Measurement of ERK 1/2 in CSF from Patients with Neuropsychiatric Disorders and Evidence for the Presence of the Activated Form

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1 Journal of Alzheimer s Disease 18 (2009) DOI /JAD IOS Press Measurement of ERK 1/2 in CSF from Patients with Neuropsychiatric Disorders and Evidence for the Presence of the Activated Form Hans-Wolfgang Klafki a,j,, Piotr Lewczuk a, Heike Kamrowski-Kruck a,j, Juan Manuel Maler a, Katharina Müller a, Oliver Peters b, Isabella Heuser b, Frank Jessen c, Julius Popp c, Lutz Frölich d, Stefanie Wolf e, Berit Prinz e, Christian Luckhaus f, Johannes Schröder g, Johannes Pantel h, Hermann-Josef Gertz i, Heike Kölsch c, Bernhard W. Müller j, Hermann Esselmann a,j, Mirko Bibl e,j, Johannes Kornhuber a and Jens Wiltfang a,j a Department of Psychiatry and Psychotherapy, University of Erlangen-Nuremberg, Erlangen, Germany b Department of Psychiatry and Psychotherapy, Charité Universitätsmedizin Berlin, Berlin, Germany c Department of Psychiatry and Psychotherapy, University of Bonn, Bonn, Germany d Division of Geriatric Psychiatry, Central Institute of Mental Health, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany e Department of Psychiatry and Psychotherapy, University of Göttingen, Göttingen, Germany f Department of Psychiatry and Psychotherapy, University of Düsseldorf, Düsseldorf, Germany g Section of Geriatric Psychiatry, University of Heidelberg, Heidelberg, Germany h Department of Psychiatry, Psychosomatic and Psychotherapy, University of Frankfurt, Germany i Department of Psychiatry, University of Leipzig, Leipzig, Germany j Present address: Department of Psychiatry and Psychotherapy, University of Duisburg-Essen, LVR-Klinikum Essen, Essen, Germany Accepted 24 April 2009 Abstract. The clinical diagnosis of neurodegenerative disorders can be supported by soluble biomarkers in cerebrospinal fluid (CSF), such as tau protein, phospho-tau, and amyloid-β peptides. In particular, increased CSF levels of phospho-tau in Alzheimer s disease appear to reflect disease specific pathological processes. We report here evidence for the presence of soluble MAP-kinase ERK1/2 in a small set of human CSF samples from patients with Alzheimer s disease, frontotemporal degeneration, and mild cognitive impairment. The level of total ERK1/2 in CSF as measured by electrochemiluminescent assay was correlated with that of total tau and phospho-tau. A small fraction of ERK1/2 in a pooled CSF sample was found to be in the doubly phosphorylated (activated) state. Our findings suggest that i) MAP kinase ERK1/2 is apparently released under neurodegenerative conditions in parallel with tau and phospho-tau and ii) in the future, it might be possible to find in CSF samples evidence for disease related alterations in brain kinase signaling pathways by use of highly sensitive and activation-state specific anti-kinase antibodies. Keywords: Alzheimer s disease, cerebrospinal fluid, ERK1/2, frontotemporal degeneration, MAP kinase, mild cognitive impairment, tau INTRODUCTION Corresponding author: Hans-Wolfgang Klafki, Department of Psychiatry and Psychotherapy, Laboratory for Molecular Neurobiology, University of Duisburg-Essen, LVR-Klinikum Essen, Virchowstr. 171, D Essen, Germany. Tel.: ; Fax: ; hans.klafki@uni-due.de. Molecular disease markers can complement the clinical diagnosis of Alzheimer s disease (AD), and they may help to identify individuals at high risk or in early stages of the disease [1,2]. Additionally, biomarkers ISSN /09/$ IOS Press and the authors. All rights reserved

2 614 H.-W. Klafki et al. / Measurement of ERK1/2 in Human CSF Samples may potentially aid in monitoring drug efficacy and they may serve in the development of novel disease modifying treatments. Biochemical changes that report pathological processes in the brain can be detected in cerebrospinal fluid (CSF). In AD patients, reduced mean CSF concentrations of soluble amyloid-β peptides ending at alanine-42 (Aβ 42 ) and increased total tau and phospho-tau proteins are well documented [3, 4]. Increased levels of total tau protein in the CSF can also be observed in several other brain disorders including Creutzfeldt-Jakob disease (CJD) [5], frontotemporal dementia (FTD) [6], and