DNA and Cell Biology

Alzheimer's disease (AD) represents the fourth leading cause of death in the U.S. and the leading cause of dementia in the elderly population. Until recently, there was little hope of finding a way to prevent the underlying brain pathology from progressing toward the inevitable conclusion of the disease. However, new immunotherapeutic approaches have been described that are based on vaccination with the β-amyloid 1-42 peptide (Aβ). The encouraging efficacy and safety of Aβ immunization in reducing neuropathology in animal models of AD has opened up new therapeutic possibilities for patients. Immunization with Aβ is aimed at reducing the Aβ-associated pathology of AD. It is hypothesized that this approach will also reduce the cascade of downstream events leading to neuronal cell loss and, ultimately, dementia. The ensuing articles in this issue describe various aspects of the Aβ immunization strategy and their potential relevance to AD treatment.

Postmortem analysis of brains of patients with Alzheimer's disease (AD) has led to diverse theories about the causes of the pathology, suggesting that this complex disease involves multiple physiological changes. In an effort to better understand the variety and integration of these changes, we generated a gene expression profile for AD brain. Comparing affected and unaffected brain regions in nine controls and six AD cases, we showed that 118 of the 7050 sequences on a broadly representative cDNA microarray were differentially expressed in the amygdala and cingulate cortex, two regions affected early in the disease. The identity of these genes suggests the most prominent upregulated physiological correlates of pathology involve chronic inflammation, cell adhesion, cell proliferation, and protein synthesis(31 upregulated genes). Conversely, downregulated correlates of pathology involve signal transduction, energy metabolism, stress response, synaptic vesicle synthesis and function, calcium binding, and cytoskeleton(87 downregulated genes). The results support several separate theories of the causes of AD pathology, as well as add to the list of genes associated with AD. In addition, approximately 10 genes of unknown function were found to correlate with the pathology.

Pathologic examination in Alzheimer's disease (AD) shows a significant correlation between β-amyloid peptide (AβP) deposition and the clinical severity of dementia. Formation ofβ-amyloid (Aβ) is a complex kinetic and thermodynamic process, dependent on peptide–peptide interactions that may be modulated by other proteins. We found that site-directed antibodies toward peptide EFRH sequences 3–6 of the N-terminal region of AβP suppress in vitro formation of Aβ and dissolve already-formed fibrillar amyloid. These so-called chaperone-like properties of monoclonal antibodies led to the development of a new immunologic approach to AD treatment. The immunization procedure, based on phages displaying the EFRH epitope as antigen, induced anti-AβP antibodies that recognized the whole AβP and exhibited antiaggregating properties similar to those of antibodies obtained by injection of Aβ fibrils. Production and performance of anti-β-amyloid antibodies in the transgenic mouse model of AD showed that these antibodies may be delivered from the periphery to the central nervous system, preventing the formation of Aβ and dissolving already-present aggregates. Moreover, immunization with Aβ protected transgenic mice from the learning and age-related memory deficits that occur in AD. These data support the hypotheses that Aβ plays a central role in AD and that site-directed antibodies that modulate Aβ conformation may provide immunotherapy of the disease.

Alzheimer's disease (AD) is a severe neurodegenerative disease for which there is currently no effective prevention or treatment. The prediction that the number of U.S. patients with AD will triple to approximately 14 million over the next 50 years underscores the urgent need to explore novel therapeutic strategies for AD. The β-amyloid protein (Aβ) accumulation and accompanying inflammation appear to play key roles in initiating the neuronal degeneration that underlies the signs and symptoms of AD. Interventions geared toward reducing Aβ accumulation and inflammatory responses should delay or prevent the onset of the clinical disease. Recently, several research groups, including ours, have shown that vaccination with Aβ results in a significant lowering of the Aβ burden in the brains of APP transgenic mice and, in some studies, improvement in their cognitive deficits. Our study described a novel approach, namely mucosal (intranasal) Aβ vaccination. Precisely how Aβ vaccination chronically lowers Aβ levels and reduces Aβ-associated pathology remains unclear. Here, we provide an overview of these studies, with particular emphasis on our work with intranasal Aβ vaccination. Examples of other intranasal vaccines and mucosal adjuvants are presented. Taken together, these data have implications for the future development of an intranasal Aβ vaccine for humans.

