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Patients with transthyretin (TTR)-type familial amyloid polyneuropathy (FAP) typically exhibit sensory dominant polyneuropathy and autonomic neuropathy. However, the molecular pathogenesis of the neuropathy remains unclear. In this study, we characterize the features of FAP TTR the substitution of lysine for glutamic acid at position 61 (E61K). This FAP was late-onset, with sensory dominant polyneuropathy, autonomic neuropathy, and cardiac amyloidosis. Interestingly, no amyloid deposits were found in the endoneurium of the four nerve specimens examined. Therefore, we examined the amyloidogenic properties of E61K TTR in vitro. Recombinant wild-type TTR, the substitution of methionine for valine at position 30 (V30M) TTR, and E61K TTR proteins were incubated at 37°C for 72 hr, and amyloid fibril formation was assessed using the thioflavin-T binding assay. Amyloid fibril formation by E61K TTR was less than that by V30M TTR, and similar to that by wild-type TTR. E61K TTR did not have an inhibitory effect on neurite outgrowth from adult rat dorsal root ganglion (DRG) neurons, but V30M TTR did. Furthermore, we studied the sural nerve of our patient by terminal deoxynucleotidyl transferase dUTP nick end labeling and electron microscopy. A number of apoptotic cells were observed in the endoneurium of the nerve by transferase dUTP nick end labeling. Chromatin condensation was confirmed in the nucleus of non-myelinating Schwann cells by electron microscopy. These findings suggest that E61K TTR is low amyloidogenic, in vitro and in vivo. However, TTR aggregates and amyloid fibrils in the DRG may cause sensory impairments in FAP because the DRG has no blood-nerve barrier. Moreover, Schwann cell apoptosis may contribute to the neurodegeneration., (© 2020 International Society for Neurochemistry.)

Transthyretin (TTR) is a homotetrameric protein in human plasma. The dissociation of the TTR tetramer and misfolding of the TTR monomer result in the formation of amyloid fibrils. Hereditary TTR amyloidosis is characterized by the extracellular deposition of amyloid fibrils containing TTR variants. The development of small molecules that kinetically stabilize the TTR tetramer is one of the effective strategies for the treatment of hereditary TTR amyloidosis. So far, several stabilizers have been discovered. Tafamidis is the only approved stabilizer for treatment of hereditary TTR amyloidosis, although two nucleic acid medicines that inhibit TTR synthesis, inotersen and patisiran, were recently approved for treatment of this disorder. In this Perspective, we seek to describe the representative kinetic stabilizers from discovery to development, interweaving the crystallographic study of the complex structures.

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., (© 2019 Ueda et al.)

Amyloidogenic transthyretin (ATTR) amyloidosis is caused by a formation of ATTR amyloid fibrils. Because ATTR misfolding triggers the formation of aggregates and amyloid fibrils, which are considered to deposit on the tissues, novel clinically effective therapeutic strategies targeted to those processes are urgently needed. In this study, to discover a new drug candidate for ATTR amyloidosis therapy, we focused on polyamidoamine dendrimer (dendrimer), a 3D-structural nanomaterial, which has a branched cationic polymer repeating polyamidoamine units. Dendrimer (G2) not only inhibited ATTR V30M amyloid fibril formation, but also reduced already formed ATTR V30M amyloid fibrils by reducing β-sheet structure of ATTR V30M protein. Moreover, intravenous administration of dendrimer (G2) reduced TTR deposition in human ATTR V30M transgenic rats. These results indicate that dendrimer (G2) may possess both inhibitory and breaking effects on ATTR V30M amyloid, suggesting that dendrimer has the potential as a dual effective agents against TTR amyloidosis.

