Inhibition of Geranylgeranylation Mediates the Effects of 3-Hydroxy-3-methylglutaryl (HMG)-CoA Reductase Inhibitors on Microglia

Abstract

Inflammatory responses involving microglia, the resident macrophages of the brain, are thought to contribute importantly to the progression of Alzheimer's disease (AD) and possibly other neurodegenerative disorders. The present study tested whether the mevalonate-isoprenoid biosynthesis pathway, which affects inflammation in many types of tissues, tonically regulates microglial activation. This question takes on added significance given the potential use of statins, drugs that block the rate-limiting step (3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase)) in mevalonate and cholesterol synthesis, in AD treatment. Both mevastatin and simvastatin caused a concentration- and time-dependent activation of microglia in cultured rat hippocampal slices. This response consisted of a transformation of the cells from a typical resting configuration to an amoeboid, macrophage-like morphology, increased expression of a macrophage antigen, and up-regulation of the cytokine tumor necrosis factor-α. Evidence for proliferation was also obtained. Statin-induced microglial changes were blocked by mevalonate but not by cholesterol, indicating that they were probably due to suppression of isoprenoid synthesis. In accord with this, the statin effects were absent in slices co-incubated with geranylgeranyl pyrophosphate, a mevalonate product that provides for the prenylation of Rho GTPases. Finally, PD98089, a compound that blocks activation of extracellularly regulated kinases1/2, suppressed statin-induced up-regulation of tumor necrosis factor-α but had little effect on microglial transformation. These results suggest that 1) the mevalonate-isoprenoid pathway is involved in regulating microglial morphology and in controlling expression of certain cytokines and 2) statins have the potential for enhancing a component of AD with uncertain relationships to other features of the disease. Inflammatory responses involving microglia, the resident macrophages of the brain, are thought to contribute importantly to the progression of Alzheimer's disease (AD) and possibly other neurodegenerative disorders. The present study tested whether the mevalonate-isoprenoid biosynthesis pathway, which affects inflammation in many types of tissues, tonically regulates microglial activation. This question takes on added significance given the potential use of statins, drugs that block the rate-limiting step (3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase)) in mevalonate and cholesterol synthesis, in AD treatment. Both mevastatin and simvastatin caused a concentration- and time-dependent activation of microglia in cultured rat hippocampal slices. This response consisted of a transformation of the cells from a typical resting configuration to an amoeboid, macrophage-like morphology, increased expression of a macrophage antigen, and up-regulation of the cytokine tumor necrosis factor-α. Evidence for proliferation was also obtained. Statin-induced microglial changes were blocked by mevalonate but not by cholesterol, indicating that they were probably due to suppression of isoprenoid synthesis. In accord with this, the statin effects were absent in slices co-incubated with geranylgeranyl pyrophosphate, a mevalonate product that provides for the prenylation of Rho GTPases. Finally, PD98089, a compound that blocks activation of extracellularly regulated kinases1/2, suppressed statin-induced up-regulation of tumor necrosis factor-α but had little effect on microglial transformation. These results suggest that 1) the mevalonate-isoprenoid pathway is involved in regulating microglial morphology and in controlling expression of certain cytokines and 2) statins have the potential for enhancing a component of AD with uncertain relationships to other features of the disease. High levels of cholesterol, and in particular of cholesterol esters (1Puglielli L. Konopka G. Pack-Chung E. Ingano L.A. Berezovska O. Hyman B.T. Chang T.Y. Tanzi R.E. Kovacs D.M. Nat. Cell Biol. 2001; 3: 905-912Crossref PubMed Scopus (384) Google Scholar), influence the generation and aggregation of β-amyloid peptides in dissociated cell cultures (2Mizuno T. Nakata M. Naiki H. Michikawa M. Wang R. Haass C. Yanagisawa K. J. Biol. Chem. 1999; 274: 15110-15114Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 3Bodovitz S. Klein W.L. J. Biol. Chem. 1996; 271: 4436-4440Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 4Urmoneit B. Turner J. Dyrks T. 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Prenylation by the mevalonate products farnesyl diphosphate and geranylgeranyl diphosphate, for example, contributes to the regulation of gene expression and cell migration in various types of tissue (18Huang K.C. Chen C.W. Chen J.C. Lin W.W. J. Biomed. Sci. 2003; 10: 396-405Crossref PubMed Google Scholar, 19Aznar S. Fernandez-Valeron P. Espina C. Lacal J.C. Cancer Lett. 2004; 206: 181-191Crossref PubMed Scopus (107) Google Scholar, 20Martin S.G. St Johnston D. Nature. 