An increasing number of reports suggest an association between COVID-19 infection and initiation or acceleration of neurodegenerative diseases (NDs) including Alzheimer’s disease (AD) and Creutzfeldt-Jakob disease (CJD). Both these diseases and several other NDs are caused by conversion of human proteins into a misfolded, aggregated amyloid fibril state. The fibril formation process is self-perpetuating by seeded conversion from preformed fibril seeds. We recently described a plausible mechanism for amyloid fibril formation of SARS-CoV-2 spike protein. Spike-protein formed amyloid fibrils upon cleavage by neutrophil elastase, abundant in the inflammatory response to COVID-19 infection. We here provide evidence of significant Spike-amyloid fibril seeded acceleration of amyloid formation of CJD associated human prion protein (HuPrP) using an in vitro conversion assay. By seeding the HuPrP conversion assay with other in vitro generated disease associated amyloid fibrils we demonstrate that this is not a general effect but a specific feature of spike-amyloid fibrils. We also showed that the amyloid fibril formation of AD associated Aβ1-42 was accelerated by Spike-amyloid fibril seeds. Of seven different 20-amino acid long peptides, Spike532 ( ₅₃₂ NLVKNKCVNFNFNGLTGTGV ₅₅₁ ) was most efficient in seeding HuPrP and Spike601 ( ₆₀₁ GTNTSNQVAVLYQDVNCTEV ₆₂₀ ) was most effective in seeding Aβ1-42, suggesting substrate dependent selectivity of the cross-seeding activity. Albeit purely in vitro , our data suggest that cross-seeding by Spike-amyloid fibrils can be implicated in the increasing number of reports of CJD, AD, and possibly other NDs in the wake of COVID-19.

Recently, it has been hypothesized that alpha-synuclein protein strain morphology may be associated with clinical subtypes of alpha-synucleinopathies, like Parkinson’s disease and multiple system atrophy. However, direct evidence is lacking due to the caveat of conformation-specific characterization of protein strain morphology. Here we present a new cell model based in vitro method to explore various alpha-synuclein (αsyn) aggregate morphotypes. We performed a spectroscopic investigation of the HEK293 cell model, transfected with human wildtype-αsyn and A53T-αsyn variants, using the amyloid fibril-specific heptameric luminescent oligomeric thiophene h-FTAA. The spectral profile of h-FTAA binding to aggregates displayed a blue-shifted spectrum with a fluorescence decay time longer than in PBS, suggesting a hydrophobic binding site. In vitro spectroscopic binding characterization of h-FTAA with αsyn pre-formed fibrils suggested a binding dissociation constant K_d < 100 nM. The cells expressing the A53T-αsyn and human wildtype-αsyn were exposed to recombinant pre-formed fibrils of human αsyn. The ensuing intracellular aggregates were stained with h-FTAA followed by an evaluation of the spectral features and fluorescence lifetime of intracellular αsyn/h-FTAA, in order to characterize aggregate morphotypes. This study exemplifies the use of cell culture together with conformation-specific ligands to characterize strain morphology by investigating the spectral profiles and fluorescence lifetime of h-FTAA, based upon its binding to a certain αsyn aggregate. This study paves the way for toxicity studies of different αsyn strains in vitro and in vivo. Accurate differentiation of specific alpha-synucleinopathies is still limited to advanced disease stages. However, early subtype-specific diagnosis is of the utmost importance for prognosis and treatment response. The potential association of αsyn aggregates morphotypes detected in biopsies or fluids to disease phenotypes would allow for subtype-specific diagnosis in subclinical disease stage and potentially reveal new subtype-specific treatment targets. Notably, the method may be applied to the entire spectrum of neurodegenerative diseases by using a combination of conformation-specific ligands in a physicochemical environment together with other types of polymorphic amyloid variants and assess the conformation-specific features of various protein pathologies.

HSP10 is a well‐known human co‐chaperone that interacts with HSP60 to comprise the HSP60/10 chaperonin complex which upholds mitochondrial proteostasis. HSP10 also demonstrates independent roles in binding to misfolded proteins and interacts with several amyloidogenic client proteins. Using a variety of biophysical and biochemical methods, we studied the interactions of HSP10 with the amyloidogenic protein α‐synuclein (α‐syn) associated with Parkinson's disease. HSP10 efficiently inhibited fibril formation of wild type (WT) and disease‐mutant A30P α‐syn at sufficient concentrations of chaperone by both binding to α‐syn monomers and by blocking secondary nucleation on fibril surfaces. However, under sub‐stoichiometric conditions, below 1:5 (HSP10:α‐syn), the chaperone sequestered multiple A30P α‐syn monomers and thereby promoted nucleation of fibril formation with a magnitude comparable to the efficacy of seeding with preformed fibrils. The fibril formation acceleration effect of the HSP10 chaperone was client‐specific as it was observed for A30P but not WT α‐syn. Our results broaden the scope of HSP10 chaperone activity and can have implications for disease onset in synucleinopathies.

Recently, it has been hypothesized that alpha-synuclein protein strain morphology may be as-sociated to clinical subtypes of alpha-synucleinopathies like Parkinson’s disease and multiple system atrophy. However, direct evidence is lacking due to a caveat of conformation-specific characterization of protein strain morphology. Here we present a new cell model based in-vitro method to explore various alpha-synuclein (αsyn) aggregates morphotypes. We performed a spectroscopic investigation of the HEK293 cell model transfected with human wildtype-αsyn and A53T-αsyn variants using the amyloid fibril specific heptameric luminescent oligomeric thio-phene h-FTAA. The spectral profile of h-FTAA binding to aggregates displayed a blue-shifted spectrum with a fluorescence decay time longer than in PBS, suggesting a hydrophobic binding site. In-vitro spectroscopic binding characterization of h-FTAA with αsyn pre-formed fibrils suggested a binding dissociation constant Kd < 100 nM. The cells expressing the A53T-αsyn and human wildtype-αsyn were exposed to recombinant pre-formed fibrils of human αsyn. The en-suing intracellular aggregates were stained with h-FTAA followed by evaluation of spectral features and fluorescence lifetime of intracellular syn/h-FTAA, in order to characterize aggregate morphotypes. This study exemplifies the use of cell-culture together with conformation-specific ligands to characterize strain morphology by investigating the spectral profiles and fluorescence lifetime of h-FTAA based upon its binding to a certain αsyn aggregate. This study paves the way for toxicity studies of different αsyn strains in-vitro and in-vivo. Accurate differentiation of specific alpha-synucleinopathies is still limited to advanced disease stages. However, early sub-type-specific diagnosis is of utmost importance for prognosis and treatment response. The po-tential association of αsyn aggregates morphotypes detected in biopsies or fluids to disease phenotypes would allow for subtype-specific diagnosis in subclinical disease stage and potentially reveal new subtypes specific treatment targets. Notably, the method may be applied to the entire spectrum of neurodegenerative diseases by using a combination of conformation-specific ligands in a physicochemical environment together with other types of polymorphic amyloid variants and assess the conformation specific features of various protein pathologies.