acute stroke [7]. Elevated total tau in CSF appears to reflect the rate of axonal and neuronal degeneration and is therefore not a specific marker for a particular neurodegenerative disorder. An early event in the development of the neurofibrillary changes in AD is the abnormal hyperphosphorylation of tau protein [8 10], and elevated concentrations of phosphorylated tau (phospho-tau) in the CSF of AD patients have been reported [11 15]. Importantly, normal phospho-tau concentrations were found in several other neurological and dementive disorders, suggesting that elevated CSF phospho-tau reflects disease specific alterations and may therefore aid in differential diagnosis of dementias or subtyping of dementias according to the neurochemical profile (reviewed in: [3,4]). The extracellular signal-regulated kinase 2 (ERK2) is one out of several kinases that have been proposed to play a role in the abnormal hyperphosphorylation of tau protein in AD (for reviews, see: [16,17]). Activated (doubly phosphorylated) forms of ERK1/2 and their upstream activating kinases MEK1/2 were shown to co-localize with early neurofibrillary changes in AD brains [18, 19]. In vitro studies indicated that ERKs can incorporate up to phosphates per molecule of tau protein at Ser-Pro and Thr-Pro motifs [20,21]. The phosphorylation sites on tau that can be phosphorylated by ERK2 include Ser422, a site which was reported to be phosphorylated in paired helical filament-tau (PHF-tau) but only weakly in biopsy-derived tau protein from human brain [22]. A recent study addressing the ability of selected kinase inhibitors to prevent abnormal tau hyperphosphorylation in cell culture and brain slice models is consistent with a possible involvement of ERK2 [23]. To test the hypothesis that in addition to phospho-tau one or more of the kinases possibly involved in abnormal tau-hyperphosphorylation might be detectable in human CSF, and may therefore have diagnostic potential, we measured soluble ERKs in a small set of human CSF samples in this pilot study. We report here that soluble ERK1/2 was detectable in human CSF from patients with AD, FTD, and mild cognitive impairment (MCI), and that its concentration was correlated with the concentrations of total tau and phospho-tau. Furthermore, we present evidence for the presence of low amounts of doubly phosphorylated (activated) ERK1/2 in pooled samples of human CSF. MATERIALS AND METHODS Patients and CSF samples CSF samples were collected from patients with different neuropsychiatric disorders as a part of the multicenter study, German Competence Network Dementias [24], according to the protocol described elsewhere [25]. Briefly, 4.5 ml of CSF was sampled into a polypropylene test tube, gently shaken immediately after collection, and centrifuged for 15 min at 1600 x g at room temperature. The CSF was aliquoted into 16 polypropylene test tubes (250 µl each) and was frozen within min after the puncture. For the analysis of tau and phospho-tau protein, the CSF was thawed only once, immediately before the measurements. In contrast, the aliquots for assaying ERK1/2 and phospho- ERK had been thawed and refrozen once, before being thawed again for ERK measurements. The 15 CSF samples that were subjected to electrochemiluminescence assays were obtained from patients with early AD (n = 4), FTD (n = 2), or MCI (n = 9). The patients (n = 15, mean age: 66.4 years, SD age: 8.2 years, 6 women and 9 men) were categorized according to clinical and neuropsychological criteria as described previously [26]. The pooled CSF samples that were used in some experiments were prepared by mixing several (usually approximately 10 20) individual CSF samples (leftovers from other studies). Antibodies Mitogen-activated protein kinase ERK2 was visualized on Western blots with phosphorylation insensitive polyclonal anti-erk2 (C-14) sc-154 (Santa Cruz Biotechnology Inc.). The activated (doubly phosphorylated) form of ERK1/2 was analyzed with polyclonal anti Active MAPK pab (Promega, Madison, USA) and with monoclonal anti-map Kinase Activated (Clone MAPK-YT) (Sigma, St. Louis, USA). For immunoprecipitation of ERK2, the agarose-conjugate of the polyclonal anti-erk2 antibody (C-14) sc-154 (Santa Cruz Biotechnology Inc.) was used.

