Inhibitory Effect of Minocycline on Amyloid b FibrilFormation and Human Microglial ActivationATOOSA FAMILIAN,1,2* RONALD S. BOSHUIZEN,4 PIET EIKELENBOOM,1,5 AND ROBERT VEERHUIS1,2,3

1Department of Psychiatry, Institute for Clinical and Experimental Neurosciences-VU (ICEN-VU), VU University Medical Center,Amsterdam, The Netherlands2Department of Pathology, Institute for Clinical and Experimental Neurosciences-VU (ICEN-VU), VU University Medical Center,Amsterdam, The Netherlands3Department of Clinical Chemistry, VU University Medical Center, Amsterdam, The Netherlands4Pepscan Systems B.V., Lelystad, The Netherlands5Department of Neurology, Academic Medical Center, University of Amsterdam, The Netherlands

KEY WORDStetracycline; cytokines; C1q; SAP; thioflavin S

ABSTRACTMinocycline, a derivative of the antibiotic tetracycline, dis-plays neuroprotective properties in various models of neuro-degenerative diseases and is now used in clinical trials,because of its relative safety and tolerability. Minocyclinepasses the blood-brain barrier and is presumed to inhibitmicroglial activation. In Alzheimer’s disease brain, a num-ber of proteins, including serum amyloid P component(SAP) and complement factors such as C1q, accumulate inamyloid b (Ab) plaques. In a previous study, SAP and C1qwere found to be required for clustering of activated micro-glia in Ab plaques. Furthermore, SAP and C1q enhancedAb fibril formation and Ab mediated cytokine release byhuman microglia in vitro. In the present study, we reportthat tetracycline and minocycline dose-dependently reduceTNF-a and IL-6 release by adult human microglia upon sti-mulation with a combination of Ab, SAP, and C1q. In addi-tion, minocycline and to a lesser extent tetracycline inhibitfibril formation of Ab as determined in a thioflavin-S-basedfluorescence test. This inhibitory effect was observed withAb alone as well as with Ab in combination with SAP andC1q. Our data suggest that minocycline and tetracycline attolerable doses can inhibit human microglial activation.This activity in part is exerted by inhibition of (SAP andC1q enhanced) Ab fibril formation. VVC 2005 Wiley-Liss, Inc.

INTRODUCTION

Amyloid plaques in Alzheimer disease (AD) brain areassociated with a locally induced, non-immune-mediated,chronic inflammatory response. Deposition of Ab is con-sidered a neuropathological hallmark of AD and is asso-ciated with clusters of activated microglia in AD affectedbrain areas (Sasaki et al., 1997; Veerhuis et al., 2003;Blasko et al., 2004; Veerhuis et al., 2004).

Clinicopathological and neuroradiological studies indi-cate that activation of microglia is a relatively earlypathologic event in the process of Alzheimer’s disease,since the presence of MHC class II-positive microglia pre-cedes the process of neuropil destruction in AD brain(Sasaki et al., 1997; Arends et al., 2000; Cagnin et al.,2001; Vehmas et al., 2003).

Clustering of activated microglia, MHC-class II positivemicroglia that express complement receptors (CD18/CD11b and c), in AD cerebral amyloid deposits is relatedto a certain degree of Ab fibril formation. Furthermore,they are seen exclusively in Ab plaques with positiveimmunostaining for SAP and C1q (Veerhuis et al., 2003).Upon activation, microglial cells secrete pro-inflammatorycytokines, such as IL-1b, IL-6, and TNF-a, nitric oxide,and reactive oxygen species (ROS), which in turn maylead to cytotoxicity and neurodegeneration (Gebicke-Haerter, 2001; Hanisch, 2002).

