of April 4, 2016.This information is current as

PathwayInnate Immune Response via MAPK

InducesτMisfolded Truncated Protein

Cente, Monika Zilkova and Michal NovakAndrej Kovac, Norbert Zilka, Zuzana Kazmerova, Martin

http://www.jimmunol.org/content/187/5/2732doi: 10.4049/jimmunol.1100216August 2011;

2011; 187:2732-2739; Prepublished online 3J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2011 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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The Journal of Immunology

Misfolded Truncated Protein t Induces Innate ImmuneResponse via MAPK Pathway

Andrej Kovac,*,†,1 Norbert Zilka,*,†,1 Zuzana Kazmerova,* Martin Cente,*

Monika Zilkova,*,† and Michal Novak*,†

Neuroinflammation plays a key role in the pathogenesis of Alzheimer’s disease and related tauopathies. We have previously shown

that expression of nonmutated human truncated t (151-391, 4R), derived from sporadic Alzheimer’s disease, induced neurofi-

brillary degeneration accompanied by microglial and astroglial activation in the brain of transgenic rats. The aim of the current

study was to determine the molecular mechanism underlying innate immune response induced by misfolded truncated t. We

found that purified recombinant truncated t induced morphological transformation of microglia from resting into the reactive

phenotype. Simultaneously, truncated t caused the release of NO, proinflammatory cytokines (IL-1b, IL-6, TNF-a), and tissue

inhibitor of metalloproteinase-1 from the mixed glial cultures. Notably, when the pure microglial culture was activated with

truncated t, it displayed significantly higher levels of the proinflammatory cytokines, suggesting a key role of microglia in the

t-mediated inflammatory response. Molecular analysis showed that truncated t increased the mRNA levels of three MAPKs

(JNK, ERK1, p38b) and transcription factors AP-1 and NF-kB that ultimately resulted in enhanced mRNA expression of IL-1b,

IL-6, TNF-a, and NO. Our results showed for the first time, to our knowledge, that misfolded truncated protein t is able to induce

innate immune response via a MAPK pathway. Consequently, we suggest that misfolded truncated protein t represents a viable

target for immunotherapy of Alzheimer’s disease. The Journal of Immunology, 2011, 187: 2732–2739.

Theprominent pathological features of Alzheimer’s diseaseand related tauopathies are t neuronal and/or glial lesionsthat correlate with clinical symptoms and disease pro-

gression (1–3). t is an intracellular microtubule-associated pro-tein that belongs to the family of the intrinsically disorderedproteins characterized by the absence of a rigid three-dimensionalstructure in their natural environment (4). However, in diseasecondition, posttranslational modifications such as truncation andhyperphosphorylation lead to t transformation from intrinsicallydisordered protein into highly ordered, soluble and insolublemisfolded structures (5–10). It has been hypothesized that en-dogenous intracellular t may be released into the extracellularspace upon neuron degeneration (11). Indeed, neuronal death isone of the major pathological hallmarks of Alzheimer’s disease(AD). It is noteworthy that neuronal loss has been linked tothe topographic distribution of neurofibrillary tangles in severalstereological studies in AD brains (12–16). Simultaneously, clini-cal research consistently demonstrated an increase in total andphospho-t in the cerebrospinal fluid of AD patients (17). All these

findings strongly pointed out that intracellular t is released intothe brain’s extracellular environment.Several independent studies showed that soluble extracellular t

may promote 1) neurotoxicity by interacting with specific recep-tors on the surface of neurons (11, 18, 19); 2) intracellular calciumincrease through M1 and M3 muscarinic receptors in neuronalcells (18); 3) synaptic impairment (20); 4) blood–brain barrierdamage (21). Recent results cast a new light on the role of in-soluble extracellular t as a transmissible agent spreading t pa-thology throughout the brain in “prion-like fashion” (22–24).Multiple lines of evidence also indicate that extracellular t pro-tein may play an important role in AD neuroinflammation. Acti-vated microglia are present in and around neurofibrillary tanglesat early (25) and at later (26–32) stages of tangle formation. Mi-croglial activation also correlates with t burden in other humantauopathies such as tangle-predominant dementia, Guamanianparkinsonism-dementia, progressive supranuclear palsy, and cor-ticobasal degeneration (33–35). Moreover, activation of microglialinked to t deposition has been documented in mice transgenic forhuman mutant t protein P301S (36, 37), R406W (38), or P301L(39) and in transgenic rats expressing human nonmutated trun-cated t (40, 41). Notwithstanding these findings, the mechanismunderlying t-mediated microglial activation has not been identi-fied. To unravel this riddle, we used recombinant truncated tprotein to induce inflammatory response in the primary microglialand mixed glial cultures. In the current study, we show for the firsttime to our knowledge that truncated t is able to activate microgliavia the MAPK pathway leading to the release of NO, proin-flammatory cytokines, and chemokines.