2003; 421: 379-384Crossref PubMed Scopus (256) Google Scholar, 21Thorpe J.L. Doitsidou M. Ho S.Y. Raz E. Farber S.A. Dev. Cell. 2004; 6: 295-302Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Several studies have used statins to investigate the roles of cholesterol and protein prenylation in the regulation of immune cell properties, including cell morphology, migration, and secretion of cytokines. The results have not been consistent; although some experiments found that statins induce proinflammatory responses (22Kiener P.A. Davis P.M. Murray J.L. Youssef S. Rankin B.M. Kowala M. Int. Immunopharmacol. 2001; 1: 105-118Crossref PubMed Scopus (125) Google Scholar), others indicate that the drugs have direct anti-inflammatory (23Weitz-Schmidt G. Trends Pharmacol. Sci. 2002; 23: 482-486Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar) and immunomodulatory (24Youssef S. Stuve O. Patarroyo J.C. Ruiz P.J. Radosevich J.L. Hur E.M. Bravo M. Mitchell D.J. Sobel R.A. Steinman L. Zamvil S.S. Nature. 2002; 420: 78-84Crossref PubMed Scopus (991) Google Scholar) actions. Whether statins also influence the activation of microglia, the resident cells mediating inflammatory responses in brain, is unknown, although the question is of evident importance for the use of the drugs in AD. Microglial activation, along with classic features of the immune response, including increases in proinflammatory cytokines and the presence of complement proteins, are found in the brains of patients with AD (see Ref. 25Akiyama H. Barger S. Barnum S. Bradt B. Bauer J. Cole G.M. Cooper N.R. Eikelenboom P. Emmerling M. Fiebich B.L. Finch C.E. Frautschy S. Griffin W.S. Hampel H. Hull M. Landreth G. Lue L. Mrak R. Mackenzie I.R. McGeer P.L. O'Banion M.K. Pachter J. Pasinetti G. Plata-Salaman C. Rogers J. Rydel R. Shen Y. Streit W. Strohmeyer R. Tooyoma I. Van Muiswinkel F.L. Veerhuis R. Walker D. Webster S. Wegrzyniak B. Wenk G. Wyss-Coray T. Neurobiol. Aging. 2000; 21: 383-421Crossref PubMed Scopus (3651) Google Scholar for a review). Possibly related to this, epidemiological studies suggest that non-steroidal anti-inflammatory drugs reduce the risk and slow the progression of the disease. On the other hand, accumulating evidence points to the conclusion that microglia help to clear extracellular amyloidogenic peptides, an activity that would presumably retard the development of AD-related pathology (26Brazil M.I. Chung H. Maxfield F.R. J. Biol. Chem. 2000; 275: 16941-16947Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Beneficial activation of microglia has thus been proposed as a rationale for therapeutic vaccination (27Schenk D.B. Yednock T. Neurobiol. Aging. 2002; 23 (683-674): 677-679Crossref PubMed Scopus (30) Google Scholar, 28Morgan D. Expert Rev. Vaccines. 2003; 2: 53-59Crossref PubMed Scopus (9) Google Scholar). The present study explored the effects of inhibiting HMG-CoA reductase on microglial morphology and expression of the cytokine TNF-α using a novel cultured brain slice preparation. This preparation has been used previously to study pathologies associated with the aged human brain (29Bednarski E. Ribak C.E. G. J. Neurosci. 17: PubMed Google Scholar, J. Ribak C.E. G. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar), including an microglial to of pathology as by J. G. 2002; PubMed Scopus Google Scholar) and J. J. K. G. Neurosci. 2001; Scholar). slices thus provide a with which to whether to statins brain inflammatory results indicate that statins microglial activation, as by transformation to macrophage morphology and up-regulation of the cytokine TNF-α, and that effect is due to suppression of the mevalonate pathway and of geranylgeranyl and of hippocampal cultures were using the of L. P.A. D. J. Neurosci. PubMed Scopus Google Scholar). were from brains of rat were to the of using a tissue and into a of with and were cell that were in with of and the 23 of the was from The cultures were with a and the was other slices in for were to mevastatin simvastatin were from for In some the were in the presence of mevalonate and cholesterol are from were to the M. J. M. J. 