J. Cleary

Natural oligomers of the amyloid-β protein specifically disrupt cognitive function

A central unresolved problem in research on Alzheimer disease is the nature of the molecular entity causing dementia. Here we provide the first direct experimental evidence that a defined molecular species of the amyloid-β protein interferes with cognitive function. Soluble oligomeric forms of amyloid-β, including trimers and dimers, were both necessary and sufficient to disrupt learned behavior in a manner that was rapid, potent and transient; they produced impaired cognitive function without inducing permanent neurological deficits. Although β-amyloidosis has long been hypothesized to affect cognition, the abnormally folded protein species associated with this or any other neurodegenerative disease has not previously been isolated, defined biochemically and then specifically characterized with regard to its effects on cognitive function. The biochemical isolation of discrete amyloid-β moieties with pathophysiological properties sets the stage for a new approach to studying the molecular mechanisms of cognitive impairment in Alzheimer disease and related neurodegenerative disorders.

The role of cell-derived oligomers of Aβ in Alzheimer's disease and avenues for therapeutic intervention

Burgeoning evidence suggests that soluble oligomers of Abeta (amyloid beta-protein) are the earliest effectors of synaptic compromise in Alzheimers disease. Whereas most other investigators have employed synthetic Abeta peptides, we have taken advantage of a beta-amyloid precursor protein-overexpressing cell line (referred to as 7PA2) that secretes sub-nanomolar levels of low-n oligomers of Abeta. These are composed of heterogeneous Abeta peptides that migrate on SDS/PAGE as dimers, trimers and tetramers. When injected into the lateral ventricle of rats in vivo, these soluble oligomers inhibit hippocampal long-term potentiation and alter the memory of a complex learned behaviour. Biochemical manipulation of 7PA2 medium including immunodepletion with Abeta-specific antibodies and fractionation by size-exclusion chromatography allowed us to unambiguously attribute these effects to low-n oligomers. Using this paradigm we have tested compounds directed at three prominent amyloid-based therapeutic targets: inhibition of the secretases responsible for Abeta production, inhibition of Abeta aggregation and immunization against Abeta. In each case, compounds capable of reducing oligomer production or antibodies that avidly bind Abeta oligomers also ameliorate the synaptotoxic effects of these natural, cell-derived oligomers.

Cyclooxygenase-2 inhibition improves amyloid-β-mediated suppression of memory and synaptic plasticity

Non-steroidal anti-inflammatory agents (NSAIDs) are associated with a marked reduction in the risk of developing Alzheimers disease, a form of dementia characterized by the accumulation of amyloid plaques containing the amyloid-beta protein (Abeta). Studies of the effects of NSAIDs upon the inflammatory response surrounding amyloid plaques and upon the generation of Abeta from the amyloid precursor protein (APP) have led to two proposed mechanisms by which NSAIDs may protect against Alzheimers disease: one, the selective lowering of Abeta42 by a subset of NSAIDs; and two, the reduction of inflammation. Although Alzheimers disease is a disorder of brain and synaptic function, the effects of NSAIDs on Abeta-mediated suppression of synaptic plasticity and memory function have never been reported. We therefore investigated how three different NSAIDs, chosen for their distinct effects on Abeta42 production and the inhibition of the cyclooxygenase (COX) isoenzymes, COX-1 and COX-2, affect memory function and synaptic plasticity. By focusing upon brain and synapse function, we made novel observations about the effects of NSAIDs on Abeta-mediated neural processes. Here we report that the selective inhibition of COX-2, but not COX-1, acutely prevented the suppression of hippocampal long-term plasticity (LTP) by Abeta. The non-selective NSAIDs, ibuprofen and naproxen, and a selective COX-2 inhibitor, MF-tricyclic, each restored memory function in Tg2576 mice over-expressing APP, and also blocked Abeta-mediated inhibition of LTP. There was no advantage of ibuprofen, a selective Abeta42-lowering agent (SALA), over the non-SALAs, naproxen and MF-tricyclic. The beneficial effects on memory did not depend upon lowered levels of Abeta42 or the inflammatory cytokines, tumour necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta). Intriguingly, improved memory function was inversely related to prostaglandin E2 (PGE2) levels. Conversely, exogenous PGE2 prevented the restorative effects of COX-2 inhibitors on LTP. The data indicate that the inhibition of COX-2 blocks Abeta-mediated suppression of LTP and memory function, and that this block occurs independently of reductions in Abeta42 or decreases in inflammation. The results lead us to propose a third possible mechanism by which NSAIDs may protect against Alzheimers disease, involving the blockade of a COX-2-mediated PGE2 response at synapses.

