Ciaran M. Regan

Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory.

Alzheimers disease constitutes a rising threat to public health. Despite extensive research in cellular and animal models, identifying the pathogenic agent present in the human brain and showing that it confers key features of Alzheimers disease has not been achieved. We extracted soluble amyloid-β protein (Aβ) oligomers directly from the cerebral cortex of subjects with Alzheimers disease. The oligomers potently inhibited long-term potentiation (LTP), enhanced long-term depression (LTD) and reduced dendritic spine density in normal rodent hippocampus. Soluble Aβ from Alzheimers disease brain also disrupted the memory of a learned behavior in normal rats. These various effects were specifically attributable to Aβ dimers. Mechanistically, metabotropic glutamate receptors were required for the LTD enhancement, and N-methyl D-aspartate receptors were required for the spine loss. Co-administering antibodies to the Aβ N-terminus prevented the LTP and LTD deficits, whereas antibodies to the midregion or C-terminus were less effective. Insoluble amyloid plaque cores from Alzheimers disease cortex did not impair LTP unless they were first solubilized to release Aβ dimers, suggesting that plaque cores are largely inactive but sequester Aβ dimers that are synaptotoxic. We conclude that soluble Aβ oligomers extracted from Alzheimers disease brains potently impair synapse structure and function and that dimers are the smallest synaptotoxic species.

GSK189254, a Novel H3 Receptor Antagonist That Binds to Histamine H3 Receptors in Alzheimer's Disease Brain and Improves Cognitive Performance in Preclinical Models

6-[(3-Cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-3-pyridinecarboxamide hydrochloride (GSK189254) is a novel histamine H3 receptor antagonist with high affinity for human (pKi = 9.59 –9.90) and rat (pKi = 8.51–9.17) H3 receptors. GSK189254 is >10,000-fold selective for human H3 receptors versus other targets tested, and it exhibited potent functional antagonism (pA2 = 9.06 versus agonist-induced changes in cAMP) and inverse agonism [pIC50 = 8.20 versus basal guanosine 5′-O-(3-[35S]thio)triphosphate binding] at the human recombinant H3 receptor. In vitro autoradiography demonstrated specific [3H]GSK189254 binding in rat and human brain areas, including cortex and hippocampus. In addition, dense H3 binding was detected in medial temporal cortex samples from severe cases of Alzheimers disease, suggesting for the first time that H3 receptors are preserved in late-stage disease. After oral administration, GSK189254 inhibited cortical ex vivo R-(–)-α-methyl[imidazole-2,5(n)-3H]histamine dihydrochloride ([3H]R-α-methylhistamine) binding (ED50 = 0.17 mg/kg) and increased c-Fos immunoreactivity in prefrontal and somatosensory cortex (3 mg/kg). Microdialysis studies demonstrated that GSK189254 (0.3–3 mg/kg p.o.) increased the release of acetylcholine, noradrenaline, and dopamine in the anterior cingulate cortex and acetylcholine in the dorsal hippocampus. Functional antagonism of central H3 receptors was demonstrated by blockade of R-α-methylhistamine-induced dipsogenia in rats (ID50 = 0.03 mg/kg p.o.). GSK189254 significantly improved performance of rats in diverse cognition paradigms, including passive avoidance (1 and 3 mg/kg p.o.), water maze (1 and 3 mg/kg p.o.), object recognition (0.3 and 1 mg/kg p.o.), and attentional set shift (1 mg/kg p.o.). These data suggest that GSK189254 may have therapeutic potential for the symptomatic treatment of dementia in Alzheimers disease and other cognitive disorders.

Intraventricular infusions of anti-neural cell adhesion molecules in a discrete posttraining period impair consolidation of a passive avoidance response in the rat.

Abstract: Intraventricular infusions of anti‐neural cell adhesion molecule (anti‐NCAM) are demonstrated to inhibit consolidation of a passive avoidance response when administered in the 6‐8‐h posttraining period. Anti‐NCAM was ineffective when administered during training or at any other time up to 10 h thereafter, and no amnesic effects were observed with absorbed anti‐NCAM or anti‐neurofilament protein. Amnesia was observed only at the 48‐h recall time, and this could not be attributed to poor antibody penetration or a prolonged residence time, as studies with 125I‐labelled anti‐NCAM in trained animals demonstrated a rapid accumulation into all brain regions, and this was marked in the olfactory bulb and hippocampus, areas showing an inherent and paradigm‐specific increase in NCAM sialylation state, respectively. The lack of an amnesic action at the 24‐h recall time is attributed to anti‐NCAM‐impaired synapse structuring becoming apparent following the paradigm‐specific increases in NCAM sialylation state.

