Inflammatory mechanisms in neurodegeneration.

This review discusses the profound connection between microglia, neuroinflammation, and Alzheimer's disease (AD). Theories have been postulated, tested, and modified over several decades. The findings have further bolstered the belief that microglia-mediated inflammation is both a product and contributor to AD pathology and progression. Distinct microglia phenotypes and their function, microglial recognition and response to protein aggregates in AD, and the overall role of microglia in AD are areas that have received considerable research attention and yielded significant results. The following article provides a historical perspective of microglia, a detailed discussion of multiple microglia phenotypes including dark microglia, and a review of a number of areas where microglia intersect with AD and other pathological neurological processes. The overall breadth of important discoveries achieved in these areas significantly strengthens the hypothesis that neuroinflammation plays a key role in AD. Future determination of the exact mechanisms by which microglia respond to, and attempt to mitigate, protein aggregation in AD may lead to new therapeutic strategies.

Aß42 Protofibrils Interact with and Are Trafficked through Microglial-Derived Microvesicles.

Microvesicles (MVs) and exosomes comprise a class of cell-secreted particles termed extracellular vesicles (EVs). These cargo-holding vesicles mediate cell-to-cell communication and have recently been implicated in neurodegenerative diseases such as Alzheimer's disease (AD). The two types of EVs are distinguished by the mechanism of cell release and their size, with the smaller exosomes and the larger MVs ranging from 30 to 100 nm and 100 nm to 1 µm in diameter, respectively. MV numbers are increased in AD and appear to interact with amyloid-ß peptide (Aß), the primary protein component of the neuritic plaques in the AD brain. Because microglial cells play such an important role in AD-linked neuroinflammation, we sought to characterize MVs shed from microglial cells, better understand MV interactions with Aß, and determine whether internalized Aß may be incorporated into secreted MVs. Multiple strategies were used to characterize MVs shed from BV-2 microglia after ATP stimulation. Confocal images of isolated MVs bound to fluorescently labeled annexin-V via externalized phosphatidylserine revealed a polydisperse population of small spherical structures. Dynamic light scattering measurements yielded MV diameters ranging from 150 to 600 nm. Electron microscopy of resin-embedded MVs cut into thin slices showed well-defined uranyl acetate-stained ring-like structures in a similar diameter range. The use of a fluorescently labeled membrane insertion probe, NBD C6-HPC, effectively tracked MVs in binding experiments, and an Aß ELISA confirmed a strong interaction between MVs and Aß protofibrils but not Aß monomers. Despite the lesser monomer interaction, MVs had an inhibitory effect on monomer aggregation. Primary microglia rapidly internalized Aß protofibrils, and subsequent stimulation of the microglia with ATP resulted in the release of MVs containing the internalized Aß protofibrils. The role of MVs in neurodegeneration and inflammation is an emerging area, and further knowledge of MV interaction with Aß may shed light on extracellular spread and influence on neurotoxicity and neuroinflammation.

Amyloid-ß42 protofibrils are internalized by microglia more extensively than monomers.

One pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid-ß peptide (Aß) in the affected brain. While there are numerous deleterious effects of Aß accumulation, there is general agreement that a sustained inflammatory response to aggregated Aß 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 Aß activate a microglia-mediated inflammatory response. One component of the response involves internalization of extracellular Aß, and this process is likely very sensitive to Aß structure. In this study we analyzed the proclivity of microglia for internalization of Aß42 monomers and protofibrils using fluorescently-labeled Aß. Both Aß42 species were labeled directly via amino linkage with an Alexa Fluor 488 tetrafluorophenyl ester (AF488-TFP) and then isolated individually by chromatography. Aß42 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-Aß42 protofibrils rapidly and in significant amounts compared to AF488-Aß42 monomers. Microglial internalization of protofibrils was dependent on time and concentration, and corresponded with tumor necrosis factor α secretion. In competition studies, unlabeled Aß42 protofibril internalization, detected by immunostaining, did not diminish AF488-protofibril uptake. Internalized AF488-Aß42 protofibrils were found widely dispersed in the cytosol with some lysosomal accumulation but little degradation. These studies highlight the sensitivity that microglia exhibit to Aß structure in the internalization process and emphasize their affinity for soluble Aß protofibrils.

Amyloid-ß(1-42) protofibrils formed in modified artificial cerebrospinal fluid bind and activate microglia.

Soluble aggregated forms of amyloid-ß protein (Aß) have garnered significant attention recently for their role in Alzheimer's disease (AD). Protofibrils are a subset of these soluble species and are considered intermediates in the aggregation pathway to mature Aß fibrils. Biological studies have demonstrated that protofibrils exhibit both toxic and inflammatory activities. It is important in these in vitro studies to prepare protofibrils using solution conditions that are appropriate for cellular studies as well as conducive to biophysical characterization of protofibrils. Here we describe the preparation and characterization of Aß(1-42) protofibrils in modified artificial cerebrospinal fluid (aCSF) and demonstrate their prominent binding and activation of microglial cells. A simple phosphate/bicarbonate buffer system was prepared that maintained the ionic strength and cell compatibility of F-12 medium but did not contain numerous supplements that interfere with spectroscopic analyses of Aß protofibrils. Reconstitution of Aß(1-42) in aCSF and isolation with size exclusion chromatography (SEC) revealed curvilinear ß-sheet protofibrils <100 nm in length and hydrodynamic radii of 21 nm. Protofibril concentration determination by BCA assay, which was not possible in F-12 medium, was more accurately measured in aCSF. Protofibrils formed and isolated in aCSF, but not monomers, markedly stimulated TNFα production in BV-2 and primary microglia and bound in significant amounts to microglial membranes. This report demonstrates the suitability of a modified aCSF system for preparing SEC-isolated Aß(1-42) protofibrils and underscores the unique ability of protofibrils to functionally interact with microglia.

Isolated amyloid-ß(1-42) protofibrils, but not isolated fibrils, are robust stimulators of microglia.

Senile plaques composed of amyloid-ß protein (Aß) are an unshakable feature of the Alzheimer's 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 Aß aggregates. The observations that microglia activation in vivo is selectively stimulated by distinct Aß deposits led us to examine what specific form of Aß is the most effective proinflammatory mediator in vitro. We report here that soluble prefibrillar species of Aß(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 Aß(1-42) in NaOH followed by dilution into F-12 media and isolation with size exclusion chromatography (SEC) revealed classic curvilinear ß-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 Aß(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 Aß(1-40) protofibrils exhibited significantly less activity than concentration-matched Aß(1-42). This report is the first to demonstrate microglial activation by SEC-purified protofibrils, and the overall findings indicate that small, soluble Aß(1-42) protofibrils induce much greater microglial activation than mature insoluble fibrils.