The 4G10 monoclonal anti-phosphotyrosine antibodies were purchased from Millipore (Billerica, MA). Anti-Lyn, anti-Syk, anti-COX-2, anti-phospho-ERK, anti-ERK2 antibody, horseradish peroxidase conjugated secondary antibodies, and protein A/G PLUS-Agarose beads were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-p38, anti-p38, anti-phospho-JNK, anti-JNK, and anti-phospho-Lyn were from Cell Signaling (Beverly, MA). Anti-CD68 antibody was from Serotec (Raleigh, NC). KC ELISA, IL-6 ELISA, and MCP-1 ELISA were from R&D Systems (Minneapolis, MN). Anti-Aβ, clone 6E10, was obtained from Covance (Emeryville, CA). Anti-oligomer antibody, A11 was purchased from Invitrogen. (Camarillo, CA). DMEM/F12, Neurobasal media and B27 supplements were purchased from Invitrogen (Rockville, MD). BSA was purchased from Serologicals Corporation (Norcross, GA).

In order to generate fibrillar and oligomeric peptides for cell stimulation, Aβ1–42 was purchased from Bachem (Torrance, CA) or American Peptide (Sunnyvale, CA). Oligomers were generated as described in Chromy et al 43. Briefly, Aβ1–42 peptide was dissolved in cold hexafluoro-2-propanol (HFIP) to a final concentration of 1 mM. The peptide was aliquoted and dried under vacuum. The aliquoted peptides were stored at -80degreeC until use. For use in cell experiments, the peptide was dissolved in DMSO to a final concentration of 5 mM then diluted to 100 μM in Ham's/F12 media. The peptide oligomers were then incubated 24 hours, 4 degree C then spun 14,000 rpm, 4 degree C, 10 min. The supernatant was collected as the oligomeric Aβ peptide. Fibrils were prepared by dissolving Aβ1–42 peptides in ddH2O to a final concentration of 250 μM then incubated at 37 degree C for 1 week 44. Fibrils were resuspended with vigorous trituration prior to removing aliquots for cell stimulation. In order to assess the peptide states under our bioassay conditions, a portion of each preparation was diluted to 20 μM in DMEM/F12 and incubated an additional 48 hours, 37 degree C for subsequent Western analysis. In order to verify that the fibril concentrations employed were accurate following the 1 week fibrillization procedure, five different aliquots of prepared fibril were quantified during different days by Bradford assay to calculate molarities of the solution. This calculated molarity was compared to the predicted molarity based upon the volume of water added to the known mass of purchased peptide used. The difference between predicted molarity and mean calculated molarities from the volume of fibril assayed varied only by 2.4% (predicted 2.5 nM; calculated 2.4 nM ± 0.3). This verified that even though fibrils formed an insoluble precipitate in the solution it was being adequately resuspended for use as a stimulant.

Microglia were derived from the brains of postnatal C57BL/6J mice as described previously 45. Neurons were cultured from cortices of embryonic day 16 (E16) mice (C57BL/6J) as described previously (Sondag and Combs 2006). For co-cultures, neurons were plated at 260 cells/mm² in 48 well plates. At day 14, media was removed and replaced with Neurobasal media supplemented with B27 components containing microglia (26 cells/mm²) and 20 μM Aβ oligomers or fibrils.

Neurons were cultured from cortices of E16 mice (C57B1/6J). Briefly, meninges-free cortices were isolated, trypsinized and plated onto 0.05 mg/mL poly-L-lysine coated tissue culture wells (260 cells/mm²) for 14 days in vitro before use. Neurons were grown in Neurobasal media with glutamine and B27 supplements (Life Technologies, Rockville, MD) to consistently provide neuronal cultures able to survive for at least one month in vitro. Neuron purity was increased up to 98–99% through a transient treatment with 1 μmol/L AraC during days 1–3. Culture purity is routinely evaluated by cell counting after immunostaining to identify the neuronal cytoskeletal protein, microtubule associated protein 2 (MAP2).

Microglia were stimulated by removing them from growth media into serum-free DMEM/F12 media (2 × 10⁶cells/mL) containing Aβ oligomer or Aβ fibril. Neurons were stimulated by removing growth media and replacing it with serum free media containing Aβ oligomer or Aβ fibril. Cells were stimulated for 5 minutes or 24 hours and total cell lysates were prepared as described below, or cells were stimulated for 24 hours at 37°C and media was collected.

