Paul T. Jantzen

A|[beta]| peptide vaccination prevents memory loss in an animal model of Alzheimer's disease

Vaccinations with amyloid-β peptide (AB) can dramatically reduce amyloid deposition in a transgenic mouse model of Alzheimers disease. To determine if the vaccinations had deleterious or beneficial functional consequences, we tested eight months of Aβ vaccination in a different transgenic model for Alzheimers disease in which mice develop learning deficits as amyloid accumulates . Here we show that vaccination with Aβ protects transgenic mice from the learning and age-related memory deficits that normally occur in this mouse model for Alzheimers disease. During testing for potential deleterious effects of the vaccine, all mice performed superbly on the radial-arm water-maze test of working memory. Later, at an age when untreated transgenic mice show memory deficits, the Aβ-vaccinated transgenic mice showed cognitive performance superior to that of the control transgenic mice and, ultimately, performed as well as nontransgenic mice. The Aβ-vaccinated mice also had a partial reduction in amyloid burden at the end of the study. This therapeutic approach may thus prevent and, possibly, treat Alzheimers dementia.

Behavioral changes in transgenic mice expressing both amyloid precursor protein and presenilin-1 mutations: lack of association with amyloid deposits.

Mutations in the amyloid precursor protein (mAPP) and in presenilin 1 (mPS1) have both been linked to increased production of the β-amyloid peptide (Aβ). Doubly transgenic mice produced by mating of a parental line carrying the “Swedish” (K670N/M671L) APP mutation with a FAD4 (M146L) mutant presenilin 1 line developed numerous fibrillar Aβ deposits by 6 months of age. Prior work demonstrated that mAPP and doubly transgenic (mAPP/mPS1) mice have deficits in Y-maze alternation behavior as early as 3 months of age. Increased activity was also apparent in the mAPP/mPS1 mice at this time point. These changes in Y-maze performance persisted in mAPP/mPS1 mice at 6 and 9 months of age. The mPS1 singly transgenic mice were not impaired on this task at any age. Six- and nine-month-old mice were also tested for spatial navigation behavior in the Morris water maze. In training trials, no differences in escape latency were detected among the four genotypes. In probe trials, no differences were detected in either the time spent in the trained quadrant or the number of platform crossings among the four groups. Histological staining for Aβ amyloid deposits indicates that all doubly transgenic mice have amyloid deposits by 6 months of age (roughly 25 mice examined thus far), yet no 3-month-old mice have been found with deposits. Aβ immunostaining confirmed that the 9-month-old mice tested behaviorally also have Aβ deposits. Thus, doubly transgenic mice exhibited changes in Y-maze performance prior to the formation of amyloid deposits, which are essentially unchanged as the deposits increase in number and size to 9 months of age. Yet these mice fail to reveal impairments in spatial navigation at 6 or 9 months in spite of the increasing plaque burden. These data indicate that Aβ deposits alone are not sufficient to cause robust spatial memory impairment in mice of this mixed background lineage and age.

Time course of the development of Alzheimer-like pathology in the doubly transgenic PS1+APP mouse.

Doubly transgenic mice expressing both a mutated amyloid precursor protein and a mutated presenilin-1 protein accumulate A(beta) deposits as they age. The early A(beta) deposits were found to be primarily composed of fibrillar A(beta) and resembled compact amyloid plaques. As the mice aged, nonfibrillar A(beta) deposits increased in number and spread to regions not typically associated with amyloid plaques in Alzheimers disease. The fibrillar, amyloid-containing deposits remained restricted to cortical and hippocampal structures and did not increase substantially beyond the 12-month time point. Even at early time points, the fibrillar deposits were associated with dystrophic neurites and activated astrocytes expressing elevated levels of glial fibrillary acidic protein. Microglia similarly demonstrated increased staining for complement receptor-3 in the vicinity of A(beta) deposits at early time points. However, when MHC-II staining was used to assess the degree of microglial activation, full activation was not detected until mice were 12 months or older. Overall, the regional pattern of A(beta) staining resembles that found in Alzheimer disease; however, a progression from diffuse A(beta) to more compact amyloid deposits is not observed in the mouse model. It is noted that the activation of microglia at 12 months is coincident with the apparent stabilization of fibrillar A(beta) deposits, raising the possibility that activated microglia might clear fibrillar A(beta) deposits at a rate similar to their rate of formation, thereby establishing a relatively steady-state level of amyloid-containing deposits.