In vitro, Ab1-42 (the major Ab species in the plaques)exerts minor stimulatory effects on the secretion of pro-inflammatory cytokines by microglia isolated from humanpost mortem brain specimens. However, a clear enhance-ment of the Ab-mediated release of cytokines (IL-6 andTNF-a) by microglia was observed when Abwas added incombination with SAP and C1q (Veerhuis et al., 2003).This may, at least in part, result from the effect of SAPand C1q on Ab fibril formation since in electron microsco-pical studies, addition of SAP and C1q to the Ab1-42 pep-tide caused an increase in fibril density with generation ofa large number of densely packed aggregates of the Ab1-42

peptide (Veerhuis et al., 2003). We recently developed anefficient method for measurement of in vitro fibril forma-tion based on the increase in fluorescence signal uponinteraction of thioflavin S with the fibrils formed by syn-thetic peptides with a high b sheet content (Boshuizenet al., 2004). This technique provides an effective tool toinvestigate in vitro formation of Ab fibrils and the role ofassociated proteins such as SAP and C1q in this process.

Minocycline, a derivative of tetracycline, is considered abeneficial neuroprotective drug in a number of centralnervous system disorders. Positive effects of this com-

Grant sponsor: European Community; Grant number: QLK3-CT-2001-00283;Grant number: QLK6-CT-1999-02004; Grant sponsor: Alzheimer Nederland; Grantnumber: V-2000-008.

*Correspondence to: Atoosa Familian, Department of Pathology, VU UniversityMedical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands.E-mail: a.familian@vumc.nl

Received 13 April 2005; Accepted 30 June 2005

DOI 10.1002/glia.20268

Published online 11 October 2005 in Wiley InterScience (www.interscience.wiley.com).

VVC 2005 Wiley-Liss, Inc.

GLIA 53:233–240 (2006)

pound were reported in models of Parkinson’s disease (Duet al., 2001; Wu et al., 2002), Huntington’s disease (Chenet al., 2000), amyotrophic lateral sclerosis (Zhu et al.,2002; Kriz et al., 2002), traumatic brain injury (SanchezMejia et al., 2001), intracerebral hemorrhage (Poweret al., 2003), and global cerebral ischemia (Arvin et al.,2002). Minocycline readily passes the blood-brain barrier,which makes it particularly suitable for treatment ofbrain disorders.

Different mechanisms have been suggested for the ben-eficial effects of minocycline as a therapeutic agent inbrain disorders (Blum et al., 2004). Since microglial acti-vation is an important feature in the pathogenesis of AD,we focused on the effects of minocycline on the Ab-induced microglial activation as a target for treatmentof AD.

A direct inhibitory effect of minocycline on proliferationand activation of rodent microglial cells through inhibi-tion of p38 mitogen-activated protein kinase (p38 MAPK)is suggested, which, in turn, can prevent neurotoxicity(Tikka et al., 2001). In contrast, the antibiotic tetracyclineand its derivatives, doxycycline and minocycline, areclaimed to bind amyloid fibrils generated by syntheticpeptides of human PrP and human Ab1-42/Ab1-40 andreduce the formation of amyloid fibrils by these peptides(Tagliavini et al., 2000; Forloni et al., 2001). Although themechanisms are not fully understood, polar interactionsand the formation of hydrogen bonds are suggested to bethe possible molecular basis for binding of tetracycline tofibrillogenic structures.

In this study, we analyzed the inhibitory effect of bothminocycline and tetracycline on the activation of humanmicroglial cell cultures, as well as on fibril formation ofAb in combination with SAP and C1q. Our data show thatthe inhibitory effects of minocycline and tetracycline onhuman microglial activation are in part due to inhibitionof (SAP and C1q enhanced) Ab1-42 fibril formation.

MATERIALS AND METHODS

Ingredients included trypsin, gentamycin, streptomy-cin, penicillin, lipopolysaccharide (LPS; E55:B5), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),poly-L-lysine (PLL; Sigma, St. Louis, MO), bovine pancrea-tic DNase I (Boehringer-Mannheim, FRG), Dulbecco’s mo-dified Eagle’s medium nutrient mixture HAM’s F-10(DMEM/HAM-F10; GIBCO-Life Technologies, Breda, TheNetherlands), and fetal calf serum (FCS; ICN Biomedi-cals, Amsterdam, The Netherlands), were obtained asindicated. Microtiter plates (96 wells) for enzyme-linkedimmunosorbent assays (ELISA) were from Nunc (Roskilde,Denmark), 48-well plates and 80-cm2 culture flasks for cellculturing fromCostar (Corning, NY).

