amyloid-beta-peptides and lucifer-yellow

Deposition of amyloid beta protein in the brain is the major pathological feature of Alzheimer's disease. Amyloid beta protein is generated from beta-amyloid precursor protein by beta-secretase and gamma-secretase. Proteolytic processing of amyloid precursor protein at the beta site by BACE1 is essential to generate amyloid beta protein. BACE1, the major beta-secretase involved in cleaving amyloid precursor protein, has been identified as a type 1 membrane-associated aspartyl protease. In this study, we found that BACE1 gene expression is controlled by a TATA-less promoter. BACE1 gene expression is tightly regulated at the transcriptional level and the transcription factor Sp1 plays an important role in regulation of BACE1 to process amyloid precursor protein generating amyloid beta protein. Furthermore, we found that BACE1 protein is ubiquitinated, and the degradation of BACE1 proteins and amyloid precursor protein processing are regulated by the ubiquitin-proteasome pathway.

Topics: Acetylcysteine; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Analysis of Variance; Aspartic Acid Endopeptidases; Cell Line, Tumor; Cloning, Molecular; Cysteine Proteinase Inhibitors; Endopeptidases; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Gene Expression Regulation; Humans; Isoquinolines; Mutagenesis; Peptide Fragments; Peptide Hydrolases; Phosphopyruvate Hydratase; Promoter Regions, Genetic; Sp1 Transcription Factor; Time Factors; Transfection

Previous studies have implicated the failure to degrade aggregated Abeta1-42 in late endosomes or secondary lysosomes as a mechanism for the accumulation of beta-amyloid in Alzheimer's disease. We examined the consequences of intracellular accumulation of Abeta1-42 on the integrity of the endosomal/lysosomal compartment by monitoring Lucifer Yellow fluorescence and the release of lysosomal hydrolases into the soluble, cytosolic fraction. In control cells, the Lucifer Yellow fluorescence is observed as punctate staining in a perinuclear distribution with no apparent cytoplasmic fluorescence, consistent with its localization in late endosomes or secondary lysosomes. After incubation with Abeta1-42 for 6 hr, a loss of lysosomal membrane impermeability is observed as evidenced by redistribution of the fluorescence to a diffuse, cytoplasmic pattern. The loss of lysosomal membrane impermeability is correlated with Abeta1-42 accumulation, since incubation of the cells with the nonaccumulating isoform of amyloid, Abeta1-40, does not induce leakage. The same results were obtained using the release of soluble lysosomal hydrolases, cathepsin D and beta-hexosaminidase, into the cytosol as an assay for the leakage of lysosomal contents. Together, our results suggest that the loss of lysosomal membrane impermeability may be an early event in Abeta pathogenesis, and provide an explanation for the miscompartmentalization of extracellular and cytoplasmic components observed in Alzheimer's disease (AD). The release of hydrolases may further cause the breakdown of the cytoskeleton and the blebbing of the plasma membrane, and the leakage of heparan sulfate glycosaminoglycans from the lysosome may ultimately promote the assembly of tau into neurofibrillary tangles (NFT).

Topics: Amyloid beta-Peptides; Cell Membrane Permeability; Cytosol; Endosomes; Fluorescent Antibody Technique; Fluorescent Dyes; Hydrolases; Isoquinolines; Kinetics; Lysosomes; Peptide Fragments; Tumor Cells, Cultured