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Inhibition of C1q-β-amyloid binding protects hippocampal cells against complement mediated toxicity
Journal of Neuroimmunology 137 (2003) 12 – 18 www.elsevier.com/locate/jneuroim
Inhibition of C1q-h-amyloid binding protects hippocampal cells against complement mediated toxicity M. Sa´rva´ri a, I. Va´go´ a, C.S. We´ber a, J. Nagy a, P. Ga´l b, M. Ma´k a, J.P. Ko´sa a, P. Za´vodszky b, T. Pa´zma´ny a,*,1 a b
Department of Molecular Biology, Gedeon Richter Ltd., Gyomroi u. 19-21, Budapest 10, POB27, 1103, Hungary Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest POB7, Hungary Received 26 September 2002; received in revised form 30 December 2002; accepted 24 January 2003
Abstract Activation of complement by h-amyloid (Ah) contributes to the pathology of Alzheimer’s disease (AD). Here, we show that C1-Inhibitor (C1-Inh) protects cultured rat hippocampal cells against h-amyloid induced complement lysis indicating a classical pathway-mediated activation mechanism. We report on screening of compound libraries to identify compounds that inhibit C1q binding to h-amyloid. Characterization of these compounds revealed that C1q possessed distinct binding sites for h-amyloid and antibodies. One selected compound protected cultured hippocampal cells against complement-dependent h-amyloid toxicity. These results provide evidence that complement has the potential to damage hippocampal cells, and C1q is a relevant target to suspend this deleterious mechanism in Alzheimer’s disease. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Alzheimer’s disease; Complement system; h-amyloid; C1q
1. Introduction Ample evidence implicates the involvement of the complement system in the pathology of Alzheimer’s disease (AD). Residential brain cells, including neurons, astrocytes and microglia, synthesize proteins of the complement system, so complement involvement does not depend on disruption of the blood –brain barrier (Gasque et al., 2000). Complement synthesis is up-regulated in AD brain (Yasojima et al., 1999) and complement proteins, including C1q are associated with senile plaques, one of the pathological hallmarks of AD (Eikelenboom and Stam, 1982; Stoltzner et al., 2000). C1q, the recognition subunit of the classical pathway of complement binds to h-amyloid (Ah), the major constituent of these plaques (Rogers et al., 1992). The interaction between C1q and Ah has three functional consequences in vitro. As a result of C1q binding, aggregation * Corresponding author. Tel.: +36-1-431-5861; fax: +36-1-260-26-43. E-mail addresses: [email protected], [email protected] (T. Pa´zma´ny). 1 Present address: Department of Immunology, Roswell Park Cancer Institute Elm & Carlton Streets, Buffalo, NY 14263, USA.
of Ah is enhanced by an order of magnitude (Webster et al., 1994). Incorporation of C1q onto amyloid fibrils blocks the uptake of Ah by microglia (Webster et al., 2000). Most importantly, C1q binding to Ah results in activation of the classical pathway of complement in an antibody independent manner (Rogers et al., 1992). It suggests that in AD pathology the classical pathway bears particular importance, although Ah activates the alternative pathway of complement via binding to C3 as well (Bradt et al., 1998). Host tissue is protected from complement lysis by soluble and membrane-bound regulators (Mu¨ller-Eberhard, 1987). Neurons in the hippocampus and cortex express low levels of most complement inhibitors, which makes them susceptible to complement-mediated damage (Yang et al., 2000; Singhrao et al., 2000). Indeed, the end product of the terminal pathway, MAC, has been detected on dystrophic neurites and damaged cells in AD brain by several groups (Rogers et al., 1992; Itagaki et al., 1994, Webster et al., 1997). These results provide solid evidence that Ah peptides, produced and accumulated in AD brain, drive-sustained activation of the complement system resulting in destruction of complement-sensitive neurons. Although the pathogenic potential of this powerful effector mechanism
0165-5728/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0165-5728(03)00040-7
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has been recognized, a complement inhibitor is still lacking from the therapeutic arsenal (Sahu and Lambris, 2000). Complement contains many potential targets for pharmaceutical intervention to inhibit pathological complement activation in AD. Since in vitro Ah activates both the classical and the alternative pathways, it is likely that in AD multiple complement pathways are activated. In order to target the right complement component, it is crucial to know which pathway is triggered primarily by Ah, leading to full activation of the complement cascade. To address this issue, we studied the effect of C1-Inhibitor (C1-Inh), a classical pathway regulator in a cell-based functional assay. C1-Inh controls activation of the C1 complex, consisting of one C1q and two C1r, C1s subunits (Schumaker et al., 1987). It is known that Ah1-40 peptide exerts enhanced cytotoxicity in the presence of human serum on cultured rat hippocampal cells (Schultz et al., 1994). Here, we report that C1-Inh protects hippocampal cells against Ah-induced complement lysis. This result suggests that in AD the classical pathway is primarily involved in initiation of the complement cascade. Therefore, we selected C1q as site for intervention and provided evidence that this recognition protein is a relevant target to selectively inhibit selfdestructive complement activation in neurodegenerative diseases including AD.
2. Materials and methods
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in PBS at room temperature for 30 min. C1q binding was carried out at room temperature for 1 h in the presence of compounds, at the concentration noted. Plates were washed three times with PBS containing 0.05% Tween20, then the amount of bound C1q was quantified by standard EIA protocol. In brief, C1q was probed with sheep anti-human C1q, then biotin labelled anti-sheep IgG and ExtrAvidinHRPO were added. TMB was used as substrate, reaction was quantified by reading OD450 nm on a Tecan Spectra ELISA reader. The same protocol was used for other direct C1q activators. For the antibody –C1q assay, plates were coated with chicken egg albumin at the concentration of 10 Ag/ml for 4 h. After removal of excess antigen, immune complexes were formed by adding 100 Al of rabbit polyclonal anti-ovalbumin (Sigma) at the dilution of 1:400. C1q binding and detection were carried out as described above. 2.3. Mass spectrometry Mass spectra were obtained on a Finnigan MAT 95SQ hybrid tandem mass spectrometer using an electrospray ionization source (Skribanek et al., 2001). N2 was applied as sheath gas, capillary was heated to 200 jC at spray voltage 3 kV and cone voltage 70 V. Ah and the compound were dissolved in 2 mM of CH3CO2NH4 – 0.5% acetic acid (v/v) 1:1 and the mixture was incubated at room temperature. Twenty microliters of the mixture was injected into the flow of the eluent, the flow rate was set to 50 Al.
