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Background: Transthyretin (TTR) amyloidosis is a progressive, life-threatening disorder caused by extracellular deposition of amyloid fibrils derived from the plasma protein TTR. Inherited forms are associated with destabilizing TTR mutations; however, recent findings indicate that amyloid formation in vivo may be promoted by disulfide bond formation between TTR subunits, suggesting oxidative stress as a potential contributor to protein misfolding and disease progression. Glutathione (GSH) is a central component of the antioxidant defense system, and disruption of GSH homeostasis can lead to the accumulation of pyroglutamate (PGA), which is detectable in plasma. Moreover, oxidative stress is frequently linked to inflammation, which may be reflected by increased indoleamine 2,3-dioxygenase 1 (IDO1) activity, observed as an elevated plasma kynurenine/tryptophan ratio.

Methods: Plasma levels of PGA, kynurenine, and tryptophan were quantified by liquid chromatography-mass spectrometry in cohorts comprising healthy TTR wild-type controls, asymptomatic carriers of the TTR-V30M mutation, and symptomatic patients with TTR-V30M amyloidosis.

Results: Symptomatic individuals had significantly elevated plasma PGA levels compared with both asymptomatic carriers and age-matched healthy controls, consistent with impaired GSH homeostasis. In parallel, the kynurenine/tryptophan ratio was increased in symptomatic TTR-V30M carriers, supporting inflammatory activation in manifest disease.

Conclusions: These findings identify disrupted GSH homeostasis and inflammatory activation as metabolic features associated with symptomatic TTR-V30M amyloidosis in vivo, supporting a link between redox imbalance and disease manifestation. Strategies aimed at restoring antioxidant homeostasis and limiting inflammatory oxidative stress may therefore warrant further investigation as approaches to delay onset or slow disease progression.

Transthyretin (TTR) amyloidosis is a progressive disorder characterized by peripheral neuropathy, autonomic dysfunction, and cardiomyopathy. The precise mechanism by which TTR misfolds and forms fibrils in vivo remains incompletely understood, posing challenges to the development of effective therapeutics. In this study, we reveal that the recently identified nonnative pathological species of TTR (NNTTR), which is enriched in the plasma of ttr-val30met gene carriers, exhibits strong amyloidogenic properties, making it a promising therapeutic target. Notably, we demonstrate that NNTTR formation is dependent on an intermolecular disulfide bond and can be promoted by oxidative conditions while being effectively suppressed by reducing agents. The formation of this disulfide bond is incompatible with the native TTR fold, thereby necessitating structural flexibility. We further show that this required flexibility can be constrained using tetramer-stabilizing drugs, thereby suppressing NNTTR formation. Interestingly, the flexibility is also hindered by macromolecular crowding, and NNTTR formation is strongly suppressed by the high protein concentration in plasma. This suppression is released upon dilution, which thus promotes NNTTR formation in areas with lower protein content, highlighting a potential link to the interstitial space, brain, and vitreous body of the eye, where TTR-amyloid is frequently observed. In summary, we demonstrate that NNTTR displays strong amyloidogenic features, underscoring its potential as a therapeutic target. We identify the redox environment and macromolecular crowding as key modulatory factors. Our findings propose a mechanistic explanation for TTR misfolding and suggest a novel therapeutic approach.