Michael R. Nichols

Toll‐like receptors 2 and 4 mediate Aβ(1–42) activation of the innate immune response in a human monocytic cell line

The primary molecules for mediating the innate immune response are the Toll‐like family of receptors (TLRs). Recent work has established that amyloid‐beta (Aβ) fibrils, the primary components of senile plaques in Alzheimer’s disease (AD), can interact with the TLR2/4 accessory protein CD14. Using antibody neutralization assays and tumor necrosis factor alpha release in the human monocytic THP‐1 cell line, we determined that both TLR2 and TLR4 mediated an inflammatory response to aggregated Aβ(1–42). This was in contrast to exclusive TLR ligands lipopolysaccharide (LPS) (TLR4) and tripalmitoyl cysteinyl seryl tetralysine (Pam3CSK4) (TLR2). Atomic force microscopy imaging showed a fibrillar morphology for the proinflammatory Aβ(1–42) species. Pre‐treatment of the cells with 10 μg/mL of a TLR2‐specific antibody blocked ∼50% of the cell response to fibrillar Aβ(1–42), completely blocked the Pam3CSK4 response, and had no effect on the LPS‐induced response. A TLR4‐specific antibody (10 μg/mL) blocked ∼35% of the cell response to fibrillar Aβ(1–42), completely blocked the LPS response, and had no effect on the Pam3CSK4 response. Polymyxin B abolished the LPS response with no effect on Aβ(1–42) ruling out bacterial contamination of the Aβ samples. Combination antibody pre‐treatments indicated that neutralization of TLR2, TLR4, and CD14 together was much more effective at blocking the Aβ(1–42) response than the antibodies used alone. These data demonstrate that fibrillar Aβ(1–42) can trigger the innate immune response and that both TLR2 and TLR4 mediate Aβ‐induced tumor necrosis factor alpha production in a human monocytic cell line.

Isolated Amyloid-β(1–42) Protofibrils, But Not Isolated Fibrils, Are Robust Stimulators of Microglia

Senile plaques composed of amyloid-β protein (Aβ) are an unshakable feature of the Alzheimers disease (AD) brain. Although there is significant debate on the role of the plaques in AD progression, there is little disagreement on their role in stimulating a robust inflammatory response within the context of the disease. Significant inflammatory markers such as activated microglia and cytokines are observed almost exclusively surrounding the plaques. However, recent evidence suggests that the plaque exterior may contain a measurable level of soluble Aβ aggregates. The observations that microglia activation in vivo is selectively stimulated by distinct Aβ deposits led us to examine what specific form of Aβ is the most effective proinflammatory mediator in vitro. We report here that soluble prefibrillar species of Aβ(1-42) were better than fibrils at inducing microglial tumor necrosis factor α (TNFα) production in either BV-2 and primary murine microglia. Reconstitution of Aβ(1-42) in NaOH followed by dilution into F-12 media and isolation with size exclusion chromatography (SEC) revealed classic curvilinear β-sheet protofibrils 100 nm in length. The protofibrils, but not monomers, markedly activated BV-2 microglia. Comparisons were also made between freshly isolated protofibrils and Aβ(1-42) fibrils prepared from SEC-purified monomer. Surprisingly, while isolated fibrils had a much higher level of thioflavin T fluorescence per mole, they were not effective at stimulating either primary or BV-2 murine microglia compared to protofibrils. Furthermore, SEC-isolated Aβ(1-40) protofibrils exhibited significantly less activity than concentration-matched Aβ(1-42). This report is the first to demonstrate microglial activation by SEC-purified protofibrils, and the overall findings indicate that small, soluble Aβ(1-42) protofibrils induce much greater microglial activation than mature insoluble fibrils.