2.1. Reagents and compound library 2.4. Cell culture Ah peptides were purchased from Bachem (Bubendorf, Switzerland). C1-Inh was a product of Centeon Pharma (Vienna, Austria). Proteins, including C-reactive protein (CRP), serum amyloid P (SAP) and C1q were commercially available from Calbiochem (LaJola, CA, USA). Sheep anti-human C1q IgG was obtained from Serotec (Oxford, UK). Myelin basic protein (MBP), secondary antibodies, substrates and the RBI library of pharmacologically active compounds were purchased from Sigma (St.Louis, MO, USA). Richter’s compound library consists of proprietary low molecular weight synthetic compounds. 2.2. Solid phase ligand-binding assays Plating and C1q binding was carried out according to published protocol (Jiang et al., 1994) with minor modifications. In brief, stock solutions of amyloid peptides for the binding assay were prepared in DMSO, and stored at 20 jC. The peptides were diluted into aqueous buffer just before use. Wells of Maxisorp microtiter plates (Nunc, Denmark) were coated overnight at 4 jC with 100 Al/well Ah peptides in 0.1 M Tris buffer, pH 8.5, at the concentration of 10 Ag/ml. After washing with PBS containing 0.05% Tween20 (Sigma) wells were blocked with 3% BSA
Primary hippocampal cultures were initiated from newborne Wistar rats (Toxi-Coop Hungary, Budapest). Hippocampi were isolated and tissue pieces were washed with Ca2 +/Mg2 + free HEPES buffered salt solution and incubated in Trypsin– EDTA solution (Sigma) for 4 min. After trituration, the cell suspension was filtered through a 70-Am mesh and sedimented at 125 g for 5 min. The cell pellet was re-suspended in D-MEM (GIBCO), containing 20% foetal bovine serum (GIBCO), 25 mM D-glucose (Sigma), 20 ng/ml NGF (Sigma) and antibiotics. Cells were plated into poly-D-lysine-coated 24-well plates (4 105 cells/well). Cultures were incubated at 37 jC in a humidified atmosphere of 5% CO2/95% air. From the third day, serum content of the culture medium was gradually decreased by replacement half of the old medium with the same amount of new one. 2.5. Cell-based functional assay Published protocol was followed to measure complement-dependent Ah toxicity (Schultz et al., 1994). In brief, a week after plating the morphology of the cells was checked and where it appeared less than optimal (e.g. high percentage of phagocytosis or poor process development) plates were not used in subsequent experiments. Prior to
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treatment, growth medium was replaced by 0.46 ml serumfree N2 medium (D-MEM supplemented with 5 Ag/ml insulin, 0.1 mg/ml transferrin, 5 Ag/ml Na-selenite, 0.02 Ag/ml progesterone, 0.1 mM putrescine, 25 mM D-glucose and antibiotics). The medium also contained 5-fluoro-5Vdeoxyuridine to stop proliferation of glial elements. Ah1-40 peptide was used as complement activator, the reverse Ah40-1 peptide was used as control. Ah peptides were dissolved before use in DMSO and diluted further with culture medium to the concentration of 0.8 mM. Fresh normal human serum was used as a complement source. Twenty-five microliters of Ah stock solution and 15 Al of fresh human serum were added to each well resulting in concentrations of 40 AM of Ah and 3% serum, respectively. The amount of C1-Inh was measured in units, 1 unit corresponds to 6 Levy and Lepow unit. Cytotoxicity was followed up to 5 days. LDH release was measured from 20 Al of supernatant using Cytotoxicity Detection Kit (Roche Diagnostics, Vienna, Austria) to evaluate cytotoxicity.
3. Results 3.1. C1-Inh protects hippocampal cells against complement-dependent b-amyloid toxicity Previously, it has been published that due to activation of the complement system, Ah1-40 peptide exerts enhanced cytotoxicity on cultured rat hippocampal cells in the presence of human serum (Schultz et al., 1994). This cell-based assay was used to study which pathway was primarily involved in initiation of the complement cascade leading to cellular damage. In preliminary experiments, we found that normal human serum and Ah1-40 individually were not cytotoxic at physiologically relevant concentrations to hippocampal cells. The threshold of Ah toxicity was generally higher than 40 AM; thereby, in subsequent experiments, we used Ah at concentrations up to 40 AM. In accordance with published results, in the presence of serum Ah1-40 exerted enhanced toxic effect in a concentration dependent manner. On the other hand, the reverse Ah40-1 peptide, in the presence of serum, showed no toxic effect at all, supporting the complement-dependent mechanism (Fig. 1A). Primary hippocampal cultures contain both neuronal and glial cells. In order to determine sensitivity of these various cell types towards complement-mediated amyloid toxicity, we cultured primary astroglial and microglial cells as described earlier (Pazmany et al., 2000). These glial cultures were treated identically as the mixed hippocampal cells with Ah1-40 in the presence and in the absence of serum. LDH release was measured to determine cytotoxicity, but no toxic effect was detected neither in the absence nor in the presence of serum (data not shown). These results suggest that the toxic effect of complement is directed against hippocampal neurons and does not affect glia.
Fig. 1. (A) Ah1-40 peptide at 40 AM concentration exerted enhanced cytotoxicity in the presence of human serum (closed triangle) on neonatal hippocampal cells. At the same concentration, the peptide alone (open triangle), or the reverse Ah40-1 peptide either in the presence (closed square) or in the absence (open square) of serum showed only residual cytotoxicity. (B) C1-Inh showed protection against complement-mediated cytotoxicity. Cells were treated with 40 AM Ah1-40 peptide in the presence of human serum and the protective effect of C1-Inh was evaluated by measuring LDH release on the fifth day. The points are averages of three independent measurements performed as described in Materials and methods.
