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• W2003898971 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. Prostaglandins Other Lipid Mediat. 1998; 55: 331-343Crossref PubMed Scopus (16) Google Scholar, 5Simons M. Keller P. De Strooper B. Beyreuther K. Dotti C.G. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6460-6464Crossref PubMed Scopus (1079) Google Scholar, 6Howland D.S. Trusko S.P. Savage M.J. Reaume A.G. Lang D.M. Hirsch J.D. Maeda N. Siman R. Greenberg B.D. Scott R.W. Flood D.G. J. Biol. Chem. 1998; 273: 16576-16582Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar) and transgenic mice (7Refolo L.M. Pappolla M.A. Malester B. LaFrancois J. Bryant-Thomas T. Wang R. Tint G.S. Sambamurti K. Duff K. Neurobiol. Dis. 2000; 7: 321-331Crossref PubMed Scopus (880) Google Scholar). Patients with high plasma cholesterol levels and cardiovascular diseases have increased risk of Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; HMG, 3-hydroxy-3-methylglutaryl; TNF-α, tumor necrosis factor-α; BrdUrd, 2′-deoxyuridine; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase.1The abbreviations used are: AD, Alzheimer's disease; HMG, 3-hydroxy-3-methylglutaryl; TNF-α, tumor necrosis factor-α; BrdUrd, 2′-deoxyuridine; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase. (8Sparks D.L. Hunsaker III, J.C. Scheff S.W. Kryscio R.J. Henson J.L. Markesbery W.R. Neurobiol. Aging. 1990; 11: 601-607Crossref PubMed Scopus (270) Google Scholar, 9Prince M. Cullen M. Mann A. Neurology. 1994; 44: 97-104Crossref PubMed Google Scholar, 10Notkola I.L. Sulkava R. Pekkanen J. Erkinjuntti T. Ehnholm C. Kivinen P. Tuomilehto J. Nissinen A. Jick H. Zornberg G.L. Jick S.S. Seshadri S. Drachman D.A. Neuroepidemiology. 1998; 17: 14-20Crossref PubMed Scopus (622) Google Scholar), and there is evidence that statins, a family of compounds that inhibit the rate-limiting enzyme in cholesterol synthesis (3-hydroxy-3-methylglutaryl coenzyme A reductase: HMG-CoA reductase), decrease the incidence of the disease (11Wolozin B. Kellman W. Ruosseau P. Celesia G.G. Siegel G. Arch. Neurol. 2000; 57: 1439-1443Crossref PubMed Scopus (1336) Google Scholar, 12Jick H. Zornberg G.L. Jick S.S. Seshadri S. Drachman D.A. Lancet. 2000; 356: 1627-1631Abstract Full Text Full Text PDF PubMed Scopus (1575) Google Scholar). These observations have led to an extensive and ongoing evaluation of statins as preventive treatments for Alzheimer's disease (13Stuve O. Youssef S. Steinman L. Zamvil S.S. Curr. Opin. Neurol. 2003; 16: 393-401Crossref PubMed Scopus (146) Google Scholar, 14Petanceska S.S. DeRosa S. Olm V. Diaz N. Sharma A. Thomas-Bryant T. Duff K. Pappolla M. Refolo L.M. J. Mol. Neurosci. 2002; 19: 155-161Crossref PubMed Scopus (146) Google Scholar, 15Crisby M. Carlson L.A. Winblad B. Alzheimer Dis. Assoc. Disord. 2002; 16: 131-136Crossref PubMed Scopus (96) Google Scholar, 16Bollen E.L. Gaw A. Buckley B.M. Arch. Neurol. 2001; 58: 1023-1024Crossref PubMed Scopus (3) Google Scholar). Mevalonate, a cholesterol precursor, the synthesis of which is blocked by statins, is converted into several bioactive compounds. Among these, the isoprenoids are of particular importance because, among other functions, they provide for the covalent addition of lipid moieties (prenylation) to regulatory proteins (17Casey P.J. Seabra M.C. J. Biol. Chem. 1996; 271: 5289-5292Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar) and thereby affect critical cell functions. 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. Lynch G. J. Neurosci. 1997; 17: 4006-4021Crossref PubMed Google Scholar, 30Bi X. Yong A.P. Zhou J. Ribak C.E. Lynch G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8832-8837Crossref PubMed Scopus (40) Google Scholar), including an intense microglial reaction to suspected causes of age-related pathology such as amyloid-β peptide uptake by neurons (31Bi X. Gall C.M. Zhou J. Lynch G. Neuroscience. 2002; 112: 827-840Crossref PubMed Scopus (95) Google Scholar) and partial lysosomal dysfunction (32Bi X. Liu J. Zhou J. Sharman K. Song Z. Lynch G. Soc. Neurosci. Abstr. 2001; 27 (Program No. 963.17)Google Scholar). Cultured slices thus provide a convenient system with which to test whether protracted exposure to statins triggers brain inflammatory reactions. Our results indicate that statins elicit microglial activation, as evidenced by transformation to macrophage morphology and up-regulation of the cytokine TNF-α, and that this effect is likely due to suppression of the mevalonate pathway and of its geranylgeranyl products. Preparation and Maintenance of Hippocampal Slice Cultures—Organotypic hippocampal cultures were prepared using the technique of Stoppini et al. (33Stoppini L. Buchs P.A. Muller D. J. Neurosci. Methods. 