Orally available compound prevents deficits in memory caused by the alzheimer amyloid-β oligomers

Despite progress in defining a pathogenic role for amyloid β protein (Aβ) in Alzheimers disease, orally bioavailable compounds that prevent its effects on hippocampal synaptic plasticity and cognitive function have not yet emerged. A particularly attractive therapeutic strategy is to selectively neutralize small, soluble Aβ oligomers that have recently been shown to mediate synaptic dysfunction.

Cognitive effects of cell-derived and synthetically derived Aβ oligomers.

Soluble forms of amyloid-β peptide (Aβ) are a molecular focus in Alzheimers disease research. Soluble Aβ dimers (≈8 kDa), trimers (≈12 kDa), tetramers (≈16 kDa) and Aβ*56 (≈56 kDa) have shown biological activity. These Aβ molecules have been derived from diverse sources, including chemical synthesis, transfected cells, and mouse and human brain, leading to uncertainty about toxicity and potency. Herein, synthetic Aβ peptide-derived oligomers, cell- and brain-derived low-n oligomers, and Aβ*56, were injected intracerebroventricularly (icv) into rats assayed under the Alternating Lever Cyclic Ratio (ALCR) cognitive assay. Cognitive deficits were detected at 1.3 μM of synthetic Aβ oligomers and at low nanomolar concentrations of cell-secreted Aβ oligomers. Trimers, from transgenic mouse brain (Tg2576), did not cause cognitive impairment at any dose tested, whereas Aβ*56 induced concentration-dependent cognitive impairment at 0.9 and 1.3μM. Thus, while multiple forms of Aβ have cognition impairing activity, there are significant differences in effective concentration and potency.

Amyloid β‐Peptides Increase Annular and Bulk Fluidity and Induce Lipid Peroxidation in Brain Synaptic Plasma Membranes

Abstract: Amyloid β‐peptides (Aβ) may alter the neuronal membrane lipid environment by changing fluidity and inducing free radical lipid peroxidation. The effects of Aβ1–40 and Aβ25–35 on the fluidity of lipids adjacent to proteins (annular fluidity), bulk lipid fluidity, and lipid peroxidation were determined in rat synaptic plasma membranes (SPM). A fluorescent method based on radiationless energy transfer from tryptophan of SPM proteins to pyrene and pyrene monomer‐eximer formation was used to determine SPM annular fluidity and bulk fluidity, respectively. Lipid peroxidation was determined by the thiobarbituric acid assay. Annular fluidity and bulk fluidity of SPM were increased significantly (p≤ 0.02) by Aβ1–40. Similar effects on fluidity were observed for Aβ25–35 (p≤ 0.002). Increased fluidity was associated with lipid peroxidation. Both Aβ peptides significantly increased (p≤ 0.006) the amount of malondialdehyde in SPM. The addition of a water‐soluble analogue of vitamin E (Trolox) inhibited effects of Aβ on lipid peroxidation and fluidity in SPM. The fluidizing action of Aβ peptides on SPM may be due to the induction of lipid peroxidation by those peptides. Aβ‐induced changes in neuronal function, such as ion flux and enzyme activity, that have been reported previously may result from the combined effects of lipid peroxidation and increased membrane fluidity.