Having the assay adopted, we examined the effect of C1Inh on Ah-mediated complement lysis. These experiments showed that C1-Inh significantly decreased complementdependent Ah cytotoxicity (Fig. 1B). The observation supported previous results demonstrating that complement activation was responsible for the enhanced Ah toxicity. Furthermore, this result suggested that inhibition of the classical complement pathway at C1 level might be neuroprotective against complement-dependent Ah cytotoxicity in AD. 3.2. Screening of RBI compound library by C1q binding assays reveals distinct binding sites for Ab and Fc on the C1q molecule Based on the above result, we screened the RBI library of pharmacologically active compounds to check the feasibility
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of finding compounds that selectively inhibit C1q binding to Ah. In preliminary experiments, we checked both Ah1-40 and Ah1-42 and got the same C1q binding. Therefore, in subsequent experiments, Ah1-42 was used for coating. Ah1-42 was immobilized onto microtiter plates and C1q binding was carried out in the presence of components of the RBI collection. Bound C1q was quantified by standard ELISA. Following the screen, compounds were selected that decreased the amount of bound C1q more than 50% at the concentration of 5 AM and were called hits. Seven hits were identified including benextramine, calmidazolium, methoctramine, milrinone, NPC-15437, tamoxifen and thioridazine, with the inhibition constant 50% (IC50) of 3, 3, 2, 3, 1, 2 and 4 AM, respectively. None of the hit compounds inhibited C1q binding to antibodies. Thereby, in accord with earlier studies, it seems feasible to selectively inhibit C1q binding to Ah, while not affecting C1q binding to antibodies (Chen et al., 1996). Amino terminal C1q A chain sequences has been implicated in binding of direct C1 activators including C-reactive protein (CRP), serum amyloid P (SAP) and Ah (Gewurz et al., 1993; Jiang et al., 1994; Chen et al., 1996). Therefore, we further screened the RBI library to identify compounds that inhibit C1q binding to these direct C1 activators including CRP, SAP and myelin basic protein (MBP). Activators were immobilized to plates, selectively bound C1q was measured and the same criteria were applied as above. The same seven hits with very similar IC50 values were found to inhibit C1q binding in the case of all direct C1 activators, as in the case of Ah. These results showed that compounds that inhibited C1q binding to Ah, did also inhibit C1q binding to other direct C1 activators, and vice versa. 3.3. Screening of Richter’s compound library to identify structures, which inhibit C1q binding to b-amyloid peptide Our proprietary compound library was screened by C1q binding assay to identify potent compounds that inhibit C1q binding to Ah. One structural class was identified to be effective in micromolar concentration. This cluster showed structural similarities to tamoxifen including a basic amine and a large planar lipophilic moiety. The most effective compound of the cluster was selected. This compound also inhibited C1q binding to other direct C1 activators including MBP, CRP (Fig. 2A) and SAP (data not shown), while it did not inhibit C1q binding to antibodies (Fig. 2B). Similar to C1q, SAP also binds to Ah in vitro (Tennent et al., 1995). In order to provide further data on the specificity of the compound, its effect was tested on SAP binding to Ah. Ah was immobilized and specifically bound SAP was measured by ELISA. Importantly, the compound did not inhibit SAP binding to Ah (Fig. 2B). These results suggested that the compound targeted the Ah binding site on the C1q molecule, which is distinct from the antibody binding site.
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Fig. 2. Characterization of the selected Richter compound by using various binding assays. (A) The compound at micromolar concentrations decreased C1q binding to various direct C1 activators such as Ah (closed circle), CRP (open triangle) and MBP (open circle), while no C1q binding was measured to the reverse Ah40-1 peptide (closed square). (B) Specificity assays such as Ah-C1q (closed circle), Ah-SAP (open square) and IgG-C1q (open triangle) showed that the compound at the same concentration did not interfere with protein – protein interactions, in general including the C1q-Fc interaction. The points are averages of six independent measurements performed as described in Materials and methods.
3.4. ESI-MS experiments do not indicate binding of the Richter compound to b-amyloid In order to provide evidence concerning the molecular target, the interaction between the compound and Ah was studied by electrospray ionization mass spectrometry (ESIMS). It is known that complex formation takes place between melatonin and Ah (Skribanek et al., 2001). Thereby for comparison and also for verification of our experimental conditions, first melatonin and Ah1-40 were incubated under native conditions to measure complex formation of melatonin with Ah (Fig. 3A). Next, the compound and Ah were incubated together under the same conditions. In the course of mass spectrometric investigation, the time dependence of complex formation was studied. The growing intensity of a complex peak with the incubation time would unambiguously prove the interaction between the two molecules. The
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abundance of the quaternary charged complex peak of melatonin and Ah at m/z 1141 showed an increasing tendency compared to the quaternary charged peak of Ah (at m/ z 1083). After 3 h of incubation, the complex peak reached about 35% relative intensity (Fig. 3A). The incubation of the Richter compound and Ah, however, gave a low intensity (less than 3%) peak at m/z 1181, detected after 3 h of incubation (Fig. 3B). This peak, according to its mass number, would correspond to the expected complex (quaternary charged peak), but it could be the result of a methodo-
Fig. 4. Inhibition of C1q-Ah binding protected neonatal rat hippocampal cells against complement-mediated Ah toxicity. Functional activity of selected Richter compound was measured in a cell-based assay including the Ah40-1 reverse peptide control as described in Materials and methods. The points are averages of three independent measurements.
logical adduct formation, as well. Our observation, that the extent of complex formation does not increase even after 24 h of incubation, suggests that there is no distinct interaction between the selected compound and Ah. Results of the binding assays and ESI-MS are in accord, indicating that the compound inhibits the Ah binding site on C1q, and sticks neither to the large carboxy-terminal hydrophobic domain, nor the amino-terminal part of Ah. 3.5. Functional characterization of compounds that inhibit C1q binding to b-amyloid Functionality of the compound with the ability to inhibit C1q binding to Ah was tested on cultured hippocampal cells. The compound showed no cytotoxicity at 1 AM concentration; hence, its functionality was studied on hippocampal cells treated with Ah1-40 and serum. The selected Richter compound protected cultured rat hippocampal cells against Ah-induced complement lysis (Fig. 4). This result provides the in vitro proof for the concept that targeting the Ah binding site on C1q protects brain cells against complement-mediated Ah cytotoxicity.
4. Discussion
Fig. 3. Demonstration of the lack of any interaction between the selected compound and Ah1-40. ESI mass spectra of the mixture of Ah1-40 and melatonin (A), and of Ah1-40 and Richter compound (B) was measured after 3 h incubation for complex formation.