Ab1-42 (Bachem, Bubendorf, Switzerland) was dissolved(stock solution; 500 lM) in pyrogen-free water (BaxterB.V. Utrecht, The Netherlands), aliquoted and stored at220�C until use. Serum amyloid P component was fromCalbiochem (La Jolla, CA). Human C1q was either fromQuidel (San Diego, CA), or isolated from the Cohn I frac-

tion of pooled human plasma, followed by cation exchangechromatography (Bio-Rex 70; Bio-Rad) and gel filtration(Bio-Gel A5; Bio-Rad) according to published protocols(Tenner et al., 1981). The C1q was homogeneous, asjudged by SDS-PAGE, and transferred from Tris-HCl buf-fer to sterile phosphate-buffered saline (PBS) by passageover a PD10 column (Pharmacia Biotech). Glycerol(GIBCO-BRL) was added to a final concentration of 40%(v/v) and the C1q preparation was subsequently passedover a 0.22-lm filter (Millipore, Bedford, MA), aliquotedand stored at 280�C. Tetracycline-HCl and minocycline-HCl were obtained from Sigma (St. Louis, MO).

Brain Tissue Specimens

Human brain specimens with a short post mortemdelay time were obtained at autopsy through the Nether-lands Brain Bank (coordinator Dr. R. Ravid). The clinicaldiagnosis of AD was neuropathologically confirmed on for-malin-fixed, paraffin-embedded tissue from differentbrain regions. Subcortical white matter and cortical graymatter from cerebral cortex specimens of AD or controlcases were collected in DMEM/HAM-F10 (1:1) containinggentamycin (Gibco, Paisley, UK; 50 lg/ml) for furthermicroglial isolation and cell culture purpose.

Cells

Microglial cells were isolated according to publishedprotocols (Veerhuis et al., 1999; De Groot et al., 2000). Inshort, cell suspensions obtained after mincing and trypsindigestion (37�C; 20 min) of brain specimens, devoid ofblood vessels and meninges, were passed through a nylon130 lmmesh filter and spun on a Percoll gradient (1.03 g/ml)at 1,250g to remove the myelin. After shock lysis of erythro-cytes in NH4Cl, the resulting cell suspension was plated inuncoated 80-cm2 flasks and grown in DMEM/HAM-F10 (1:1)containing 10% (v/v) FCS, 2 mM L-glutamine, penicillin(100 IU/ml), and streptomycin (50 lg/ml) and, additionally,25 lg/ml granulocyte-monocyte colony-stimulating factor(rHuGM-CSF; Leucomax, Sandoz, Uden, The Netherlands).Isolated cells were then cultured at 37�C, 5% CO2 for 7–10 days before stimulation experiments. Microglial cells hadeither a bipolar or an amoeboid morphology.

Stimulation Experiments

Stimulation experiments were performed as describedpreviously (Veerhuis et al., 1999). Isolated microglial cellsafter trypsinization and scraping with a cell scraper(Costar, Corning, NY), were transferred to 48-well platesat cell densities of 2 3 104 cell/well, and allowed to settlefor 24 h. Before the start of the experiment, appropriateamounts of Ab1-42 peptides alone or in combination withSAP and C1q in DMEM/HAM-F10 were incubated withdifferent concentrations of tetracycline-HCl (Sigma) orminocycline-HCl (Sigma) for 1 h at room temperature.

