Microglial phagocytosis induced by fibrillar β-amyloid is attenuated by oligomeric β-amyloid: implications for Alzheimer's disease
© Pan et al; licensee BioMed Central Ltd. 2011
Received: 29 November 2010
Accepted: 30 June 2011
Published: 30 June 2011
Reactive microglia are associated with β-amyloid (Aβ) deposit and clearance in Alzhiemer's Disease (AD). Paradoxically, entocranial resident microglia fail to trigger an effective phagocytic response to clear Aβ deposits although they mainly exist in an "activated" state. Oligomeric Aβ (oAβ), a recent target in the pathogenesis of AD, can induce more potent neurotoxicity when compared with fibrillar Aβ (fAβ). However, the role of the different Aβ forms in microglial phagocytosis, induction of inflammation and oxidation, and subsequent regulation of phagocytic receptor system, remain unclear.
We demonstrated that Aβ(1-42) fibrils, not Aβ(1-42) oligomers, increased the microglial phagocytosis. Intriguingly, the pretreatment of microglia with oAβ(1-42) not only attenuated fAβ(1-42)-triggered classical phagocytic response to fluorescent microspheres but also significantly inhibited phagocytosis of fluorescent labeled fAβ(1-42). Compared with the fAβ(1-42) treatment, the oAβ(1-42) treatment resulted in a rapid and transient increase in interleukin 1β (IL-1β) level and produced higher levels of tumor necrosis factor-α (TNF-α), nitric oxide (NO), prostaglandin E2 (PGE2) and intracellular superoxide anion (SOA). The further results demonstrated that microglial phagocytosis was negatively correlated with inflammatory mediators in this process and that the capacity of phagocytosis in fAβ(1-42)-induced microglia was decreased by IL-1β, lippolysaccharide (LPS) and tert-butyl hydroperoxide (t-BHP). The decreased phagocytosis could be relieved by pyrrolidone dithiocarbamate (PDTC), a nuclear factor-κB (NF-κB) inhibitor, and N-acetyl-L-cysteine (NAC), a free radical scavenger. These results suggest that the oAβ-impaired phagocytosis is mediated through inflammation and oxidative stress-mediated mechanism in microglial cells. Furthermore, oAβ(1-42) stimulation reduced the mRNA expression of CD36, integrin β1 (Itgb1), and Ig receptor FcγRIII, and significantly increased that of formyl peptide receptor 2 (FPR2) and scavenger receptor class B1 (SRB1), compared with the basal level. Interestingly, the pre-stimulation with oAβ(1-42) or the inflammatory and oxidative milieu (IL-1β, LPS or t-BHP) significantly downregulated the fAβ(1-42)-induced mRNA over-expression of CD36, CD47 and Itgb1 receptors in microglial cells.
These results imply that Aβ oligomers induce a potent inflammatory response and subsequently disturb microglial phagocytosis and clearance of Aβ fibrils, thereby contributing to an initial neurodegenerative characteristic of AD. Antiinflammatory and antioxidative therapies may indeed prove beneficial to delay the progression of AD.
Microglial phagocytosis has been proposed as an Aβ-lowering mechanism of Aβ immunization in Alzhiemer's Disease (AD) . Microglia interact with fibrillar Aβ through the cell surface receptor system  that promote the clearance and phagocytosis of fAβ. The functional components of the receptor system include the scavenger receptor CD36, CD47 (integrin-associated protein), β1 integrin (Itgb1) [2–4], macrophage scavenger receptor class A (SRA) and class B (SRB) , receptor for advanced glycation end products (RAGE) [6, 7], and the formyl peptide receptor (FPR) . Exogenous microglial lateral ventricle transplantation has been shown to increase Aβ clearance in AD model rats . Bone marrow-derived microglia can also efficiently restrict amyloid deposits . These findings indicate the potential of exogenous and healthy microglia for therapeutic approach to AD. However, an enigma still remains: Why are those entocranial resident microglia surrounding plaques "activated" but unable to trigger an effective phagocytic response to engulf and degrade fibrillar Aβ deposits in ADż Recent evidence indicates that dysfunctional microglia is associated with aging [11, 12]. Human brains containing high Aβ loads show a significantly higher degree of microglial dystrophy than nondemented, amyloid-free brains. Also, microglial cell senescence is exacerbated by amyloid [11, 12]. Therefore, microglial degeneration may affect its phagocytosis and serve as an important factor in AD pathogenesis.