Accumulating evidence suggests that complement contributes significantly to the pathology of AD (McGeer and McGeer, 2001). It is likely that both the classical and alternative pathways are activated by Ah in AD brain. Following activation, complement promotes local inflammation and facilitates destruction through opsonization and lysis (Mu¨ller-Eberhard, 1987; Frank and Fries, 1991). C5a, released during activation of the complement cascade, induces expression of interleukin-1h and interleukin-6 in Ah primed monocytes (O’Barr and Cooper, 2000). In turn, proinflammatory cytokines enhance the expression of early
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complement proteins, but not C1-Inhibitor (Veerhuis et al., 1999). As C1-Inh is the major regulator of the classical pathway, the imbalance between activation signals and regulatory forces in AD brain may lead to uncontrolled complement activation in the presence of Ah. Although it is difficult to discern which pathway initiated activation of the cascade, it is a crucial information to elaborate an anticomplement therapy for the treatment of AD. The importance of C1 in Ah-induced complement activation has been underscored by a recent observation that C1, reconstituted from purified subcomponents of C1q, C1r and C1s, is directly activated by Ah (Tacnet-Delorme et al., 2001). Here, we demonstrate that C1-Inh protects hippocampal cells against complement-mediated Ah toxicity (Fig. 1B). This result supports the view that in vivo Ah activates the complement system primarily through C1q. Therefore, we selected C1q as molecular target and identified compounds that inhibit C1q binding to Ah. One compound with this characteristic also protected hippocampal cells against complement-mediated Ah toxicity (Fig. 4). Comparison of the two protection curves reveals different characteristics between C1-Inh and the Richter compound, that may be due to their different mechanism of action, their different target sites (C1r and C1s serine proteases in the case of C1-Inh vs. C1q in the case of the Richter compound), or their different molecular properties (large protein vs. small organic compound) or the combination of these. This result gives strong support to the view that in vivo Ah activates the complement system primarily through C1q, thereby inhibition of the classical pathway should result in complete shutoff of Ah-mediated complement activation in vivo which may have therapeutic value in AD. Although inhibition of C1q –Ah binding is a particularly attractive target for pharmaceutical intervention, research suffers from the lack of structural data on C1q and from the controversial data on the Ah binding site on C1q. Some papers have provided data indicating that the collagen-like region contains the Ah binding site on the C1q molecule (Jiang et al., 1994; Chen et al., 1996; Velazquez et al., 1997). However, a recent study claims that instead of the collagen-like region, the globular heads participate in the binding of Ah (Tacnet-Delorme et al., 2001). Due to the complexity of C1q structure (Reid and Porter, 1976; Kishore and Reid, 2000), the binding site for Ah has not been determined at amino acid level yet. Although localization of the Ah binding site on C1q is out of the scope of this study, small synthetic compounds often provide valuable information on binding sites of proteins. Screening of compound libraries, using C1q binding assays, revealed that compounds that inhibited C1q binding to Ah, did not affect C1q binding to IgG antibodies in immune complex. This result supports the view that C1q possess distinct binding sites for Ah and the Fc part of antibodies. On the other hand, compounds that inhibited C1q binding to Ah did also inhibit C1q binding to other direct activators including CRP, SAP and MBP, and vice versa.
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IC50 values of the compounds for various direct C1 activators were very similar (Fig. 2A). Based on these results, it is likely that C1q possess the same binding site for direct C1 activators. In the literature, several studies suggest that the collagen-like region binds some direct C1 activators including CRP (Jiang et al., 1992; Gewurz et al., 1993), SAP (Ying et al., 1993; Zahedi, 1996) and DNA (Uwatoko and Mannik, 1990). Accumulating evidence suggests the role of pathological complement activation in experimental autoimmune encephalomyelitis (EAE), mouse model of multiple sclerosis as well. Astrocyte-targeted expression of sCrry, soluble form of a mouse complement receptor, prevents EAE (Davoust et al., 1999), while B cell-deficient mice are susceptible to EAE and develop a chronic-sustained disease similar to that seen in wild type mice (Hjelmstro¨m et al., 1998). Myelin proteins, including myelin basic protein and myelin oligodendrocyte glycoprotein, bind to C1q and activate the classical complement pathway (Vanguri and Shin, 1986; Johns and Bernard, 1997). These results implicate myelin proteins to induce complement activation in an antibody independent manner, which contributes to the pathology of EAE. Therefore, it seems relevant to test the most potent compounds in various models of this human disease. This can provide further information on the role of pathological complement activation in various forms of multiple sclerosis and reveal the benefits of its inhibition via C1q. Summing up, we have demonstrated that complement inhibition at C1 level suspends complement-mediated Ah cytotoxicity. This result highlights the importance of the classical pathway and its recognition molecule C1q in the pathology of AD. Result of the screening of compound libraries shows that C1q possess distinct binding sites for direct activators and Fc part of antibodies. Selected compound targeting the Ah binding site on the C1q molecule protects complement sensitive neurons against Ah-mediated complement lysis. On the other hand, our screening results suggest that it is likely that C1q possess the same binding site for direct C1 activators including Ah and MBP. Since recent EAE studies implicate myelin induced complement activation in the pathology of the disease, this result warrants the study of the selected compound in EAE models. Acknowledgements We thank Irini Bozonasz and Piroska Unghy for skillful technical assistance. References Bradt, B., Kolb, W.P., Cooper, N.R., 1998. Complement-dependent proinflammatory properties of the Alzheimer’s disease h-peptide. J. Exp. Med. 188, 431 – 438.
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Schumaker, V.N., Zavodszky, P., Poon, P.H., 1987. Activation of the first component of complement. Annu. Rev. Immunol. 5, 21 – 42. Singhrao, S.K., Neal, J.W., Rushmere, N.K., Morgan, B.P., Gasque, P., 2000. Spontaneous classical pathway activation and deficiency of membrane regulators render human neurons susceptible to complement lysis. Am. J. Pathol. 157, 905 – 918. Skribanek, Z., Balaspiri, L., Mak, M., 2001. Interaction between synthetic amyloid-h-peptide (1 – 40) and its aggregation inhibitors studied by electrospray ionization mass spectrometry. J. Mass Spectrom. 36, 1226 – 1229. Stoltzner, S.E., Grenfell, T.J., Mori, C., Wisniewski, K.E., Wisniewski, T.M., Selkoe, D.J., Lemere, C.E., 2000. Temporal accrual of complement proteins in amyloid plaques in Down’s syndrome with Alzheimer’s disease. Am. J. Pathol. 156, 489 – 499. Tacnet-Delorme, P., Chevallier, S., Arlaud, G.J., 2001. Beta-amyloid fibrils activate the C1 complex of complement under physiological conditions: evidence for a binding site for A beta on the C1q globular region. J. Immunol. 