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We prepared monomers, oligomers, and fibrils of Aβ, which promoted the interaction between NLRP3 and each form of Aβ and analyzed the interaction between NLRP3 and ASC induced by each form of Aβ in a cell-free system with the amplified luminescent proximity homogeneous assay. We also confirmed the physiological relevance in a cell-based assay using human embryonic kidney 293T cells and human peripheral mononuclear cells.

Monomers, oligomers, and fibrils of Aβ were successfully prepared. Aβ oligomers and fibrils interacted with NLRP3. Aβ oligomers and fibrils induced the interaction between NLRP3 and ASC. However, Aβ monomers did not interact with NLRP3 or induce interaction between NLRP3 and ASC in the cell-free system, and IL-1β was not secreted according to the cell-based assay.

We previously developed the NLRP3 inflammasome in a cell-free system to detect intrinsic and extrinsic directly interacting ligands [5]. The NALP3 inflammasome was reported to be a sensor of phagocytosed Aβ [6]; however, Aβ did not induce an interaction with NLRP3 or apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) in the cell-free system [5].

Currently, numerous ligands, including Aβ, have been reported to activate the NLRP3 inflammasome; however, none were confirmed at the molecular level in previous reports. Thus, the purpose of our study was to clarify whether Aβ oligomers directly or indirectly activate the NLRP3 inflammasome. In order to confirm direct interactions, we used the cell-free system in this study.

pDONR221-NLRP3 and pDONR221-ASC were inserted into pEU-E01-GW-bls-STOP and pEU-E01-GW-STOP, respectively, for cell-free protein expression, as previously reported [5]. The entire cDNA of GFP was derived from pEU-E01-bls-GFP. The open reading frame of GFP without a stop codon was modified in a two-step polymerase chain reaction using the following primer sets:

C-terminal biotinylated full-length NLRP3 (NLRP3-FL-Btn), N-terminal FLAG-tagged full-length ASC (FLAG-ASC-FL), and FLAG-tagged CARD-only ASC (FLAG-ASC-CARD) are schematically indicated in Fig. 2a. Using wheat germ cell-free system-specific expression plasmids, NLRP3-FL-Btn, FLAG-ASC-FL, and FLAG-ASC-CARD proteins were also synthesized, as described previously [5].

Next, we investigated whether Aβ monomers, oligomers, and fibrils interacted with NLRP3 under cell-free conditions with ALPHA. Direct interactions between NLRP3-FL-Btn and Aβ oligomers or Aβ fibrils were observed in a dose-dependent manner (Fig. 2b, oligomers and fibrils, respectively). However, no interaction between NLRP3-FL-Btn and Aβ monomers was observed (Fig. 2b, monomers). We further confirmed the interaction between NLRP3-FL-Btn and Aβ fibrils. The ALPHA signals of the interaction gradually increased with 5.4 μg/mL of Aβ fibrils and decreased at higher concentrations of Aβ fibrils (Fig. 2c).

Next, we tested whether Aβ induced the interaction between NLRP3-FL-Btn and FLAG-ASC-FL. Both Aβ oligomers and Aβ fibrils significantly induced the interaction between NLRP3-FL-Btn and FLAG-ASC-FL in the cell-free system (Fig. 2d, oligo and fibrils, respectively). Positive controls of poly(I:C) also induced the interaction between NLRP3-FL-Btn and FLAG-ASC-FL (Fig. 2d, poly(I:C)).

The cell-based assay using 293T cell expression plasmids revealed that Aβ oligomers at 10 and 100 nM, but not monomers or fibrils, were able to activate the reconstituted-intracellular NLRP3 inflammasome leading to IL-1β secretion (Fig. 3a). In a cell-based assay using 293T cells, Aβ fibrils did not induce IL-1β secretion, different from the cell-free system (Fig. 1).

To test the specificity of the cell-free assay system, the interactions between NLRP3-Btn or GFP-Btn and 4.5 μM Aβ were assessed. The ALPHA-positive ratio of the interaction between NLRP3-Btn and Aβ was much higher than that between GFP-Btn and Aβ (Additional file 1: Figure S1). Therefore, NLRP3 was able to specifically recognize Aβ in comparison with GFP (Additional file 1: Figure S1).