1991; 37: 173-182Crossref PubMed Scopus (2513) Google Scholar). Briefly, hippocampi were harvested from brains of 9–12-day-old Sprague-Dawley rat pups under sterile conditions. Slices (400-μm thick) were cut perpendicular to the long axis of hippocampus using a McIllwain tissue chopper and collected into a cutting medium consisting of minimum Eagle's medium with Earle's salts (Invitrogen), 25 mm HEPES, 10 mm Tris base, 10 mm glucose, and 3 mm MgCl2 (pH 7.2). Slices were positioned onto 30-mm cell culture inserts (Millicell-CM, Millipore, Bedford, MA) that were placed in 6-well culture trays with 1 ml of growth medium/well (growth medium: 50% basal medium eagle, 25% Earle's balanced salt solution, 25% horse serum, and the following supplements: 136 mm NaCl, 2 mm CaCl2, 2.5 mm MgSO4, 5mm NaHCO3, 3 mm glutamine, 40 mm glucose, 0.5 mm ascorbic acid, 20 mm HEPES buffer (pH 7.3 at 23 °C), 1 mg/liter insulin, 25 mg/liter penicillin). Each of the above components was purchased from Sigma. The cultures were incubated at 36 °C with a 5% CO2-enriched atmosphere, and the medium was changed every other day until use, generally 12–14 days later. Hippocampal slices maintained in vitro for 12–14 days were exposed to medium containing mevastatin (0.01–50 μm), simvastatin (0.01–10 μm; both were from Calbiochem), or vehicle (Me2SO, 0.1%) for 0–6 days. In some experiments, the aforementioned chemicals were applied in the presence of mevalonate (100 and 500 μm), cholesterol (100 μm; both are from Sigma; these concentrations were chosen according to the literature (34Eberlein M. Heusinger-Ribeiro J. Goppelt-Struebe M. Br. J. Pharmacol. 2001; 133: 1172-1180Crossref PubMed Scopus (117) Google Scholar, 35Ikeda U. Shimpo M. Ikeda M. Minota S. Shimada K. J. Cardiovasc. Pharmacol. 2001; 38: 69-77Crossref PubMed Scopus (45) Google Scholar)), or of inhibitors of the mitogen-activated protein kinase (MAPK) kinase (MEK), PD98059 (25 μm), or the p38 mitogen-activated protein kinase (MAPK) kinase, SB233580 (20 μm; both were purchased from Calbiochem). For assessment of overall cell proliferation, 5-bromo-2′-deoxyuridine (BrdUrd) (10 μm; Sigma) was added to culture medium 24 h before the slices were fixed. Immunocytochemistry—Following treatment, slices were thoroughly washed with 0.1 m sodium phosphate-buffered saline, fixed for 12–16 h in cold 0.1 m phosphate buffer (pH 7.2) containing 4% paraformaldehyde, cryoprotected in 20% sucrose for 1–2 h, and then carefully removed from the insert membranes. Serial sections (25-μm thick) were cut parallel to the broad face of the explant, using a freezing microtome. Immunocytochemistry was performed using the standard avidin-biotin horseradish peroxidase complex (ABC) method using the reagents and instructions of the VECTASTAIN® Elite ABC kit from Vector Laboratories (Burlingame, CA). Briefly, free-floating sections were preincubated with 10% normal horse serum in phosphate buffer for 1 h at room temperature. Sections were then incubated with the monoclonal antibody ED-1 (1:1000), with the monoclonal anti-CD11b antibody OX-42 (1:1000; both were from Serotec, Oxford), or with a polyclonal goat against TNF-α antibody (1:1000; Sigma) in 5% normal horse serum overnight at 4 °C. Sections were washed in phosphate buffer, incubated in biotinylated anti-mouse IgG (for ED-1 and OX-42) or anti-goat IgG (for TNF-α) (both used at 1:400) for 2–3 h, washed in phosphate buffer, incubated in the avidin-biotin complex solution for 45 min, and then processed for diaminobenzidine reaction. After final rinses in phosphate-buffered saline, sections were mounted on SuperFrost Plus slides (Fisher Scientific), air-dried, dehydrated in a series of graded ethanol, and coverslipped (clearing solvent; Stephens Scientific, Kalamazoo, MI) with Permount (Fisher Scientific). For BrdUrd staining, DNA was denatured with 2 m HCl for 30 min at 37 °C, and the antigens were unmasked by incubation with 0.1% pepsin. Sections were blocked in 10% normal horse serum (room temperature for 1 h) followed by incubation with monoclonal mouse anti-BrdUrd (1:200; Sigma). Subsequently, tissues were further processed