The 5-HT(6) receptor antagonist SB-271046 reverses scopolamine-disrupted consolidation of a passive avoidance task and ameliorates spatial task deficits in aged rats.

The highly potent and selective 5-HT6 receptor antagonist SB-271046 [5-chloro-N-(4-methoxy-3-piperazin-1-yl-phenyl)-3-methyl-2-benzothiophenesulfonamide] has previously been demonstrated to improve retention significantly in a spatial water maze paradigm in adult rats. However, SB-271046 did not have any effect on task acquisition. As these apparently contradictory findings may be reconciled by a prime influence of SB-271046 on memory consolidation, the ability of this compound to reverse the discrete temporal action of a cholinergic antagonist in the 6-h period following passive avoidance training was investigated. SB-271046, given orally, by gavage, 30 min prior to training Wistar rats in a step-through, light–dark passive avoidance task, was found to reverse significantly the amnesia produced by administering scopolamine (0.8 mg/kg, intraperitoneal) in the 6-h post-training period. The effect was dose-dependent over a range of 3–20 mg/kg. Further, we investigated the cognition-enhancing effects of chronic SB-271046 administration (10 or 20 mg/kg/day; 40 days) on the acquisition and consolidation of a water maze spatial learning task in a population of 20-month-old Wistar rats with age-related learning deficits. Drug treatment progressively and significantly decreased platform swim angle and escape latencies over the five sequential trials on four consecutive daily sessions compared to vehicle-treated controls. SB-271046 also improved task recall as measured by significant increases in the searching of the target quadrant on post-training days 1 and 3, when the animals would have been substantially drug-free. This significant improvement of task recall suggests SB-271046, in addition to inducing symptomatic cognition-enhancing actions, also attenuates age-related decline in neural function.

Repetitive and Transient Increases in Hippocampal Neural Cell Adhesion Molecule Polysialylation State Following Multitrial Spatial Training

Abstract: Polysialylated neurons, located at the inner border of the dentate granule cell layer, have been demonstrated to exhibit time‐dependent change in their frequency at 10–12 h following training in the Morris water maze, a spatial learning paradigm. Such a change was not observed in animals required to locate a visible platform or in those rendered amnesic with scopolamine. This frequency response was capable of rapid reactivation following further training stimuli in a manner that was independent of circadian influence. These learning‐associated modulations in neural cell adhesion molecule (NCAM) polysialylation state did not increase in magnitude despite improved performance, suggesting their activation is required for processing information rather than contributing to previously stored, task‐associated memory. An increase in NCAM polysialylation appears to be a universal learning response to both spatial and nonspatial paradigms as similar time‐dependent changes occurred following training in a one‐trial, step‐through, passive avoidance response subsequent to water maze training.

Memory consolidation induces a transient and time-dependent increase in the frequency of neural cell adhesion molecule polysialylated cells in the adult rat hippocampus.

Abstract: Animals trained in a passive avoidance task exhibit a transient time‐dependent increase in hippocampal neural cell adhesion molecule (NCAM) polysialylation at 12–24 h following the initial learning trial. Using immunocytochemical techniques with a monoclonal antibody that specifically recognises NCAM‐polysialic acid homopolymers, a distinct population of granule‐like cells, at the border of the granule cell layer and the hilus in the dentate gyrus of the adult rat hippocampus, has been demonstrated to exhibit time‐dependent change in frequency at 10–12 h following the initial learning of a one‐trial, step‐through, passive avoidance response. These changes were paradigm specific as they failed to occur in those animals rendered amnesic with scopolamine. These polysialylated dentate neurons are not de novo granule cell precursors as administration of 5‐bromo‐2′‐deoxyuridine every 2 h from the point of learning to the 12‐h posttraining time showed no significant difference between trained and passive animals in the small number of heterogeneously distributed, labelled cells. These findings directly identify a morphological substrate of memory, implied by previous correlative and interventive studies on NCAM function.

Ultrastructural analysis reveals avoidance conditioning to induce a transient increase in hippocampal dentate spine density in the 6 hour post-training period of consolidation.

