Aging and transthyretin-related amyloidosis: pathologic examinations in pulmonary amyloidosis.

Although aging is closely related with the onset of senile systemic amyloidosis (SSA) caused by wild-type transthyretin (TTR), the effect of aging on amyloid formation has remained unclear in familial amyloidotic polyneuropathy (FAP), caused by variant- and wild-type TTR. The aim of this study was to elucidate the effects of aging and/or other factors in FAP on amyloid formation in the lung, one of the most important target organs of amyloid deposition in SSA. Pulmonary amyloid distribution was determined using 19 autopsied lung samples from patients with FAP amyloidogenic TTR (ATTR) V30M, the most common type of FAP. Amyloid deposition was observed around the walls of the bronchi/ bronchioles, the pulmonary arteries, and the pulmonary veins, while no amyloid deposits could be found around the lymphatics. In addition, amyloid deposition in the alveolar regions was a characteristic finding in aged patients with FAP ATTR V30M (average ages of the patients with amyloid positive vs. negative: 50.55 +/- 8.75 vs. 39.75 +/- 4.17 years old, p < 0.005), similar to the finding in one SSA patient. These results suggest that aging could play an important role in the progression of pulmonary amyloid formation in FAP ATTR V30M.

Muscular amyloid angiopathy with amyloidgenic transthyretin Ser50Ile and Tyr114Cys.

Among patients with familial amyloid polyneuropathy (FAP), those with transthyretin Val30Met mainly show distally predominant weakness and atrophy, whereas some FAP patients, including those with transthyretin Ser50Ile and Tyr114Cys, show muscle weakness and atrophy that is dominant proximally, simulating myopathy. To clarify the cause of proximally dominant muscular atrophy in patients with FAP transthyretin Ser50Ile and Tyr114Cys, we investigated the distinctive features of muscle specimens of patients with FAP, 3 of who had Val30Met, 2 Ser50Ile, and 2 Tyr114Cys transthyretin. All specimens showed transthyretin amyloid around blood vessels and perimysium, and neurogenic denervation patterns. The amount of amyloid around the vessels was much greater in patients with FAP Ser50Ile and Tyr114Cys than in Val30Met patients. Muscular amyloid angiopathy may contribute to motor nerve injury that, in turn, may lead to amyotropic changes in patients with FAP Ser50Ile and Tyr114Cys.

A novel tool for detecting amyloid deposits in systemic amyloidosis in vitro and in vivo.

We synthesized (trans,trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB) and used this compound to detect amyloid fibrils in autopsy and biopsy samples from patients with localized amyloidosis, such as familial prion disease, and systemic amyloidosis, such as familial amyloidotic polyneuropathy, amyloid A (AA) amyloidosis, light chain (AL) amyloidosis, and dialysis-related amyloidosis. BSB showed reactions in all Congo red-positive and immunoreactive regions of the samples examined in the study, and some amyloid fibrils in the tissues could be detected more precisely with BSB than with the other methods. In the mouse model of AA amyloidosis, injected BSB reacted with amyloid in all regions in the serial sections in which Congo red staining was positive. A highly sensitive 27-MHz quartz crystal microbalance analysis revealed that BSB showed a significant affinity for amyloid fibrils purified from familial amyloidotic polyneuropathy and dialysis-related amyloidosis samples and suppressed formation of transthyretin amyloid in vitro. These results suggest that BSB may become a valuable tool for detection of amyloid deposits in amyloidosis and of the mechanism of amyloid formation.

Role of VLDL/chylomicron in amyloid formation in familial amyloidotic polyneuropathy.

