Cryo-EM confirms a common fibril fold in the heart of four patients with ATTRwt amyloidosis.

ATTR amyloidosis results from the conversion of transthyretin into amyloid fibrils that deposit in tissues causing organ failure and death. This conversion is facilitated by mutations in ATTRv amyloidosis, or aging in ATTRwt amyloidosis. ATTRv amyloidosis exhibits extreme phenotypic variability, whereas ATTRwt amyloidosis presentation is consistent and predictable. Previously, we found unique structural variabilities in cardiac amyloid fibrils from polyneuropathic ATTRv-I84S patients. In contrast, cardiac fibrils from five genotypically different patients with cardiomyopathy or mixed phenotypes are structurally homogeneous. To understand fibril structure's impact on phenotype, it is necessary to study the fibrils from multiple patients sharing genotype and phenotype. Here we show the cryo-electron microscopy structures of fibrils extracted from four cardiomyopathic ATTRwt amyloidosis patients. Our study confirms that they share identical conformations with minimal structural variability, consistent with their homogenous clinical presentation. Our study contributes to the understanding of ATTR amyloidosis biopathology and calls for further studies.

Amyloid fibril polymorphism in the heart of an ATTR amyloidosis patient with polyneuropathy attributed to the V122Δ variant.

ATTR amyloidosis is a phenotypically heterogeneous disease characterized by the pathological deposition of transthyretin in the form of amyloid fibrils into various organs. ATTR amyloidosis may stem from mutations in variant (ATTRv) amyloidosis, or aging in wild-type (ATTRwt) amyloidosis. ATTRwt generally manifests as a cardiomyopathy phenotype, whereas ATTRv may present as polyneuropathy, cardiomyopathy, or mixed, in combination with many other symptoms deriving from secondary organ involvement. Over 130 different mutational variants of transthyretin have been identified, many of them being linked to specific disease symptoms. Yet, the role of these mutations in the differential disease manifestation remains elusive. Using cryo-electron microscopy, here we structurally characterized fibrils from the heart of an ATTRv patient carrying the V122Δ mutation, predominantly associated with polyneuropathy. Our results show that these fibrils are polymorphic, presenting as both single and double filaments. Our study alludes to a structural connection contributing to phenotypic variation in ATTR amyloidosis, as polymorphism in ATTR fibrils may manifest in patients with predominantly polyneuropathic phenotypes.

ATTRv-V30M Type A amyloid fibrils from heart and nerves exhibit structural homogeneity.

ATTR amyloidosis is a systemic disease characterized by the deposition of amyloid fibrils made of transthyretin, a protein integral to transporting retinol and thyroid hormones. Transthyretin is primarily produced by the liver and circulates in blood as a tetramer. The retinal epithelium also secretes transthyretin, which is secreted to the vitreous humor of the eye. Because of mutations or aging, transthyretin can dissociate into amyloidogenic monomers triggering amyloid fibril formation. The deposition of transthyretin amyloid fibrils in the myocardium and peripheral nerves causes cardiomyopathies and neuropathies, respectively. Using cryo-electron microscopy, here we determined the structures of amyloid fibrils extracted from cardiac and nerve tissues of an ATTRv-V30M patient. We found that fibrils from both tissues share a consistent structural conformation, similar to the previously described structure of cardiac fibrils from an individual with the same genotype, but different from the fibril structure obtained from the vitreous humor. Our study hints to a uniform fibrillar architecture across different tissues within the same individual, only when the source of transthyretin is the liver. Moreover, this study provides the first description of ATTR fibrils from the nerves of a patient and enhances our understanding of the role of deposition site and protein production site in shaping the fibril structure in ATTRv-V30M amyloidosis.

Cryo-EM confirms a common fibril fold in the heart of four patients with ATTRwt amyloidosis.

ATTR amyloidosis results from the conversion of transthyretin into amyloid fibrils that deposit in tissues causing organ failure and death. This conversion is facilitated by mutations in ATTRv amyloidosis, or aging in ATTRwt amyloidosis. ATTRv amyloidosis exhibits extreme phenotypic variability, whereas ATTRwt amyloidosis presentation is consistent and predictable. Previously, we found an unprecedented structural variability in cardiac amyloid fibrils from polyneuropathic ATTRv-I84S patients. In contrast, cardiac fibrils from five genotypically-different patients with cardiomyopathy or mixed phenotypes are structurally homogeneous. To understand fibril structure's impact on phenotype, it is necessary to study the fibrils from multiple patients sharing genotype and phenotype. Here we show the cryo-electron microscopy structures of fibrils extracted from four cardiomyopathic ATTRwt amyloidosis patients. Our study confirms that they share identical conformations with minimal structural variability, consistent with their homogenous clinical presentation. Our study contributes to the understanding of ATTR amyloidosis biopathology and calls for further studies.

