended to be potentiated by XLi093 in doses of 10 mg/kg, and especially 20 mg/kg. Diazepam decreased acquisition and recall in the water maze, with a minimum effective dose of 1.5 mg/kg. Both antagonists reversed the thigmotaxis induced by 2 mg/kg diazepam throughout the test, suggesting that both GABA(A) receptor subtypes participate in BZ effects on the procedural component of the task. Diazepam-induced impairment in the declarative component of the task, as assessed by path efficiency, the latency and distance before finding the platform across acquisition trials, and also by the spatial parameters in the probe trial, was partially prevented by both, 15 mg/kg beta-CCt and 10 mg/kg XLi093. Combining a BZ with beta-CCt results in the near to control level of performance of a cognitive task, without sedation, and may be worth testing on human subjects.

Subtype selectivity of α+β− site ligands of GABAA receptors: identification of the first highly specific positive modulators at α6β2/3γ2 receptors

GABAA receptors are the major inhibitory neurotransmitter receptors in the mammalian brain and the target of many clinically important drugs interacting with different binding sites. Recently, we demonstrated that CGS 9895 (2‐(4‐methoxyphenyl)‐2H‐pyrazolo[4,3‐c]quinolin‐3(5H)‐one) elicits a strong and subtype‐dependent enhancement of GABA‐induced currents via a novel drug‐binding site at extracellular αx+βy− (x = 1–6, y = 1–3) interfaces. Here, we investigated 16 structural analogues of CGS 9895 for their ability to modulate GABA‐induced currents of various GABAA receptor subtypes.

The role of α1 and α5 subunit-containing GABAA receptors in motor impairment induced by benzodiazepines in rats.

Benzodiazepines negatively affect motor coordination and balance and produce myorelaxation. The aim of the present study was to examine the extent to which populations of &ggr;-aminobutyric acid A (GABAA) receptors containing &agr;1 and &agr;5 subunits contribute to these motor-impairing effects in rats. We used the nonselective agonist diazepam and the &agr;1-selective agonist zolpidem, as well as nonselective, &agr;1-subunit and &agr;5-subunit-selective antagonists flumazenil, &bgr;CCt, and XLi093, respectively. Ataxia and muscle relaxation were assessed by rotarod and grip strength tests performed 20 min after intraperitoneal treatment. Diazepam (2 mg/kg) induced significant ataxia and muscle relaxation, which were completely prevented by pretreatment with flumazenil (10 mg/kg) and &bgr;CCt (20 mg/kg). XLi093 antagonized the myorelaxant, but not the ataxic actions of diazepam. All three doses of zolpidem (1, 2, and 5 mg/kg) produced ataxia, but only the highest dose (5 mg/kg) significantly decreased the grip strength. These effects of zolpidem were reversed by &bgr;CCt at doses of 5 and 10 mg/kg, respectively. The present study demonstrates that &agr;1 GABAA receptors mediate ataxia and indirectly contribute to myorelaxation in rats, whereas &agr;5 GABAA receptors contribute significantly, although not dominantly, to muscle relaxation but not ataxia.

PWZ-029, AN INVERSE AGONIST SELECTIVE FOR α5 GABAA RECEPTORS, IMPROVES OBJECT RECOGNITION, BUT NOT WATER-MAZE MEMORY IN NORMAL AND SCOPOLAMINE-TREATED RATS

Inverse agonism at the benzodiazepine site of α(5) subunit-containing GABA(A) receptors is an attractive approach for the development of putative cognition-enhancing compounds, which are still far from clinical application. Several ligands with binding and/or functional selectivity for α(5) GABA(A) receptors have been synthesized and tested in a few animal models. PWZ-029 is an α(5) GABA(A) selective inverse agonist whose memory enhancing effects were demonstrated in the passive avoidance task in rats and in Pavlovian fear conditioning in mice. In the present study we investigated the effects of PWZ-029 administration in novel object recognition test and Morris water maze, in normal and scopolamine-treated rats. All the three doses of PWZ-029 (2, 5 and 10 mg/kg) improved object recognition after the 24-h delay period, as shown by significant differences between the exploration times of the novel and old object, and the respective discrimination indices. PWZ-029 (2 mg/kg) also successfully reversed the 0.3 mg/kg scopolamine-induced deficit in recognition memory after the 1-h delay. In the Morris water maze test, PWZ-029 (5, 10 and 15 mg/kg) did not significantly influence swim patterns, either during five acquisition days or during the treatment-free probe trial. PWZ-029 (2, 5 and 10 mg/kg) also proved to be ineffective in the reversal of the 1mg/kg scopolamine-induced memory impairment in the water maze. The present mixed results encourage use of a variety of tests and experimental conditions in order to increase the predictability of preclinical testing of selective α(5) GABA(A) inverse agonists.