3 H.-W. Klafki et al. / Measurement of ERK1/2 in Human CSF Samples 615 Protein precipitation with trichloroacetic acid and desoxycholate Per sample, 250 µl of human CSF was pipetted into a 1.5 ml reaction tube. Insoluble material was removed by centrifugation at x g for 5 min. The supernatant was transferred into a fresh tube, mixed with 2.5 µl of 2% (w/v) of sodium-desoxycholate and incubated on ice for 30 min. 75 µl of 50% (w/v) trichloroacetic acid was added (final concentration: 11.5% TCA) and the sample was mixed and incubated for at least 60 min on ice. After 30 min centrifugation at 4 C and x g, the supernatant was discarded. The resulting pellet was washed once with 250 µl of acetone (prechilled at -20 C) and air-dried. SDS-PAGE and Western blotting The samples were dissolved in SDS-sample buffer (final: 62.5 mm Tris-HCl, ph 6.8, 10% (v/v) glycerol, 2% (w/v SDS), 100 mm DTT, 0.02% (w/v) bromophenolblue), heated to 95 C for 5 min and separated on 10%T/2,7%C SDS-polyacrylamide gels [27]. The separated proteins were blotted onto PVDF membranes (Millipore Immobilon-P 0.45 µm) by semi-dry transfer (Hoefer Semiphor) at 1 ma/cm2 for 60 min with 25 mm Tris, 192 mm glycine, 20% (v/v) methanol [28]. After blotting, the membranes were rinsed with deionized H 2 O, blocked with a mixture of 1 vol of 2 x RotiBlock (Carl Roth GmbH, Karlruhe, Germany) and 1 vol of ECL-Advance blocking reagent (GE Healthcare) (2% in PBS/0.075% Tween-20 (PBS-T)), and incubated with primary antibody dilutions at 4 C overnight. In some of the earlier experiments, the blocking was done with either RotiBlock or ECL-Advance blocking reagent, without mixing the two. The membranes were washed with PBS-T (2 short rinses plus 3 10 min) and incubated with horseradish-peroxidase conjugated secondary antibodies for 45 min at room temperature. In some cases, the secondary horseradish-peroxidase anti-mouse IgG antibody was replaced by a biotinylated anti-mouse IgG antibody (Vector Laboratories) for 60 min at room temperature followed by a washing step and subsequent incubation with streptavidinperoxidase (GE Heathcare) for another 60 min. After washing (see above), the blots were developed with ECL-Plus TM or ECL-Advance TM (GE Healthcare) chemiluminescent substrates according to the manufacturer s instructions. Prior to reprobing with a different antibody, the membranes were stripped for min at 37 C with 0.2 M glycine/hcl, ph 2.5 containing 0.05% Tween-20. Phosphoprotein enrichment by metal affinity chromatography Several individual CSF samples (leftovers from other studies) were pooled and subjected to multiaffinity chromatography on an IgY 12 LC 10 column (Beckman Coulter) for removal of highly abundant proteins, such as Albumin and IgG. 5.2 ml of the unbound fraction (corresponding to 2.4 ml of the original pooled CSF sample) was concentrated to 0.2ml with Vivaspin VS MWCO spin concentrators (Vivascience AG, Germany). The concentrated sample was mixed with 1.5 ml of loading buffer (buffer A from phosphoprotein enrichment kit, BD Clontech) and loaded onto a phosphoprotein enrichment mini column with closed column outlet. The column was prepared from a commercial phosphoprotein enrichment column (BD Clontech Phosphoprotein enrichment kit), and contained 1/5 of the original column bed volume (bed volume: 0.2 ml). After 50 min incubation at 4 C with agitation, the column was fixed in an upright position and the gel bed was allowed to settle. The column inlet and outlet were opened and the unbound material (flow through) was collected. After 5 washes with 1 ml of loading buffer, the phosphoprotein-enriched fraction was eluted with ml of elution buffer (buffer B, BD Clontech phosphoprotein enrichment kit). For Western blot analysis, aliquots (200 µl, each) of selected fractions were subjected to protein precipitation with TCA and desoxycholate, as described above. ERK2 immunoprecipitation 0.5 ml of pooled human CSF samples was mixed with 55 µl of 10x Complete protease inhibitor cocktail in ultrapure H 2 O (Roche), 2.75 µl of 1 M NaF, 2.75 µl of 200 mm activated orthovanadate, 1.1 µl of 0.5 M EDTA and 25 µl of anti-erk2-agarose