ABSTRACT
BACE is an aspartyl protease that cleaves the amyloid precursor protein (APP) at the beta-secretase cleavage site and is involved in Alzheimer's disease. The aim of our study was to determine whether BACE affects the processing of the APP homolog APLP2. To this end, we developed BACE knockout mice with a targeted insertion of the gene for beta-galactosidase. BACE appeared to be exclusively expressed in neurons as determined by differential staining. BACE was expressed in specific areas in the cortex, hippocampus, cerebellum, pons, and spinal cord. APP processing was altered in the BACE knockouts with Abeta levels decreasing. The levels of APLP2 proteolytic products were decreased in BACE KO mice, but increased in BACE transgenic mice. Overexpression of BACE in cultured cells led to increased APLP2 processing. Our results strongly suggest that BACE is a neuronal protein that modulates the processing of both APP and APLP2.
Subject(s)
Amyloid beta-Protein Precursor/metabolism , Aspartic Acid Endopeptidases/metabolism , Brain Chemistry/genetics , Brain/enzymology , Nerve Tissue Proteins/metabolism , Alzheimer Disease/enzymology , Alzheimer Disease/genetics , Alzheimer Disease/physiopathology , Amyloid Precursor Protein Secretases , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Aspartic Acid Endopeptidases/deficiency , Aspartic Acid Endopeptidases/genetics , Brain/pathology , Brain/physiopathology , Cells, Cultured , Disease Models, Animal , Down-Regulation/genetics , Endopeptidases , Genes, Reporter/genetics , Genetic Vectors/genetics , Humans , Mice , Mice, Knockout , Mice, Transgenic , Nerve Tissue Proteins/genetics , Neurons/enzymology , Neurons/pathology , Transfection , beta-Galactosidase/geneticsABSTRACT
FE65 binds to the Alzheimer amyloid precursor protein (APP), but the function of this interaction has not been identified. Here, we report that APP and FE65 are involved in regulation of cell movement. APP and FE65 colocalize with actin and Mena, an Abl-associated signaling protein thought to regulate actin dynamics, in lamellipodia. APP and FE65 specifically concentrate with beta 1-integrin in dynamic adhesion sites known as focal complexes, but not in more static adhesion sites known as focal adhesions. Overexpression of APP accelerates cell migration in an MDCK cell wound--healing assay. Coexpression of APP and FE65 dramatically enhances the effect of APP on cell movement, probably by regulating the amount of APP at the cell surface. These data are consistent with a role for FE65 and APP, possibly in a Mena-containing macromolecular complex, in regulation of actin-based motility.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Protein Precursor/metabolism , Cell Movement/physiology , Cytoskeletal Proteins , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Actins/metabolism , Amyloid beta-Protein Precursor/pharmacology , Animals , Carrier Proteins/metabolism , Cell Adhesion/physiology , Cell Compartmentation/physiology , Cell Line , Cell Movement/drug effects , Dogs , Focal Adhesions/metabolism , Humans , Integrin beta1/metabolism , Macromolecular Substances , Microfilament Proteins , Nerve Tissue Proteins/pharmacology , Nuclear Proteins/pharmacology , Protein Binding/physiology , Protein Transport/physiology , Pseudopodia/metabolismABSTRACT
The principal component of Alzheimer's amyloid plaques, Abeta, derives from proteolytic processing of the Alzheimer's amyloid protein precursor (APP). FE65 is a brain-enriched protein that binds to APP. Although several laboratories have characterized the APP-FE65 interaction in vitro, the possible relevance of this interaction to Alzheimer's disease has remained unclear. We demonstrate here that APP and FE65 co-localize in the endoplasmic reticulum/Golgi and possibly in endosomes. Moreover, FE65 increases translocation of APP to the cell surface, as well as both alphaAPPs and Abeta secretion. The dramatic (4-fold) FE65-dependent increase in Abeta secretion suggests that agents which inhibit the interaction of FE65 with APP might reduce Abeta secretion in the brain and therefore be useful for preventing or slowing amyloid plaque formation.
Subject(s)
Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Nerve Tissue Proteins/metabolism , Nuclear Proteins/metabolism , Animals , Binding Sites , Biological Transport , Cell Membrane/metabolism , Cells, Cultured , Dogs , Enzyme-Linked Immunosorbent Assay , Neurons/metabolism , Organelles/metabolism , Phosphorylation , Protein Binding , Rabbits , Rats , TransfectionABSTRACT
In order to localize amyloid protein precursor (APP) in nerve terminals, we have immunoisolated vesicular organelles from nerve terminal preparations using antibodies to Rab5 and synaptophysin. These immunoisolates were then analyzed by electron microscopy and by immunoblotting. The synaptophysin immunoisolates represented a nearly homogeneous population of small synaptic vesicles, with less than 10% contamination by other organelles, and very little APP. In contrast, Rab5 immunoisolates contained, in addition to small synaptic vesicles, substantial numbers of large uni- and bilamellar vesicles and high levels of APP. Thus, it appears that nerve terminal APP is contained predominantly in large vesicular organelles, distinct from synaptic vesicles and from the synaptic vesicle recycling pathway.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Protein Precursor/chemistry , GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/metabolism , Synaptic Vesicles/chemistry , Animals , Brain Chemistry , PC12 Cells , Rats , rab5 GTP-Binding ProteinsABSTRACT
The N-methyl-D-aspartate (NMDA)/phencyclidine (PCP) receptor from rat forebrain was solubilized with sodium cholate and purified by affinity chromatography on amino-PCP-agarose. A 3700-fold purification was achieved. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol revealed four major bands of Mr 67,000, 57,000, 46,000, and 33,000. [3H]Azido-PCP was irreversibly incorporated into each of these bands after UV irradiation. The dissociation constant (Kd) of [1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) binding to the purified NMDA/PCP receptor was 120 nM. The maximum specific binding (Bmax) for [3H]TCP binding was 3.3 nmol/mg of protein. The pharmacological profile of the purified receptor complex was similar to that of the membranal and soluble receptors. The binding of [3H]TCP to the purified receptor was modulated by the NMDA receptor ligands glutamate, glycine, and NMDA.