Materials and Methodst protein purification

Human truncated t protein was purified from Escherichia coli bacteriallysates according to a published procedure (42) with modification. Briefly,the bacterial lysates were purified two times on cation-exchange HiTrap SPSepharose HP columns. The fractions that contained truncated t protein

*Institute of Neuroimmunology, Slovak Academy of Sciences, Center of Excellencefor Alzheimer’s Disease and Related Disorders, 845 10 Bratislava, Slovak Republic;and †Axon Neuroscience, 1030 Vienna, Austria

1A.K. and N.Z. contributed equally to this work.

Received for publication January 21, 2011. Accepted for publication June 28, 2011.

This work was supported by Axon Neuroscience and by research grants from theSlovak Research and Development Agency of Slovak Republic (Grant APVV-0631-07 to N.Z., Grants APVV-0603-06 and APVV LPP-0363-06 to M.N., and GrantAPVV LPP 0043-09 to M.C.).

Address correspondence and reprint requests to Prof. Michal Novak, Institute ofNeuroimmunology, Slovak Academy of Sciences, Dubravska 9, 845 10 Bratislava,Slovak Republic. E-mail address: Michal.Novak@savba.sk

Abbreviations used in this article: AD, Alzheimer’s disease; iNOS-2, inducible NOsynthase-2; LAL, Limulus amebocyte lysate; MS, mass spectrometry; TIMP-1, tissueinhibitor of metalloproteinase-1.

Copyright� 2011 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/11/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100216

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were pooled and purified by size-exclusion chromatography on a HiLoadSuperdex 200 26/60 column. Then, the buffer was exchanged for DTT freebuffer, and the protein was further immunoaffinity purified using the DC25mAb column. Finally, the protein was concentrated on a cation-exchangeHiTrap SP Sepharose HP column. To prevent the oxidation, prepared tprotein was stored in PBS under argon atmosphere at270˚C. The purity oftruncated t protein was routinely checked by SDS gel electrophoresis fol-lowed by Coomassie blue, silver staining, or Western blot with DC25 Ab.

MALDI-TOF

Mass spectrometry analysis of recombinant protein was performed at theBruker Daltonics facility (Bremen, Germany) on an HCT Ultra ion trap andan Ultraflex III MALDI-TOF/TOF (sinapinic acid as matrix). In someexperiments, prepared t protein was subjected to nano liquid chromatog-raphy (Easy nLC; Proxeon) before the MALDI-TOF analysis.

Detection of bacterial endotoxin in t protein using the Limulusamebocyte lysate test

To avoid any biases, we outsourced the testing of our t samples for thepresence of the endotoxin to a certified company (Biont GmbH) that used theLimulus amebocyte lysate (LAL) kinetic turbidimetric method. The LALmethod is based on monitoring of the rate of the development of turbidity,which is inversely proportional to the concentration of endotoxin in thesample. Unknown concentration of endotoxin in the sample was determinedby interpolating the onset time of the sample in the linear regression of thecalibration curve. Reagents used for the test were purchased fromAssociatesof Cape Cod. Control standard endotoxin (lyophilized endotoxin derivedfrom E. coli 0113:H10) was used as a standard. All other material used (e.g.,buffer, LAL reagent water, LAL reagent, glass tubes, tips) were endotoxin-free (Associates of Cape Cod). Detection and quantification of bacterialendotoxin in t protein by LAL test was hindered by interference in thesample. To overcome the inhibiting activity of t protein, a special treatmentof the sample was required. The combination dilution–heating procedurewas developed for testing and validating the sample based on the methodrecommended by Associates of Cape Cod for the kinetic turbidimetricmethod. The end-dilution used (1:180) was chosen based on the results ofpreliminary experiments taking into account the maximum valid dilution(1:1000). The sample was diluted only with LAL reagent water. The pH ofthe sample after dilution met the requirements for testing (pH 6–8). Therange of the calibration curve used was 0.001–1.000 EU/ml with the samplespiked with 0.5 EU/ml as positive product control. After treatment of thesample (dilution 1:180, heating for 30 min at 60˚C), all the requirements forthe validity of the assay (correlation coefficient$0.98) and the requirementsof the sample results (pH 6–8, spike recovery 50–200%) were met.