234 FAMILIAN ET AL.

This extra step was designed to analyze whether the inhi-bitory effect of these drugs on Ab fibril formation resultsin a decline in microglial activation. For this, Ab1-42 at afinal concentration of 10 lM, alone or in combination with85 nM SAP and 5 nM C1q was incubated in DMEM/HAM-F10 containing tetracycline-HCl or minocycline-HCl infinal concentrations of 25 lM or 250 nM. As a positive con-trol, LPS was also incubated for 1 h at room temperaturebefore addition to the cells at a final concentration of100 ng/ml. No detrimental effects of peptide and C1q orSAP treatment on cell viability, as assessed in a MTTassay, were observed in control experiments. Culturesupernatants from microglia were collected 24 h after thestart of the treatment, centrifuged to remove cells, ali-quoted, and stored at 220�C, until assayed for the pre-sence of cytokines.

ELISA

Cytokine levels in cell culture supernatants were mea-sured in commercial enzyme immunoassay (ELISA) kitsaccording to the instructions of the manufacturer (San-quin, Amsterdam, The Netherlands, for IL-6 and TNF-a.These are sandwich ELISAs in which 96-well ELISAplates (PolySorp; Nunc, Roskilde, Denmark) are coatedwith IgG fractions of cytokine-specific monoclonal antibo-dies in 50 mM carbonate buffer, pH 9.6 at room tempera-ture for 18 h, and subsequently blocked by incubationwith phosphate-buffered saline (PBS) containing 2% (v/v)milk for 45 min. Standards, human recombinant cyto-kines, and samples are diluted in PBS 2% (v/v) milk.Washing in between incubation steps was with PBScontaining 0.05% Tween 20. Bound cytokines weredetected after subsequent incubations with the respectivebiotinylated antibodies and with streptavidin poly-HRP(CLB; Amsterdam). 3,5,30,50-tetramethylbenzidine (TMB;Sigma) at a concentration of 100 lg/ml in 0.11 M sodiumacetate pH 5.5 containing 0.003% (v/v) H2O2 (100 ll finalvolume) was used as a substrate. Color development wasstopped by adding an equal volume of 2 M H2SO4, afterwhich the absorption at 450 nm was determined in amicroculture plate reader (Titertek Multiscan; Flow labs).Detection limits of the ELISAs were 0.2 and 1 pg/ml forIL-6 and TNF-a, respectively.

Thioflavin S-Based Quantification of AbFibril Formation

The extent of fibril formation of Ab1-42 peptides wasdetermined in a thioflavin S-based microtiter plate test(Boshuizen et al., 2004). Ab peptides were mixed witheither 50 mM phosphate buffer, pH 5 alone, or phosphatebuffer containing SAP and C1q, or in combination withtetra- or minocycline. After incubation at room tempera-ture for 1 h, mixtures were transferred to gelatin-coatedwells of a 96F Maxisorb white microwell plate (Nunc, Ros-kilde, Denmark) and allowed to dry overnight at 37�C.Subsequently, 50 ll of a 0.2-mg/ml thioflavin S solution

was added to each well and left to incubate at room tempfor 90 min, after which the fluorescence signal wasdetected in a Wallac Victor 1420 Multilabel counter(Perkin-Elmer, Wellesley, MA) with the following settings:excitation 450 nm, emission 535 nm and lamp energy10,000 (arbitrary units).

Statistical Analysis

SPSS (release 9.0.1) for Windows was used for statisti-cal analysis. Analysis of variance (ANOVA) followed byBonferoni’s test was used to determine significance of dif-ferences between levels of Ab fibril formation and cyto-kine release by microglial cells in control and treatedgroups. P-values of <0.05 were considered significant.

RESULTSEffects of Tetracycline and Minocyclineon Human Microglial Activation in Vitro

Similar to our previous results (Veerhuis et al., 2003),Ab1-42 (10 lM) on its own had minor stimulatory effectson microglial cells isolated from post mortem brain speci-mens, whereas addition of SAP and C1q to Ab led to a sig-nificant increase in secretion of the cytokines IL-6 andTNF-a. Now, we investigated the effect of tetracyclineand minocycline, effective neuroprotective compoundsknown to interfere with Ab fibril formation, on Ab/SAP/C1q-mediated cell activation of human microglia.