Sample buffer containing no SDS or β-mercaptoethanol was added to Aβ oligomers or Aβ fibrils. Unheated samples were resolved on a 15% polyacrylamide gel in the absence of SDS. Western blotting was performed as described below.

To perform immunoprecipitations, cells were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer. Lysates were then vortexed, incubated on ice for 15 min, briefly pulse sonicated, and centrifuged (10 min, 4 °C) to remove insoluble material. Primary antibody (1 μg/mg protein) was added to the equal protein amounts from supernatants and incubated 4 h at 4°C. Protein A/G beads (Santa Cruz) were added and incubated an additional 4 hr at 4°C. Beads were washed three times with lysis buffer, and immunoprecipitates were resolved and Western blotted as described below.

Western blotting of cell or tissue lysate was done as described previously 45. Briefly, ice cold RIPA buffer was used to lyse cells. Lysates were sonicated and centrifuged (14,000 × g, 4°C, 10 min) to remove insoluble material. Protein concentrations were quantitated and proteins were resolved by SDS-PAGE and transferred to PVDF membranes. Western blots were blocked and incubated in primary antibodies. Blots were washed followed by incubation with HRP-conjugated secondary antibodies. Blots were washed again followed by detection with enhanced chemiluminescence (Pierce, Rockford, IL).

Media was collected from microglia following 24 hour stimulation. Levels of mouse interleukin-6, KC, and MCP-1 in the media were determined using commercially available ELISA kits according to the manufacturer's protocol.

In order to define the peptides used in our system, we first assessed the state of our oligomer and fibril preparations. To confirm that the Aβ1–42 peptide maintained the appropriate fibrillar or oligomeric conformation throughout the duration of stimulation, we incubated the peptide in media alone at 37 degree C for 48 hours (Fig. 1A). As reported by others, the peptide forms high molecular weight SDS-stable oligomers with incubation but does not adopt a fibrillar conformation 46. The bulk of the peptide in the oligomeric preparation migrated in the molecular weight range of dimer/trimers (Fig. 1A). As expected, the fibrillar peptide in spite of increasing amounts of loaded protein, did not resolve into the separating gel and remained in the stacking portion of the gel (Fig. 1B). Upon subjecting the peptide to non-denaturing gel electrophoresis (Fig. 1C), we determined again that the fibril was too large to resolve into the separating gel. However, under these conditions the bulk of the oligomers ran as a ~24 kD species rather than the dimer/trimer that was present under the denaturing conditions, suggesting that the dimer/trimers were a result of larger multimer disassembly during the SDS-PAGE. It is likely the 24 kDa form that was observed under the non-denaturing conditions is a more representative formation of the peptide presented to cells during our experimental studies. As further evidence of the oligomeric versus fibrillar properties of our two preparations we performed dot blot Westerns using anti-Aβ antibody, 6E10, versus anti-oligomer antibody, A11 47, to verify that minimal oligomeric peptide was in our fibrillar preparation (Fig. 1D).

We next compared the ability of fibrillar and oligomeric forms of Aβ1–42 to stimulate primary microglia by treating our cultures with increasing doses of the peptides. Prior work has demonstrated that fibrillar Aβ stimulates a specific tyrosine kinase-based activation response in microglia leading to acquisition of a reactive, neurotoxic phenotype 484950. In order to determine whether Aβ oligomers stimulate a comparable response, we first assayed changes in protein phosphotyrosine levels following stimulation. Western blot analysis revealed a dose dependent and qualitatively similar increase in protein phosphotyrosine levels, an indirect measure of tyrosine kinase activity. Interestingly, the oligomers (Aβ_o) were a qualitatively more potent stimulant than the fibrils (Aβ_f) (Fig. 1E).

To continue comparing the activation profile, we tested whether Aβ-mediated stimulation of tyrosine kinases led to subsequent activation of MAPKs since we, as well as others, have demonstrated that Aβ_f also stimulate increased MAP kinase activities in microglia 49515253. Aβ_f stimulated increased levels of the active, phosphorylated form of extracellular signal-regulated kinase (ERK) but not p38 or c-Jun N-terminal kinase (JNK) (Fig. 2A). In contrast, Aβ_o stimulated increased activity of p38 MAPK (Fig. 2A). To further characterize the signaling response associated with Aβ stimulation, we sought to identify proteins that were tyrosine phosphorylated upon stimulation. Previous studies demonstrated the Src family, non-receptor tyrosine kinases Lyn and Syk regulate MAPK activation during Aβ stimulation of monocytes 49. Therefore, we assessed activation of these specific tyrosine kinases via assaying their phosphorylation state upon stimulation. As expected, levels of the phosphorylated form of both Lyn and Syk increased upon Aβ stimulation, however the activation was specific to the oligomeric form of the peptide (Fig. 2A, B). Collectively, these data demonstrate that the different peptide conformations stimulate similar but distinct signaling responses in microglia.