conjugate (Santa Cruz Biotechnology Inc. # SC154-AC). A second sample was prepared in the same way and was spiked with additional 240 pg of His-tagged activated recombinant ERK2 (Calbiochem # ). The samples were incubated overnight at 4 C on a shaker. The agarose-immuncomplexes were harvested by 1 min centrifugation at 2000 RPM in a table top centrifuge and washed 1x with 0.5 ml of 10 mm Tris- HCl, ph 7.4. To check for the efficiency of the procedure, the unbound fraction (supernatant) was mixed with 11 µl of 1 M Tris-HCl, ph 7.5 plus 25 µl of fresh anti-erk2-agarose slurry and incubated at 4 Cona shaker for an additional 2 hours. The immuncomplexes

4 616 H.-W. Klafki et al. / Measurement of ERK1/2 in Human CSF Samples were recovered as described. For SDS-PAGE/Western blot analysis, the immunoprecipitates were mixed with 25 µl of 1x SDS-sample buffer and heated to 95 C for 5 min. Per lane, 12 µl of the eluate was loaded onto a 10%T/2, 7%C SDS gel. ERK1/2 / perk Duplex Total and phospho-erk1/2 in human CSF samples with and without spiking with recombinant His-tagged ERK2 were measured with the Multi-Spot Phospho (T/Y:202/204; 185/187)/Total ERK1/2 assay (Meso Scale Discovery, Gaithersburg, USA). In a pilot experiment, a pooled CSF sample was spiked with 480 ng/ml of recombinant activated Histagged ERK2 (Calbiochem). Starting from this spiked CSF sample, serial dilutions were prepared in 1x duplex-assay buffer (final concentrations in the assay: 104 mm NaCl, 13.9 mm Tris-HCl, ph 7.5, 0.7 mm EDTA, 0.7 mm EGTA, 0.7 % Triton-X-100 plus protease inhibitors and phosphatase inhibitors). Aliquots of 50 µl each were analyzed in duplicate with the multispot phospho/total ERK1/2 assay according to the manufacturer s instructions. For assaying endogenous phospho- and total ERK1/2 in human CSF samples, 15 aliquots of human CSF samples (leftovers from a different study that had been thawed and refrozen before) were thawed, and 135 µl of each sample was mixed with a 10 x assay buffer concentrate (see above). 50 µl aliquots were tested for the presence of phospho- and total ERK1/2 in duplicates. Assay performance of ERK1/2 Monoplex for the analysis of CSF samples To evaluate the linearity of the signal, a pool prepared from several individual CSF samples was spiked with recombinant His-tagged ERK2, and serial dilutions were prepared in assay buffer, essentially as described above. 50 µl aliquots were measured in duplicates with total ERK1/2 High bind chemiluminescent assay (Meso Scale Discovery, Gaithersburg, USA). For the analysis of intra-assay variability, two different CSF samples were mixed with 10x monoplex- assay buffer concentrate (final concentrations in the assay: 150 mm NaCl, 20 mm Tris-HCl, ph 7.5, 1 mm EDTA, 1 mm EGTA, 1% Triton-X-100 plus protease inhibitors, and phosphatase inhibitors) and 50 µl aliquots were tested with 10 replicates in the same assay plate. For testing the influence of differences in preanalytical sample handling, two different CSF samples were measured after 1x thawing, 5x freeze-thawing and after 2 h of incubation at room temperature. The 10x assay buffer concentrate was added directly prior to the assay, i.e., after freeze-thawing and 2 h incubation at room temperature, respectively. ELISA measurements of total tau and phospho-tau pt181 For all CSF samples used in this study, routine neurochemical dementia diagnostics including ELISA measurements of total tau and ptau181 (Innogenetics, Ghent, Belgium) was performed according to the protocols described elsewhere [29]. Statistical analysis Statistical tests were performed with GraphPad Prism 5.01 software (GraphPad Software Inc., The normal distribution of the total tau, phospho-tau, and total ERK values was analyzed with the D Agostino-Pearson omnibus K2 Test. No evidence for deviations from normal distribution was observed, and therefore the correlations between total ERK1/2 in CSF and total tau and phospho-tau were calculated with Pearson s correlation tests. A level of p<0.05 was regarded as statistically significant; the actual p levels are given for descriptive purposes. RESULTS Evidence for the presence