To determine tolerable levels, isolated microglia wereincubated with increasing concentrations of tetracyclineand minocycline for 24 h and cell viability was assessedby LDH release and MTT assay. Neither compound hadtoxic effects on microglia at concentrations of �25 lM. Incomparison with minocycline, toxic effects of tetracyclineon microglia started at a lower concentration. An increasein release of LDH by microglia was detectable upon incu-bation with tetracycline at concentrations of 80 lM andhigher. Cytotoxic effects on incubation with minocyclinewere observed at concentrations higher than 250 lM(Fig. 1). Comparable results were obtained upon incuba-tion of neuroblastoma cells with minocycline and tetracy-cline (data not shown).

To measure the effect of these compounds on microglialactivation, we incubated 10 lM Ab1-42 alone or in combi-nation with SAP and C1q together with various amountsof tetracycline or minocycline up to 25 lM, a concentra-tion that, based on the toxicity experiments, was consid-ered nontoxic for the microglial cells (Fig. 1). Mixtureswere pre-incubated for 1 h before incubation with cells toallow Ab1-42 peptides to form fibrils. To assess microglialactivation, the levels of secreted stimulatory cytokines,IL-6 and TNF-a, in the cell supernatants was determinedby ELISA. Co-incubation of tetra- or minocycline withAb1-42 peptides alone did not affect cytokine release bymicroglia (data not shown). However, both compoundsshowed inhibitory effects on the microglial cytokinerelease induced by the complex of Ab, SAP and C1q. The

235MINOCYCLINE INHIBITS HUMAN GLIAL ACTIVATION

secretion of IL-6 and TNF-a by microglia exposed tothe combination of Ab with SAP and C1q was signifi-cantly reduced upon incubation with tetra- or minocyclineat a concentration of 25 lM (molar ratio of 2.5:1 to Ab)(Fig. 2). A lower dosage of tetracycline or minocycline(250 nM), comparable to the molar ratio 0.025:1 to Ab,resulted only in reduction of TNF-a release. Microglialsecretion of IL-6 did not change significantly (Fig. 2).

Assessment of Ab1-42 Fibril Formation

Formation of Abb fibrils

Using the thioflavin S-based fluorescence assay, we ana-lyzed the formation of Ab1-42 fibrils. Compared with bufferalone, an increase in fluorescence levels, which indicatesformation of Ab fibrils, was observed at 10 lM Ab andfurther increased at higher concentrations of Ab (data notshown). A concentration of 25 lM Ab1-42 was chosen forfurther studies on fibril formation, because of its accepta-ble signal to noise ratio (1.96 0.3; average6 SD, n5 12).

Effect of SAP and C1q on Abb fibril formation

Next, Ab fibril formation in the presence of SAP and C1qwas assessed by thioflavin S assay. Fluorescence levelssignificantly increased upon addition of SAP and C1q toAb (Fig. 3). Incubation of SAP and C1q in the absence ofAb peptide did not significantly change the fluorescencesignal, compared with buffer alone (data not shown).

Inhibitory effect of tetracycline and minocycline

To assess the effect of tetracycline and its derivative, mino-cycline, on Ab fibril formation, the generation of fluores-cence signal by 25 lM Ab alone or together with SAP andC1q in the presence of increasing concentrations of tetra-cycline or minocycline was determined in our assay. Var-ious concentrations of tetracycline or minocycline result-ing in tetracycline/Ab or minocycline/Ab molar ratios of0.025, 0.25, 2.5, and 8 were tested. In the titration experi-ment, inhibition of Ab fibril formation in the presence ofSAP and C1q was observed upon co-incubation with mino-cycline at molar ratios of 8:1 and 2.5:1; i.e., 53% and 16%,

Fig. 1. Effect of tetracycline and minocycline on microglial toxicity.Microglial cells (2 3 104 cells/well) were incubated with various concen-trations of tetracycline or minocycline (up to molar ratio 250:1 to Ab) at37�C for 24 h. Viability of the cells after exposure to different concen-trations of tetracycline and minocycline was determined by measure-ment of LDH release and cellular capacity to reduce MTT activity. Via-bility was not impaired by minocycline at doses up to 80 lM, whereastetracycline had no harmful effect up to 25 lM. Data are given as meanand SD of triplicate samples.