Glial cell cultures

Rat mixed glial cell cultures were prepared according to McCarthy and deVellis (43). Cerebral cortices of newborn rats (0–1 d old) were dissected,stripped of their meninges, and mechanically dissociated by repeatedpipetting followed by passage through a nylon mesh. Cells were plated in 96-well plates and 75-cm2 flasks precoated with poly-L-lysine (10 mg/ml) andcultivated in DMEM containing 10% FCS and 2 mM L-glutamine (all fromLife Technologies Invitrogen) at 37˚C, 5% CO2 in a water-saturated atmo-sphere. Themediumwas changed twice a week. Cultures reached confluenceafter 8–10 d in vitro and were used between 14 and 20 d in vitro.

Primary microglial cultures were isolated from mixed glial cultures byagitating the flask for 2 h at 200 rpm. To remove contaminating cells notbelonging to microglia, the isolated cells were washed 30 min after plating.The cells were maintained in astrocyte-conditioned medium and were usedfor experiments after 24 h in culture. The purity of microglial cell culturesisolated by this procedure was .95% (CD11b/CD18 staining).

Cell stimulation

For cell stimulation, glial cell culture mediumwas replaced with serum-freeDMEM supplemented with N2 supplement and L-glutamine (Life Tech-nologies Invitrogen). Cultures were stimulated with truncated t protein orLPS (O26:B6; Sigma) for 24 h. Polymyxin B (cell culture-tested; Sigma),SB203580 (a specific p38 MAPK inhibitor; Calbiochem), and PD98059 (aspecific p44/42 inhibitor; Calbiochem) were added to the culture 30 minbefore cell stimulation. Experiments were performed with eight replicatesper condition.

Nitrite assay

Nitrite (downstream product of NO) was measured in culture supernatantsas an indicator of NO production. Nitrite production was assessed by Griessreaction. Briefly, 50 ml cell culture medium was incubated with 100 ml

Griess reagent A [1% sulfanilamide (Sigma), 5% phosphoric acid] for 5min, followed by addition of 100 ml Griess reagent B (0.1% N-(1-naph-thyl)ethylenediamine; Sigma) for 5 min. The absorbance was determinedat 540 nm using a microplate reader (PowerWave HT; Bio-Tek).

ELISA cytokines

Concentrations of cytokines and chemokines secreted to the culture mediawere measured by commercial ELISA kits [IL-1b, TNF-a, IL-6, tissueinhibitor of metalloproteinase-1 (TIMP-1) (R&D Systems), and MCP-1(Invitrogen)] according to the manufacturer’s protocol.

Immunocytochemistry

Glial cultures were plated on glass coverslips (12-mm diameter). Afterbeing exposed to the experimental conditions, cells were washed in PBS andfixed with cold acetone–ethanol for 10 min at 4˚C. Cells were blocked with5% BSA in PBS and then incubated with anti-CD68 (Serotec, Oxford,U.K.), anti-CD11b/CD18 (Serotec) and anti-GFAP (Dako, Hamburg,Germany) Abs followed by incubation with corresponding Alexa 488-conjugated goat anti-rabbit and Alexa 546-conjugated goat anti-mouse Abs(Invitrogen-Molecular Probes, Eugene, OR). Finally, slides were mountedin fluorescence mounting media and photographed with an Olympus IX 71Fluoview laser scanning confocal microscope.

