Only amyloidogenic intermediates of transthyretin induce apoptosis

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Abstract

In diseases like Alzheimer's disease and familial amyloidotic polyneuropathy (FAP) amyloid deposits co-localize with areas of neurodegeneration. FAP is associated with mutations of the plasma protein transthyretin (TTR). We can here show an apoptotic effect of amyloidogenic mutants of TTR on a human neuroblastoma cell line. Toxicity could be blocked by catalase indicating a free oxygen radical dependent mechanism. The toxic effect was dependent on the state of aggregation and unexpectedly mature fibrils from FAP-patients who failed to exert an apoptotic response. Morphological studies revealed a correlation between toxicity and the presence of immature amyloid. Thus, we can show that toxicity is associated with early stages of fibril formation and propose that mature full-length fibrils represent an inert end stage, which might serve as a rescue mechanism.

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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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    Abbreviations: TTR, transthyretin; SDI, succinate dehydrogenase inhibition; LDH, lactodehydrogenase; TUNEL, TdT-mediated dUTP nick end labelling; PARP, poly(ADP-ribose) polymerase.

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