Idan Rakover

Immunotherapy against APP β-Secretase Cleavage Site Improves Cognitive Function and Reduces Neuroinflammation in Tg2576 Mice without a Significant Effect on Brain Aβ Levels

Background/Objectives: Active and passive immunization methodologies against amyloid-β (Aβ) are employed to clear and reduce cerebral Aβtowards treatment of Alzheimer’s disease (AD) patients. The therapeutic potential of these antibodies in AD patients is limited because of adverse inflammatory reactions and cerebral hemorrhage, which are associated with the treatment. We propose a novel approach to inhibit Aβ production via antibodies against the β-secretase cleavage site of the amyloid precursor protein (APP). Such an approach limits APP processing by β-secretase, mainly through the endocytic pathway, and overcomes some of the limitations of BACE inhibition. Anti-APP β-site antibodies, tested in a cellular model expressing wild-type APP, were found to bind full-length APP, internalize into the cells and interfere with BACE activity, inhibiting both intra- and extracellular Aβ peptide formation. Methods: We investigated the effect of anti-β-site antibodies in an AD animal model regarding antibody efficacy, as well as possible adverse effects in the brain and periphery that may result from antibody treatment. Results/Conclusions: Here, we show that long-term systemic administration of anti-APP β-site antibodies to Tg2576 transgenic mice improved mouse cognitive functions associated with a reduction in both brain inflammation and the incidence of microhemorrhage. Furthermore, antibody treatment did not induce any peripheral autoimmunity responses. In spite of the beneficial effects observed in antibody-treated mice, brain Aβ levels were not altered as a result of antibody treatment.

Antigen-specific therapy of EAE via intranasal delivery of filamentous phage displaying a myelin immunodominant epitope.

The presence of anti-myelin antibodies (Abs) in patients with early multiple sclerosis (MS) and in MS animal models has led to renewed interest in the role of B cells, plasma cells and their products in the pathogenesis of the disease, and in their therapeutic potential. Here, we present a novel strategy based on filamentous phage display of the myelin oligodendrocyte glycoprotein immunodominant epitope (MOG 36-44) fused to the main coat protein. Filamentous phages are well characterized, both structurally and genetically. We found that the fibrous shape of the phage (1000 nm long and 6 nm wide) enables penetration into the central nervous system (CNS) when administered nasally. Thus, intranasal treatment of experimental autoimmune encephalomyelitis (EAE) in mice, with phage MOG, showed improved neuronal function, reduced levels of proinflammatory cytokines, particularly monocyte chemoattractant protein 1 (MCP-1), interferon gamma (IFN-gamma) and IL-6, but no change in IL-10 or IL-12 levels. Moreover, the treatment induced depletion of the autoantibodies against MOG and prevented demyelination resulting in improved clinical scores and the reduced inflammation in the CNS and periphery in EAE mice compared to untreated sick animals.

Beneficial effect of antibodies against β- secretase cleavage site of APP on Alzheimer's-like pathology in triple-transgenic mice.

The toxicity of amyloid β and tau, the two hallmark proteins in Alzheimer’s disease (AD), has been extensively studied individually. Recently new data suggest their possible interactions and synergistic effects in the disease. In this study, we investigate the ability of antibodies against the β secretase cleavage site on APP, named BBS1, to affect tau pathology, besides their well established effect on intracellular Aβ and amyloid load. For this purpose we treated the triple transgenic mice model of AD (3x Tg-AD) with mAb BBS1 intracerebroventricularly, using mini osmotic pumps for one month. The experimental data demonstrated reduction in total and phosphorylated tau levels, explained by significant reduction in GSK3β which phosphorylates tau on sites recognized by antibodies against PHF1 and AT-8. The treatment increased the cognitive capabilities and reduced the brain inflammation levels which accompany AD pathology. The data showing that tau pathology was significantly reduced by BBS1 antibodies suggest a close interaction between tau and Aβ in the development of AD, and may serve as an efficient novel immunotherapy against both hallmarks of this disease.

Effect of Antibodies against beta secretase cleavage site on cerebral amyloid angiopathy

Background: Cerebral amyloid angiopathy (CAA) is a neurological condition in which amyloid protein builds up on the walls of the arteries in the brain, increasing the risk of hemorrhagic stroke. Deposits of amyloidb (Ab)protein are found in thewalls of medium and small-sized leptomeningeal and cortical arteries, arterioles and, less frequently, in capillaries and veins of the central nervous system, causing the CAA pathology in Alzheimer’s mice models. We developed an antibody which binds the beta-secretase cleavage site on the amyloid precursor protein (APP) and inhibits amyloidbeta processing, reducing Ab levels via the endocytic pathway, which was effective in Alzheimer’s diseased mice models. Methods: We treated APP London mutated Tg mice and APP/IndTg mice with beta-secretase blocker (BBS) antibody intraperitoneum (IP) for two months, after which we analyzed microhemorrhage levels and the accumulation of Ab deposits in the blood vessels of the treated animals compared to non-relevant antibody. Results: We showed a reduction in the Ab deposits in the arteries and veins in Alzheimer’s mouse model and CAA mouse models as a result of treatment with the beta secretase inhibitor. Microhemorrage and inflammationmarkers were reduced as a result of the treatment indicating the treatment efficacy. Conclusions: The results may indicate the ability of BBS antibody treatment to inhibit amyloid angiopathy in Alzheimer’s and CAA patients, as well as vascular dementia.

O2-05-02

clearance of amyloid beta peptides by immunological or chemical means as a method of treatment for Alzheimer’s disease (AD). One of the new findings that immerged from these immunological studies was that passive immunization with amyloid beta peptide antibodies resulted in an efflux of amyloid beta peptide from the brain into the plasma (DeMattos et al., Proc Natl Acad Sci USA 98:8850-8855, 2001; DeMattos et al., Science 295: 2264-2267, 2002.) producing what was termed a peripheral “sink effect”. An elegant extension of this “sink effect” concept was described by Matsuoka et al., (J Neurosci. 23:29-33, 2003) who used two amyloid beta peptide-binding compounds, ganglioside GM1 and gelsolin, to bind plasma amyloid beta peptide in hAPP transgenic mice, resulting in a lowering of brain amyloid beta peptide levels by 50% or more. Objective: We have tested the peripheral sink effect using an amyloid peptide degrading enzyme. Methods: We have expressed the amyloid peptide degrading peptidase neprilysin, or an inactive form of neprilysin, on leukocytes in hAPP transgenic mice. Results and Conclusions: Expression of neprilysin activity on leukocytes declined over a four-month period. Mice expressing active neprilysin at two months of age and sacrificed at 7 months of age showed a dramatic decrease in diffuse amyloid deposits in their brain relative to mice expressing inactive neprilysin. These studies demonstrate the use of peripheral neprilysin as a way to prevent amyloid deposition in brain. This work was supported in part by grant AG024899 from the National Institute on Aging.