Destruction of Amyloid Fibrils of a β2-Microglobulin Fragment by Laser Beam Irradiation

This research was originally published in the Journal of Biological Chemistry. Daisaku Ozawa, Hisashi Yagi, Tadato Ban, Atsushi Kameda, Toru Kawakami, Hironobu Naiki and Yuji Goto. Destruction of Amyloid Fibrils of a β2-Microglobulin Fragment by Laser Beam Irradiation. J. Biol. Chem. 2009; 284, 1009-1017. © the American Society for Biochemistry and Molecular Biolog

Innovative launch opportunity for Micro/Nano-satellite by using one and only function on Kibo/ISS

The Japan Aerospace Exploration Agency (JAXA) developed an innovative system called the JEM Small Satellite Orbital Deployer (J-SSOD) to deploy satellites into orbit from the Japanese Experiment Module (“Kibo”), which is one of the ISS modules, by taking advantage of its one and only function of having both an airlock and a robotic arm. The satellites are delivered to Kibo as part of the spaceship cargo load, thereby mitigating the launch mechanical environment and increasing launch opportunities. In 2012, we successfully deployed five satellites on the first J-SSOD mission, which opened new capabilities for Kibo/ISS utilization and contributed to broadening the possibilities for the Micro/Nano-satellites. At present, satellite deployers other than J-SSOD that use Kibo include the NanoRacks CubeSat Deployer (NRCSD) and Cyclops (Space Station Integrated Kinetic Launcher for Orbital Payload Systems). As of May 2017, 193 satellites have been successfully deployed from Kibo. This paper introduces an overview of this innovative launch opportunity and related international cooperation

Endocytosed 2-Microglobulin Amyloid Fibrils Induce Necrosis and Apoptosis of Rabbit Synovial Fibroblasts by Disrupting Endosomal/Lysosomal Membranes: A Novel Mechanism on the Cytotoxicity of Amyloid Fibrils.

Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients. In dialysis-related amyloidosis, β2-microglobulin (β2-m) amyloid fibrils deposit in the osteoarticular tissue, leading to carpal tunnel syndrome and destructive arthropathy with cystic bone lesions, but the mechanism by which these amyloid fibrils destruct bone and joint tissue is not fully understood. In this study, we assessed the cytotoxic effect of β2-m amyloid fibrils on the cultured rabbit synovial fibroblasts. Under light microscopy, the cells treated with amyloid fibrils exhibited both necrotic and apoptotic changes, while the cells treated with β2-m monomers and vehicle buffer exhibited no morphological changes. As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay. Interestingly, when the cells were incubated with amyloid fibrils for several hours, many endosomes/lysosomes filled with amyloid fibrils were observed under confocal laser microscopy and electron microscopy, Moreover, some endosomal/lysosomal membranes were disrupted by intravesicular fibrils, leading to the leakage of the fibrils into the cytosol and adjacent to mitochondria. Inhibition of actin-dependent endocytosis by cytochalasin D attenuated the toxicity of amyloid fibrils. These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid fibrils is described

Destruction of Amyloid Fibrils of Keratoepithelin Peptides by Laser Irradiation Coupled with Amyloid-specific Thioflavin T*

Mutations in keratoepithelin are associated with blinding ocular diseases, including lattice corneal dystrophy type 1 and granular corneal dystrophy type 2. These diseases are characterized by deposits of amyloid fibrils and/or granular non-amyloid aggregates in the cornea. Removing the deposits in the cornea is important for treatment. Previously, we reported the destruction of amyloid fibrils of β2-microglobulin K3 fragments and amyloid β by laser irradiation coupled with the binding of an amyloid-specific thioflavin T. Here, we studied the effects of this combination on the amyloid fibrils of two 22-residue fragments of keratoepithelin. The direct observation of individual amyloid fibrils was performed in real time using total internal reflection fluorescence microscopy. Both types of amyloid fibrils were broken up by the laser irradiation, dependent on the laser power. The results suggest the laser-induced destruction of amyloid fibrils to be a useful strategy for the treatment of these corneal dystrophies

β2-m amyloid fibrils induce apoptosis of HIG-82 cells as measured by the TUNEL assay.