Abundant proinflammatory cytokines, chemokines, complement products, and oxygen radicals are presented in AD brains [13, 14]. The binding of Aβ peptide to cell surface receptors induces proinflammatory gene expression and subsequently cytokines production . Aβ seems to modulate these events all the time and interact with proinflammatory cytokines in a synergistic manner  to induce neuronal damage via reactive oxygen species (ROS)-dependent pathways . ROS scavengers such as catalase obviously reduce the activation of nuclear factor kappa-B (NF-κB), a transcription factor mediating immune and inflammatory responses , and subsequently decrease the elevated Aβ-induced IL-1β level . Accordingly, strategies to suppress oxidative stress and NF-κB activation may attenuate neuroinflammation and neuronal damage, which will be beneficial to AD treatment.
The processing of β-amyloid precursor protein (APP) by β- and γ-secretases produces Aβ peptides, of which Aβ(1-42) is especially biochemical active for its spontaneous proneness to oligomerization and fibrillation. Soluble Aβ oligomers rather than Aβ fibrils have been observed as the primary pathological species at early time points preceding fibril formation . However, from APP processing to Aβ plaque formation, the specific role of Aβ oligomers and fibrils in mediating microglial activation is still unclear. Particularly, how do Aβ oligomers induce the generation of oxidative stress, inflammatory response and subsequently affect phagocytosis of Aβ fibrils in microgliaż Thus, the effect of Aβ components at different stages on microglia functions needs to be clarified so as to produce promising strategies to retard the early AD-related pathological affairs.
Here we investigated the differential effect of Aβ(1-42) oligomers versus fibrils on the viability of microglia, the expressions of inflammatory mediators, and phagocytosis function in microglia. Particularly, we applied the central Aβ components at the early and terminal stage (oligomers and fibrils, respectively) combined with some pharmacological agents to treat microglia in a proper sequential design, in order to study the role of Aβ components at different stages in microglial phagocytosis and cell surface components of the phagocytic receptor system, including CD36, CD47, integrin β1, SRA, SRB1, RAGE, FPR2, as well as the classical phagocytic receptors, the Ig receptors (FcγR I and FcγRIII). We further gained insights into the impact of anti-inflammation and anti-oxidation on oligomeric Aβ-activated microglial cells in NF-κB signaling.
Effects of oligomeric versus fibrillar Aβ(1-42) on microglial cell viability
Aβ(1-42) fibrils, not oligomers, enhanced phagocytic activity in a dose- and time-dependent manner in microglial cells
Differential impact of oligomeric versus fibrillar Aβ(1-42) on the expressions of inflammatory mediators in microglial cells
Oligomeric Aβ(1-42) attenuated fAβ(1-42)-stimulated microglial phagocytosis
Since phagocytosis of fAβ is central to the role of activated microglia in AD, we further tested the effect of oAβ-prestimulated microglia on the phagocytic response to fAβ itself. oAβ(1-42)-stimulated and -unstimulated microglia with fluorescent labeled fAβ were incubated for 30 min, and cell-associated fluorescence intensity was measured (Figure 4D-F). Cells pre-stimulated with oAβ(1-42) (1.0 μM), compared with unstimulated cells, showed a respective decrease in uptaking of labeled fAβ (53.6% for 3 h and 78.2% for 12 h) (P < 0.001) (Figure 4D, F). Moreover, the inhibition of oAβ(1-42) on microglial internalizating fAβ displayed a time-dependent effect (P < 0.05) (Figure 4F). The simultaneous view of the entire cell (x-y, x-z and x-z plane) showed fluorescent labeled fAβ localizing in the cytoplasm. (Figure 4E)
These results suggest that the early stage component of Aβ, oligomers, can impair the microglial phagocytic function, and may subsequently impact the capacity of microglia to clear terminal fibrils Aβ or tissue debris in the brain.