RNA extraction and quantitation by quantitative real-time PCR

Rat primary microglial cells (3 3 106) grown in 6-well plates were brieflywashed with 1 ml prewarmed PBS. Total RNAwas isolated using RNeasyMini Kit according to the manufacturer’s recommendations (Qiagen,Hilden, Germany). The genomic DNA was removed by DNase I digestionduring the RNA purification. RNAwas eluted into 40 ml RNase-free water.The integrity of isolated total RNA samples was determined with anAgilent 2100 Bioanalyzer using an RNA 6000 Nano Labchip kit (AgilentTechnologies, Waldbronn, Germany). For transcriptomic analysis, high-quality RNA samples were used (RNA integrity number = 8.0 to 9.5).Synthesis of the first strand was carried out using the High Capacity cDNAReverse Transcription Kit (Applied Biosystems, Foster City, CA) ac-cording to the manufacturer’s protocol. Briefly, 10 ml of the 23 reversetranscription mastermix was mixed with RNA sample (1 mg/10 ml) andcDNA was synthesized. Levels of mRNA were determined using quanti-tative real-time PCR with b-actin as a reference. TaqMan gene expressionassays (Applied Biosystems) were used for the determination of expressionlevels of several kinases, transcription factors, and target inflammatorygenes using an oligonucleotide probe with a 59 fluorescent reporter label(FAM for target genes and VIC for reference gene) and a 39 quencherdye (NFQ): JNK1, Rn01218952_m1; p38a, Rn00578842_m1; p38b,Rn01407663_g1; ERK1,Rn00820922_g1; ERK2,Rn00587719_m1;NFkB1,Rn01399583_m1; NFkB2, Rn01413849_g1; c-Jun, Rn00572991_s1; c-Fos,Rn02396759_m1; IL-1b, Rn99999009_m1; IL-6, Rn01410330_m1; TNF-a,Rn00562055_m1; inducible NO synthase-2 (iNOS-2), Rn00561646_m1;and reference b-actin, Rn00667869_m1. Composition of the quantitativePCR reaction (25 ml) was as follows: 12.5 ml 23 TaqMan gene expressionmastermix; 1.25 ml 203 target FAM- or VIC-labeled TaqMan primerassay; 10.25 ml nuclease-free H2O and 1 ml cDNA sample (50 ng/ml).PCR reactions were performed in duplicate under the following conditions:50˚C for 2 min, 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 s and60˚C for 1 min. Comparative ddCt analysis was performed to comparegene expression between control and truncated t-treated rat primarymicroglial cells. Results are expressed as a fold change of mRNA levelin truncated t-treated cells compared with nontreated control primarymicroglia. Genes with a fold change $2 were defined as differentiallyexpressed.

Data analysis

Values are presented as the means 6 SEM. Statistical analysis was per-formed using a one-way ANOVA (GraphPad Prism). Tukey’s multiplecomparison test was used for post hoc comparison. Differences at p, 0.05were accepted as statistically significant.

ResultsPurification and proteomic characterization of recombinanttruncated t

To avoid bacterial macromolecular contamination, we includeda DC25 immunoaffinity purification step in the preparation ofrecombinant truncated t with the highest purity. The results shownin Fig. 1A represent the silver, Coomassie blue, and DC25 staining

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of the immunoaffinity-purified t protein. The protein band corre-sponding with 32 kDa represents truncated t protein. The dis-crepancy between the molecular mass of truncated t determinedon SDS-PAGE (32 kDa) and its exact theoretical mass (25 kDa)could be explained by its nonglobular character. Another minorprotein band was observed around 29 kDa and represents a smallt fragment that could not be separated during the DC25 im-munoaffinity purification. To confirm protein, primary structureand purity mass spectrometry experiments were performed. Adirect infusion experiment performed on an HCT Ultra ion traprevealed the presence of two major peaks with corresponding m/zof 16,645.8 and 25,415.3 (Fig. 1B). These were in the next ex-periment separated by use of nano liquid chromatography andsubjected to MALDI in-source decay analysis. The obtained se-quences were compared with the sequence of truncated t protein.The 16-kDa species turns out to be a truncated t species coveringthe sequence part [1–161], and the major 25-kDa peak refers to theexpected 25-kDa recombinant truncated t sequence. No otherprotein peaks were observed within mass spectrometry (MS)spectra indicating very high protein purity.

