A New Approach for the Treatment of Alzheimer’s Disease

Alzheimer's Disease and its Molecular Basis

Alzheimer's disease (AD) is the most frequent cause of dementia in Western societies accounting for 60 to 70% of cases. The WHO estimates that there are at present over 35 million people living with dementia worldwide and as the world population ages, the frequency is expected to double by 2030 and triple by 2050. Nonetheless, there are still no available treatments that stop or reverse the progression of AD, which worsens as it progresses, and eventually leads to death. Pathologically, the disease is characterized by two types of lesions in the brain: extracellular senile plaques consisting primarily of amyloid precursor protein (APP)-derived amyloid-Β (AΒ) peptide and intracellular neurofibrillary tangles consisting largely of hyperphosphorylated microtubule-associated tau protein. APP can get cleaved by secretases to give AΒ of a variety of lengths but this process also leaves the APP intracellular domain (AICD) which itself can undergo further cleavage by caspases giving rise to, e.g., APP-C31 the 31 amino acid C-terminal fragment of APP. Full-length APP is subject to caspase cleavage too, resulting again in generation of the APP-C31 peptide as well as APPΔC31, the protein that remains after cutting off its C-terminal 31 amino acids. APPΔC31 has been found in higher levels in the brains of patients with AD as compared to controls and is markedly increased in early stages of AD. Production of APP-C31, that may be mediated by AΒ interaction with full-length APP, resulting in dimerization and caspase cleavage, is toxic to cells and can finally lead to neuronal cell death by apoptosis. Thus, induction of APP-C31 production may play a pivotal role in synapse loss which represents the basis for cognitive impairment in AD. Consequently, inhibition of this cleavage could be a new target for the treatment of AD.

Enzo’s APPΔC31 Specific Antibody and ELISA Kit

In order to more accurately and sensitively quantify caspase cleaved APP in cell and tissue lysates, Karen S. Poksay and colleagues from the Buck Institute for Research on Aging, the Sanford Burnham Prebys Medical Discovery Institute and the University of California in Los Angeles partnered with Enzo Life Sciences. We developed both a cleavage site-specific (neo epitope) polyclonal antibody that recognizes the C-terminus of APPΔC31 resulting from the caspase cleavage as well as an ELISA (ENZ-ABS445 and ADI-900-227, respectively). Various immunoblot analyses showed that Enzo’s anti-APPΔC31 antibody is specific for caspase-cleaved APP, and does not react with full-length APP. Also, in the ELISA used for the authors’ screening and validation studies, a second N-terminal APP-specific antibody was used so that only APPΔC31 is detected which could be confirmed by analyzing HEK 293T cell lysates overproducing either APPΔC31 or full-length APP.

Lead Compound Search for APPΔC31 Production Inhibition

Using CHO cells stably transfected with wildtype human APP (CHO-7W cells) and treated with activated simvastatin to stimulate the caspase cleavage of APP several, compound libraries including Enzo’s protease inhibitor, natural products and autophagy libraries were screened in an AlphaLISA based on Enzo’s anti-APPΔC31 antibody. By applying a hit criterion of at least 70% inhibition of APPΔC31 production, a total of 91 hits out of the 2243 compounds screened were found. Based on structure potential, therapeutic hits were further validated for inhibition of APPΔC31 induction in cerivastatin-treated CHO-7W cells and analyzed with Enzo’s APPΔC31-specific ELISA kit. Lead inhibitors were also tested in murine ex vivo organotypic hippocampal slice cultures by determining APPΔC31 levels with the ELISA kit from Enzo in combination with Enzo’s AMP’d Signal Amplification kit to increase sensitivity. By this approach, several compounds could be identified that inhibited APPΔC31 production and rescued cell death in a dose-dependent manner that fell into several classes including SERCA inhibitors, inhibitors of Wnt signaling, and calcium channel antagonists. Taken together, this study might open up new horizons for investigating, understanding and ultimately treating this fatal disease that Dr. Alzheimer described already more than hundred years ago.


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