Correlation between cognitive deficits and Aβ deposits in transgenic APP+PS1 mice

Doubly transgenic mAPP+mPS1 mice (15-16 months) had impaired cognitive function in a spatial learning and memory task that combined features of a water maze and a radial arm maze. Nontransgenic mice learned a new platform location each day during 4 consecutive acquisition trials, and exhibited memory for this location in a retention trial administered 30 min later. In contrast, transgenic mice were, on average, unable to improve their performance in finding the hidden platform over trials. The cognitive performance of individual mice within the transgenic group were inversely related to the amount of Abeta deposited in the frontal cortex and hippocampus. These findings imply that mAPP+mPS1 transgenic mice develop deficits in cognitive ability as Abeta deposits increase. These data argue that radial arm water maze testing of doubly transgenic mice may be a useful behavioral endpoint in evaluating the functional consequences of potential AD therapies, especially those designed to reduce Abeta load.

Number of Aβ Inoculations in APP+PS1 Transgenic Mice Influences Antibody Titers, Microglial Activation, and Congophilic Plaque Levels

There have been several reports on the use of beta-amyloid (Abeta ) vaccination in different mouse models of Alzheimers disease (AD) and its effects on pathology and cognitive function. In this report, the histopathologic findings in the APP+PS1 doubly transgenic mouse were compared after three, five, or nine Abeta inoculations. The number of inoculations influenced the effects of vaccination on Congo red levels, microglia activation, and anti-Abeta antibody titers. After three inoculations, the antibody titer of transgenic mice was substantially lower than that found in nontransgenic animals. However, after nine inoculations, the levels were considerably higher in both genotypes and no longer distinguishable statistically. The number of inoculations influenced CD45 expression, an indicator of microglial activation. There was an initial upregulation, which was significant after five inoculations, but by nine inoculations, the extent of microglial activation was equivalent to that in mice given control vaccinations. Along with this increased CD45 expression, there was a correlative reduction in staining by Congo red, which stains compact plaques. When data from the mice from all groups were combined, there was a significant correlation between activation of microglia and Congo red levels, suggesting that microglia play a role in the clearance of compact plaque.

Amyloid-β vaccination, but not nitro-nonsteroidal anti-inflammatory drug treatment, increases vascular amyloid and microhemorrhage while both reduce parenchymal amyloid

Vaccination with Abeta(1-42) and treatment with NCX-2216, a novel nitric oxide releasing flurbiprofen derivative, have each been shown separately to reduce amyloid deposition in transgenic mice and have been suggested as potential therapies for Alzheimers disease. In the current study we treated doubly transgenic amyloid precursor protein and presenilin-1 (APP+PS1) mice with Abeta(1-42) vaccination, NCX-2216 or both drugs simultaneously for 9 months. We found that all treatments reduced amyloid deposition, both compact and diffuse, to the same extent while only vaccinated animals, with or without nonsteroidal anti-inflammatory drug (NSAID) treatment, showed increased microglial activation associated with the remaining amyloid deposits. We also found that active Abeta vaccination resulted in significantly increased cerebral amyloid angiopathy and associated microhemorrhages, while NCX-2216 did not, in spite of similar reductions in parenchymal amyloid. Co-administration of NCX-2216 did not attenuate this effect of the vaccine. This is the first report showing that active immunization can result in increased vascular amyloid and microhemorrhage, as has been observed with passive immunization. Co-administration of an NSAID agent with Abeta vaccination does not substantially modify the effects of Abeta immunotherapy. The difference between these treatments with respect to vascular amyloid development may reflect the clearance-promoting actions of the vaccine as opposed to the production-modifying effects proposed for flurbiprofen.

