Structural alterations in the most unstable transthyretin tetramer favor its aggregation in physiological conditions

Transthyretin (TTR) is a 55kDa transporter of thyroxine in the blood and cerebrospinal fluid (CSF). Among all variants, A25T is the most unstable tetramer associated with leptomeningeal amyloidosis (LA). Aiming to study A25T stability and aggregation, we first observed, using high hydrostatic pressure, that A25T dissociates easily than the unstable L55P variant. A25T was also the most amyloidogenic, aggregating in conditions where wt and L55P remained soluble. Using HPLC and western blotting, we showed that A25T aggregated in human plasma and CSF, forming amyloid fibrils that co‐aggregated with unknown proteins. We solved the crystal structures of the wt and A25T and the comparative analysis shed light into the mechanism behind A25T amyloidogenicity: an expanded tetramer which is stabilized by a lower number of H‐bonds and hydrophobic interactions. In the presence of T4 and flufenamic acid, the structure of A25T was similar to that displayed by the wt protein and aggregation was 100% inhibited in CSF. Since LA symptoms suggest inflammation, microglia cultures were incubated with A25T aggregates and underwent activation, phagocytosis and secreted nitric oxide and IL‐1 beta. Also, exposure of media from only A25T aggregates‐activated microglia to primary neurons induce neuronal death. In conclusion, altered A25T tetramer rapidly forms amyloid fibrils which can drive inflammation. Support: CAPES and FAPERJ