Misfolded proteins in amyloid plaques in transgenic Alzheimer’s disease mouse brains are visualized directly without labeling.
Amyloid- peptide (AP) that accumulates in the Alzheimer's diseased brain is derived from proteolytic processing of the amyloid precursor protein (APP) by means of -and ␥-secretases. The -secretase APP cleaving enzyme (BACE), which generates the N terminus of AP, has become a target of intense research aimed at blocking the enzyme activity, thus reducing AP and, subsequently, plaque formation. The search for specific inhibitors of -secretase activity as a possible treatment for Alzheimer's disease intensified with the discovery that BACE may be involved in processing other non-APP substrates. The presence of the APP-BACE complex in early endosomes highlights the cell surface as a potential therapeutic target, suggesting that interference in APP-BACE interaction at the cell surface may affect amyloid- production. We present here a unique approach to inhibit AP production by means of antibodies against the -secretase cleavage site of APP. These antibodies were found to bind human APP overexpressed by CHO cells, and the formed immunocomplex was visualized in the early endosomes. Indeed, blocking of the -secretase site by these antibodies interfered with BACE activity and inhibited both intracellular and extracellular AP formation in these cells.Alzheimer's disease ͉ -secretase site ͉ monoclonal antibodies ͉ amyloid -peptide production ͉ endocytic pathway A lzheimer's disease (AD) is characterized by the accumulation of senile plaques in the brain extracellular space and by intraneuronal accumulation of neurofibrillary tangles. The senile plaques are composed of deposited amyloid- peptides (APs), which are derived from the enzymatic processing of a type I transmembrane protein called amyloid precursor protein (APP) (1). The -secretase APP cleaving enzyme (BACE) generates the N terminus of the AP peptide and produces a membrane-bound C-terminal fragment (CTF), C99. This membrane-bound product serves as a substrate for ␥-secretase complex processing, which releases amyloid peptides of 40 or 42 aa. Pharmacologic and cell biology studies demonstrated that the three major enzymatic activities involved in APP processing, ␣--and ␥-secretases, are distinct in their subcellular localization and in their respective cleavage products (2, 3). It was shown that -secretase activity must reside both in the endosomes (4) and in the secretory pathway (5). Antibody uptake and biotinylation studies showed that most cell surface-located BACE is reinternalized into the early endosomal compartments, from where it can recycle back to the cell surface or can later be retrieved to endosomal͞lysosomal compartments and͞or to the trans-Golgi network (6, 7). The endocytic pathway, responsible for internalization and initial processing of cell surface APP in endosomes, is well established (4,(8)(9)(10). Indeed, the mutagenesis of the APP internalization signal (11) and expression of the dominantnegative dynamin mutant that prevents endocytosis in the transfected cells (10) reduced both AP 40 and AP 42 secretion levels. Recen...
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.
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