The transthyretin amyloidoses are fatal protein misfolding disorders characterized by the extracellular deposition of aggregates and amyloid fibrils derived from the plasma homotetrameric protein transthyretin (TTR). Aging is a major risk factor for both the senile (wild-type) and genetic (mutant TTR) forms of these disorders. It is not known which aspects of the aging process contribute to the onset of the TTR amyloidoses. Protein carbonylation is an oxidative modification common of old age and it is associated with age-related neurodegenerative illnesses such as Alzheimer's and Parkinson's diseases. We previously established in vitro that recombinant carbonylated TTR (car-TTR) is less stable and has higher propensity to aggregate than non-oxidized TTR. We also demonstrated that the only therapy currently under clinical trials based on TTR tetramer stabilization is significantly less efficient for car-TTR than for non-oxidized TTR. The implications of these studies are that if car-TTR levels increase with age, they will promote the onset of the amyloidoses; moreover, tetramer stabilization therapy may not be sufficient to treat these disorders. Although car-TTR has been detected in plasma of healthy individuals, no systematic studies exist with respect to its changing abundance with age or with the presence of TTR amyloidoses. Our working hypotheses are: 1) Car-TTR content in plasma increases with age; 2) Car-TTR content in plasma is higher in TTR amyloid patients than in healthy controls. Our goals are to establish whether an increase in car-TTR in plasma is associated with age and/or with overt TTR amyloidoses. To accomplish these goals we will exploit our already established methods to quantify total TTR in plasma and optimize our working protocols to specifically quantify car-TTR in the same samples. The experimental design includes the following groups: i) Healthy humans aged 20 to 72 years; ii) Patients with TTR amyloidosis and age-matched healthy controls; iii) Healthy young and old transgenic mice over-expressing human wild-type TTR; iv) Transgenic mice over-expressing human wild-type TTR, with and without histologically confirmed TTR deposition. Finding a correlation between car-TTR content with age and/or the positive diagnosis of TTR amyloidosis, together with the already established fact that car-TTR is more prone to aggregation than non-oxidized TTR, will indicate that this type of age-related oxidative modification may play a role in the onset of the TTR aggregation disorders. It will also open a new therapeutic approach to prevent disease onset consisting in anti-oxidant therapy to preclude car-TTR formation. We will formally test the causal hypothesis, i.e. car-TTR formation is involved in the onset of the TTR amyloidoses, and the novel therapeutic strategy in vivo in a forthcoming R01 proposal. Finally, the results of our studies can have direct implications to the understanding of the initiation of other age-related diseases of protein aggregation where the precursor amyloidogenic proteins might be susceptible to the same type of oxidative modifications.
The transthyretin amyloidoses are age-related fatal disorders of systemic protein aggregation that result in organ dysfunction and death. We will determine whether the levels of oxidative carbonylation of transthyretin in biological samples are associated with age and disease diagnosis. The studies will help to understand whether this form of protein oxidation is a factor that modulates the onset of these disorders and will open new avenues for therapeutic intervention.
