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Originally published In Press as doi:10.1074/jbc.M600627200 on May 25, 2006

J. Biol. Chem., Vol. 281, Issue 30, 20842-20850, July 28, 2006
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Fibrillar beta-Amyloid-stimulated Intracellular Signaling Cascades Require Vav for Induction of Respiratory Burst and Phagocytosis in Monocytes and Microglia*

Brandy Wilkinson1, Jessica Koenigsknecht-Talboo, Christian Grommes, C. Y. Daniel Lee, and Gary Landreth

From the Alzheimer Research Laboratory, Department of Neuroscience, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

Received for publication, January 23, 2006 , and in revised form, May 23, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Microglial interaction with extracellular beta-amyloid fibrils (fAbeta) is mediated through an ensemble of cell surface receptors, including the B-class scavenger receptor CD36, the {alpha}6beta1-integrin, and the integrin-associated protein/CD47. The binding of fAbeta to this receptor complex has been shown to drive a tyrosine kinase-based signaling cascade leading to production of reactive oxygen species and stimulation of phagocytic activity; however, little is known about the intracellular signaling cascades governing the microglial response to fAbeta. This study reports a direct mechanistic link between the fAbeta cell surface receptor complex and downstream signaling events responsible for NADPH oxidase activation and phagosome formation. The Vav guanine nucleotide exchange factor is tyrosine-phosphorylated in response to fAbeta peptides as a result of the engagement of the microglia fAbeta cell surface receptor complex. Co-immunoprecipitation studies demonstrate an Abeta-dependent association between Vav and both Lyn and Syk kinases. The downstream target of Vav, the small GTPase Rac1, is GTP-loaded in an Abeta-dependent manner. Rac1 is both an essential component of the NADPH oxidase and a critical regulator of microglial phagocytosis. The direct role of Vav in fAbeta-stimulated intracellular signaling cascades was established using primary microglia obtained from Vav–/– mice. Stimulation of Vav–/– microglia with fAbeta failed to generate NADPH oxidase-derived reactive oxygen species and displayed a dramatically attenuated phagocytic response. These findings directly link Vav phosphorylation to the Abeta-receptor complex and demonstrate that Vav activity is required for fAbeta-stimulated intracellular signaling events upstream of reactive oxygen species production and phagosome formation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Considerable attention has been focused on inflammatory processes in the etiology of Alzheimer disease (AD)2 (1, 2). Many of these inflammatory events center around the senile plaques, which are primarily composed of extracellular beta-amyloid (Abeta) and are surrounded by activated microglia. Although a variety of proinflammatory molecules are elaborated by activated glial cells, free radical-mediated oxidative stress has been hypothesized to be a primary source of neuronal damage found in the AD brain (3, 4). The accumulation of Abeta deposits is associated with several markers of oxidative stress, including lipid peroxidation (5, 6), nucleic acid oxidation (7), and protein oxidation (8). Oxidative damage is observed early in the progression of AD (9, 10) and can be detected prior to fibrillar Abeta (fAbeta) deposition in both the human brain (11) and animal models of the disease (9). The initiating events leading to Alzheimer disease remain unknown; however, these findings suggest that oxidative damage plays an early critical role in the pathogenesis of AD.

Microglia have been postulated to be a potential source of oxidative stress in response to Abeta peptides (2, 12). Microglia are phagocytes of myeloid origin and represent the principal immune effector cells in the brain. It has recently been appreciated that "resting" or "quiescent" microglia are highly dynamic and constantly extend their processes to survey their microenvironment presumably allowing them to react quickly to local injury or invading pathogens (13, 14). Phenotypically activated microglia are found clustered adjacent to Abeta plaques (15, 16), and microglia exposed to fAbeta release cytokines, neurotoxins, and both reactive oxygen (ROS) and nitrogen species (2, 12). It is hypothesized that the sustained microglial proinflammatory response results in the production of ROS that are ultimately responsible for the oxidative damage observed in both the AD brain and animal models of the disease.

We have described previously a multireceptor cell surface complex for fAbeta on microglia consisting of CD36, {alpha}6beta1 integrin, CD47, and the class A scavenger receptor (17). Engagement of this receptor complex initiates tyrosine kinase-based signaling cascades (17–19) and a fAbeta-stimulated respiratory burst leading to release of superoxide anion (17, 19–24). Furthermore, our recent findings provided evidence that in vitro microglia are capable of mounting a phagocytic response to fAbeta through engagement of the aforementioned ensemble of cell surface receptors (25).