Increased Fibrillar β-Amyloid in Response to Human C1q Injections into Hippocampus and Cortex of APP+PS1 Transgenic Mice

Human C1q when injected directly into hippocampus and cortex of doubly transgenic APP+PS1 mice results in the increase of Congo red-positive fibrillar deposits. Although there was no significant change in overall area stained for Aβ total, qualitatively it appeared that there was less diffuse Aβ in C1q-treated mice versus vehicle. There was no apparent change in astroglial or microglial activation caused by injection of C1q with respect to vehicle injections. These effects of C1q were only found in 50% BUB/BnJ mice, a strain with higher serum complement activity than other mouse lines. These in vivo data were consistent with the effects of C1q to increase fibrillogenesis of Aβ in vitro. In conclusion, complement protein C1q, believed to be involved in the pathogenesis of Alzheimers disease in humans, can cause increased fibrillogenesis in the APP+PS1 mouse model of amyloid deposition.

Nucleus basalis lesions in neonate rats induce a selective cortical cholinergic hypofunction and cognitive deficits during adulthood

SummaryIbotenic acid was infused into the nucleus basalis magnocellularis (nBM) of 2-day old rats to eliminate immature cholinergic neurons before they develop functional synaptic connections in the neocortex. For bilaterally lesioned neonates, cognitive testing was initiated 2 months after lesioning and animals were sacrificed at 8 or 12 months of age. Lesioned animals exhibited a marked deficit in the retention of passive avoidance behavior, as well as in the acquisition of 2-way active avoidance behavior. Lesioned animals also made significantly more alternation errors than control animals in the Lashley III spatial maze and showed severe impairments in general learning, reference memory and working memory during 17-arm radial maze testing. For all 4 tasks, neonatally lesioned animals did not show any recovery to the performance level of control animals. Histological analysis of the subcortex from lesioned animals during adulthood revealed: (1) a substantial reduction in acetylcholinesterase-positive cells (presumably cholinergic) within the nucleus basalis, (2) decreased acetylcholinesterase staining in neocortex and (3) a gliosis essentially restricted to the globus pallidus. Surrounding brain regions were apparently not damaged as a direct result of excitotoxin infusion. Neurochemically, neonate nBM lesioning produced a long term cholinergic hypofunction as evidenced by significant reductions of 25% and 18% in frontal cortex chorine acetyltransferase (CAT) activity at 12 and 8 months of age, respectively. By contrast, prefrontal cortical concentrations of biogenic amines and their metabolites were unaffected, thus indicating a degree of neurochemical specificity for these neonatal nBM lesions. The persistant cortical cholinergic hypofunction in lesioned animals may be related to the long term deficits in learning/memory abilities that were also observed. It is suggested that neonatal nBM lesioning could provide a useful animal model for elucidating the plasticity of the developing brain after cortical anervation.

The Association of Microglial Activation and Amyloid Reduction in APP+PS1 Transgenic Mice

Abstract: One increasingly dominant hypothesis regarding the pathogenesis of Alzheimer dementia is the inflammationhypothesis. In brief, this hypothesis argues that at least some of the neurodegeneration found in this disease issecondary to excessive activation of microglia and astrocytes, resulting in secretion of pro-inflammatory mediators,activation of the complement cascade and degeneration of synapses and neurons. The APP+PS1 transgenic mouse isa model of As amyloid deposition that results in a phenotype resembling some but not all aspects of Alzheimers. Ourgroup has evaluated a number of manipulations designed to both exacerbate and ameliorate the microglial activationin this transgenic model, ranging from LPS injections, administration of anti-As antibodies and treatment with anti-inflammatory drugs. Contrary to our original predictions that microglial activation should exacerbate the Alzheimerphenotype in these mice, we find that treatments that cause microglial activation are associated with reduced amyloidloads. These data are discussed in the context of differences between the murine and human immune systems andqualitative differences in the As deposits found in these mouse models compared to human specimens.