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The amyloid precursor protein (APP) of Alzheimer's disease has been shown to stimulate neurite outgrowth in vitro. The effect of APP on neurite outgrowth can be enhanced if APP is presented to neurons in substrate-bound form, in the presence of heparan sulfate proteoglycans. To identify specific heparan sulfate proteoglycans that bind to APP, conditioned medium from neonatal mouse brain cells was subjected to affinity chromatography with recombinant APP695 as a ligand. Glypican bound strongly to the APP affinity column. Purified glypican bound to APP with an equilibrium dissociation constant of 2.8 nM and inhibited APP-induced neurite outgrowth from chick sympathetic neurons. The effect of glypican was specific for APP, as glypican did not inhibit laminin-induced neurite outgrowth. Furthermore, treatment of cultures with 4-methylumbelliferyl-beta-D-xyloside, a competitive inhibitor of proteoglycan glycanation, inhibited APP-induced neurite outgrowth but did not inhibit laminin-induced neurite outgrowth. This result suggests that endogenous proteoglycans are required for substrate-bound APP to stimulate neurite outgrowth. Secreted glypican may act to inhibit APP-induced neurite outgrowth in vivo by competing with endogenous proteoglycans for binding to APP.

The importance of the amyloid precursor-protein (APP) in the pathogenesis of Alzheimer's disease (AD)became apparent through the identification of distinct mutations in the APP gene, causing early onset familial AD with the accumulation of a 4-kDa peptide fragment ([Beta]A4) in amyloidplaques and vascular deposits. However, the physiological role of APP is still unclear. In this work, Drosophila melanogasteris used as a model system to analyze the function of APP by expressing wild-type and various mutant forms of human APP in fly tissue culture cells as well as in transgenic fly lines. After expression of full-length APP forms, secretion of APP but not of [Beta]A4 was observed in both systems. By using SPA4CT, a short APP form in which the signal peptide was fused directly to the [Beta]A4 region, transmembrane domain, and cytoplasmictail, we observed [Beta]A4 release in flies and fly-tissue culture cells. Consequently, we showed a [Gamma]-secretase activity in flies. Interestingly, transgenic flies expressing full-length forms of APP have a blistered-wing phenotype. As the wing is composed of interacting dorsal and ventral epithelial cell layers, this phenotype suggests that human APP expression interferes with cell adhesion/signaling pathways in Drosophila, independently of [Beta]A4 generation.

The amyloid precursor protein (APP) plays a crucial role in the pathogenesis of Alzheimer's disease. During intracellular transport APP undergoes a series of proteolytic cleavages that lead to the release either of an amyloidogenic fragment called [Beta]-amyloid (A[Beta]) or of a nonamyloidogenic secreted form consisting of the ectodomain of APP ([APP.sub.sec]). It is A[Beta] that accumulates in the brain lesions that are thought to cause the disease. By reducing the cellular cholesterol level of living hippocampal neurons by 70% with lovastatin and methyl-[Beta]-cyclodextrin, we show that the formation of A[Beta] is completely inhibited while the generation of [APP.sub.sec] is unperturbed. This inhibition of A[Beta] formation is accompanied by increased solubility in the detergent Triton X-100 and is fully reversible by the readdition of cholesterol to previously depleted cells. Our results show that cholesterol is required for A[Beta] formation to occur and imply a link between cholesterol, A[Beta], and Alzheimer's disease.

Cerebral plaques containing [Beta]-amyloid ([Beta]A4) represent an invariant pathological feature of Alzheimer disease (AD). [Beta]A4 is proteolytically generated from its parent molecule, amyloid precursor protein (APP). In non-neuronal cells [Beta]A4 has been shown to be secreted via a pH-sensitive and endocytosis-dependent pathway, and this process, when occurring in the brain, is considered to play an important role in AD. In neurons the mechanisms of [Beta]A4 production are not known. Here we have analyzed these mechanisms by expressing human APP and its mutant versions in hippocampal neurons using the Semliki forest virus system. We show that these cells initially generate two pools of [Beta]A4, an extracellular and an intracellular, and only the extracellular pool is produced via a pH-sensitive and endocytosis-dependent pathway. Thus, hippocampal neurons are able to utilize an alternate pathway to produce intracellular [Beta]A4. We also show that a common feature of two types of APP mutations ('Swedish' and 'London') implicated in early-onset AD is their increased production of C-terminally elongated [Beta]A4 ([[Beta].sub.42]), both intra- and extracellularly. Since neurons are the only cells that produce substantial levels of intracellular [Beta]A4 and also the main victims in AD, these findings may provide an important link between [Beta]A4 and neurodegeneration.

A[β.sub.1-40], a major component of Alzheimer's disease cerebral amyloid, is present in the cerebro spinal fluid and remains relatively soluble at high concentrations (less than or equal to 3.7 mM). Thus, physiological factors which induce Aβ amyloid formation could provide clues to the pathogenesis of the disease. It has been shown that human Aβ specifically and saturably binds zinc. Here, concentrations of zinc above 300 nM rapidly destabilized human A[β.sub.1-40] solutions, inducing tinctorial amyloid formation. However, rat Aβ.sub. zinc less avidly and is immune to these effects, perhaps explaining the scarcity with which these animals form cerebral Aβ amyloid. These data suggest a role for cerebral zinc metabolism in the neuropathogenesis of Alzheimer's disease., The role of Aβ amyloid formation in the pathogenesis of Alzheimer's disease (AD) has been underscored by the discovery of mutations in the Alzheimer amyloid protein precursor (APP) close to [...]