Although recent findings have provided valuable insights into the role microglia play in the proinflammatory events observed in AD, the intracellular signaling molecules responsible for the initiation of these responses remain to be elucidated. Specifically, it is unknown how the fAbeta receptor complex is linked to the small GTPase Rac1, which is a critical element in signaling to both the NADPH oxidase (26, 27) and the phagocytic machinery (28, 29). One candidate molecule that could potentially link the fAbeta cell surface receptor complex to downstream signaling events is the proto-oncogene Vav. Vav is one of the most well characterized guanine nucleotide exchange factors (GEFs) for Rac1. We report that Vav phosphorylation is mediated through the fAbeta cell surface receptor complex, and our studies further demonstrate that Vav is an essential component of the fAbeta-stimulated intracellular signaling pathway that leads to NADPH oxidase activation and ROS production. We also report a critical role for Vav in the initiation of a phagocytic response to Abeta peptides.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Abeta peptides corresponding to the human Abeta amino acids 25–35 and 1–42 were purchased from American Peptide Co. (Sunnyvale, CA). The method used to fibrillarize Abeta peptides has been well characterized (30, 31). Abeta peptides were fibrillized by reconstitution in sterile distilled water followed by incubation at 37 °C for 1 week. The initial peptide concentration reflects that of the monomeric peptides comprising the Abeta fibrils. The composition of the fibrillar solutions may include Abeta oligomers as well as fibrils.

The 4N1K and RHD peptides were purchased from Bachem (Philadelphia) and reconstituted in sterile distilled water. Fucoidan was purchased from Sigma and reconstituted in sterile distilled water. Glutathione S-transferase (GST)-CD36 peptide was a gift from Dr. Maria Febbraio (The Cleveland Clinic Foundation, Cleveland, OH). Invasin 195 was a gift from Dr. Ingo Autenrieth (University of Tubingen, Germany). PAK-PBD beads were purchased from Cytoskeleton (Denver, CO). The anti-Rac and the anti-phospho-Tyr antibody 4G10 were obtained from Upstate%20Biotechnology">Upstate Biotechnology, Inc. (Waltham, MA). The anti-Vav, -Syk, and -Lyn antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Affinity-purified horseradish peroxidase-conjugated sheep anti-mouse and donkey anti-rabbit antibodies were from Amersham Biosciences. Nitro blue tetrazolium chloride (NBT) was purchased from Roche Applied Science. Piceatannol was purchased from Roche Applied Science. Nile red fluorospheres (1 µM microspheres) were purchased from Molecular Probes (Eugene, OR).

Tissue Culture—Human THP-1 monocytes (American Type Culture Collection, Manassas, VA) were grown in RPMI 1640 medium (Whittaker Bioproducts, Walkersville, MD) containing 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 5 x 10–5 M 2-mercaptoethanol, 5 mM HEPES, and 15 µg/ml gentamycin in 5% CO2. THP-1 monocytes are used in these assays as they grow in suspension and do not attach to the tissue culture substrate through integrin-based adhesive mechanisms, allowing dissection of Abeta fibril-dependent signaling mechanisms in the absence of high basal levels of tyrosine kinase-based integrin signaling. Responses of THP-1 monocytes to fAbeta faithfully replicate the responses of primary microglia (17, 18, 25, 32–34). Primary microglia were derived from postnatal day 1–3 mouse brains as described previously (18, 32–34).

Cell Stimulation and Immunoprecipitations—THP-1 monocytes were collected and resuspended in Hanks' balanced salt solution for 30 min at 37 °C. Cells were then stimulated with fAbeta-(25–35) or fAbeta-(1–42) peptides for 3 min at 37 °C. For analysis of the elements of the fAbeta receptor, cells were incubated with fucoidan, 4N1K, GST-CD36, invasin 195, or RHD peptide for 30 min at 37 °C prior to stimulation with Abeta peptides. Cells were collected by centrifugation and lysed in Triton buffer (1% Triton X-100, 20 mM Tris, pH 7.5, 100 mM NaCl, 40 mM NaF, 1 mM EDTA, 1 mM EGTA, and 1 mM Na3VO4). The insoluble material was removed by centrifugation at 10,000 x g for 12 min at 4 °C. Protein concentration of cell lysates was determined by the Bradford method (35). Aliquots of the cellular lysates (500 µg/ml) were added to protein A-agarose (30 µl) with the anti-Vav, -Lyn, or -Syk primary antibodies (2 µg of primary antibody/mg of lysate) and incubated with rocking for 2 h at 4 °C. Immune complexes were washed three times in Triton buffer; sample buffer was added, and samples were boiled for 5 min. Samples were resolved on 9 or 12% SDS-polyacrylamide gels and Western-blotted to polyvinylidene difluoride membranes. Blots were probed with either anti-phospho-Tyr (1:1000), -Vav (1:1000), -Syk (1:1000), or -Lyn (1:1000) antibodies overnight at 4 °C. The protein was detected by enhanced chemiluminescence. Blots were stripped and reprobed with the appropriate primary antibody as a loading control. Band intensities were quantified using NIH Image 1.62 software (Bethesda, MD). All experiments were performed a minimum of three times. Values statistically different from controls were calculated using a one-way ANOVA, and the Tukey-Kramer multiple comparisons test was used to determine p values.

