Integrative Proteomics: Functional and Molecular Characterization of a

Integrative Proteomics: Functional and Molecular Characterization of a Particular Glutamate-Related Neuregulin Isoform Simone Schillo,† Vojislav Pejovic´ ,† Christian Hunzinger,† Torsten Hansen,| Slobodan Poznanovic´ ,† Jo1 rg Kriegsmann,| Werner J. Schmidt,‡ and Andre´ Schrattenholz*,†,§ ProteoSys AG, Carl-Zeiss-Str. 51, 55129 Mainz, Germany, Institute of Physiological Chemistry and Pathobiochemistry, Johannes-Gutenberg University Medical School, Duesbergweg 6, 55099 Mainz, Germany, Department Neuropharmacology, University Tu ¨ bingen, Mohlstr. 54/1, D-72074 Tu ¨ bingen, Germany, and Institute of Pathology, Johannes-Gutenberg University Medical School, Langenbeckstr. 1, 55131 Mainz, Germany Received January 24, 2005

Glutamate is the major excitatory neurotransmitter in the mammalian brain and is related to memory by calcium-conducting receptors. Neuregulins have emerged as long-term modulating molecules of synaptic signaling by glutamate receptors, playing a role in some cognition/memory-related disorders and moreover being part of transient functional microdomains, called lipid rafts. Here we characterize one specific isoform of neuregulin as a central biomarker for glutamate-related signaling, integrating results from in vitro and in vivo models by a differential functional and proteomic approach. Keywords: neuregulin • learning • memory • radial maze • glutamate • NMDA receptor • hippocampal neurons • differential proteomics • calcium transient

1. Introduction The molecular events underlying the formation of memory and related synaptic plasticity are very complex and involve an ever increasing number of interacting modular components, which moreover are potentially organized in functional spatiotemporal microdomains such as cholesterol-rich membrane rafts.1-5 A rise in postsynaptic Ca2+ via activation of NMDA (N-methyl-D-aspartate)-type of glutamate receptors is crucial at a very basic and initial stage in some memory-related longterm modifications of synaptic machinery,6,7 whereas neuregulins (NRG) appear to be organizing later stages.8,9 The underlying changes involve gene transcription, but also posttranslational modifications of proteins, like glycosylation, phosphorylation and proteolysis and appear to regulate protein assembly and microenvironment, providing flexibility on different time-scales. Here we focus on certain aspects of the roles of NRG und NMDA-receptors in two related experimental paradigms: (i) in mammals, the CA1 region of the hippocampus provides the model region of choice for initial stages of memory formation. We show the correlation of a specific neuregulin isoform with NMDA receptor mediated calcium transients in primary hippocampal neurons by a differential proteomic analysis; (ii) in an animal model of learning, we * To whom correspondence should be addressed. PD Dr. Andre´ Schrattenholz, CSO; ProteoSys AG, 55129 Mainz, Carl-Zeiss-Str. 51, Germany. Tel.: +49-6131-5019215. Fax: +49-6131-5019211. E-mail: andre.schrattenholz@ proteosys.com. † ProteoSys AG. ‡ University Tu ¨ bingen. § Institute of Physiological Chemistry and Pathobiochemistry, JohannesGutenberg University Medical School. | Institute of Pathology, Johannes-Gutenberg University Medical School.

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Journal of Proteome Research 2005, 4, 900-908

Published on Web 03/29/2005

further quantified the correlation of this particular neuregulin isoform with memory performance, by Western blots of 2-dimensional gels and staining with anti-neuregulin antibodies. Moreover, we used the antibody to quantify the expression of that particular neuregulin in post mortem slices from hippocampi of Alzheimer disease patients. Taken together, we show that just one particular isoform of neuregulin-β 1 (NRG1 Type 1 β) is associated with activity- and memory-related processes, and that many other isoforms of the same protein are not or much less clearly. We discuss potential physiological implications.

2. Experimental Section 2.1. Primary Cell Culture. Cell cultures were prepared using hippocampi from female neonatal Sprague-Dawley rats as described.10 Neuronal cells were plated on glass cover slips coated with poly-L-lysine at a density ranging from 75 000 to 150 000 cm2. 2.2. Fura-2 Ca2+ Imaging. Calcium transients in neurons after glutamate stimulation were measured and quantified as described previously.11,12 After loading cells with the fluorescent dye fura-2 AM (2 µM, Molecular Probes, Leiden, The Netherlands) for 40 min, for imaging an inverted light microscope (Axiovert 100, ZEISS, Germany) equipped with an UV-illumination source (75 W XBO lamp, OSRAM, Germany) was used. Acquisition and analysis of the data were performed by using MetaFluor software (Universal Imaging Corporation). Only cells identified as neurons by immunostaining (Supporting Information) and those whose calcium levels returned to the resting state after a first stimulation were taken into account. 2.3. Stimulation of Neurons. Every group of cells was stimulated twice for 30 s (one pair of images being acquired 10.1021/pr050012p CCC: $30.25