In order to identify directly stimulating endogenous ligands, we previously developed a reconstituted NLRP3 inflammasome in a cell-free system [5, 13]. In this study, we successfully prepared Aβ oligomers and Aβ fibrils (Fig. 1) and found that oligomerized Aβ directly interacted with NLRP3 and induced the interaction of NLRP3 and ASC in the cell-free system, which may be an initial event in NLRP3 inflammasome activation (Fig. 2). Although the cell-free system cannot fully reflect physiological events, this study provides evidence that helps clarify the AD pathogenesis, being consistent with a report that Aβ causes lysosomal damage, inducing ROS-mediated NLRP3 inflammasome activation [9].

Under cell-free conditions, Aβ oligomers and fibrils, but not monomers, interacted with NLRP3 (Fig. 2). As for inflammasome-related intracellular pattern recognition receptors, NLRP3 and AIM2 were reported to recognize poly(I:C) and poly(dA:dT), both of which are large-molecular-weight nucleic acid polymers, to assemble the NLRP3 and AIM2 inflammasomes [14,15,16,17]. NLRC4 was reported to recognize flagellin, which can oligomerize to form fibrils to assemble the NLRC4 inflammasome [18, 19]. Pyrin was reported to recognize actin, which can oligomerize to form fibrils to assemble the pyrin inflammasome [20, 21]. In this context, fibril formation is considered to play an important role in inflammasome formation. Indeed, fibril formation was reported to be based on conformational changes in vitro [22]. According to a study of Aβ, Aβ monomers show lower radius of gyration than the Aβ oligomers, which may be related to the binding capacity of NLRP3, resulting in NLRP3 inflammasome activation [23]. This hypothesis may explain why the Aβ monomers did not interact with NLRP3.

Unlike LPS, which is a well-known strong IL-1β inducer, Aβ oligomers induced even low-level IL-1β secretion from NLRP3 inflammasome-reconstituted-293T cells and human MNCs (Fig. 3a, b). On the other hand, Aβ fibrils were unable to induce such IL-1β secretion in the cell-based assay, being different from the cell-free assay (Fig. 3). We speculate that because Aβ forms a large complex from oligomers to fibrils, it can only reach intracellular NLRP3 in very low amounts. This may explain why little activated caspase-1 from PMCs was detected on immunoblotting (Fig. 3c).

We next examined the specificity of the cell-free system for NLRP3 versus green fluorescence protein (GFP) and found that NLRP3 was able to more specifically recognize Aβ as compared with GFP (Additional file 1: Figure S1); however, this does not rule out redundant functions of inflammasomes.

We also investigated the effects of the known NLRP3 inhibitors MCC950 and isoliquiritigenin [24, 25] on the interaction between FLAG-NLRP3-FL and Aβ in the cell-free system; however, there was no significant inhibition (Additional file 2: Figures S2A and S2B). These data suggest that there may be targets of inhibitors other than the Aβ recognition site.

Induced pluripotent stem cells (iPSCs) have been generated from somatic cells by transgenic expression of Oct4 (Pou5f1), Sox2, Klf4 and Myc. A major difficulty in the application of this technology for regenerative medicine, however, is the delivery of reprogramming factors. Whereas retroviral transduction increases the risk of tumorigenicity, transient expression methods have considerably lower reprogramming efficiencies. Here we describe an efficient piggyBac transposon-based approach to generate integration-free iPSCs. Transposons carrying 2A peptide-linked reprogramming factors induced reprogramming of mouse embryonic fibroblasts with equivalent efficiencies to retroviral transduction. We removed transposons from these primary iPSCs by re-expressing transposase. Transgene-free iPSCs could be identified by negative selection. piggyBac excised without a footprint, leaving the iPSC genome without any genetic alteration. iPSCs fulfilled all criteria of pluripotency, such as pluripotency gene expression, teratoma formation and contribution to chimeras. piggyBac transposon-based reprogramming may be used to generate therapeutically applicable iPSCs.

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