Rac Activation Assay—THP-1 cell lysates were subjected to affinity precipitation using the specific interaction of the Rac GTPase with its downstream effector, the PBD of PAK (36). The PBD-PAK is bound to glutathione-agarose beads and was used according to the manufacturer's instructions (Cytoskeleton, Denver, CO). Following stimulation with fAbeta-(25–35) (60 µM/63.6 µg/ml), cells were collected and lysed with Mg2+ lysis buffer (25 mM Tris, pH 7.5, 5 mM MgCl2, 0.15 M NaCl, 1% Igepal (Nonidet P-40), 5% sucrose). PBD-PAK beads were added to 1 mg/ml cell lysate and incubated with rocking for 1 h at 4°C. Beads were collected by centrifugation and resuspended in sample buffer. Samples were subjected to 12% SDS-PAGE immunoblot analysis using an anti-Rac antibody (1:1000). Aliquots of the cell lysates (40 µg/lane) were run as protein loading controls. The protein was detected by enhanced chemiluminescence, and band intensities were quantified using NIH Image 1.62 software (Bethesda, MD). Values statistically different from controls were calculated using a one-way ANOVA, and the Tukey-Kramer multiple comparisons test was used to determine p values.

Cellular Fractionation—For these experiments we utilized a cell fractionation protocol described previously (37). Briefly, THP-1 cells (6 x 106 cells) were collected and resuspended in Hanks' balanced salt solution for 30 min at 37 °C. Cells were then stimulated for 0 or 10 min with fAbeta-(25–35) (60 µM) and lysed by incubation in relaxation buffer (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1.25 mM EGTA, and 10 mM PIPES, pH 7.3) on ice for 15 min followed by 10 s of sonication. Cells were cleared by centrifugation at 500 x g for 5 min at 4 °C. The supernatant was then centrifuged for 1 h at 110,000 x g at 4 °C in a Beckman Coulter SW50.1 rotor. The resulting supernatant was removed and saved as the "cytosolic" fraction, and the membrane pellet was resuspended in relaxation buffer. Lysates were then subjected to 12% SDS-PAGE immunoblot analysis using an anti-Rac antibody (1:1000) to determine the relative amount of Rac in each fraction. The membrane marker flotillin (1:1000) was used to assess the efficacy of the fractionation procedure. These experiments were performed four times (n = 4), and the data were analyzed by Student's t test with a confidence interval of 99%.

Measurement of Superoxide Production—Intracellular superoxide radical generation was assayed by analyzing nitro blue tetrazolium (NBT) reduction (19, 38). Superoxide generation is determined at the microscopic level by the presence of an insoluble formazan precipitant, which appear as dark purple/black granules within the cell (39). For these experiments primary microglial cells from either Vav–/– mice or Vav+/+ mice (SV129) were harvested as described previously. The Vav–/– mice (40) were a generous gift from Dr. Juan Rivera (NIAMS, National Institutes of Health, Bethesda) and Dr. Victor Tybulewicz (National Institute for Medical Research, London, UK). Microglia were plated in 24-well plates overnight in DMEM/F-12 without serum. The following day, the media were removed and replaced with NBT (1 mg/ml) in DMEM/F-12 with or without fAbeta-(25–35) (60 µM) at 37 °C for 30 min. Phorbol 12-myristate 13-acetate (PMA; 390 nM)) was used as a positive control for the stimulation of ROS production (17, 19, 24). Cells were then fixed with 2% paraformaldehyde for 15 min at 4 °C. Three random fields of cells (>100 cells) were counted by a blind observer on an inverted microscope. The assay was performed in triplicate. Values statistically different from controls were calculated using a one-way ANOVA, and the Tukey-Kramer multiple comparisons test was used to determine p values.

Phagocytosis Assay—The phagocytosis assay was performed as described previously (25, 41). Briefly, primary murine microglia cells were collected and plated into 24-well plates overnight in DMEM/F-12 without serum. The cells were then stimulated by the addition of 60 µM fAbeta-(25–35) for 30 min. Nile red fluorospheres (1 µM microspheres) were added to the cells for 30 min. The uptake of fluorescent microspheres was used as a marker of fluid phase phagocytosis. Cells were then fixed with 2% paraformaldehyde, and three random fields of cells (>100 cells) were counted on an inverted microscope. The assay was performed in triplicate. Values statistically different from controls were calculated using a one-way ANOVA, and the Tukey-Kramer multiple comparisons test was used to determine p values.