Post-translational modification of amyloid a protein in patients with AA amyloidosis.

AA amyloidosis is a disease caused by extracellular deposition of insoluble ß-pleated sheet fibrils composed of amyloid A (AA) protein, an amino (N)-terminal fragment of serum amyloid A (SAA). The deposits disrupt tissue structure and compromise organ function. Although the disease is systemic, deposition in kidney glomeruli is the most common manifestation. The leading cause of AA amyloidosis is sustained or recurrent inflammation accompanied by elevated levels of SAA. Factors determining the conversion of SAA to AA amyloid fibrils have yet to be fully resolved. Herein, we present liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis of AA proteins purified from eight patients with AA amyloidosis. For the first time, post-translational modifications (PTM), including carbamylation, acetylation and oxidation, were identified on AA peptides; all eight samples showed some degree of PTM. The amyloid in 6 samples comprised peptides derived from SAA1 with few or none from SAA2, while the other two samples contained both SAA1- and SAA2-derived peptides. N-terminal AA peptides beginning with Arg1 as well as AA peptides starting with Ser2 were present in five of the eight samples, while all or nearly all of the N-terminal peptides in the other three samples lacked Arg1. These data demonstrate that multiple species of AA amyloid proteins can comprise the subunits in amyloid fibrils and raise the possibility that PTM may play a role in fibrillogenesis.

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 transgenic mouse model reproduces human hereditary systemic amyloidosis.

Amyloidoses are rare life-threatening diseases caused by protein misfolding of normally soluble proteins. The fatal outcome is predominantly due to renal failure and/or cardiac dysfunction. Because amyloid fibrils formed by all amyloidogenic proteins share structural similarity, amyloidoses may be studied in transgenic models expressing any amyloidogenic protein. Here we generated transgenic mice expressing an amyloidogenic variant of human apolipoprotein AII, a major protein of high density lipoprotein. According to amyloid nomenclature this variant was termed STOP78SERApoAII. STOP78SER-APOA2 expression at the physiological level spontaneously induced systemic amyloidosis in all mice with full-length mature STOP78SER-ApoAII identified as the amyloidogenic protein. Amyloid deposits stained with Congo red were extracellular, and consisted of fibrils of approximately 10 nm diameter. Renal glomerular amyloidosis was a major feature with onset of renal insufficiency occurring in mice older than six months of age. The liver, heart and spleen were also greatly affected. Expression of STOP78SER-APOA2 in the liver and intestine in mice of the K line but not in other amyloid-laden organs showed they present systemic amyloidosis. The amyloid burden was a function of STOP78SER-APOA2 expression and age of the mice with amyloid deposition starting in two-month-old high-expressing mice that died from six months onwards. Because STOP78SER-ApoAII conserved adequate lipid binding capacity as shown by high STOP78SER-ApoAII amounts in high density lipoprotein of young mice, its decrease in circulation with age suggests preferential deposition into preformed fibrils. Thus, our mouse model faithfully reproduces early-onset hereditary systemic amyloidosis and is ideally suited to devise and test novel therapies.

Carbamylation of the amino-terminal residue (Gly1) of mouse serum amyloid A promotes amyloid formation in a cell culture model.

Amyloid A (AA) amyloidosis is a fatal protein deposition disease afflicting a small percentage of patients with chronic inflammation. Factors other than inflammation that determine development of AA amyloidosis remain largely unknown. The subunit protein comprising AA amyloid fibrils is derived from serum amyloid A (SAA), specifically its amino-terminal portion. In this in vitro study, carbamylation of residues in this region (primarily Gly1 but also Lys24) was shown to markedly increase amyloid-forming propensity as judged by extensive accumulation of amyloid in cell cultures. Contrastingly, no amyloid deposition occurred in cultures given SAA having a noncarbamylated amino terminus. Carbamylation, known to occur during uremia or inflammation, merits investigation as a potential determinant of AA amyloid fibril formation.

Hereditary systemic immunoglobulin light-chain amyloidosis.

