We reported previously that the carbohydrate domain of the amyloid precursor protein is involved in amyloid precursor protein (APP)-APP interactions. Functional in vitro studies suggested that this interaction occurs through the collagen binding site of APP. The physiological significance remained unknown, because it is not understood whether and how APP dimerization occurs in vivo. Here we report that cellular APP exists as homodimers matching best with a two-site model. Consistent with our published crystallographic data, we show that a deletion of the entire sequence after the kunitz protease inhibitor domain did not abolish APP homodimerization, suggesting that two domains are critically involved but that neither is essential for homodimerization. Finally, we generated stabilized dimers by expressing mutant APP with a single cysteine in the ectodomain juxtamembrane region. Mutation of Lys 624 to cysteine produced ϳ6 -8-fold more A␤ than cells expressing normal APP. Our results suggest that amyloid A␤ production can in principle be positively regulated by dimerization in vivo. We suggest that dimerization could be a physiologically important mechanism for regulating the proposed signal activity of APP.

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␥-Protocadherins (␥-pcdhs) are type I membranespanning glycoproteins, widely expressed in the mammal and required for survival. These cell adhesion molecules are expressed from a complex locus comprising 22 functional variable exons arranged in tandem, each encoding extracellular, transmembrane and intracellular sequence, and three exons for an invariant Cterminal domain (␥-ICD). However, the signaling mechanisms that lie downstream of ␥-pcdhs have not been elucidated. Here we report that ␥-pcdhs are subject to presenilin-dependent intramembrane cleavage (PS-IP), accompanied by shedding of the extracellular domain. The cleaved intracellular domain (␥-ICD) translocates to the cell nucleus and was detected in subsets of cortical neurons. Notably, gene-targeted mice lacking functional ␥-ICD sequence showed severely reduced ␥-pcdh mRNA levels and neonatal lethality. Most importantly, inhibition of ␥-secretase decreased ␥-pcdh locus expression. Luciferase reporter assays demonstrated that ␥-pcdh promoter activity is increased by ␥-ICD. These results reveal an intracellular signaling mechanism for ␥-pcdhs and identify a novel vital target for the ␥-secretase complex.Cadherins represent a large superfamily of transmembrane glycoproteins, sharing common structural features, but exhibiting differential adhesive binding specificities (1). Characterized best are the so-called classic cadherins, including E-, R-, N-, and P-cadherin, and the remainder forms subgroupings collectively referred to as protocadherins (pcdhs).1 Classic cadherins are characterized by an ectodomain consisting of 5 extracellular cadherin-like (EC) repeats, followed by a single transmembrane domain and a highly conserved C-terminal cytoplasmic region. Classic cadherins engage mostly in homophilic adhesive binding triggered by Ca 2ϩ ions intercalating between the EC domains to produce a rigidified, rod-like ectodomain (2). Intracellularly, classic cadherins associate with different proteins, including catenins, that regulate their adhesive properties or mediate downstream signaling (3), which is important for establishing brain structure and connectivity including synaptic structure, function and plasticity (4 -9). Besides their well documented signaling function via catenins, E-and N-cadherin have been shown to undergo presenilin dependent intramembrane proteolysis (PS-IP), which consists of matrix protease-mediated cleavage of the ectodomain and the characteristic release of a soluble cytoplasmic domain by the activity of the ␥-secretase complex and its catalytic constituents presenilin-1/2 (10, 11). The released cytoplasmic cadherin domains can assume signaling function in the cytoplasm and/or nucleus, in analogy to the cytoplasmic domains of prominent targets of ␥-secretase, Notch, APP, ErbB-4, and SREBP-1 (12-15).In comparison to classic cadherins, pcdhs differ in the number of EC domains and have divergent intracellular domains. Recently, Ͼ50 novel type-I transmembrane pcdhs have been described (16,17), of which most, if not all, are expressed in...

