Guy R. Seabrook

APP Processing and Synaptic Function

A large body of evidence has implicated Abeta peptides and other derivatives of the amyloid precursor protein (APP) as central to the pathogenesis of Alzheimers disease (AD). However, the functional relationship of APP and its proteolytic derivatives to neuronal electrophysiology is not known. Here, we show that neuronal activity modulates the formation and secretion of Abeta peptides in hippocampal slice neurons that overexpress APP. In turn, Abeta selectively depresses excitatory synaptic transmission onto neurons that overexpress APP, as well as nearby neurons that do not. This depression depends on NMDA-R activity and can be reversed by blockade of neuronal activity. Synaptic depression from excessive Abeta could contribute to cognitive decline during early AD. In addition, we propose that activity-dependent modulation of endogenous Abeta production may normally participate in a negative feedback that could keep neuronal hyperactivity in check. Disruption of this feedback system could contribute to disease progression in AD.

Age-related cognitive deficits, impaired long-term potentiation and reduction in synaptic marker density in mice lacking the β-amyloid precursor protein

Mutations in the beta-amyloid precursor protein are strongly associated with some cases of familial Alzheimers disease. The normal physiological role of beta-amyloid precursor protein in the brain was evaluated in a cross-sectional analysis of mice deficient in beta-amyloid precursor protein. Compared with wild-type control mice the beta-amyloid precursor protein-null mice developed age-dependent deficits in cognitive function and also had impairments in long-term potentiation. In addition, the brains of the beta-amyloid precursor protein-null mice had marked reactive gliosis in many areas, especially in the cortex and hippocampus. A subpopulation of mice (n = 15) died prematurely (between three and 18 months of age). Analysis of another six mice from the same population that were showing weight loss and hypolocomotor activity exhibited a marked reactive gliosis as detected by immunoreactivity for glial fibrillary acidic protein and a profound loss of immunoreactivities for the presynaptic terminal vesicle marker proteins synaptophysin and synapsin and the dendritic marker microtubule-associated protein-2 in many brain areas, but most predominantly in the cortex and hippocampus. These results suggest that normal beta-amyloid precursor protein may serve an essential role in the maintenance of synaptic function during ageing. A compromise of this function of the beta-amyloid precursor protein may contribute to the progression of the memory decline and the neurodegenerative changes seen in Alzheimers disease.

An inverse agonist selective for alpha5 subunit-containing GABAA receptors enhances cognition.

α5IA is a compound that binds with equivalent subnanomolar affinity to the benzodiazepine (BZ) site of GABAA receptors containing an α1, α2, α3, or α5 subunit but has inverse agonist efficacy selective for the α5 subtype. As a consequence, the in vitro and in vivo effects of this compound are mediated primarily via GABAA receptors containing an α5 subunit. In a mouse hippocampal slice model, α5IA significantly enhanced the θ burst-induced long-term potentiation of the excitatory postsynaptic potential in the CA1 region but did not cause an increase in the paroxysmal burst discharges that are characteristic of convulsant and proconvulsant drugs. These in vitro data suggesting that α5IA may enhance cognition without being proconvulsant were confirmed in in vivo rodent models. Hence, α5IA significantly enhanced performance in a rat hippocampal-dependent test of learning and memory, the delayed-matching-to-position version of the Morris water maze, with a minimum effective oral dose of 0.3 mg/kg, which corresponded to a BZ site occupancy of 25%. However, in mice α5IA was not convulsant in its own right nor did it potentiate the effects of pentylenetetrazole acutely or produce kindling upon chronic dosing even at doses producing greater than 90% occupancy. Finally, α5IA was not anxiogenic-like in the rat elevated plus maze nor did it impair performance in the mouse rotarod assay. Together, these data suggest that the GABAA α5-subtype provides a novel target for the development of selective inverse agonists with utility in the treatment of disorders associated with a cognitive deficit.

Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation

The forebrain cholinergic system promotes higher brain function in part by signaling through the M1 muscarinic acetylcholine receptor (mAChR). During Alzheimers disease (AD), these cholinergic neurons degenerate, therefore selectively activating M1 receptors could improve cognitive function in these patients while avoiding unwanted peripheral responses associated with non-selective muscarinic agonists. We describe here benzyl quinolone carboxylic acid (BQCA), a highly selective allosteric potentiator of the M1 mAChR. BQCA reduces the concentration of ACh required to activate M1 up to 129-fold with an inflection point value of 845 nM. No potentiation, agonism, or antagonism activity on other mAChRs is observed up to 100 μM. Furthermore studies in M1−/− mice demonstrates that BQCA requires M1 to promote inositol phosphate turnover in primary neurons and to increase c-fos and arc RNA expression and ERK phosphorylation in the brain. Radioligand-binding assays, molecular modeling, and site-directed mutagenesis experiments indicate that BQCA acts at an allosteric site involving residues Y179 and W400. BQCA reverses scopolamine-induced memory deficits in contextual fear conditioning, increases blood flow to the cerebral cortex, and increases wakefulness while reducing delta sleep. In contrast to M1 allosteric agonists, which do not improve memory in scopolamine-challenged mice in contextual fear conditioning, BQCA induces β-arrestin recruitment to M1, suggesting a role for this signal transduction mechanism in the cholinergic modulation of memory. In summary, BQCA exploits an allosteric potentiation mechanism to provide selectivity for the M1 receptor and represents a promising therapeutic strategy for cognitive disorders.

L-655,708 enhances cognition in rats but is not proconvulsant at a dose selective for α5-containing GABAA receptors

The in vitro and in vivo properties of L-655,708, a compound with higher affinity for GABA(A) receptors containing an alpha5 compared to an alpha1, alpha2 or alpha3 subunit have been examined further. This compound has weak partial inverse agonist efficacy at each of the four subtypes but, and consistent with the binding data, has higher functional affinity for the alpha5 subtype. In a mouse hippocampal slice model, L-655,708 was able to enhance the long-term potentiation produced by a theta burst stimulation, consistent with a potential role for the alpha5 subtype in processes involving synaptic plasticity, such as learning and memory. When administered in a formulation specifically designed to achieve relatively constant plasma drug concentrations, and therefore maintain selective occupancy of alpha5- compared to alpha1-, alpha2- and alpha3-containing receptors (75+/-4% versus 22+/-10%, respectively), L-655,708 did not alter the dose of pentylenetetrazole required to induce seizures, indicating that the inverse agonist effects of L-655,708 at the alpha5 subtype are not associated with a proconvulsant liability. In the Morris water maze, L-655,708 enhanced performance not only during acquisition but also in a probe trial, demonstrating that this compound has cognition enhancing effects. These data further support the potential of alpha5-containing GABA(A) receptors as a target for novel cognition enhancing drugs.

Functional Properties of the High-Affinity TRPV1 (VR1) Vanilloid Receptor Antagonist (4-Hydroxy-5-iodo-3-methoxyphenylacetate ester) Iodo-Resiniferatoxin

We have synthesized iodinated resiniferatoxin bearing a 4-hydroxy-5-iodo-3-methoxyphenylacetate ester (I-RTX) and have characterized its activity on rat and human TRPV1 (VR1) receptors, as well as in behavioral assays of nociception. In whole cell patch-clamp recordings from transfected cells the functional activity of I-RTX was determined. Currents activated by capsaicin exhibited characteristic outward rectification and were antagonized by capsazepine and I-RTX. On rat TRPV1 the affinity of I-RTX was 800-fold higher than that of capsazepine (IC50 = 0.7 and 562 nM, respectively) and 10-fold higher on rat versus human receptors (IC50 = 0.7 and 5.4 nM, respectively). The same difference was observed when comparing the inhibition of [3H]RTX binding to rat and human TRPV1 membranes for both RTX and I-RTX. Additional pharmacological differences were revealed using protons as the stimulus. Under these conditions capsazepine only partly blocked currents through rat TRPV1 receptors (by 70 to 80% block), yet was a full antagonist on human receptors. In contrast, I-RTX completely blocked proton-induced currents in both species and that activated by noxious heat. I-RTX also blocked capsaicin-induced firing of C-fibers in a rat in vitro skin-nerve assay. Despite this activity and the high affinity of I-RTX for rat TRPV1, only capsazepine proved to be an effective antagonist of capsaicin-induced paw flinching in rats. Thus, although I-RTX has limited utility for in vivo behavioral studies it is a high-affinity TRPV1 receptor antagonist that will be useful to characterize the functional properties of cloned and native vanilloid receptor subtypes in vitro.