Identification of novel positive allosteric modulators and null modulators at the GABAA receptor α+β- interface.

GABAA receptors are the major inhibitory neurotransmitter receptors in the mammalian brain and the target of many clinically important drugs interacting with different binding sites. Recently, we demonstrated that CGS 9895 (2‐(4‐methoxyphenyl)‐2H‐pyrazolo[4,3‐c]quinolin‐3(5H)‐one) acts as a null modulator (antagonist) at the high affinity benzodiazepine binding site, but in addition elicits a strong enhancement of GABA‐induced currents via a novel drug binding site at the extracellular α+β− interface. Here, we investigated 32 structural analogues of CGS 9895 for their ability to mediate their effects via the α1+β3− interface of GABAA receptors.

Ethanol, not detectably metabolized in brain, significantly reduces brain metabolism, probably via action at specific GABA(A) receptors and has measureable metabolic effects at very low concentrations

Ethanol is a known neuromodulatory agent with reported actions at a range of neurotransmitter receptors. Here, we measured the effect of alcohol on metabolism of [3‐13C]pyruvate in the adult Guinea pig brain cortical tissue slice and compared the outcomes to those from a library of ligands active in the GABAergic system as well as studying the metabolic fate of [1,2‐13C]ethanol. Analyses of metabolic profile clusters suggest that the significant reductions in metabolism induced by ethanol (10, 30 and 60 mM) are via action at neurotransmitter receptors, particularly α4β3δ receptors, whereas very low concentrations of ethanol may produce metabolic responses owing to release of GABA via GABA transporter 1 (GAT1) and the subsequent interaction of this GABA with local α5‐ or α1‐containing GABA(A)R. There was no measureable metabolism of [1,2‐13C]ethanol with no significant incorporation of 13C from [1,2‐13C]ethanol into any measured metabolite above natural abundance, although there were measurable effects on total metabolite sizes similar to those seen with unlabelled ethanol.

A Review of the Updated Pharmacophore for the Alpha 5 GABA(A) Benzodiazepine Receptor Model

An updated model of the GABA(A) benzodiazepine receptor pharmacophore of the α5-BzR/GABA(A) subtype has been constructed prompted by the synthesis of subtype selective ligands in light of the recent developments in both ligand synthesis, behavioral studies, and molecular modeling studies of the binding site itself. A number of BzR/GABA(A) α5 subtype selective compounds were synthesized, notably α5-subtype selective inverse agonist PWZ-029 (1) which is active in enhancing cognition in both rodents and primates. In addition, a chiral positive allosteric modulator (PAM), SH-053-2′F-R-CH3 (2), has been shown to reverse the deleterious effects in the MAM-model of schizophrenia as well as alleviate constriction in airway smooth muscle. Presented here is an updated model of the pharmacophore for α5β2γ2 Bz/GABA(A) receptors, including a rendering of PWZ-029 docked within the α5-binding pocket showing specific interactions of the molecule with the receptor. Differences in the included volume as compared to α1β2γ2, α2β2γ2, and α3β2γ2 will be illustrated for clarity. These new models enhance the ability to understand structural characteristics of ligands which act as agonists, antagonists, or inverse agonists at the Bz BS of GABA(A) receptors.

Cognition-Impairing Effects of Benzodiazepine-Type Drugs: Role of GABAA Receptor Subtypes in an Executive Function Task in Rhesus Monkeys

The present studies evaluated the role of α1 and α5 subunit-containing GABAA receptors (α1GABAA and α5GABAA receptors, respectively) in the ability of benzodiazepine (BZ)-type drugs to alter performance in the cognitive domain of executive function. Five adult female rhesus monkeys (ages of 9-17years old) were trained on the object retrieval with detours (ORD) task of executive function. For the ORD task, the monkeys were required to retrieve food items from a clear box with one open end that was rotated to different positions along with varying placements of food. When the non-selective BZ triazolam and the α1GABAA-preferring agonists zolpidem and zaleplon were evaluated in the ORD task, deficits in performance occurred at doses that did not increase the latency of monkeys to initiate responding and/or increase the percentage of reaches that were incorrect (i.e., reaches in which food was not obtained). Cognition-impairing effects of triazolam and zolpidem in ORD were blocked by the α1GABAA-preferring antagonist, βCCT, whereas the α5GABAA-preferring antagonist XLi-093 blocked the effects of triazolam but not zolpidem. While these findings suggest a role for both α1GABAA and α5GABAA receptor mechanisms, α1GABAA receptor mechanisms appear to be sufficient for impairments in executive function induced by BZ-type drugs.