Human recombinant truncated t was not contaminated withbacterial LPS

To exclude the possibility that the glial cultures were activated bythe residual contamination with LPS, we tested NO release in thepresence of polymyxin B, which is widely used as an inhibitor ofLPS. We treated our mixed glial cultures with truncated t with orwithout the presence of polymyxin B (10 mg/ml) (Fig. 2). Therewas no significant effect of polymyxin B preincubation on trun-cated t-stimulated NO release (7.65 6 0.23 versus 6.38 6 0.25, n= 16) by the cells suggesting that this effect is specific to truncatedt. As we have expected, the effect of LPS was completely abol-ished by the presence of polymyxin B (7.0 6 0.48 versus 1.99 60.08, n = 16). Polymyxin B alone has no effect on NO release. Toidentify possible endotoxin contamination in the sample, an LALkinetic turbidimetric test was also performed. We found that thecontent of bacterial endotoxin in tested samples of t protein wasunder detection limit of the assay (,0.001 EU/ml).

Truncated t protein stimulates microglial transformation fromresting to reactive phenotype

Activation of microglia is characterized by typical morphologicalchanges. The activated microglia develops enlarged cell processes,

which gives the cell a bushy appearance. In the late phase ofactivation, they became brain macrophages with typical roundshape. To demonstrate whether truncated t induces morphologicalchanges in vitro, a mixed glial cell culture model was used.Nonstimulated microglia in mixed glial cell culture showed rest-ing ramified morphology similar to the situation in vivo (Fig. 3A,3C). Stimulation of mixed glial cultures with truncated t protein(1 mM) for 24 h led to significant morphological changes. Theseinclude loss of cellular branching and transition from ramified toameboid (activated) state (Fig. 3B, 3D).

Misfolded truncated t protein induced cytokine in mixed glialcultures

To characterize activation of microglia triggered by human trun-cated t protein further, we measured the release of several cyto-kines and tissue inhibitor of metalloproteinases using ELISAassays. The results showed that stimulation of mixed glial cultureswith truncated t protein already at 100 nM concentration resultedin significant release of proinflammatory cytokines such as IL-1b(Fig. 4A, n = 2), IL-6 (Fig. 4B, n = 2), and TNF-a (Fig. 4C, n = 2)and of TIMP-1 (Fig. 4D, n = 2).

Microglial activation induced by truncated t is mediated byMAPK pathways

Many key cellular responses to extracellular stimuli are mediatedby kinase and phosphatase cascades. One of the most importantkinase families involved in immune response is the MAPK family.To identify a potential MAPK signaling pathway activated bytruncated t, we incubated primary mixed glial cultures with orwithout preincubation with ERK1/ERK2 and p38 MAPK inhib-itors. Our results clearly showed that truncated t-induced NOproduction was significantly blocked by pretreatment with theERK1/ERK2 MAPK inhibitor (PD98059) at 5 mM and 50 mM(p , 0.001, Fig. 5A). Similarly, treatment with the p38 MAPKinhibitor (SD202190) at concentrations above 0.2 mM and 2 mMmarkedly reduced NO production (p , 0.001, Fig. 5B). Notably,none of the tested inhibitors was able to abrogate completely theeffect of truncated t activation suggesting that both kinases, p38and ERK1/ERK2, are activated simultaneously.

FIGURE 1. The proteomic analyses of the purity of the human truncated

t. SDS-PAGE, WB, and MS analysis of immunoaffinity-purified truncated

t. To get truncated t with the highest purity, we included in the purification

step a DC25 immunoaffinity column. A, Silver staining, Coomassie blue

staining, and immunoblotting with DC25 Ab revealed the presence of one

major band with the apparent molecular mass ∼32 kDa. B, Electrospray

ionization (ESI)+ mass spectrum after software deconvolution showed

a major peak representing human truncated t (25 kDa) and a small t

fragment (16 kDa; aa 1–161).

FIGURE 2. Polymyxin B does not reduce NO release from glial cells

treated with truncated t protein. Polymyxin B inhibited LPS activity;

however, it did not have a significant effect on truncated t inflammatory

activity. Cells were preincubated with or without 10 mg/ml polymyxin B

for 30 min at 37˚C before truncated t (5 mM) or LPS (1 mg/ml) was added.

After 24 h, the NO production was measured by the Griess reaction. Values

represent the means 6 SD of three experiments conducted in quadrupli-

cate. ***p , 0.001 (versus no polymyxin B). PMX, polymyxin B.