167, 6374 – 6381. Tennent, G.A., Lovat, L.B., Pepys, M.B., 1995. Serum amyloid P component prevents proteolysis of the amyloid fibrils of Alzheimer disease and systemic amyloidosis. Proc. Natl. Acad. Sci. U. S. A. 92, 4299 – 4303. Uwatoko, S., Mannik, M., 1990. The location of binding sites on C1q for DNA. J. Immunol. 144, 3484 – 3488. Vanguri, P., Shin, M.L., 1986. Activation of complement by myelin: Identification of C1q binding proteins of human myelin from the central nervous tissue. J. Neurochem. 46, 1535 – 1541. Veerhuis, R., Janssen, I., De Groot, C.J.A., Van Muiswinkel, F.L., Hack, C.E., Eikelenboom, P., 1999. Cytokines associated with amyloid plaques in Alzheimer’s disease brain stimulate human glial and neuronal cultures to secrete early complement proteins, but not C1-Inhibitor. Exp. Neurol. 160, 289 – 299. Velazquez, P., Cribbs, D.H., Poulos, T.L., Tenner, A.J., 1997. Aspartate residue 7 in amyloid h-protein is critical for classical complement pathway activation: implications for Alzheimer’s disease pathogenesis. Nat. Med. 3, 77 – 79. Webster, S., O’Barr, S., Rogers, J., 1994. Enhanced aggregation and h structure of amyloid h peptide after co-incubation with C1q. J. Neurosci. Res. 39, 448 – 456. Webster, S., Lue, L.F., Brachova, L., Tenner, A.J., McGeer, P.L., Terai, K., Walker, D.G., Bradt, B., Cooper, N.R., Rogers, J., 1997. Molecular and cellular characterization of the membrane attack complex, C5b-9, in Alzheimer’s disease. Neurobiol. Aging 18, 415 – 421. Webster, S., Yang, A.J., Margol, L., Garzon-Rodriguez, W., Glabe, C.G., Tenner, A.J., 2000. Complement component C1qmodulates the phagocytosis of Ah by microglia. Exp. Neurol. 161, 127 – 138. Yang, B., Li, R., Meri, S., Rogers, J., Shen, Y., 2000. Deficiency of complement defense protein CD59 may contribute to neurodegeneration in Alzheimer’s disease. J. Neurosci. 20, 7505 – 7509. Yasojima, K., Schwab, C., McGeer, E.G., McGeer, P.L., 1999. Up-regulated production and activation of the complement system in Alzheimer’s disease brain. Am. J. Pathol. 154, 927 – 936. Ying, S.C., Gewurz, A.T., Jiang, H., Gewurz, H., 1993. Human serum amyloid P component oligomers bind and activate the classical complement pathway via residues 14 – 26 and 76 – 92 of the A chain collagenlike region of C1q. J. Immunol. 150, 169 – 176. Zahedi, K., 1996. Characterization of the binding of serum amyloid P to type IV collagen. J. Biol. Chem. 271, 14897 – 14902.
Inhibition of C1q-h-amyloid binding protects hippocampal cells against complement mediated toxicity M. Sa´rva´ri a, I. Va´go´ a, C.S. We´ber a, J. Nagy a, P. Ga´l b, M. Ma´k a, J.P. Ko´sa a, P. Za´vodszky b, T. Pa´zma´ny a,*,1 a b
Department of Molecular Biology, Gedeon Richter Ltd., Gyomroi u. 19-21, Budapest 10, POB27, 1103, Hungary Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest POB7, Hungary Received 26 September 2002; received in revised form 30 December 2002; accepted 24 January 2003
Abstract Activation of complement by h-amyloid (Ah) contributes to the pathology of Alzheimer’s disease (AD). Here, we show that C1-Inhibitor (C1-Inh) protects cultured rat hippocampal cells against h-amyloid induced complement lysis indicating a classical pathway-mediated activation mechanism. We report on screening of compound libraries to identify compounds that inhibit C1q binding to h-amyloid. Characterization of these compounds revealed that C1q possessed distinct binding sites for h-amyloid and antibodies. One selected compound protected cultured hippocampal cells against complement-dependent h-amyloid toxicity. These results provide evidence that complement has the potential to damage hippocampal cells, and C1q is a relevant target to suspend this deleterious mechanism in Alzheimer’s disease. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Alzheimer’s disease; Complement system; h-amyloid; C1q
1. Introduction Ample evidence implicates the involvement of the complement system in the pathology of Alzheimer’s disease (AD). Residential brain cells, including neurons, astrocytes and microglia, synthesize proteins of the complement system, so complement involvement does not depend on disruption of the blood –brain barrier (Gasque et al., 2000). Complement synthesis is up-regulated in AD brain (Yasojima et al., 1999) and complement proteins, including C1q are associated with senile plaques, one of the pathological hallmarks of AD (Eikelenboom and Stam, 1982; Stoltzner et al., 2000). C1q, the recognition subunit of the classical pathway of complement binds to h-amyloid (Ah), the major constituent of these plaques (Rogers et al., 1992). The interaction between C1q and Ah has three functional consequences in vitro. As a result of C1q binding, aggregation * Corresponding author. Tel.: +36-1-431-5861; fax: +36-1-260-26-43. E-mail addresses: [email protected], [email protected] (T. Pa´zma´ny). 1 Present address: Department of Immunology, Roswell Park Cancer Institute Elm & Carlton Streets, Buffalo, NY 14263, USA.
of Ah is enhanced by an order of magnitude (Webster et al., 1994). Incorporation of C1q onto amyloid fibrils blocks the uptake of Ah by microglia (Webster et al., 2000). Most importantly, C1q binding to Ah results in activation of the classical pathway of complement in an antibody independent manner (Rogers et al., 1992). It suggests that in AD pathology the classical pathway bears particular importance, although Ah activates the alternative pathway of complement via binding to C3 as well (Bradt et al., 1998). Host tissue is protected from complement lysis by soluble and membrane-bound regulators (Mu¨ller-Eberhard, 1987). Neurons in the hippocampus and cortex express low levels of most complement inhibitors, which makes them susceptible to complement-mediated damage (Yang et al., 2000; Singhrao et al., 2000). Indeed, the end product of the terminal pathway, MAC, has been detected on dystrophic neurites and damaged cells in AD brain by several groups (Rogers et al., 1992; Itagaki et al., 1994, Webster et al., 1997). These results provide solid evidence that Ah peptides, produced and accumulated in AD brain, drive-sustained activation of the complement system resulting in destruction of complement-sensitive neurons. Although the pathogenic potential of this powerful effector mechanism
0165-5728/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0165-5728(03)00040-7
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has been recognized, a complement inhibitor is still lacking from the therapeutic arsenal (Sahu and Lambris, 2000). Complement contains many potential targets for pharmaceutical intervention to inhibit pathological complement activation in AD. Since in vitro Ah activates both the classical and the alternative pathways, it is likely that in AD multiple complement pathways are activated. In order to target the right complement component, it is crucial to know which pathway is triggered primarily by Ah, leading to full activation of the complement cascade. To address this issue, we studied the effect of C1-Inhibitor (C1-Inh), a classical pathway regulator in a cell-based functional assay. C1-Inh controls activation of the C1 complex, consisting of one C1q and two C1r, C1s subunits (Schumaker et al., 1987). It is known that Ah1-40 peptide exerts enhanced cytotoxicity in the presence of human serum on cultured rat hippocampal cells (Schultz et al., 1994). Here, we report that C1-Inh protects hippocampal cells against Ah-induced complement lysis. This result suggests that in AD the classical pathway is primarily involved in initiation of the complement cascade. Therefore, we selected C1q as site for intervention and provided evidence that this recognition protein is a relevant target to selectively inhibit selfdestructive complement activation in neurodegenerative diseases including AD.