Oligomeric Aβ(1-42) attenuated fAβ-stimulated phagocytosis, which was correlated with the elevated inflammatory mediators
Two types of Aβ(1-42) elicited the intracellular level of superoxide anion (SOA) in microglial cells: effects of NAC and PDTC
PDTC or NAC rescues oligomeric Aβ-elicited phagocytosis impairment in the inflammatory and oxidative milieu
Stimulation of microglial cells with IL-1β, LPS or t-BHP significantly reduced phagocytosis of fluorescence labeled fAβ to 18%, 26% and 30% of that of unstimulated cells, respectively (P < 0.001) (Figure 7B, C). Interestingly, as expected, the pretreatment of cells with PDTC markedly relieved IL-1β, LPS, t-BHP or Aβ oligomers-impaired phagocytosis (P < 0.05) (Figure 7C). NAC pretreatment also rescued t-BHP-impaired phagocytosis of fAβ (P < 0.05) (Figure 7C). Similar results were obtained with NAC counteracting IL-1β, LPS or Aβ oligomers-impaired phagocytosis, but differences were not statistically found (Figure 7C). Neither PDTC nor NAC treatment alone had affect on microglial phagocytosis (data not shown).
Collectively, these findings further support that Aβ oligomers-induced proinflammatory mediators and oxidative milieu negatively regulate microglial phagocytic function.
Oligomeric Aβ, the inflammatory and oxidative milieu regulate gene expressions of Aβ-related cell surface receptors in microglial cells
These data indicate that microglia, when early exposed in the oligomeric Aβ or proinflammatory cytokines and oxidative milieu, have decreased the expression of Aβ-related cell surface receptors, and thereby may have decreased the capacity of microglia to bind and subsequently clear Aβ.
Role of NF-κB signaling in the expression of inflammatory mediators in oAβ-stimulated microglial cells
The elucidation of the mechanism by which microglial phagocytosis is regulated, may help identify the etiology of Aβ desposit and therapeutic targets in aggregation-prone protein-associated neurodegenerative diseases such as Alzheimer's disease. The present study reports an important and exciting finding that extracellular oligomeric Aβ(1-42) suppresses the phagocytic function of microglia triggered by fibrillar Aβ. Our findings support the hypothesis that microglial dysregulation by oligomeric Aβ-elicited proinflammatory and oxidative stress milieu hampers clearance of fibrillar Aβ deposits, thereby leading to an initial neurodegenerative process characteristic of AD.
Oligomeric Aβ plays a critical role in the pathogenesis of AD. It is believed to contribute to early impairment of cognitive functions such as learning and memory [20, 25]. In neuronal cells, it inhibits neuronal viability 10-fold more than fibrillar Aβ . In microglial cells, the current findings reveal that oligomeric Aβ (≥ 5.0 μM) is more cytotoxic than fibrillar Aβ, suggesting that Aβ oligomers play a critical role in non-neuronal toxicity, whereby may lead to glial dysfunction.
Both pro-inflammatory cytokines and oxidative damage are observed early in the progression of AD [14, 26] and can be detected prior to fibrillar Aβ deposition in AD brain . Microglial activation can also be detected in vivo in around 50% of patients with mild cognitive impairment (MCI) , suggesting microglial activation is an early affair that involves progressive damage to immune system of AD patients. Our present data illustrate the phenotypic complexity of reactive microglia. Microglial cells treated with the two conformations of Aβ showed different profile changes of morphology and inflammatory mediators, including IL-1β, TNF-α, NO and PGE2. The Aβ-elicited microglial inflammatory responses were characterized by a conformation dependent manner. These results are in accordance with the works of Heuschling and colleagues [29, 30]. They further demonstrated that the formylpeptide receptor 2 (FPR2) might mediate oAβ signaling and activate c-Jun and NF-κB pathway, which is also consistent with our current data that oAβ significantly upregulated the mRNA expression of FPR2, as well as our recent report  that Aβ oligomers can trigger a potent inflammatory response in microglia through NF-κB and JNK signaling. However, compared with their studies, our current data clearly displayed that the kinetics' profiles of time course and dose response of inflammatory mediators induced by the two forms of Aβ in microglial cells. Aβ oligomers resulted in a rapid and transient increase in IL-1β level. Compared with Aβ fibrils, they produced higher levels of TNF-α, NO, PGE2 and intracellular superoxide anion (SOA). In contrast, a higher concentration and a longer stimulating time were required for Aβ fibrils to induce microglial activation. Taken together, our present findings highlight the viewpoint that, at the early stage of AD, small diffusible oligomers activate microglia, leading to a more potent induction of inflammation, whereas fibrillar Aβ or plaques sustain the chronic inflammation at the terminal stage of AD pathogenesis. More importantly, our findings also reveal that the time course response of inflammatory mediators in microglia is correlated with the oAβ-impaired microglial phagocytosis stimulated by fibrillar Aβ.