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Microglial primary culture released cytokines after treatmentwith truncated t protein

Next, we analyzed production of proinflammatory cytokines byprimary microglia cultures using different concentrations of humantruncated t protein (0.1–1 mM).As shown in Fig. 6, there was a significant increase in proin-

flammatory cytokine IL-1b (Fig. 6A, n = 2), IL-6 (Fig. 6B, n = 2),

and TNF-a (Fig. 6C, n = 2) production upon addition of truncatedt protein. The levels of the cytokines released after treatment withtruncated t were significantly higher compared with the levelsreleased by treated astroglia–microglia culture. On the contrary,activated microglia did not express TIMP-1. Notably, the 0.1 mMtruncated t was able to induce all tested proinflammatory cyto-kines suggesting that truncated t was immunogenic already atnanomolar concentration.

Human truncated t stimulates activation of microglia throughNF-kB- and MAPK-dependent pathways

We have demonstrated that human truncated t protein (151–391,4R) induces morphological changes and distinct activation of ratprimary microglia. To identify the underlying molecular mecha-nisms involved in the truncated t-induced activation of microglia,we have analyzed the gene expression of several kinases, tran-scription factors, and target inflammatory genes. Quantitative real-time PCR analysis revealed upregulated mRNA expression ofJNK1 (2.6-fold), p38b (2.3-fold), and ERK1 (2.2-fold) kinasesupon treatment of microglia with 1 mM human truncated t protein.No difference in the mRNA levels was observed in the case ofp38a (1.4-fold change) and ERK2 (1.1-fold change) kinase (Fig.7A). Notably, peak upregulation of MAPK mRNA expression wasdetermined after 6 h, whereas prolonged incubation (12 h) showedthat the levels of mRNA either gradually decreased (ERK1) orremained stable (JNK1, p38b). These data indicate that JNK1,p38b, and ERK1 are the key players in the MAPK signaling oftruncated t-induced activation of microglia.To identify downstream transcription factors involved in the

truncated t-induced activation of microglia, we analyzed mRNAlevels of NF-kB and AP-1 factors. We found upregulated mRNAexpression of c-Jun (8.7-fold) and c-Fos (3.8-fold), the integralcomponents of AP-1 transcription factor, in microglia after 1 h oftruncated t treatment. Levels of these factors decreased graduallyin the course of time. Notably, increased levels of NFkB1 (7-fold)and NFkB2 (16-fold) were determined after 6 h of treatment (Fig.7B). These data clearly indicate the sequence of molecular eventsupon the truncated t treatment, where elevation of AP-1 tran-scription factor precedes the NF-kB signaling.

FIGURE 4. Truncated t stimulates release of inflammatory mediators from primary rat mixed glial culture. Mixed glial cell cultures were stimulated for 12

h with truncated t protein (0.1–1 mM). Media were collected, and IL-1b (A), IL-6 (B), TNF-a (C), and TIMP-1 (D) concentrations were determined via

commercial ELISA. Already at 0.1 mM concentration, truncated t induced significant release of proinflammatory cytokines and TIMP-1. LPS (1 mg/ml) was

used as a positive control. Values of nitrite accumulation from treated cells represent themean6 SEMof two independent experiments conducted in duplicate.

FIGURE 3. Truncated t induced morphological transformation of mi-

croglia in mixed glial culture. A and C, In nonstimulated mixed glial

culture, microglia displayed morphology of the resting glia. B and D, Glial

culture treated with truncated t induces significant change of microglia

morphology characterized by loss of cellular branching and transformation

to ameboid shape (white arrows). Double immunofluorescent staining of

complement receptor 3 (A, B; red color, ALEXA546), CD68 (C, D; red

color, ALEXA546), and GFAP (A–D; green color, ALEXA488) Abs. Scale

bars, 20 mm.

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Activation of MAPK-dependent pathways and NF-kB signalingresulted in distinct elevation of target inflammatory genes alreadyafter 1 h of treatment (IL-1b, 17.8-fold; IL-6, 9.3-fold; TNF-a,111.7-fold; iNOS-2, 2.1-fold). This effect was either sustained(TNF-a, 104-fold) or further increased (IL-1b, 322-fold; IL-6,11,044-fold; iNOS-2, 11,520-fold) after 6 h of treatment. Pro-longed (12 h) incubation of microglia in the presence of humantruncated t leads to additional elevation of iNOS-2 (28,323-fold)and IL-6 (15,950-fold) levels; however, the levels of TNF-a (26-fold) and IL-1b (170-fold) are gradually decreasing comparedwith those at 6-h incubation (Figs. 7C, 8).

