27 July 2007. Statin supporters got a boost last week when researchers reported that simvastatin may reduce the risk for AD by an impressive 50 percent (see ARF related news story). But does this have anything to do with cholesterol? While statins are adept at lowering this lipid in the blood, the link between serum cholesterol and AD is tenuous at best, and in fact reduced plasma cholesterol may even be a risk for dementia (see Stewart et al., 2007). Likewise, there is no strong link between brain cholesterol and AD (see Wood et al., 2005) and it is unclear if statins, which poorly penetrate the blood-brain barrier (BBB), even lower brain cholesterol. But there is more to the statin story than steroids. In the July 23 Journal of Biological Chemistry, Gary Landreth and colleagues report that small amounts of statins, about the same found in the brain after oral administration, are sufficient to alter the biology of another group of essential lipids, isoprenoids. Even at these low, physiological doses, statins reduce isoprenylation of key proteins, such as Rac, and reduce production of Aβ by cultured neurons, according to the study. The results may help explain why simvastatin, the most BBB penetrable, reduces the risk for AD.
“We felt that the epidemiological effects of statins are robust but the link to hypercholesterolemia is not absolutely clear, so we’ve been exploring isoprenoid-dependent effects thinking this may be where the biological effects are,” said Landreth, Case Western Reserve University, Cleveland, Ohio, in an interview with ARF. Isoprenoid synthesis, like that of cholesterol, depends on the enzyme HMG-CoA reductase, which is inhibited by statins. The isoprenoids, including geranylgeranyl pyrophosphate and farnesyl pyrophosphate, modify small GTPases, such as those of the Rac and Rab families, regulating their integration into cell membranes and their interactions with protein partners. Rab family members have been linked to trafficking of amyloid-β (Aβ) precursor protein (see McConlogue et al., 1996) and more recently to an APP signaling pathway that regulates apoptosis (see Laifenfeld et al., 2007). “The big surprise was that statins preferentially affect only a subset of these G proteins,” said Landreth.
The researchers discovered the differential effect on GTPases when they administered physiologically relevant doses of statins to cultured neurons. “One of the things that really struck us was that despite statins being the most heavily prescribed drugs in the world, nobody really knew what concentrations they reached in the brain until earlier this year,” said Landreth. It turns out that with normal dosing statins reach the 300-500 nM range in the brain, but few studies have been done on statins at those concentrations, Landreth said. To address this, first author Stephen Ostrowski and colleagues examined the effect of a range of statin concentrations on cultured neurons. “What Steve was able to show was that if you lower the statin concentration down into the physiological range, you only affect a limited subset of proteins. Rac1 and Rab1b, in particular, appear to be exquisitely sensitive to statins at those concentration ranges,” said Landreth.
Ostrowski and colleagues found that at high (10 micromolar) doses, statins reduce the electrophoretic mobility of Rab family proteins, which are modified by two geranylgeranyl groups. Rho and Rac mobility was unaffected, which probably reflects the fact that these proteins are modified by only one lipid moiety. But both simvastatin and lovastatin reduced the cell membrane association of all GTPases tested, including Rac, Rho A, Cdc42, and Rab family members (Rab1b, Rab4, Rab5b, and Rab6). However, at lower concentrations (200 nanomolar), simvastatin impaired only the association of Rac and Rab1b with the cell membrane—by about 40 percent in the case of Rac. “The mechanistic basis for why these nominally similar proteins are so dramatically different in their sensitivity to drugs is not at all clear, but what that says is, in vivo, the biology of statins will hinge on a relatively small group of G proteins, rather than being due to a class effect,” said Landreth.
How might statins affect AD pathology? Previously, work from Sam Gandy’s group at the Thomas Jefferson University, Philadelphia, showed that statins might actually increase APP synthesis in neurons (see ARF related news story). Ostrowski and colleagues were able to confirm that finding, but they also found that the effect is limited to N2a-Swe cells, which harbor a construct driving expression of human APP with the Swedish mutation. In several other cell types, including wild-type N2a and H4 neuroglioma cells, statins had no effect on APP synthesis. “We don’t have a good explanation for that,” said Landreth, but he suggested it may be an artifact of the cell line and that it will help clear up some controversy in the literature. Bob Vassar’s group at Northwestern University, Chicago, had failed to find increased synthesis of APP in statin-treated cells, for example (see ARF related news story).
Though Vassar’s group found that statins had no effect on APP synthesis, they did find that the drugs increased intracellular levels of APP. The current paper supports that finding. Ostrowski and colleagues found that statins affect APP trafficking. They found that in N2a wild-type cells, high doses of simvastatin or lovastatin increased the cellular amount of APP and also of α and β C-terminal fragments (CTFs), the products of α- and β-secretase, respectively. The APP accumulation could be reversed by adding mevalonate, an isoprenoid precursor, or geranylgeranyl pyrophosphate, but not cholesterol. When the authors specifically inhibited Rho family proteins with Toxin A, APP levels were unaltered, suggesting that the statin effect is not mediated through Rho GTPases. But statins also reduced the secretion of Aβ by about 40 percent. “We conclude, that as APP is trafficked within the cell through Rab-dependent mechanisms, it is likely that inhibition of Rab isoprenylation by statins alters APP trafficking leading to APP accumulation,” write the authors.
The story is slightly more complex, however, because the authors found that in H4 cells expressing human APP (H4.APPWT and H4.HPLAP), statins reduced the level of CTFs, as did Toxin A, suggesting that Rho contributes to CTF stability. To investigate this, the authors treated H4 cells with Toxin A or statins in the presence of either the proteasome inhibitor MG132 or the lysosomal inhibitor chloroquine. The latter blocked Toxin A-induced CTF degradation, suggesting that Rho family proteins are involved in processing CTFs through the lysosome. “RhoA appears to be constitutively suppressing lysosomal processing of CTFs and when you remove RhoA you accelerate lysosomal degradation,” said Landreth. He also noted that though purely a phenomenological finding, this is one that he plans to pursue. While it may not lead to new therapeutic targets—researchers have tried unsuccessfully to develop farnesyltransferase inhibitors for cancer, for example—it should at least help explain the pleiotropic effects of statins.—Tom Fagan.
Ostrowski SM, Wilkinson BL, Golde TE, Landreth G. Statins reduce amyloid-beta production through inhibition of protein isoprenylation. J. Biol. Chem. 2007, July 23. Abstract