Amyloid-(β(1―42) Fibrillar Precursors Are Optimal for Inducing Tumor Necrosis Factor-α Production in the THP-1 Human Monocytic Cell Line

Pathological studies have determined that fibrillar forms of amyloid-beta protein (Abeta) comprise the characteristic neuritic plaques in Alzheimers disease (AD). These studies have also revealed significant inflammatory markers such as activated microglia and cytokines surrounding the plaques. Although the plaques are a hallmark of AD, they are only part of an array of Abeta aggregate morphologies observed in vivo. Interestingly, not all of these Abeta deposits provoke an inflammatory response. Since structural polymorphism is a prominent feature of Abeta aggregation both in vitro and in vivo, we sought to clarify which Abeta morphology or aggregation species induces the strongest proinflammatory response using human THP-1 monocytes as a model system. An aliquot of freshly reconstituted Abeta(1-42) in sterile water (100 microM, pH 3.6) did not effectively stimulate the cells at a final Abeta concentration of 15 microM. However, quiescent incubation of the peptide at 4 degrees C for 48-96 h greatly enhanced its ability to induce tumor necrosis factor-alpha (TNFalpha) production, the level of which surprisingly declined upon further aggregation. Imaging of the Abeta(1-42) aggregation solutions with atomic force microscopy indicated that the best cellular response coincided with the appearance of fibrillar structures, yet conditions that accelerated or increased the level of Abeta(1-42) fibril formation such as peptide concentration, temperature, or reconstitution in NaOH/PBS at pH 7.4 diminished its ability to stimulate the cells. Finally, depletion of the Abeta(1-42) solution with an antibody that recognizes fibrillar oligomers dramatically weakened the ability to induce TNFalpha production, and size-exclusion separation of the Abeta(1-42) solution provided further characterization of an aggregated species with proinflammatory activity. The findings suggested that an intermediate stage Abeta(1-42) fibrillar precursor is optimal for inducing a proinflammatory response in THP-1 monocytes.

The influence of gold surface texture on microglia morphology and activation

Microglial cells play a critical role in the propagation of neuroinflammation in the central nervous system. Microglia sense and respond to environmental signals including chemical, physical and biological cues from the surrounding cell/tissue components. In this project, our goal was to examine the effects of surface texture on BV-2 microglia morphology and function by comparing flat and nanoporous gold (np-Au) surfaces to the more conventional glass. The biocompatibility of np-Au with microglia was evaluated using functional cell assays and high resolution imaging with scanning electron microscopy (SEM). Microglia seeded on glass, ultra-flat gold (UF-Au), ultra-thin (UT) np-Au and np-Au monolith were adherent to all surfaces and their viability was not compromised as assessed by multiple toxicity assays. SEM revealed detailed morphological characteristics of adherent microglia and indicated few dramatic changes as a result of the different surfaces. Microglia proliferation was hampered by np-Au monolith but less by UT np-Au and not at all on UF-Au or glass. Microglial activation, measured by tumor necrosis factor α (TNFα) production, was fully functional (and equivalent) on all gold surfaces compared to glass. The present findings should help further the understanding of basic microglia biology on textured surfaces and more fully evaluate np-Au as a multi-functional biocompatible material. The knowledge obtained and technology developed will have a significant impact in the fabrication of nanoelectronic devices, chemical sensor development, porous nanostructured materials for BioMEMs/NEMs integration, and functional biomaterial coatings for drug delivery.

APP Regulates Microglial Phenotype in a Mouse Model of Alzheimer's Disease.

Prior work suggests that amyloid precursor protein (APP) can function as a proinflammatory receptor on immune cells, such as monocytes and microglia. Therefore, we hypothesized that APP serves this function in microglia during Alzheimers disease. Although fibrillar amyloid β (Aβ)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP−/−) microglial cultures, oligomeric Aβ was unable to stimulate increased secretion from mAPP−/− cells. This was consistent with an ability of oligomeric Aβ to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aβ produced less microgliosis in mAPP−/− mice compared with wild-type mice. The mAPP−/− mice crossed to an APP/PS1 transgenic mouse line demonstrated reduced microgliosis and cytokine levels and improved memory compared with wild-type mice despite robust fibrillar Aβ plaque deposition. These data define a novel function for microglial APP in regulating their ability to acquire a proinflammatory phenotype during disease. SIGNIFICANCE STATEMENT A hallmark of Alzheimers disease (AD) brains is the accumulation of amyloid β (Aβ) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aβ stimulation of microglial activation is one source of brain inflammatory changes during disease. Aβ is a cleavage product of the ubiquitously expressed amyloid precursor protein (APP) and is able to self-associate into a wide variety of differently sized and structurally distinct multimers. In this study, we demonstrate both in vitro and in vivo that nonfibrillar, oligomeric forms of Aβ are able to interact with the parent APP protein to stimulate microglial activation. This provides a mechanism by which metabolism of APP results in possible autocrine or paracrine Aβ production to drive the microgliosis associated with AD brains.

