The amyloid precursor protein (APP) undergoes constitutive shedding by a protease activity called a-secretase. This is considered an important mechanism preventing the generation of the Alzheimer's disease amyloid-b peptide (Ab). a-Secretase appears to be a metalloprotease of the ADAM family, but its identity remains to be established. Using a novel a-secretase-cleavage site-specific antibody, we found that RNAi-mediated knockdown of ADAM10, but surprisingly not of ADAM9 or 17, completely suppressed APP a-secretase cleavage in different cell lines and in primary murine neurons. Other proteases were not able to compensate for this loss of a-cleavage. This finding was further confirmed by mass-spectrometric detection of APPcleavage fragments. Surprisingly, in different cell lines, the reduction of a-secretase cleavage was not paralleled by a corresponding increase in the Ab-generating b-secretase cleavage, revealing that both proteases do not always compete for APP as a substrate. Instead, our data suggest a novel pathway for APP processing, in which ADAM10 can partially compete with c-secretase for the cleavage of a C-terminal APP fragment generated by b-secretase. We conclude that ADAM10 is the physiologically relevant, constitutive a-secretase of APP.

Analysis of murine cerebrospinal fluid (CSF) by quantitative mass spectrometry is challenging because of low CSF volume, low total protein concentration, and the presence of highly abundant proteins such as albumin. We demonstrate that the CSF proteome of individual mice can be analyzed in a quantitative manner to a depth of several hundred proteins in a robust and simple workflow consisting of single ultra HPLC runs on a benchtop mass spectrometer. The workflow is validated by a comparative analysis of BACE1؊/؊ and wild-type mice using label-free quantification. The protease BACE1 cleaves the amyloid precursor protein (APP) as well as several other substrates and is a major drug target in Alzheimer's disease. We identified a total of 715 proteins with at least 2 unique peptides and quantified 522 of those proteins in CSF from BACE1؊/؊ and wild-type mice. Several proteins, including the known BACE1 substrates APP, APLP1, CHL1 and contactin-2 showed lower abundance in the CSF of BACE1؊/؊ mice, demonstrating that BACE1 substrate identification is possible from CSF. Additionally, ectonucleotide pyrophosphatase 5 was identified as a novel BACE1 substrate and validated in cells using immunoblots and by an in vitro BACE1 protease assay. Likewise, receptor-type tyrosine-protein phosphatase N2 and plexin domain-containing 2 were confirmed as BACE1 substrates by in vitro assays. Taken Cerebrospinal fluid (CSF) 1 consists of interstitial fluid that is in continuous exchange with the central nervous system and the peripheral blood system. It represents the only body fluid in humans that is in direct contact with brain tissue and accessible in a routine clinical setting. Thus, the easy accessibility from the periphery renders CSF perfectly suited to study pathologic neurological processes (1). Human CSF has a relatively low protein content (ϳ 0.4 mg/ml), but features a highly diverse proteome. It is thus increasingly studied by modern mass spectrometry based proteomics (2). The proteomic analysis of human CSF typically involves various protein concentration and fractionation steps as well as the depletion of highly abundant proteins, such as serum albumin. This allows the identification of several hundred up to 2600 proteins from several milliliters of human CSF (3).Mice are the most popular animal model in preclinical research, because of their similarity to humans in genetics and physiology, their unlimited supply and their ease of genetic engineering. The study of their CSF can provide valuable insights into disease mechanisms and biomarker discovery Author Contributions: The manuscript was written by Bastian Dislich and Stefan F. Lichtenthaler. The study was guided and planned by Bastian Dislich, Sebastian Hogl, Melanie Meyer-Luehmann and Stefan F. Lichtenthaler. Bastian Dislich established the mass spectrometry of murine CSF and performed the proteomic analysis of the BACE1Ϫ/Ϫ CSF. Felix Wohlrab performed all other experiments, except for the CSF extraction, which was performed by Teresa Bachhuber, and the analysis of...