Several members of a family died from renal failure as a result of systemic amyloidosis. Extensive studies to detect previously documented gene mutations associated with amyloidosis failed to identify a causative factor. In search of the genetic basis for this syndrome, amyloid fibrils were isolated from renal tissue of a member of the kin who died while on renal dialysis. Amino acid sequencing of isolated amyloid protein identified sequences compatible with the constant region of the immunoglobulin κ light-chain. Isolation and characterization of κ light-chain protein from serum of an affected member of the kindred revealed mutation in the constant region of κ light-chain, with cysteine replacing serine at amino acid residue 131. This mutation (Ser131Cys) was confirmed by DNA analysis, which identified a single-base change of cytosine to guanine at the second position of codon 131 of the κ light-chain gene (TCT131TGT). DNA analysis of members of the extended family revealed transmission of the Ser131Cys mutation and association with systemic amyloidosis. This amyloid light-chain (AL) amyloidosis, which is a hereditary type of amyloidosis and not the result of a monoclonal plasma cell dyscrasia, may be misdiagnosed and lead to inappropriate chemotherapy.

Characterization of rat serum amyloid A4 (SAA4): a novel member of the SAA superfamily.

The serum amyloid A (SAA) family of proteins is encoded by multiple genes, which display allelic variation and a high degree of homology in mammals. The SAA1/2 genes code for non-glycosylated acute-phase SAA1/2 proteins, that may increase up to 1000-fold during inflammation. The SAA4 gene, well characterized in humans (hSAA4) and mice (mSaa4) codes for a SAA4 protein that is glycosylated only in humans. We here report on a previously uncharacterized SAA4 gene (rSAA4) and its product in Rattus norvegicus, the only mammalian species known not to express acute-phase SAA. The exon/intron organization of rSAA4 is similar to that reported for hSAA4 and mSaa4. By performing 5'- and 3'RACE, we identified a 1830-bases containing rSAA4 mRNA (including a GA-dinucleotide tandem repeat). Highest rSAA4 mRNA expression was detected in rat liver. In McA-RH7777 rat hepatoma cells, rSAA4 transcription was significantly upregulated in response to LPS and IL-6 while IL-1α/ß and TNFα were without effect. Luciferase assays with promoter-truncation constructs identified three proximal C/EBP-elements that mediate expression of rSAA4 in McA-RH7777 cells. In line with sequence prediction a 14-kDa non-glycosylated SAA4 protein is abundantly expressed in rat liver. Fluorescence microscopy revealed predominant localization of rSAA4-GFP-tagged fusion protein in the ER.

Human SAA1-derived amyloid deposition in cell culture: a consistent model utilizing human peripheral blood mononuclear cells and serum-free medium.

Amyloid A (AA) amyloidosis is a fatal disease caused by extracellular deposition of fibrils derived from serum AA (SAA). AA amyloid fibril formation has previously been modeled in macrophage cultures using highly amyloidogenic mouse SAA1.1, but attempts to do the same with human SAA invariably failed. Our objective was to define conditions that support human SAA-derived amyloid formation in peripheral blood mononuclear cell (PBMC) cultures. Two conditions were found to be critical - omission of fetal calf serum and use of StemPro34, a lipid-enriched medium formulated for hematopoietic progenitor cells. Cultures maintained in serum-free StemPro34 and provided with recombinant human SAA1 in the complete absence of amyloid-enhancing factor exhibited amyloid deposition within 7 d. Amyloid co-localized with cell clusters that characteristically included cells of fibrocytic/dendritic morphology as well as macrophages. These cells formed networks that appeared to serve as scaffolding within and upon which amyloid accumulated. Cells in amyloid-forming cultures demonstrated increased adherence, survival and expression of extracellular matrix components. Of the three human SAA1 isoforms, SAA1.3 showed the most extensive amyloid deposition, consistent with it being the most prevalent isoform in Japanese patients with AA amyloidosis. Attesting to the reproducibility and general applicability of this model, amyloid formation has been documented in cultures established from eight PBMC donors.

Antisense oligonucleotide suppression of serum amyloid A reduces amyloid deposition in mice with AA amyloidosis.