for immunostaining following the aforementioned procedures. Methods for Quantification of Microglia Structural Features—Images of ED-1, TNF-α, and BrdUrd immunostaining in the stratum pyramidale of CA1 region and in the polymorph layer of the dentate gyrus of cultured hippocampal slices were visualized using a Zeiss microscope (Axioskop 2; ×10 objective, 0.75 optovar). Images of tissue sections were digitized and scanned with a Zeiss digital photo camera (AxioCam Hrc) and an automated image-scanning program, KS 400 (Zeiss). An automated in-house computer program was used to enhance image contrast, extract ED-1, TNF-α, or BrdUrd immunopositive particles, and measure their relevant parameters. Computed measures consisted of: the number of particles (immunolabeled cells), total area, and average area per particle. Statistical significance was determined by one-way analysis of variance using Prism V. 3. Immunoblotting—Electrophoresis and immunoblotting were carried out following conventional procedures. Control and experimental slices were collected in ice-cold 10 mm Tris-HCl harvest buffer consisting of 10 mm Tris, 0.32 m sucrose, 2 mm EDTA, 2 mm EGTA, and 0.1 mm leupeptin, pH 7.4, centrifuged, and sonicated after resuspension in lysis buffer (10 mm Tris/HCl, 1 mm EDTA, 1 mm EGTA, 0.1% protein inhibitor mixture (Sigma), pH 7.4). Proteins (40–60 μg) from each sample were denatured by boiling for 5 min in sample buffer (2% SDS, 50 mm Tris-HCl (pH 6.8), 10% 2-mercaptoethanol, 10% glycerol, and 0.1% bromphenol blue) and separated by electrophoresis on SDS-polyacrylamide gels (10%), after which the proteins were transferred to nitrocellulose membranes. Nitrocellulose membranes were incubated with polyclonal antibodies against TNF-α (1:5000) or monoclonal antibodies against diphosphorylated ERK1/2 (1:10,000; both antibodies are from Sigma) for a 12–16-h incubation at room temperature; the blots were then stained to less than maximum intensity with anti-IgG-alkaline phosphatase conjugates, using a nitro-blue tetrazolium substrate system (Bio-Rad). Images were analyzed by densitometry using the NIH Image system (version 1.60). Preparation of Membrane Fractions—Membrane fractions were prepared as described previously (36Bi X. Bi R. Baudry M. Methods Mol. Biol. 2000; 144: 203-217PubMed Google Scholar). Briefly, hippocampal slices treated with mevastatin (0.1, 1, 5 μm) or vehicle only (0.1% Me2SO) were homogenized in lysis buffer and centrifuged at 25,000 × g for 20 min at 4 °C. Membrane and cytoplasm proteins were denatured in sample buffer and resolved by SDS-PAGE; immunoblotting for Rho was carried out as described above using an anti-Rho antibody (1:1000; Upstate Biotechnology, Lake Placid, NY). Statistical Analysis—All results are expressed as means ± S.E. analysis of variance and two-tail Student's t tests were performed for statistical analysis as appropriate. p < 0.05 was considered to be statistically significant. Statins Activate Microglia in Cultured Hippocampal Slices— Mevastatin and simvastatin, two inhibitors of the rate-limiting enzyme HMG-CoA reductase, were used to test for interactions between cholesterol metabolism and microglia activation. The antibody ED-1, which predominately labels activated macrophages and microglia (37Woods A.G. Poulsen F.R. Gall C.M. Neuroscience. 1999; 91: 1277-1289Crossref PubMed Scopus (41) Google Scholar, 38Kato H. Kogure K. Araki T. Itoyama Y. Brain Res. 1995; 694: 85-93Crossref PubMed Scopus (71) Google Scholar, 39Finch C.E. Morgan T.E. Rozovsky I. Xie Z. Weindruch R. Prolla T. Streit W.J. Microglia in the Regenerating and Degenerating Nervous System. Springer-Verlag New York Inc., New York2002: 275-305Google Scholar), recognized few cells in untreated cultured slices (Fig. 1A). The immunopositive elements had a thin cell body with twisted fine processes (Fig. 1A, inset), a morphology corresponding to that associated with resting microglia. Six-day treatment with 10 μm mevastatin caused a large increase in the number of ED-1-labeled cells (Fig. 1D), along with pronounced morphological changes. As shown in Fig. 1D (inset), cells in the treated slices were amoeboid, notably devoid of processes, and in general resembled macrophage-like mononuclear cells. These effects could not be detected at 0.1 μm (Fig. 1B), were evident at 1.0 μm (Fig. 1C), and were maximal at 10 μm (Fig. 1D). Higher concentrations (≥25 μm) decreased the number of stained