Synergistic amplification of {beta}-amyloid- and interferon-{gamma}-induced microglial neurotoxic response by the senile plaque component chromogranin A

Gilad Twig,1,2,3 Solomon A. Graf,1,2 Mark A. Messerli,2 Peter J. S. Smith,2 Seung H. Yoo,4 and Orian S. Shirihai1,2

1Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston; 2BioCurrents Research Center, Program in Molecular Physiology, Marine Biological Laboratory, Woods Hole, Massachusetts; 3Department of Physiology, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel; and 4National Creative Research Initiative Center for Secretory Granule Research and Department of Biochemistry, Inha University College of Medicine, Incheon, Korea

Submitted 30 June 2004 ; accepted in final form 23 August 2004


    ABSTRACT
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Activation of the microglial neurotoxic response by components of the senile plaque plays a critical role in the pathophysiology of Alzheimer's disease (AD). Microglia induce neurodegeneration primarily by secreting nitric oxide (NO), tumor necrosis factor-{alpha} (TNF{alpha}), and hydrogen peroxide. Central to the activation of microglia is the membrane receptor CD40, which is the target of costimulators such as interferon-{gamma} (IFN{gamma}). Chromogranin A (CGA) is a recently identified endogenous component of the neurodegenerative plaques of AD and Parkinson's disease. CGA stimulates microglial secretion of NO and TNF{alpha}, resulting in both neuronal and microglial apoptosis. Using electrochemical recording from primary rat microglial cells in culture, we have shown in the present study that CGA alone induces a fast-initiating oxidative burst in microglia. We compared the potency of CGA with that of {beta}-amyloid ({beta}A) under identical conditions and found that CGA induces 5–7 times greater NO and TNF{alpha} secretion. Coapplication of CGA with {beta}A or with IFN{gamma} resulted in a synergistic effect on NO and TNF{alpha} secretion. CD40 expression was induced by CGA and was further increased when {beta}A or IFN{gamma} was added in combination. Tyrphostin A1 (TyrA1), which inhibits the CD40 cascade, exerted a dose-dependent inhibition of the CGA effect alone and in combination with IFN{gamma} and {beta}A. Furthermore, CGA-induced mitochondrial depolarization, which precedes microglial apoptosis, was fully blocked in the presence of TyrA1. Our results demonstrate the involvement of CGA with other components of the senile plaque and raise the possibility that a narrowly acting agent such as TyrA1 attenuates plaque formation.

Alzheimer's disease; oxidative burst; apoptosis; nitric oxide; tyrphostin A1


ACTIVATION OF MICROGLIA, macrophages that reside in brain, plays a crucial role in the pathophysiology of neurodegenerative diseases such as Parkinson's disease, amyotrophic lateral sclerosis, and, in particular, Alzheimer's disease (AD) (19). Senile plaques, the histological hallmark of AD, represent a site of inflammation where immune cells, activated by {beta}-amyloid ({beta}A) and other plaque components, produce cytotoxic agents that result in neurodegeneration. The immune effectors of the plaque are microglia, which have been shown to play a dual role in the progression of the disease (40). While activated microglia in the senile plaque secrete neurotoxic agents, they can also act as phagocytic cells, removing amyloid fibrils and retarding further neuronal degradation. Agents that influence microglial response to {beta}A were shown to be important modifiers of AD progression. These mediators can be divided into protoxic mediators, such as interferon-{gamma} (IFN{gamma}), and prophagocytic mediators, such as tumor growth factor-{beta} (TGF{beta}) (1, 41). Such interactions exemplify the dynamic nature of the senile plaque and emphasize the need to explore the complex interaction of the multiple components that make up the plaque.

