A detergent-insoluble membrance compartment contains Aβ in vivo

Abstract

Ordered assembly of the amyloid-beta protein (A beta) into amyloid fibrils is a critical step in Alzheimer's disease (AD). To release the amyloidogenic peptide A beta from the Alzheimer amyloid precursor protein (APP), two secretases act sequentially: first, beta-secretase cleaves close to the membrane within the ectodomain and then gamma-secretase cuts within the transmembrane domain. The sites of gamma-secretase cleavage are after residues 40 or 42 of A beta. Except in those rare cases of AD caused by a mutation, levels of secreted A beta are not elevated; thus, the secretory pathway may be unaffected, and factors other than the extracellular concentration of A beta may contribute to the aggregation properties of the peptide. A beta is also present in intracellular compartments. The two gamma-secretase cleavage products, A beta42 and A beta40, were found in different compartments: A beta42 in the endoplasmic reticulum (ER)/intermediate compartment, and A beta40 in the trans-Golgi network (TGN). The cellular compartments that harbor A beta are target sites for therapeutic intervention. Here we report that in the brain, the principal compartment in which A beta resides is a detergent-insoluble glycolipid-enriched membrane domain (DIG). Also present in the DIG fractions are the endoproteolytic fragments of presenilin-1 (PS1) and APP. The presence of these proteins, which all contribute to the generation of A beta, indicates that the DIG fraction is probably where the intramembranous cleavage of APP occurs.

Amyloid precursor protein, although partially detergent-insoluble in mouse cerebral cortex, behaves as an atypical lipid raft protein

“…Thus, although APP, alkaline phosphodiesterase I, aminopeptidase A and clathrin are present in DIGs isolated from mouse cerebral cortex, they do not behave as typical DIG proteins. The observation that altering the protein\ detergent ratio used during solubilization of the membranes can alter the apparent distribution of APP and other atypical DIGs proteins in the sucrose density gradients may account for the apparently conflicting reports on the presence of APP in DIGs [27,31,32,[35][36][37][38]. One possible explanation to reconcile these conflicting data is that APP, alkaline phosphodiesterase I and clathrin are ineffectively solubilized contaminants of the DIGs fraction.…”

“…More recent studies have reported that a minor proportion of the total cellular APP is located in DIGs isolated either from APP-transfected rat hippocampal neurons [37] or from rat brain cortical grey matter [31]. It has been reported also that a large proportion of Aβ, along with various levels of presenilin-1 and presenilin-2, are present in DIGs isolated from either brain tissue or neuronal cell cultures [31,32,40]. The presence of these Alzheimer's-disease-related proteins in DIGs has led to the hypothesis that lipid rafts may be a site for the proteolytic processing of APP [31,36].…”

“…Essentially all of the GPI-anchored alkaline phosphatase activity resided in the DIGs (fractions 6-10 of the sucrose gradient) irrespective of the protein\detergent ratio used during solubilization ( Figure 1B). As caveolin-1 is absent from neuronal tissue, flotillin, which has been shown previously to be present in DIGs isolated from bovine cortical grey matter [30], rat cortical grey matter [31] and human neuroblastoma SH-SY5Y cells [32], was employed as a transmembrane protein marker for the detection of DIGs in the sucrose gradients. Flotillin was present exclusively in the DIGs region of the sucrose gradient (fractions 6-10), irrespective of the protein\detergent ratio used during solubilization ( Figure 1C).…”

“…Despite the absence of caveolin, DIGs can be isolated from neuronal sources and are found to contain a number of GPI-anchored proteins, such as alkaline phosphatase, 5h-nucleotidase, F3 protein, the prion protein [24][25][26][27], tyrosine kinases, and α-and β-subunits of heterotrimeric G-proteins [28,29]. Another protein, flotillin, has been found to be present exclusively in DIGs isolated from neuronal sources and can, therefore, be used in place of caveolin as a transmembrane polypeptide-anchored marker for these membrane domains [30][31][32].…”

“…Currently there are conflicting reports as to whether APP is present in lipid rafts, where its processing by the secretases may occur. Some studies have reported that a minor amount of APP is present in DIGs [31,[35][36][37], while others have failed to detect it in such fractions [27,32,38].…”