Figure 1
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  		FIGURE 1.
Fibrillar Abeta stimulates tyrosine phosphorylation of Vav. A, THP-1 monocytes (5 x 106 cells) were stimulated with fAbeta-(25–35) or fAbeta-(1–42) peptides for 3 min. Vav was immunoprecipitated (IP) from cell lysates with an anti-Vav antibody and analyzed by Western blot (WB) analysis using the anti-phospho-Tyr antibody 4G10. Blots were stripped and reprobed with an anti-Vav antibody as a protein-loading control. B, time course of Vav protein-Tyr phosphorylation in THP-1 cells treated with fAbeta-(25–35) (60 µM). C, analysis of band intensity of Western blots of phosphorylated Vav normalized to Vav protein levels and expressed as relative density. The data shown are reflective of pooled data from three independent studies. **, p < 0.01 at 5 min; *, p < 0.05 at 10 min as compared with 0 min.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Stimulation of Vav-Tyr Phosphorylation in THP-1 Monocytes by Fibrillar Abeta Peptides—Vav can act as a Rac-specific GEF upon its phosphorylation by Src family tyrosine kinases. We tested whether fAbeta engagement of the microglia led to Vav tyrosine phosphorylation. Here we report the Tyr phosphorylation of the Vav protein following fAbeta stimulation with either fibrillar Abeta-(1–42) or Abeta-(25–35) peptides (Fig. 1A). fAbeta treatment resulted in the transient phosphorylation of Vav, which was maximal after 5 min (Fig. 1, B and C). These findings are consistent with our previous findings that demonstrated that fAbeta peptides, Abeta-(1–40), Abeta-(1–42), and Abeta-(25–35), stimulate tyrosine kinase-based signaling in both THP-1 monocytes and microglia (17–19, 21). These findings confirmed that the magnitude of stimulation between these different Abeta peptides was qualitatively similar, and at this concentration (60 µM) the fibrillar Abeta-(25–35) peptide most effectively drives intracellular signaling (17, 21) as it contains the biologically active C-terminal beta-pleated sheet domain that is necessary for fibril formation (42).

Engagement of the Microglial Fibrillar Abeta Cell Surface Receptor Complex Is Required for Vav Phosphorylation—We have recently shown that microglia employ a multireceptor cell surface complex to detect and respond to Abeta fibrils. These receptor elements act in concert to stimulate intracellular signaling cascades as well as initiate a novel type of phagocytosis in microglia (17, 25). Importantly, we have shown that perturbation of the interaction of fAbeta with individual subunits abrogates the signaling and phagocytic response from the complex as a whole. To determine whether fAbeta engagement of this receptor ensemble was responsible for the observed Vav tyrosine phosphorylation, we tested the effect of antagonists to each of the individual receptor subunits. The participation of the class A and B scavenger receptors was assessed using fucoidan, which is an antagonist of these receptors (43). The role of the class B scavenger CD36 was directly assessed using GST-CD36, which is composed of the 93–120-amino acid extracellular binding domain of CD36 coupled with a GST tag. This fusion protein is designed to competitively inhibit the binding of ligands to cell surface CD36 (44). We found that fucoidan and GST-CD36 both inhibited Vav phosphorylation in response to fAbeta stimulation in THP-1 monocytes (Fig. 2, A and B). To evaluate the contribution of the {alpha}6beta1 integrin, we employed two different antagonists specifically directed toward the beta1 integrin. First, the RHD peptide, which contains an epitope that binds to beta1 integrins (45), dramatically reduced Vav phosphorylation in response to fAbeta (Fig. 2C). The second beta1 integrin antagonist was derived from invasin protein (invasin 195), which is encoded by the enteropathogenic Yersinia (46), and acts to functionally block beta1 integrin activation (47). We demonstrate that invasin 195 also inhibited fAbeta-stimulated Vav phosphorylation in THP-1 monocytes (Fig. 2D). The CD47 antagonist 4N1K, which is derived from the cell-binding domain of thrombospondin, blocks intracellular signaling by directly binding to CD47 (48). Incubation of THP-1 monocytes with 4N1K inhibited Abeta fibril stimulation of Vav phosphorylation (Fig. 2E). Taken together, these results establish that fAbeta engagement of the fAbeta cell surface receptor complex initiates Vav phosphorylation and activation.


Figure 2
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  		FIGURE 2.
The Abeta receptor complex mediates the stimulation of Vav protein-Tyr phosphorylation. A–E, THP-1 cells were preincubated with fucoidan (A, 300 µg/ml), GST-CD36 (B, 100 pM), RHD peptide (C, 100 µg/ml), invasin 195 (D, 5 µg/ml), or 4N1K (E, 100 µM) for 30 min prior to stimulation with fAbeta-(25–35) (60 µM) for 3 min. Blots were stripped and reprobed with an anti-Vav antibody as a protein-loading control (Ctrl). Individual graphs are reflective of pooled data from three independent experiments of the relative density ratio of Vav protein-Tyr phosphorylation. *, p < 0.05; **, p < 0.01 compared with control. IP, immunoprecipitation; WB, Western blot.

 


Figure 3
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  		FIGURE 3.
Fibrillar Abeta-stimulation leads to Vav association with both Lyn and Syk kinases. THP-1 cell were stimulated with fAbeta-(25–35) (60 µM) for 3 min, and cell lysates were immunoprecipitated (IP) with either an anti-Lyn (A) or and anti-Syk (B) antibody. Immunoprecipitates were resolved by 9% SDS-PAGE and analyzed by Western blot (WB) using an anti-Vav antibody. Blots were stripped and reprobed with the appropriate antibody as a protein-loading control.