 2005 American Chemical Society

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Activity- and Memory-Related Neuregulin Isoform

every five seconds), with 30 min between ligand applications. Stimulation buffer for the Ca2+-imaging experiments contained, in mM: 125 NaCl, 5 KCl, 6 CaCl2, 0.8 MgCl2, 5 glucose, 20 HEPES, pH 7.3, and to which was added (in µM) 50 L-glutamate, 10 glycine (Sigma), 10 bicuculline, 25 AP-5, 10 CNQX, 500 (S)MCPG and 10 nifedipine (Tocris), as explained elsewhere in the text. Washing buffer was (in mM): 125 NaCl, 5 KCl, 2 KH2PO4, 2 CaCl2, 1 MgCl2, 5 glucose, 20 HEPES; pH 7.3. 2.4. Immunochemistry. Primary hippocampal neurons have been labeled by means of indirect immunofluorescence. Primary antibody used in these experiments was directed against R-Tau protein as neuronal marker (IgG, rabbit, polyclonal), diluted 1:100. Secondary antibody (goat, IgG, anti-rabbit, Texas Red-coupled, Jackson Research) was diluted 1:200. 2.5. Western Blotting and Immunodetection of NRG-β Isoforms. 2D gels were electroblotted onto nitrocellulose membranes using a standard procedure.13 Unspecific binding sites were blocked by incubation in TBST (20 mM Tris pH 7.4, 175 mM NaCl, 3.5 mM KCl, 0.1% Tween-20) containing 4% BSA. After incubation with the first antibody (anti-HRG-β, goat polyclonal IgG; Santa Cruz Biotech 1:500 in blocking buffer for Western blots from primary hippocampal culture proteins; antiNeuregulin Ab-2; rabbit polyclonal antibody, Fa. Neomarkers, 1:500 in blocking buffer for proteins extracted from hippocampi of test animals after radial maze experiments) overnight at room temperature, the blots were washed three times in TBST and antibody-antigen complexes were visualized by secondary antibodies coupled to alkaline phosphatase as described13 (we used anti-goat-IgG and anti-rabbit-IgG, whole molecule alkaline phosphatase-conjugates, Sigma, each at 1:1000 in blocking buffer, each for 1h at room temperature). The quantitative analysis of Western blots was performed using the program Investigator HT Analyzer Version 2.20, by NonLinear Dynamics Ltd., Genomic Solutions, Huntington, UK. 2.6. Immunohistochemistry of HRG-β. 4 µm paraffin sections from human brain samples were mounted on silanecoated slides, dewaxed and rehydrated through gradient alcohols. Staining with monoclonal antibodies against human HRG-β (polyclonal goat anti-human HRG-β, Santa Cruz) was performed using the Avidin-Biotin Complex (ABC-) technique. As control, goat serum (Vector, Burlingame, CA) and a specific blocking peptide (Santa Cruz Biotechnology) were used to replace the primary antibodies. ABC technique was performed as follows: Slides were pretreated by microwave processing (600 W three times 5 min; slides incubated in 10 mM Citrate buffer). They were then treated with an avidin/biotin blocking kit (Vector). After that, nonspecific binding of immunoglobulins was blocked by incubation with 4% nonfat dried bovine milk (Heurler, Radolfzell, Germany)/2% normal rabbit serum (Vector) in Tris buffer (pH 7.6). Primary antibodies diluted 1:10 and negative controls (in the case of goat serum diluted 1:5, in the case of blocking peptide diluted 1:2 in 2% nonfat dried bovine milk) were then incubated overnight at 4 °C, followed by a 30-min incubation with biotinylated rabbit anti-goat IgG (Vector). Slides were then covered with ABC kit (1:200 dilution; Vector) for 30 min. The color reaction was performed using the new fuchsin method. Endogenous alkaline phosphatase was blocked by adding levamisole (Sigma) to the substrate solution. Color development was stopped under microscopic control by immersing the slides in Tris buffer (pH 7.6). Finally, slides were counterstained with Mayer’s hematoxylin (Merck, Darmstadt, Germany) and mounted in Kaiser’s glycerol gelatin (Merck).

