Only amyloidogenic intermediates of transthyretin induce apoptosis☆
Section snippets
Materials and methods
Enzymes and oligonucleotides. Enzymes were purchased from New England Biolabs (Hertfordshire, England), oligonucleotides from Life Technologies (Paisley, UK), Aβ(1–40)-peptide, and the catalase from Sigma–Aldrich (St. Louis, MO, USA).
In vitro mutagenesis. The TTR mutants were produced using PCR-site-directed mutagenesis [20]. The TTRV14N, V16E mutant was constructed by the substitution of two valines for asparagine and glutamic acid in positions 14 and 16, respectively, in the A-strand using
TTR toxicity
The well-established SDI (succinate dehydrogenase inhibition) test was used as a screening test of the TTR mutants, using the Alzheimer Aβ(1–40)-peptide as a positive control. Fig. 1 show that both the TTRV14N, V16E and the TTRG53S, E54D, L55S mutants showed a clear toxic effect in the same molar range as Aβ(1–40).
In contrast TTRwt did not cause any toxic response nor did the TTRV30M mutant (Fig. 1, left panel). More surprisingly, amyloid extracted from TTRV30M patients was atoxic up to 8 days
Discussion
Neuronal degeneration closely associated to amyloid fibril plaques is a significant feature in different forms of amyloidosis. In the case of Alzheimer's disease or FAP the lesions have correlated with expression lipid membrane peroxidation indicative of oxidative stress [14]. Both in vivo and in vitro evidence has been presented that amyloid induces apoptosis, although this has been controversial in the case of the Aβ-peptide [15], [27]. Reports on molecular mechanisms concerning receptors and
Acknowledgements
This work was supported by the Swedish Medical Research Council (009107), the Danish Medical Council (9802572), a BioMed2 grant (BMH4-CT98-3689), the Medical Faculty, Umeå University, Swedish Society for Medical Research, the Danish Association against Rheumatism, Lundbeckfonden (Denmark) the Patient Association FAMY, Skellefteå, and the Amyl Foundation, Piteå, Sweden. Thanks to Bengt Hallberg for good advice.
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Pro-oxidative effects of aggregated transthyretin in human Schwannoma cells
2013, NeuroToxicologyCitation Excerpt :Some possibilities include (i) contributions of reactive species to the damage of TTR itself, making it more toxic (as has been reported for amyloid-β and Alzheimer's disease: Atamna and Boyle, 2006), (ii) effects on cell membrane structure and function, a known effect of reactive chemical species including hydrogen peroxide, e.g., endocytic function (Cheng and Vieira, 2006; Kano et al., 2011), (iii) damage to other cellular components, e.g., to enzymes that may further compromise energy metabolism (Aoi and Sakuma, 2011; Choi et al., 2004). These and other pathologic mechanisms may also contribute to death of neurons and other cells in relation to amyloidogenic pro-oxidative damage; for example, amyloidogenic TTR has been reported to induce apoptosis in neuroblastoma cells, and this effect was dependent on ROS (Andersson et al., 2002). Furthermore, it will be of interest in future studies to determine if all of the cellular oxidative damage and subsequent pathologic effects are initiated at the cell membrane through interaction with exogenous agTTR, or whether some of the agTTR molecular species are internalized and initiate toxic effects from intracellular compartments.
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Abbreviations: TTR, transthyretin; SDI, succinate dehydrogenase inhibition; LDH, lactodehydrogenase; TUNEL, TdT-mediated dUTP nick end labelling; PARP, poly(ADP-ribose) polymerase.