The highly sulfated domains of heparan sulfate (HS), alias HS S-domains, are made up of repeated trisulfated disaccharide units [iduronic acid (2S)-glucosamine (NS, 6S)] and are selectively remodeled by extracellular endoglucosamine 6-sulfatases (Sulfs). Although HS S-domains are critical for signal transduction of several growth factors, their roles in amyloidoses are not yet fully understood. Herein, we found HS S-domains in the kidney of a patient with transthyretin amyloidosis. In in vitro assays with cells stably expressing human Sulfs, heparin, a structural analog of HS S-domains, promoted aggregation of transthyretin in an HS S-domain-dependent manner. Interactions of cells with transthyretin fibrils and cytotoxicity of these fibrils also depended on HS S-domains at the cell surface. Furthermore, glypican-5, encoded by the susceptibility gene for nephrotic syndrome GPC5, was found to be accumulated in the transthyretin amyloidosis kidney. Our study, thus, provides a novel insight into the pathologic roles of HS S-domains in amyloidoses, and we propose that enzymatic remodeling of HS chains by Sulfs may offer an effective approach to inhibiting formation and cytotoxicity of amyloid fibrils., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Transthyretin-related amyloidosis is a slowly progressive disorder caused by deposition of insoluble amyloid plaques formed by fibrillization of mutant or defective transthyretin (TTR) monomers that leads to neurodegeneration and organ failure. Thus, any compound exhibiting TTR amyloid formation inhibitory activity or TTR amyloid fibril disrupting activity might be a potential candidate for the development of therapies for these disorders. Our aim in this study was the evaluation of the TTR amyloid fibril disrupting potential of extracts of leaves and immature fruits of two Juglans plants, i.e., Juglans mandshurica var. sachalinensis and Juglans mandshurica var. cordiformis . The TTR amyloid fibril disrupting activity was measured by Thioflavin-T (ThT) assay and PROTEOSTAT ® Protein aggregation assay methods. A fifty percent acetone extract of the fruits of Juglans mandshurica var. cordiformis showed strong amyloid fibril disrupting activity, and was further fractionated using different solvents. Ethyl acetate and n -butanol fractions showed significant activity in both assays. Syringic acid was isolated and identified as main compound in both of these fractions; however, it did not show any activity. Furthermore, some of the previously reported compounds from Juglans plants including naphthoquinone derivatives and phenolic compounds were evaluated to identify the potential bioactive compounds. Among them, juglone, a naphthoquinone derivative showed promising activity. However, juglone also showed strong cytotoxicity in HEK293 cells. Thus, future studies should focus on the isolation and identification of naphthoquinone derivatives or other compounds from Juglans plan ts with potent bioactivity and low cytotoxicity.

Hereditary transthyretin (TTR)-related amyloidosis is caused by mutations in the TTR gene. The mutations destabilize the tetramer and/or monomer of TTR, and thus the stabilization of TTR is a key strategy for the treatment of TTR-related amyloidosis. In this review, we summarized the natural products and synthetic compounds that have been shown to inhibit the amyloidogenesis of TTR. The stabilizers and/or the amyloid fibril disrupters isolated from natural sources may become lead compounds for the treatment of TTR-related amyloidosis.

Lewy bodies, mainly composed of α-synuclein (αS), are pathological hallmarks of Parkinson's disease and dementia with Lewy bodies. Epidemiological studies showed that green tea consumption or habitual intake of phenolic compounds reduced Parkinson's disease risk. We previously reported that phenolic compounds inhibited αS fibrillation and destabilized preformed αS fibrils. Cumulative evidence suggests that low-order αS oligomers are neurotoxic and critical species in the pathogenesis of α-synucleinopathies. To develop disease modifying therapies for α-synucleinopathies, we examined effects of phenolic compounds (myricetin (Myr), curcumin, rosmarinic acid (RA), nordihydroguaiaretic acid, and ferulic acid) on αS oligomerization. Using methods such as photo-induced cross-linking of unmodified proteins, circular dichroism spectroscopy, the electron microscope, and the atomic force microscope, we showed that Myr and RA inhibited αS oligomerization and secondary structure conversion. The nuclear magnetic resonance analysis revealed that Myr directly bound to the N-terminal region of αS, whereas direct binding of RA to monomeric αS was not detected. Electrophysiological assays for long-term potentiation in mouse hippocampal slices revealed that Myr and RA ameliorated αS synaptic toxicity by inhibition of αS oligomerization. These results suggest that Myr and RA prevent the αS aggregation process, reducing the neurotoxicity of αS oligomers. To develop disease modifying therapies for α-synucleinopathies, we examined effects of phenolic compounds on α-synuclein (αS) oligomerization. Phenolic compounds, especially Myricetin (Myr) and Rosmarinic acid (RA), inhibited αS oligomerization and secondary structure conversion. Myr and RA ameliorated αS synaptic toxicity on the experiment of long-term potentiation. Our results suggest that Myr and RA prevent αS aggregation process and reduce the neurotoxicity of αS oligomers. Phenolic compounds are good candidates of disease modifying drugs for α-synucleinopathies., (© 2015 International Society for Neurochemistry.)