AA amyloid patients who experience disease progression and develop renal failure have not received sufficient benefit from agents that treat inflammation or infection. We have begun to explore the potential application of antisense oligonucleotides (ASOs) to specifically suppress SAA production and thereby reduce amyloid deposition. Proof-of-concept experiments conducted in mice initially examined ASO ability to reduce serum levels of SAA during an acute inflammatory response. Peak SAA levels in ASO-treated mice were reduced as much as 65% relative to levels in saline-treated mice. The extent of suppression was dose-dependent and influenced by the time interval between ASO administration and inflammatory stimulation. Subsequent experiments tested whether ASO suppression of SAA was sufficient to mitigate amyloid deposition. Amyloidosis was induced by amyloid-enhancing factor and silver nitrate injection; ASO treatment was initiated 1 week later and continued 1× or 3× per week; inflammation was re-triggered by subsequent injection(s) of silver nitrate; mice were sacrificed after 4-5 weeks. Examination of tissues by Congo red staining and SAA/AA immunohistochemistry revealed consistently less amyloid in the organs of ASO-treated mice compared to saline-treated counterparts. These findings provide rationale for further investigation of SAA-specific ASOs as a potential therapy for AA amyloidosis.

Intravenous cell therapy for acute renal failure with serum amyloid A protein-reprogrammed cells.

Serum amyloid A protein (SAA), a prominent component of the acute-phase response, is strongly expressed in developing and repairing kidneys and promotes tubulogenesis. Accordingly, we reprogrammed relatively undifferentiated NRK52E cells with the mouse SAA1.1 gene and transplanted SAA-positive and -negative cells into rats with acute renal failure. We found that SAA-positive cells accelerated renal recovery in three models of acute renal failure: gentamicin nephrotoxicity, cisplatin-mediated renal injury, and ischemia-reperfusion renal injury. The dramatic improvement of renal failure was demonstrable within 2 days, consistent with an early paracrine effect. However, abundant donor cells were also found integrated in the healing tubular architecture after 7 days. We conclude that infusions of SAA-positive cells promote renal recovery after acute renal failure and offer a potentially powerful and novel therapy of renal failure.

Suppression of choroid plexus transthyretin levels by antisense oligonucleotide treatment.

Leptomeningeal amyloidosis associated with mutations in transthyretin (TTR) is a rare but fatal form of amyloidosis. Dementia and intracerebral haemorrhage are prominent features of this disease for which no specific therapy is known. In previous studies, we have shown that antisense oligonucleotides (ASOs) specific for human TTR could inhibit hepatic synthesis of TTR in mice transgenic for a human amyloid-associated TTR and may offer a medical means of treating systemic TTR amyloidosis. Parenteral administration of TTR-specific ASO, however, had no effect on the expression of TTR by the choroid plexus, which is believed to be the source of the amyloid protein in patients who have leptomeningeal amyloidosis. In the present study, mice transgenic for the human TTR amyloid-associated mutation Ile84Ser were treated by administration of TTR-specific ASO (50 microg or 75 microg per day) via an osmotic pump into the cerebral ventricular system over a 4-week period. Intraventricular administration of TTR-specific ASO significantly reduced choroid human TTR mRNA levels, and these findings correlated with decreased TTR in choroid plexus epithelial cells as demonstrated by immunohistochemistry. Suppression of choroid TTR expression by intraventricular administered ASO may offer a medical means of treating leptomeningeal amyloidosis.

Acute-phase response protein serum amyloid A stimulates renal tubule formation: studies in vitro and in vivo.

Serum amyloid A protein (SAA) surges 1,000-fold in the blood of acute-phase animals, and yet its function during these acute events remains unknown. We report herein that SAA stimulates a developmental program in cultured NRK-52E cells that culminates in differentiated and functional tubules that feature a proximal tubule phenotype. We also found strong SAA expression in states of tubule formation (in utero stage) and regeneration (recovery from ischemia-reperfusion injury). These data lend support to a novel view of a more localized renal acute-phase reaction, where renal SAA may act as a paracrine or autocrine molecule that promotes tubule formation during development and repair.

Leukocyte chemotactic factor 2: A novel renal amyloid protein.

Renal amyloid deposits can often be seen in primary amyloidosis (immunoglobulin light chain disease) or in secondary forms such as reactive amyloidosis as well as in several hereditary forms where a variety of mutant proteins 'precipitate' as amyloid plaques. However, in rare cases, amyloidosis may be identified by renal biopsy, but no definitive diagnosis could be made. We have isolated amyloid fibrils from such a case in which the patient presented with nephrotic syndrome and subsequent azotemia requiring hemodialysis. Evaluation for amyloid deposition in other organ systems was negative and immunohistochemical analysis of the kidney deposits for known contributing proteins was unrevealing. Biochemical analysis of the fibrils identified a new amyloid subunit protein, leukocyte chemotactic factor 2, originally identified as a possible chemotactic and growth factor. A monoclonal antibody to this protein reacted specifically with the amyloid deposits in the glomeruli and interstitium by immunohistochemistry. This study emphasizes the importance of biochemical characterization of amyloid present in renal biopsies.