Phagocytosis, a macrophage function critical for the uptake and degradation of infectious agents and senescent cells, contributes to the immune and inflammatory response and performs homeostatic activity in the normal CNS . Microglia are competent phagocytes and are efficient in phagocytic uptake of amyloid aggregates [1, 32, 33] and senile plaques themselves  when examined in vitro. However, the limited clearance of dysfunction of microglia is characteristic of several neurodegenerative diseases . In AD patients, the phagocytic function of peripheral blood mononuclear cells has been found to be impaired , suggesting that phagocytic function is also defective in AD.
The present study finds that an early stage component of Aβ, oligomers, is able to impair microglial phagocytic function and subsequently disturbs the capacity of terminal Aβ fibrils clearance. Its intriguing findings elucidate the mechanisms through which oAβ works: (1) oAβ stimulation downregulates the mRNA expressions of phagocytosing fAβ-related receptors such as CD36, integrin β1, as well as the classical phagocytic receptors, the Ig receptor (FcγRIII), whereas oAβ upregulates the expression of cell surface receptor genes (such as FPR2) which can induce a potent microglial proinflammatory response; (2) the prestimulation of oAβ or the inflammatory and oxidative milieu (IL-1β, LPS or t-BHP) significantly attenuates the fAβ-induced mRNA over-expression of CD36, CD47 and Itgb1 receptors. Therefore, these findings firstly provide a probable explanation for why "activated" microglia surrounding plaques lose their capacity to phagocytose Aβ deposits effectively during the terminal stage of AD brain. Furthermore, our observation also raises an intriguing question whether the initial microglial dysfunction induced by Aβ oligomers results in a decline in microglial-mediated clearance of tissue debris and microbes, e.g., viruses, bacteria, fungi, thereby, at the terminal stage of AD, increasing the risk of encephalic infectious disease, e.g., encephalitis.
It has been reported that microglia internalize oAβ through a nonsaturable, fluid phase macropinocytic mechanism that is distinct from receptor-mediated endocytosis , whereas microglia interact with fAβ through a characterized Aβ cell surface receptor complex comprising the B-class scavenger receptor CD36, α6β1 integrin, and CD47 . In the present study, the fluorescent microsphere was used as a marker of fluid phase phagocytosis. We focused not only on the changes of microglial phagocytic function after the inducement with different Aβ forms (fAβ and oAβ) in a proper order, but also on the uptaking of fAβ itself by microglial cells. Our present data reveal that both oAβ and the inflammatory and oxidative milieu (IL-1β, LPS or t-BHP) significantly attenuated the fAβ-induced over-expression of FcγRIII gene and support the notion that for fluorescent microsphere itself, microglial phagocytosis, distinct from the internalization pathways of Aβ, may work through the mechanisms mediated by the classical phagocytic receptors, the Ig receptors (FcRγI and FcγRIII) or complement receptors .