α5-GABAA receptors negatively regulate MYC-amplified medulloblastoma growth

Neural tumors often express neurotransmitter receptors as markers of their developmental lineage. Although these receptors have been well characterized in electrophysiological, developmental and pharmacological settings, their importance in the maintenance and progression of brain tumors and, importantly, the effect of their targeting in brain cancers remains obscure. Here, we demonstrate high levels of GABRA5, which encodes the α5-subunit of the GABAA receptor complex, in aggressive MYC-driven, “Group 3” medulloblastomas. We hypothesized that modulation of α5-GABAA receptors alters medulloblastoma cell survival and monitored biological and electrophysiological responses of GABRA5-expressing medulloblastoma cells upon pharmacological targeting of the GABAA receptor. While antagonists, inverse agonists and non-specific positive allosteric modulators had limited effects on medulloblastoma cells, a highly specific and potent α5-GABAA receptor agonist, QHii066, resulted in marked membrane depolarization and a significant decrease in cell survival. This effect was GABRA5 dependent and mediated through the induction of apoptosis as well as accumulation of cells in S and G2 phases of the cell cycle. Chemical genomic profiling of QHii066-treated medulloblastoma cells confirmed inhibition of MYC-related transcriptional activity and revealed an enrichment of HOXA5 target gene expression. siRNA-mediated knockdown of HOXA5 markedly blunted the response of medulloblastoma cells to QHii066. Furthermore, QHii066 sensitized GABRA5 positive medulloblastoma cells to radiation and chemotherapy consistent with the role of HOXA5 in directly regulating p53 expression and inducing apoptosis. Thus, our results provide novel insights into the synthetic lethal nature of α5-GABAA receptor activation in MYC-driven/Group 3 medulloblastomas and propose its targeting as a novel strategy for the management of this highly aggressive tumor.

Allosteric Modulation of GABAA Receptor Subtypes: Effects on Visual Recognition and Visuospatial Working Memory in Rhesus Monkeys

Non-selective positive allosteric modulators (PAMs) of GABAA receptors (GABAARs) are known to impair anterograde memory. The role of the various GABAAR subtypes in the memory-impairing effects of non-selective GABAAR PAMs has not been fully elucidated. The current study assessed, in rhesus monkeys, effects of modulation of α1, α2/3, and α5GABAARs on visual recognition and spatial working memory using delayed matching-to-sample (DMTS) and self-ordered spatial search (SOSS) procedures, respectively. The DMTS procedure (n=8) involved selecting a previously presented ‘sample’ image from a set of multiple images presented after a delay. The SOSS procedure (n=6) involved touching a number of boxes without repeats. The non-selective GABAAR PAM triazolam and the α1GABAA preferential PAMS zolpidem and zaleplon reduced accuracy in both procedures, whereas the α5GABAA preferential PAMs SH-053-2′F-R-CH3 and SH-053-2′F-S-CH3, and the α2/3GABAA preferential PAM TPA023B were without effects on accuracy or trial completion. The low-efficacy α5GABAAR negative allosteric modulator (NAM) PWZ-029 slightly increased only DMTS accuracy, whereas the high-efficacy α5GABAAR NAMs RY-23 and RY-24 did not affect accuracy under either procedure. Finally, the slopes of the accuracy dose-effect curves for triazolam, zolpidem, and zaleplon increased with box number in the SOSS procedure, but were equivalent across DMTS delays. The present results suggest that (1) α1GABAARs, compared with α2/3 and α5GABAARs, are primarily involved in the impairment, by non-selective GABAAR PAMs, of visual recognition and visuospatial working memory in nonhuman primates; and (2) relative cognitive impairment produced by positive modulation of GABAARs increases with number of locations to be remembered, but not with the delay for remembering.