2.7. Proteomics. 2D-PAGE, silver staining of gels, spot picking and mass spectrometry were performed as described previously11,14 using Immobiline DryStrips (pH 4-7, Amersham Pharmacia Biotech). ESI-MS sequencing was carried out using a Finnegan Mat (San Jose, USA) LCQ ion trap mass spectrometer. The resulting peptide mass fingerprints were searched against the non redundant NCBI Protein Sequence Database using Mascot Server software v.1.8. (Matrix Science, London, UK). 2.8. Neurotransmitter Release. Neurons from primary hippocampal culture were cultivated and stimulated as described above. Fraction collection of supernatants from stimulated neurons was performed using computer-assisted synchronization. Samples were put on ice immediately and kept frozen at -80 °C until further treatment. Subsequently, samples were purified as described elsewhere15 and derivatized by orthophthaldialdehyde (OPA) or 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) using commercially available kits (Sigma for OPA, Waters for AQC). RP-HPLC-FLD of fluorescent amino acid (OPA) or amine derivatives (AQC) was performed using either an Agilent Chemstation 1100 with a Lichrosorb RP C18 column (200 × 4.6 mm; particle size: 5 µm; Merck) or a Waters Alliance 2690 Module with an XTerra MS C18 column (150 × 3.0 mm, particle size: 5 µm; Waters), employing gradient elution protocols described elsewhere.15,16 Separated components were detected by a fluorescence detector or, in some cases, by mass spectrometry (MS-ESI) using a ZMD 2000 module (Waters). The amount of column eluate introduced to the ion source was set to approximately 0.1 mL/min with a graduated Micro-Splitter Valve (Upchurch Scientific). Area under curve (AUC) of peaks was used for quantification. 2.9. Learning in the 8-Arm Radial Maze. Nine Sprague Dawley rats were grouped as follows: -Group 1 N ) 3 treated i.p. with saline (1 mL/kg) control 1 -Group 2 N ) 3 treated with saline (1 mL/kg) control 2 -Group 3 N ) 3 treated i.p. with MK-801 0.1 mg/kg (approximately 1 mL/kg) Treatments were applied 1 h before the maze experiments. Groups 2 and 3 were learning in the maze as described previously.17,18 Group 1 was put into the laboratory, but not into the maze, as a control. In the maze task, 4 randomly chosen arms were baited. The baiting pattern remained constant for the same rat. Different rats had different baiting patterns. Each rat was tested once a week over 5 weeks and performed 10 runs per day and each rat started from an arbitrarily chosen, but unbaited arm. Starting from the test day 2 errors were measured. The total number, i.e., working-, plus reference-memory errors will be displayed in results and time needed for completing the task; time per arm visit. After 5 weeks of learning, all animals were killed, the hippocampi were removed, and the hippocampal proteins were analyzed using 2D gels and Western blots with anti NRG-I as described above.

3. Results 3.1. Properties of Glutamate Induced Calcium Transients. Primary hippocampal neurons were first stimulated for 30 s with a buffer containing L-glutamate, glycine, and bicuculline at concentrations of 50, 10, and 10 µM, respectively. The amplitudes of the Fura-2 fluorescence signals (proportional to cytosolic calcium concentrations) obtained were compared to amplitudes obtained 30 min afterward during a second stimulation. Under these conditions, usually an increase of Ca2+ Journal of Proteome Research • Vol. 4, No. 3, 2005 901

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Figure 1. LTP-analogous potentiation of glutamate-induced Ca2+ transients in hippocampal neurons. A representative group of neurons, during the first (a) and the second (b) glutamate stimulation (50 µM, 30 s, 30 min intermission). Ratio values were calibrated to give intracellular Ca2+ concentrations (nM) as described in the Methods section. The control (a) shows amplitudes of approximately 425 nM, whereas approximately 700 nM are reached 30 min after the first stimulus (b). The statistics of the effect is shown in C: The second response (∆R ) R2max - R1max) is stronger and faster than the first one (blue, squares: 1st response (1st min), n ) 36; red, circles: 2nd response (31st min), n ) 36; error bars are sem). The pharmacological analysis of the effect is shown in d. It is mainly NMDA receptor-mediated, as demonstrated by blockade with the specific antagonists AP-5 (25 µM, n ) 86) or MK-801 (10 µM, data not shown), which abolish or slightly reverse the effect to a depotentiation upon second stimulus. Also blockade of metabotropic glutamate receptors by 500 µM S-MCPG (n ) 43) prevented potentiated second responses. CNQX (10 µM, n ) 69), a specific inhibitor of nonNMDA ionotropic glutamate receptors only slightly reduces potentiations of Ca2+ transients, i.e., AMPA- and kainate receptors do not contribute significantly, which is also true for L-type voltage-gated Ca2+ channels, because 10 µM nifedipine (n ) 41) does not show a significant effect. Normalized ∆R values obtained under such conditions were compared to the one measured in the control group (n ) 116), stimulated with 50 µM Glu alone, error bars are sem. Table 1 summarizes statistical details of these experiments. Table 1. Pharamacology and Statistical Analysis of Glutamate-Induced Ca2+ Transients in Hippocampal Neuronsa 1

2

3

4

5

6

7

8

9

10

stimulus

mean

s.e.m.

N

% PN

%NN

%DN

t-value

p-value

significance

E E/AP-5 E/CNQX E/MCPG E/Nif

100 -29.29 61.50 18.72 59.50

15.62 17.51 18.07 29.85 22.36

116 85 69 43 41

69 34.1 65.9 37.2 73.2

3.4 0 2.9 2.3 4.9

27.6 65.9 31.9 60.5 21.9

-5.4732 -1.5648 -2.5855 -1.3742

1.32 × 10-9 0.1193 0.0106 0.1713

+++ + -

a Rows 1-5 show basic statistical data for the pharmacology of Ca2+ influx potentiation. Values for ∆R (R values: I 340/I380) were normalized against the mean ∆R value from the control experiments (E: glutamate only. E/AP-5, E/CNQX, E/MCPG, and E/Nif represent experiments with the coapplication of AP-5 (25 µM), CNQX (10 µM), MCPG (500 µM), and nifedipine (10 µM), respectively. S.e.m. is standard error of mean, N is number of the cells. In rows 6-8 the relative numbers of de-(DN), non-(NN), and potentiated neurons (PN, in percent) for the various stimulation conditions are given. Cells were considered potentiated or depotentiated if their R values after stimulation were above or below the (mean ( s.d.) interval of the cell values prior to stimulation. Statistical comparison of the normalized ∆R values in the coapplication experiments (E/APV, E/CNQX, E/MCPG, E/Nif) against the control (E) is shown in rows 9-11, using the independent t-test at the significance level of 0.05 (highly significant: +++, significant: +, not significant: -).

