In a recent study posted to the bioRxiv preprint* server, researchers in Sweden investigated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein amyloid as a possible triggering factor for protein misfolding and amyloid formation in neurodegenerative diseases (NDs).

Neurodegenerative diseases reminiscent of Creutzfeldt-Jakob disease (CJD) and Alzheimer’s disease (AD) are accelerated by the conversion of human proteins into misfolded amyloid fibril aggregates. This process is self-perpetuating through seeding from preformed fibril seeds. SARS-CoV-2 S, abundant within the inflammatory response, is a plausible mechanism for amyloid fibril formation.

Study: SARS-CoV-2 Spike amyloid fibrils specifically and selectively accelerates amyloid fibril formation of human prion protein and the amyloid β peptide. Image Credit: Design_Cells / Shutterstock

*Necessary notice: bioRxiv publishes preliminary scientific reports that usually are not peer-reviewed and, due to this fact, mustn’t be considered conclusive, guide clinical practice/health-related behavior, or treated as established information.

Concerning the study

In the current study, researchers performed cross-seeding neurodegenerative disease-related proteins with S protein amyloid fibrils.

Within the in vitro environment, seeding studies were carried out by cross-seeding human prion protein (HuPrP)- and the Aβ1-42 protein-derived with seven distinct amyloid protein fibrils obtained from SARS-CoV-2 S peptides. The researchers examined whether SARS-CoV-2 S protein-derived amyloid protein accelerates the human A peptide and HuPrP fibril formation. Thus, prefabricated fibrils from seven SARS-CoV-2 Wuhan-Hu-1 strain S amyloidogenic sequences (spike seeds)-derived peptides, each 20 amino-acid long, were added, matching the starting protein sequence of the S glycoprotein.

E. Coli was used to make human PrP. To check seeding effect specificity, 10% seed concentrations of various amyloidogenic proteins reminiscent of Aβ1-42, TTR, insulin, and lysozyme were introduced. ThT fluorescence changes with time were used to observe fibrillation. To slow the fibrillation rate of the unseeded HuPrP response and dissolve the S protein peptides, hexafluoro-isopropanol (HFIP) was used. The heterologous seed may be a protein that differs from the substrate by a single point mutation, the identical protein from a special species, or one other protein related to the identical illness.

The seeding effect of S protein amyloids was studied using Aβ1-42 as a substrate. Replicate response samples were obtained for inspection using transmission electron microscopy (TEM) while ThT was monitoring Aβ1-42 fibril development. To match the best concentration of S protein peptide amyloids, the team added control seeds to HuPrP at 0.010 mg/mL concentration. The positive control was the fibrillar type of HuPrP90-231 at 0.0010 mg/mL concentration.

Results

Using an in vitro conversion experiment, the researchers discovered that S protein-amyloid fibril-seeded amyloid production of CJD-associated HuPrP was significantly accelerated. The HuPrP conversion assay was seeded with different in vitro-generated disease-associated amyloid fibrils, and the Aβ1-42 protein was exposed to a panel of control seeds, but no fibril development was accelerated for insulin, lysozyme, or TTR. The findings indicated seeding as a novel property of S protein-amyloid fibrils somewhat than a universal impact.

Positive controls included Aβ1-42 protein fibrils and an amalgamation of seven fibrillated S glycoprotein peptides, and each controls indicated seeding motion. Further, S protein-amyloid fibril seeds enhanced amyloid fibril development in AD-associated Aβ1-42. Spike532 (532NLVKNKCVNFNFNGLTGTGV551) was most successful in seeding HuPrP, whereas Spike601 (601GTNTSNQVAVLYQDVNCTEV620) was simplest in seeding Aβ1-42, demonstrating substrate-dependent selectivity of the cross-seeding activity.

The event of Aβ1-42 fibrils was enhanced by all seven S protein amyloids. Nevertheless, like with HuPrP seeding, there was a substantial differential in seeding efficiency across the varied S protein amyloids, with Aβ1-42 being more attentive to Spike601. The exponential phase of growth was finished for all seeded samples at 120 minutes within the TEM study, whereas the unseeded specimen, lacking fibrillar components but wealthy in oligomeric structures, continued to be at first nucleation phase. Amyloid fibrils were seen in all S peptide amyloid-seeded samples.

The CJD-linked HuPrP and AD-linked Aβ1-42 peptides showed cross-seeding vulnerability with SARSCoV-2 S glycoprotein amyloids. Spike532 amyloid considerably shortened the lag period of amyloid production for HuPrP by a greater extent than other amyloid peptides, but Spike192 amyloid protein didn’t significantly reduce the lag period.

Spike532 seeding significantly reduced the lag period of HuPrP protein fibrillation by 80%, from 712 to 135 minutes on average. The ten-fold spike seed dilution nearly eliminated the seeding abilities of the S protein amyloids. Nevertheless, Spike532 amyloid almost retained its ability to scale back the lag period, with only a ten% reduction in the flexibility by the dilution.

The 0.010 mg/mL SARS-CoV-2 S protein mixture reduced lag duration from 6.75 hours to five.1 hours (25%). Insulin fibrils reduced the lag period of HuPrP protein fibrillation considerably, while TTR, lysozyme, and the Aβ1-42 protein fibrils didn’t. The authors didn’t propose a mechanistic explanation for the lower Aβ1-42/40 ratio amongst SARS-CoV-2-positive individuals, but Aβ1-42 accumulation may be one. All seven investigated SARS-CoV-2 S peptide amyloids significantly reduced the duration of fibril generation, especially in an already swift, unseeded response. Spike532 was probably the most efficient at HuPrP seeding, while Spike601 was probably the most efficient for Aβ1-42 seeding.

Overall, the study findings showed that SARS-CoV-2 S glycoprotein amyloid fibrils preferentially and specifically enhanced the production of HuPrP and amyloid peptide amyloid fibrils. The findings, albeit solely in vitro, indicated that S protein-amyloid fibril cross-seeding could also be involved within the growing variety of cases of CJD, AD, and maybe other NDs within the aftermath of COVID-19.

*Necessary notice: bioRxiv publishes preliminary scientific reports that usually are not peer-reviewed and, due to this fact, mustn’t be considered conclusive, guide clinical practice/health-related behavior, or treated as established information.