A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH.

Transthyretin (TTR) is a major amyloidogenic protein associated with hereditary (ATTRm) and nonhereditary (ATTRwt) intractable systemic transthyretin amyloidosis. The pathological mechanisms of ATTR-associated amyloid fibril formation are incompletely understood, and there is a need for identifying compounds that target ATTR. C-terminal TTR fragments are often present in amyloid-laden tissues of most patients with ATTR amyloidosis, and on the basis of in vitro studies, these fragments have been proposed to play important roles in amyloid formation. Here, we found that experimentally-formed aggregates of full-length TTR are cleaved into C-terminal fragments, which were also identified in patients' amyloid-laden tissues and in SH-SY5Y neuronal and U87MG glial cells. We observed that a 5-kDa C-terminal fragment of TTR, TTR81-127, is highly amyloidogenic in vitro, even at neutral pH. This fragment formed amyloid deposits and induced apoptosis and inflammatory gene expression also in cultured cells. Using the highly amyloidogenic TTR81-127 fragment, we developed a cell-based high-throughput screening method to discover compounds that disrupt TTR amyloid fibrils. Screening a library of 1280 off-patent drugs, we identified two candidate repositioning drugs, pyrvinium pamoate and apomorphine hydrochloride. Both drugs disrupted patient-derived TTR amyloid fibrils ex vivo, and pyrvinium pamoate also stabilized the tetrameric structure of TTR ex vivo in patient plasma. We conclude that our TTR81-127-based screening method is very useful for discovering therapeutic drugs that directly disrupt amyloid fibrils. We propose that repositioning pyrvinium pamoate and apomorphine hydrochloride as TTR amyloid-disrupting agents may enable evaluation of their clinical utility for managing ATTR amyloidosis.

A novel age-related venous amyloidosis derived from EGF-containing fibulin-like extracellular matrix protein 1.

Most intractable tissue-degenerative disorders share a common pathogenic condition, so-called proteinopathy. Amyloid-related disorders are the most common proteinopathies and are characterized by amyloid fibril deposits in the brain or other organs. Aging is generally associated with the development of these amyloid-related disorders, but we still do not fully understand how functional proteins become pathogenic amyloid deposits during the human aging process. We identified a novel amyloidogenic protein, named epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1), in massive venous amyloid deposits in specimens that we obtained from an autopsied patient who died of gastrointestinal bleeding. Our postmortem analyses of additional patients indicate that EFEMP1 amyloid deposits frequently developed in systemic venous walls of elderly people. EFEMP1 was highly expressed in veins, and aging enhanced venous EFEMP1 expression. In addition, biochemical analyses indicated that these venous amyloid deposits consisted of C-terminal regions of EFEMP1. In vitro studies showed that C-terminal regions formed amyloid fibrils, which inhibited venous tube formation and cell viability. EFEMP1 thus caused a novel age-related venous amyloid-related disorder frequently found in the elderly population. Understanding EFEMP1 amyloid formation provides new insights into amyloid-related disorders occurring during the aging process. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Extreme Adhesion Activity of Amyloid Fibrils Induces Subcutaneous Insulin Resistance.

Insulin-derived amyloidoma, also called an insulin ball, is a skin-related complication of insulin therapy caused by repeated insulin injections at the same site, where native folded insulin changes into amyloid fibrils and forms a mass with a granulomatous reaction. Insulin-derived amyloidoma is a clinically important condition because of its association with subcutaneous insulin resistance, but the precise effect and mechanism of the insulin absorption impairment have not been clarified. We generated insulin-derived amyloidomas in mouse skin, with the amyloidomas large enough to perform insulin tolerance tests in the mass by repeated injections of highly concentrated insulin amyloid fibrils. We demonstrated that the insulin-derived amyloidomas inhibit insulin absorption. By simultaneous administration of insulin and insulin amyloid fibrils, we showed that this effect is due to the amyloid fibril itself in the absence of a granulomatous reaction. In vitro studies revealed that insulin amyloid fibrils have extremely strong adhesion to native human insulin and various insulin analogs. Furthermore, we showed that native insulin that had adhered to insulin amyloid forms amyloid fibrils at physiological pH. These results suggest that the extreme adhesion of insulin amyloid to native insulin is the main mechanism of impaired insulin absorption and amyloidoma growth.

New simple and quick method to analyze serum variant transthyretins: direct MALDI method for the screening of hereditary transthyretin amyloidosis.

BACKGROUND: Hereditary transthyretin amyloidosis (ATTRv amyloidosis) is caused by a variant transthyretin (TTR), which is a serum protein secreted by the liver. Mass spectrometry (MS) is a useful tool that can detect variant TTRs in serum samples from patients with ATTRv amyloidosis. We previously reported several mass spectrometric methods to detect variant TTRs in serum samples. Those methods require cumbersome immunoprecipitation with anti-TTR antibodies and significant time to analyze the variant TTRs. In our study here, we developed a new simple and quick method to detect variant TTRs in serum samples by means of matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) MS without immunoprecipitation (direct MALDI). METHODS: By using direct MALDI, we analyzed 288 serum samples obtained from patients who were clinically suspected having amyloidosis to investigate the usefulness of this direct MALDI method to detect variant TTRs in serum samples. RESULTS: The method completed the process within 30 min. We successfully identified variant TTRs in serum samples from patients, except for a few patients with TTR Glu61Lys and Glu89Gln mutations because of the small mass shift of those variant TTRs from wild-type TTR. We also found that the mass shifts of variant TTRs measured by direct MALDI corresponded to theoretical mass changes. CONCLUSION: Our results suggest that the direct MALDI method is useful for the screening of ATTRv amyloidosis.