The Abeta1-42M35C mutated amyloid peptide Abeta1-42 and the 25-35 fragment fail to mimic the subtype-specificity of actions on recombinant human nicotinic acetylcholine receptors (alpha7, alpha4beta2, alpha3beta4).

Alzheimer's disease (AD) is a neurodegenerative condition involving accumulation of the beta-amyloid peptide, Abeta1-42. Previously we have shown that amyloid peptides (Abeta1-42, Abeta1-40) have different actions on the three major brain nicotinic acetylcholine receptor (nAChR) subtypes (alpha7, alpha4beta2 and alpha3beta4). The methionine in position 35 of Abeta (M35) has been shown to be important in the toxicity of Abeta and the 25-35 fragment can mimic some of the actions of the Abeta1-42 peptide. However, the extent to which this mutant and the fragment mimic subtype selectivity is unknown. Two-electrode voltage-clamp electrophysiology has been used to study the actions on alpha7, alpha4beta2 and alpha3beta4 recombinant nAChRs expressed in Xenopus laevis oocytes of full length Abeta1-42, and Abeta peptide fragments, scrambled peptides, and the Abeta1-42 peptide containing mutations of the methionine in position 35. The Abeta25-35 fragment did not display subunit specificity. Abeta1-42 with an M35C mutation showed similar subtype-specificity to wild-type Abeta1-42. However, Abeta1-42 with an M35V substitution reduced the peak amplitude of ACh-induced currents recorded from alpha4beta2 nAChRs, but did not affect those recorded from alpha7 or alpha3beta4. These results indicate that the amino acid in position 35 of Abeta1-42 is an important determinant of the subtype-specificity of this peptide on human recombinant alpha7, alpha4beta2 and alpha3beta4 nAChRs and that the 25-35 fragment fails to mimic all of the actions of the full-length peptide.

Contribution of specific binding to the central benzodiazepine site to the brain concentrations of two novel benzodiazepine site ligands.

The in vivo occupancy of brain benzodiazepine binding sites by compounds A and B was measured using a [(3)H]Ro 15-1788 binding assay and related to plasma and brain drug concentrations. The plasma concentration associated with 50% occupancy was higher for compound A than compound B (73 and 3.7 nM, respectively), however, there was little difference in the brain concentrations required (73 and 63 nM). Both compounds showed a non-linear relationship between plasma and brain concentrations such that above brain concentrations of approximately 100 nM increasing plasma concentrations did not result in a concomitant increase in brain concentrations. This is consistent with brain concentrations being dependent on a saturable compartment which was postulated to be the benzodiazepine binding site-containing GABA(A) receptors. This hypothesis was tested in alpha1H101R mice, in which the alpha1 subunit of the GABA(A) receptor is rendered insensitive to benzodiazepine binding resulting in an approximate 50% reduction in the total benzodiazepine-containing GABA(A) receptor population. It was shown that the Occ(50) brain concentrations in the alpha1H101R animals was lower (17 nM) than in wild type mice (63 nM), as was the plateau concentration in the brain (105 and 195 nM, respectively). These data suggest measured concentrations of compounds A and B in brain tissue are dependent on receptor expression with a minimal contribution from unbound and non-specifically bound compound.

Selective labelling of diazepam-insensitive GABAA receptors in vivo using [3H]Ro 15-4513.

Classical benzodiazepines (BZs), such as diazepam, bind to GABAA receptors containing alpha1, alpha2, alpha3 or alpha5 subunits that are therefore described as diazepam-sensitive (DS) receptors. However, the corresponding binding site of GABAA receptors containing either an alpha4 or alpha6 subunit do not bind the classical BZs and are therefore diazepam-insensitive (DIS) receptors; a difference attributable to a single amino acid (histidine in alpha1, alpha2, alpha3 and alpha5 subunits and arginine in alpha4 and alpha6). Unlike classical BZs, the imidazobenzodiazepines Ro 15-4513 and bretazenil bind to both DS and DIS populations of GABAA receptors. In the present study, an in vivo assay was developed using lorazepam to fully occupy DS receptors such that [3H]Ro 15-4513 was then only able to bind to DIS receptors. When dosed i.v., [3H]Ro 15-4513 rapidly entered and was cleared from the brain, with approximately 70% of brain radioactivity being membrane-bound. Essentially all membrane binding to DS+DIS receptors could be displaced by unlabelled Ro 15-4513 or bretazenil, with respective ID50 values of 0.35 and 1.2 mg kg(-1). A dose of 30 mg kg(-1) lorazepam was used to block all DS receptors in a [3H]Ro 15-1788 in vivo binding assay. When predosed in a [3H]Ro 15-4513 binding assay, lorazepam blocked [3H]Ro 15-4513 binding to DS receptors, with the remaining binding to DIS receptors accounting for 5 and 23% of the total (DS plus DIS) receptors in the forebrain and cerebellum, respectively. The in vivo binding of [3H]Ro 15-4513 to DIS receptors in the presence of lorazepam was confirmed using alpha1H101R knock-in mice, in which alpha1-containing GABAA receptors are rendered diazepam insensitive by mutation of the histidine that confers diazepam sensitivity to arginine. In these mice, and in the presence of lorazepam, there was an increase of in vivo [3H]Ro 15-4513 binding in the forebrain and cerebellum from 4 and 15% to 36 and 59% of the total (i.e. DS plus DIS) [3H]Ro 15-4513 binding observed in the absence of lorazepam.