| Primary (AL) Amyloidosis is a disease of protein misfolding and clearance in which a plasma cell dyscrasia produces an immunoglobulin light chain, which aggregates and deposits as insoluble fibrils in various tissues and disrupts the physiology of organs such as heart, kidney, lung, intestines and peripheral nervous system. Cellular responses to amyloidogenic light chains (AL-LCs) are not well understood. Our lab has shown that cellular internalization of AL-LCs occurs within 4 hours and that there is an increase in glycosaminoglycan (GAG) synthesis and sulfation. To determine the mechanism of internalization, we used live cell confocal imaging of fluorescently conjugated AL-LCs. AL-LC internalization was mass and concentration dependent. Disruption of membrane lipid rafts with filipin III did not alter internalization of AL-LCs. However, when a decrease in membrane fluidity was induced using methyl-beta-cyclodextrin, decreased temperature or cytochalasin D, internalization was inhibited or reduced. The inhibition experiments were run in parallel with membrane markers (DiI and FM 4-64) and endocytic markers (caveolin, transferrin, and cholera toxin B). Co-localization with membrane was not detected. Furthermore, removal of GAGs or their sulfation did not alter internalization of AL-LC. AL-LC did co-localize with the classical pinocytosis marker dextran. Internalized AL-LCs were found within vesicles and their motility was inhibited by nocodazole, a microtubule inhibitor. AL-LCs localized to lysosomes, but not to Golgi, mitochondria, endoplasmic reticulim or nucleus. Proteasomal inhibitors MG132 and bortezomib interrupted cellular processing of AL-LCs. Cellular response to AL-LC was marked by a decrease in mitochondrial function and phosphorylated extracellular signal-regulated kinase (pERK), but no apoptosis or change in [Ca2+]i. Internalized and secreted AL-LC undergoes a shift towards a less hydrophobic species. Together this data indicates that cells internalize AL-LC by non-specific fluid phase endocytosis and that cytosolic and secreted AL-LCs are modified over time. We predict that changes in the cellular response are specific to AL-LC and contribute to cytotoxicity and fibril formation. |