Concepts underlying memory consolidation invoke change in synapse structure and function. Such concepts relate to change in connectivity pattern enabled by increased synapse number, change in synaptic configuration resulting from overproduction and selective pruning, or structural change in synapse transmission zones. This study undertook the unbiased estimation of learning associated change in dendritic spine number on granule cells in the hippocampal dentate gyrus. Rats were trained to acquire a passive avoidance response after which spine number in the mid-molecular layer of the dorsal dentate gyrus were estimated at increasing post-training times. This showed there to be an increase in spine density with time after training which was initiated at 3 h, and maximal at 6 h. The increase at this latter time was not detected in passive control animals. At 72 h post-training spine density was seen to return to basal levels. These results are consistent with the various models for synapse connectivity change in memory formation whether they relate to altered number or connectivity pattern.

Intraventricular infusions of antibodies to amyloid-β-protein precursor impair the acquisition of a passive avoidance response in the rat

Intraventricular infusions of an antiserum raised against a 14 amino acid residue in the extracellular domain of amyloid-beta-protein precursor significantly decreased stepdown latency, at both 24 h and 48 h recall times, in rats trained to avoid an electroshock by remaining on a platform. The antiserum was effective when infused up to 2.5 h following training and no retention deficit was noted when it was administered at 4h or 6h after training. An antiserum generated against a 17 amino acid residue of the A4 amyloid peptide had no effect on learning. Thus the amyloid precursor protein, which is aberrantly processed in Alzheimers disease, appears to be directly involved in memory formation.

A Synthetic Peptide Ligand of Neural Cell Adhesion Molecule (NCAM) IgI Domain Prevents NCAM Internalization and Disrupts Passive Avoidance Learning

Abstract: The neural cell adhesion molecule (NCAM) mediates cell adhesion and signal transduction through trans‐homophilic‐ and/or cis‐heterophilic‐binding mechanisms. Intraventricular infusions of anti‐NCAM have revealed a functional requirement of NCAM for the consolidation of memory in rats and chicks in a specific interval 6‐8 h after training. We have now extended these studies to a synthetic peptide ligand of NCAM (C3) with an affinity for the IgI domain and the capability of inhibiting NCAM‐mediated neurite outgrowth in vitro. Intraventricular administration of a single 5 μg bolus of C3 strongly inhibited recall of a passive avoidance response in adult rats, when given during training or in the 6‐8‐h posttraining period. The effect of C3 on memory consolidation was similar to that obtained with anti‐NCAM as the amnesia was not observed until the 48‐h recall time. The unique amnesic action of C3 during training could be related to disrupted NCAM internalization following training. In the 3‐4‐h posttraining period NCAM 180, the synapse‐associated isoform, was down‐regulated in the hippocampal dentate gyrus. This effect was mediated by ubiquitination and was prevented by C3 administration during training. These findings indicate NCAM to be involved in both the acquisition and consolidation of a passive avoidance response in the rat. Moreover, the study provides the first in vivo evidence for NCAM internalization in learning and identifies a synthetic NCAM ligand capable of modulating memory processes in vivo.

Spatial learning activates neural cell adhesion molecule polysialylation in a corticohippocampal pathway within the medial temporal lobe

Abstract: Transient and time‐dependent modulations of neural cell adhesion molecule (NCAM) polysialylation in the dentate gyrus of the rodent hippocampus are a feature of spatial and nonspatial forms of learning. In the hippocampal formation, polysialic acid immunoreactivity was localized to granule‐like cells and their mossy fibre axons. We now demonstrate the latter to extend to the CA3 region where apparent recurrent and Schaffer collaterals were labelled. The axons of the CA1 pyramidal cell layer were immunopositive, as was the subiculum that they innervate. Layers I and III of the entorhinal cortex stained intensely for polysialic acid; however, these were not visible in the more lateral aspect of this region and were replaced by a single band of immunopositive neurons that extended to include the perirhinal and piriform cortices. After Morris water maze training, the number of polysialylated neurons within the entorhinal cortex exhibited a two‐ to threefold increase at the 10–12‐h posttraining time with respect to that observed immediately after training. This increase was task specific, as no change was observed in freely swimming animals or those required to locate a visible platform. These results suggest the presence of a corticohippocampal pathway involved in the eventual consolidation of memory.