Cryo-EM structure of ex vivo fibrils associated with extreme AA amyloidosis prevalence in a cat shelter

Amyloidosis is a systemic disease characterized by the extracellular deposition of misfolded proteins forming amyloid fibrils. AA amyloidosis, specifically, is caused by the misfolding of serum amyloid A protein (SAA), an acute-phase protein involved in inflammation. While AA amyloidosis is observed in humans and animals, some captive animal populations, such as cheetahs and DSH cats in shelters, exhibit extremely high prevalence rates (70% and 57-73%, respectively). This raises the hypothesis of prion-like transmission, where amyloid fibrils can spread between individuals. To better understand the structural basis of this high prevalence in cats, researchers used cryo-electron microscopy (cryo-EM) to determine the high-resolution structure of AA amyloid fibrils extracted from the kidney of a DSH cat with renal failure from a shelter with an exceptionally high prevalence of AA amyloidosis. This structure can then be compared to previously known amyloid structures from other species to identify key differences that might correlate with transmissibility. Understanding the structure is crucial for developing potential therapeutic interventions and gaining insight into the mechanisms driving disease transmission.

Previous research has shown that AA amyloidosis is a common type of amyloidosis in animals and humans, characterized by the deposition of misfolded serum amyloid A protein (SAA). Studies have demonstrated the cross-β sheet architecture of amyloid fibrils. While human and mouse SAA amyloid structures have been determined, significant structural differences between the two species exist despite substantial sequence similarity. The high prevalence of AA amyloidosis in captive cheetahs has been linked to a potential prion-like mechanism of transmission, involving the spread of amyloid fibrils between animals. The finding of SAA in cat bile suggests a potential fecal-oral transmission route, which has also been reported for cheetahs. Understanding the structural basis of AA amyloidosis and its potential for prion-like transmission is essential for developing effective treatment strategies.

A female DSH cat exhibiting clinical signs of renal failure and AA amyloidosis was euthanized, and kidney tissue was collected. Histology and immunofluorescence confirmed the presence of amyloid deposits, which were subsequently extracted. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified the major components as SAA residues 19–111. Cryo-EM was employed to determine the structure of the amyloid fibrils. Specifically, a 4 μL droplet of the fibril sample was applied onto a C-flat grid, blotted, and plunge-frozen in liquid ethane. Cryo-EM data collection was performed using a Talos Arctica 200 kV microscope with a Falcon 3 direct electron detector. Image processing and 3D reconstruction were carried out using RELION 3.1. The final model was built de novo into the cryo-EM map and refined using Coot, Chimera-Isolde, and Phenix. Various bioinformatic tools were used for structural analysis, including calculation of solvation free energy and dissociation energy cost to assess fibril stability. The amino acid sequence of the cat AA amyloid was also compared to sequences from cheetahs. Statistical analysis included comparison of obtained cryo-EM image processing and LC-MS/MS results.

The cryo-EM structure of the cat AA amyloid fibrils revealed a characteristic cross-β architecture composed of two identical 76-residue-long protofilaments. Each protofilament consists of 11 β-strands arranged in an extended hairpin structure. The structure features a unique fold distinct from previously determined human and mouse SAA amyloid structures, despite greater than 70% sequence homology. A crucial aspect is an eight-residue insert unique to feline SAA, which contributes to the increased stability of the amyloid fibrils. This insert is located at the inter-protomer interface, increasing the buried surface area and contributing to the fibril’s stability. Staggered ionic locks and hydrophobic clusters further stabilize the intra- and inter-protomer interfaces. Analysis using structure-based solvation free energy (ΔGsol) calculations indicated a higher stability for the cat AA amyloid compared to human and mouse SAA amyloids. Similarly, the dissociation energy cost (ΔGdiss) for cat AA amyloid was also significantly higher. Importantly, the amino acid sequence of AA amyloid from a cheetah is 99% identical to the cat sequence, also including the eight-residue insert. This strong structural similarity between cat and cheetah AA amyloids supports the hypothesis of potential prion-like transmission of this amyloid disease.

The unique structural features of the feline AA amyloid, particularly the eight-residue insert and its distinct fold, likely contribute to the high prevalence of AA amyloidosis in cat shelters. The increased stability of the feline amyloid fibrils, as indicated by the ΔGsol and ΔGdiss calculations, suggests that these fibrils may be more resistant to degradation and clearance by the host, thereby promoting disease progression. The high degree of sequence similarity between cat and cheetah AA amyloids further supports the potential for prion-like transmission, where the increased stability might facilitate transmission via a fecal-oral route. The high prevalence in cat shelters, in stark contrast to the low prevalence in privately-owned cats, highlights the role of environmental factors and potential transmission mechanisms in crowded populations.

This study provides the first high-resolution structure of ex vivo AA amyloid from a spontaneously occurring case in a domestic animal from a man-made habitat. The unique structural features of the feline AA amyloid, particularly the eight-residue insert, contribute to increased fibril stability and may facilitate transmission in crowded environments. Future research should focus on investigating the precise mechanisms of amyloid transmission and exploring the potential for developing therapeutic strategies targeting the unique structural features of the feline amyloid fibrils.

The study focuses on a single cat, limiting the generalizability of the findings. While the cheetah amyloid sequence is highly similar, the structure remains uncharacterized and might differ. Although Proline-66 is modeled as a cis isomer, the resolution of the cryo-EM map does not definitively confirm this isomerization. Further studies with larger sample sizes and characterization of other affected animals are required to strengthen the conclusions.