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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

Inhibition of endocytosis by cytochalasin D attenuates the toxicity of β2-m amyloid fibrils.HIG-82 cells preincubated with Ham’s F12 medium containing 0 to 1.0 μg/ml cytochalasin D (CytoD) for 2 hrs, were incubated with the medium containing vehicle buffer or 100 μg/ml β2-m fibrils in the presence of CytoD for 2 days. LDH releasing assay (A) and MTT reduction assay (B) were performed as described in Materials and Methods. Data normalized to positive control and vehicle in LDH releasing assay and MTT reduction assay, respectively, were presented as mean ± SD of three independent experiments. Statistical analysis was performed by Student’s unpaired t-test. *P < 0.05, **P < 0.01.
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pone.0139330.g009: Inhibition of endocytosis by cytochalasin D attenuates the toxicity of β2-m amyloid fibrils.HIG-82 cells preincubated with Ham’s F12 medium containing 0 to 1.0 μg/ml cytochalasin D (CytoD) for 2 hrs, were incubated with the medium containing vehicle buffer or 100 μg/ml β2-m fibrils in the presence of CytoD for 2 days. LDH releasing assay (A) and MTT reduction assay (B) were performed as described in Materials and Methods. Data normalized to positive control and vehicle in LDH releasing assay and MTT reduction assay, respectively, were presented as mean ± SD of three independent experiments. Statistical analysis was performed by Student’s unpaired t-test. *P < 0.05, **P < 0.01.

Mentions: To further confirm the contribution of endosomes/lysosomes to the cytotoxicity of β2-m amyloid fibrils, we finally examined the effect of CytoD on the cytotoxicity of β2-m amyloid fibrils. CytoD, a kind of fungal toxin from Zygosporium mansonii, disrupts actin polymerization and inhibits actin-dependent endocytosis at 1.0 μg/ml [49, 50]. As shown in Fig 9, CytoD dose-dependently attenuated the cytotoxicity of β2-m amyloid fibrils in both the LDH releasing assay (33.1 ± 3.4%, 33.2 ± 5.2%, 24.4 ± 2.4% and 20.0 ± 1.3% of positive control for 0, 0.1, 0.5 and 1.0 μg/ml CytoD, respectively; 0 vs. 1.0 μg/ml CytoD, P<0.01; 0 vs. 0.5 and 0.1 vs. 1.0 μg/ml CytoD, P<0.05) and the MTT reduction assay (59.7 ± 9.9%, 58.6. ± 10.2%, 74.8 ± 6.8% and 78.1 ± 5.0% of the negative control for 0, 0.1, 0.5 and 1.0 μg/ml CytoD, respectively; 0 vs. 1.0 and 0.1 vs. 1.0 μg/ml CytoD, P<0.05).


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)

Inhibition of endocytosis by cytochalasin D attenuates the toxicity of β2-m amyloid fibrils.HIG-82 cells preincubated with Ham’s F12 medium containing 0 to 1.0 μg/ml cytochalasin D (CytoD) for 2 hrs, were incubated with the medium containing vehicle buffer or 100 μg/ml β2-m fibrils in the presence of CytoD for 2 days. LDH releasing assay (A) and MTT reduction assay (B) were performed as described in Materials and Methods. Data normalized to positive control and vehicle in LDH releasing assay and MTT reduction assay, respectively, were presented as mean ± SD of three independent experiments. Statistical analysis was performed by Student’s unpaired t-test. *P < 0.05, **P < 0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139330.g009: Inhibition of endocytosis by cytochalasin D attenuates the toxicity of β2-m amyloid fibrils.HIG-82 cells preincubated with Ham’s F12 medium containing 0 to 1.0 μg/ml cytochalasin D (CytoD) for 2 hrs, were incubated with the medium containing vehicle buffer or 100 μg/ml β2-m fibrils in the presence of CytoD for 2 days. LDH releasing assay (A) and MTT reduction assay (B) were performed as described in Materials and Methods. Data normalized to positive control and vehicle in LDH releasing assay and MTT reduction assay, respectively, were presented as mean ± SD of three independent experiments. Statistical analysis was performed by Student’s unpaired t-test. *P < 0.05, **P < 0.01.
Mentions: To further confirm the contribution of endosomes/lysosomes to the cytotoxicity of β2-m amyloid fibrils, we finally examined the effect of CytoD on the cytotoxicity of β2-m amyloid fibrils. CytoD, a kind of fungal toxin from Zygosporium mansonii, disrupts actin polymerization and inhibits actin-dependent endocytosis at 1.0 μg/ml [49, 50]. As shown in Fig 9, CytoD dose-dependently attenuated the cytotoxicity of β2-m amyloid fibrils in both the LDH releasing assay (33.1 ± 3.4%, 33.2 ± 5.2%, 24.4 ± 2.4% and 20.0 ± 1.3% of positive control for 0, 0.1, 0.5 and 1.0 μg/ml CytoD, respectively; 0 vs. 1.0 μg/ml CytoD, P<0.01; 0 vs. 0.5 and 0.1 vs. 1.0 μg/ml CytoD, P<0.05) and the MTT reduction assay (59.7 ± 9.9%, 58.6. ± 10.2%, 74.8 ± 6.8% and 78.1 ± 5.0% of the negative control for 0, 0.1, 0.5 and 1.0 μg/ml CytoD, respectively; 0 vs. 1.0 and 0.1 vs. 1.0 μg/ml CytoD, P<0.05).

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