α-Synuclein fibril-induced inclusion spread in rats and mice correlates with dopaminergic Neurodegeneration

Abstract

Proteinaceous inclusions in neurons, composed primarily of α-synuclein, define the pathology in several neurodegenerative disorders. Neurons can internalize α-synuclein fibrils that can seed new inclusions from endogenously expressed α-synuclein. The factors contributing to the spread of pathology and subsequent neurodegeneration are not fully understood, and different compositions and concentrations of fibrils have been used in different hosts. Here, we systematically vary the concentration and length of well-characterized α-synuclein fibrils and determine their relative ability to induce inclusions and neurodegeneration in different hosts (primary neurons, C57BL/6J and C3H/HeJ mice, and Sprague Dawley rats). Using dynamic-light scattering profiles and other measurements to determine fibril length and concentration, we find that femptomolar concentrations of fibrils are sufficient to induce robust inclusions in primary neurons. However, a narrow and non-linear dynamic range characterizes fibril-mediated inclusion induction in axons and the soma. In mice, the C3H/HeJ strain is more sensitive to fibril exposures than C57BL/6J counterparts, with more inclusions and dopaminergic neurodegeneration. In rats, injection of fibrils into the substantia nigra pars compacta (SNpc) results in similar inclusion spread and dopaminergic neurodegeneration as injection of the fibrils into the dorsal striatum, with prominent inclusion spread to the amygdala and several other brain areas. Inclusion spread, particularly from the SNpc to the striatum, positively correlates with dopaminergic neurodegeneration. These results define biophysical characteristics of α-synuclein fibrils that induce inclusions and neurodegeneration both in vitro and in vivo, and suggest that inclusion spread in the brain may be promoted by a loss of neurons.

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

“…In addition to pS129 aSyn pathology, the pre-formed fibrils (PFFs) trigger synaptic dysfunction, perturbations in cell excitability, and cell death in cell lines overexpressing disease-related proteins as well as in primary neuronal cultures from wild-type mice [32–33]. In vivo, intracerebral injection of aSyn PFFs into the dorsal striatum results in dysregulation of striatal dopamine release, neurodegeneration in the SNpc, and behavioral deficits in rodents overexpressing disease-related proteins or non-transgenic rodents [29–30, 34–35]. The development and validation of this model has led to a new animal model for PD research that provides many advantages over other models.…”

“…Recombinant aSyn monomers may be generated in house using protocols such as those described in Volpicelli-Daley, Luk, & Lee (2014) [31], Abdelmotilib et al, 2017 [35], or Fares et al, 2016 [44]. Monomeric aSyn protein specifically-formulated to generate aSyn PFFs may also be purchased from commercial sources (Appendix A).…”

“…Specifically, samples of aSyn monomers incubated at 37°C, room temperature, or 4°C without shaking fail to form the elongated fibrillar aggregates of aSyn that adopt the beta-sheet conformation and amyloidogenic properties [47]. It is recommended to shake the aSyn monomers at 37°C in a sealed incubator or shaker with a temperature controlled lid to prevent evaporation, with verification of turbidity after incubation to confirm the presence of higher molecular weight species of aSyn such as the aSyn fibrils [31, 35, 47]. The MJFF protocol for fibril generation can be found in Appendix A, with additional protocols in the cited literature [29–35, 41–48].…”

“…Importantly, the sonicated aSyn PFF samples are heterogenous in nature and it remains unclear which specific form of aggregated aSyn—be it the short fibrils or oligomers generated during sonication—are seeding the majority of the pathology. However, previous studies have shown that sonication must result in majority of aSyn PFFs at 50nm or smaller to consistently seed pS129 aSyn pathology in culture or after injection [35, 47–48]. Failure to sonicate samples prior to use or failure to sufficiently sonicate the large fibrils to smaller components will reduce pathogenicity or even result in a lack of pathology [35, 47–48].…”

“…However, previous studies have shown that sonication must result in majority of aSyn PFFs at 50nm or smaller to consistently seed pS129 aSyn pathology in culture or after injection [35, 47–48]. Failure to sonicate samples prior to use or failure to sufficiently sonicate the large fibrils to smaller components will reduce pathogenicity or even result in a lack of pathology [35, 47–48]. To prevent this issue, it is suggested to test multiple sonication parameters when first establishing the model in one’s lab.…”