{beta}A is thought to be the principal component of senile plaque, and its secretion by neurons is thought to initiate plaque formation (14). Exposure of microglia to {beta}A elicits a neurotoxic response in which nitric oxide (NO) and tumor necrosis factor-{alpha} (TNF{alpha}) released by microglia are the key effectors (8). While {beta}A alone is a moderate inducer of such a response, its effect is subject to synergistic amplification in the presence of cytokines, such as IFN{gamma} (23). The mechanism underlying this effect was shown to be the induction of expression of a tyrosine kinase receptor protein, CD40 (25). CD40 signal transduction is central to a number of chronic inflammatory diseases, including rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis (35). Using a CD40 ligand-deficient animal, Tan et al. (33) demonstrated that {beta}A activation of microglia is CD40 dependent. In addition, agents that attenuate the synergistic neurotoxic effect of IFN{gamma}, such as TGF{beta}1 and interleukin-4, were shown to downregulate the expression of CD40 in microglia (23, 39).

A recently described component of the senile plaque that has gained attention in the past few years is the neurosecretory protein chromogranin A (CGA). A number of histological studies have demonstrated its existence in the majority of human senile plaques and have correlated this finding with the preclinical stage of AD (3, 10, 21, 29, 42). A key step in the progression of AD and other neurodegenerative diseases is the production of reactive oxygen species (ROS) by microglia (20, 27, 30). This is illustrated by the reduction in neurodegeneration in patients with AD and in animal models involving antioxidant drug treatment (20). In the present study, we show for the first time that CGA quickly induces a ROS efflux from microglia. In addition to ROS, CGA strongly stimulates microglia to produce NO and TNF{alpha}, which cause neuronal cell death in mixed microglia-neuron cultures treated with CGA (46, 34, 37). We demonstrate that CGA acts synergistically with either {beta}A or IFN{gamma} to stimulate microglia and that this effect can be fully prevented by a selective tyrosine kinase inhibitor, tyrphostin A1 (TyrA1) (12). Furthermore, we demonstrate that IFN{gamma} and {beta}A can act together with CGA to strongly upregulate CD40 expression.

In addition to eliciting a microglial neurotoxic phenotype, CGA induces microglial mitochondrial depolarization and, subsequently, apoptosis (17, 18). Reduction in microglial mass and accumulation of microglial debris is thought to inhibit amyloid clearance by phagocytosis and enhance recruitment of additional immune effectors to the site. Therefore, CGA might both enhance the microglial neurotoxic effect and diminish microglial phagocytic activity in senile plaques. These findings implicate CGA as an important stimulator of senile plaque development and stress the need to evaluate its comparative potency and relevance to other plaque components, in particular {beta}A.


    MATERIALS AND METHODS
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Materials. CGA was purified from bovine adrenal medullary chromaffin cells as described previously (43). Other material was purchased from the following vendors: TyrA1, Alexis Biochemicals (San Diego, CA); IFN{gamma}, Chemicon International (Temecula, CA); {beta}A, Bachem (Torrance, CA); and catalase, Sigma-Aldrich (St. Louis, MO).

Microglial cultures. Microglial cells were isolated from newborn rats as described previously in detail (13). Briefly, brains were cleared from meninges and blood vessels and then minced with a razor blade. Cells were dissociated by performing trypsinization for 10 min (0.25% trypsin EDTA; Invitrogen, Carlsbad, CA) in phosphate buffer with DNase at 37°C. The dissociated cells were washed twice in PBS supplemented with 10% fetal calf serum (FCS) and transferred to 75-cm2 flasks (Costar, Corning, NY) at a density of 2 x 107 cells/flask. Cells were maintained in 5% CO2 at 37°C in DMEM supplemented with 0.37% NaHCO3 and 10% heat-treated FCS. Medium was changed every 3 days. After 6 days, cells growing on the top of the confluent cell layer were removed by shaking and then plated onto glass coverslips (Nunc Intermed, Naperville, IL) at a concentration of 3 x 106 cells/ml. To remove nonadherent cells and any remaining floating debris, each coverslip was gently washed after 20–30 min. This procedure was shown to result in cultures containing 97% microglial cells (31).

