Energetic Characteristics of the New Transthyretin Variant A25T May Explain Its Atypical Central Nervous System Pathology

Abstract

SUMMARY:Transthyretin (TTR) is a tetrameric protein that must misfold to form amyloid fibrils. Misfolding includes rate-limiting tetramer dissociation, followed by fast tertiary structural changes that enable aggregation. Amyloidogenesis of wild-type (WT) TTR causes a late-onset cardiac disease called senile systemic amyloidosis. The aggregation of one of Ͼ 80 TTR variants leads to familial amyloidosis encompassing a collection of disorders characterized by peripheral neuropathy and/or cardiomyopathy. Prominent central nervous system (CNS) impairment is rare in TTR amyloidosis. Herein, we identify a new A25T TTR variant in a Japanese patient who presented with CNS amyloidosis at age 42 and peripheral neuropathy at age 44. The A25T variant is the most destabilized and fastest dissociating TTR tetramer published to date, yet, surprising, disease onset is in the fifth decade. Quantification of A25T TTR in the serum of this heterozygote reveals low levels relative to WT, suggesting that protein concentration influences disease phenotype. Another recently characterized TTR CNS variant (D18G TTR) exhibits strictly analogous characteristics, suggesting that instability coupled with low serum concentrations is the signature of CNS pathology and protects against early-onset systemic amyloidosis. The low A25T serum concentration may be explained either by impaired secretion from the liver or by increased clearance, both scenarios consistent with A25T's low kinetic and thermodynamic stability. Liver transplantation is the only known treatment for familial amyloid polyneuropathy. This is a form of gene therapy that removes the variant protein from serum preventing systemic amyloidosis. Unfortunately, the choroid plexus would have to be resected to remove A25T from the CSF-the source of the CNS TTR amyloid. Herein we demonstrate that small-molecule tetramer stabilizers represent an attractive therapeutic strategy to inhibit A25T misfolding and CNS amyloidosis. Specifically, 2-[(3,5-dichlorophenyl)amino]benzoic acid is an excellent inhibitor of A25T TTR amyloidosis in vitro. (Lab Invest 2003, 83:409 -417).

Quantification of the Thermodynamically Linked Quaternary and Tertiary Structural Stabilities of Transthyretin and Its Disease-Associated Variants: The Relationship between Stability and Amyloidosis

“…ERAD occurs to a greater extent in the liver than in the choroid plexus, partially explaining the CNS phenotype of this amyloid disease (12,13). That this is one of the few disease-associated TTR variants that is substantially degraded by the cellular secretory pathway supports the fact the quaternary and tertiary structural stability of A25T are significantly compromised. …”

“…The process of amyloidogenesis is causatively linked to numerous human diseases, many of which are associated with neurodegeneration (1-3). To date, all of the TTR variants associated with familial amyloidosis that have had their folding energetics characterized-21 of them-are destabilized relative to WT TTR, based on the observation that their denaturation transitions occur at lower concentrations of urea than WT TTR (7)(8)(9)(10)(11)(12)(13)(14)(15). TTR is tetrameric under physiological conditions.…”

“…The binding of the small molecule resveratrol to the TTR tetramer was used to assess the quaternary structure of TTR as a function of urea concentration and TTR concentration, as previously described (7,12). Resveratrol binding to tetrameric TTR (occupying at least one of the two thyroxine-binding sites) results in a blue shift in the fluorescence spectrum of resveratrol and a concomitant large increase in its fluorescence quantum yield.…”

“…NIH-PA Author Manuscript NIH-PA Author Manuscript compared and integrated with known dissociation and unfolding rates (7,8,12,13,18,47), in order to gain insight into factors that may lead to amyloid disease. Another advantage of the method is that stability parameters can be derived using only tryptophan fluorescence data.…”

“…A number of autosomal dominant TTR amyloidoses have also been described that are associated with the deposition of variant transthyretin. These disorders have been subcategorized as familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), or central nervous system selective amyloidosis (CNSA), depending on the tissue(s) affected by deposition of the mutant TTR proteins (8,12,13,28,29).The liver secretes TTR into the blood, whereas the choroid plexus secretes TTR into the CSF -secreted TTR from the liver and the choroid appear to be the source of amyloidogenic transthyretin in the periphery and the brain, respectively. Gene therapy mediated by replacing a disease-associated variant TTR/WT TTR expressing liver by a WT TTR/WT TTR expressing liver through transplantation has proven useful as a strategy for treating familial amyloid polyneuropathy (30).…”