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Several anti-amyloid β (Aβ) antibodies are under evaluation for the treatment of Alzheimer's disease (AD). Clinical studies using the N-terminal-directed anti-Aβ antibody bapineuzumab have demonstrated reduced brain PET-Pittsburg-B signals, suggesting the reduction of Aβ plaques, and reduced levels of total and phosphorylated tau protein in the CSF of treated AD patients. Preclinical studies using 3D6 (the murine form of bapineuzumab) have demonstrated resolution of Aβ plaque and vascular burdens, neuritic dystrophy, and preservation of synaptic density in the transgenic APP mouse models. In contrast, few studies have evaluated the direct interaction of this antibody with synaptotoxic soluble Aβ species. In the current report, we demonstrated that 3D6 binds to soluble, synaptotoxic assemblies of Aβ(1-42) and prevents multiple downstream functional consequences in rat hippocampal neurons including changes in glutamate AMPA receptor trafficking, AD-type tau phosphorylation, and loss of dendritic spines. In vivo, we further demonstrated that 3D6 prevents synaptic loss and acutely reverses the behavioral deficit in the contextual fear conditioning task in transgenic mouse models of AD, two endpoints thought to be linked to synaptotoxic soluble Aβ moieties. Importantly C-terminal anti-Aβ antibodies were ineffective on these endpoints. These results, taken with prior studies, suggest that N-terminal anti-Aβ antibodies effectively interact with both soluble and insoluble forms of Aβ and therefore appear particularly well suited for testing the Aβ hypothesis of AD.

In addition to parenchymal amyloid-beta (Abeta) plaques, Alzheimer's disease (AD) is characterized by Abeta in the cerebral vasculature [cerebral amyloid angiopathy (CAA)] in the majority of patients. Recent studies investigating vascular Abeta (VAbeta) in amyloid precursor protein transgenic mice have suggested that passive immunization with anti-Abeta antibodies may clear parenchymal amyloid but increase VAbeta and the incidence of microhemorrhage. However, the influences of antibody specificity and exposure levels on VAbeta and microhemorrhage rates have not been well established, nor has any clear causal relationship been identified. This report examines the effects of chronic, passive immunization on VAbeta and microhemorrhage in PDAPP mice by comparing antibodies with different Abeta epitopes (3D6, Abeta(1-5); 266, Abeta(16-23)) and performing a 3D6 dose-response study. VAbeta and microhemorrhage were assessed using concomitant Abeta immunohistochemistry and hemosiderin detection. 3D6 prevented or cleared VAbeta in a dose-dependent manner, whereas 266 was without effect. Essentially complete absence of VAbeta was observed at the highest 3D6 dose, whereas altered morphology suggestive of ongoing clearance was seen at lower doses. The incidence of microhemorrhage was increased in the high-dose 3D6 group and limited to focal, perivascular sites. These colocalized with Abeta deposits having altered morphology and apparent clearance in the lower-dose 3D6 group. Our results suggest that passive immunization can reduce VAbeta levels, and modulating antibody dose can significantly mitigate the incidence of microhemorrhage while still preventing or reducing VAbeta. These observations raise the possibility that Abeta immunotherapy can potentially slow or halt the course of CAA development in AD that is implicated in vascular dysfunction.

The behavioral and biochemical impact of active immunization against human beta-amyloid (Abeta) was assessed using male transgenic (Tg) mice overexpressing a human mutant amyloid precursor protein (heterozygous PDAPP mice) and littermate controls. Administration of aggregated Abeta42 occurred at monthly intervals from 7 months ("prevention") or 11 months ("reversal"), followed by double-blind behavioral training at 16 months on a cued task, then serial spatial learning in a water maze. Using a 2 x 2 design, with Abeta42 adjuvanted with MPL-AF (adjuvant formulation of monophosphoryl lipid A) or MPL-AF alone, PDAPP mice were impaired compared with non-Tg littermates on two separate measures of serial spatial learning. Immunization caused no overall rescue of learning but limited the accumulation of total Abeta and Abeta42 levels in cortex and hippocampus by up to 60%. In immunized PDAPP mice, significant negative correlations were observed between hippocampal and cortical Abeta levels and learning capacity, particularly in the prevention study, and correlations between learning capacity and antibody titer. Moreover, a subset of PDAPP mice with very low Abeta levels (hippocampal Abeta levels of <6000 ng/g or cortical Abeta levels of <1000 ng/g) was indistinguishable from non-Tg controls. Mice in the prevention study were also rescued from cognitive impairment more effectively than those in the reversal study. The combination of variability in antibody response and differential levels of Abeta accumulation across the population of immunized PDAPP mice may be responsible for success in cognitive protection with only a subset of these animals, but the similarity to the findings of certain human vaccination trials is noteworthy.

Immunotherapy targeting the amyloid beta (Abeta) peptide is a novel therapy under investigation for the treatment of Alzheimer's disease (AD). A clinical trial using Abeta(1-42) (AN1792) as the immunogen was halted as a result of development of meningoencephalitis in a small number of patients. The cytokine TGF-beta1 is a key modulator of immune responses that is increased in the brain in AD. We show here that local overexpression of TGF-beta1 in the brain increases both meningeal and parenchymal T lymphocyte number. Furthermore, TGF-beta1 overexpression in a mouse model for AD [amyloid precursor protein (APP) mice] leads to development of additional T cell infiltrates when mice were immunized at a young but not old age with AN1792. Notably, only mice overproducing both Abeta (APP mice) and TGF-beta1 experienced a rise in T lymphocyte number after immunization. One-third of infiltrating T cells were CD4 positive. We did not observe significant differences in B lymphocyte numbers in any of the genotypes or treatment groups. These results demonstrate that TGF-beta1 overproduction in the brain can promote T cell infiltration, in particular after Abeta(1-42) immunization. Likewise, levels of TGF-beta1 or other immune factors in brains of AD patients may influence the response to Abeta(1-42) immunization.