Measurement of hydrogen peroxide generation. Hydrogen peroxide release from microglial cells was measured electrochemically using a self-referencing sensor with a platinum-tipped microelectrode coated with cellulose acetate as described previously (36). H2O2 was oxidized at +0.6 V against a Ag-AgCl reference electrode. The H2O2 concentration difference was measured at two positions 10 µm apart and orthogonal to the cell surface. Measurements were performed at 10-µm steps away from the cell surface. Cells were kept at 37°C throughout the experiment. The H2O2 flux was calculated according to the modified Fick equation:

where flux (J) is equal to the product of the diffusion coefficient (D) of H2O2 and the concentration difference of H2O2 (dI·S) over a known distance (dx).

NO production by microglial cells. NO in cell culture supernatants was measured spectrochemically using the Griess reagent (Sigma) with sodium nitrite as the standard and a NanoDrop spectrophotometer (NanoDrop, Wilmington, DE) (11). Data were corrected for the background levels of nitrite in the cell-free medium.

TNF{alpha} measurement. TNF{alpha} was quantified in cell culture supernatants using an enzyme-linked immunosorbent assay kit (Pierce Biotechnology, Rockford, IL) with a solid phase-bound antibody to capture the rat TNF{alpha} from standards and samples. The reporter antibody was horseradish peroxidase-conjugated anti-rat TNF{alpha}. Bound TNF{alpha} was visualized with hydrogen peroxide and tetramethylbenzidine and read at 450 nm. Results were converted to nanograms per milliliter of TNF{alpha} using a standard curve.

Western blot analysis. Gradient 4–15% gels were obtained from Bio-Rad (Hercules, CA). Anti-CD40, Abcam, was used at a dilution of 1:1,000, and secondary HRP-conjugated mouse anti-rabbit antibody (Invitrogen) was used at a 1:5,000 dilution. As a control, we used an antibody against the abundant mitochondrial protein ABCB10 (Research Genetics, Huntsville, AL). Bands were visualized using LumilightPLUS chemiluminescence (Roche, Indianapolis, IN).

Mitochondrial membrane potential. The relative mitochondrial membrane potential ({Delta}{Psi}m) was recorded using the ratiometric voltage-sensitive dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Molecular Probes, Eugene, OR). At low {Delta}{Psi}m, JC-1 exists primarily as green fluorescent monomers (527-nm emission) and at highly negative {Delta}{Psi}m as orange/red fluorescent aggregates (590-nm emission). A Zeiss LSM 510 META was used for confocal microscopic imaging with a 488-nm excitation laser. Cells were incubated in 5 µM JC-1 for 15 min at 37°C to load the dye and then washed three times with 37°C culture medium.

Statistics. The dose-response curve of CGA and {beta}A was fitted to the following hyperbolic function:

where R is the level of secretion (NO or TNF{alpha}) that was elicited by a concentration, C, of a given activator ({beta}A or CGA). R0 is the baseline release level elicited when no substance is added (C = 0). Rmax reflects the maximal level of secretion. AC50 is the concentration needed to elicit 50% of Rmax. The exponent n reflects the steepness of the dose-response curve. This equation was also used to fit the dose-response effect of TyrA1. In this case, Rmax reflects the maximal inhibitory effect on NO or TNF{alpha} release, where C and IC50 (rather than AC50) denote the concentration of TyrA1 used and the concentration needed to elicit 50% of the maximal inhibitory effect. All fitting procedures and statistical tests were conducted using KaleidaGraph software (Synergy Software, Reading, PA).


    RESULTS
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CGA induces an oxidative burst in primary microglia. CGA-induced H2O2 secretion from microglia was monitored using electrochemical detection with a microelectrode and electrode self-referencing as described previously (36). The self-referencing approach is based on electrochemical detection of a gradient of H2O2 that surrounds cells in culture. This method is advantageous for three reasons. 1) It enables the noninvasive determination of the H2O2 gradient surrounding single microglia cells with high spatial and temporal resolution. 2) It is not influenced by ROS generated in the solution by direct release from proteins and protein aggregates as reported previously for {beta}A (32). 3) It is not sensitive to NO, another free radical that is released by these cells (see below).