The "inflammation hypothesis" stresses that hyperactive microglia are the primary cause of AD-associated neurotoxicity. In contrast, we propose that AD is caused not only by hyperactive but also by dysfunctional microglia. Microglial cells generate potentially damaging cytokines, nitric oxide, oxygen free radicals, and arachidonic acid derivatives, which could be mediators of the so-called secondary damage . Dysfunctional microglia also show a significant reduction in the expression of their Aβ-binding receptors and Aβ-degrading enzymes, but maintain their ability to produce proinflammatory cytokines in AD [38, 39]. These cytokines may in turn act in an autocrine manner and promote Aβ production by stimulating β- and γ-secretases and/or reduce Aβ clearance by reducing expression of Aβ-binding receptors and Aβ-degrading enzymes [38, 39]. Therefore, together with our present results, we propose that, in a pathological condition like AD, oligomeric Aβ firstly triggers a rapid and potent inflammation and subsequently fibrillar Aβ sustains a chronic inflammatory environment, which suppresses the activation of phagocytic machinery, thereby affecting the ability of microglia to handle potentially toxic compounds, inhibiting clearance of fAβ and plaques, inducing a secondary immune response, and in turn aggravating brain inflammation. Antiinflammatory and antioxidative therapy can restore the functions of microglia, promote their capacity to clear Aβ, and decrease the production proinflammatory mediators, which may indeed be very helpful to delay the progression of AD.
A previous study reports that Aβ(1-42) fibrillization is a controlling factor in potentiating phagocytosis , which is attenuated by proinflammatory cytokines , and anti-inflammatory mediators, e.g., IL-4 treated microglia, enhancing the uptake and degradation of Aβ (1-42) . In this study, our results firstly reveal that microglial phagocytosis was negatively correlated with oligomeric Aβ-induced inflammatory mediators and ROS. IL-1β, LPS and t-BHP all decreased the phagocytosis of fAβ induced-microglia, which could be relieved by a nuclear factor-κB (NF-κB) inhibitor (PDTC), as well as a free radical scavenger (NAC), suggesting that impaired phagocytosis by oAβ is mediated through NF-κB signaling dependent-inflammation and oxidative stress mechanism in microglial cells. Thereby, our results support a model in which the induction of oligomeric Aβ in microglia promotes oxidative damage and autocrine proinflammatory cytokine, which contributes to glial dysregulation and suppresses activation of the phagocytic machinery at the early stage of AD.
ROS is critical for inflammatory gene expression, including iNOS, in glial cells . Microglia as a robust source of ROS increase oxidative stress and contribute to their dysregulation in AD. In this study, the elevated phagocytic response triggered by fAβ(1-42) occurred early after initial 30 min of incubation and kept at a high level for 3~6 h, but slowly declined at 12 h over time, which also indicates that the elevated ROS induced by fAβ itself may counteract with or negatively regulate the effect of fAβ-elicited phagocytosis. In addition, the pretreatment of PDTC or NAC mostly blocked the relocation of NF-κB and production of proinflammatory cytokines, whereas the same treatment did not restore the full capacity of phagocytic activity, suggesting that there may be other pathways involved in the process. Together with our current results, therefore, it is likely that there is a functionally relevant crosstalk between those different inflammatory events, e.g., secretion of ROS, cytokines, and phagocytosis.
Our findings illustrate that the exposure of microglial cells to oAβ(1-42) produces the maximal response of TNF-α, PGE2, and nitrite release at 24 h but that of IL-1β at 3 h, which supports that IL-1β is an immediate-response molecule and key immunoregulator at an early AD stage [43–45]. And the microglial proinflammatory response in AD may begin before the appearance of plaques in response to oligomeric Aβ. The intervention to prevent microglia activation should commence long before the appearance of Aβ deposits.
The present study demonstrates that Aβ oligomers induce a potent inflammatory response, and subsequently disturb microglial phagocytic function preceding Aβ fibrils formation at an early AD stage. This provides a strong support for a novel view that β-amyloid conformation as an important determinant factor encourages sequential and progressive damage to the brain's immune system at different stages of AD pathogenesis. The present study also supports that anti-inflammatory and anti-oxidative therapies may facilitate the recovery of phagocytosis, the clearance of tissue debris and microbes, and the removal of fibrillar Aβ, and eventually ameliorate the pathologies of AD brain.
