Molecular and Biological Compatibility with Host Alpha-Synuclein Influences Fibril Pathogenicity

Abstract

Summary The accumulation and propagation of misfolded α-Synuclein (α-Syn) is a central feature of Parkinson’s disease (PD) and other synucleinopathies. Molecular compatibility between a fibrillar seed and its native protein state is a major determinant of amyloid self-replication. We show that cross-seeded aggregation of human (Hu) and mouse (Ms) α-Syn is bidirectionally restricted. Although fibrils formed by Hu-Ms-α-Syn chimeric mutants can overcome this inhibition in cell-free systems, sequence homology poorly predicts their efficiency in inducing α-Syn pathology in primary neurons or after intracerebral injection into wildtype mice. Chimeric α-Syn fibrils demonstrate enhanced or reduced pathogenicities compared to wildtype Hu- or Ms-α-Syn fibrils. Furthermore, α-Syn mutants induced to polymerize by fibrillar seeds inherit the functional properties of their template, suggesting transferable pathogenic and non-pathogenic states likely influence the initial engagement between exogenous α-Syn seeds with endogenous neuronal α-Syn. Thus, transmission of synucleinopathies is regulated by biological processes in addition to molecular compatibility.

Selective lowering of synapsins induced by oligomeric α-synuclein exacerbates memory deficits

“…Our results indicated that larger o-αSyn species lost their ability to reduce synapsins when introduced into SNCA-null neurons lacking αSyn, whereas smaller o-αSyn assemblies did not depend on endogenous αSyn to lower synapsins, reminiscent of prion-like mechanisms. In agreement with earlier reports (35,40,62), these findings therefore suggested the presence of different functional conformers of αSyn with species requiring template assembly and others that did not. On the one hand, the fact that both αSyn aggregates trigger the same cellular change is surprising, as one would perhaps predict differential alterations in neuronal biology induced by each species.…”

Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson’s Disease in Rodents

“…When aSyn fibrils have formed, the ThT assay should result in readings 20–100 folder higher for human aSyn PFFs as compared to human aSyn monomers (Figure 5A). Human aSyn PFFs will also result in higher levels of ThT than murine aSyn PFFs, leading to greater subtlety in differences between monomers and PFFs in murine aSyn as compared to human aSyn [51. A protocol for the ThT assay can also be found in Appendix A.…”

“…Therefore, investigators should remember to use the proposed quality control methods in Figure 5A–G as general pass/fail criteria whereby failure suggests the need to remake the PFFs and success suggests likely (but not guaranteed) pathology when introduced in vivo. For pilot studies, analysis at 30 dpi is generally enough time to induce robust pS129 pathology in the rat [34] and mouse [51] brain after striatal injection, although rates of pathology may differ based on the species of aSyn PFFs, the rodent strain, the target structure, and the aSyn PFF dose, among other factors.…”

“…Generally, greater homology between the host and species of recombinant aSyn protein will result in greater pathology in vitro and in vivo . This has been demonstrated at the level of seeding recombinant protein as human aSyn PFFs are much more effective at templating amyloid-formation of human aSyn monomers than mouse aSyn monomers when measured by the ThT kinetic seeding assay [44, 51]. Similarly, mouse aSyn PFFs seed amyloid-structure formation in mouse aSyn monomers more effectively than human aSyn monomers [51].…”

“…This has been demonstrated at the level of seeding recombinant protein as human aSyn PFFs are much more effective at templating amyloid-formation of human aSyn monomers than mouse aSyn monomers when measured by the ThT kinetic seeding assay [44, 51]. Similarly, mouse aSyn PFFs seed amyloid-structure formation in mouse aSyn monomers more effectively than human aSyn monomers [51]. The importance of species homology between the aSyn PFF and host was confirmed in vitro with higher levels of pS129 aSyn staining, soluble aSyn, and insoluble aSyn in mouse primary hippocampal neuron cultures following incubation with mouse aSyn PFFs versus human aSyn PFFs [51], although other studies have reported relatively similar levels of seeding between mouse and human aSyn PFFs in vitro [35].…”