Based upon the differences observed in the stimulated signaling response we predicted that the resultant, reactive phenotypes would also be unique. An abundance of literature has described the reactive, secretory microglial phenotype stimulated by Aβ_f while little is known regarding Aβ_o effects on these cells 3940. Our signaling results above suggested that the resultant phenotypes would differ between Aβ_o and Aβ_f stimulations. We first compared protein levels of two typical reactive microglial marker proteins, cyclooxygenase-2 (COX-2) and CD68. In addition, we assessed protein levels of CD45, a tyrosine phosphatase demonstrating increased microglial-like immunoreactivity in AD brains 54. Oligomeric but not fibrillar Aβ stimulated increased protein levels of both COX-2 and CD68 following 24 hr stimulation, while neither form of the peptide stimulated a change in CD45 levels (Fig. 2C). Based upon this difference, we predicted that the two peptide conformations would stimulate distinct secretory profiles. Using results from a preliminary cytokine/chemokine array profile (Fig. 3A) we elected to quantify secretion of interleukin-6 (IL-6), keratinocyte chemoattractant chemokine (KC, mouse homolog to IL-8), and monocyte chemoattractant protein-1 (MCP-1) following stimulation. Both Aβ_o and Aβ_f stimulated increased IL-6 and KC secretion. However, Aβ_o stimulated a significantly greater amount of IL-6 (Fig. 3A) while Aβ_f stimulated a significantly greater amount of KC (Fig 3B). Interestingly, Aβ_o stimulated a decrease in MCP-1 secretion while Aβ_f had no effect (Fig. 3C). In agreement with the differences observed in stimulated signaling response, these data confirm that the resultant reactive, secretory phenotypes stimulated by Aβ_o or Aβ_f are distinct.

We next compared whether the two peptide conformations stimulated microglia to acquire a neurotoxic phenotype. Several prior reports have demonstrated that Aβ_f stimulate microglia to become neurotoxic 364955 while this remains unclear for Aβ_o. Using our working concentrations responsible for the stimulatory effects described above we first verified that neither Aβ_o nor Aβ_f were toxic to microglia (Fig. 4A). We then tested the direct toxicity of the oligomers and fibrils to primary cortical neuron cultures as there have been conflicting reports about the toxicity of the oligomers in vitro 4356. After quantitating released LDH into the media it appeared that the oligomers did not stimulate neuronal toxicity under our assay conditions. However, the same assay demonstrated significant fibril dependent toxicity in a dose-dependent manner, as expected (Fig. 4B). Because we have identified several proinflammatory molecules that are secreted by microglia in response to Aβ1–42 stimulation, we tested whether the different conformations of the peptide stimulated microglia to secrete neurotoxic species when they were co-cultured with neurons. As expected, the fibrils stimulated microglia to increase neuron death during a 24 hour stimulation in mixed cultures above that induced by direct neuronal toxicity of the peptide alone (Fig. 4C). Although the oligomers were not toxic alone, as determined by LDH release quantitation, they did stimulate neuronal death in the presence of microglia (Fig. 4C). However, microglia can basally secrete LDH that could act as a confound in our LDH quantitation data and LDH release may not accurately identify cells dying by means other than necrotic death. Therefore, the ability of oligomeric Aβ to kill neurons independent and in the presence of microglia was again examined using a cell counting measure to determine viability. In addition, a longer time period of stimulation, 48 hours, was employed. These data demonstrated that oligomeric peptide alone was toxic to neurons at this longer time period and stimulated a greater degree of cell death when microglia were co-cultured with the neurons (Fig. 4D). This suggests that the longer time period of stimulation or the different assay of viability are required to accurately determine Aβo effects on neurons in our cultures. Collectively, these data demonstrated that the two peptide conformations stimulate distinct effects on neuron viability both directly and indirectly through microglial activation.