We examined the affinity of transthyretin (TTR) for lipoproteins and the effect of lipoproteins on TTR-related amyloidogenesis using serum samples from healthy volunteers and patients with familial amyloidotic polyneuropathy (FAP) ATTRVal30Met. In both volunteers and patients, TTR levels were highest in the VLDL fraction containing chylomicrons (VLDL/CM) and next highest in the HDL fraction. Levels were lowest in the LDL fraction. Mass spectrometric analyses of TTR spectra revealed significant TTR association with VLDL/CM and the levels of variant TTR were decreased in the FAP patients. Examination of the affinity of wild-type and variant TTRs for lipoprotein via a quartz crystal microbalance (QCM) revealed the highest affinity of both proteins for VLDL/CM. In in vitro amyloid formation test measured with thioflavin T and electron microscopy, in the presence of VLDL/CM, amyloid formation of TTR was enhanced more than in the presence LDL or in the absence of lipoprotein species. These results suggest that TTR should be highly associated especially with VLDL/CM and amyloidogenicity of TTR should be enhanced around the adipocytes.

Identification of an aging-related spherical inclusion in the human brain.

Inclusions, such as corpora amylacea, axonal spheroids and ubiquitin-positive granular structures, are present in aged brains. We found a phosphorylated tau-positive inclusion in brain tissues obtained from 13 non-demented subjects and five patients with Alzheimer's disease. This inclusion was spherical and 3-20 microm in size. It was most frequently detected in the hippocampal CA1 region and in the prosubiculum but was not present in the white matter. The density of this inclusion increased significantly with aging and decreased after the occurrence of neurofibrillary tangles. The presence of the inclusion was confirmed using immunoelectron microscopy. These findings show a possibility that the inclusion is a novel aging-related structure in the human brain.

Disulfide-bond formation in the transthyretin mutant Y114C prevents amyloid fibril formation in vivo and in vitro.

The Y114C mutation in human transthyretin (TTR) is associated with a particular form of familial amyloidotic polyneuropathy. We show that vitreous aggregates ex vivo consist of either regular amyloid fibrils or disordered disulfide-linked precipitates that maintain the ability to bind Congo red. Furthermore, we demonstrate in vitro that the ATTR Y114C mutant exists in three forms: one unstable but nativelike tetrameric form, one highly aggregated form in which a network of disulfide bonds is formed, and one fibrillar form. The disulfide-linked aggregates and the fibrillar form of the mutant can be induced by heat induction under nonreduced and reduced conditions, respectively. Both forms are recognized by the amyloid specific antibody MAB(39-44). In a previous study, we have linked exposure of this epitope in TTR to a three-residue shift in beta-strand D. The X-ray crystallographic structure of reduced tetrameric ATTR Y114C shows a structure similar to that of the wild type but with a more buried position of Cys10 and with beta-mercaptoethanol associated with Cys114, verifying the strong tendency for this residue to form disulfide bonds. Combined with the ex vivo data, our in vitro findings suggest that ATTR Y114C can lead to disease either by forming regular unbranched amyloid fibrils or by forming disulfide-linked aggregates that maintain amyloid-like properties but are unable to form regular amyloid fibrils.

A novel localized amyloidosis associated with lactoferrin in the cornea.

We report a novel localized amyloidosis associated with lactoferrin. To elucidate the precursor protein of corneal amyloidosis associated with trichiasis, we analyzed amyloid deposits from three patients by histopathology and biochemistry. Amyloid deposits showed immunoreactivity, confirmed by electron microscopy, for only anti-human lactoferrin antibody. Electrophoresis of amyloid fibrils revealed lactoferrin with and without sugar chains; N-terminal sequence analysis revealed full-length lactoferrin and a truncated tripeptide of N-terminal amino acids, Gly-Arg-Arg. Carboxymethylated wild-type lactoferrin formed amyloid fibrils in vitro. Lactoferrin gene analysis in the three patients revealed a Glu561Asp mutation in all of the patients and a compound heterozygote of Ala11Thr and Glu561Asp mutations in one patient. A heterozygotic Glu561Asp mutation appeared in 44.8% of healthy Japanese volunteers, suggesting that the mutation may not be an essential mutation for amyloid formation (p = 0.104). Results thus suggest that lactoferrin is this precursor protein.