(214a) Effect of Engineered Transthyretin Mutants On Beta-Amyloid Aggregation and Toxicity

Alzheimer’s disease (AD) is the most prevalent age-associated neurodegenerative disease. Some of the characteristic features associated with AD are extracellular senile plaques, intraneuronal neurofibrillary tangles, and extensive neuronal cell death.  The plaques consist mostly of the 4kDa peptide β-amyloid (Aβ).  Aβ spontaneously self-assembles through a multistep process into soluble oligomers and fibrillar aggregates.  Numerous in vitro and transgenic animal studies have causally linked Aβ aggregation to neurotoxicity.  The current paradigm is that the soluble oligomeric intermediates are the most toxic form of Aβ.

Transthyretin (TTR) is a homotetrameric transport protein present in both blood and cerebrospinal fluid (CSF). Upregulation of TTR expression in Tg2576 mice has been linked to protection against the toxic effects of Aβ deposition. This and other observations suggest that TTR serves as a natural anti-AD compouond, and motivate our detailed study of TTR-Aβ association.

Through enzyme-linked immunoassays, we demonstrated that Aβ binds directly to TTR; more Aβ binds if it is aggregated, or if TTR is in its monomeric form. To further identify the interaction region of TTR, we used crosslinking plus tandem mass spectrometry, and peptide arrays. These data show that there are two domains on TTR, strand G and EF helix/loop, that interact with Aβ. Scanning alanine mutagenesis was used to further pinpoint residues important for binding. Alanine mutants L82A (on the EF loop) and L110A (on strand G) bound significantly less Aβ compared to wildtype TTR; thus we conclude that Aβ binding is mediated through these bulky hydrophobic residues. Two other alanine mutants, S85A and L17A, showed increased binding to Aβ. S85A was less stable as a tetramer than wildtype; increased Aβ binding is postulated to be a direct consequence of reduced quaternary stability. Enhancement of binding in L17A is postulated to arise from reduced steric restriction to the interior L110 site.

The next set of experiments are aimed at connecting TTR binding to TTR’s effect on Aβ aggregation and toxicity. Surface plasmon resonance was used to determine the kinetics and equilibrium of Aβ binding to TTR (wildtype and mutants). Via dynamic light scattering, we characterized the effect of TTR on Aβ aggregate growth. S85A and L17A were the most effective, completely inhibiting growth of Aβ aggregates and thus showing a causal relationship between binding and inhibition of aggregation.  In in vitro assays, TTR inhibited Aβ toxicity in a dose-dependent manner; efficacy of mutants was linked to the strength of binding. Importantly, inhibition was observed at substoichiometric TTR: Aβ ratios. Our data can be explained by postulating that TTR sequesters Aβ toxic oligomers. These results open new avenues for AD prevention by bolstering or supplementing TTR’s natural protective action.