 
Vav Associates with Both Lyn and Syk Kinases in Fibrillar Abeta-stimulated THP-1 Monocytes—Microglia and THP-1 cells respond to fAbeta by activating the Src family kinase Lyn as well as the tyrosine kinase Syk, and these kinases have been reported previously to be required for proximal signaling events in the microglial response to Abeta fibrils (17–19, 25). Interestingly, both Lyn and Syk have been shown to stimulate Vav phosphorylation in response to a variety of other stimuli (49, 50). Therefore, we sought to determine whether Lyn or Syk associate with Vav following stimulation with fAbeta. Either Lyn or Syk was immunoprecipitated from THP-1 monocytes stimulated with or without fAbeta. The immunoprecipitants were transferred to immunoblots and probed with a Vav antibody. The immunoblots revealed that Lyn (53–56-kDa protein doublet) and Syk (80-kDa) co-immunoprecipitated with Vav following fAbeta stimulation (Fig. 3, A and B). To further establish a role for either Lyn or Syk kinases in Vav phosphorylation, inhibitors directed against either Src or Syk were analyzed. Pretreatment of THP-1 cells with either piceatannol, a Syk inhibitor, or PP2, an Src inhibitor, resulted in an inhibition of Abeta-stimulated Vav tyrosine phosphorylation (Fig. 4, A and B). These findings directly link Vav to proximal fAbeta receptor signaling events.

Rac1 Is Activated and Translocates to the Membrane in fAbeta-stimulated THP-1 Monocytes—We wanted to establish whether the downstream target of Vav, the small GTPase Rac1, also displayed a fAbeta-dependent activation. Rac1, the predominant isoform in myeloid lineage cells (51), is a member of the Rho family of small monomeric GTPases and is an integral component of the active NADPH oxidase (52). Although fAbeta-stimulated ROS production has been shown previously (17, 19, 24), direct evidence for activation of Rac by Abeta peptides has not been demonstrated. We demonstrate that THP-1 cells stimulated with fAbeta exhibit a robust increase in Rac GTP loading. Affinity precipitation assays reveal that GTP-bound Rac is increased within 5 min of fAbeta treatment, and the levels of Rac-GTP return to near base-line levels within 30 min (Fig. 5, A and B).


Figure 4
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  		FIGURE 4.
Inhibition of either Src kinase or Syk kinase activity inhibits tyrosine phosphorylation of Vav. A, THP-1 cells were pretreated with either PP2 (10 µM) or piceatannol (25 µg/ml; PICE) for 30 min prior to stimulation with fAbeta-(25–35) (60 µM) for 3 min. Vav was immunoprecipitated (IP) from cell lysates with an anti-Vav antibody and analyzed by Western blot (WB) analysis using the anti-phospho-Tyr antibody 4G10. Blots were stripped and reprobed with an anti-Vav antibody as a protein-loading control (Ctrl). B, analysis of band intensity of Western blots of phosphorylated Vav normalized to Vav protein levels and expressed as relative density. *, p < 0.05 compared with control.

 


Figure 5
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  		FIGURE 5.
Time course of Rac GTP loading following stimulation with fAbeta. A, THP-1 cells stimulated with fAbeta-(25–35) (60 µM) for 0, 1, 5, 10, 15, or 30 min were subject to a Rac affinity precipitation assay that captures only GTP-bound Rac. Cell lysates were analyzed by immunoblot analysis using an anti-Rac antibody. Cell lysate samples (40 µg/lane) were run in parallel as protein loading controls. B, analysis of the relative density ratio of Rac-GTP loading. The data shown are reflective of pooled data from three independent studies. *, p < 0.05 at 5 and 10 min as compared with 0 min.

 
Following GDP/GTP exchange and conversion into an active conformation, Rac must translocate to the plasma membrane where it interacts with other NADPH oxidase subunits to form the active oxidase complex and produce superoxide. Rac also fulfills a number of other functions related to cytoskeletal organization and presumably activated by other GEFs; thus the cells exhibit a significant basal level of active Rac associated with the membrane. Important to our studies, fAbeta stimulation results in a 2-fold increase in Rac membrane association (Fig. 6, A and B), which is reflective of fAbeta-specific actions in stimulating NADPH oxidase activity and phagosome formation.


Figure 6
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  		FIGURE 6.
Rac exhibits fAbeta-dependent translocation to the membrane. A, THP-1 monocytes were stimulated with fAbeta-(25–35) (60 µM) for 0 or 10 min. Cytosolic (C) and membrane (M) fractions (10 µg of protein/lane for both fractions) were analyzed by immunoblot analysis using an anti-Rac antibody. Cell fractions were also immunoblotted with an anti-flotillin antibody to assess the efficacy of the fractionation procedure. WB, Western blot. B, analysis of the ratio of membrane-bound versus cytosolic Rac. Data are reflective of pooled results from four independent experiments and were analyzed by Student's t test. *, p < 0.05 at 10 min as compared with 0 min.