Beta-amyloid(1–40) effects on behavior and memory

Beta amyloid 1-40 is a primary protein in plaques found in the brains of patients with Alzheimers disease. There is evidence that unaggregated soluble beta-amyloid may be neurotoxic and may have behavioral effects on some types of memory. In the current study, the 1-40 fragment of beta-amyloid protein (beta A4), or vehicle, was bilaterally injected into the rostral hippocampus of rats performing under stable food-maintained schedules of reinforcement or under a delayed conditional discrimination procedure. Under the first procedure, rats were trained to stability under a multiple fixed interval 15 s, fixed ratio 30 reinforcement schedule. This reinforcement schedule has proven sensitive to low-dose drug effects. Acute bilateral hippocampal beta A4 (1.0, 2.0 and 3.0 microliters of 10(-3) M) administration did not significantly alter responding, compared to vehicle, under either reinforcement condition. Following the acute single-injection regimen, rats were administered chronic daily beta A4 (1 microliter of 10(-3) M), bilaterally, for 15 days. No significant changes in lever-pressing performance were observed during the chronic injection regimen, but performance declined significantly 30 days after termination of the chronic daily regimen. Histological examination revealed three of six rats showed positive reactions under Thioflavin S staining in and around the area of cannulae termination. The second assessment employed a delayed conditional discrimination procedure to evaluate the effects of intrahippocampal injections of beta A4 on short-term working memory. This conditional discrimination procedure assesses appropriate responding, dependent on a previously presented stimulus, after delays of various lengths have been imposed between the stimulus and the response opportunity.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of an exogenous β-amyloid peptide on retention for spatial learning

Three experiments assessed the effects of β-amyloid 1–40 (βA4) on spatial learning in Sprague-Dawley rats. In Experiment 1, rats were trained on a signaled footshock avoidance in a Y-maze. Rats received a single injection of βA4 or vehicle in both sides of the hippocampus immediately after the fifth trial. The βA4 group took significantly longer than the vehicle group to learn to avoid the shock when trained to criterion 1 week later, suggesting a detrimental effect of βA4 on memory consolidation. Experiment 2 used a food reinforcer rather than shock relief under procedures similar to Experiment 1. Again, the βA4 group took longer to learn the maze to criterion. This shows that the effect in Experiment 1 was not specific to shock-maintained learning. In Experiment 3, rats were trained to retrieve a food pellet from each arm of an eight-arm radial maze. After training to criterion, β4 or vehicle was administered intrahippocampally 30 min before the daily session for 26 sessions. There were no acute or chronic effects of βA4 injection on radial maze performance, and no aggregation of βA4 or significant necrosis was observed upon postmortem histological analysis. These experiments suggest that single injections of βA4 impair memory consolidation, but repeated injections of βA4 over an extended period do not affect well-learned behavior.

Naloxone effects on sucrose-motivated behavior

The opioid system plays an important role in feeding. In general, opioid agonists typically increase feeding and opioid antagonists decrease feeding in nonfood restricted animals. In food restricted animals the effects of these drugs are substantially reduced. Opioid antagonists have shown a marked effectiveness at reducing consumption of sweet foods. Explanations for this robust effect have typically focused on drug induced changes in taste, taste perception, or palatability. The current study relates the effects of the opioid antagonist naloxone on motivation to obtain different sucrose concentrations to the drugs effects on unrestricted sucrose solution consumption. Changes in motivation to respond were assessed under a progressive ratio reinforcement schedule (PR) which required increased response cost for each successive unit of sucrose solution. Motivation, as measured by the PR, increased as sucrose concentration increased and naloxone produced a dose-dependent decrease in motivation to respond for a given sucrose concentration. Thus, the effectiveness of naloxone was indirectly related to strength of the sucrose concentration. Under unrestricted access to sucrose solutions, naloxone reduced consumption greatest under the higher concentrations. The data suggest at least part of naloxones effects on sweet tasting food may be mediated through endogenous opioid reward systems that are reflected in measures of motivation.

Oligomers of the amyloid-β protein disrupt working memory: Confirmation with two behavioral procedures

Converging lines of evidence suggest that oligomers of amyloid-beta play a role in the cognitive impairment characteristic of Alzheimers disease, but only three studies have provided experimental evidence of such impairment. To provide additional information about the effects of these oligomers on memory, the present study examined the memory of groups of rats exposed to ICV injections of the culture media (CM) of Chinese Hamster Ovary cells that were (7PA2) and were not (CHO-) transfected with a human mutation of amyloid precursor protein that appears to cause early-onset Alzheimers disease. The 7PA2 CM, which contained concentrations of soluble amyloid-beta oligomers physiologically relevant to those found in human brain, significantly disrupted working memory in rats tested in a radial-arm maze. In contrast, CHO- CM, which did not contain such oligomers, had no effect on memory. The disruptive effects of 7PA2-derived amyloid-beta oligomers, evident 2h after exposure, disappeared within a day. These findings are compared to results from 7PA2 CM tested under a complex procedure thought to measure aspects of executive function. The results confirm the disruptive effects of low-n amyloid-beta oligomers and extend them to a well-established rat model of memory.