A platinum-tipped microelectrode was placed at known distances from the surface of microglial cells to measure the H2O2 gradient generated in the cell's vicinity along axes perpendicular to the cell surface. Figure 1 shows a summary of these gradient measurements. In the presence of medium alone, no H2O2 gradient could be detected. Addition of CGA to a final concentration of 15 nM generated a stable gradient of H2O2 around the cell. The H2O2 was most concentrated near the cell surface and decreased to nearly immeasurable levels at an average distance of 25.7 µm (3.7 µm ± SD) from the cell surface. H2O2 release could be observed as early as 4 min after exposure of the cells to CGA. Washing out the CGA by perfusion of the dish with CGA-free medium slightly reduced the magnitude of the flux but did not bring it to baseline. The signal could be fully abolished by perfusing the cells with medium containing a combination of CGA and catalase, which verified that the probe was measuring H2O2. Subsequent washing out of the catalase and CGA unmasked the ongoing oxidative burst because catalase did not enter the cells in significant amounts. Treating microglia with the carrier solution alone containing heat-treated CGA did not induce any production of H2O2 (data not shown). In four experiments for each condition, we found that 1) the average response time for detecting a measurable change (5 fA) was 6.5 min (range, 4–12 min) and 2) after addition of CGA, the duration of activity of the oxidative burst exceeded our 30-min measurement window.



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Fig. 1. Chromogranin A (CGA) induces release of H2O2 from microglia in culture. Electrochemical detection in conjunction with self-referencing was used to record a gradient of H2O2 along an axis perpendicular to the cell surface. Unstimulated cells were used as control and displayed an undetectable H2O2 gradient (x), as did cells stimulated with heat-treated CGA (not shown). A significant H2O2 gradient is formed after addition of 15 nM CGA ({bullet}) and is completely attenuated when catalase (0.1 mg/ml) was added to the bath medium ({blacksquare}). After these manipulations, CGA and catalase were washed out by replacing the entire bath medium with the one in the control state ({circ}). Note that the gradient is only mildly diminished compared with the initial application of CGA alone ({bullet}).

 
CGA potency and synergism with {beta}A. The secretion of NO and TNF{alpha} were determined in response to increasing concentrations of CGA and {beta}A. Figure 2 shows the average ± SE dose-response curves measured for NO and TNF{alpha}. Our results demonstrate that CGA is a more potent stimulator of microglial neurotoxic response than is {beta}A. CGA at 10–3 the concentration of {beta}A elicited greater microglial secretion of NO and TNF{alpha}. The stimulant concentrations capable of eliciting 50% of the maximal secretory response (activation concentration, AC50) of NO were 16.8 nM for CGA and 22.3 µM for {beta}A. The AC50 values for CGA- and {beta}A-induced TNF{alpha} release were 13.2 nM and 9.1 µM, respectively. Exposure of microglia to increasing concentrations of heat-treated CGA did not elicit a significant change in the secretion of NO or TNF{alpha} (Fig. 2). As CGA and {beta}A coexist in the senile plaque, we tested the combined effect of the two on the release of NO and TNF{alpha} from microglia. The agents were added at concentrations that independently elicited low levels of microglial NO and TNF{alpha} secretions (Fig. 2). Treatment of the microglial cells with the combination of CGA and {beta}A resulted in NO and TNF{alpha} release that was significantly greater than the sum of the effects of either agent alone. Such a synergistic response indicates that both CGA and {beta}A target related induction pathways.



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Fig. 2. A: dose-response relationship of CGA-induced release of nitric oxide (NO) and tumor necrosis factor-{alpha} (TNF{alpha}) from microglia in culture. Release of NO and TNF{alpha} were measured 24 h after exposing microglia to incremental concentrations of CGA and {beta}-amyloid ({beta}A). Each point on the graph reflects an average of 3–6 experiments (bars indicate SE). The data points were fitted to a hyperbolic function (see Statistics) to derive the maximal secretory response (Rmax) and the concentration needed to elicit 50% of the maximal secretory response (AC50). Asterisk at 50 mM {beta}A indicates that the degree of NO and TNF{alpha} release was significantly higher with CGA (15 nM, 22.5 nM, 27.5 nM) than with {beta}A using an unpaired t-test. P < 0.0001. For emphasis, the inset shows the dose-response curves (NO and TNF{alpha}) elicited by {beta}A of higher resolution. B: CGA acts synergistically with {beta}A to activate microglia. Microglia were incubated with {beta}A (18 µM), with CGA (7.5 nM), or with both {beta}A (18 µM) and CGA (7.5 nM), and the release of NO and TNF{alpha} was measured after 24 h. Results of 5 experiments are plotted with SE. *P < 0.005, significant change of NO or TNF{alpha} release compared with the control state. **P < 0.005, coapplication of CGA and {beta}A elicited a significantly higher NO and TNF{alpha} release than the sum of the releases by independently stimulated cells.