 
Fibrillar Abeta-stimulated Reactive Oxygen Species Production Is Inhibited in Vav–/– Primary Microglia—In light of our findings that Vav is phosphorylated in response to Abeta stimulation, we hypothesized that Vav might be a necessary upstream intermediate in the intracellular signaling pathway leading to NADPH oxidase assembly and subsequent ROS production. To examine the role of Vav in Abeta-stimulated respiratory burst, we utilized primary murine microglia obtained from Vav–/– mice. Analysis of intracellular superoxide production was monitored by the reduction of NBT. We observed that superoxide production was severely attenuated in fAbeta-stimulated Vav–/– microglia when compared with fAbeta-stimulated Vav+/+ microglia (Fig. 7). Vav–/– microglia produced significant levels of ROS in response to PMA indicating that the deletion of Vav does not inhibit the function of the NADPH oxidase in response to an alternate stimulus. These data demonstrate an obligatory role for Vav activity in fAbeta-stimulated intracellular signaling pathways leading to effective NADPH oxidase assembly and respiratory burst in primary microglia.

The Genetic Deletion of Vav Inhibits the fAbeta-induced Phagocytic Response in Primary Microglia—In an effort to establish whether Vav plays a broad role in response to fAbeta stimulation of microglia, we analyzed the role Vav might play in the intracellular signaling events that link fAbeta engagement of the cell surface receptor complex to the phagocytic machinery. Both Vav and Rac1 have been implicated in both cytoskeletal alterations (53) and membrane ruffling (54) observed during phagocytosis. We sought to identify whether genetic deletion of Vav activity upstream of its effector, Rac1, would alter the fAbeta-induced phagocytic response. Primary microglia from Vav knock-out (Vav–/–) and wild type (Vav+/+) mice were evaluated for their ability to phagocytose microspheres. Vav–/– microglia treated with fAbeta-(25–35) failed to mount a phagocytic response when compared with Vav+/+ microglia (Fig. 8). These findings establish Vav as an essential proximal component of the fAbeta-stimulated signaling pathway that catalyzes Rac1-dependent phagosome formation.


Figure 7
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  		FIGURE 7.
Vav is required for ROS production in primary microglia stimulated with fAbeta. Neonatal primary microglia obtained from Vav–/– mice or Vav+/+ (SV129) mice were incubated in serum-free DMEM/F-12 containing NBT and stimulated with fAbeta-(25–35) (60 µM) or PMA (390 nM) for 30 min. Superoxide anion generation as monitored by the presence of insoluble formazan was visualized on a Leica DMIRB inverted microscope. Three random fields of cells (>100 cells) were counted by a blind observer. These experiments are reflective of pooled data from three independent studies. **, p < 0.01 compared with control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study extends our previous efforts to elucidate the intracellular signaling mechanisms responsible for microglial proinflammatory responses to fibrillar Abeta peptides. The dissection of proximal intracellular signaling cascades is essential for understanding how fAbeta at the cell surface is coupled to the release of proinflammatory secretory products from microglia. Fibrillar Abeta engages an ensemble of cell surface receptors that initiate intracellular signaling cascades that may utilize similar signaling molecules before diverging to assemble the NADPH oxidase and the phagosome.

A primary goal of these experiments was to evaluate potential signaling intermediates upstream of the NADPH oxidase. Of particular interest was a group of signaling molecules that act as guanine nucleotide exchange factors (GEFs) for Rac. These molecules are known to activate the NADPH oxidase subunit Rac1 by facilitating GDP/GTP exchange on Rac1. Several Rac-GEFs have been identified, including Vav (28, 55), Tiam1 (56), Dock180/Elmo (57), and Eps8/E3b1/Sos-1 (58). We chose to analyze Vav activity because it has been the most tightly linked to the activation of the NADPH oxidase (52). Vav is a multidomain protein composed of adjacent Dbl homology (DH) and pleckstrin homology domains that are common to almost all Rho family GEFs (59). The DH domain is responsible for the catalysis of GDP/GTP exchange (60). The DH and pleckstrin homology domains are bordered on the C terminus by a zinc finger motif, a short proline-rich region, and an Src homology (SH) SH3-SH2-SH3 domain and bordered on the N terminus by a calponin homology domain and an acidic region. Tyrosine phosphorylation of Vav is critical for activation of its GEF activity, and recent studies have demonstrated that tyrosine phosphorylation of Tyr-174 relieves N-terminal autoinhibition between the acidic region and the DH domain (61). Notably, Crespo et al. (62) have demonstrated that Vav tyrosine phosphorylation is necessary for activation of its exchange activity toward Rac1 and subsequent activation of downstream targets. In this study, we establish that Vav displays a fAbeta-dependent phosphorylation on tyrosine residues, and this tyrosine phosphorylation is contingent upon fibril engagement of the microglia fAbeta cell surface receptor complex.


Figure 8
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  		FIGURE 8.
Vav is essential for fAbeta-stimulated phagocytosis in primary murine microglia. Neonatal primary microglial cells from Vav–/– mice or Vav+/+ (SV129) mice were treated with fAbeta-(25–35) (60 µM) for 30 min before the addition of microspheres for an additional 30 min. The ingestion of fluorescent microspheres was visualized on an inverted microscope, and three random fields of cells (>100 cells) were counted to determine the percentage of phagocytic cells. The data are reflective of pooled data from three independent experiments. **, p < 0.01 compared with control.

