Involvement of oxidative stress-induced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer's disease

Abstract

Alzheimer's disease (AD) is an age-related disorder characterized by deposition of amyloid beta-peptide (Abeta) and degeneration of neurons in brain regions such as the hippocampus, resulting in progressive cognitive dysfunction. The pathogenesis of AD is tightly linked to Abeta deposition and oxidative stress, but it remains unclear as to how these factors result in neuronal dysfunction and death. We report alterations in sphingolipid and cholesterol metabolism during normal brain aging and in the brains of AD patients that result in accumulation of long-chain ceramides and cholesterol. Membrane-associated oxidative stress occurs in association with the lipid alterations, and exposure of hippocampal neurons to Abeta induces membrane oxidative stress and the accumulation of ceramide species and cholesterol. Treatment of neurons with alpha-tocopherol or an inhibitor of sphingomyelin synthesis prevents accumulation of ceramides and cholesterol and protects them against death induced by Abeta. Our findings suggest a sequence of events in the pathogenesis of AD in which Abeta induces membrane-associated oxidative stress, resulting in perturbed ceramide and cholesterol metabolism which, in turn, triggers a neurodegenerative cascade that leads to clinical disease.

Fig. 1.

Levels of ceramides and cholesterol increase in the brain during normal aging. (A and B) Representative electrospray tandem MS spectrograms showing relative levels of different ceramides in samples of cerebral cortex from a 3-month-old (A) and a 25-month-old (B) mouse. (C) Levels of sphingomyelin C24:0, ceramide C24:0, galactosyl-ceramide, and free cholesterol in samples of cerebral cortex from 3-, 6-, and 25-month-old mice. Values are the mean and SEM (five mice of each age). *, P < 0.01 compared with the value for 3-month-old mice. ##, P < 0.01 compared with the value for 6-month-old mice and P < 0.001 compared with the value for 3-month-old mice.

Fig. 2.

Levels of ceramides and cholesterol increase in the brain in association with oxidative stress in AD. (A) Levels of sphingomyelin C24:0, ceramide C24:0, and free cholesterol in samples of middle frontal gyrus and cerebellum from AD- and age-matched control patients. (B) Levels of HNE adducts in samples of middle frontal gyrus and cerebellum from AD- and age-matched control patients. Values are the mean and SEM (seven control and seven AD patients). **, P < 0.01; ***, P < 0.001 compared with the value for control subjects and with the value in cerebellum of AD patients.

Fig. 3.

Levels of membrane-associated ceramides and free cholesterol are increased in association with disease severity in AD. Levels of sphingomyelins (A), ceramides and sulfatide (B), and free cholesterol (C) were quantified in isolated membrane raft preparations of frontal cortex from AD patients with mild, moderate, and severe cognitive impairment and age-matched control patients. Values are the mean and SEM. *, P < 0.05; **, P < 0.01 compared with the value for control subjects; #, P < 0.01 compared with the value for patients with moderate disease severity.

Fig. 4.

Pathways of metabolism of sphingomyelin, ceramide, and cholesterol, their modulation by oxidative stress, and their possible roles in neuronal death in AD. The production of sphingomyelin from serine and palmitoyl CoA is catalyzed by the enzyme serine palmitoyl CoA-transferase (SPT). Ceramides are synthesized as precursors to sphingomyelin and are also generated by the hydrolysis of sphingomyelin by sphingomyelinases (SMase). Exposure of cells to reactive oxygen species and Aβ induces ceramide production. CAPK, ceramide-activated protein kinase; CAPP, ceramide-activated protein phosphatase.

Fig. 5.

Inhibitors of ceramide production protect neurons against death induced by amyloid β-peptide and membrane oxidative stress. (A) Cultured hippocampal neurons were pretreated for 30 min with 10 or 50 nM ISP-1 or vehicle and were then exposed for 24 h to 5 μMAβ1–42, 1 μM HNE, or saline (control), and the numbers of neurons with apoptotic nuclei were quantified. Values are the mean and SE of determinations made in four to eight separate cultures. (B) Model for the role of altered membrane lipid metabolism in the pathogenesis of AD. The aging process, in combination with genetic and environmental factors, results in increased membrane-associated oxidative stress and increased production and accumulation of neurotoxic forms of Aβ, which itself exacerbates oxidative stress. The oxidative stress perturbs cholesterol metabolism and activates sphingomyelinases, resulting in increased ceramide production. The increased levels of ceramide and cholesterol may trigger synaptic dysfunction and neuronal death.