 
Interaction with IFN{gamma}: a key component of the senile plaque. IFN{gamma} is found in senile plaques and has been shown to potentiate {beta}A-induced microglial activation (1). We tested whether IFN{gamma} also potentiates CGA-induced microglial activation by determining the dose-response curves of NO and TNF{alpha} release after incubation of microglia with CGA in the presence and absence of IFN{gamma} (Fig. 3). By itself, this concentration of IFN{gamma} elicited a small but significant release of NO (4.3 ± 1.5 µM/24 h) and TNF{alpha} (0.45 ± 0.2 ng/ml/24 h) compared with the untreated cells (NO, 1.8 ± 1.3 µM/24 h; TNF{alpha}, 0.16 ± 0.3 ng/ml/24 h). Addition of 150 U/ml IFN{gamma} increased the potency of CGA-induced microglial activation, shifting leftward the dose-response curves for NO and TNF{alpha} secretion. In the presence of IFN{gamma} (150 U/ml), the AC50 value for NO release was reduced from 16.8 to 6.6 nM, and the AC50 value for TNF{alpha} release was reduced from 13.2 to 6.3 nM. The effect of the synergistic interaction of CGA and IFN{gamma} on NO and TNF{alpha} secretion was statistically significant (P < 0.001) at all CGA concentrations >4 nM.



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Fig. 3. Interferon-{gamma} (IFN{gamma}) enhanced CGA-induced release of NO (A) and TNF{alpha} (B) from primary cultured microglia. Results summarize 5 experiments. The dose-response curves of CGA were fitted to a hyperbolic function (see Statistics) in the presence or absence of 150 U/ml IFN{gamma} (solid and dashed curves, respectively) and are shown for the IFN{gamma} group with SE. *P < 0.001, significant difference between cultures treated with the same concentrations of CGA, with or without 150 U/ml IFN{gamma}.

 
Induction of CD40 during microglial activation. The linked activity of CGA with {beta}A and with IFN{gamma} suggests a possible role for CD40 in CGA-induced microglial activation. To test for this possibility, we determined the effect of CGA alone and in combination with either IFN{gamma} or {beta}A on CD40 expression. Treatment of microglia with 7.5 nM CGA resulted in the induction of CD40 detected by Western blot analysis (Fig. 4A). Expression of CD40 was further upregulated when CGA was supplemented with IFN{gamma} at 150 U/ml. Similarly, the combination of CGA and {beta}A resulted in an even further increase in CD40 expression compared with each agent alone (Fig. 4B). In this experiment, we used CGA at concentrations that elicited approximately a half-maximal response (15 nM).



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Fig. 4. Western blots showing that CGA, {beta}A, and IFN{gamma} induced the expression of CD40 in cultured isolated microglia. A: exposure of microglia to CGA (7.5 nM) for 24 h resulted in the upregulation of CD40 (n = 5). Addition of IFN{gamma} at 150 U/ml further increased the level of CD40 (n = 3). B: microglia cultured for 24 h in the presence and absence of CGA, with and without {beta}A. Note that when added in combination, {beta}A and CGA exerted an additive effect on CD40 expression (n = 3). ABCB10 is an abundant mitochondrial protein and is used here as a control for the total amount of protein loaded on the gel.

