Amyloid A Amyloidosis

Abstract

Amyloid and AA Amyloidosis

Amyloidosis is a general term for a wide array of protein misfolding diseases caused by aggregation and deposition of abnormal fibrils in tissue, leading to organ dysfunction.⁷ Amyloid fibrils are composed of polypeptides arranged in a filamentous cross-β structure—put another way, peptides in stacked β-sheets oriented perpendicular to a long axis.^5,9,12,14 The abnormal and insoluble fibrils are the result of overproduction, mutation, fragmentation, seeding, and/or other alteration of the normal and soluble parent protein.⁵⁷ Amyloidoses can be classified as local or systemic and primary or secondary. Amyloid diseases are also categorized by whether they are sporadic, heritable, or infectious (prions). More than 30 amyloid diseases are recognized in people and 10 in animals.⁴⁶

Classic gross and histologic lesions of systemic amyloidosis are well known to pathologists. Grossly, affected tissues appear enlarged, firm, and waxy. Histologically, amyloid appears as extracellular proteinaceous deposits that are eosinophilic and glassy. Congo red dye imparts a red to orange appearance to amyloid, with apple-green birefringence under polarized light. Amyloid can be further confirmed using electron microscopy to identify 7.5- to 10-nm diameter unbranched fibrils in affected tissue.

Amyloid A (AA) amyloidosis is the most common amyloid disease of animals. AA amyloidosis is a secondary amyloidosis that is an uncommon consequence of persistent or cyclic elevation of the acute phase protein serum amyloid A (SAA) associated with chronic inflammatory disease.⁴⁷ SAA is not a single protein but a family of proteins encoded by a highly conserved gene family containing up to 5 SAA genes, depending on the species. SAA1 and SAA2 are the acute phase SAA genes upregulated in response to proinflammatory cytokines. Specifically, SAA transcription is increased in response to proinflammatory cytokines interleukin (IL)–6, IL-1β, and tumor necrosis factor (TNF)–α binding their respective receptors on hepatocytes.⁵² Protein complexes modulated by nuclear factor (NF)–κB and CCAAT enhancer-binding protein (C/EBP)-β and C/EBP-δ induce SAA transcription through NF-κB and C/EBP binding motifs in the SAA1 and SAA2 promoters.^{4,22,23,25,43,52} SAA circulates bound to high-density lipoprotein (HDL) and participates in the immune response and cholesterol transport.^2,3,13,29 Under proinflammatory conditions, circulating SAA levels can increase up to 1000-fold.^3,34 AA amyloidosis occurs when SAA is elevated for a prolonged period and aggregates to form AA amyloid in tissue. Deposition of AA amyloid is often widespread, with kidney, spleen, and liver among the most commonly affected organs, and death is often due to renal failure.^21,30

Genetic Risk Factors for AA Amyloidosis

The triggers and predisposing factors for amyloid aggregation remain incompletely understood in human and veterinary medicine. Many people and animals have chronic inflammatory conditions and prolonged elevation of SAA in serum, yet not all develop AA amyloidosis. Allelic variation and mutations have been associated with greater risk for AA amyloidosis in studies of those with chronic inflammatory diseases. In people with rheumatoid arthritis, variants SAA1.1 (Caucasian) and SAA1.3 (Japanese) and a single nucleotide polymorphism (SNP) at –13(C/T) in the 5′UTR of SAA1 are associated with a greater likelihood of developing AA amyloidosis.^6,35,36,58

In this issue of Veterinary Pathology, Kamiie et al²⁸ present findings that implicate a variant SAA2 gene sequence in AA amyloid disease susceptibility in pigs. The variant was identified using protein sequencing by mass spectrometry of formalin-fixed paraffin-embedded (FFPE), amyloid-laden tissue from a pig with AA amyloidosis and confirmed as a variant of SAA2 by gene sequencing. The variant SAA2 lacked 2 amino acids in the N-terminal domain compared with wild-type SAA2. The study analyzed the characteristics of fibrils generated from synthetic peptides of variant SAA2 compared with wild-type SAA2 and reveals differing fibril morphology. Moreover, fibrils from the variant SAA2 peptides imparted their morphology to fibrils from wild-type SAA2 in in vitro fibrillization studies, suggesting that variant SAA2 can act as a seed or scaffold for wild-type SAA2 to fibrilize. As the variant sequence has only been identified in pigs with AA amyloidosis, the presence of variant SAA2 may influence AA aggregation in pigs. The study is an informative example of how sequence and fibril analysis in a single case can more broadly inform pathogenesis.

