Transthyretin variants impact blood–nerve barrier and neuroinflammation in amyloidotic neuropathy

Abstract Hereditary transthyretin amyloidosis with polyneuropathy (ATTRv-PN) is a neurodegenerative disease caused by mutations in the gene encoding transthyretin (TTR). Although amyloid deposition is pathognomonic for diagnosis, this pathology in nervous tissues cannot fully account for nerve degeneration, implying additional pathophysiology for neurodegeneration, which, however, has not yet been elucidated fully. In this study, neuroinflammation in ATTRv-PN was investigated by examining nerve morphometry, the blood–nerve barrier and macrophage infiltration in the sural nerves of ATTRv-PN patients and the sciatic nerves of a complementary mouse system, i.e. the humanized knock-in hTTRA97S mouse. The direct effects of mutant TTR proteins were evaluated in these hTTRA97S mice in vivo and in a human umbilical vein endothelial cell (HUVEC) model in vitro. This case–control and cross-sectional study included 19 patients [17 men; 62.9 ± 3.9 years of age; familial amyloid polyneuropathy (FAP) stage 1, n = 11; FAP stage 2, n = 7; FAP stage 3, n = 1] with p.Ala117Ser (A97S) and 46 patients (39 men; 65.3 ± 11.4 years of age; FAP stage 1, n = 31; FAP stage 2, n = 12; FAP stage 3, n = 3) with p.Val50Met (V30M). Both genotypes had elevated protein in the CSF: 88.9% (16 cases in 18 patients) in A97S and 51.1% (23 cases in 45 patients) in V30M. The myelinated nerve fibres in sural nerves were markedly depleted in ATTRv-PN, and the myelinated nerve fibre density was inversely correlated with CSF protein, implying leakage of the blood–nerve barrier. The tight junction ultrastructure of the endoneurial microvessels in sural nerves was impaired, as indicated by the reduced expression of zonula occludens-1 (ZO-1). The cultured HUVECs that were not transfected with any TTR gene variant presented reduced ZO-1 expression when exposed to mutant recombinant TTR of A97S or V30M compared with wild-type TTR. Increased infiltration of macrophages with expression of the inflammasome maker, NLR family pyrin domain containing 3 (NLRP3), suggested that polarization to the pro-inflammatory M1 lineage was robust in the sural nerves of ATTRv-PN patients and the sciatic nerves of hTTRA97S mice compared with those of controls and wild-type mice. In parallel, the mRNA expression of interleukin-1β was greater in the sural nerves of ATTRv-PN than in those of the controls. In conclusion, the disrupted blood–nerve barrier attributable to mutant TTR protein resulting in an increased CSF protein level was associated with nerve degeneration in ATTRv-PN via the infiltration of inflammatory macrophages and the production of inflammatory cytokines.