 
Tyrphostin A1 effect on microglial activation. TyrA1 has been shown to inhibit the CD40-induced tyrosine kinase cascade (12). We therefore investigated the effect of TyrA1 on the CGA-induced microglial secretion of NO and TNF{alpha} (Fig. 5A). TyrA1 exhibited a dose-dependent inhibition of CGA (15 nM)-induced activity, reducing the release of NO and TNF{alpha} secretion with IC50 values of 4.8 µM for NO and 6.6 µM for TNF{alpha}. In addition, TyrA1 inhibited the net effect of IFN{gamma} (150 U/ml), {beta}A (18 µM), and CGA (15 nM) with IC50 values of 4 µM for NO and 3.1 µM for TNF{alpha}.



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Fig. 5. Tyrphostin A1 (TyrA1) inhibits the effect of IFN{gamma}, {beta}A, and CGA on primary cultured microglia. A: effect of TyrA1 on release of NO and TNF{alpha} from activated microglia. Incremental concentrations of TyrA1 were coapplied with CGA (15 nM, open squares) or with a cocktail (CGA 15 nM, {beta}A18 µM, IFN{gamma} 150 U/ml, closed squares), and the release of NO or TNF{alpha} was measured after 24 h. Each data point is an average of 4 experiments. Each set of data points at incremental concentration of TyrA1 was fitted to a hyperbolic function (see Statistics) to derive the concentration needed to achieve 50% inhibition in the release values (IC50). The IC50 values of TyrA1 for NO secretion were 4.35 µM and 3.95 µM for the cocktail and CGA groups, respectively. For TNF{alpha} release, the IC50 values were 6.60 µM and 3.20 µM for the cocktail and CGA group, respectively. B: TyrA1 prevents CGA-induced mitochondrial depolarization. Confocal microscopic photomicrographs of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide-stained microglia in culture. Red staining indicates active mitochondria. Mitochondria that are green and which do not express any red color are depolarized. Note that mitochondria in cells exposed to CGA are depolarized. This effect is prevented when CGA is added to the culture in combination with TyrA1 (n = 5). C: TyrA1 does not alter CD40 expression. Western blot analysis of microglia exposed to CGA in the presence or absence of TyrA1. Porin is presented as a control for load of the gel (n = 3).

 
CGA has been shown to induce microglial apoptosis with an early phase of moderate mitochondrial depolarization (18). To test whether the downstream cascade involving CD40 is also responsible for microglial apoptosis, we monitored {Delta}{Psi}m in microglia exposed to CGA alone or in combination with TyrA1. We used the ratiometric membrane potential-sensitive dye JC-1 and confocal microscopy. After 24 h in the presence of CGA (15 nM), an apparent mitochondrial depolarization could be seen (Fig. 5B, middle) compared with the control state (Fig. 5B, top). However, when added in combination with 17 µM of TyrA1, CGA (15 nM) had no effect on {Delta}{Psi}m.

Western blot analysis of the expression of CD40 after treatment with CGA in combination with TyrA1 showed no effect of the latter on CD40 protein level (Fig. 5C). This finding is in agreement with those of previous studies in showing that TyrA1 inhibits the downstream signal transduction cascade of CD40 rather than influencing its expression (12).


    DISCUSSION
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CGA is found in pathological structures of a number of neurodegenerative diseases, including AD, Parkinson's disease, and Pick's disease (42). Previous studies demonstrated its capacity to activate microglia and induce neuronal as well as microglial damage (6, 34, 37). In this study, we have demonstrated for the first time that CGA can quickly activate microglia to produce ROS intermediates. We compared the long-term effect of CGA and {beta}A on microglia and demonstrated that, in culture, CGA is a more potent activator than {beta}A. Furthermore, we have shown that CGA can synergize with {beta}A and IFN{gamma} to augment the production of NO and TNF{alpha}. The microglial response to the combination of CGA and {beta}A includes the upregulation of CD40 protein expression. The inhibition of the CD40 tyrosine kinase cascade using TyrA1 completely abolished CGA-induced NO, TNF{alpha} secretion, and subsequent mitochondrial depolarization.