of soluble ERK1/2 in human CSF To test whether soluble MAP kinase ERK1/2 was detectable in human CSF, we subjected five human CSF samples to TCA/DOC protein precipitation and analyzed the precipitated proteins by Western- Immunoblot. A protein with an apparent molecular mass of 42 kda was readily detected in each of the 5 samples with the phosphorylation-insensitive pan-anti ERK2 antibody (Fig. 1A). Additional protein bands of approximately 55 and 25 kda were presumably due to human IgG heavy and light chains that cross reacted with the secondary perodixase labeled anti rabbit IgG antibody. On a parallel blot probed with polyclonal anti activated MAP kinase antibody, the same pattern of background protein bands was visible, but no clear evidence for doubly phosphorylated (activated) ERK1/2 was obtained (Fig. 1B).

5 H.-W. Klafki et al. / Measurement of ERK1/2 in Human CSF Samples 617 Fig. 1. Western blot detection of ERK2 in TCA/DOC precipitated proteins from 5 different human CSF samples. Per lane, a sample corresponding to 100 µl of original CSF sample was loaded. A) Western blot probed with polyclonal anti-erk2 antibody in combination with peroxidase labelled secondary anti-rabbit IgG antibody. B) Parallel blot probed with polyclonal anti-active MAP kinase antibody in combination with peroxidase labelled secondary anti-rabbit IgG antibody. Molecular masses of MagicMark TM Protein standards are indicated on the left side. A small fraction of ERK1/2 in CSF is doubly phosphorylated/activated Following the removal of highly abundant proteins, such as albumin and IgG, from a pooled CSF sample, the remaining proteins were fractionated on a phosphoprotein enrichment column. Samples of the unbound material and of the phosphoprotein-enriched eluate fractions were analyzed by immunoblotting. Doubly phosphorylated (activated) ERK1/2 was clearly detected by a monoclonal anti-activated MAP kinase antibody in the eluate fractions E2-E4, which were expected to be enriched in phosphoproteins (Fig. 2A). A parallel blot that was probed with a phosphorylationinsensitive polyclonal pan-anti ERK2 antibody confirmed the presence of a small amount of ERK2 in the phosphoprotein-enrichedfraction, and revealed that most of the ERK2 protein in the CSF pool was in the unphosphorylated (inactive) state (Fig. 2B). From a series of Western blots of different human CSF pools and serial dilutions of recombinant His-tagged ERK2 protein, we estimated the concentration of ERK2 in pooled CSF samples to be in the range of roughly pg/ml (data not shown). To confirm this estimate, an aliquot of a CSF pool was spiked with recombinant His-ERK2 at a final concentration of 480 pg/ml and subjected to anti-erk2 immunoprecipitation. For comparison, a second aliquot of the same CSF pool without addition of His-ERK2 was processed in parallel. Western blot analysis of the immunoprecipitates revealed background staining with IgG H-and L-chains with biotinylated anti-mouse IgG antibody and peroxidase conjugated streptavidin in the absence of a primary antibody (Fig. 3A). Immunodetection with monoclonal anti-activated ERK1/2 antibody clearly detected the recombinant (activated) His-tagged ERK2 in the immunoprecipitate from the spiked sample (apparent Mr 46 kda) but only faint bands of 42 kda corresponding to the endogenous ERK protein in the CSF pool (Fig. 3B). After stripping the membrane and reprobing with pan anti-erk2 antibody, both the endogenous and the recombinant His-tagged ERK2 proteins were clearly visualized (Fig. 3C). The concentration of the endogenous total ERK2 in the sample appeared to exceed that of the recombinant His-tagged ERK2 that was added to one of the two aliquots. Importantly, only a minor fraction of endogenous CSF ERK2 seemed to be in the activated (doubly phosphorylated) form, which is in agreement with the findings from the phosphoprotein enrichment procedure described above. Detection of ERK1/2 by MSD multispot phospho/total ERK1/2 assay kit To test the suitability of a commercial phospho/total ERK1/2 duplex chemiluminescence assay for measuring ERKs in CSF, a pooled CSF sample was spiked with 480 