levels as illustrated in Figure 1a, showing a representative group of neurons during the 9th second of the first (Figure 1a, left) and second (Figure 1b, right) stimulation, was observed. The mean increase in ratio signal (∆R) and the pharmacological analysis of the potentiation (∆R) are shown in Figure 1c,d. This increase is mainly dependent on NMDA and metabotropic glutamate receptors, since coapplication of appropriate an902

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tagonists (25 µM AP-5 and 500 µM S-MCPG, respectively) prevents this type of potentiation. Blockade of AMPA/kainate glutamate receptors by CNQX (10 µM) and L-type voltage-gated Ca2+ channels by nifedipine (10 µM) had no significant effect (Figure 1d). Table 1 summarizes statistical details of these experiments, which characterize two different activity-dependent functional states of hippocampal neurons. This type of


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Figure 2. GABA-release from hippocampal neurons is decreased during potentiated Ca2+ transients. Hippocampal neurons release a complex mixture of low molecular weight compounds during stimulations, including amino acids, some of which are indicators for the physiological mode of neurons; (a) Typical HPLC chromatogram of the superfusate collected during cellular responses to glutamate stimuli, as shown in Figure 1; here, amino acids were derivatized with o-phthaldialdehyde. (b) Simultaneously with glutamate-induced potentiation of Ca2+ transients, 30 min after the first stimulation, the release of GABA is significantly reduced, in contrast to control conditions. This result was confirmed by ESI-MS analysis of fractions collected from the experiments, where supernatants were derivatized with AQC: GABA was identified by retention time and mass: the m/z ratio of GABA (AQC derivative) is 274.1. (c) Statistical summary of GABA release quantification during glutamate stimulations of hippocampal neurons (n ) 18).

activity is induced by glutamate and effects are mediated by calcium signals via NMDA receptors. 3.2. Analysis of Neurotransmitter Release Synchronized to Glutamate Induced Calcium Transient. Simultaneously to the recording of neuronal calcium transients in these two functional states, the superfusing extracellular solution was collected and fractionated using an integrated microscopic assembly with a flow chamber, a synchronized fraction collector and an agent application device. Thus, changes in the molecular profile of released neurotransmitters could be correlated directly to calcium fluxes. During stimulations, as shown in Figure 2, hippocampal neurons release a complex mixture of low molecular weight compounds, including amino acids. Figure 2a shows a typical HPLC profile after fluorescent detection of amino acids derivatized with o-phthaldialdehyde (OPA): The amount of some released amino acids changes significantly during consecutive stimulations, i.e., their concentrations are correlated kinetically to second and potentiated Ca2+ transients and related protein expression changes. Quantification of GABA-OPA at a retention time of 21.8 min reveals that GABA release is reduced by over 40%, which is not the case under control conditions (Figure 2b). This result was confirmed by ESI-MS analysis of fractions derivatized with

AQC:16 GABA-AQC was identified at 26.3 min and m/z ratio of 274.1 (Figure 2b). Figure 2c gives a statistical summary of GABA release quantification during glutamate stimulations of hippocampal neurons (n ) 18). This result is supplying a further functional and activity-dependent parameter discriminating the two states of hippocampal neurons during first and second stimulation. The physiological significance of these functional changes after glutamate-induced neuronal activity was not investigated further. But here, we see a correlation of a time-dependent effect of the main excitatory neurotransmitter glutamate, mediated by calcium transients and NMDA receptors, with a decrease of the main inhibitory neurotransmitter GABA. 3.3. Functional Proteomics. Hippocampal cells from the two functional states described above plus material from an additional experiment with a blocker of NMDA receptors, MK801, were harvested and subjected to a differential proteomic analysis. Two-dimensional PAGE with pH 4-7 IEF gradients, reproducibly revealed a set of a few thousand proteins on silver stained gels, which remained mostly unchanged under all conditions. This is demonstrated in the upper part of Figure 3, showing three synthetic gels (a, control; b, glutamate; c, glutamate + MK-801; pI 4-7 gradients, each averaging 15 individual experiments). In the lower part, enlarged frames Journal of Proteome Research • Vol. 4, No. 3, 2005 903

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Figure 3. Glutamate-induced changes in hippocampal proteins analyzed by 2-D electrophoresis. Primary hippocampal neurons were stimulated according to the protocol described in the methods section. In the upper part three synthetic gels, each averaging 15 individual gel images are shown: In contrast to the nonstimulated (a ) control) and the MK-801 (10 µM) blocked controls (c ) glutamate+MK801) there are a few protein changes associated with neurons reacting to the second stimulus with increased Ca2+ transients (b ) glutamate), one of the most conspicuous ones is marked by frames. As indicated by an arrow in the lower panels a-c of individual representative gels and labeled Neu-β, it is a fragment of NRG1 (32 kDa and pI value of 4.5; n ) 15).