A number of studies support an important role for ROS in the pathophysiology of AD and demonstrate beneficial effects of antioxidants on the progression of the disease (30). Neurons in the vicinity of the senile plaque show increased levels of oxidized proteins, lipids, and DNA. As the prime effectors in the brain inflammatory response, microglia are thought to be the major contributors of ROS, including NO and O2 (7, 28). It is unclear whether any of the senile plaque components are direct stimulants of microglial oxidative bursting, during which they release hydrogen peroxide. While some investigators have demonstrated that {beta}A may potentiate the effect of oxidative burst inducers such as phorbol 12-myristate 13-acetate (PMA) (22, 38), others have found that {beta}A is a direct inducer of H2O2 production in a variety of immune cells (9). However, it is important to note that the authors of these latter reports used ROS-sensitive fluorescent dyes that do not differentiate between NO and O2. In addition, Tabner et al. (32) showed that {beta}A can readily liberate detectable amounts of hydroxyl radicals in culture media with no cells, which could account for the conflicting reports. The technique we used detects the gradient of specifically cell-derived H2O2. It is not influenced by H2O2, which is not cell derived or derived from other ROS such as NO. Our results indicate that CGA activates the microglial production of ROS within minutes, while heat-treated CGA had no effect. This is the most immediate effect reported for CGA in microglia. Although the heat-induced structural changes in CGA that are responsible for the loss of its effect in microglia are not yet clear, boiling has been shown to change the conformation of CGA (43). Compared with the H2O2 gradient measured around microglial cells stimulated with the robust stimulator PMA (130 nM), CGA (15 nM) elicited a flux ~40% as large (36).

The potential relevance of CGA to the pathophysiology of AD has been the subject of several investigations. The ability of CGA to elicit microglial activation and produce NO and TNF{alpha} suggests that CGA may play a role similar to that of {beta}A and act in concert with {beta}A to induce the production of these neurotoxic agents.

Variations in culture conditions and stimulation protocols that were used by researchers at different laboratories make it difficult to assess the significance of the effect of CGA as a newly characterized factor. In the present study, we used identical experimental conditions to compare the capacity of CGA with {beta}A to induce NO and TNF{alpha} secretion in microglia. The concentrations of CGA and {beta}A used in this study are comparable to those described before (6, 33). We found that the maximal CGA effect on NO production is 6–7 times that of {beta}A and that its maximal effect on TNF{alpha} secretion is 5–6 times that of {beta}A. Moreover, CGA half-maximal activity (AC50) was reached at 1,000-fold lower concentrations compared with {beta}A. Since TNF{alpha} was shown to stimulate NO production, it is possible that the observed enhancement of NO production is indirectly mediated through the potentiation of TNF{alpha} secretion (8). Kingham et al. (17) examined {Delta}{Psi}m and caspase activity after exposure of microglia to 10 nM CGA. Under these conditions, the earliest mitochondrial depolarization occurred after 24 h and was upstream of caspase 1 activation (18). The mechanism is thought to involve downregulation of mitochondrial oxidative phosphorylation directly or indirectly by NO (18, 26). Thus the higher capacity of CGA to induce NO production may explain why, as opposed to {beta}A, CGA is an inducer of microglial apoptosis.

We show further that CGA and {beta}A act together to induce considerable microglial activation. The production of both TNF{alpha} and NO production is synergistically enhanced by the coapplication of the two agents. This finding is of importance to the pathophysiology of the early stage of a developing senile plaque, in which CGA is more commonly found (10). In this case, CGA may potentiate the neurotoxic effect of as yet nonpathological concentrations of {beta}A. Potentiation of inflammatory mediators is a common feature of cytokine receptors that share similar downstream elements. Of relevance to AD is the remarkable effect of IFN{gamma} on {beta}A-induced microglial activation (1). IFN{gamma} acts by inducing CD40, a key initiator of the neuroinflammatory cascade in AD (24, 25). When added in combination with CGA, IFN{gamma} significantly shifted the AC50 of CGA to a lower concentration. This effect on NO production was more pronounced, possibly because of the combined effect of IFN{gamma} and the TNF{alpha} secreted by the stimulated cells. Induction of CD40 expression is thought to underlie the synergistic effect of IFN{gamma} with {beta}A. This suggests that CGA may itself be an inducer of microglial CD40 expression and/or may act through the upregulation of CD40 cascade.