Materials and methods
Dulbecco's modified Eagel's medium (DMEM), DMEM-F12, Hanks' balanced salt solution (HBSS), and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY); phenol red-free F12 medium from PromoCell (Heidelberg, Germany); and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), LPS (from Escherichia coli serotype O111: B4), tert-butyl hydroperoxide (t-BHP), N-Acetyl-L-cysteine (NAC), nitroblue tetrazolium (NBT), and hexafluoroisopropanal (HFIP) from Sigma-Aldrich (St. Louis, MO). Aβ(1-42) peptide was purchased from Quality Controlled Biochemicals, Inc. (QCB, Hopkinton, MA); Lyophilized HilyteFluor™488 labeled Aβ(1-42) peptide was provided by AnaSpec (Freemont, CA, USA). NF-κB inhibitor, pyrrolidone dithiocarbamate (PDTC) from Calbiochem (La Jolla, CA, USA); IL-1β from R & D Systems (Minneapolis, MN); and Nile red fluorospheres and AlexaFluor488-phalloidin from Molecular Probes (Eugene, OR). Primary rabbit ployclonal antibody to mouse NF-κB p65 was from Cell signaling (Berverly, MA); 4',6-diamidine-2'-phenylindole, dihydrochloride(DAPI), and Westernblot Chemiluminescent Detection System (LumiGLO system) from KPL (Gaithersburg, MD); TNF-α and IL-1β ELISA kits from BioSource (Camarillo, CA); PGE2 EIA kit from Cayman (Ann Arbor, MI); TriPure Isolation Reagent and FastStart Universal SYBR Green Master (ROX) from Roche Diagnostics GmbH (Mannheim, Germany); and RevertAid™ First Strand cDNA Synthesis Kit from Fermentas (Shenzhen, China).
BV-2 cells culture
The immortalized Murine BV-2 microglial cells were maintained in Dulbucco's modified Eagle medium (DMEM) supplemented with 5% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin, and were kept at 37°C in humidified 5% CO2/95% air. The cells were passaged every three days when growing up to 75% confluence.
Primary microglia culture
Microglia were derived from postnatal day 1 (P1) mouse brains (C57BL/6). Briefly, meninges-free cortices from P1 mice were isolated and trypsinized. Cells were plated onto tissue culture plastic in DMEM-F12 with L-glutamine containing 10% FBS and fed every three days. After 14 d, the cultures were shaken vigorously (120 min; 260 r.p.m. on a rotary shaker) to remove microglia.
Preparation of Aβ(1-42)
Aβ(1-42) was prepared as previously described [21, 46]. Briefly, lyophilized Aβ(1-42) peptide was initially monomerized by dissolving it to a final concentration of 1 mM in 100% hexafluoroisopropanal (HFIP) and separated into liquots in sterile microcentrifuge tubes. Then HFIP was evaporated under vacuum in a SpeedVac, and the peptide film was stored dessicated at -20°C until use. For the oligomers assembly, the peptide film was resuspended in dimethylsulfoxide (DMSO) to 5 mM with water bash ultrasonic for 10 min, then diluted to a final concentration of 100 μM in phenol red-free F-12 media, and incubated at 4°C for 24 h. To induce fibril formation, Aβ(1-42) was resuspended in sterile MinQ H2O and incubated for 1 week at 37°C.
Cell viability Assay--MTT assay
MTT is converted in living cells to formazan, which has a specific absorption maximum. Cells were treated with Aβ(1-42) for indicated time periods and were further incubated for 4 h after the culture medium was changed to a medium containing 0.5 mg/ml MTT. Then, they were added with solubilization solution (10% SDS, 5% isopropanol in 0.012 M HCl) and incubated at 37°C in humidified 5% CO2/95% air for overnight. The absorbance of the supernatant was measured at 570 nm on an automated microtiter plate reader. Data were expressed as the mean percentage of viable cell versus control.
Microglial cells were collected and 1 × 105 cells were plated in 24-well plates overnight. The medium was changed to serum-free DMEM, and 3 h later, the cells were incubated in the presence or absence of the revulsants (IL-1β, LPS, oAβ(1-42) or t-BHP) with or without inhibitors (PDTC or NAC) for indicated time periods. The fluorescent microspheres, as a marker of fluid phase phagocytosis, were then added to the treated cells for 30 min after having been washed in PBS containing 0.1% BSA. Cells were then fixed with 4% paraformaldehyde, and three random fields of cells (>100 cells) were counted under an inverted fluorescent microscope.
