Effector function of anti-pyroglutamate-3 Aß antibodies affects cognitive benefit, glial activation and amyloid clearance in Alzheimer's-like mice.

BACKGROUND: Pyroglutamate-3 Aß (pGlu-3 Aß) is an N-terminally truncated and post-translationally modified Aß species found in Alzheimer's disease (AD) brain. Its increased peptide aggregation propensity and toxicity make it an attractive emerging treatment strategy for AD. We address the question of how the effector function of an anti-pGlu-3 Aß antibody influences the efficacy of immunotherapy in mouse models with AD-like pathology. METHODS: We compared two different immunoglobulin (Ig) isotypes of the same murine anti-pGlu-3 Aß mAb (07/1 IgG1 and 07/2a IgG2a) and a general N-terminal Aß mAb (3A1 IgG1) for their ability to clear Aß and protect cognition in a therapeutic passive immunotherapy study in aged, plaque-rich APPSWE/PS1ΔE9 transgenic (Tg) mice. We also compared the ability of these antibodies and a CDC-mutant form of 07/2a (07/2a-k), engineered to avoid complement activation, to clear Aß in an ex vivo phagocytosis assay and following treatment in APPSLxhQC double Tg mice, and to activate microglia using longitudinal microPET imaging with TSPO-specific 18F-GE180 tracer following a single bolus antibody injection in young and old Tg mice. RESULTS: We demonstrated significant cognitive improvement, better plaque clearance, and more plaque-associated microglia in the absence of microhemorrhage in aged APPSWE/PS1ΔE9 Tg mice treated with 07/2a, but not 07/1 or 3A1, compared to PBS in our first in vivo study. All mAbs cleared plaques in an ex vivo assay, although 07/2a promoted the highest phagocytic activity. Compared with 07/2a, 07/2a-k showed slightly reduced affinity to Fcγ receptors CD32 and CD64, although the two antibodies had similar binding affinities to pGlu-3 Aß. Treatment of APPSLxhQC mice with 07/2a and 07/2a-k mAbs in our second in vivo study showed significant plaque-lowering with both mAbs. Longitudinal 18F-GE180 microPET imaging revealed different temporal patterns of microglial activation for 3A1, 07/1, and 07/2a mAbs and no difference between 07/2a-k and PBS-treated Tg mice. CONCLUSION: Our results suggest that attenuation of behavioral deficits and clearance of amyloid is associated with strong effector function of the anti-pGlu-3 Aß mAb in a therapeutic treatment paradigm. We present evidence that antibody engineering to reduce CDC-mediated complement binding facilitates phagocytosis of plaques without inducing neuroinflammation in vivo. Hence, the results provide implications for tailoring effector function of humanized antibodies for clinical development.

Immunotherapy targeting pyroglutamate-3 Aß: prospects and challenges.

Immunization against amyloid-ß (Aß) peptides deposited in Alzheimer's disease (AD) has shown considerable therapeutic effect in animal models however, the translation into human Alzheimer's patients is challenging. In recent years, a number of promising Aß immunotherapy trials failed to reach primary study endpoints. Aside from uncertainties in the selection of patients and the start and duration of treatment, these results also suggest that the mechanisms underlying AD are still not fully understood. Thorough characterizations of protein aggregates in AD brain have revealed a conspicuous heterogeneity of Aß peptides enabling the study of the toxic potential of each of the major forms. One such form, amino-terminally truncated and modified pyroglutamate (pGlu)-3 Aß peptide appears to play a seminal role for disease initiation, qualifying it as novel target for immunotherapy approaches.

Blockage of CR1 prevents activation of rodent microglia.