Probing the amyloid-β(1–40) fibril environment with substituted tryptophan residues

A signature feature of Alzheimers disease is the accumulation of plaques, composed of fibrillar amyloid-beta protein (Abeta), in the brain parenchyma. Structural models of Abeta fibrils reveal an extensive beta-sheet network with a hydrophobic core extending throughout the fibril axis. In this study, phenylalanines in the Abeta(1-40) sequence were substituted with tryptophan residues at either position 4 (F4W) or 19 (F19W) to probe the fibril environment. The F4W substitution did not alter self-assembly kinetics, while the F19W change slightly lengthened the lag phase without hindering fibril formation. The tryptophan fluorescence of Abeta(1-40) F19W, but not Abeta(1-40) F4W, underwent a marked blue shift during fibril formation and this shift was temporally correlated with thioflavin T binding. Isolated Abeta(1-40) F19W fibrils exhibited the largest fluorescence blue shifts consistent with W19 insertion into the Abeta(1-40) fibril inner core and direct probing of the substantially hydrophobic environment therein.

Oligomeric amyloid-β(1–42) induces THP-1 human monocyte adhesion and maturation

Amyloid-beta (Abeta) is a naturally occurring 40- or 42-residue peptide fragment with a primary role in Alzheimers disease (AD). Aggregated Abeta accumulates as both dense core plaques and diffuse deposits in the brains of AD patients. Abeta plaques are surrounded by activated microglia, some of which are believed to be derived from peripheral blood monocytes that have infiltrated the central nervous system and differentiated into phagocytes in response to Abeta. We have modeled this process using THP-1 human monocytes and found Abeta(1-42) to be as effective as phorbol myristate acetate at differentiating THP-1 monocytes based on cell adhesion, fibronectin binding, CD11b cell-surface expression, and morphological changes. Cell adhesion studies and atomic force microscopy imaging revealed an inverse correlation between Abeta(1-42)-induced monocyte maturation and aggregation progression. Freshly reconstituted Abeta(1-42) solutions were the most effective, yet continued aggregation reduced, and eventually abolished, the ability to induce monocyte adhesion. Abeta(1-40), lower aggregation concentrations of Abeta(1-42), and an aggregation-restricted Abeta(1-42) L34P mutant had little effect on monocyte adhesion under the same conditions as Abeta(1-42). These findings implicated an oligomeric, but not monomeric or fibrillar, Abeta(1-42) aggregation species in the monocyte maturation process. The rapidly-formed Abeta(1-42) oligomers were distinct from Abeta-derived diffusible ligands which did not elicit significant THP-1 monocyte adhesion. These data demonstrate that a specific oligomeric Abeta(1-42) aggregation species can potently initiate the THP-1 monocyte maturation process.

Amyloid-β(1-42) protofibrils stimulate a quantum of secreted IL-1β despite significant intracellular IL-1β accumulation in microglia.

Neuroinflammation is a characteristic feature of the Alzheimers disease (AD) brain. Significant inflammatory markers such as activated microglia and cytokines can be found surrounding the extracellular senile plaques predominantly composed of amyloid-β protein (Aβ). Several innate immune pathways, including Toll-like receptors (TLRs) and the NLRP3 inflammasome, have been implicated in AD inflammation. Aβ plays a primary role in activating these pathways which likely contributes to the progressive neurodegeneration in AD. In order to better understand the complexities of this interaction we investigated the inflammatory response of primary microglia to Aβ(1-42) protofibrils. Aβ(1-42) protofibrils triggered a time- and MyD88-dependent process that produced tumor necrosis factor alpha (TNFα) and interleukin-1β (IL-1β) mRNA, and intracellular pro and mature forms of IL-1β protein. The accumulation of both IL-1β forms indicated that Aβ(1-42) protofibrils were able to prime and activate the NLRP3 inflammasome. Surprisingly, Aβ-induced accumulation of intracellular mature IL-1β did not translate into greater IL-1β secretion. Instead, we found that Aβ elicited a quantized burst of secreted IL-1β and this process occurred even prior to Aβ priming of the microglia suggesting a basal level of either pro or mature IL-1β in the cultured primary microglia. The IL-1β secretion burst was rapid but not sustained, yet could be re-evoked with additional Aβ stimulation. The findings from this study demonstrated multiple sites of IL-1β regulation by Aβ(1-42) protofibrils including TLR/MyD88-mediated priming, NLRP3 inflammasome activation, and modulation of the IL-1β secretory process. These results underscore the wide-ranging effects of Aβ on the innate immune response.