Phagocytic efficiency was determined by referring to Koenigsknecht et al. . Briefly, the phagocytic efficiency was based on a weighted average of ingested microspheres per cell. The number of cells containing microspheres, the number of microspheres per cell, and the total number of cells were counted respectively. Phagocytic efficiency(%) = (1 × X1+2 × X2+3 × X3....+n × X n )/the total number of cells × 100%. X n represents the number of cells containing n microspheres (n = 1, 2, 3, ..., up to a maximum of 6 points for more than 5 microspheres ingested per cell).
The treated cells were rinsed with PBS before being fixed in 4% paraformaldehyde (PFA) and washed again in PBS. The cells were then incubated at room temperature with 0.1% Triton X-100 buffer for 5 min and washed again in PBS. AlexaFluor488 phalloidin (1:50 diluted in PBS) was added to the coverslips and incubated at room temperature protected from light for 20 min. Finally, coverslips were then incubated with DAPI (1:1000) for double staining. The coverslips were mounted on glass slides. The association of AlexaFluor488-labeled phalloidin was viewed under an inverted fluorescent microscope.
Fluorescence labeled fAβ(1-42) phagocytosis assay
HilyteFluor™488 labeled Aβ(1-42) was aggregated according to the above fibril-forming condition. Primary microglia or BV-2 cells were plated at a density of 1 × 105 cells/well of a 24-well plate, then were pre-stimulated without or with oAβ(1-42) (1.0 μM) for 3 h or 12 h, LPS (1.0 μg/ml) for 12 h, t-BHP (100 μM) for 1 h and IL-1β (20 ng/ml) for 18 h. Stimulated or unstimulated cells were incubated with Hilyte-488 labeled fAβ(1-42) for 30 min at 37°C. Cells were washed and fixed in 4% PFA. Evaluation of Hilyte-488 labeled fAβ(1-42) phagocytosis in microglia was performed using a confocal microscope (Leica TCS SP5). A photomultiplier module was used to combine confocal with phase-contrast images to provide simultaneous views of the fluorescent fAβ and the entire cell to distinguish between phagocytosed fluorescent fAβ and fAβ adhered to cell surface. We counted intracellular fluorescent fAβ in three different experiments and analyzed 50 cells for each experiment. Data acquirement and analysis were performed with Leica Microsystems software (LAS AF Lite Version:1.8.1 build 1390).
Measurement of TNF-α, IL-1β, PGE2and nitrite levels
Microglial cells were stimulated with oligomeric and fibrillar Aβ(1-42) (1.0 μM) for 1, 2, 3, 6, 12, and 24 h. Or cells were stimulated with that of Aβ(1-42) (0.2~10 μM) for indicated time (TNF-α, NO and PGE2 for 24 h, or IL-1β for 3 h). The supernatants were collected and stored at -80°C until assays for TNF-α, IL-1β and PGE2 were performed. TNF-α, IL-1β and PGE2 levels were detected by mouse TNF-α, IL-1β ELISA kits and PGE2 EIA kit according to the procedures provided by the manufacturers. Accumulated nitrite (NaNO2) accumulation in the medium was used as an indicator of NO production as previously described . The isolated supernatants were mixed with an equal volume of Greiss reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2% phosphoric acid) and incubated at room temperature for 15 min. NaNO2 was used to generate a standard curve, and nitrite production was determined by measuring optical density at 540 nm. In the above studies with drugs (including PDTC and NAC), care was taken to ensure that cell viability was not altered under the concentrations of inhibitors used.