The identification of differentially regulated proteins in animal models of psychiatric diseases is essential for a comprehensive analysis of associated psychopathological processes. Mass spectrometry is the most relevant method for analyzing differences in protein expression of tissue and body fluid proteomes. However, standardization of sample handling and sample-to-sample variability are problematic. Stable isotope metabolic labeling of a proteome represents the gold standard for quantitative mass spectrometry analysis. The simultaneous processing of a mixture of labeled and unlabeled samples allows a sensitive and accurate comparative analysis between the respective proteomes. Here, we describe a cost-effective feeding protocol based on a newly developed 15N bacteria diet based on Ralstonia eutropha protein, which was applied to a mouse model for trait anxiety. Tissue from 15N-labeled vs. 14N-unlabeled mice was examined by mass spectrometry and differences in the expression of glyoxalase-1 (GLO1) and histidine triad nucleotide binding protein 2 (Hint2) proteins were correlated with the animals' psychopathological behaviors for methodological validation and proof of concept, respectively. Additionally, phenotyping unraveled an antidepressant-like effect of the incorporation of the stable isotope 15N into the proteome of highly anxious mice. This novel phenomenon is of considerable relevance to the metabolic labeling method and could provide an opportunity for the discovery of candidate proteins involved in depression-like behavior. The newly developed 15N bacteria diet provides researchers a novel tool to discover disease-relevant protein expression differences in mouse models using quantitative mass spectrometry.

Depression and anxiety disorders affect a great number of people worldwide. Whereas singular factors have been associated with the pathogenesis of psychiatric disorders, growing evidence emphasizes the significance of dysfunctional neural circuits and signaling pathways. Hence, a systems biology approach is required to get a better understanding of psychiatric phenotypes such as depression and anxiety. Furthermore, the availability of biomarkers for these disorders is critical for improved diagnosis and monitoring treatment response. In the present study, a mouse model presenting with robust high versus low anxiety phenotypes was subjected to thorough molecular biomarker and pathway discovery analyses. Reference animals were metabolically labeled with the stable 15 N isotope allowing an accurate comparison of protein expression levels between the high anxiety-related behavior versus low anxiety-related behavior mouse lines using quantitative mass spectrometry. Plasma metabolomic analyses identified a number of small molecule biomarkers characteristic for the anxiety phenotype with particular focus on myo-inositol and glutamate as well as the intermediates involved in the tricarboxylic acid cycle. In silico analyses suggested pathways and subnetworks as relevant for the anxiety phenotype. Our data demonstrate that the high anxietyrelated behavior and low anxiety-related behavior mouse model is a valuable tool for anxiety disorder drug discovery efforts. Molecular & Cellular Proteomics 10: 10.1074/mcp.M111.008110, 1-11, 2011.For an improved understanding of the etiology of complex diseases such as psychiatric disorders the elucidation of molecular pathways is critical. In this regard biomarker information can deliver valuable data not only on individual molecular entities but at the same time on pathways critical for disease pathobiology, thus yielding important information for the development of therapeutic agents.Animal models have the capability to mimic certain aspects of complex disorders and thereby untangle complicated phenotypes such as anxiety, which can be measured in the mouse with the help of the elevated plus maze (EPM) 1 and other anxiety tests (1). In earlier studies we have identified proteome differences in a mouse model of extremes in trait anxiety that are qualitative and quantitative in nature. Whereas the enzyme enolase phosphatase was found as a different isoform in high (HAB) versus low (LAB) anxiety-related behavior mice, another enzyme, glyoxalase-1 (Glx1), showed altered expression levels between the two lines (1, 2). Our approach thus considers the two poles of the continuum "anxiety": vulnerability of individuals with high risk scores as well as resilience of individuals with low risk scores at the often neglected "other end" of the continuum of polygenic liability (3).In order to analyze the proteomes of the HAB and LAB mouse lines in greater detail, we have used a comprehensive and accurate proteomics platform that involves metabolic labeling of live animals with stable isotopes follo...