Fig. 2. Effect of tetracycline and minocycline on Ab-mediated micro-glial activation. Ab alone or in combination with SAP and C1q wasallowed to form fibrils in absence or presence of tetracycline or minocy-cline for 60 min at room temperature. At the end of pre-incubation thesamples were incubated with 2 3 104 microglia cells/well at 37�C for24 h. The secretion of pro-inflammatory cytokines IL-6 and TNF-a tothe supernatants of the cells were measured by ELISA . Both tetracy-cline and minocycline had inhibitory effects on microglial stimulationat the molar ratio 2.5:1 to Ab. At the lower dose (molar ratio 0.025:1 toAb), only the production of TNF-a was decreased by these compounds.The data are given as mean and SEM of quantitative ELISA for IL-6(7 independent experiments each in triplicate, n 5 21) and for TNF-a(five independent experiments each in triplicate, n 5 15). The amountof secreted TNF-a was below the ELISA detection limit for TNF-a intwo experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

236 FAMILIAN ET AL.

respectively. At molar ratios of 0.25:1 and 0.025:1 fluores-cence levels corresponding to the degree of Ab fibril forma-tion remained unchanged (Fig. 4). Therefore, further thio-flavin S experiments were carried out using tetracyclineand minocycline at molar ratios of 2.5:1 and 8:1 to Ab.Both tetracycline and minocycline were able to signi-ficantly inhibit fibril formation at the molar ratio of 8:1 toAb. However, at molar ratio of 2.5:1 only minocycline alsoshowed a significant inhibitory effect (Fig. 5).

DISCUSSION

Although various animal studies have provided evi-dence for beneficial effects of minocycline in models of

chronic neurodegenerative disorders, the exact mode ofaction of minocycline is unknown. A neuroprotective func-tion via interaction with the apoptosis machinery such asupregulation of expression of the anti-apoptotic protein,Bcl-2 and reduction in cleavage/activation of Bid has beensuggested (Wang et al., 2004; Wang et al., 2003). Otherstudies point to an indirect neuroprotective effect for min-ocycline as a consequence of inhibition of microglial acti-vation (Tikka et al., 2001,). Up to now, all studies investi-gating the effects of minocycline on microglial cells areperformed on rodent microglia.

In this study, we investigated the effect of minocyclineon Ab-mediated activation of human microglial cells iso-lated from post mortem brain specimens. Because the iso-lation of human microglial cells yield a limited number ofcells, selection of the most relevant dosages for tetracy-cline and minocycline to be tested on cultured microglialcells was essential.

Fig. 3. Measurement of Ab fibril formation by thioflavin S assay.The degree of fibril formation of Ab1-42 peptide alone or in combinationwith SAP and C1q was determined by a thioflavin-based fluorescenceassay. The levels of fluorescence reflect the amounts of Ab fibril forma-tion. Addition of SAP and C1q to Ab enhances the fluorescence whichindicates higher amounts of Ab fibrils. Data are expressed as mean andSD of arbitrary units from triplicate samples. ***P < 0.001.

Fig. 4. Inhibitory effect of minocycline on Ab fibril formation in thio-flavin S-based fluorescence assay. Dose-dependent inhibition of minocy-cline on fibril formation in a mixture of 25 lM Ab together with SAPand C1q in absence or presence of indicated amounts up to 200 lM(molar ratio 8:1 to Ab) is demonstrated. Data present average arbitraryunit of every sample after subtraction of the corresponding buffer back-ground fluorescence (AU-CBG) 6 standard deviation of three wells.