Modified NBT assay for superoxide anion (SOA)
A modified assay for the intracellular conversion of nitroblue tetrazolium (NBT) to formazan by superoxide anion (O2-) was used to measure the production of reactive oxygen species [48, 49]. In brief, 0.1% NBT was added to the media at the end of the treatment periods. As negative controls, BV-2 microglial cells were pretreated with 5.0 mM NAC 1 h prior to oligomeric or fibrillar Aβ(1-42) treatment. As a positive control, BV-2 cells were treated with 100 μM t-BHP for 60 min [50, 51]. After incubation for 45 min at 37°C, the treated cells were washed twice with warm PBS, then once with methanol, and air-dried. The NBT deposited inside the cells was then dissolved with 240 μl of 2 M potassium hydroxide (KOH) and 280 μl of dimethylsulfoxide (DMSO) with gentle shaking for 10 min at room temperature. The dissolved NBT solution was then transferred to a 96-well plate and absorbance was read on a microplate reader at 630 nm. Meanwhile, the cells were allowed to adhere to glass cover slips placed in a 6-well flat culture plate. After similar treatments, NBT incubation, washing and fixing with methanol were carried out. The cells containing blue formazan particles (NBT-positive cells) were pictured under a microscope.
Quantitative real-time PCR
Total RNA was extracted from the treated mouse BV-2 microglial cells using a commercially available assay (TriPure Isolation Reagent, Roche) according to the manufacturer's protocol. First-strand cDNA was synthesized with the use of 1 μg of total RNA (RevertAid™ First Strand cDNA Synthesis Kit, Fermentas). The quantitative PCR was performed with Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR Green to detect the amplification products. Reactions were as follows: 50°C for 2 minutes, 95°C for 10 minutes, and then 40 cycles of 95°C for 15 seconds followed by 60°C for 1 minute. Relative quantification of mRNA expression was calculated by the comparative cycle threshold (Ct) method after the target genes levels were normalized to expression of GAPDH housekeeping control gene for each sample. The fold difference in gene expression between treated groups was calculated as follows: fold difference = 2-ddCt, where ddCt = (Ct-target - Ct-GAPDH)treated sample - (Ct-target - Ct-GAPDH)control sample. Designed primers sequences [see Additional file 1, Table S1] were as follows: GAPDH, CD36, CD47, integrin β1 (Itgb1), scavenger receptor A (SRA), scavenger receptor B1(SRB1), RAGE, FPR2, FcγR I and FcγRIII.
The treated cells were first resuspended with cold hypotonic buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and 0.5 mM PMSF], followed by vigorous vortex for 15 s before standing at 4°C for 10 min and incubated for an additional 5 min after addition of 10% Nonidet P-40. The cytoplasmic protein was contained in the supernatant following centrifugation (6,000 × g, 4°C, 10 min). The pelleted nuclei were resuspended in cold buffer B [20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 1 mM PMSF] and incubated for 20 min on ice, and nuclear lysates were then centrifuged at 14,000 × g at 4°C for 5 min. Supernatants containing the solubilized nuclear proteins were stored at -80°C for NF-κB assay.
The treated cells were washed with ice-cold PBS and then were incubated for 20 min with lysis buffer containing 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 0.1% SDS, 0.5% (w/v) sodium deoxycholate, 1% Triton-X 100, 1 mM PMSF, 60 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 μg/ml pepstatin. Then the cells lysates were centrifuged at 12000 × g for 10 min. Nuclear and cytoplasmic extracts were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The membranes were probed with NF-κB p65 subunits antibody (1:750) to determine the efficiency of nucleocytoplasmic separation. Quantification of the band density was determined by densitometric analysis.
Data were shown by the means ± S.E. of at least three independent experiments. Statistical differences between values were determined by ANOVA followed by Tukey post hoc test, the partial correlation analyses by Pearson test. Significance level was set at P < 0.05.
We thank Dr. Mary Jo Ladu (University of Illinois at Chicago) for excellent technical assistance in identifying Aβ conformations. We extend our thanks to Mr. Huang Hongzhi for proofreading the manuscript and Mrs. Lin ling (Centre of Neurobiology of Fujian Medical University) for assistance with analysis of confocal microscopy. This work was supported by National Natural Science Grant of China (31040034), Fujian Province Natural Science Grant (2010J05063), Outstanding Young Persons' Research Program for Higher Education of Fujian Province, China (JA10123), Major Project of Fujian Science and Technology Bureau (2009D061) and Key Program of Scientific Research of Fujian Medical University (09ZD002).
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