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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.

Okoshi T, Yamaguchi I, Ozawa D, Hasegawa K, Naiki H - PLoS ONE (2015)

Bottom Line: Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients.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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pathology, Department of Pathological Sciences, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Endocytosed β2-m amyloid fibrils leak from endosomes/lysosomes into the cytosol.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.
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pone.0139330.g008: Endocytosed β2-m amyloid fibrils leak from endosomes/lysosomes into the cytosol.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.

Mentions: To investigate the mechanism of cytotoxicity mediated by amyloid fibrils, we next performed the ultrastructural analysis with transmission electron microscopy. When HIG-82 cells were incubated with Ham’s F12 medium containing vehicle buffer for 6 hrs, the cellular outline was clear and smooth. The oval nucleus with thin nuclear membrane, fine and homogenous chromatin, and a few nucleoli was observed (Fig 7A). In contrast, when HIG-82 cells were incubated with Ham’s F12 medium containing 100 μg/ml amyloid fibrils for 2 hrs, they were covered with amyloid fibrils (Fig 7B). Moreover, a part of the plasma membrane covered with amyloid fibrils was found to invaginate and fuse to form an endocytic vesicle containing amyloid fibrils (Fig 7B, inset). There were many intracytoplasmic endosomes/lysosomes filled with amyloid fibrils (Fig 7B) and some endosomal/lysosomal membranes were disrupted by intravesicular fibrils (Fig 7C). When HIG-82 cells were incubated with amyloid fibrils for 6 hrs, the cytoplasm was filled with endosomes/lysosomes containing abundant amyloid fibrils and some endosomes/lysosomes were found to fuse with each other (Fig 7E). Very importantly, the endocytosed amyloid fibrils leaked from endosomal/lysosomal vesicles into the cytosol and were contiguous with actin filaments (Fig 8A and 8D), and some amyloid fibrils were found adjacent to mitochondria (Fig 8B, 8C, 8E, and 8F). Nuclear deformation, shrinkage, and chromatin condensation at the nuclear rim (Fig 7F) and partial disruption of plasma membranes were also observed (Fig 7D).


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.

Okoshi T, Yamaguchi I, Ozawa D, Hasegawa K, Naiki H - PLoS ONE (2015)

Endocytosed β2-m amyloid fibrils leak from endosomes/lysosomes into the cytosol.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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4589361&req=5

pone.0139330.g008: Endocytosed β2-m amyloid fibrils leak from endosomes/lysosomes into the cytosol.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.
Mentions: To investigate the mechanism of cytotoxicity mediated by amyloid fibrils, we next performed the ultrastructural analysis with transmission electron microscopy. When HIG-82 cells were incubated with Ham’s F12 medium containing vehicle buffer for 6 hrs, the cellular outline was clear and smooth. The oval nucleus with thin nuclear membrane, fine and homogenous chromatin, and a few nucleoli was observed (Fig 7A). In contrast, when HIG-82 cells were incubated with Ham’s F12 medium containing 100 μg/ml amyloid fibrils for 2 hrs, they were covered with amyloid fibrils (Fig 7B). Moreover, a part of the plasma membrane covered with amyloid fibrils was found to invaginate and fuse to form an endocytic vesicle containing amyloid fibrils (Fig 7B, inset). There were many intracytoplasmic endosomes/lysosomes filled with amyloid fibrils (Fig 7B) and some endosomal/lysosomal membranes were disrupted by intravesicular fibrils (Fig 7C). When HIG-82 cells were incubated with amyloid fibrils for 6 hrs, the cytoplasm was filled with endosomes/lysosomes containing abundant amyloid fibrils and some endosomes/lysosomes were found to fuse with each other (Fig 7E). Very importantly, the endocytosed amyloid fibrils leaked from endosomal/lysosomal vesicles into the cytosol and were contiguous with actin filaments (Fig 8A and 8D), and some amyloid fibrils were found adjacent to mitochondria (Fig 8B, 8C, 8E, and 8F). Nuclear deformation, shrinkage, and chromatin condensation at the nuclear rim (Fig 7F) and partial disruption of plasma membranes were also observed (Fig 7D).

Bottom Line: Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients.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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pathology, Department of Pathological Sciences, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus