Simvastatin strongly reduces levels of Alzheimer's disease beta -amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo

Abstract

Recent epidemiological studies show a strong reduction in the incidence of Alzheimer's disease in patients treated with cholesterol-lowering statins. Moreover, elevated Abeta42 levels and the varepsilon4 allele of the lipid-carrier apolipoprotein E are regarded as risk factors for sporadic and familial Alzheimer's disease. Here we demonstrate that the widely used cholesterol-lowering drugs simvastatin and lovastatin reduce intracellular and extracellular levels of Abeta42 and Abeta40 peptides in primary cultures of hippocampal neurons and mixed cortical neurons. Likewise, guinea pigs treated with high doses of simvastatin showed a strong and reversible reduction of cerebral Abeta42 and Abeta40 levels in the cerebrospinal fluid and brain homogenate. These results suggest that lipids are playing an important role in the development of Alzheimer's disease. Lowered levels of Abeta42 may provide the mechanism for the observed reduced incidence of dementia in statin-treated patients and may open up avenues for therapeutic interventions.

Figure 1

Cholesterol depletion reduces secretory and intracellular Aβ levels in hippocampal neurons expressing SFV-APP695 or the “Swedish” mutant SFV-APP695sw. Hippocampal neurons were grown for 2 days in the presence of lovastatin/mevalonate. Next, cells were infected with SFV-APP695 or SFV-APP695sw and were treated after infection with 5 mM CDX for 10 min; control cells were not treated. After an additional 3.5 h, cells were lysed and whole-cell lysates were analyzed. (a) Reduced Aβ levels from APP695-expressing cells. Immunoprecipitation and Western blot analysis of Aβ from untreated cells and lovastatin/mevalonate- and CDX-treated cells (depletion). Lanes 1, 5, 8, and 10 show Aβ40 precipitates (antibody G2–10); lanes 2, 4, and 7 show Aβ42 precipitates (antibody G2–11); and lanes 3 and 6 show Aβ from directly loaded conditioned media. Intracellular Aβ was below detection limit without immunoprecipitation. Please note that different sample volumes were loaded for Aβ40 and Aβ42. Lanes 11–13 show calibration Aβ samples for quantification. Aβ was detected with human anti-Aβ-1–16 (W02). A strong reduction of secreted Aβ is observed in lanes 1–3 and 7 and 8 as compared with untreated cells in lanes 4–6 and 9 and 10. (b) Reduced Aβ levels and effect on C-terminal fragments from APP695sw. Cells were metabolically labeled for 2.5 h after infection. The lower gel shows reduced levels of total secretory Aβ from treated vs. untreated cells (immunoprecipitated with antibody B7/6). The upper gel shows immunoprecipitated APP C-terminal fragments from cell homogenates detected by antibody B12/4. Fragments presumably generated by α- and β-secretase cleavage are indicated. Unlike peripheral cells, hippocampal neurons prefer β-secretase-mediated cleavage of APP over α-secretase-mediated cleavage; this effect is enhanced by the use of the preferentially β-secretase-cleaved APP695sw (APP_sw). Upon cholesterol depletion, levels of β-cleaved fragment were highly reduced, indicating an inhibition of β-secretase activity. (c) Quantification of intracellular and secretory Aβ40 and Aβ42 levels (n = 7, n = 9, respectively). (d) Quantification of APP695sw C-terminal fragments and secretory Aβ (n = 7, n = 9, respectively). (e) Quantitative calibration standard used for Western blot detection, lanes 11–13 in a. (f) Lovastatin and simvastatin without CDX are sufficient to reduce Aβ levels from primary mixed cortical neurons. Mixed cortical neurons were grown for 3 days in the presence of lovastatin or simvastatin (S or L) and mevalonate. After infection with SFV-APP695, cells were treated with 5 mM CDX for 10 min (SC or LC), not treated with CDX (S or L), or left untreated (0). After further 3.5 h cells were lysed. (Upper) Aβ40 or Aβ42 detected from conditioned media. (Lower) Aβ40 or Aβ42 detected from cell lysates (n = 6). A strong reduction of intracellular and secretory Aβ40 and Aβ42 was observed.

Figure 2

In vivo treatment of guinea pigs with simvastatin reduced total Aβ, Aβ40, and Aβ42 levels in guinea pig CSF. Aβ levels recovered after treatment was stopped. CSF was collected at day 0 and 3 from guinea pigs. After this, animals were given a simvastatin-containing diet and CSF samples were drawn twice weekly. (a) After 3 weeks simvastatin-containing food was replaced by standard diet. Aβ levels declined for the first 3 weeks and recovered subsequently. In control animals Aβ levels remained constant (n = 5). (b) In a second experiment animals were treated as before for 3 weeks. A continuous decline in all Aβ levels was found. In control animals Aβ levels remained constant. For clarity, Aβ42 and Aβ40 levels of control animals are given in a and Aβ42 and Aβ40 level measurement was extended for control animals to 6 weeks as in the previous experiment (n = 6). (c) Western blot determination of CSF Aβ levels after 3 weeks of treatment. (d) Aβ tissue levels. Aβ was precipitated with W02 antibody from detergent-lysed cortex samples. Simvastatin-treated animals showed a strong reduction of brain tissue Aβ levels equivalent to the reduction observed in the CSF in a (n = 4). Mean level of control animals was set to 100% (±22% SD; n = 6). As expected from the CSF results, the recovery animals had normal Aβ levels (n = 6). The reduction in treated as compared with control animals was significant (P = 0.012).