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
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ABSTRACT |
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Alzheimer's disease; oxidative burst; apoptosis; nitric oxide; tyrphostin A1
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
A elicits a neurotoxic response in which nitric oxide (NO) and tumor necrosis factor-
(TNF
) released by microglia are the key effectors (8). While
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
(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
A activation of microglia is CD40 dependent. In addition, agents that attenuate the synergistic neurotoxic effect of IFN
, such as TGF
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, which cause neuronal cell death in mixed microglia-neuron cultures treated with CGA (46, 34, 37). We demonstrate that CGA acts synergistically with either
A or IFN
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
and
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 A.
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MATERIALS AND METHODS |
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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 2030 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:
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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 measurement.
TNF
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
from standards and samples. The reporter antibody was horseradish peroxidase-conjugated anti-rat TNF
. Bound TNF
was visualized with hydrogen peroxide and tetramethylbenzidine and read at 450 nm. Results were converted to nanograms per milliliter of TNF
using a standard curve.
Western blot analysis. Gradient 415% 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 (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
m, JC-1 exists primarily as green fluorescent monomers (527-nm emission) and at highly negative
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 A was fitted to the following hyperbolic function:
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RESULTS |
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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, 412 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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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).
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DISCUSSION |
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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 A may potentiate the effect of oxidative burst inducers such as phorbol 12-myristate 13-acetate (PMA) (22, 38), others have found that
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
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 suggests that CGA may play a role similar to that of
A and act in concert with
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 A to induce NO and TNF
secretion in microglia. The concentrations of CGA and
A used in this study are comparable to those described before (6, 33). We found that the maximal CGA effect on NO production is 67 times that of
A and that its maximal effect on TNF
secretion is 56 times that of
A. Moreover, CGA half-maximal activity (AC50) was reached at 1,000-fold lower concentrations compared with
A. Since TNF
was shown to stimulate NO production, it is possible that the observed enhancement of NO production is indirectly mediated through the potentiation of TNF
secretion (8). Kingham et al. (17) examined
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
A, CGA is an inducer of microglial apoptosis.
We show further that CGA and A act together to induce considerable microglial activation. The production of both TNF
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
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
on
A-induced microglial activation (1). IFN
acts by inducing CD40, a key initiator of the neuroinflammatory cascade in AD (24, 25). When added in combination with CGA, IFN
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
and the TNF
secreted by the stimulated cells. Induction of CD40 expression is thought to underlie the synergistic effect of IFN
with
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 was shown to involve the activation of NF-
B, which binds the CD40 promoter, leading to increased CD40 transcription (2, 25). IFN
activated NF-
B by stimulating the secretion of TNF
, resulting in autocrine activation of NF-
B. Our results indicate that CD40 can be induced to a moderate level in the absence of IFN
directly by CGA or
A. In the case of CGA, we show that CD40 induction does not require TNF
production, because TyrA1, which completely abolished TNF
secretion, did not affect CD40 induction. This finding also is supported by the absence of correlation between the capacity of TNF
production and CD40 expression exhibited by CGA- or
A-treated cells. A possible IFN
/TNF
bypassing mechanism is identified in the work of Kang et al. (16), who showed that microglial NF-
B can be activated by ROS. Since CGA-induced ROS generation is rapid, it is likely to be independent of TNF
synthesis and secretion and thus through TNF
-independent activation of NF-
B.
Previous investigations have shown that in microglia, the initiation of TNF and NO secretion requires both the ligation of CD40 receptor and the activation of CCAAT/enhancer-binding protein-
by IFN
(15). Clearly, as opposed to
A, CGA can induce a substantial NO release without IFN
, supporting again the possibility that CGA might interact with downstream components of the IFN
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-B translocation to the nucleus (12). Application of TyrA1 completely inhibited the combined effect of
A, CGA, and IFN
on NO and TNF
secretion. This finding emphasizes the overall theme suggesting the use of a common pathway for all three stimulators. Coapplication of TyrA1 resulted in restored
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, TGF1 promotes amyloid clearance and reduces plaque formation. Conversely, IFN
, which induces CD40, is a strong potentiator of the neurotoxic response induced by
A. In this context, our results place CGA together with IFN
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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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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