Secreted Human Amyloid Precursor Protein Binds Semaphorin 3a and Prevents Semaphorin-Induced Growth Cone Collapse

The amyloid precursor protein (APP) is well known for giving rise to the amyloid-β peptide and for its role in Alzheimer's disease. Much less is known, however, on the physiological roles of APP in the development and plasticity of the central nervous system. We have used phage display of a peptide library to identify high-affinity ligands of purified recombinant human sAPPα695 (the soluble, secreted ectodomain from the main neuronal APP isoform). Two peptides thus selected exhibited significant homologies with the conserved extracellular domain of several members of the semaphorin (Sema) family of axon guidance proteins. We show that sAPPα695 binds both purified recombinant Sema3A and Sema3A secreted by transfected HEK293 cells. Interestingly, sAPPα695 inhibited the collapse of embryonic chicken (Gallus gallus domesticus) dorsal root ganglia growth cones promoted by Sema3A (Kd≤8·10−9 M). Two Sema3A-derived peptides homologous to the peptides isolated by phage display blocked sAPPα binding and its inhibitory action on Sema3A function. These two peptides are comprised within a domain previously shown to be involved in binding of Sema3A to its cellular receptor, suggesting a competitive mechanism by which sAPPα modulates the biological action of semaphorins

Impairment of Adolescent Hippocampal Plasticity in a Mouse Model for Alzheimer's Disease Precedes Disease Phenotype

The amyloid precursor protein (APP) was assumed to be an important neuron-morphoregulatory protein and plays a central role in Alzheimer's disease (AD) pathology. In the study presented here, we analyzed the APP-transgenic mouse model APP23 using 2-dimensional gel electrophoresis technology in combination with DIGE and mass spectrometry. We investigated cortex and hippocampus of transgenic and wildtype mice at 1, 2, 7 and 15 months of age. Furthermore, cortices of 16 days old embryos were analyzed. When comparing the protein patterns of APP23 with wildtype mice, we detected a relatively large number of altered protein spots at all age stages and brain regions examined which largely preceded the occurrence of amyloid plaques. Interestingly, in hippocampus of adolescent, two-month old mice, a considerable peak in the number of protein changes was observed. Moreover, when protein patterns were compared longitudinally between age stages, we found that a large number of proteins were altered in wildtype mice. Those alterations were largely absent in hippocampus of APP23 mice at two months of age although not in other stages compared. Apparently, the large difference in the hippocampal protein patterns between two-month old APP23 and wildtype mice was caused by the absence of distinct developmental changes in the hippocampal proteome of APP23 mice. In summary, the absence of developmental proteome alterations as well as a down-regulation of proteins related to plasticity suggest the disturption of a normally occurring peak of hippocampal plasticity during adolescence in APP23 mice. Our findings are in line with the observation that AD is preceded by a clinically silent period of several years to decades. We also demonstrate that it is of utmost importance to analyze different brain regions and different age stages to obtain information about disease-causing mechanisms

A Comprehensive Functional Analysis of Ancestral Human Signal Peptides

Abstract With the sequencing of the Neandertal genome, it has become possible to identify amino acid substitutions that occurred on the human lineage since its separation from the Neandertal lineage. Conceptually, it will therefore be possible to functionally analyze all such amino acid substitutions in the future. Here, we analyze the function of substitutions that occurred during recent human evolution in N-terminal signal peptides. We develop a high-throughput flow cytometrybased assay to analyze signal peptide efficiency as the ratio of surface to total reporter protein per live cell. Such ratios differed significantly among signal peptides derived from different human genes. However, no modern human signal peptide differed significantly from its ancestral counterpart, an observation compatible with the predictions of the neutral theory of molecular evolution

Confocal microscopic dual-laser dual-polarization FRET (2polFRET) at the acceptor side for correlating rotations at different distances on the cell surface

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Solution Studies and Structural Model of the Extracellular Domain of the Human Amyloid Precursor Protein

ABSTRACT The amyloid precursor protein (APP) is the precursor of the �-amyloid peptide (A�), which is centrally related to the genesis of Alzheimer’s disease (AD). In addition, APP has been suggested to mediate and/or participate in events that lead to neuronal degeneration in AD. Despite the fact that various aspects of the cell biology of APP have been investigated, little information on the structure of this protein is available. In this work, the solution structure of the soluble extracellular domain of APP (sAPP, composing 89 % of the amino acid residues of the whole protein) has been investigated through a combination of size-exclusion chromatography, circular dichroism, and synchrotron radiation small-angle x-ray scattering (SAXS) studies. sAPP is monomeric in solution (65 kDa obtained from SAXS measurements) and exhibits an anisometric molecular shape, with a Stokes radius of 39 or 51 Å calculated from SAXS or chromatographic data, respectively. The radius of gyration and the maximum molecular length obtained by SAXS were 38 Å and 130 Å, respectively. Analysis of SAXS data further allowed building a structural model for sAPP in solution. Circular dichroism data and secondary structure predictions based on the amino acid sequence of APP suggested that a significant fraction of APP (30 % of the amino acid residues) is not involved in standard secondary structure elements, which may explain the elongated shape of the molecule recovered in our structural model. Possible implications of the structure of APP in ligand binding and molecular recognition events involved in the biological functions of this protein are discussed