Discussiont protein represents a key player in the pathogenesis of AD andrelated tauopathies (2, 3). Under physiological circumstances, tprotein is viewed as an intracellular cytoplasmic protein. However,it has been detected in extracellular biological fluids, such ashuman cerebrospinal fluid. Moreover, its levels in the cerebro-spinal fluid are significantly elevated in AD patients, and it is usedas a biomarker (17). t is able to accumulate in the extracellularspace usually as a consequence of neuronal death and may sig-nificantly contribute to neurodegeneration (11). In the currentstudy, we report for the first time to our knowledge that misfoldedtruncated t protein is a potent inflammatory mediator.In this study, we focused on truncated t protein, which has been

shown to be a driving force behind neurofibrillary degeneration in

transgenic rats expressing misfolded truncated t (44, 45). We havepreviously demonstrated that in the transgenic rat brain, neurofi-brillary lesions and axonal degeneration are closely associated withthe distribution of reactive microglia and macrophages (40). Toidentify the potentially crucial role of misfolded truncated t inneuroinflammation, we have treated microglia–astroglia culturewith this pathological form of t. To avoid the possible bacterialprotein contamination, we enriched the purification procedure us-ing pan-t mAb DC25 for immunoaffinity purification. Mass spec-trometry analysis of purified t revealed the presence of two majorpeaks with corresponding m/z 16,645.8 and 25,415.3, which rep-resent the minor 16-kDa t form [1–161] and the major 25-kDa formof misfolded t (151-391, 4R), respectively. No other protein peakswere observed within the MS spectra demonstrating the absence ofbacterial protein contaminants. The results presented earlier dem-onstrate that LPS is a frequent and functionally significant con-taminant in many commercial-grade preparations of proteins andpeptides used commonly in research on microglial activation (46).To exclude LPS contamination, we have tested truncated t in theabsence and/or presence of the LPS inhibitor polymyxin B. Wefound that polymyxin B did not eliminate the microglial activationinduced by truncated t. Moreover, the LAL test confirmed theabsence of bacterial endotoxins in our t samples.The majority of our experiments were done on the mixed glial

cultures that allow us towork with highly ramifiedmicroglia, whichis considered to be the predominant microglial phenotype in thenormal brain (47). Microglial ramification is better developed (48–51), and the microglia is less susceptible to exterior signals whencocultured with astrocytes than a pure microglial culture (47, 52,

FIGURE 5. Truncated t activates microglial cells via p38 and ERK1/

ERK2 MAPK pathways. PD98059 (A; p44/42 kinase inhibitor, 5 and 50

mM) and SB202190 (B; p38 kinase inhibitor, 0.2 and 2 mM) were added to

the culture medium 30 min before treatment with truncated t (1 mM). After

24 h, NO secretion was quantified by the Griess reaction. Values represent

the mean 6 SEM of two experiments conducted in quadruplicate. ***p ,0.001 (versus truncated t-stimulated cells without pretreatment).

FIGURE 6. Primary rat microglia released proinflammatory cytokines

after stimulation with truncated t. Primary microglia isolated from 20-d-

old mixed glial cultures was stimulated with truncated t protein (0.1–1

mM). Media were collected, and IL-1b (A), IL-6 (B), and TNF-a (C)

concentrations were determined via commercial ELISA. Data represent the

values from two independent experiments performed in duplicate.

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53). Furthermore, astrocytes play pivotal roles in microglial dif-ferentiation mainly by secreting insoluble factors such as fibro-nectin and laminin (54). Using mixed glial cultures, we found thathuman misfolded truncated t significantly changed the microglialmorphology from the resting to a reactive phenotype. This mor-phological change was accompanied by a release of proin-flammatory cytokines (IL-1b, IL-6, TNF-a). The pure microglialcultures showed even higher inflammatory response. In contrast,microglia did not upregulate the expression of TIMP-1 suggestingthat astrocytes are also involved in t-mediated immune response.Thus, truncated t is capable of direct activation of inflammatoryintracellular signaling pathways in microglia and astroglia as well.Previously, it has been demonstrated that the p38 and ERK1/