Transthyretin (TTR) is a homotetrameric protein involved in human hereditary amyloidoses. The discovery and development of small molecules that inhibit the amyloid fibril formation of TTR is one of the therapeutic strategies for these diseases. Herein, we discovered that γ-mangostin (γ-M) is an effective inhibitor against the amyloid fibril formation of V30M amyloidogenic TTR. In-vitro binding assays revealed that γ-M was the most potent of the selected xanthone derivatives, and it bound to the thyroxine (T4)-binding sites and stabilized the TTR tetramer. X-ray crystallographic analysis revealed the diagonal binding mode of γ-M and the two binding sites of chloride ions at the T4-binding site. One of the chloride ions was replaced with a water molecule in the α-mangostin complex, which is a methylated derivative of γ-M. The stronger inhibitory potency of γ-M could be explained by the additional hydrogen bonds with the chloride ion. The present study establishes γ-M as a novel inhibitor of TTR fibrillization.

Transthyretin (TTR) amyloid fibril formation, which is triggered by the dissociation of tetrameric TTR, appears to be the causative factor in familial amyloidotic polyneuropathy and senile systemic amyloidosis. Binding of thyroxine (T(4)), a native ligand of TTR, stabilizes the tetramer, but the bioavailability of T(4) for TTR binding is limited due to the preferential binding of T(4) to globulin, the major T(4) carrier in plasma. Here, we show that Cr(3+) increased the T(4)-binding capacity of wild-type (WT) and amyloidogenic V30M-TTR. Moreover, we demonstrate that Cr(3+) and T(4) cooperatively suppressed in vitro fibril formation due to the stabilization of WT-TTR and V30M-TTR.

Transthyretin (TTR) is one of more than 30 amyloidogenic proteins, and the amyloid fibrils found in patients afflicted with ATTR amyloidosis are composed of this protein. Wild‐type TTR amyloids accumulate in the heart in senile systemic amyloidosis (SSA). ATTR amyloidosis occurs at a much younger age than SSA, and the affected individuals carry a TTR mutant. The naturally occurring amyloidogenic Y116S TTR variant forms more amyloid fibrils than wild‐type TTR. Thus, the Y116S mutation reduces the stability of the TTR structure. A neutron diffraction experiment on Y116S TTR was performed to elucidate the mechanism of the changes in structural stability between Y116S variant and wild‐type TTR through structural comparison. Large crystals of the Y116S variant were grown under optimal crystallization conditions, and a single 2.4 mm3 crystal was ultimately obtained. This crystal was subjected to time‐of‐flight (TOF) neutron diffraction using the IBARAKI biological crystal diffractometer (iBIX) at the Japan Proton Accelerator Research Complex, Tokai, Japan (J‐PARC). A full data set for neutron structure analysis was obtained in 14 days at an operational accelerator power of 500 kW. A new integration method was developed and showed improved data statistics; the new method was applied to the reduction of the TOF diffraction data from the Y116S variant. Data reduction was completed and the integrated intensities of the Bragg reflections were obtained at 1.9 Å resolution for structure refinement. Moreover, X‐ray diffraction data at 1.4 Å resolution were obtained for joint neutron–X‐ray refinement. [ABSTRACT FROM AUTHOR]