First Demonstration of Cerebrospinal Fluid and Plasma Aβ Lowering with Oral Administration of a β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Inhibitor in Nonhuman Primates

β-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic Aβ42 peptides in Alzheimers disease. Although previous mouse studies have shown brain Aβ lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of Aβ in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC50 ∼ 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain Aβ levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma Aβ levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPPβ, Aβ40, Aβ42, and plasma Aβ40 levels. CSF Aβ42 lowering showed an EC50 of ∼20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF Aβ lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

Schizophrenia and L-745, 870, a novel dopamine D4 receptor antagonist

The discovery of a novel high-affinity and selective dopamine D4 receptor antagonist, L-745,870, and the results of clinical trials with this compound are reviewed. Despite several lines of evidence which suggest that a selective D4 receptor antagonist may be an effective antipsychotic agent with a lower propensity to induce extrapyramidal side-effects, L-745,870 was ineffective as an antipsychotic in humans.

Antinociceptive activity of NK1 receptor antagonists: non-specific effects of racemic RP67580

1 Release of substance P in the dorsal horn is considered a primary event in the perception of pain. The profile of racemic RP67580, a non‐peptide antagonist at the NK1 (substance P) receptor, was examined in a range of antinociception tests on rodents. 2 At doses up to 30 mg kg−1, s.c. racemic RP67580 exhibited antinociceptive activity in writhing and formalin paw tests in mice and gerbils. Acetic acid induced writhing and the licking response to formalin were reduced to 40–50% of the level observed in vehicle‐treated animals (P < 0.05). However, this agent was not active in mouse tail flick, rat paw pressure or rat and guinea‐pig formalin paw tests. 3 Like racemic RP67580, the calcium channel blockers nifedipine (30 mg kg−1, i.p.) and verapamil (10 or 20 mg kg−1, s.c.) inhibited the response to formalin by approximately 60% in gerbils (P < 0.05 compared with vehicle‐treated animals). 4 Evidence for calcium channel antagonist activity of RP67580 was obtained in vitro. Racemic RP67580 inhibited calcium entry into depolarized strips of guinea‐pig ileum longitudinal muscle myenteric plexus (apparent KB = 587 ± 115 nm), inhibited [3H]‐diltiazem binding to rabbit skeletal membranes (IC50 = 298 nm) and depressed high threshold calcium currents in neurones cultured from rat cortex (10% inhibition at 10 μm). 5 These findings indicate that the acute antinociceptive effects of RP67580 may not be attributable to a specific interaction with NK1 receptors and may be mediated via calcium channel blockade.

LRRTM3 promotes processing of amyloid-precursor protein by BACE1 and is a positional candidate gene for late-onset Alzheimer's disease

Rare familial forms of Alzheimers disease (AD) are thought to be caused by elevated proteolytic production of the Aβ42 peptide from the β-amyloid-precursor protein (APP). Although the pathogenesis of the more common late-onset AD (LOAD) is not understood, BACE1, the protease that cleaves APP to generate the N terminus of Aβ42, is more active in patients with LOAD, suggesting that increased amyloid production processing might also contribute to the sporadic disease. Using high-throughput siRNA screening technology, we assessed 15,200 genes for their role in Aβ42 secretion and identified leucine-rich repeat transmembrane 3 (LRRTM3) as a neuronal gene that promotes APP processing by BACE1. siRNAs targeting LRRTM3 inhibit the secretion of Aβ40, Aβ42, and sAPPβ, the N-terminal APP fragment produced by BACE1 cleavage, from cultured cells and primary neurons by up to 60%, whereas overexpression increases Aβ secretion. LRRTM3 is expressed nearly exclusively in the nervous system, including regions affected during AD, such as the dentate gyrus. Furthermore, LRRTM3 maps to a region of chromosome 10 linked to both LOAD and elevated plasma Aβ42, and is structurally similar to a family of neuronal receptors that includes the NOGO receptor, an inhibitor of neuronal regeneration and APP processing. Thus, LRRTM3 is a functional and positional candidate gene for AD, and, given its receptor-like structure and restricted expression, a potential therapeutic target.