ERK2 (p44/42) families of MAPK pathways play a prominentrole in activation of the microglial cell in chronic neurodegener-ative diseases such as AD (55–58) and Parkinson’s disease (59,60). Both p38 and ERK1/ERK2 MAPK activation has been shownto be essential for IL-1, IL-6, and TNF-a expression and NO re-lease (61). Similarly, exposure of microglia to truncated t resultedin the generation of NO and in elevation of IL-1b, IL-6, andTNF-a expression. These observations led us to test whether

MAPK family members are involved in the t-induced signal trans-duction cascade. Using specific inhibitors of ERK1/ERK2 kinase(PD98059) and p38 kinase (SB202190), we found that MAPKpathways were involved in microglia inflammatory response in-duced by truncated t. Previously, it has been shown that activationof p38 and ERK1/ERK2 MAPK contributes to the activation oftranscription factors such as AP-1 or NF-kB, which in turninduces the release of proinflammatory cytokines and other in-flammatory molecules (61, 62). Our transcriptomic results clearlyshow that human truncated t induced upregulation of mRNAexpression of several MAPKs (JNK1, p38b, ERK1) and tran-scription factors (c-Jun, c-Fos, NFkB1, NFkB2) that further in-crease transcription of proinflammatory genes ultimately leadingto the release of proinflammatory cytokines IL-1b, IL-6, andTNF-a (Fig. 8).Multiple membrane receptors have been implicated in microglial

activation and intracellular signal transduction pathways (63). Inparticular, the class B scavenger receptor CD36 has been shown tomediate the microglial proinflammatory response to other proteinsinvolved in neurodegeneration, amyloid b (64–66) and a-synu-clein (67). Other cell surface proteins suggested to function as Abreceptors include the class A scavenger receptors SRA (68, 69)and B1 (70), the receptor for advanced glycation end products(71), heparin sulfate proteoglycans (72, 73), the serpin enzymecomplex (74), and many more. Identification of the receptors fortruncated t in microglial cells would be a further important steptoward understanding the mechanism of the extracellular t in-flammatory cascade.Previously, it has been shown that diseased-modified neuro-

nal proteins are able to activate microglial cells at higher con-centrations than those of bacterial endotoxins: amyloid b acti-vates microglia at the concentration 0.2–1 mM (69, 75, 76) anda-synuclein at the concentration 0.1–1 mM (77, 78). Similarly, theactive concentration of misfolded t was in the same range between0.1 and 1 mM. Furthermore, t concentration in the brain remainsa matter of debate. Several authors showed that the concentrationof intraneuronal t can reach 1–2 mM (11, 79, 80, 81), whereasothers argue that it could be even higher, 5–10 mM (82). Fur-thermore, Gomez-Ramos et al. (11) estimated that t in the ex-tracellular space would be ∼130 nM. In this study, we showed thatt is able to activate microglia at the concentration of 100 nM,

FIGURE 7. Human truncated t causes activation of microglia through

NF-kB- andMAPK-dependent pathways. Rat primary microglial cells were

incubated in the presence of 1 mM human truncated t protein (151-391, 4R)

for various time points. Quantitative real-time PCR analysis revealed an

upregulation of mRNA expression of MAPKs: JNK1, p38b, and ERK1 (A).

Simultaneously, increased mRNA levels were determined for c-Jun and c-

Fos (1 h) and NFkB1 and NFkB2 (6 h) transcription factors (B). Activation

of NF-kB- and MAPK-dependent pathways resulted in elevation of the

target inflammatory genes’ mRNA levels (IL-1b, IL-6, TNF-a, iNOS-2)

(C). Results are expressed as fold change over nontreated control cells

considering 2-fold change as significant. Real-time PCR results are repre-

sentative of three individual experiments. Error bars show 6SD.

FIGURE 8. Schematic illustration of t-mediated microglial activation.

An illustration demonstrates that truncated t activates microglial cells by

inducing the release of proinflammatory mediators via MAPK pathways.

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which indicates that extracellular t could be a potent inducer ofneuroinflammation in AD and related tauopathies.In conclusion, our study revealed that soluble truncated t protein

acts as a potent innate inflammatory stimulus. Moreover, we pro-vide new insights into the understanding of inflammatory pathwaysactivated by misfolded truncated t protein in AD and relatedtauopathies. We suggest that misfolded truncated t-mediated in-flammatory response represents a viable target for immunotherapyof human AD.

DisclosuresThe authors have no financial conflicts of interest.

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