Endocytosed β2-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

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.

Molecular pathogenesis of human amyloidosis: Lessons from β2‐microglobulin‐related amyloidosis

“…We assessed the effect of β2‐m amyloid fibrils on the cultured synovial fibroblasts derived from rabbit periarticular soft tissue (HIG‐82 cells) . We found that endocytosed amyloid fibrils cause both necrosis (plasma membrane disruption) and apoptosis of the cells by disrupting endosomal/lysosomal membranes with the leakage of the fibrils into the cytosol and adjacent to mitochondria (Fig.…”

Iowa Mutant Apolipoprotein A-I (ApoA-IIowa) Fibrils Target Lysosomes

“…Thus, our results suggest that apoA-I Iowa fibrils may be internalized by cells and transported to lysosomes in a dynamin- and actin dynamics-dependent, but caveolin-independent, manner. Similarly, dynasore and cytochalasin D reportedly reduced the cytotoxicity of β 2 -microglobulin amyloid fibrils, possibly by inhibiting fibril internalization 18 57 . Nonphagocytic cells internalized beads having a diameter of 5.5 μM in a clathrin- and dynamin-dependent fashion 58 .…”

“…Pinocytosis-dependent and dynamin-dependent endocytoses were implicated in the internalization and cytotoxicity of β 2 -microglobulin amyloid fibrils 18 57 . In the present study, cytochalasin D and dynasore inhibited degradation of apoA-I Iowa fibrils, whereas nystatin did not.…”

Amyloidosis-inducing activity of blood cells in mouse AApoAII amyloidosis

“…It is possible that the phagocytosis of AApoAII amyloid fibrils by monocytes/macrophages contributed to the transmission of amyloidosis when the cells were transfused to recipients. Tissue cells could be damaged by endocytosis of amyloid fibrils in various amyloidoses [ 22 , 35 ]. Also, Aβ was demonstrated to bind to RBCs [ 23 ].…”