AA amyloid aggregation can clearly be influenced by the SAA gene and protein sequence. It is worth noting that genetic elements outside the coding domains of the SAA gene(s) can influence SAA transcription and affect disease. In cheetahs, a species with a high prevalence of AA amyloidosis, a point mutation in the putative NF-κB binding site in the SAA1 promoter results in increased SAA transcription under proinflammatory conditions in vitro and elevated serum SAA in vivo.^15,42,60 Familial Mediterranean fever (FMF) is an autosomal recessive, heritable form of AA amyloidosis in humans associated with 60 different mutations in the pyrin gene. Pyrin acts through the caspase-1 pathway on secretion of IL-1β and activation of NF-κB, both of which modulate SAA transcription.^8,49 In Chinese Shar-pei dogs, a possible model for FMF, a potential modifier locus for AA amyloidosis has been identified on chromosome 14 using genome-wide SNP analysis.^11,40

Further investigation is warranted to elucidate the underlying molecular mechanisms behind AA amyloidosis in the many other animals with a high prevalence of AA amyloidosis. Not only Shar-pei dogs but Abyssinian and Siamese cats and black-footed cats have familial or suspected heritable AA amyloidosis.^24,26,38,51 Black-footed ferrets and island foxes are highly in-bred or lack heterozygosity, and an underlying genetic predisposition for AA amyloidosis is strongly suggested.^18,19,44 In the era of high-throughput gene sequencing and proteomics, in-depth analysis of sequence differences, differential gene expression, and protein profiling holds high promise for identifying disease-associated genetic variants, transcriptional differences, and protein-protein associations that affect disease.

Protein Sequencing by Mass Spectrometry

The utilization of mass spectrometry to identify, type, and sequence amyloid in tissue and tissue sections has grown in the past decade and can be paired with immunohistochemistry (IHC) to increase diagnostic accuracy.²⁰ Owing to the availability of excellent monoclonal antibodies for humans, IHC is often sufficient for a definitive identification of AA amyloidosis. However, such antibodies do not exist for all animal species, and cross-reactivity with human and mouse antibodies is not universal. Mass spectrometry offers the ability to not only identify the type of amyloid in tissue but also sequence the protein and identify the potential variants or isoforms, as done by Kamiie et al.²⁸ A reference proteome is needed, which are increasingly available across species through public databases (http://www.uniprot.org).¹

Beyond the use of mass spectrometry as a diagnostic tool, the research applications are vast, including protein profiling of amyloids and their associated proteins and identifying novel mutations not present in databases with the use of algorithms.¹⁰ Amyloid can be isolated from fresh tissue or from FFPE tissues. Mass spectrometry has been used to sequence AA amyloid in island foxes, identify the amyloid in equine recurrent uveitis as AA, and identify SAA3 as the causative agent of amyloid deposition in the uterus of goats.^16,17,41 The use of laser capture microdissection (LCM) allows isolation of pure amyloid aggregates from specific microanatomical sites from frozen or FFPE sections.^45,54 This technique readily lends itself to characterize amyloid proteins in distinct microenvironments and add to the understanding of how protein-protein interactions influence amyloid tissue tropism. While there are challenges to mass spectrometry, including cost of equipment and expertise in data interpretation, proteomic analysis of amyloidosis in veterinary species is an open field with great potential for discovery.