2. Materials and methods
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in PBS at room temperature for 30 min. C1q binding was carried out at room temperature for 1 h in the presence of compounds, at the concentration noted. Plates were washed three times with PBS containing 0.05% Tween20, then the amount of bound C1q was quantified by standard EIA protocol. In brief, C1q was probed with sheep anti-human C1q, then biotin labelled anti-sheep IgG and ExtrAvidinHRPO were added. TMB was used as substrate, reaction was quantified by reading OD450 nm on a Tecan Spectra ELISA reader. The same protocol was used for other direct C1q activators. For the antibody –C1q assay, plates were coated with chicken egg albumin at the concentration of 10 Ag/ml for 4 h. After removal of excess antigen, immune complexes were formed by adding 100 Al of rabbit polyclonal anti-ovalbumin (Sigma) at the dilution of 1:400. C1q binding and detection were carried out as described above. 2.3. Mass spectrometry Mass spectra were obtained on a Finnigan MAT 95SQ hybrid tandem mass spectrometer using an electrospray ionization source (Skribanek et al., 2001). N2 was applied as sheath gas, capillary was heated to 200 jC at spray voltage 3 kV and cone voltage 70 V. Ah and the compound were dissolved in 2 mM of CH3CO2NH4 – 0.5% acetic acid (v/v) 1:1 and the mixture was incubated at room temperature. Twenty microliters of the mixture was injected into the flow of the eluent, the flow rate was set to 50 Al.
2.1. Reagents and compound library 2.4. Cell culture Ah peptides were purchased from Bachem (Bubendorf, Switzerland). C1-Inh was a product of Centeon Pharma (Vienna, Austria). Proteins, including C-reactive protein (CRP), serum amyloid P (SAP) and C1q were commercially available from Calbiochem (LaJola, CA, USA). Sheep anti-human C1q IgG was obtained from Serotec (Oxford, UK). Myelin basic protein (MBP), secondary antibodies, substrates and the RBI library of pharmacologically active compounds were purchased from Sigma (St.Louis, MO, USA). Richter’s compound library consists of proprietary low molecular weight synthetic compounds. 2.2. Solid phase ligand-binding assays Plating and C1q binding was carried out according to published protocol (Jiang et al., 1994) with minor modifications. In brief, stock solutions of amyloid peptides for the binding assay were prepared in DMSO, and stored at 20 jC. The peptides were diluted into aqueous buffer just before use. Wells of Maxisorp microtiter plates (Nunc, Denmark) were coated overnight at 4 jC with 100 Al/well Ah peptides in 0.1 M Tris buffer, pH 8.5, at the concentration of 10 Ag/ml. After washing with PBS containing 0.05% Tween20 (Sigma) wells were blocked with 3% BSA
Primary hippocampal cultures were initiated from newborne Wistar rats (Toxi-Coop Hungary, Budapest). Hippocampi were isolated and tissue pieces were washed with Ca2 +/Mg2 + free HEPES buffered salt solution and incubated in Trypsin– EDTA solution (Sigma) for 4 min. After trituration, the cell suspension was filtered through a 70-Am mesh and sedimented at 125 g for 5 min. The cell pellet was re-suspended in D-MEM (GIBCO), containing 20% foetal bovine serum (GIBCO), 25 mM D-glucose (Sigma), 20 ng/ml NGF (Sigma) and antibiotics. Cells were plated into poly-D-lysine-coated 24-well plates (4 105 cells/well). Cultures were incubated at 37 jC in a humidified atmosphere of 5% CO2/95% air. From the third day, serum content of the culture medium was gradually decreased by replacement half of the old medium with the same amount of new one. 2.5. Cell-based functional assay Published protocol was followed to measure complement-dependent Ah toxicity (Schultz et al., 1994). In brief, a week after plating the morphology of the cells was checked and where it appeared less than optimal (e.g. high percentage of phagocytosis or poor process development) plates were not used in subsequent experiments. Prior to
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treatment, growth medium was replaced by 0.46 ml serumfree N2 medium (D-MEM supplemented with 5 Ag/ml insulin, 0.1 mg/ml transferrin, 5 Ag/ml Na-selenite, 0.02 Ag/ml progesterone, 0.1 mM putrescine, 25 mM D-glucose and antibiotics). The medium also contained 5-fluoro-5Vdeoxyuridine to stop proliferation of glial elements. Ah1-40 peptide was used as complement activator, the reverse Ah40-1 peptide was used as control. Ah peptides were dissolved before use in DMSO and diluted further with culture medium to the concentration of 0.8 mM. Fresh normal human serum was used as a complement source. Twenty-five microliters of Ah stock solution and 15 Al of fresh human serum were added to each well resulting in concentrations of 40 AM of Ah and 3% serum, respectively. The amount of C1-Inh was measured in units, 1 unit corresponds to 6 Levy and Lepow unit. Cytotoxicity was followed up to 5 days. LDH release was measured from 20 Al of supernatant using Cytotoxicity Detection Kit (Roche Diagnostics, Vienna, Austria) to evaluate cytotoxicity.
3. Results 3.1. C1-Inh protects hippocampal cells against complement-dependent b-amyloid toxicity Previously, it has been published that due to activation of the complement system, Ah1-40 peptide exerts enhanced cytotoxicity on cultured rat hippocampal cells in the presence of human serum (Schultz et al., 1994). This cell-based assay was used to study which pathway was primarily involved in initiation of the complement cascade leading to cellular damage. In preliminary experiments, we found that normal human serum and Ah1-40 individually were not cytotoxic at physiologically relevant concentrations to hippocampal cells. The threshold of Ah toxicity was generally higher than 40 AM; thereby, in subsequent experiments, we used Ah at concentrations up to 40 AM. In accordance with published results, in the presence of serum Ah1-40 exerted enhanced toxic effect in a concentration dependent manner. On the other hand, the reverse Ah40-1 peptide, in the presence of serum, showed no toxic effect at all, supporting the complement-dependent mechanism (Fig. 1A). Primary hippocampal cultures contain both neuronal and glial cells. In order to determine sensitivity of these various cell types towards complement-mediated amyloid toxicity, we cultured primary astroglial and microglial cells as described earlier (Pazmany et al., 2000). These glial cultures were treated identically as the mixed hippocampal cells with Ah1-40 in the presence and in the absence of serum. LDH release was measured to determine cytotoxicity, but no toxic effect was detected neither in the absence nor in the presence of serum (data not shown). These results suggest that the toxic effect of complement is directed against hippocampal neurons and does not affect glia.