from representative individual gels show the area of the most conspicuous and persistent change, labeled as “Neu-β”. Under given conditions, only few other protein changes were consistently and reproducibly associated with glutamate-induced potentiation. Thus, we focus on a more complete analysis of this particular protein, integrating further mechanistically related biological samples and extending to further isoforms of the same protein by staining Western blots of 2D gels with appropriate antibodies. The spot density of this protein was always significantly increased after the second glutamate stimulation (quantitative statistic analysis was performed with similar results as described in detail for Western blots in Figure 4). It was identified by mass spectroscopy using MALDI-TOF mass fingerprinting and ESI-MS peptide sequencing.13 The identified protein “Neuβ”, deduced from 11 peptides covering 39% (118/304 aas) of the amino acid sequence, proved to be a member of the NRG-1 family of heregulins (see Table 2 and Figure 5 for details). The identity of the protein was confirmed by sequencing selected peptides using ion trap electrospray mass spectroscopy of the unseparated peptide mixture previously analyzed by MALDITOF. The ESI-MS analysis of m/z 798.4 generated a set of y7, y6, y5, y4 and of b4, b5, b6, b7 ions, which were sufficient to identify the peptide 88-94 (sequence in Figure 5). ESI-MS analysis of m/z 842.7 gave a set of y7, y6, y5, y4, y3 and of b4, b5, b6, b7 ions, confirming the sequence of the peptide at position 81-87 (sequence in Figure 5). These peptide sequences are conserved in neuregulins R and β, but in the central nervous 904


system neuregulins β are predominant.8,9 The best match would be a fragment of splice variant beta2 of NRG-1 (Figure 5). The identity of neuregulin β was proven independently by staining Western blots from 2-dimensional gels with a specific antibody against this protein (no cross reaction with neuregulin R, not shown). As shown in figs. 4a and b, the procedure detects about 30 different isoforms of neuregulin β present. Only the one at an approximately pI of 4.5 was detectable by silver stain (i.e., the concentrations of the other isoforms are below 1 ng/ spot). Statistics is shown in Figure 4c: the average spot density for this particular neuregulin β isoform is 361 (a.u.) after second stimulation with glutamate and 172 for controls (n ) 5 for each condition). The theoretical pI, calculated from neuregulin sequence is around 9.0, whereas in our experiment the specific activity-dependent isoform is found at a pI of 4.5, pointing to some post-translational modification as a response to glutamate stimulation, which was not analyzed in more detail. 3.4. Western Blot Staining for NRG-1 of 2D Gels from Hippocampal Proteins of Test Animals after Performance in a Behavioral Learning Experiment. To further investigate whether this activity-related NRG-1 isoform is also conspicuous in memory-related processes, a set of test rats were trained in a radial maze learning paradigm. Figure 6a shows representative Western blots (2D gels in the same setting as for primary culture) from hippocampal proteins of rat #3 (right) after 5 weeks of learning and a control rat (left), which was not exposed to the baited radial maze. In the lower part 6b, the near perfect inverse correlation of the NRG-1 (32kD, pI 5) spot


Figure 4. Identification of neuregulin β isoforms in hippocampal neurons by immunochemistry, The substantial increase of this particular fragment of NRG1 (32 kDa and pI value of 4.5) associated with increased Ca2+ transients, does not extend to other isoforms. (a) 2D Western blot of hippocampal control neurons stained with a specific antibody against neuregulin β. The frame of pI- and molecular weight values is chosen according to conditions of Figure 3. (b) A similar Western blot from stimulated hippocampal cells. Potentiated Ca2+ transients are associated with a substantial increase in the concentration of NRG1 at 32 kD and a pI of 4.5. Other isoforms detected on these blots do not appear to be clearly correlated with the stimulation. (c) Statistical summary of spot densities of the activity-specific neuregulin β isoform at a molecular weight of 32 kD and a pI of 4.5: average values are 361 (a.u.) for prestimulated neurons and 172 for controls (n ) 5 for each condition). Table 2. MALDI-TOF and ESI (*) Mass Spectrometry Peptide Fingerprinting of Peptide Mixture Obtained after In-Gel Digestion of Neuregulin β: 11 Peptides Covering 39% of the Amino Acid Sequence Strongly Indicate a Crucial Role of Neuregulin β in Glutamate-Induced Long Term Changes of Hippocampal Neurons Neuregulin-β (Rattus norvegicus): 11 Peptides cover 39% (118/304 aas) start end expected measured residue residue mass mass

