The processing and metabolism of amyloid precursor protein (APP) is a major interest in Alzheimer disease (AD) research, because not only amyloid β (Aβ) peptide, but also cellular or mitochondrial APP are intimately involved in cellular dysfunction and AD pathogenesis. Here we demonstrate that APP is directly and efficiently cleaved by the HtrA2 serine protease in vitro and in vivo. Using several APP mutants and N-terminal amino acid sequencing, we identified that the HtrA2-mediated APP cleavage product is the C161 fragment encompassing amino acids 535-695 of APP695. The immunofluorescence and subcellular fractionation studies indicate that APP is partly colocalized with HtrA2 in the mitochondria where HtrA2 can cleave APP under normal conditions. The HtrA2-cleaved C161 fragment was detected in the cytosolic fraction; therefore, we postulate that the C161 fragment is released into the cytosol after cleavage of APP by HtrA2. Interestingly, the level of C161 was remarkably decreased in motor neuron degeneration (mnd2) mice in which the serine protease activity of HtrA2 was greatly reduced. These results show that the protease activity of HtrA2 is essential for the production of C161 and that processing of APP into C161 is a natural event occurring under normal physiological conditions. Our study suggests that the direct cleavage of mitochondrial APP by HtrA2 may prevent mitochondrial dysfunction caused by accumulation of APP and that the regulation of HtrA2 protease activity may be a therapeutic target in AD.
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