Fig. 1. (A) Ah1-40 peptide at 40 AM concentration exerted enhanced cytotoxicity in the presence of human serum (closed triangle) on neonatal hippocampal cells. At the same concentration, the peptide alone (open triangle), or the reverse Ah40-1 peptide either in the presence (closed square) or in the absence (open square) of serum showed only residual cytotoxicity. (B) C1-Inh showed protection against complement-mediated cytotoxicity. Cells were treated with 40 AM Ah1-40 peptide in the presence of human serum and the protective effect of C1-Inh was evaluated by measuring LDH release on the fifth day. The points are averages of three independent measurements performed as described in Materials and methods.
Having the assay adopted, we examined the effect of C1Inh on Ah-mediated complement lysis. These experiments showed that C1-Inh significantly decreased complementdependent Ah cytotoxicity (Fig. 1B). The observation supported previous results demonstrating that complement activation was responsible for the enhanced Ah toxicity. Furthermore, this result suggested that inhibition of the classical complement pathway at C1 level might be neuroprotective against complement-dependent Ah cytotoxicity in AD. 3.2. Screening of RBI compound library by C1q binding assays reveals distinct binding sites for Ab and Fc on the C1q molecule Based on the above result, we screened the RBI library of pharmacologically active compounds to check the feasibility
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of finding compounds that selectively inhibit C1q binding to Ah. In preliminary experiments, we checked both Ah1-40 and Ah1-42 and got the same C1q binding. Therefore, in subsequent experiments, Ah1-42 was used for coating. Ah1-42 was immobilized onto microtiter plates and C1q binding was carried out in the presence of components of the RBI collection. Bound C1q was quantified by standard ELISA. Following the screen, compounds were selected that decreased the amount of bound C1q more than 50% at the concentration of 5 AM and were called hits. Seven hits were identified including benextramine, calmidazolium, methoctramine, milrinone, NPC-15437, tamoxifen and thioridazine, with the inhibition constant 50% (IC50) of 3, 3, 2, 3, 1, 2 and 4 AM, respectively. None of the hit compounds inhibited C1q binding to antibodies. Thereby, in accord with earlier studies, it seems feasible to selectively inhibit C1q binding to Ah, while not affecting C1q binding to antibodies (Chen et al., 1996). Amino terminal C1q A chain sequences has been implicated in binding of direct C1 activators including C-reactive protein (CRP), serum amyloid P (SAP) and Ah (Gewurz et al., 1993; Jiang et al., 1994; Chen et al., 1996). Therefore, we further screened the RBI library to identify compounds that inhibit C1q binding to these direct C1 activators including CRP, SAP and myelin basic protein (MBP). Activators were immobilized to plates, selectively bound C1q was measured and the same criteria were applied as above. The same seven hits with very similar IC50 values were found to inhibit C1q binding in the case of all direct C1 activators, as in the case of Ah. These results showed that compounds that inhibited C1q binding to Ah, did also inhibit C1q binding to other direct C1 activators, and vice versa. 3.3. Screening of Richter’s compound library to identify structures, which inhibit C1q binding to b-amyloid peptide Our proprietary compound library was screened by C1q binding assay to identify potent compounds that inhibit C1q binding to Ah. One structural class was identified to be effective in micromolar concentration. This cluster showed structural similarities to tamoxifen including a basic amine and a large planar lipophilic moiety. The most effective compound of the cluster was selected. This compound also inhibited C1q binding to other direct C1 activators including MBP, CRP (Fig. 2A) and SAP (data not shown), while it did not inhibit C1q binding to antibodies (Fig. 2B). Similar to C1q, SAP also binds to Ah in vitro (Tennent et al., 1995). In order to provide further data on the specificity of the compound, its effect was tested on SAP binding to Ah. Ah was immobilized and specifically bound SAP was measured by ELISA. Importantly, the compound did not inhibit SAP binding to Ah (Fig. 2B). These results suggested that the compound targeted the Ah binding site on the C1q molecule, which is distinct from the antibody binding site.
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Fig. 2. Characterization of the selected Richter compound by using various binding assays. (A) The compound at micromolar concentrations decreased C1q binding to various direct C1 activators such as Ah (closed circle), CRP (open triangle) and MBP (open circle), while no C1q binding was measured to the reverse Ah40-1 peptide (closed square). (B) Specificity assays such as Ah-C1q (closed circle), Ah-SAP (open square) and IgG-C1q (open triangle) showed that the compound at the same concentration did not interfere with protein – protein interactions, in general including the C1q-Fc interaction. The points are averages of six independent measurements performed as described in Materials and methods.
3.4. ESI-MS experiments do not indicate binding of the Richter compound to b-amyloid In order to provide evidence concerning the molecular target, the interaction between the compound and Ah was studied by electrospray ionization mass spectrometry (ESIMS). It is known that complex formation takes place between melatonin and Ah (Skribanek et al., 2001). Thereby for comparison and also for verification of our experimental conditions, first melatonin and Ah1-40 were incubated under native conditions to measure complex formation of melatonin with Ah (Fig. 3A). Next, the compound and Ah were incubated together under the same conditions. In the course of mass spectrometric investigation, the time dependence of complex formation was studied. The growing intensity of a complex peak with the incubation time would unambiguously prove the interaction between the two molecules. The
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abundance of the quaternary charged complex peak of melatonin and Ah at m/z 1141 showed an increasing tendency compared to the quaternary charged peak of Ah (at m/ z 1083). After 3 h of incubation, the complex peak reached about 35% relative intensity (Fig. 3A). The incubation of the Richter compound and Ah, however, gave a low intensity (less than 3%) peak at m/z 1181, detected after 3 h of incubation (Fig. 3B). This peak, according to its mass number, would correspond to the expected complex (quaternary charged peak), but it could be the result of a methodo-
Fig. 4. Inhibition of C1q-Ah binding protected neonatal rat hippocampal cells against complement-mediated Ah toxicity. Functional activity of selected Richter compound was measured in a cell-based assay including the Ah40-1 reverse peptide control as described in Materials and methods. The points are averages of three independent measurements.
logical adduct formation, as well. Our observation, that the extent of complex formation does not increase even after 24 h of incubation, suggests that there is no distinct interaction between the selected compound and Ah. Results of the binding assays and ESI-MS are in accord, indicating that the compound inhibits the Ah binding site on C1q, and sticks neither to the large carboxy-terminal hydrophobic domain, nor the amino-terminal part of Ah. 3.5. Functional characterization of compounds that inhibit C1q binding to b-amyloid Functionality of the compound with the ability to inhibit C1q binding to Ah was tested on cultured hippocampal cells. The compound showed no cytotoxicity at 1 AM concentration; hence, its functionality was studied on hippocampal cells treated with Ah1-40 and serum. The selected Richter compound protected cultured rat hippocampal cells against Ah-induced complement lysis (Fig. 4). This result provides the in vitro proof for the concept that targeting the Ah binding site on C1q protects brain cells against complement-mediated Ah cytotoxicity.
