Inhibition of amyloid precursor protein processing by β-secretase through site-directed antibodies

Abstract

Amyloid-␤ peptide (A␤P) 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 A␤P, has become a target of intense research aimed at blocking the enzyme activity, thus reducing A␤P 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 A␤P 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 A␤P 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 (A␤Ps), 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 A␤P 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 A␤P 40 and A␤P 42 secretion levels. Recen...

Update on Alzheimer's Disease Therapy and Prevention Strategies

“…An alternative immunotherapy approach consists of sitedirected Abs that affect the BACE-APP complex by targeting the β-secretase cleavage site of APP. Therefore, anti-APP Abs would preserve BACE activity directed toward non-APP substrates and selectively interfere with the BACE-APP complex, inhibiting both intracellular and extracellular Aβ formation (23). One challenge in the use of an immunotherapeutic approach for neurodegenerative disease therapy is limited blood-brain barrier penetration of conventional Abs and immune effector activation, but these challenges can often be addressed through protein engineering.…”

“…Crenezumab binds to the central epitope region of Aβ [12][13][14][15][16][17][18][19][20][21][22][23] , which seems to be most responsible for aggregation. Importantly, crenezumab contains a human IgG4 backbone that translates into reduced effector function on microglia, promotion of microglial Aβ phagocytosis, and reduced proinflammatory response.…”

“…Solanezumab (Eli Lilly & Co.) ( Table 1) binds to soluble monomers and reduces Aβ 42 levels by shifting the equilibrium toward the production of shorter and less toxic Aβ species, demonstrating the peripheral sink effect discussed above. Solanezumab recognizes a central epitope region Aβ [16][17][18][19][20][21][22][23][24] and, interestingly, the ApoE4 genotype seems not to affect drug action. Phase III trials are ongoing to confirm solanezumab's efficacy as a disease-modifying agent and possible disease-prevention agent.…”

“…Clinical data indicate that crenezumab reduces plaques with no vasogenic edema risk, and it is currently being tested in a phase II trial and in a five-year prevention trial. Gantenerumab (Chugai Pharmaceutical Co., Ltd., Roche) ( Table 1) is the first fully human anti-Aβ IgG1 that appears to recognize both the N-terminal tail (Aβ [3][4][5][6][7][8][9][10][11][12] ) and the central region (Aβ [18][19][20][21][22][23][24][25][26][27] ). It has moderate binding affinity to monomers and oligomers and potently binds to and degrades fibrils (39).…”

Immunotherapy for Alzheimer disease

“…Antibody approaches are also being applied in efforts to block secretase cleavage to generate β-amyloid. 20 Finally, there remains some worry that β-amyloid peptides have an as yet unknown normal biological function, although cumulative immunotherapy and other therapeutic studies in animal models have provided sufficient support for the continued pursuit of β-amyloid lowering as a treatment for AD. Immunotherapy for Alzheimer disease…”