<p>After HIG-82 cells were incubated with Ham’s F12 medium containing vehicle buffer or 100 μg/ml β2-m fibrils or r-β2-m monomer for 2 days, TUNEL assay was performed as described in Materials and Methods. (A) The representative fluorescence images of TUNEL and DAPI double staining. The original magnification was x100. (B) The percentage of apoptotic cells to total cells. Data were presented as a dot plot of the ratios of five independent experiments with the mean value. Statistical analysis was performed by Mann-Whitney U-test. *P < 0.05.</p

β2-m amyloid fibrils are internalized and sorted to lysosomes.

<p>HIG-82 cells incubated with Ham’s F12 medium containing vehicle buffer, 10 μg/ml β2-m monomer, or 10 μg/ml β2-m fibrils for 12 hrs were stained for lysosomes (red), β2-m (green), and nuclei (blue), and observed with the confocal laser microscope as described in Materials and Methods. When the cells were incubated with fibrils (right column), green fluorescence indicating β2-m fibrils were observed inside the cells in a granular pattern, as well as on the surface of the cells. Importantly, some green-colored granules containing β2-m fibrils were merged with red-colored lysosomes. The scale bars are 10 μm long.</p

Inhibition of β2-Microglobulin Amyloid Fibril Formation by α2-Macroglobulin*

The relationship between various amyloidoses and chaperones is gathering attention. In patients with dialysis-related amyloidosis, α2-macroglobulin (α2M), an extracellular chaperone, forms a complex with β2-microglobulin (β2-m), a major component of amyloid fibrils, but the molecular mechanisms and biological implications of the complex formation remain unclear. Here, we found that α2M substoichiometrically inhibited the β2-m fibril formation at a neutral pH in the presence of SDS, a model for anionic lipids. Binding analysis showed that the binding affinity between α2M and β2-m in the presence of SDS was higher than that in the absence of SDS. Importantly, SDS dissociated tetrameric α2M into dimers with increased surface hydrophobicity. Western blot analysis revealed that both tetrameric and dimeric α2M interacted with SDS-denatured β2-m. At a physiologically relevant acidic pH and in the presence of heparin, α2M was also dissociated into dimers, and both tetrameric and dimeric α2M interacted with β2-m, resulting in the inhibition of fibril growth reaction. These results suggest that under conditions where native β2-m is denatured, tetrameric α2M is also converted to dimeric form with exposed hydrophobic surfaces to favor the hydrophobic interaction with denatured β2-m, thus dimeric α2M as well as tetrameric α2M may play an important role in controlling β2-m amyloid fibril formation

β2-m amyloid fibrils are endocytosed into endosomes/lysosomes, leading to the disruption of their membranes.

<p>Representative electron micrographs of HIG-82 cells taken as described in Materials and Methods. HIG-82 cells were incubated with Ham’s F12 medium containing vehicle buffer for 6 hrs (A), or 100 μg/ml β2-m fibrils for 2 hrs (B, C) or 6 hrs (D-F) as described in Materials and Methods. The inset in (B) is a higher magnification of the box. (B, D) HIG-82 cells were covered with amyloid fibrils. Note that a part of the plasma membrane invaginated and fused to form an endocytic vesicle containing amyloid fibrils (inset in B). (C, E) Many endosomes/lysosomes were filled with amyloid fibrils, and some endosomal/lysosomal membranes were disrupted by intravesicular fibrils. (F) Nuclear deformation, shrinkage, and chromatin condensation at the nuclear rim were also observed. The scale bars are 5 μm long in A, B, D and F and 1 μm long in C and E.</p

Endocytosed β2-m amyloid fibrils leak from endosomes/lysosomes into the cytosol.

<p>Representative electron micrographs of HIG-82 cells incubated with Ham’s F12 medium containing 100 μg/ml β2-m fibrils for 6 hrs as described in Materials and Methods. Images were taken as described in Materials and Methods. (D-F) Higher magnifications of the boxes in A-C, respectively. Note that the endocytosed amyloid fibrils leaked from endosomal/lysosomal vesicles into the cytosol (A, D), and some fibrils were found adjacent to mitochondria (B, C, E, F). The scale bars are 500 nm long in A-C and 200 nm long in D-F.</p