The Small Molecule Alpha-Synuclein Aggregator, FN075, Enhances Alpha-Synuclein Pathology in Subclinical AAV Rat Models

Abstract

Animal models of Parkinson’s disease, in which the human α-synuclein transgene is overexpressed in the nigrostriatal pathway using viral vectors, are widely considered to be the most relevant models of the human condition. However, although highly valid, these models have major limitations related to reliability and variability, with many animals exhibiting pronounced α-synuclein expression failing to demonstrate nigrostriatal neurodegeneration or motor dysfunction. Therefore, the aim of this study was to determine if sequential intra-nigral administration of AAV-α-synuclein followed by the small α-synuclein aggregating molecule, FN075, would enhance or precipitate the associated α-synucleinopathy, nigrostriatal pathology and motor dysfunction in subclinical models. Rats were given unilateral intra-nigral injections of AAV-α-synuclein (either wild-type or A53T mutant) followed four weeks later by a unilateral intra-nigral injection of FN075, after which they underwent behavioral testing for lateralized motor functionality until they were sacrificed for immunohistological assessment at 20 weeks after AAV administration. In line with expectations, both of the AAV vectors induced widespread overexpression of human α-synuclein in the substantia nigra and striatum. Sequential administration of FN075 significantly enhanced the α-synuclein pathology with increased density and accumulation of the pathological form of the protein phosphorylated at serine 129 (pS129-α-synuclein). However, despite this enhanced α-synuclein pathology, FN075 did not precipitate nigrostriatal degeneration or motor dysfunction in these subclinical AAV models. In conclusion, FN075 holds significant promise as an approach to enhancing the α-synuclein pathology in viral overexpression models, but further studies are required to determine if alternative administration regimes for this molecule could improve the reliability and variability in these models.

Intra-striatal infusion of the small molecule alpha-synuclein aggregator, FN075, fails to enhance Parkinsonism in a subclinical AAV-alpha-synuclein rat model.

“…Disappointingly, when FN075 was injected into the striatum, across four rostro-caudal sites, this did not drive the AAV-mediated α-synuclein pathology further, with no increase in pS129 or aggregated α-synuclein, and no precipitation of nigrostriatal neurodegeneration or motor dysfunction. This is in contrast to our previous study in which FN075 injection into the substantia nigra did significantly increase pathological variants of α-synuclein after AAV vector overexpression (Kelly et al, 2021).…”

“…However, this approach is not without its limitations with consistency and replicability in the generation and application of preformed α-synuclein fibrils being a significant challenge for many researchers (Polinski et al, 2018). As an alternative to this, we recently turned to a small molecule approach where the α-synuclein aggregator, FN075, was sequentially injected into the substantia nigra of rats what had been previously injected with AAV-α-synuclein at the same site (Kelly et al, 2021). Although this showed promise with FN075 significantly increasing the levels of pathological pS129-α-synuclein induced by the AAV-α-synuclein vector, ultimately, this neither enhanced nigrostriatal neurodegeneration nor motor dysfunction.…”

“…The reason why intra-striatal administration of FN075 did not have any effect on AAV-α-synuclein-mediated pathology is not known. Thus far, FN075 has only been administered in vivo in four published studies -in Drosophila (Pokrzywa et al, 2017), mice (Chermenina et al, 2015) and rats (Olsen et al, 2019, Kelly et al, 2021. In the Pokrzywa et al (2017) study, feeding FN075 to wild type flies had no impact on their motor function, while in α-synuclein overexpressing flies, the compound significantly reduced locomotor activity and lifespan.…”

“…Free-floating immunohistochemistry was conducted using the streptavidin-biotin peroxidase method as previously described (Kelly et al, 2021). In brief, sections were quenched in a solution containing 3% hydrogen peroxide and 10% methanol in distilled water to eliminate endogenous peroxidase activity.…”

“…We recently demonstrated the potential of this molecule for enhancing viral overexpression models by sequentially injecting the rat substantial nigra with AAV-α-synuclein followed 4 weeks later by FN075. FN075 significantly increased AAV-mediated pathological α-synuclein protein levels with a significant increase in protein phosphorylated at serine 129 (pS129-α-synuclein; (Oueslati, 2016, Xu et al, 2015) in rat substantia nigra (Kelly et al, 2021). However, despite this enhanced α-synuclein pathology this did not enhance nigrostriatal degeneration or motor dysfunction.…”