 
The SH2 domain of Vav mediates its interaction with activated protein-tyrosine kinases, presumably by binding phosphotyrosine residues. Several protein-tyrosine kinases have been shown to facilitate Vav phosphorylation (59). In fAbeta-stimulated microglia, Vav phosphorylation is likely contingent on its interaction with either Src family kinases or Syk kinase. Indeed, elements of the fAbeta receptor complex are found to physically associate with Src family kinases and Syk (17). We have demonstrated previously a dramatic reduction in fAbeta-stimulated ROS production in THP-1 cells pretreated with the Syk inhibitor piceatannol (19). Furthermore, Bianca et al. (24) have demonstrated a similar reduction in ROS production in both monocytes and microglia treated with the Src family-selective tyrosine kinase inhibitor PP1. These findings verify the necessity for both of these kinases in the signaling pathways leading to NADPH oxidase assembly. We have also demonstrated previously that inhibition of either of these tyrosine kinases results in inhibition of the phagocytic response to fAbeta (25). In this study, we demonstrate a fAbeta-dependent interaction between Vav and the Src family kinase Lyn and the tyrosine kinase Syk, and inhibition of kinase activity leads to a dramatic reduction in Vav tyrosine phosphorylation. This association provides a mechanistic link between Vav and the most proximal fAbeta-stimulated intracellular signaling events.

The NADPH oxidase normally plays an essential role in innate immunity. The oxidase is found associated with phagocyte membranes where it can facilitate the destruction of invading microorganisms by releasing Formula into the phagocytic vesicle (63, 64). This released Formula serves as a precursor for additional ROS that are rapidly formed, including hydrogen peroxide, hydroxyl radical, peroxynitrite, and other oxidants that aid in the killing of the invading pathogen (27). Importantly, excessive production of these ROS is cytotoxic and can damage tissue adjacent to sites of inflammatory action (65); consequently, NADPH oxidase assembly is a highly regulated process (27, 63). The NADPH oxidase is a multicomponent enzyme system that is composed of two integral membrane proteins, p22phox and gp91phox (together know as cytochrome b558), and three essential cytosolic components, p47phox, p67phox, and Rac1. A fourth nonessential cytosolic component, p40phox, may serve in a regulatory capacity. The activation of the PHOX subunits is regulated through signaling pathways that are independent from Rac activation and translocation (27, 66, 67). Following their phosphorylation, the cytosolic PHOX subunits translocate to the membrane where they interact to assemble the functional oxidase complex (26, 63). The importance of the NADPH oxidase in host defense is illustrated by the hereditary loss-of-function disorder, chronic granulomatous disease, which results from mutations in any of these individual subunits. Chronic granulomatous disease patients are subject to recurrent bacterial and fungal infections and a reduced life expectancy.

In the AD brain, microglial NADPH oxidase-derived ROS are believed to be a primary source of oxidative damage (19, 22–24). Previous studies have demonstrated the translocation of the p47phox and p67phox subunits from the cytosol to the membrane in both human AD brain tissue (68) and fAbeta-stimulated monocytes and microglia (24). We extended these findings to document the ability of fAbeta to activate parallel signaling pathways leading to Rac1 GTP loading. In resting cells, Rac is held inactive in the cytosol through an interaction between its C-terminal prenyl moiety and the effector region of the GDP dissociation inhibitor, RhoGDI. The timing of Rac translocation in relation to its activation remains unclear; however, recent findings suggest Rac moves to the membrane prior to its GTP loading (28). During activation, Rac undergoes GDP/GTP exchange facilitated by a GEF and becomes dissociated from RhoGDI. GTP-loaded Rac is then free to interact with other components of the NADPH oxidase where it is an integral and essential component for ROS production (52, 69). Our findings demonstrate that Rac1 translocates to the plasma membrane and is GTP-loaded in a fAbeta-dependent manner. The temporal activation of Rac1 is also similar to the activation time course identified for Vav. We hypothesize that following fAbeta stimulation Vav acts as the GEF for Rac1 in microglia, thereby facilitating the participation of Rac in ROS formation. We directly examined the role Vav plays in upstream fAbeta-stimulated signaling pathways leading to NADPH oxidase assembly by analyzing ROS production in Vav-deficient primary microglia. Our data confirm that Vav is indeed required for fAbeta-dependent intracellular signaling cascades leading to functional NADPH oxidase assembly. Taken together, these results provide evidence that fAbeta drives assembly of the microglial NADPH oxidase through an ensemble of cell surface receptors that initiate a tyrosine kinase-Vav-Rac1-based signaling cascade (Fig. 9).