Although the pathogenesis of Alzheimer's disease (AD) is not fully understood, growing evidence indicates that the deposition of β-amyloid (Aβ) and the local reactions of various cell types to this protein play major roles in the development of the disease. Immunization with the Aβ 1-42 peptide has been reported to decrease Aβ deposits in the brains of mutant amyloid precursor protein (APP/V717F) transgenic (tg) mice (Schenk et al. Immunization with amyloid-β attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature 1999;400:173–177). We have replicated this finding in APPswe/PS1ΔE9 tg mice, which also develop Aβ deposits in the brain. The immunized animals developed high titers of antibodies against Aβ 1-42 in serum, and Aβ deposits in the brains were significantly reduced. Using surface-enhanced laser desorption/ionization (SELDI) mass spectrometry and ProteinChip^® technology, we detected trends toward increased soluble Aβ peptide in the brain and a decrease in assayable Aβ peptide in the serum of immunized compared with control animals. This last finding raises the possibility that anti-Aβ antibodies in the periphery sequester Aβ peptides or target them for degradation and in this way contribute to the enhanced Aβ clearance from the brain in immunized animals.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by overproduction of β-amyloid (Aβ), which is formed from amyloid precursor protein (APP), with the subsequent pathologic deposition of Aβ in regions of the brain important for memory and cognition. Recently, vaccination of murine models of AD that exhibit Aβ deposition has halted or delayed the usual progression of the pathology of AD. Our group has demonstrated that vaccination of a doubly transgenic mouse model (expressing mutant APP and presenilin-1) with the Aβ 1-42 peptide protects these mice from the memory deficits they would ordinarily develop. This report further characterizes the Aβ 1-42 peptide vaccine in mice. Anti-Aβ response time course analysis indicated that at least three vaccinations (each 100 μg) were necessary to elicit a significant anti-Aβ titer. Subsequent vaccinations resulted in half-maximal antibody titers of at least 10,000, and these titers were maintained for at least 5 months after the final boost. Peptide binding competition studies indicated that the highest humoral responses are generated against the N terminus of the Aβ peptide. Also, measurement of specific murine Ig isotypes in Aβ-vaccinated mice demonstrated a predominant IgG₁ and IgG_2b response, suggesting a type 2 (Th2) T-helper cell immune response, which drives humoral immunity. Finally, lymphocyte proliferation assay experiments using Aβ peptides and splenocytes from vaccinated mice demonstrated that the vaccine specifically stimulates T-cell epitopes present within the Aβ peptide.

There have been several reports on the use of β-amyloid (Aβ ) vaccination in different mouse models of Alzheimer's 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 Aβ inoculations. The number of inoculations influenced the effects of vaccination on Congo red levels, microglia activation, and anti-Aβ 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.

Long-term vaccinations with human β-amyloid peptide 1-42 (Aβ1-42) have recently been shown to prevent or markedly reduce Aβ deposition in the PDAPP transgenic model of Alzheimer's disease (AD). Using a similar protocol to vaccinate 7.5-month-old APP (Tg2576) and APP+PS1 transgenic mice over an 8-month period, we previously reported modest reductions in brain Aβ deposition at 16 months. In these same mice, Aβ vaccinations had no deleterious behavioral effects and, in fact, benefited the mice by providing partial protection from age-related deficits in spatial working memory in the radial arm water maze task (RAWM) at 15.5 months. By contrast, control-vaccinated transgenic mice exhibited impaired performance throughout the entire RAWM test period at 15.5 months. The present study expands on our initial report by presenting additional behavioral results following long-term Aβ vaccination, as well as correlational analyses between cognitive performance and Aβ deposition in vaccinated animals. We report that 8 months of Aβ vaccinations did not reverse an early-onset balance beam impairment in transgenic mice. Additionally, in Y-maze testing at 16 months, all mice showed comparable spontaneous alternation irrespective of genotype or vaccination status. Strong correlations were nonetheless present between RAWM performance and extent of "compact" Aβ deposition in both the hippocampus and the frontal cortex of vaccinated APP+PS1 mice. Our results suggest that the behavioral protection of long-term Aβ vaccinations is task specific, with preservation of hippocampal-associated working memory tasks most likely to occur. In view of the early short-term memory deficits exhibited by AD patients, Aβ vaccination of presymptomatic AD patients could be an effective therapeutic to protect against such cognitive impairments.