cells, with the remainder having features of dying microglia (Fig. 1, E and F). Typical results obtained with OX-42, a monoclonal antibody against a second microglial marker (Cd11b), are described in Fig. 2. In accord with earlier work (40Huh Y. Jung J.W. Park C. Ryu J.R. Shin C.Y. Kim W.K. Ryu J.H. Neurosci. Lett. 2003; 349: 63-67Crossref PubMed Scopus (33) Google Scholar), this antibody provides a near complete image of resting microglia, as can be seen by the labeling of fine details in the ramified processes (Fig. 2A, arrows). Six-day treatment with 10 μm mevastatin greatly increased the density of labeling, causing the normally unlabeled cell bodies (Fig. 2A, arrowheads) to become almost completely filled with immunoreactive products (Fig. 2B). Moreover, the thin twisted processes found in resting cells were either altogether absent or reduced to stubby, unramified elements (Fig. 2B, arrows). These effects, although more dramatic, were comparable with those obtained with ED-1. Fig. 3 summarizes the changes in the number of ED-1-positive cells and the area they occupied in field CA1 and dentate gyrus of statin-treated slices. Mevastatin at 0.1 μm had no significant effect on the number (1.1 ± 0.1 times control, p > 0.1, n = 19) or size (total area) of immunopositive microglia (Fig. 3, A and B). Both measures were significantly increased at 1.0 μm. The number of labeled cells doubled (2.1 ± 0.2 times control, p < 0.001, n = 19), whereas the area they occupied increased by 197 ± 18%. As shown in the figure, the effects of the statin at 10 μm appeared to be larger in the pyramidal cell fields than in the dentate gyrus. This variation was highly significant for both number and area when measured at higher concentrations (at 5 μm, p = 0.003 for area and 0.048 for number; at 10 μm, p < 0.0001 for area and p = 0.0014 for number; Mann-Whitney test). Higher concentrations elicited smaller increases in cell number than were found at 10 μm (data not shown), confirming the impression gained from survey micrographs. A comparison of Fig. 3, A and B, suggested that statins had a larger effect on the area occupied by activated microglia than they did on cell number. A test of this point is summarized in Fig. 3C. The ratio of the two measures was significantly greater than that found in untreated slices at all mevastatin concentrations greater than 0.1 μm and, as shown, increased in a dose-dependent manner. This provides evidence that mevastatin not only increased the number of immunopositive microglia but also enlarged individual cells. The above-described morphological changes were evident at 48 h and reached a maximum at 96 h (not shown). Cell Division in Statin-treated Slices—Microglia retain the capacity to divide and migrate in adult brain, and this could account for their increased numbers in statin-treated slices. This hypothesis was tested by analyzing BrdUrd (10 μm) uptake in control and mevastatin-treated slices. Fig. 4 summarizes the results obtained following a six-day treatment. Significant differences from control slices were not observed with 0.01 or 0.1 μm concentrations, but treatment with 1 μm caused a significant increase in the number of BrdUrd-labeled cells in both CA1 and dentate gyrus (p < 0.01 for both regions). The results for the numbers of BrdUrd-labeled cells were variable, and the increases in this measure appeared to be smaller than those for the numbers of ED-1-positive cells. Unexpectedly, mevastatin at 5 and 10 μm had smaller effects than at 1 μm and did not increase cell numbers above control values (Fig. 4). These results suggest that the increase in ED-1-immunopositive microglia produced by low concentrations of mevastatin is in part due to proliferation and in part to activation, and thus labeling, of the extant population of microglia. Only the latter effect was obtained with higher concentrations of the statin. Statins Up-regulate TNF-α Expression—Activation of macrophages, including microglia, involves up-regulation of cytokines as well as the morphological changes described above. The release of these toxic agents is generally assumed to mediate the contribution of inflammation to the progression of various diseases, including AD; thus, it was of interest to determine whether statin-induced changes in microglia extended to cytokine expression. Fig. 5 describes results for TNF-α, an important central nervous system cytokine. Very few cells were immunopositive