Amyloidogenic Processing Of APP Research Articles

Splice variants of the β-site APP-cleaving enzyme BACE1 in human brain and pancreas

BACE is the β-secretase responsible for the first step in amyloidogenic processing of the amyloid precursor protein APP. We have identified two BACE isoforms, BACE1B and BACE1C, lacking 25 and 44 amino acids, respectively. Whereas the BACE1B transcript is present in human pancreas and brain, the BACE1C transcript is found in pancreas only. In transfected cells both BACE1A, which encodes the originally described full-length BACE1 protein and the close homolog BACE2 localized mainly to post-Golgi membranes. In contrast, the two shorter isoforms were found in the endoplasmic reticulum only, and they did not display β-secretase activity. Using RNase protection we in addition show that the major pancreatic transcript is BACE1A. This suggests that the known absence of β-secretase activity in the pancreas is not due to a missing BACE1A transcript.

Presenilin-1 P264L Knock-In Mutation: Differential Effects on Aβ Production, Amyloid Deposition, and Neuronal Vulnerability

The pathogenic mechanism linking presenilin-1 (PS-1) gene mutations to familial Alzheimer's disease (FAD) is uncertain, but has been proposed to include increased neuronal sensitivity to degeneration and enhanced amyloidogenic processing of the beta-amyloid precursor protein (APP). We investigated this issue by using gene targeting with the Cre-lox system to introduce an FAD-linked P264L mutation into the endogenous mouse PS-1 gene, an approach that maintains normal regulatory controls over expression. Primary cortical neurons derived from PS-1 homozygous mutant knock-in mice exhibit basal neurodegeneration similar to their PS-1 wild-type counterparts. Staurosporine and Abeta1-42 induce apoptosis, and neither the dose dependence nor maximal extent of cell death is altered by the PS-1 knock-in mutation. Similarly, glutamate-induced neuronal necrosis is unaffected by the PS-1P264L mutation. The lack of effect of the PS-1P264L mutation is confirmed by measures of basal- and toxin-induced caspase and calpain activation, biochemical indices of apoptotic and necrotic signaling, respectively. To analyze the influence of the PS-1P264L knock-in mutation on APP processing and the development of AD-type neuropathology, we created mouse lines carrying mutations in both PS-1 and APP. In contrast to the lack of effect on neuronal vulnerability, cortical neurons cultured from PS-1P264L homozygous mutant mice secrete Abeta42 at an increased rate, whereas secretion of Abeta40 is reduced. Moreover, the PS-1 knock-in mutation selectively increases Abeta42 levels in the mouse brain and accelerates the onset of amyloid deposition and its attendant reactive gliosis, even as a single mutant allele. We conclude that expression of an FAD-linked mutant PS-1 at normal levels does not generally increase cortical neuronal sensitivity to degeneration. Instead, enhanced amyloidogenic processing of APP likely is critical to the pathogenesis of PS-1-linked FAD.

Expression of β-Amyloid Precursor Protein-CD3γ Chimeras to Demonstrate the Selective Generation of Amyloid β1–40and Amyloid β1–42 Peptides within Secretory and Endocytic Compartments

Amyloid beta-protein (Abeta) is the main constituent of amyloid fibrils found in senile plaques and cerebral vessels in Alzheimer's disease (AD) and is derived by proteolysis from the beta-amyloid precursor protein (APP). We have analyzed the amyloidogenic processing of APP using chimeric proteins stably transfected in Chinese hamster ovary cells. The extracellular and transmembrane domains of APP were fused to the cytoplasmic region derived from the CD3 gamma chain of the T cell antigen receptor (CD3gamma). CD3gamma contains an endoplasmic reticulum (ER) retention motif (RKK), in the absence of which the protein is targeted to lysosomes without going through the cell surface (Letourneur, F., and Klausner, R.D. (1992) Cell 69, 1143-1157). We used the wild-type sequence of CD3gamma to create an APP chimera predicted to remain in the ER (gammaAPP(ER)). Deletion of the RKK motif at the C terminus directed the protein directly to the lysosomes (gammaAPP(LYS)). A third chimera was created by removing both lysosomal targeting signals in addition to RKK (gammaAPP(DeltaDelta)). This last construct does not contain known targeting signals and consequently accumulates at the cell surface. We show by immunofluorescence and by biochemical methods that all three APP chimeras localize to the predicted compartments within the cell, thus providing a useful model to study the processing of APP. We found that Abeta(1-40) is generated in the early secretory and endocytic pathways, whereas Abeta(1-42) is made mainly in the secretory pathway. More importantly, we provide evidence that, unlike in neuronal models, both ER/intermediate compartment- and endocytic-derived Abeta forms can enter the secretable pool. Finally, we directly demonstrate that lysosomal processing is not involved in the generation or secretion of either Abeta(1-40) or Abeta(1-42).

Metabolites of the beta-amyloid precursor protein generated by beta-secretase localise to the trans-Golgi network and late endosome in 293 cells.

Deposition of beta-amyloid occurs in the brains of all sufferers of Alzheimer's disease. beta-amyloid is proteolytically derived from the beta-amyloid precursor protein by as yet unidentified enzymes termed secretases. We have generated and characterised antisera to the carboxy-terminal domain and beta-secretase cleavage site of the Alzheimer's amyloid precursor protein. The beta-secretase cleavage event occurs at the extreme N-terminus of the beta-amyloid peptide. Our antiserum to the N-terminus of the beta-amyloid peptide (NT beta 4) specifically recognises beta-secretase cleaved species as opposed to intact beta APP. NT beta 4 specifically immunoprecipitates a 13 kDa fragment of beta APP (p13) which is potentially amyloidogenic. We have used these antisera in confocal laser scanning immunofluorescence microscopy to localise the intracellular location of potentially amyloidogenic beta APP processing fragments such as p13. Using a number of marker antisera of known intracellular location, we have defined the major location of beta APP fragments possessing the Asp-1 N-terminus of beta-amyloid as the trans-Golgi network or late endosome on the basis of colocalisation with a monoclonal antibody to the cation-independent mannose-6-phosphate receptor. The colocalisation was further investigated using brefeldin A which demonstrated that the p13 fragment and mannose-6-phosphate receptor are trafficked by alternative pathways from the trans-Golgi network.