Mechanisms involved in amyloid induced cytotoxicity

Detta är en avhandling från Umeå : Molekylärbiologi

Sammanfattning: Amyloidoses comprise a group of diseases where normal or mutated protein precipitates into amyloid fibrils. The deposition of fibrils causes dysfunction of organs and toxicity to nervous tissue. Up to date, 24 different proteins and peptides are known to be able to form amyloid fibrils. The most well known are Amyloid beta peptide and Prione protein causing Alzheimer’s disease and Creutzfeld Jacob’s disease respectively.The aims of this thesis were to investigate the structural properties of cytotoxic amyloid and examine the mechanisms involved. The model protein mostly used in the studies was the plasma protein transthyretin (TTR). Familial Amyloidotic Polyneuropathy (FAP) is a hereditary, autosomal-dominant neurodegenerative disease caused by point mutations in the TTR gene. One of the most common variants of FAP is a mutation in position 30 where alanine is exchanged for methonine. This gives rise to “Skellefteåsjukan” in Sweden.TTR is secreted into the plasma as a tetramer. Point mutations destabilize the tetramer leading to disassembled monomers, which undergo partial denaturation as an initiation step to aggregation and amyloid fibril formation. In vivo amyloidogenesis takes a long time and does not occur until late in adult life. Most of the clinical TTR mutations do not form amyloid in vitro under physiological conditions. We have created amyloidogenic TTR mutants that are prone to aggregate and form fibrils under physiological conditions. This provides us with a model system on the cellular level for studies of the mechanisms of amyloid associated cytotoxicity as we can control the aggregation process and capture defined stages in the TTR amyloidogenic pathway.We used Atomic Force Microscopy (AFM) to follow the morphology of aggregates during fibril formation. Initially, amorphous aggregates were formed that subsequently matured into fibrillar structures, denoted protofilaments. This observation was interpreted as an optimisation of ß-strand registers. In addition we identified a correlation between the presence of early-formed aggregates of TTR and cytotoxicity. The toxic response was mediated via an apoptotic mechanism.We were not able to more carefully determine the structure and size of the toxic TTR species. To address this problem we turned to another amyloidogenic protein, equine lysozyme (EL). Intermediate samples corresponding to the aggregation and growth phase of amyloid fibrils of EL were collected. These samples were subjected to cytotoxicity assays as well as monomeric starting material and mature amyloid fibrillar species. The results clearly showed that the soluble oligomers were cytotoxic in contrast to the monomers and fibrils. Our data indicate that the toxic properties of the oligomers are size dependent.In this thesis we asked the question whether all mutated forms of TTR can be expressed and secreted or if there is a selection against the most aggressive mutations in vivo? We transfected hematopoetic K562 cells with wild type or mutant TTR, with or without the N-terminal signal peptide, responsible for secretion, to generate both extra- and intracellular TTR. We show that the post-translational quality control of the cells does not allow intracellular mutant TTR outside the secretory pathway, possibly due to the cytotoxic effects, while translocated to the secretory pathway made it escape the quality control permitting secretion and amyloid formation outside the cells.We have further analyzed the cytotoxic mechanisms induced by TTR oligomers with a focus on intracellular apoptotic signalling pathways. We show that TTR oligomers bind to the surface of the target cells but are not taken up, that is in contrast to mature fibrils that do not bind them at all. The apoptotic response occurred in a caspase-independent and a free radical dependent way.