ng/ml of recombinant His-tagged ERK2. This was approximately 500 to 2000 times the estimated ERK1/2 concentration in human CSF samples (see above). Starting from the spiked CSF pool, serial dilutions were prepared in 1x assay buffer and analyzed in duplicates with the multispot phospho/total ERK1/2 assay. Linear correlations between His-ERK2 concentration and mean signal were observed for both total ERK1/2 and phosphorylated ERK1/2 readout (Fig. 4A, B) with concentrations ranging from pg/ml. On the same assay plate, a set of 15 human CSF samples from the German Competence Net Dementia collection was analyzed. Prior to measurement, each CSF

6 618 H.-W. Klafki et al. / Measurement of ERK1/2 in Human CSF Samples Table 1 Evaluation of intra-assay variation for ERK1/2 monoplex measurement of CSF samples CSF-sample 1 CSF sample 2 Background mean counts SD Range n % CV SD: Standard deviation; % CV: Coefficient of variation Fig. 2. Evidence for activated (doubly phosphorylated) ERK1/2 in pooled CSF. Following the removal of highly abundant proteins from a pool prepared from several individual CSF samples, the remaining proteins were fractionated on a phosphate metal affinity column. Selected fractions from the unbound material (flow through and wash fractions) and from the phospho-protein enriched eluate fractions were analyzed by Western blotting. A) Activated (doubly phosphorylated) ERK1/2 was detected in the eluate fractions E2, E3, and E4 with a monoclonal antibody against the activated form. B) A parallel blot was immunostained with a phosphorylation-insensitive anti-erk2 antibody. The presence of small amounts of ERK2 in the phosphoprotein enriched material was confirmed. Furthermore, the blot indicated that the major fraction of ERK2 in pooled human CSF was unphosphorylated. M: Marker proteins, FT: Flow through, W1: Wash fraction 1, W5: Wash fraction 5, E1-E7: selected eluate fractions (phosphoprotein-enriched material), IgY: Pooled CSF sample after removal of highly abundant proteins by multiaffinity chromatography on an IgY-LC10 column. Note that the blot does not truly reflect the relative distribution of ERKs between the unbound and the phosphoprotein enriched fractions, since 33% of each eluate fraction was loaded, but only 3.9% of the flow through and 6.7% of the wash fractions. sample was mixed with a 10x assay buffer concentrate to ensure comparable assay conditions. With each one of the 15 samples, we obtained counts well above background level for the detection of total ERK1/2. By comparison with the His-ERK2 dilutions, we estimated the concentrations of total CSF ERK1/2 in the 15 samples to range from 108 pg/ml to 468 pg/ml with a mean value of 260 pg/ml and a standard deviation of The signals measured for phospho-erk1/2 in all 15 CSF samples were close to or in some cases even below background and were therefore not considered for evaluation Total ERK1/2 levels in CSF are correlated with phospho- and total tau concentrations The CSF concentrations of total tau and phospho-tau in aliquots from the same 15 CSF samples were determined with commercial ELISA. Total ERK1/2 counts as well as total tau and phospho-tau concentrations in the 15 CSF samples passed D Agostino-Pearson omnibus K2 test for normal distribution (p = 0.55 for ERK1/2). The ERK1/2 measurements were significantly correlated with the CSF levels of both phosphotau and total tau (Fig. 4C and D). The total ERK signals

7 H.-W. Klafki et al. / Measurement of ERK1/2 in Human CSF Samples 619 Fig. 3. Western blot analysis of immunoprecipitated ERKs from pooled human CSF. A) Western blot detection with biotinylated anti-mouse IgG antibody and streptavidin- peroxidase in the absence of a primary antibody detected immunoglobulin heavy and light chains. Lane 1: Mr standards, lane 2: Immunoprecipitate from a human CSF pool. 3: IP-control for remaining ERK in the supernatant after immunoprecipitation (see description of method). 4: Immunoprecipitate from a human CSF pool spiked with 480 pg/ml of recombinant activated His-tagged human ERK2. 