Neuroprotective Secreted Amyloid Precursor Protein Acts by Disrupting Amyloid Precursor Protein Dimers*S⃞

The amyloid precursor protein (APP) is implied both in cell growth and differentiation and in neurodegenerative processes in Alzheimer disease. Regulated proteolysis of APP generates biologically active fragments such as the neuroprotective secreted ectodomain sAPPα and the neurotoxic β-amyloid peptide. Furthermore, it has been suggested that the intact transmembrane APP plays a signaling role, which might be important for both normal synaptic plasticity and neuronal dysfunction in dementia. To understand APP signaling, we tracked single molecules of APP using quantum dots and quantitated APP homodimerization using fluorescence lifetime imaging microscopy for the detection of Förster resonance energy transfer in living neuroblastoma cells. Using selective labeling with synthetic fluorophores, we show that the dimerization of APP is considerably higher at the plasma membrane than in intracellular membranes. Heparan sulfate significantly contributes to the almost complete dimerization of APP at the plasma membrane. Importantly, this technique for the first time structurally defines the initiation of APP signaling by binding of a relevant physiological extracellular ligand; our results indicate APP as receptor for neuroprotective sAPPα, as sAPPα binding disrupts APP dimers, and this disruption of APP dimers by sAPPα is necessary for the protection of neuroblastoma cells against starvation-induced cell death. Only cells expressing reversibly dimerized wild-type, but not covalently dimerized mutant APP are protected by sAPPα. These findings suggest a potentially beneficial effect of increasing sAPPα production or disrupting APP dimers for neuronal survival

Semaphorin 3A specifically binds sAPPα.

<p>(A) Wells in a 96-well plate were coated with either 50 ng sAPPα₆₉₅ (APP), 1 µg BSA or 50 ng Semaphorin 3A (Sema3A), blocked with 1% BSA and overlayed with different concentrations of Sema3A or sAPPα₆₉₅ (as indicated below the lanes). After washing, binding was determined by probing with anti-Sema3A antibody. The graph shows concentration-dependent binding of Sema3A to sAPPα₆₉₅ by densitometric quantification of the immunodots using NIH Image J. Bars represent averages of three independent experiments performed in triplicate each. The blot illustrates a representative experiment. (B) Wells in a 96-well plate were coated with 100 ng sAPPα₆₉₅ (APP), blocked with 1% BSA and incubated with increasing volumes of conditioned medium containing alkaline phosphatase-conjugated Semaphorin 3A (Sema3A-AP). After washing, binding was determined by measuring alkaline phosphatase activity. Bars represent averages of three independent experiments performed in triplicate. (C) Streptavidin-coated beads were incubated with biotinylated sAPPα₆₉₅ (bAPP) and Sema3A-AP-conditioned medium or control (non-transfected) medium. After washing, binding was evaluated by measuring alkaline phosphatase activity associated with the beads. Bars represent averages of three independent experiments.</p

sAPPα modulates the biological activity of Sema3A.

<p>Growth cone morphology was examined in control chick DRG explants (A,E) and in explants challenged for 30 minutes with 0.8 nM Sema3A (B,F), Sema3A+75 nM sAPPα₆₉₅ (C,G) or Sema3A+sAPPα₆₉₅+75 nM each of peptides ARSHPAM and LTASLLI (D,H). After fixation and neurofilament immunostaining, growth cone morphology was examined using fluorescence (upper panels) or phase contrast microscopy (lower panels). Scale bars: 10 µm. Representative images (A–H) illustrate on average two growth cones, but more than 700 growth cones were evaluated in each experimental condition. Panel I shows results of quantitative analysis of the percentage of collapsed growth cones (as determined from phase contrast images) in each experimental condition. Bars correspond to means ± SEM of different ganglia. Each experimental condition was replicated 3–6 times in independent experiments using different DRG cultures. Asterisks represent statistically significant differences (**p<0.01; ***p<0.001; ANOVA followed by Bonferroni post-hoc test).</p

sAPPα-binding peptides selected by phage display are homologous to members of the human semaphorin family.

<p>(A) Sequence alignment of the LRSHPLG and TFASVMT peptides with members of the human semaphorin family; identical residues are shown in red and conservative amino acid replacements are in blue. Sequence alignment was performed using ClustALL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022857#pone.0022857-Larkin1" target="_blank">[56]</a> (B) Ribbon representation of the structure of the receptor binding module of Semaphorin 3A. The amino acid sequences homologous to the LRSHPLG (blue; ARSHPAM) and TFASVMT (red; LTASLLI) peptides are highlighted. Residues involved in receptor specificity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022857#pone.0022857-Koppel1" target="_blank">[39]</a> are shown in yellow and red. The figure was generated using RasMol and the atomic coordinates for Sema3A (Protein Data Bank accession code 1Q47; ref. 40).</p