BackgroundAlzheimer’s disease is a neurodegenerative disease characterized by the interstitial deposition of amyloid β (Aβ) plaque, which is thought to be related to chronic neuroinflammation. Aβ is known to make fibrils via oligomers from monomers. Aβ has been reported to activate the NLRP3 inflammasome in infiltrated macrophages. NLRP3, an intracellular pattern recognition receptor, has been reported to recognize numerous pathogens and/or metabolites and form complexes with adopter protein ASC to make the inflammasome, an interleukin (IL)-1β-processing platform. Although reactive oxygen species from mitochondria have been reported to be involved in the activation of the NLRP3 inflammasome in microglial cells upon the deposition of Aβ, whether Aβ directly or indirectly activates the NLRP3 inflammasome remains unclear.MethodsWe 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.ResultsMonomers, 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.ConclusionOligomerized Aβ originating from non-toxic Aβ monomers directly interacted with NLRP3, leading to the activation of the NLRP3 inflammasome. This may be an attractive target for the treatment of Alzheimer’s disease.Electronic supplementary materialThe online version of this article (10.1186/s41232-018-0085-6) contains supplementary material, which is available to authorized users.

Recent findings revealed that type 2 diabetes mellitus (T2D) is a chronic inflammatory disease and an islet amyloid polypeptide (IAPP)/amylin, is deposited within pancreatic islets. IAPP/amylin has been reported to activate NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome in infiltrated macrophages. NLRP3, an intracellular pattern recognition receptor, has been shown to recognize pathogens and/or metabolites and complexes with the adopter protein apoptosis-associated speck-like protein containing a caspase-recruitment domain ASC to form a huge complex, called an inflammasome, an interleukin (IL)-1β-processing platform. Although reactive oxygen species (ROS) were reported to be involved in activation of NLRP3 inflammasome, we were hypothesized that IAPP could directly activate NLRP3 inflammasome, leading to islets β-cell death. We analyzed expression of the inflammasome components ASC, NLRP3, caspase-1, IL-1β, IAPP/amylin, and insulin immunohistochemically in Langerhans’ islets of autopsy cases. The initial event of NLRP3 inflammasome activation was assessed using a cell-free system consisting of NLRP3 and ASC with the amplified luminescent proximity homogeneous assay. IAPP/amylin deposition in Langerhans’ islets was detected and significantly correlated with expressions of IL-1β and ASC. IAPP/amylin directly interacted with NLRP3 and initiated an interaction between NLRP3 and ASC in a cell-free system. The deposition of IAPP/amylin in β-cells of Langerhans’ islets may act together with the expression level of an inflammasome component, ASC, to regulate IL-1β processing, and directly lead to the dysfunction of β-cells. The interaction between IAPP/amylin and NLRP3 could be an attractive drug target to avoid both inflammation and β-cell death for T2D therapy.

Introduction Nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3), an intracellular pattern recognition receptor, recognizes various pathogen-associated molecular pattern and/or damage-associated molecular pattern molecules to constitute inflammasome that act as an interleukin (IL)-1β processing platform. Injected insulin is reported to induce focal amyloidosis and the formation of subcutaneous lumps called insulin balls, but the formation of subcutaneous lumps and the underlying cytotoxic mechanism has not been elucidated. Methods Amyloid formation was evaluated by thioflavin T spectroscopic assay and scanning electron microscopy. Binding between insulin amyloid fibrils and NLRP3 was evaluated by immunoprecipitation followed by native polyacrylamide gel electrophoresis. Inflammasome activation was evaluated by immunofluorescence speck formation called “ASC speck” and Western blotting. IL-1β secretion in culture supernatants of peripheral blood mononuclear cells was evaluated by enzyme-linked immunosorbent assay. Cytotoxicity was measured by lactate dehydrogenase release assay. Results Insulin amyloid fibrils interact directly with NLRP3, resulting in NLRP3 inflammasome activation and pyroptotic cell death. Conclusion Insulin ball formation and cytotoxicity may be associated with NLRP3 inflammasome activation followed by pyroptotic cell death.

A combinational method for the separation and the degradation of amyloid‐β fibrils in the biological samples is developed to suppress the amyloid‐β fibrils content. The complexes of the Congo red dye and amyloid‐β fibrils migrate to the anode in a non‐denaturing agarose electrophoresis owing to its negative charge, thus separating the complex containing amyloid‐β fibrils from the human plasma proteins and the amyloid‐β monomer. The separated amyloid‐β fibrils exhibited birefringence under a polarizing microscope and immunoreactivity to the anti‐amyloid‐β antibody. The digestion of 0.23 nmol of the amyloid‐β fibrils using nattokinase at 37°C for 24 h increased with the amount of enzyme up to 9 pmol, beyond which the digestion was independent of the enzyme concentration. The digestion of 0.23 nmol of the amyloid‐β fibrils with 22.5 pmol of nattokinase at 37°C increased with time up to 6 h, following which, it became independent of time. The extraction of the separated amyloid‐β fibrils from the agarose gel accompanied the removal of major plasma proteins from the spiked plasma and amyloid‐β fibrils. The combination of the non‐denaturing agarose gel electrophoresis and the dye staining would be applicable to examine formation and degradation processes of amyloid‐β fibrils after their separation from the biological samples.