Development of ß-sheet structure in Aß aggregation intermediates diminishes exposed hydrophobic surface area and enhances proinflammatory activity.

Three decades of research, both in vitro and in vivo, have demonstrated the conformational heterogeneity that is displayed by the amyloid ß peptide (Aß) in Alzheimer's disease (AD). Understanding the distinct properties between Aß conformations and how conformation may impact cellular activity remain open questions, yet still continue to provide new insights into protein misfolding and aggregation. In particular, there is interest in the group of soluble oligomeric prefibrillar Aß species comprising lower molecular weight oligomers up to larger protofibrils. In the current study, a number of strategies were utilized to separate Aß protofibrils and oligomers and show that the smaller Aß oligomers have a much different conformation than Aß protofibrils. The differences were consistent for both Aß40 and Aß42. Protofibrils bound thioflavin T to a greater extent than oligomers, and were highly enriched in ß-sheet secondary structure. Aß oligomers possessed a more open structure with significant solvent exposure of hydrophobic domains as determined by tryptophan fluorescence and bis-ANS binding, respectively. The protofibril-selective antibody AbSL readily discerned conformational differences between protofibrils and oligomers. The more developed structure for Aß protofibrils ultimately proved critical for provoking the release of tumor necrosis factor α from microglial cells. The findings demonstrated a dependency on ß-sheet structure for soluble Aß aggregates to cause a microglial inflammatory response. The Aß aggregation process yields many conformationally-varied species with different levels of ß-structure and exposed hydrophobicity. The conformation elements likely determine biological activity and pathogenicity.

Visualization of Ceramide-Associated Proteins in Ceramide-Rich Platforms Using a Cross-Linkable Ceramide Analog and Proximity Ligation Assays With Anti-ceramide Antibody.

Ceramide-rich platforms (CRPs) mediate association of proteins with the sphingolipid ceramide and may regulate protein interaction in membrane contact sites to the cytoskeleton, organelles, and infectious pathogens. However, visualization of ceramide association to proteins is one of the greatest challenges in understanding the cell biology of ceramide. Here we introduce a novel labeling technique for ceramide-associated proteins (CAPs) by combining photoactivated cross-linking of a bioorthogonal and bifunctional ceramide analog, pacFACer with proximity ligation assays (PLAs). pacFACer cross-linked to CAPs is covalently attached to a fluorophore using click chemistry. PLAs use antibodies to: (1) the candidate CAP and the fluorophore (PLA1); and (2) the CAP and ceramide (PLA2). PLA1 shows the subcellular localization of a particular CAP that is cross-linked to pacFACer, while PLA2 tests if the cross-linked CAP forms a complex with endogenous ceramide. Two proteins, tubulin and voltage-dependent anion channel 1 (VDAC1), were cross-linked to pacFACer and showed PLA signals for a complex with ceramide and pacFACer, which were predominantly colocalized with microtubules and mitochondria, respectively. Binding of tubulin and VDAC1 to ceramide was confirmed by coimmunoprecipitation assays using anti ceramide antibody. Cross-linking to pacFACer was confirmed using click chemistry-mediated attachment of biotin and streptavidin pull-down assays. Inhibition of ceramide synthases with fumonisin B1 (FB1) reduced the degree of pacFACer cross-linking and complex formation with ceramide, while it was enhanced by amyloid beta peptide (Aß). Our results show that endogenous ceramide is critical for mediating cross-linking of CAPs to pacFACer and that a combination of cross-linking with PLAs (cross-link/PLA) is a novel tool to visualize CAPs and to understand the regulation of protein interaction with ceramide in CRPs.

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

UNLABELLED: 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 Alzheimer's disease. Although fibrillar amyloid ß (Aß)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP(-/-)) microglial cultures, oligomeric Aß was unable to stimulate increased secretion from mAPP(-/-) cells. This was consistent with an ability of oligomeric Aß to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aß 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 Aß 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 Alzheimer's disease (AD) brains is the accumulation of amyloid ß (Aß) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aß stimulation of microglial activation is one source of brain inflammatory changes during disease. Aß 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 Aß 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 Aß production to drive the microgliosis associated with AD brains.

CD47 does not mediate amyloid-ß(1-42) protofibril-stimulated microglial cytokine release.

Neuroinflammation triggered by accumulation of amyloid-ß protein (Aß) is a significant component of the Alzheimer's disease (AD) brain. Senile plaques composed of Aß attract and activate microglia cells resulting in cytokine secretion and a proinflammatory environment. The mechanism by which Aß activates microglia is complex and involves numerous cellular components. One receptor potentially involved in Aß recognition and the ensuing microglia proinflammatory response is CD47. Since there is significant interest in soluble aggregated Aß species, we sought to determine if CD47 plays a key role in microglia cytokine release stimulated by soluble Aß(1-42) protofibrils. Pretreatment of primary murine microglia with the CD47 antagonist peptide 4N1K significantly and potently inhibited both tumor necrosis factor-α (TNFα) and interleukin-1ß (IL-1ß) secretion stimulated by Aß(1-42) protofibrils. 4N1K displayed toxicity to the microglia but only at concentrations much higher than the observed inhibition. Surprisingly, 4N1K also potently inhibited TNFα secretion triggered by lipopolysaccharide which is not known to signal through CD47. Treatment of the microglia with a neutralizing anti-CD47 antibody failed to block the Aß protofibril response even though comparable samples were completely inhibited by 4N1K. Finally, Aß(1-42) protofibrils stimulated similar levels of secreted TNFα production in both wild-type and CD47(-/-) microglia and 4N1K still potently inhibited the Aß protofibril response even in the CD47(-/-) microglia. The overall findings demonstrated that the microglial proinflammatory response to Aß(1-42) protofibril is not dependent on CD47 and that 4N1K exhibits CD47-independent inhibitory activity.

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

Accumulation of aggregated amyloid-ß protein (Aß) is an important feature of Alzheimer's disease. There is significant interest in understanding the initial steps of Aß aggregation due to the recent focus on soluble Aß oligomers. In vitro studies of Aß 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 Aß across a broad range of sizes. We have observed the presence of these fibrillar elements at very early stages of Aß incubation. Using a dot blot assay, OC-reactivity was found in size exclusion chromatography (SEC)-purified Aß(1-42) monomer fractions immediately after isolation (early-stage). The OC-reactivity was not initially observed in the same fractions for Aß(1-40) or the aggregation-restricted Aß(1-42) L34P but was detected within 1-2weeks of incubation. Stability studies demonstrated that early-stage OC-positive Aß(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 Aß(1-42) solution at 4°C. Within 6-8days the OC-positive Aß42 aggregates were resistant to SDS denaturation. The progression to, and development of, SDS resistance for Aß(1-42) occurred prior to thioflavin T fluorescence. In contrast, Aß(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 Aß aggregation and suggest that it may be possible to target early-stage aggregates before they develop significant stability.