The importance of the complement system in Alzheimer's disease (AD) pathogenesis has been emphasized through recent genome wide association studies. However, the cellular and molecular role of these complement proteins is not fully understood. Microglia express complement receptors and the activation of specific receptors may increase Aß clearance and reduce neurodegeneration. Here, we investigated the contribution of complement receptor 1 (CR1), the second most significant hit in GWAS studies, on microglia to neuronal damage. We show that microglia displaying an activated phenotype demonstrate an increase in CR1 expression. We also provide evidence that activation of microglial CR1 was detrimental to neurons and this correlated with an increase in microglial intracellular superoxide generation, and tumour necrosis factor-α (TNFα) and interleukin-1 ß (IL-1ß) secretion. Amyloid-ß 42 (Aß1-42)-treated microglia displayed an increased ability to phagocytose dextran beads following antibody blockage of CR1 but a decreased capacity to phagocytose fluorescent-tagged Aß1-42. Together, these results indicate that microglial CR1 plays a role in the neuronal death observed in AD and investigating this further may provide a possible strategy to control neurotoxicity in the AD brain.

Initial assessment of the pathogenic mechanisms of the recently identified Alzheimer risk Loci.

Recent genome wide association studies have identified CLU, CR1, ABCA7 BIN1, PICALM and MS4A6A/MS4A6E in addition to the long established APOE, as loci for Alzheimer's disease. We have systematically examined each of these loci to assess whether common coding variability contributes to the risk of disease. We have also assessed the regional expression of all the genes in the brain and whether there is evidence of an eQTL explaining the risk. In agreement with other studies we find that coding variability may explain the ABCA7 association, but common coding variability does not explain any of the other loci. We were not able to show that any of the loci had eQTLs within the power of this study. Furthermore the regional expression of each of the loci did not match the pattern of brain regional distribution in Alzheimer pathology. Although these results are mainly negative, they allow us to start defining more realistic alternative approaches to determine the role of all the genetic loci involved in Alzheimer's disease.

Microglia, Alzheimer's disease, and complement.

Microglia, the immune cell of the brain, are implicated in cascades leading to neuronal loss and cognitive decline in Alzheimer's disease (AD). Recent genome-wide association studies have indicated a number of risk factors for the development of late-onset AD. Two of these risk factors are an altered immune response and polymorphisms in complement receptor 1. In view of these findings, we discuss how complement signalling in the AD brain and microglial responses in AD intersect. Dysregulation of the complement cascade, either by changes in receptor expression, enhanced activation of different complement pathways or imbalances between complement factor production and complement cascade inhibitors may all contribute to the involvement of complement in AD. Altered complement signalling may reduce the ability of microglia to phagocytose apoptotic cells and clear amyloid beta peptides, modulate the expression by microglia of complement components and receptors, promote complement factor production by plaque-associated cytokines derived from activated microglia and astrocytes, and disrupt complement inhibitor production. The evidence presented here indicates that microglia in AD are influenced by complement factors to adopt protective or harmful phenotypes and the challenge ahead lies in understanding how this can be manipulated to therapeutic advantage to treat late onset AD.

Complement receptor 1 (CR1) and Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease and it poses an ever-increasing burden to an aging population. Several loci responsible for the rare, autosomal dominant form of AD have been identified (APP, PS1 and PS2), and these have facilitated the development of the amyloid cascade hypothesis of AD aetiology. The late onset form of the disease (LOAD) is poorly defined genetically, and up until recently the only known risk factor was the ε4 allele of APOE. Recent genome-wide association studies (GWAS) have identified common genetic variants that increase risk of LOAD. Two of the genes highlighted in these studies, CLU and CR1, suggest a role for the complement system in the aetiology of AD. In this review we analyse the evidence for an involvement of complement in AD. In particular we focus on one gene, CR1, and its role in the complement cascade. CR1 is a receptor for the complement fragments C3b and C4b and is expressed on many different cell types, particularly in the circulatory system. We look at the evidence for genetic polymorphisms in the gene and the possible physiological effects of these well-documented changes. Finally, we discuss the possible impact of CR1 genetic polymorphisms in relation to the amyloid cascade hypothesis of AD and the way in which CR1 may lead to AD pathogenesis.