for TNF-α in control slices, but this changed dramatically after a 6-day treatment with mevastatin (10 μm). As shown (Fig. 5A, bottom panels), a substantial proportion of the cells in statin-treated slices were positive for the cytokine, and these cells had the amoeboid morphology associated with activated microglia. Comparison of ED-1 and TNF-α staining on the same sections showed that nearly all TNF-α-positive cells were also labeled by ED-1, and this was confirmed in merged images. Quantitative analyses indicated that the threshold for statin-induced increases in the cytokine was below 0.1 μm (Fig. 5B). The incidence of labeled cells at this concentration was greater than in control slices (CA1: 4.2 ± 1.6 times control, n = 9 versus n = 13), and this difference was highly significant (p = 0.013, U test, two tails). The effects at 1.0 μm were less variable, but not markedly greater, than at 0.1 μm and highly significant. Higher statin concentrations produced very large (10–25-fold) increases in the number of TNF-α-positive microglia (Fig. 5B). The number of TNF-α labeled cells in the pyramidal cell region was smaller than that for ED-1 labeled cells, suggesting that only a subset of activated microglia expressed the cytokine, as expected from the micrographs in Fig. 5A. Interestingly, the regional differences in the numbers of ED-1-positive cells were not observed with TNF-α-positive cells, as is evident in the CA1 versus dentate gyrus comparison shown in Fig. 5B. This suggests that field CA1 has a population of statin-sensitive microglia that is not present in the dentate gyrus. Statin-induced changes in the levels of TNF-α were further analyzed by immunoblotting. Levels of the cytokine in cultured slices maintained for 2–3 weeks under control conditions were undetectable (Fig. 6A, cont), and a similar conclusion was reached using reverse transcription-PCR. 2X. Bi, J. Zhou, and G. Lynch, unpublished observation. Six-day incubations with mevastatin or simvastatin (10 μm) induced a marked increase in TNF-α levels (Fig. 6A, Mev and Sim); quantitative analysis revealed an approximately 10-fold increase in TNF-α levels induced by mevastatin (Fig. 6B). Immunoblot experiments showed that statin-induced increases in the cytokine were evident after 2 days and approached a maximum 2 days later (Fig. 6C), a time course comparable with that for the number of ED-1 immunopositive microglia. Links between Mevalonate-Isoprenoid Biosynthesis and Microglia—HMG-CoA reductase is an early step in cholesterol synthesis and, as might be expected from this, statins block the synthesis of several intermediary, biologically active steroids, in addition to reducing cholesterol production. Replacement treatments were used to determine whether reductions in an intermediate product, rather than reduced cholesterol levels, were involved in statin-induced microglia changes. Western blot analysis showed that mevalonate, an immediate product of HMG-CoA reductase, by itself had no effect on TNF-α levels but completely blocked mevastatin-induced increases in the cytokine (Fig. 6, A and B, Mnt and Mev/Mnt). The effects of replacement on statin-induced microglial transformation were analyzed by immunohistochemistry. Fig. 7, A and B, are, respectively, photomicrographs of ED-1-immunostained sections through a control slice and a slice treated with mevastatin for 6 days; massive changes in microglial morphology and an apparent increase in the numbers of labeled cells can be seen. As shown in Fig. 7C, adding 100 μm cholesterol to the culture medium did not detectably alter the changes in ED-1-immunopositive microglia produced by a 6-day treatment with mevastatin. The labeled cells were densely labeled and ovalshaped; note also that the twisted dendritic processes typical of resting microglia were almost completely absent. In marked contrast, mevalonate (500 μm) completely blocked micr" @default.
• W2003898971 created "2016-06-24" @default.
• W2003898971 creator A5012477004 @default.
• W2003898971 creator A5015041062 @default.
• W2003898971 creator A5044957913 @default.
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• W2003898971 date "2004-11-01" @default.
• W2003898971 modified "2023-10-05" @default.
• W2003898971 title "Inhibition of Geranylgeranylation Mediates the Effects of 3-Hydroxy-3-methylglutaryl (HMG)-CoA Reductase Inhibitors on Microglia" @default.
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