Western blot analysis of CGA-treated microglia confirmed that CD40 can be induced by CGA alone. CD40 induction by IFN{gamma} was shown to involve the activation of NF-{kappa}B, which binds the CD40 promoter, leading to increased CD40 transcription (2, 25). IFN{gamma} activated NF-{kappa}B by stimulating the secretion of TNF{alpha}, resulting in autocrine activation of NF-{kappa}B. Our results indicate that CD40 can be induced to a moderate level in the absence of IFN{gamma} directly by CGA or {beta}A. In the case of CGA, we show that CD40 induction does not require TNF{alpha} production, because TyrA1, which completely abolished TNF{alpha} secretion, did not affect CD40 induction. This finding also is supported by the absence of correlation between the capacity of TNF{alpha} production and CD40 expression exhibited by CGA- or {beta}A-treated cells. A possible IFN{gamma}/TNF{alpha} bypassing mechanism is identified in the work of Kang et al. (16), who showed that microglial NF-{kappa}B can be activated by ROS. Since CGA-induced ROS generation is rapid, it is likely to be independent of TNF{alpha} synthesis and secretion and thus through TNF{alpha}-independent activation of NF-{kappa}B.

Previous investigations have shown that in microglia, the initiation of TNF{alpha} and NO secretion requires both the ligation of CD40 receptor and the activation of CCAAT/enhancer-binding protein-{beta} by IFN{gamma} (15). Clearly, as opposed to {beta}A, CGA can induce a substantial NO release without IFN{gamma}, supporting again the possibility that CGA might interact with downstream components of the IFN{gamma} signal transduction pathway.

Although used largely as an inactive tyrphostin, TyrA1 was recently shown to be a fairly specific inhibitor of CD40 cascade in macrophages, inhibiting CD40 ligand-induced NF-{kappa}B translocation to the nucleus (12). Application of TyrA1 completely inhibited the combined effect of {beta}A, CGA, and IFN{gamma} on NO and TNF{alpha} secretion. This finding emphasizes the overall theme suggesting the use of a common pathway for all three stimulators. Coapplication of TyrA1 resulted in restored {Delta}{Psi}m. This effect of TyrA1 can be explained by the TyrA1-induced reduction in NO production.

Microglial function in AD has two faces, and the CD40 pathway plays a role in directing the cellular response to stimulants toward beneficial or detrimental outcomes. Studies by Wyss-Coray et al. (41) showed that by reducing CD40 expression, TGF{beta}1 promotes amyloid clearance and reduces plaque formation. Conversely, IFN{gamma}, which induces CD40, is a strong potentiator of the neurotoxic response induced by {beta}A. In this context, our results place CGA together with IFN{gamma} as a CD40 inducer, directing the microglial response toward a neurotoxic pathway. Our results demonstrate the linkage of CGA with other components of the plaque and raise the possibility that a selective inhibitor such as TyrA1 can attenuate plaque formation, both by inhibiting secretion of neurotoxins and by restoring microglial viability. As such, TyrA1 might serve as a pharmaceutical candidate drug targeting the inflammatory components of AD.


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G. Twig was supported by a fellowship from the Grass Foundation. S. Graf was supported by a fellowship from the Neal W. Cornell Endowed Research Fund. This project was funded by the Grass Foundation and by National Institutes of Health Grant P41RR001395-21.


    ACKNOWLEDGMENTS
 
We thank Sarah Haigh for help with protein analysis. We thank Drs. Dani Dagan, Barbara Ehrlich, Jeffrey Laskin, Steve Zotolli, Richard Shader, Ayse Dosemeci, and Gal Yaniv for their critical review of the manuscript. We thank the fellows, trustees, and directors of the Grass Foundation of 2001 for valuable discussions throughout the time this work was conducted.


    FOOTNOTES
 

Address for reprint requests and other correspondence: O. S. Shirihai, Dept. of Pharmacology, School of Medicine, Tufts Univ., 136 Harrison Ave., Boston, MA 02111 (E-mail: Orian.Shirihai{at}tufts.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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