Fig. 5. Inhibitory effects of minocycline (A) or tetracycline (B) onfibril formation of 25 lM Ab in absence or presence of SAP and C1q.Both compounds inhibited formation of Ab fibrils. Inhibitory effect ofminocycline on Ab fibril formation was significant at the molar ratios of2.5:1 and 8:1 to Ab. In contrast, tetracycline had an inhibitory effectonly at the higher concentration (1:8). Data are given as arbitrary unit(mean and SD) for the samples and corresponding backgrounds (thefluorescence measurement of every sample in absence of Ab as control).n 5 9 (3 independent experiments performed each in triplicate). Therewas no significant difference between the fluorescence levels of corre-sponding backgrounds. *P < 0.05, **P < 0.01, ***P < 0.001.

237MINOCYCLINE INHIBITS HUMAN GLIAL ACTIVATION

The administered dose of minocycline in animal stu-dies varies between 20–100 mg/kg/day either intrave-nously (IV) or intraperitoneally (IP). Intravenous admin-istration of minocycline of �25 mg/kg was reported to besafe and did not alter the physiological parameters of theexperimental animals (Colovic and Caccia, 2003; Xu et al.,2004; Blum et al., 2004). However, at doses exceeding25 mg/kg, a reduction in body weight, as well as toxiceffects such as skin irritation and a rise in the levels ofhepatic enzymes, were observed (Bocker et al., 1991;Smith et al., 2003; Blum et al., 2004). Plasma levels ofminocycline after IV administration of 20 mg/kg in ratsreached a peak concentration of 28.8 mg/L (Fagan et al.,2004; Xu et al., 2004), which is equivalent to 60 lM ofthis compound. Diffusion of minocycline through theblood-brain barrier results in brain and cerebrospinalfluid (CSF) levels of �30–50% of that in the systemiccirculation in both rat and human (Saivin and Houin,1988; Smith et al., 2003; Colovic and Caccia, 2003; Faganet al., 2004). Hence, the concentration of minocycline inthe brain upon receiving 20 mg/kg IV can reach a maxi-mum level of 20–30 lM. In the present study, no toxiceffects on human microglial (Fig. 1) or neuroblastomacells (data not shown) exposed to minocycline or tetracy-cline up to 25 lM were observed, as determined by MTTassay and LDH release. Similar toxicity levels wereobserved in another in vitro culture model using the hu-man gingival epithelioid S-G cell line (Babich and Tipton,2002). Altogether, 25 lM minocycline is considered a non-toxic concentration that equals the attainable concentra-tion in brain tissue.

As a marker for glial activation, we determined theamounts of secreted pro-inflammatory cytokines, IL-6 andTNF-a, in the supernatants of microglial cells stimulatedwith Ab1-42. Similar to our previous study (Veerhuis et al.,2003), stimulation of human microglial cells with 10 lMAb1-42, a dose that is not toxic for the cells, together withSAP and C1q, led to a significant enhancement in secre-tion of IL-6 and TNF-a. Simultaneous incubation of Ab,SAP, C1q, and 25 lM minocycline or tetracycline, corre-sponding to a molar ratio of 2.5:1 for minocycline or tetra-cycline to Ab, significantly reduced the secretion of bothIL-6 and TNF-a (Fig. 2). Because inhibitory effects of min-ocycline on proliferation and activation of rat microgliacould also be observed at a concentration of 200 nM(Tikka and Koistinaho, 2001), we also investigated theeffects of tetracycline and minocycline at a 250 nM dosageon the Ab-induced cytokine release by human microglialcells, corresponding to a molar ratio of 0.025:1 to Ab. Noinhibitory effects on Ab/SAP/C1q-mediated release of IL-6was observed upon incubation with this dosage of eithercompound. However, at this dosage, tetracycline still sig-nificantly reduced the Ab/SAP/C1q-mediated secretion ofTNF-a. Although not significant, a trend to decreaseTNF-a levels was observed upon incubation with minocy-cline at 250 nM. Such a dissimilar effect of minocycline onTNF-a and IL-6 has also been shown before on other celltypes (Celerier et al., 1996; Kloppenburg et al., 1996;Ledeboer et al., 2005). The different effects of minocyclineand tetracycline on IL-6 and TNF-a secretion may be due

to inhibition of different pathways involved in the produc-tion of cytokines by the activated glial cells.