4. Discussion
Fig. 3. Demonstration of the lack of any interaction between the selected compound and Ah1-40. ESI mass spectra of the mixture of Ah1-40 and melatonin (A), and of Ah1-40 and Richter compound (B) was measured after 3 h incubation for complex formation.
Accumulating evidence suggests that complement contributes significantly to the pathology of AD (McGeer and McGeer, 2001). It is likely that both the classical and alternative pathways are activated by Ah in AD brain. Following activation, complement promotes local inflammation and facilitates destruction through opsonization and lysis (Mu¨ller-Eberhard, 1987; Frank and Fries, 1991). C5a, released during activation of the complement cascade, induces expression of interleukin-1h and interleukin-6 in Ah primed monocytes (O’Barr and Cooper, 2000). In turn, proinflammatory cytokines enhance the expression of early
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complement proteins, but not C1-Inhibitor (Veerhuis et al., 1999). As C1-Inh is the major regulator of the classical pathway, the imbalance between activation signals and regulatory forces in AD brain may lead to uncontrolled complement activation in the presence of Ah. Although it is difficult to discern which pathway initiated activation of the cascade, it is a crucial information to elaborate an anticomplement therapy for the treatment of AD. The importance of C1 in Ah-induced complement activation has been underscored by a recent observation that C1, reconstituted from purified subcomponents of C1q, C1r and C1s, is directly activated by Ah (Tacnet-Delorme et al., 2001). Here, we demonstrate that C1-Inh protects hippocampal cells against complement-mediated Ah toxicity (Fig. 1B). This result supports the view that in vivo Ah activates the complement system primarily through C1q. Therefore, we selected C1q as molecular target and identified compounds that inhibit C1q binding to Ah. One compound with this characteristic also protected hippocampal cells against complement-mediated Ah toxicity (Fig. 4). Comparison of the two protection curves reveals different characteristics between C1-Inh and the Richter compound, that may be due to their different mechanism of action, their different target sites (C1r and C1s serine proteases in the case of C1-Inh vs. C1q in the case of the Richter compound), or their different molecular properties (large protein vs. small organic compound) or the combination of these. This result gives strong support to the view that in vivo Ah activates the complement system primarily through C1q, thereby inhibition of the classical pathway should result in complete shutoff of Ah-mediated complement activation in vivo which may have therapeutic value in AD. Although inhibition of C1q –Ah binding is a particularly attractive target for pharmaceutical intervention, research suffers from the lack of structural data on C1q and from the controversial data on the Ah binding site on C1q. Some papers have provided data indicating that the collagen-like region contains the Ah binding site on the C1q molecule (Jiang et al., 1994; Chen et al., 1996; Velazquez et al., 1997). However, a recent study claims that instead of the collagen-like region, the globular heads participate in the binding of Ah (Tacnet-Delorme et al., 2001). Due to the complexity of C1q structure (Reid and Porter, 1976; Kishore and Reid, 2000), the binding site for Ah has not been determined at amino acid level yet. Although localization of the Ah binding site on C1q is out of the scope of this study, small synthetic compounds often provide valuable information on binding sites of proteins. Screening of compound libraries, using C1q binding assays, revealed that compounds that inhibited C1q binding to Ah, did not affect C1q binding to IgG antibodies in immune complex. This result supports the view that C1q possess distinct binding sites for Ah and the Fc part of antibodies. On the other hand, compounds that inhibited C1q binding to Ah did also inhibit C1q binding to other direct activators including CRP, SAP and MBP, and vice versa.
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IC50 values of the compounds for various direct C1 activators were very similar (Fig. 2A). Based on these results, it is likely that C1q possess the same binding site for direct C1 activators. In the literature, several studies suggest that the collagen-like region binds some direct C1 activators including CRP (Jiang et al., 1992; Gewurz et al., 1993), SAP (Ying et al., 1993; Zahedi, 1996) and DNA (Uwatoko and Mannik, 1990). Accumulating evidence suggests the role of pathological complement activation in experimental autoimmune encephalomyelitis (EAE), mouse model of multiple sclerosis as well. Astrocyte-targeted expression of sCrry, soluble form of a mouse complement receptor, prevents EAE (Davoust et al., 1999), while B cell-deficient mice are susceptible to EAE and develop a chronic-sustained disease similar to that seen in wild type mice (Hjelmstro¨m et al., 1998). Myelin proteins, including myelin basic protein and myelin oligodendrocyte glycoprotein, bind to C1q and activate the classical complement pathway (Vanguri and Shin, 1986; Johns and Bernard, 1997). These results implicate myelin proteins to induce complement activation in an antibody independent manner, which contributes to the pathology of EAE. Therefore, it seems relevant to test the most potent compounds in various models of this human disease. This can provide further information on the role of pathological complement activation in various forms of multiple sclerosis and reveal the benefits of its inhibition via C1q. Summing up, we have demonstrated that complement inhibition at C1 level suspends complement-mediated Ah cytotoxicity. This result highlights the importance of the classical pathway and its recognition molecule C1q in the pathology of AD. Result of the screening of compound libraries shows that C1q possess distinct binding sites for direct activators and Fc part of antibodies. Selected compound targeting the Ah binding site on the C1q molecule protects complement sensitive neurons against Ah-mediated complement lysis. On the other hand, our screening results suggest that it is likely that C1q possess the same binding site for direct C1 activators including Ah and MBP. Since recent EAE studies implicate myelin induced complement activation in the pathology of the disease, this result warrants the study of the selected compound in EAE models. Acknowledgements We thank Irini Bozonasz and Piroska Unghy for skillful technical assistance. References Bradt, B., Kolb, W.P., Cooper, N.R., 1998. Complement-dependent proinflammatory properties of the Alzheimer’s disease h-peptide. J. Exp. Med. 188, 431 – 438.
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