A two-site ELISA and a bioassay were used to detect NGF-like activity in human brain tissue. Both assays detected mouse NGF and recombinant human NGF with approximately equal sensitivity, whereas the antibodies showed little cross-reactivity with the recombinant human proteins NT-3 and brain-derived neurotrophic factor. NGF-like activity was detected in fresh human cortical samples obtained from epileptic patients, with the highest activity observed in the right hemisphere of men. NGF-like activity was subsequently measured in autopsy samples of frontal and occipital cortex from patients with Alzheimer's disease (AD) and from individuals with no history or pathological evidence of AD. Based on both the ELISA and the bioassay measurements, NGF-like activity was significantly elevated in both brain regions in AD. These results demonstrate the feasibility of detecting NGF-like activity in both fresh and postmortem human brain tissue and further suggest that AD is characterized by increased, rather than decreased, levels of cortical beta-NGF. The AD-related increase in NGF may be a consequence of degenerative changes in the basal forebrain cholinergic system.
We previously demonstrated that in Chinese hamster ovary cells scavenger receptor, class B, type I-dependent selective cholesteryl ester uptake occurs in caveolae. In the present study we hypothesized that cholesteryl ester is transported from caveolae through the cytosol to an internal membrane by a caveolin chaperone complex similar to the one we originally described for the transport of newly synthesized cholesterol. Caveolae are plasma membrane domains found in most types of cells and are identified biochemically by the presence of a 22-kDa protein called caveolin (1). Caveolin plays a pivotal role in the formation, structural integrity, and function of caveolae (1). Caveolin has multiple functions, but the function of caveolin relevant to the present studies is its role in the trafficking of intracellular sterol. Caveolin can directly bind to cholesterol (2, 3), and in an earlier study, we demonstrated that acylation of caveolin was required for the binding of cholesterol to caveolin (4). We speculated that the acylation of caveolin, which occurs adjacent to the hydrophobic membrane domain of caveolin, forms a binding pocket that sequesters cholesterol from the aqueous environment (4). These studies were extended to demonstrate that caveolin is part of a lipid-protein chaperone complex that transports newly synthesized cholesterol from the endoplasmic reticulum directly to caveolae (2). The lipid-protein complex consists of cholesterol, caveolin, heat shock protein 56 (HSP56), 1 cyclophilin 40, and cyclophilin A. Acylation of caveolin at cysteine residues 143 and 156, but not at 133, was required for cholesterol to associate with caveolin (4) and for the assembly of the lipid-protein complex. The lipid-protein complex rapidly (ϳ10 min) transported newly synthesized cholesterol to caveolae where it remained for some time before diffusing throughout the plasma membrane (2) or was effluxed to extracellular acceptors (5, 6). Pharmacological disruption of the lipid-protein complex with cyclosporin A or rapamycin prevented the rapid translocation of newly synthesized cholesterol to caveolae and resulted in a net decrease in the mass of cholesterol associated with caveolae over time. The decrease in caveolae cholesterol mass was presumably caused by the diffusion of cholesterol from caveolae to the rest of the plasma membrane without replenishing the cholesterol from intracellular stores.We recently demonstrated that caveolae in CHO cells are also involved in the selective uptake of exogenous cholesteryl esters from HDL (7,8). Selective uptake refers to the uptake of cholesteryl esters without the internalization and degradation of the entire lipoprotein particle (9). The scavenger receptor, class B, type I (SR-BI) is a physiological receptor for HDL that facilitates the selective uptake of HDL cholesteryl esters (10). Babitt et al. (11) demonstrated that SR-BI is preferentially localized to caveolae in CHO cells, and we recently demonstrated that HDL-derived cholesteryl ester is initially transferred to caveolae...
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