Seeding and the Prion-Like Hypothesis

The mechanism whereby soluble circulating SAA converts to insoluble aggregated AA fibrils is unknown. Proposed mechanisms include (1) overproduction of the parent SAA protein, (2) mutation resulting in an amyloid-prone SAA protein, (3) enzymatic cleavage to an amyloid-prone protein fragment, and/or (4) seeding and nucleation.^53,57 Seeding and nucleation have been nicely reviewed by Jucker and Walker.²⁷ Briefly, nucleation is the aggregation of misfolded protein fragments into a seed. The seed acts as a scaffold onto which monomers or oligomers (dimers, trimers, etc) of the native protein aggregate, adopting the misfolded conformation and elongating into a growing fibril. The growing fibril can fragment, spontaneously or by cleavage, and generate new seeds, thus becoming self-propagating, or “prion-like.” The introduction of a preformed seed greatly reduces the time required for aggregation, both in vitro and in animal models.

Animal models of AA amyloidosis are well established and use seeding to accelerate disease and study aggregation in vivo. In mice given an inflammatory stimulus, for example, subcutaneous injections of silver nitrate (AgNO₃) or lipopolysaccharide (LPS), serum levels of SAA peak within 1 day and AA amyloid deposition occurs in ≤1 month. The intravenous administration of amyloid enhancing factor (AEF) as a seed decreases the time to amyloid deposition to as little as 2 days. The seed in AEF is the AA fibril itself.³² The accelerated, although less efficient, deposition of AA amyloid has also been shown in mice by cross-seeding with AA fibrils from other species, such as cow, chicken, cheetah, and cat, and from exogenous sources such as silk and fibrils derived from bacteria and fungus.^31,33,37,59

The role of amyloid seeding in AA amyloidosis is examined in this issue of Veterinary Pathology by Watanabe et al.⁵⁶ The authors demonstrate AA amyloid deposition, clearance, and reinduction in the previously reported interleukin 1 receptor antagonist knockout (IL-1raKO) mouse, a model for human rheumatoid arthritis.⁵⁵ In the current study, the authors induce AA amyloidosis with administration of AEF and an inflammatory stimulus (AgNO₃). They then temporally correlate anatomic location and degree of AA amyloid deposits during initial deposition, clearance, and reinduction, with serum SAA levels and serum oligomer levels. They demonstrate that amyloid is partially cleared from tissue following cessation of the inflammatory stimulus and that AA amyloid is redistributed and deposition is more severe upon reinduction with an additional inflammatory stimulus. Notably, the redistribution reliably includes glomerular amyloid deposition, a site of deposition often lacking in other animal models of AA amyloidosis, but one that better represents human end-stage renal disease from AA amyloidosis.³⁹

The authors propose a model of organ-to-organ spread of AA amyloid oligomers through the serum based on pattern of deposition relative to blood flow, the temporal relationship between serum dimer/oligomer levels following initial deposition and during clearance, and the demonstration of AEF-like activity of serum containing SAA oligomers. The model is supported by previous studies that demonstrate tissue redistribution of amyloid deposition on reinduction of amyloidosis and travel through serum of AA aggregates or cleavage products in monocytes or cell-free in exosomes that have AEF activity.^48,50 The present study provides clues as to the possible pathogenesis of early, clinically undetected AA amyloid deposition in contrast to the later, more clinically relevant deposition in glomeruli. The reproducible pattern of tissue deposition in the reinduced phase of AA amyloid deposition could also represent a model system in which to study tissue tropism.

In summary, AA amyloidosis is a debilitating, often fatal misfolded protein disease widely recognized across animal species. Recent advances in our knowledge about the influence of seeding and genetic variation on disease progression have added to our understanding of pathogenesis, but there is still much that is unknown. Seeding of AA amyloidosis has been shown in vitro and only in animal models with experimentally induced elevation of SAA. It remains to be seen if seeding is a naturally occurring phenomenon.

SAA gene and protein sequence can play a role in disease. Identification of the underlying molecular mechanism will require examination of the multitude of components governing the inflammatory stimulus, the acute phase response, the SAA transcription pathway, and the downstream protein-protein interactions of SAA. The application of high-throughput gene sequencing and proteomic analyses to AA amyloidosis in veterinary species will undoubtedly uncover new pathways and genetic variants that will contribute to understanding the pathogenesis of AA amyloidosis and amyloid disease as a whole.

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