Cellular events associated with the initial phase of AA amyloidogenesis: insights from a human monocyte model.

Reactive amyloidosis is a systemic protein deposition disease that develops in association with chronic inflammation. The deposits are composed of extracellular, fibrillar masses of amyloid A (AA) protein, an N-terminal fragment of the acute-phase serum protein serum amyloid A (SAA). The pathogenic conversion of SAA into amyloid has been studied in two human cell culture models, peritoneal cells and peripheral blood monocytes. Human monocyte cultures proved more robust than either mouse or human peritoneal cells at initiating amyloid formation in the absence of a preformed nidus such as amyloid-enhancing factor and particularly well suited for examination of individual cells undergoing amyloid formation. Amyloid-producing monocyte cultures were stained with Congo red and Alcian blue for detection of amyloid and glycosaminglycans, respectively; immunocytochemistry was performed to identify SAA/AA, CD68, CD14, lysosomal protein Lamp-1, and early endosomal protein EEA1. SAA interaction with monocytes was also visualized directly via fluorescence confocal microscopy. Amyloid was initially detected only in intracellular vesicles, but with time was seen extracellularly. Morphologic changes in lysosomes were noted during the early phase of amyloid formation, suggesting that exocytosis of fibrils may occur via lysosome-derived vesicles. Cultures engaged in amyloid formation remained metabolically active; no cytotoxic effects were observed. Mimicking in vivo phenomena, amyloid formation was accompanied by increased glycosaminoglycan content and C-terminal processing of SAA. The ability of human monocytes to endocytose and intracellularly transform SAA into amyloid via a mechanism that requires and maintains, rather than compromises, metabolic activity distinguishes them as a useful model for probing earliest events in the disease process.

Aging in heterozygous Dnmt1-deficient mice: effects on survival, the DNA methylation genes, and the development of amyloidosis.

We previously reported that heterozygous DNA methyltransferase 1-deficient (Dnmt1(+/-)) mice maintain T-cell immune function and DNA methylation levels with aging, whereas controls develop autoimmunity, immune senescence, and DNA hypomethylation. We therefore compared survival, cause of death, and T-cell DNA methylation gene expression during aging in Dnmt1(+/-) mice and controls. No difference in longevity was observed, but greater numbers of Dnmt1(+/-) mice developed jejunal apolipoprotein AII amyloidosis. Both groups showed decreased Dnmt1 expression with aging. However, expression of the de novo methyltransferases Dnmt3a and Dnmt3b increased with aging in stimulated T cells from control mice. MeCP2, a methylcytosine binding protein that participates in maintenance DNA methylation, increased with age in Dnmt1(+/-) mice, suggesting a mechanism for the sustained DNA methylation levels. This model thus provides potential mechanisms for DNA methylation changes of aging, and suggests that changes in DNA methylation may contribute to some forms of amyloidosis that develop with aging.

Targeted suppression of an amyloidogenic transthyretin with antisense oligonucleotides.

Transthyretin (TTR) amyloidosis, the most common form of hereditary systemic amyloidosis, is characterized clinically by adult-onset axonal neuropathy and restrictive cardiomyopathy. More than 85 mutations in transthyretin have been found to cause this hereditary disease. Since essentially all circulating TTR is of hepatic origin, orthotopic liver transplantation has been used as the only specific form of therapy. Unfortunately, in many patients amyloid deposition continues after orthotopic liver transplantation, indicating that mutant TTR is no longer required for progression of the disease after tissue deposits have been initiated. As a first step toward medical treatment of this disease, we have employed antisense oligonucleotides (ASOs) to inhibit hepatic expression of TTR. A transgenic mouse model carrying the human TTR Ile84Ser mutation was created and shown to express high levels of human mutant transthyretin. TTR ASOs suppressed hepatic TTR mRNA levels and serum TTR levels by as much as 80%. Suppression of hepatic synthesis of transthyretin may offer a medical treatment for transthyretin systemic amyloidosis.