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.