1 11 269 88 81 232 102 57 17 70 16

5 16 274 94 87 239 117 72 38 87 38

650.3 645.4 796.4 798.5 842.4 1036.5 1790.1 1999.2 2214.1 2229.1 2342.2

650.8 644.9 796.0 798.4 842.7 1036.9 1790.7 1998.9 2214.6 2229.6 2343.1

sequence

MSERK GKGKKK QKLHDR IQKKPGK* NKPENIK* AEELYQKR ASLADSGEYMCKVISK CETSSEYSSLRFKWFK DRGSRGKPGPAEGDPSPALPPR WFKNGNELNRKNKPENIK KDRGSRGKPGPAEGDPSPALPPR

density with the total errors committed by the individual animals is shown. Animals 1, 4, and 7 were group 3, as described in the methods section, their learning success was inhibited by NMDA receptor antagonist MK-801, the average NRG-1 (32kD, pI 5) spot densities (a.u.) for the three groups are 9919 ( 769 for group 2 (exposed to maze and learning); 6712 ( 896 for group 3 (exposed to maze, learning impaired by MK-801); and 5477 ( 607 for group 1 (controls not exposed to the maze). Here we see a correlation between a memoryrelated performance in vivo and the concentration of the NRG-1 (32kD, pI 5) isoform in the hippocampus, which is the

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Figure 5. Position of neuregulin peptides from the potentiationdependent isoform identified by mass spectrometry: The sequence of NRG-1 (splice variant β-2) is given in black and underlined. In red are corresponding peptides identified in the present study by MALDI peptide mass fingerprints and ESI-MS sequencing. The black frames point out the positions of the various domains present in the ectodomain of NRG-1 proteins. Position numbers for domains or peptides are given in brackets (numbering according to supposed splice variant β-2). No further peptides could be found from the cytoplasmatic domain, apparently the potentiation-dependent isoform described here has only a very short cytoplasmatic tail, in accordance with apparent molecular weight in 2D gels.

brain structure which is assumed to be responsible for the underlying mechanisms. These mechanisms are thought to be related to the type of in vitro activity examined in the cell culture experiments described above (glutamate, calcium, NMDA recptors).19,20 In Figure 7, results from experiments using tissue slices from post mortem human brains are shown. Paraffin sections from each of 15 Alzheimer patient’s brains and age-matched controls were stained accordingly. As control, goat serum and a specific blocking peptide were used to replace the primary antibodies. Regions without obvious neuronal loss or extensive plaque or tangle formation were chosen in order to avoid artifacts due to different numbers of neurons. In the upper part three representative individual hippocampal slices are shown, A: an age-matched control brain; B: an Alzheimer patient’s brain, and C: a negative control, using a blocking peptide for antineuregulin-β staining of a section from an age-matched control tissue. The experiment reveals that the protein appears to be present in much higher concentrations in age-matched controls. Statistics of expression of neuregulin-β in tissue sections from Alzheimer brains and age-matched controls is presented in the lower part, Figure 7b, showing that on average the relative intensities in Alzheimer sections are 0.155 (a.u.), as Journal of Proteome Research • Vol. 4, No. 3, 2005 905

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Figure 6. Expression of the activity-dependent neuregulin-β isoform in sections from rat hippocampi (CA1 region) is correlateted to learning success, in an 8-arm radial maze test. Three groups of animals were investigated: Rats 3, 6 and 2 were trained a baited maze; control rats 7, 4, and 1 were trained in the baited maze, but learning was impaired by MK-801. In the center of the upper part, a representative 2D gel of hippocampal sections from rats after training, stained for total proteins is shown as an overview. Left: A representative Western blot stained for neuregulin-β, from a control animal which did not learn; Right: A corresponding Western blot from an animal after learning The overall expression pattern of neuregulin-β isoforms is surprisingly similar to the pattern obtained in vitro. Only an isoform at 32 kDa and pI value of 4.5, very similar to the one from the in vitro experiment, is again present in considerably higher concentration in animals which have learned. In vitro and in vivo results correspond very closely. Figure 6, lower part: Individual analysis of rats from the learning paradigm reveals a clear correlation of learning success and concentration of the special NRG1 isoform (32 kDa and pI value of 4.5): In average the number of total errors committed by the animals in the maze decreased by about 65% after training, these individual values of total errors in the fifth and last maze exposure committed after five weeks of training, are indicated in purple. The individual quantities of spot densities of the special memory-related NRG1 isoform (32 kDa and pI value of 4.5) are shown in blue and demonstrate that the concentration of this isoform increased consistently with measured learning success, as expressed by decreasing numbers of total errors committed.

compared to 0.603 in age-matched controls, n ) 15 for both groups. The expression of neuregulin-β in post mortem human hippocampi of the sample groups investigated here is 3.89fold higher in those which did not have disease-related memory deficits prior to death.

4. Discussion In the first series of experiments, we differentially analyzed effects of glutamate-induced calcium transients in primary rat 906


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Figure 7. Statistics of expression of neuregulin-β in tissue sections from Alzheimer brains and age-matched controls: Paraffin-embedded tissue sections were stained with a specific antibody against the protein. The areas were chosen from regions without obvious neuronal loss or extensive plaque or tangle formation. (A) Age-matched control; (B) Alzheimer brain; (C) Negative control using a blocking peptide for anti-neuregulin-β staining of a section from an age-matched control tissue section. The statistical chart in the lower part of the figure shows that on average the relative intensities in Alzheimer sections are 0.155, as compared to 0.603 in age-matched controls, n ) 15 for both groups. The expression of neuregulin-β in the sample groups investigated is 3.89-fold higher in the Ammon’s horn regions of human hippocampi with assumed better prior learning performance.

