Discussion
To date, the predominant approach in Parkinson’s disease animal research involves the use of models which utilise neurotoxins that selectively target the dopaminergic neurons in the nigrostriatal pathway, when administered either systemically or cerebrally (Beal, 2010, Bezard and Przedborski, 2011, Trigo-Damas et al., 2018). While these models have significantly contributed to our understanding of various facets of Parkinson’s disease, they have at times led to drugs being initially hailed as neuroprotective only to fail to live up to expectations when clinically tested. Although widely used for evaluating potential novel therapeutic interventions, these models are generally not optimal as they are not entirely representative of the neuropathological changes that occur in Parkinson’s disease. The mechanism by which they cause cell death, the rapidity of disease induction, and absence of α-synuclein pathology are all major limitations. A more representative model, encapsulating the changes occurring during the disease state can be induced using viral vector-mediated overexpression of α-synuclein (Cenci and Bjorklund, 2020, Volpicelli-Daley et al., 2016, Van der Perren et al., 2015). In this model, the widespread accumulation of α-synuclein in the nigrostriatal pathway can induce a gradual loss in dopaminergic neurons (Lo Bianco et al., 2002, Kirik et al., 2002). Nevertheless, a significant drawback to this model is its pronounced variability in disease manifestation, along with a protracted development of pathology. Thus, it is necessary for future studies to develop novel models reflecting the disease state in a more reliable and consistent manner.
Optimising the current viral vector models thereby preserving its benefits while mitigating its limitations is an attractive option. This has been attempted by integrating it with other relevant toxic insults associated with Parkinson’s disease. Björklund, Parmar and colleagues demonstrated the potential of preformed fibrils to synergise with AAV-α-synuclein triggering a Lewy body-like pathology capable of progressive loss of dopaminergic neurons (Bjorklund et al., 2022, Thakur et al., 2017). Not only did this elicit impaired motor behaviour, but also evoked a pronounced inflammatory response, manifesting in microglia activation and lymphocyte infiltration. 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.
Although α-synuclein is ubiquitously expressed throughout the nervous system, it is primarily located in presynaptic terminals (Burre et al., 2018) including the nigrostriatal dopaminergic terminals in the striatum. Here, α-synuclein is thought to play a physiological role in synaptic vesicle trafficking (Burre, 2015) and a pathophysiological role in nigrostriatal die-back in the early stages of Parkinson’s disease (Murphy and McKernan, 2022, Wong et al., 2019, Tofaris, 2022, Sharma and Burre, 2023, Tagliaferro and Burke, 2016). This provided the rationale for the present study where FN075 was injected into the striatum (rather than the substantia nigra) following AAV-mediated overexpression of α-synuclein.
As expected, intra-nigral administration of AAV-α-synuclein led to overexpression of the protein throughout the nigrostriatal pathway with pronounced staining seen in the ventral midbrain and striatum. Furthermore, there was a significant AAV-mediated increase in both phosphorylated and aggregated forms of α-synuclein. Despite this pathology, but in line with the well-established variability associated with this model (Kirik et al., 2002), this did not lead to any overt nigrostriatal neurodegeneration or motor deficits. 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).
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. In the Chermenina et al. (2015) study, the effects of intra-nigral and intra-striatal administration of FN075 were compared in mice, and it was found that nigral administration, but not striatal administration, led to nigral cell loss. Importantly, this FN075-induced nigral degeneration was not observed in α-synuclein knock-out mice, confirming that it was an α-synuclein-mediated effect. This study in mice provided the rationale for our own two studies where we injected FN075 into the substantia nigra in rats, both of which were focussed on dual exposure models (Olsen et al., 2019, Kelly et al., 2021). In contrast to the mouse study where nigral FN075 induced nigrostriatal degeneration in its own right, in our rat studies, the molecule did not cause any nigrostriatal neurodegeneration when injected on its own. However, it had pronounced synergistic effects when injected after either AAV-α-synuclein (Kelly et al., 2021) or a viral inflammagen (Poly I:C) where it precipitated α-synuclein aggregation, neuroinflammation, nigrostriatal neurodegeneration and motor impairment (Olsen et al., 2019). Given the evidence for a synergism between FN075 and other Parkinsonian challenges, it is surprising that synergistic effects were not seen in this study. However, this could be reflective of the anterograde/retrograde transfer of FN075 and/or FN075-templated α-synuclein aggregates between the nigrostriatal terminals in the striatum and their cell bodies in the substantia nigra. Although is it known that preformed α-synuclein fibrils can travel in both anterograde and retrograde direction in the nigrostriatal neurons (Uchihara and Giasson, 2016), there is no information available on the axonal transit of FN075-templated α-synuclein fibrils.
In summary, this approach to modelling Parkinson’s disease, involving the combination of nigral AAV-α-synuclein and striatal FN075, was unsuccessful in inducing nigrostriatal neurodegeneration and motor dysfunction, two cardinal features of the human condition. Nevertheless, there remains the possibility that alternative administration regimes for FN075 (for example higher doses or repeated doses) may yet be found to improve the reliability and variability of viral-induced α-synuclein overexpression models.