Figure 9
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  		FIGURE 9.
Model of intracellular Vav signaling following Abeta fibril interaction with the microglial cell surface receptor complex. Fibrillar engagement of an ensemble of cell surface receptors results in the initiation of a tyrosine kinase-based signaling cascade. Tyrosine phosphorylation of the Vav-GEF results in the activation of its downstream effector the Rac1 GTPase. Rac activity leads to both NADPH oxidase-derived ROS production and phagosome formation through Rac-dependent actin cytoskeleton reorganization.

 
It remains plausible that in the AD brain fAbeta-induced oxidative events are mediated by cell types other than microglia. Indeed, astrocytes have been shown recently to have a functional NADPH oxidase (70), and fAbeta stimulation elicits activation leading to neuronal death in a co-culture model (71). However, it remains unclear what the quantitative contributions of astrocytes are in Abeta-induced ROS production.

There is compelling evidence implicating a Vav-Rac interaction in formation of the phagosome in Fc{gamma}R-mediated (type I) phagocytosis (29, 72). Vav mutants lacking their catalytic domain (DH domain) responsible for GEF activity abolished the Fc{gamma}R-mediated activation of Rac and consequently reduced the phagocytic response of macrophages (28). The activation of both Vav and Rac has also been implicated in the novel beta1-integrin-dependent phagocytic mechanism used to internalize the bacteria Yersinia (47). In the AD brain, microglia exhibit a proinflammatory phenotype, yet fail to mount an effective phagocytic response to Abeta plaques (73); however, microglia have a well established ability to elicit a phagocytic response to both Abeta fibrils and isolated senile plaques in vitro (74–76). We have recently reported that in vitro microglia use a beta1-integrin-dependent phagocytic mechanism to engulf Abeta fibrils analogous to the phagocytic response elicited by Yersinia (25). Intracellular signaling resulting in phagosome formation is also mediated through the fAbeta cell surface receptor complex (25) suggesting that similar signaling pathways might be involved.

In this study, we have established a definitive role for Vav in the fAbeta-initiated signaling pathway leading to phagosome formation. Microglia from Vav-null mice have an attenuated phagocytic response to Abeta fibrils. Coupled with the Vav activation data described above, these results would indicate that intracellular signaling cascades initiated by fibril engagement of a cell surface receptor complex requires Vav activity for a phagocytic response to fAbeta.

We provide conclusive evidence that Vav plays a pivotal role in Abeta-stimulated intracellular signaling cascades to either the NADPH oxidase or the phagocytic machinery in microglia. The convergence of these signaling pathways may suggest a level of regulation proximal to Vav phosphorylation. It is possible that the signaling cascades responsible for Vav activation dictate how different pools of Vav are directed toward downstream targets. For example, Vav phosphorylation in response to Fc{gamma}R stimulation was unaffected in Syk-deficient macrophages (77) suggesting that alternative kinases may activate Vav. It is plausible that upstream signaling pathways governing phagosome formation may be redundant as we have demonstrated previously that both inhibitors of Syk (piceatannol) and Src family kinases (PP2 and LY294002) dramatically inhibit fAbeta-induced phagocytosis (25). Interestingly, Cougoule et al. (72) have recently shown that Src controls the recruitment of Vav and Rac to nascent phagosomes, although Syk is necessary for Rac GTP loading.

This study identifies an important tyrosine kinase-Vav-Rac signaling pathway in fAbeta-stimulated microglia (Fig. 9). These novel findings will allows us to examine new alternative therapeutic targets that will facilitate Abeta clearance and suppress the oxidative stress and subsequent neuronal damage observed in the AD brain.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant AG16740, the Blanchette Hooker Rockefeller Foundation, and the American Health Assistance Foundation (to G. E. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported in part by a Ruth L. Kirschstein National Research Service Award, National Institutes of Health Grant F32 AG24031. To whom correspondence should be addressed: Dept. of Neuroscience, Case Western Reserve University, SOM E504, 2109 Adelbert Rd., Cleveland, OH 44106. Tel.: 216-368-3435; Fax: 216-368-3079; E-mail: blw9{at}po.cwru.edu.

2 The abbreviations used are: AD, Alzheimer disease; fAbeta, fibrillar beta-amyloid; ROS, reactive oxygen species; GEF, guanine nucleotide exchange factor; NBT, nitro blue tetrazolium chloride; GST, glutathione S-transferase; GDI, guanine nucleotide dissociation inhibitor; SH, Src homology; PAK, p21-activated protein kinase; PMA, phorbol 12-myristate 13-acetate; PBD, p21-binding domain; DH, Dbl homology; ANOVA, analysis of variance; PIPES, 1,4-piperazinediethanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Maria Febbraio and Ingo Autenrieth for their generous gift of reagents. We also thank Drs. Juan Rivera and Victor Tybulewicz for the Vav–/– mice.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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L. M. Stuart, S. A. Bell, C. R. Stewart, J. M. Silver, J. Richard, J. L. Goss, A. A. Tseng, A. Zhang, J. B. E. Khoury, and K. J. Moore
CD36 Signals to the Actin Cytoskeleton and Regulates Microglial Migration via a p130Cas Complex
J. Biol. Chem., September 14, 2007; 282(37): 27392 - 27401.
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