hippocampal neurons, a common and relatively simple in vitro model. On the protein level, we focused on one particular change correlated to these functional effects, which were relatively independent of AMPA receptors and L-type voltagegated calcium channels, but were depending on activation of NMDA and metabotropic glutamate receptors. Ca2+ entry into postsynaptic neurons through NMDA-receptors is a critical trigger in the induction of LTP,21,22 which currently provides the best model for very basic and initial processes of memory formation. LTP has been induced chemically by conditions similar to those of our in vitro experiment:23,24 The mostly CA1like properties of potentiation obtained in our experiments (Table 1) probably reflect pyramidal CA1 neurons as most abundant cell type in the hippocampus.25 The decrease in GABA concentrations upon the second stimulation (Figure 3) can be interpreted (a) as inhibition of GABA release and/or (b) as increased GABA reuptake. Both possibilities have been reported to play a role in hippocampal LTP/LTD in memory-related mechanisms, where strong glutamate stimulation not only potentiates transmission at synapses of pyramidal neurons, but also leads to depotentiation of GABAergic interneurons.26 The possibility (b) is supported by observations that activity of hippocampal GABA transporter GAT1 is increased by direct tyrosine phosphorylation,27 and that tyrosine kinase inhibitors prevent induction of pre- and postsynaptic LTP.28 In the experiments presented here no further emphasis was put on the question of underlying mechanisms (likely to involve tyrosine phosphorylation) or whether the observed effects are pre- or postsynaptic.


The differential proteomic results indicate a crucial role of neuregulin β, since the distinct concentration increase of the particular isoform at 32 kD and pI of 4.5 was consistently and reproducibly associated with NMDA receptor-dependent calcium transients. The number of experiments which were performed at the level of primary hippocampal cell culture was in the range of 50 2D gels with pI range 4-7, used either for mass spectrometry or anti-NRG-1 staining of corresponding Western blots. In the further analysis, we focused on this protein, because neuregulins (NRGs) have been implicated previously in activityor memory-related molecular synaptic events. They are cellcell signaling proteins that are ligands for receptor tyrosine kinases of the ErbB family. Their family of proteins is encoded by four genes (nrg-1 to nrg-4). NRGs each contain an epidermal growth factor (EGF)-like domain. The NRG1 proteins, which moreover may possess Ig-like and glycosylated domains, play essential roles in the nervous system, heart, and breast. There is also evidence for involvement of NRG signaling in the development and function of several other organ systems and in human disease, including the pathogenesis of schizophrenia, Morbus Alzheimer and breast cancer.8,9,29 They are widely expressed in the central and peripheral nervous systems, where they control cellular proliferation and differentiation, as well as mediating migration between nerve and Schwann cells, glia and oligodendrocytes. Our in vitro results, showing a consistent increase in the concentration of one particular NRG-1 β isoform (MW 32 kD, pI 4.5) in hippocampal cells as a result of glutamate stimulation, support findings of an important role of neuregulin β in synaptic activity and plasticity. The surprisingly complex pattern of NRG β isoforms, as apparent from Figures 4 and 6, must essentially arise from a diversity of post-translational modifications: approximately a dozen alternative splicing variants are supposed to be generated from the gene nrg-1. The cellular processing of NRG-1’s by proteolysis can release ectodomains as paracrine signals,8,30,31 their kinetics in turn depending on internal sequence-dependent, as well as external (e.g., calcium-) signals in feed-back types of regulation.32 Figure 5 summarizes our findings on the background of what has been reported previously: The NRG-1 peptides identified here all reside in the ectodomain, with the exception of one peptide just next to the transmembrane region on the proposed intracellular side, fitting best to a truncated fragment of splice variant beta2 of NRG-1. The molecular weight resulting from such a previously unknown NRG-1 species with an extremely short cytoplasmatic tail (preventing the release of the ectodomain) would be appropriate for the apparent molecular mass found in our study, the quite acidic pI points to further posttranslational modifications. Within the scope of this work we have not put emphasis on determining the site of such possible modification. Our data fit well to previous functional studies showing that NRG-1 β1 induces a specific expression of the NMDA receptor NR2C subunit,32 under conditions requiring previous activation of NMDA receptors and being related to mechanisms relevant for memory, like LTP.33 We further examined the expression of the NRG-1 isoform found by in vitro experiments in a memory-related animal experiment, which moreover included a control linking the performance of the test animals to NMDA receptors (MK-801 blockade of learning success in the radial maze). Surprisingly, in corresponding 2D-Western blots of hippocampi of test animals after 5 weeks of learning, the expression pattern looked very similar: the NRG-1 (32kD, pI 5) isoform concentration was