Alzheimer's disease neuropathology is characterized by key features that include the deposition of the amyloid beta peptide (Abeta) into plaques, the formation of neurofibrillary tangles, and the loss of neurons and synapses in specific brain regions. The loss of synapses, and particularly the associated presynaptic vesicle protein synaptophysin in the hippocampus and association cortices, has been widely reported to be one of the most robust correlates of Alzheimer's disease-associated cognitive decline. The beta-amyloid hypothesis supports the idea that Abeta is the cause of these pathologies. However, the hypothesis is still controversial, in part because the direct role of Abeta in synaptic degeneration awaits confirmation. In this study, we show that Abeta reduction by active or passive Abeta immunization protects against the progressive loss of synaptophysin in the hippocampal molecular layer and frontal neocortex of a transgenic mouse model of Alzheimer's disease. These results, substantiated by quantitative electron microscopic analysis of synaptic densities, strongly support a direct causative role of Abeta in the synaptic degeneration seen in Alzheimer's disease and strengthen the potential of Abeta immunotherapy as a treatment approach for this disease.

Transgenic (Tg) mouse models overexpressing amyloid precursor protein (APP) develop senile plaques similar to those found in Alzheimer's disease in an age-dependent manner. Recent reports demonstrated that immunotherapy is effective at preventing or removing amyloid-beta deposits in the mouse models. To characterize the mechanisms involved in clearance, we used antibodies of either IgG1 (10d5) or IgG2b (3d6) applied directly to the brains of 18-month-old Tg2576 or 20-month-old PDAPP mice. Both 10d5 and 3d6 led to clearance of 50% of diffuse amyloid deposits in both animal models within 3 d. Fc receptor-mediated clearance has been shown to be important in an ex vivo assay showing antibody-mediated clearance of plaques by microglia. We now show, using in vivo multiphoton microscopy, that FITC-labeled F(ab')2 fragments of 3d6 (which lack the Fc region of the antibody) also led to clearance of 45% of the deposits within 3 d, similar to the results obtained with full-length 3d6 antibody. This result suggests that direct disruption of plaques, in addition to Fc-dependent phagocytosis, is involved in the antibody-mediated clearance of amyloid-beta deposits in vivo. Dense-core deposits that were not cleared were reduced in size by approximately 30% with full-length antibodies and F(ab')2 fragments 3 d after a topical treatment. Together, these results indicate that clearance of amyloid deposits in vivo may involve, in addition to Fc-dependent clearance, a non-Fc-mediated disruption of plaque structure.

The PDAPP transgenic mouse overexpresses human amyloid precursor protein V717F (PDAPP minigene) and develops age-related cerebral amyloid-beta protein (Abeta) deposits similar to senile plaques in Alzheimer's disease. We find age-related cortical and limbic Abeta deposition that begins at 8 months and progresses to cover 20-50% of the neuropil in cingulate cortex, entorhinal cortex, and hippocampus of 18-month-old heterozygotic animals. The regional patterns of transgene expression and amyloid deposition suggest that Abeta deposits occur at the terminals of overexpressing neurons. Amyloid deposition is associated with dystrophic neurites and extensive gliosis. However, stereological analysis shows that there is no overt neuronal loss in entorhinal cortex, CA1 hippocampal subfield, or cingulate cortex through 18 months of age. In addition, there is no apparent loss of mRNA encoding neuronal synaptic, cytoskeletal, or metabolic proteins. Thus, widespread Abeta deposition in 18-month-old heterozygotic mice produces neuritic alterations and gliosis without widespread neuronal death.

Overexpression of mutated human amyloid precursor protein (hAPP717V-->F) under control of platelet-derived growth factor promoter (PDAPP minigene) in transgenic (tg) mice results in neurodegenerative changes similar to Alzheimer's disease (AD). To clarify the pathology of these mice, we studied images derived from laser scanning confocal and electron microscopy and performed comparisons between PDAPP tg mice and AD. Similar to AD, neuritic plaques in PDAPP tg mouse contained a dense amyloid core surrounded by anti-hAPP- and antineurofilament-immunoreactive dystrophic neurites and astroglial cells. Neurons were found in close proximity to plaques in PDAPP tg mice and, to a lesser extent, in AD. In PDAPP tg mice, and occasionally in AD, neuronal processes contained fine intracellular amyloid fibrils in close proximity to the rough endoplasmic reticulum, coated vesicles, and electron-dense material. Extracellular amyloid fibrils (9-11 nm in diameter) were abundant in PDAPP tg and were strikingly similar to those observed in AD. Dystrophic neurites in plaques of PDAPP tg mouse and AD formed synapses and contained many dense multilaminar bodies and neurofilaments (10 nm). Apoptotic-like figures were present in the tg mice. No paired helical filaments have yet been observed in the heterozygote PDAPP tg mice. In summary, this study shows that PDAPP tg mice develop massive neuritic plaque formation and neuronal degeneration similar to AD. These findings show that overproduction of hAPP717V-->F in tg mice is sufficient to cause not only amyloid deposition, but also many of the complex subcellular degenerative changes associated with AD.