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Biol. 2000; Google Scholar). hippocampal slices with mevastatin were in and for and proteins were in and by for Rho was as using an results are as of and were for as was to in and of the rate-limiting enzyme HMG-CoA were used to for cholesterol and microglia activation. The which macrophages and microglia A.G. F.R. 1999; PubMed Scopus Google Scholar, H. K. T. Y. PubMed Scopus Google Scholar, C.E. I. R. T. Streit in the and Scholar), cells in cultured slices The had a cell with a morphology to that associated with resting with mevastatin caused a in the of cells along with in cells in the slices were amoeboid, of and in macrophage-like These effects not were evident and were the of with the features of microglia and results with a a microglial are in In accord with Y. C. Neurosci. Lett. 2003; PubMed Scopus Google Scholar), provides a of resting microglia, as by the of in the with mevastatin increased the of the cell to with products the found in resting cells were absent to These although were with with the changes in the of cells and the they in and of slices. had effect on the of microglia A and Both were increased The of cells the they increased by in the the effects of the statin to in the cell in the This was for and for and for for and for increases in cell were found not the from A of A and that statins had a effect on the by microglia they on cell A of is in The of the was that found in slices mevastatin as increased in a This provides evidence that mevastatin not increased the of microglia but also The changes were evident and a Cell in the to and in brain, and for increased in slices. This was tested by in and slices. the results a treatment. from slices were not with but with caused a in the of cells in and for The results for the of cells were and the increases in to for the of mevastatin and had effects and not cell These results suggest that the in microglia by of mevastatin is in due to proliferation and in to activation, and thus of the of the effect was with of the TNF-α of including microglia, up-regulation of cytokines as as the changes The of is to the of inflammation to the progression of various including was of to whether statin-induced changes in microglia to cytokine results for TNF-α, an cells were for TNF-α in but a with mevastatin a of the cells in slices were for the and cells had the morphology associated with of and TNF-α on the that cells were also by and was in that the for statin-induced increases in the cytokine was The incidence of cells was in slices and was The effects were but not and statin increases in the of microglia The of TNF-α cells in the cell was that for that a of microglia the as from the in the in the of cells were not with as is evident in the in This that has a of microglia that is not present in the Statin-induced changes in the levels of TNF-α were by of the cytokine in cultured slices for were and a conclusion was using J. and G. with mevastatin simvastatin a in TNF-α levels and an in TNF-α levels by mevastatin experiments that statin-induced increases in the cytokine were evident and a a with that for the of and reductase is an step in cholesterol synthesis as from this, statins block the synthesis of several in addition to cholesterol treatments were used to whether in an cholesterol were involved in statin-induced microglia that an product of HMG-CoA by had effect on TNF-α levels but blocked increases in the cytokine A and and The effects of on statin-induced microglial transformation were by A and of a slice and a slice with mevastatin for changes in microglial morphology and an in the of cells in cholesterol to the not the changes in microglia by a with The cells were and also that the typical of resting microglia were In mevalonate blocked microglial transformation Cell were and were is the of farnesyl diphosphate and of geranylgeranyl diphosphate, compounds that provide for protein of isoprenoids the and Rho of that critical regulatory roles in inflammatory and immune slices with farnesyl not the effects of mevastatin not but treatments with geranylgeranyl caused a of the microglial with geranylgeranyl had evident effect on microglia but the changes by mevastatin with also blocked the of TNF-α by mevastatin A and regulates Rho activity by controlling to in the statin treatments used in the present study were to a decrease in the levels of Rho proteins in cultured slices. experiments suggest that the mevalonate pathway, by and possibly Rho activation, a influence microglial effects are blocked by geranylgeranyl and by of Rho from and cultured hippocampal slices were with mevastatin in the presence of geranylgeranyl for and for with are from to from hippocampal slices with mevastatin were for using results were from In of the critical they in cell and in particular in proliferation and G.L. 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In have found that HMG-CoA reductase induce microglial transformation and expression of the cytokine TNF-α, features in Statin-induced changes were blocked by mevalonate and geranylgeranyl of activation suppressed the effects of statin on gene regulation effects on These results provide the evidence that protein possibly of of Rho is involved in regulating microglial morphology and cytokine with statins to microglial activation of mevalonate-isoprenoid