Stability of early-stage amyloid-β(1-42) aggregation species.

Accumulation of aggregated amyloid-β protein (Aβ) is an important feature of Alzheimers disease. There is significant interest in understanding the initial steps of Aβ aggregation due to the recent focus on soluble Aβ oligomers. In vitro studies of Aβ aggregation have been aided by the use of conformation-specific antibodies which recognize shape rather than sequence. One of these, OC antiserum, recognizes certain elements of fibrillar Aβ across a broad range of sizes. We have observed the presence of these fibrillar elements at very early stages of Aβ incubation. Using a dot blot assay, OC-reactivity was found in size exclusion chromatography (SEC)-purified Aβ(1-42) monomer fractions immediately after isolation (early-stage). The OC-reactivity was not initially observed in the same fractions for Aβ(1-40) or the aggregation-restricted Aβ(1-42) L34P but was detected within 1-2weeks of incubation. Stability studies demonstrated that early-stage OC-positive Aβ(1-42) aggregates were resistant to 4M urea or guanidine hydrochloride but sensitive to 1% sodium dodecyl sulfate (SDS). Interestingly, the sensitivity to SDS diminished over time upon incubation of the SEC-purified Aβ(1-42) solution at 4°C. Within 6-8days the OC-positive Aβ42 aggregates were resistant to SDS denaturation. The progression to, and development of, SDS resistance for Aβ(1-42) occurred prior to thioflavin T fluorescence. In contrast, Aβ(1-40) aggregates formed after 6days of incubation were sensitive to both urea and SDS. These findings reveal information on some of the earliest events in Aβ aggregation and suggest that it may be possible to target early-stage aggregates before they develop significant stability.

Amyloid-β42 protofibrils are internalized by microglia more extensively than monomers

One pathological hallmark of Alzheimers disease (AD) is the accumulation of amyloid-β peptide (Aβ) in the affected brain. While there are numerous deleterious effects of Aβ accumulation, there is general agreement that a sustained inflammatory response to aggregated Aβ contributes to progressive neurodegeneration in AD and microglial cells play a significant role in this process. Our laboratory and others have shown that small soluble aggregates of Aβ activate a microglia-mediated inflammatory response. One component of the response involves internalization of extracellular Aβ, and this process is likely very sensitive to Aβ structure. In this study we analyzed the proclivity of microglia for internalization of Aβ42 monomers and protofibrils using fluorescently-labeled Aβ. Both Aβ42 species were labeled directly via amino linkage with an Alexa Fluor 488 tetrafluorophenyl ester (AF488-TFP) and then isolated individually by chromatography. Aβ42 protofibrils retained their size and morphological properties after labeling but monomers had a much higher stoichiometry of labeling compared to protofibrils. Primary murine microglia internalized AF488-Aβ42 protofibrils rapidly and in significant amounts compared to AF488-Aβ42 monomers. Microglial internalization of protofibrils was dependent on time and concentration, and corresponded with tumor necrosis factor α secretion. In competition studies, unlabeled Aβ42 protofibril internalization, detected by immunostaining, did not diminish AF488-protofibril uptake. Internalized AF488-Aβ42 protofibrils were found widely dispersed in the cytosol with some lysosomal accumulation but little degradation. These studies highlight the sensitivity that microglia exhibit to Aβ structure in the internalization process and emphasize their affinity for soluble Aβ protofibrils.