research articles correlated perfectly to learning success. To our knowledge, there are only few distinct molecular parameters, which so clearly correlate in a relatively simple activity-related in vitro paradigm and an in vivo memory-related behavioral model. The next step, extending these finding to human disease with memory deficits is far from systematic and complete, but shows that in Alzheimer patients’ brains the expression of NRG-1 is significantly less pronounced in hippocampi than in agematched controls. Here, no further distinction of isoforms could be performed. Obviously changes in one memory-related isoform, even if predominant, cannot completely explain the result, there must be an overall decrease of all NRG-1 isoforms. In some NRG-1-stains of slices from Alzheimer brains there appears to be a diffuse background stain, absent in agematched controls. We were also able to detect higher amounts of NRG-1 in a few cerebrospinal fluid samples from Alzheimer’s patients as compared to age-matched controls. However, these effects, which would point to a general loss of NRG-1 in Alzheimer’s disease, were not statistically significant, further studies should clarify these questions. Taken together, we think we could show that a functionally well-controlled differential proteomics analysis of relatively simple in vitro models can generate surrogate biomarker information, not available on the nucleic acid level.34,35 Even though a surprisingly large number of functional molecules, splice variants, and post-translationally modified proteins interact dynamically in complex protein expression profiles, here the NRG-1 (32 kD, pI 5) isoform appears to be a crucial molecule, not only related to “glutamate-NMDA receptorcalcium” signaling and thus to basic mechanisms such as LTP/ LTD, but also to memory in a NMDA receptor-related learning paradigm. As the primary hippocampal neurons and the hippocampi taken from rats after learning, the last experiment is observing NRG-1 changes in hippocampus, a brain region which is considered crucial for early steps in memory consolidation, also in humans. The staining of post mortem slices from human brains again indicates a difference with regard to prior learning performance, but no further analysis of the correlation of NRG-1 abundance in the Ammon’s horn of hippocampi to Alzheimer-disease-related β-amyloid processing and τ-hyperphosphorylation, nor a more detailed NRG-1 isoform analysis and quantification of the human material were performed within the scope of this study. In a broader perspective, there is LTP/LTD, modulated by the main excitatory neurotransmitter glutamate and its respective NMDA receptors. These receptors are calcium-conducting channels which moreover are coincidence detectors, by simultaneously reacting to voltage and glutamate, and thus the only molecule fitting a basic requirement of the only memoryrelated theory, the Hebbian synapse, which today is generally accepted and accommodating most present experimental data.36-38 The functional assay used here to differentially affect hippocampal neurons were under control of exactly these effectors, yielding precisely tuned protein extracts for the corresponding differential proteomics experiments. The most conspicuous and consistent change was the NRG-1 (32 kD, pI 5) isoform. In the next steps, we focused on this protein, not only because neuregulin has recently been shown to decrease GABAergic neurotransmission,39 which fits to what we found in vitro, but in particular because NRG-1β isoforms have increasingly been implicated from a genetic level in human diseases of the central nervous system.29,40 Moreover, the Journal of Proteome Research • Vol. 4, No. 3, 2005 907

research articles emerging role of NRG in the organization of sphingolipid/ cholesterol-rich membrane domains41-43 is intriguingly pointing to integrative properties of NRG’s in normal and pathophysiological signaling thoughout the bodies of mammals. Briefly, in these so-called lipid rafts NRG, heparansulfate binding proteoglycans and ErbB receptors assemble in activity- and localization-dependent mechanisms, thus regulating the subsequent signaling by tyrosine phosphorylation and calcium.42-45 In the central nervous system, synaptic activity is the prime motive force that guides synaptic formation, and also regenerative and protective mechanisms, but is also involved in corresponding pathophysiological events leading to disease or dysfunction. The question about the underlying molecular has recently become focused on regulatory factors that act on tyrosine kinase receptors on both sides of the synaptic interface. The neuregulins, existing both as membrane-bound and soluble forms through alternatively splicing and potentially posttranslational modifications, not only promote or modulate the local expression of the major ligand-gated ion channels such as acetylcholine, GABAA and NMDA receptors, but also appear to be affecting β-amyloid processing proteases, like secretases. In processes relevant for regeneration, heparinbinding forms of neuregulin accumulate to high levels in the synaptic basal lamina through the developmentally programmed expression of heparan sulfate proteoglycans, thus providing a sustained source of neuregulin to the most active synapses. They interact always with ErbB recptor tyrosine kinases. Recently, certain NRG isoforms have been discussed in neuroprotective mechanisms and neuroregeneration, and there appears to be transport through the blood brain barrier for soluble NRG isoforms.46 The time-resolved analysis of dynamic cellular events on the level of immediate protein profiles, provides a direct resource for discovery and development of novel surrogate biomarkers. The horizontal integration of several mechanistically related in vitro and in vivo models and biopsies on the molecular level, serves to more effectively and directly prevalidate these biomarkers and respective interventions than possible by conventional approaches.34,35,47,48

Acknowledgment. We thank A. Maelicke for discussions, J. Godovuc-Zimmermann for assistance with mass spectrometry, K. Schroer for assistance with data analysis, A. Cabuk and V. Pondeljak for technical assistance. Note Added after ASAP Publication. This manuscript was originally published on the Web (03/29/05) with several errors in Table 2. The version published 04/05/05 and in print is correct.

Supporting Information Available: Potentiated Ca2+ transients occur exclusively in hippocampal neurons. The glutamate-induced calcium influx of a group of stimulated cells is monitored by Fura-2 measurement (left part). After the functional experiment, the neuronal character of the very same group of cells is shown by immunostaining with an antibody against R-Tau protein, a specific neuronal marker (right part). This material is available free of charge via the Internet at http://pubs.acs.org.

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