5: IP-control for remaining ERK in the supernatant from 4. B) The added recombinant activated His-tagged ERK2 with an apparent Mr of 46 kda was readily detected in the immunoprecipitate from a spiked human CSF pool with an antibody specific for doubly phosphorylated ERK1/2 (lane 4). Only a faint band corresponding to the endogenous doubly phosphorylated 42 kda ERK1/2 was observed (B, lanes 2 and 4). C) Reprobing of the membrane with a phosphorylation-insensitive anti-erk2 antibody indicated that the total amount of immunoprecipitated endogenous ERK2 in the pooled CSF sample exceeded that of the recombinant His-ERK that was added to one of the aliquots (lane 4). The sample amount loaded per lane corresponded to approximately 240 µl of CSF (with or without spiking) that was subjected to immunoprecipitation with 12 µl of anti ERK2-agarose beads. observed in the three diagnostic groups are shown in Fig. 4E. Performance of ERK1/2 monoplex assay for measuring human CSF samples: Intra-assay variation and influence of pre-analytical sample handling The findings described so far indicated that the CSF concentration of activated ERK1/2 was below detection sensitivity of the duplex assay, while total ERK1/2 in CSF was readily detectable and correlated with total tau and phospho-tau. To evaluate the suitability of a total ERK1/2 monoplex chemiluminescence assay for future testing of a larger set of CSF samples, a series of control experiments was done. Measurements of two different CSF samples with 10 replicates resulted in coefficients of variations smaller than 5% for the raw counts, indicating acceptable intra-assay variation (Table 1). With recombinant His-ERK2 protein at concentrations ranging from 75 to 2400 pg/ml, the monoplex assay was linear (r 2 = 0.998, p<0.0001). At the lowest tested concentration of 75 pg/ml, the signalto-noise ratio (calculated as [mean signal-mean background]/standard deviation of background counts) was 45, which is well above the limit of quantification as defined by a signal-to-noise ratio > 10:1 [30]. To analyze the influence of differences in preanalytical sample handling, two different CSF samples were measured in duplicates after 1x thawing, 5x freeze thawing and after 2 hours incubation at room temperature (Fig. 4F). Both preanalytical 5x freeze-thawing and 2 hours of incubation at room temperature resulted in slightly increased mean ERK signals in the monoplex assay. For statistical evaluation of these effects, a larger sample would be required, nevertheless, the observations indicate that standardized sample handling procedures should be applied. DISCUSSION One of the most urgent fields in current pharmacological research is certainly the search for novel therapeutics with strong disease modifying properties for the treatment of AD. An important prerequisite for being able to start any dementia therapy in an early disease stage will be an improved early and differential diagnosis of neuropsychiatric disorders. Clinical dementia diagnosis can be supported by biomarkers that

8 620 H.-W. Klafki et al. / Measurement of ERK1/2 in Human CSF Samples Fig. 4. Quantification of total and phospho-erk1/2 by chemiluminescence assays. A) and B): Detection of recombinant His-tagged activated ERK2 by phospho/total ERK1/2 duplex assay was linear in the tested concentration range. Mean values and linear regression from measurements of a serial dilution of recombinant His-tagged ERK2 in duplicates are shown for A) total ERK1/2 and B) doubly phosphorylated ERK1/2. C) and D): Total ERK1/2 measurements in human CSF samples correlate with CSF levels of total Tau and phospho-tau ps181 as measured by ELISA. E) Total ERK signals in CSF samples from patients with MCI, AD and FTD. The bars represent the mean signals in the 3 groups. F) Influence of preanalytical sample treatment on ERK1/2 signals in CSF samples: 2 different individual CSF samples were measured with ERK1/2 monoplex assay after 1x thawing, 5x freeze-thawing and after 2 hours incubation at room temperature. The bars refer to the mean counts of the duplicate reads and the error bars indicate the range. report pathological changes in the brain. Increased concentrations of phosphorylated tau protein in CSF from AD patients (for review, see e.g. [1]) are presumably related to the imbalance of kinase and phosphatase activities in AD brains that are responsible for abnormal tau hyperphosphorylation and ultimately NFT (neurofibrillary tangle) formation. We report here that MAP kinase ERK1/2, which is one out of several kinases that have been implicated in pathological tau-hyperphosphorylation, can be detected in human

