Novel self-replicating α-synuclein polymorphs that escape ThT monitoring can spontaneously emerge and acutely spread in neurons

Abstract

The conformational strain diversity characterizing α-synuclein (α-syn) amyloid fibrils is thought to determine the different clinical presentations of neurodegenerative diseases underpinned by a synucleinopathy. Experimentally, various α-syn fibril polymorphs have been obtained from distinct fibrillization conditions by altering the medium constituents and were selected by amyloid monitoring using the probe thioflavin T (ThT). We report that, concurrent with classical ThT-positive products, fibrillization in saline also gives rise to polymorphs invisible to ThT (τ−). The generation of τ− fibril polymorphs is stochastic and can skew the apparent fibrillization kinetics revealed by ThT. Their emergence has thus been ignored so far or mistaken for fibrillization inhibitions/failures. They present a yet undescribed atomic organization and show an exacerbated propensity toward self-replication in cortical neurons, and in living mice, their injection into the substantia nigra pars compacta triggers a synucleinopathy that spreads toward the dorsal striatum, the nucleus accumbens, and the insular cortex.

Alpha-Synuclein and Mitochondrial Dysfunction in Parkinson’s Disease: The Emerging Role of VDAC

“…Similarly, injections of PFFs into WT mice and non-human primates induce progressive synucleinopathy [ 54 , 55 ]. Taken together, these and other data suggest that when exogenously added, αSyn fibrils are taken up by neurons and act as “seeds” (i.e., conformational template) for the aggregation of endogenous monomeric αSyn [ 53 ]. However, some issues have to be taken into account.…”

“…Furthermore, depending on the disease stage, α-Syn aggregates have been found in different brain regions of PD patients suggesting a prion-like cell-to-cell transmission [ 49 , 50 , 51 ]. To this end, the exogenous addition of preformed αSyn fibrils (PFFs) to cells in culture recapitulates the self-assembly process of endogenous αSyn into amyloid fibrils [ 52 , 53 ]. Similarly, injections of PFFs into WT mice and non-human primates induce progressive synucleinopathy [ 54 , 55 ].…”

Similar neuronal imprint and absence of cross-seeded partner fibrils in α-synuclein aggregates from MSA and Parkinson’s disease brains

“…In both cases, the catabolism of IBs released by the dying host cells in the extracellular space could then cause a local leak of small IB fragments and α-syn fibrils from the IB mass, fragments that in turn would be capable of being taken up by the neighboring cells in which they would trigger a de novo aggregation of endogenous α-syn, the formation of novel IBs, and so on. Indeed, in mice, the intracerebral injection of IBs extracted from human DLB brains leads to the development and to the spread of a synucleinopathy with neuropathological and cytological features that are strikingly identical to the one observed after injections of pure recombinant α-syn fibrils 16,17 . Thus, if this IB-dependent intercellular spread mechanism holds, it is tempting to speculate that the composition of IBs present in a host cell could reflect the history of the spread, i.e., show proteins of the donor cell transferred together with the IB fragment seeds and eventually associated with the α-syn fibrils mass in the neo-formed IB.…”

“…We purified and isolated aggregated α-syn and its associated insoluble proteomes from PD and MSA brains using their sarkosyl-insolubility. We used Sarkospin, a previously developed procedure for purifying pathological protein aggregates by sedimentation 17,28,29 . By adapting Sarkospin to α-syn, we sought to specifically isolate the aggregated forms of the protein from their physiological monomeric and oligomeric counterparts.…”

Protein resonance assignment by solid-state NMR based on 1H-detected 13C-based double-quantum spectroscopy at fast MAS

“…Two-dimensional (2D) 1 H-detected correlation experiments such as 2D 13 C- 1 H spectra offer a powerful spectroscopic tool to monitor chemical shift perturbations. These spectral “fingerprints” have the potential to become the new routine experiment for SSNMR investigations of proteins, in analogy to the 15 N- 1 H HSQC experiment for solution NMR, as we recently demonstrated to rapidly compare amyloid fibril conformation of various α-synuclein fibril polymorphs based on 2D 15 N/ 13 C- 1 H spectra (De Giorgi et al 2020). Although few minutes to hours are required to obtain these spectral fingerprints, a tremendous time is still required to perform the so-called resonance assignment step, usually based on 3D experiments to establish triple resonance connectivity’s between 1 H, 13 C and 15 N atoms(Barbet-Massin et al 2014)(Penzel et al 2015)(Fricke et al 2017).…”