Transthyretin (TTR)-related familial amyloidotic polyneuropathy, which is induced by amyloidogenic transthyretin (ATTR), is characterized by systemic accumulation of amyloid fibrils. Although it is believed that protein misfolding of monomeric form of TTR is a rate-limiting step for TTR amyloid formation, no effective therapy targeting this misfolding step is available. Our recent studies revealed that cyclodextrins (CyDs), cyclic oligosaccharides composed of glucose units, might interact with TTR and prevent the protein misfolding. In this study, we focused on and elucidated the inhibitory effect of 6-O- a-(4-O-α-d-Glucuronyl)-d-glucosyl-β-CyD (GUG-β-CyD) on TTR amyloid formation. Tryptophan (Trp) fluorescence and 1H-NMR spectroscopy analyses indicated that GUG-β-CyD stabilized TTR conformation via interaction with the hydrophobic amino acids of TTR. Moreover, GUG-β-CyD suppressed TTR deposition in transgenic rats possessing a human ATTR V30M gene in vivo. Collectively, these data indicate that GUG-β-CyD may inhibit TTR misfolding by stabilizing its conformation, which, in turn, suppresses TTR amyloid formation. [ABSTRACT FROM AUTHOR]

Abstract: Aims: Transthyretin (TTR)-related familial amyloidotic polyneuropathy (FAP) is characterized by the systemic accumulation of amyloid fibrils caused by amyloidogenic. Our previous studies demonstrated that albumin played a role in the inhibition of TTR amyloid-formation. The aim of this study was to evaluate the effect of albumin on TTR disposition and tissue deposition in vivo. Main methods: For pharmacokinetic studies, recombinant wild-type TTR (rTTR) and recombinant amyloidogenic TTR Val30Met (rATTR V30M) were labeled with iodine and administered to Sprague–Dawley rats and analbuminemia rats (NAR: Nagase Analbuminemia Rats). The deposition of ATTR V30M was also analyzed by immunohistochemistry in the transgenic (Tg) rats possessing a human ATTR V30M gene (ATTR V30M Tg rats) and NAR possessing a human ATTR V30M gene (ATTR V30M Tg NAR). Key findings: The presence of albumin had no effect on the tissue distribution of either rTTR or rATTR V30M. However, more ATTR V30M was deposited in the hearts, stomachs and small intestines of ATTR V30M Tg NAR rats, compared to ATTR V30M Tg rats. Significance: Although the disposition of TTR and ATTR V30M was unaffected by the presence of albumin, the deposition of ATTR V30M in some organs was apparently increased in the absence of albumin compared to the presence of albumin. These results show that albumin would contribute to suppressing the tissue deposition of TTR in pathogenesis of FAP, but does not affect the disposition of TTR. [Copyright &y& Elsevier]

Amyloid fibril formation is associated with protein misfolding disorders, including neurodegen- erative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases. Familial amyloid polyneuro- pathy (FAP) is a hereditary disease caused by a point mutation of the human plasma protein, transthyretin (TTR), which binds and transports thyroxine (T4). TTR variants contribute to the pathogenesis of amyloi- dosis by forming amyloid fibrils in the extracellular environment. A recent report showed that epigalloca- techin 3-gallate (EGCG), the major polyphenol component of green tea, binds to TFR and suppresses TTR amyloid fibril formation. However, structural analysis of EGCG binding to TTR has not yet been conducted. Here we first investigated the crystal structure of the EGCG-V3OM `FrR complex and found novel binding sites distinct from the thyroxine binding site, suggesting that EGCG has a mode of action different from those of previous chemical compounds that were shown to bind and stabilize the TTR tetramer structure. Furthermore, EGCG induced the oligomerization and monomer suppression in the cellular system of clinically reported TTR variants. Taken together, these findings suggest the possibility that EGCG may be a candidate compound for FAP therapy. [ABSTRACT FROM AUTHOR]