Metabolite-initiated protein misfolding may trigger Alzheimer's disease

Abstract

Anfinsen showed that a protein's fold is specified by its sequence. Although it is clear why mutant proteins form amyloid, it is harder to rationalize why a wild-type protein adopts a native conformation in most individuals, but it misfolds in a minority of others, in what should be a common extracellular environment. This discrepancy suggests that another event likely triggers misfolding in sporadic amyloid disease. One possibility is that an abnormal metabolite, generated only in some individuals, covalently modifies the protein or peptide and causes it to misfold, but evidence for this is sparse. Candidate metabolites are suggested by the recently appreciated links between Alzheimer's disease (AD) and atherosclerosis, known chronic inflammatory metabolites, and the newly discovered generation of ozone during inflammation. Here we report detection of cholesterol ozonolysis products in human brains. These products and a related, lipid-derived aldehyde covalently modify A␤, dramatically accelerating its amyloidogenesis in vitro, providing a possible chemical link between hypercholesterolemia, inflammation, atherosclerosis, and sporadic AD. Anfinsen's classic experiments demonstrated that a protein's amino acid sequence specifies its conformation (1). These ideas were extended to explain the misfolding susceptibility of mutant proteins associated with a growing number of familial amyloid diseases (2-5). Although it is thus clear why mutant proteins might be more susceptible to misfolding, it is harder to understand why a wild-type protein or peptide adopts a native conformation in some individuals but it misfolds in others in what should be a common extracellular environment, leading to sporadic amyloid diseases. This discrepancy suggests that other events likely trigger misfolding in sporadic amyloid disease, but their nature remains elusive.The misfolding of secreted amyloid ␤ peptides (A␤) 39-43 residues in length is linked by a plethora of evidence to the pathology of Alzheimer's disease (AD) (6, 7). A␤ misfolding occurs when the soluble, monomeric, extracellular ensemble of extended conformations and low M r oligomers is transformed first into spherical assemblies, then into a number of intermediates, and lastly into fibrillar cross ␤-sheet quaternary structures known as amyloid (8)(9)(10)(11)(12). Amyloid fibrils and related structures recruit soluble A␤ to the aggregate by a seeded polymerization mechanism (10). The direct neurotoxicity of A␤ aggregates (8, 13) combined with their role in mediating chronic inflammation by microglia (14) and complement cascade activation (15) suggests that aggregation then mediates inflammation (16), which in turn promotes aggregation, in a vicious cycle of AD pathology.It is known that atherosclerosis and AD share many risk factors, including hypercholesterolemia and inflammation. The apoE-4 allele, which exacerbates hypercholesterolemia, has been linked to AD by data from both epidemiological and transgenic mouse studies (17)(18)(19)(20). It has also recently been shown t...

Analysis of oxysterols by electrospray tandem mass spectrometry

“…It is not known if some of these occur naturally, and there is a need for methods to make comprehensive studies of the nature and levels of oxysterols in tissues and body fluids. This is illustrated by the recent identification of novel oxysterols, formed from cholesterol by the action of ozone, in human tissues [27][28][29][30]. Evidence has been presented for their involvement in the mediation of inflammatory processes associated with the development of atherosclerotic lesions [28], and in the initiation of protein misfolding as occurs in several neurological diseases [29].…”

“…This is illustrated by the recent identification of novel oxysterols, formed from cholesterol by the action of ozone, in human tissues [27][28][29][30]. Evidence has been presented for their involvement in the mediation of inflammatory processes associated with the development of atherosclerotic lesions [28], and in the initiation of protein misfolding as occurs in several neurological diseases [29]. Of these products, 3␤-hydroxy-5-oxo-5,6-secocholestan-6-al (5,6-seco-sterol), a ketoaldehyde, and its aldol condensation product both contain a reactive aldehyde group (Scheme 1).…”

“…Of these products, 3␤-hydroxy-5-oxo-5,6-secocholestan-6-al (5,6-seco-sterol), a ketoaldehyde, and its aldol condensation product both contain a reactive aldehyde group (Scheme 1). Incubation of 5,6-secosterol, or its aldol, with amyloid ␤ peptide (A␤) leads to amyloidogenesis, possibly by condensation with amino groups in the peptide [29]. The levels of the 5,6-secosterol and its aldol were surprisingly high in brain of patients with Alzheimer's disease and healthy subjects [combined seco-sterol levels ϳ0.44 pmol/mg (ϳ180 ng/g) and ϳ0.35 pmol/mg (ϳ150 ng/g), respectively].…”

Site-specific modification of Alzheimer's peptides by cholesterol oxidation products enhances aggregation energetics and neurotoxicity

“…We have proposed that reversible covalent modification of A␤ by small molecule oxidation products, in combination with factors like adsorption to the extracellular matrix or membranes (15), can explain the ability of A␤ to form amyloid at physiologic concentrations (16)(17)(18). Small molecule oxidation products are generated when reactive oxygen species react with double bonds, including those of hydrophobic membrane components (19).…”

“…1 A) (24), covalently modify A␤ and increase its amyloidogenicity (16)(17)(18)(25)(26)(27). Prior data indicated that cholesterol oxidation products 1 and 2, denoted 1(2) hereafter because they interconvert via an aldol/retro-aldol reaction (16,18,24), react with A␤ via Schiff base formation at the N-terminal ␣-amine of Asp-1 (D1) and the side-chain -amines of Lys-16 (K16) and Lys-28 (K28) (18). Compounds 1 and 2 have been detected by us (18,28) and others (29) in ex vivo human and rat brain samples at combined concentrations of up to 400 pg/mg of wet brain (Ϸ1 M concentration).…”