9 H.-W. Klafki et al. / Measurement of ERK1/2 in Human CSF Samples 621 CSF samples from patients with AD, FTD, and MCI by Western blot and by commercial chemiluminescence assays. The CSF levels of ERK1/2 protein were correlated with those of total tau protein and of phospho-tau protein, suggesting that under neurodegenerative conditions ERK1/2 is released into the CSF in parallel with tau. Future studies including non-demented controls and a larger sample size will be required to evaluate whether measuring ERK concentrations in CSF may be helpful for diagnostic purposes and may therefore have clinical relevance. MAP kinases ERK1and ERK2 are activated by double phosphorylation at a conserved TEY motif by the dual-specificity protein kinases MEK1 or MEK2 [31]. Specific antibodies directed against the doubly phosphorylated (activated) form of MAP kinases are commercially available and allow for the investigation of the activation state of ERK1/2 in biological samples. Starting from a pool prepared from several individual human CSF samples, we enriched phosphorylated proteins and found clear evidence for the presence of a small fraction of ERK2 to be in the activated state. Our findings suggest that it might be possible to find evidence for disease related alterations of kinase signaling cascades by analyzing CSF samples for the presence of specific kinases that are regulated by phosphorylation and dephosphorylation. ERK1/2 and the activated form have been reported previously in CSF samples from patients with meningitis, but were not detected in CSF samples from several control patients including three cases with dementia [32]. However, in the study by Pollak and colleagues [32], the CSF samples were examined by Western blot without prior cell separation. While the control samples were acellular, all of the ERK1/2 positive CSF samples from menigitis patients contained cells, suggesting that the presence of ERK1/2 in these samples was possibly related to elevated cell counts. Our first attempt to quantify doubly phosphorylated ERK1/2 in a small set of CSF samples from patients with neuropsychiatric disorders was not successful. As it turned out, the sensitivity of the commercial duplex chemiluminescent assay we employed was not sufficient. Nevertheless, we believe that our findings that i) soluble ERK1/2 in human CSF samples appears to be correlated with total- and phospho-tau and ii) a small fraction of ERK1/2 in pooled CSF seems to be doubly phosphorylated may open a new window into studying brain kinase signaling states, provided that other kinases can be found as well in CSF and that sufficient detection sensitivity for activation state specific antibodies can be reached. Interesting targets in addition to ERK1/2 for this kind of approach may include Jun-N-terminal kinase/stress activated protein kinases (JNK/SAPKs), p38 MAP kinase, and glycogen synthase kinase 3 (GSK3). Although it is too early to judge whether alterations of ERKs in CSF can be expected to correlate with the clinical status of patients or may aid in differential diagnosis of neuropsychiatric disorders, we believe that the findings of our current methodological study strongly support further testing with a larger sample size. Importantly, in future studies non-demented controls should be included to evaluate the clinical value of this kind of measurements. ACKNOWLEDGMENTS This work was supported by the following grants from the German Federal Ministry of Education and Research (BMBF): Kompetenznetz Demenzen (01 GI 0420), HBPP-NGFN2 (01 GR 0447), NeuroAllianz, and by the EU grants cneupro (contract no. LSHM- CT ), and neurotas (contract no. LSHB- CT ). 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