Several mechanisms may contribute to neuroprotectiveactivities of minocycline such as inhibition of caspase 1and 3 expression, reduction in generation of induciblenitric oxide synthase (iNOS) and COX-2, and inhibition ofp38 MAPK (Yrjanheikki et al., 1998; Yrjanheikki et al.,1999; Chen et al., 2000; Tikka et al., 2001).

Another possible mode of action may be through inhibi-tion of Ab fibril formation. Tetracycline and its deriva-tives, doxycycline and minocycline, have a tendency tobind fibrillogenic structures such as synthetic peptides ofhuman PrP and human Ab 1-42/Ab1-40 and via polar inter-actions reduce formation of amyloid fibrils (Tagliaviniet al., 2000; Forloni et al., 2001).

In this study, we quantified the degree of fibril forma-tion of Ab peptides using thioflavin S-based fluorescencetechnique. As already seen with electron microscopy, Abfibril formation significantly increased in the presence ofSAP and C1q. Co-incubation of minocycline and Abinhibited Ab fibril formation. Comparable inhibitoryeffects of minocycline on Ab fibril formation were ob-served in the absence or presence of SAP and C1q at aminimum molar ratio of 2.5:1 to Ab. In contrast, tetracy-cline did not inhibit Ab fibril formation at this molarratio, although a significant inhibitory effect wasobserved at the higher concentration. Hence, minocyclineexerts a dual inhibitory effects on microglial activation, adirect inhibitory effect at low dosage and an extra inhibi-tory effect by prevention of Ab aggregation at higherdosage. Nevertheless, the required concentration of min-ocycline for inhibition of Ab fibril formation in vitro ishigher than the circulating concentration of minocyclineupon administration.

In human trials for rheumatoid arthritis, acne vulgaris,and Huntington’s disease, long-term treatment with min-ocycline of �200 mg/day (comparable to 3 mg/kg) wasreported to be safe and tolerable (Goulden et al., 1996;O’Dell et al., 1999, 2001; Bonelli et al., 2004). Recenthuman trials with this compound on chronic neurologicaldisorders also pointed to beneficial effects of minocyclinein amyotrophic lateral sclerosis, Huntington’s disease,and multiple sclerosis at similar dosages (Gordon et al.,2004; Thomas et al., 2004; Metz et al., 2004). Increaseddosage of minocycline up to 400 mg/day was well toleratedby the majority of the patients, although a trend towardincreased risk of gastrointestinal problems was observed(Gordon et al., 2004). In humans, peak plasma concentra-tion of 6.2 mg/L is obtained after a 200 mg of IV minocy-cline (Saivin and Houin, 1988), which is comparable to12.8 lM. Up to now, there are no data available on theplasma concentration upon administration of 400 mg/day,although with regard to the linear relation between peakplasma concentration and administered dose in animalexperiments (Colovic and Caccia, 2003), a twofold in-crease in peak plasma concentration could be expected.Therefore, it is likely that inhibitory effects of minocyclineon aggregation and/or fibril formation of Ab at higheradministrated doses may contribute to prevention of ADplaque formation.

238 FAMILIAN ET AL.

Amyloid plaque formation and microglial activationproceed neurodegenerative changes in the pathogenesisof AD (Arends et al., 2000; Vehmas et al., 2003; Veerhuiset al., 2003). Since minocycline is able to prevent Ab fibrilformation as well as Ab-induced microglial activation, thiscompound may act on initial stages of the disease process.

ACKNOWLEDGMENTS

The authors thank the Netherlands Brain Bank (coor-dinator Dr. R. Ravid) for supplying the human CNS tis-sue, and Dr. A. Williams (EC project coordinator, Depart-ment of Veterinary Pathology and Infectious Diseases,Royal Veterinary College, Hatfield, UK) for helpful scien-tific discussions.

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