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

Numerous challenges hinder the development of neuroprotective treatments for Parkinson’s disease, with a regularly identiﬁed issue being the lack of clinically relevant animal models. Viral vector overexpression of α-synuclein is widely considered the most relevant model, however this has been limited by high variability and inconsistency. One potential method of optimisation is pairing it with a secondary insult such as FN075, a synthetic molecule demonstrated to accelerate α-synucleinopathy. Thus, the aim of this study was to investigate if sequential infusion of AAV- α-synuclein and FN075 into the rat brain can replicate α-synucleinopathy, nigrostriatal pathology and motor dysfunction associated with Parkinson’s disease. Rats received a unilateral injection of AAV- α-synuclein (or AAV-GFP) into two sites in the substantia nigra, followed 4 weeks later by unilateral injection of FN075 (or vehicle) into the striatum. Animals underwent behavioural testing every 4 weeks until sacriﬁce at 20 weeks, followed by immunohistochemistry assessment post-mortem. As anticipated, AAV- α-synuclein led to extensive overexpression of human α-synuclein throughout the nigrostriatal pathway, as well as elevated levels of pathological and aggregated forms of the protein. However, the sequential administration of FN075 into the striatum did not exacerbate any of the α-synuclein pathology. Furthermore, despite the extensive α-synuclein pathology, neither administration of AAV- α-synuclein nor FN075, alone or in combination, was suﬃcient to induce dopaminergic degeneration or motor deﬁcits. In conclusion, this approach did not replicate the key characteristics of Parkinson’s disease, and further studies are required to create more representational models for testing of novel compounds and treatments for Parkinson’s disease.


Introduction
Parkinson's disease is a devastating neurological condition affecting millions of people worldwide.Despite decades of research, current treatments are purely symptomatic and there remains no preventative or curative therapy.Thus, there is an unmet clinical need for a disease-modifying treatment capable of slowing or inhibiting the course of the disease for patients suffering from Parkinson's disease.There are many issues impeding the development of a neuroprotective treatment, but one key factors has been consistently identified, is the lack of clinically-relevant animal models to test treatment efficacy (Beal, 2010).
Presently, many of the established animal models of Parkinson's disease rely on the administration of a toxin either systemically or directly into the brain.Two of the earliest and most notable Parkinson's disease models are induced by 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).These are the most commonly used for drug discovery and research because of their rapid development, simplicity, reproducibility and cost effectiveness (Beal, 2010, Bezard and Przedborski, 2011, Trigo-Damas et al., 2018).While certainly these models have been invaluable in Parkinson's disease research, they are not without their limitations, primarily that toxin-based models lack key neuropathological changes relevant to the disease state, such as α-synuclein pathology, and that they are usually not progressive in nature.
A few years after a major breakthrough in understanding Parkinson's pathology -where the α-synuclein protein was identified as the main component of Lewy bodies and implicated in the disease pathogenesis (Spillantini et al., 1998) -the viral vector α-synuclein overexpression models of Parkinson's disease were first introduced (Lo Bianco et al., 2002, Kirik et al., 2002).Deniz Kirik and colleagues utilised adenoassociated viral (AAV) vectors injected into the substantia nigra of rats to overexpress either wild-type or A53T mutated human α-synuclein (Kirik et al., 2002).As a consequence of the protein overexpression, they observed a significant loss, albeit highly variable, of dopaminergic neurons in the nigrostriatal pathway was capable of producing motor defects when exceeding 50-60% dopaminergic degeneration.They also found α-synuclein positive inclusions and dystrophic neurites, similar to those observed in patients with Parkinson's disease.This model has been employed in other animals such as mice (St Martin et al., 2007) and primates (Kirik et al., 2003) with comparable success.
Although this model holds many advantages, has a strong etiological background, and is capable of mimicking a progressive disease state in aged animals, it has its limitations.Primarily, the model suffers from a highly variable and inconsistent disease manifestation, as well as a pathology that is slow to develop (Van der Perren et al., 2015).Many factors can be attributed to the disparity between results such as vector, serotype, promotor and titre (Volpicelli-Daley et al., 2016, Van der Perren et al., 2011, Van der Perren et al., 2015).However, these variations do not explain the discrepancies of neurodegeneration observed within singular studies (Kirik et al., 2002).Therefore, the considerable drawbacks restricting this model signify that enhanced viral α-synuclein-based Parkinson's disease models are necessary.
One approach to enhancing the viral α-synuclein model may be to sequentially inject AAV-α-synuclein and the novel α-synuclein aggregator, FN075, into the nigrostriatal pathway.FN075 is a peptidomimetic small molecule that has been shown to accelerate α-synuclein aggregation, and the fibrils formed by this process are structurally similar to those formed by naturally occurring aggregation (Horvath et al., 2012, Cegelski et al., 2009).In mice, FN075 caused impairments in motor function and nigrostriatal degeneration 6 months after a single intracerebral injection (Chermenina et al., 2015), while in rats, sequential exposure to the viral mimetic, Poly I:C, followed by FN075, exacerbated the neurodegeneration, neuroinflammation and motor impairment caused by the inflammagen (Olsen et al., 2019).
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.Following this, we hypothesised that administration of FN075 into the nigrostriatal terminals in the striatum rather than into the cell bodies in the substantia nigra might be a more promising approach.This hypothesis was primarily based on the fact that α-synuclein is strongly enriched in synaptic terminals (Burre, 2015, Sharma and Burre, 2023, Burre et al., 2018) and is thought to contribute to the terminal degeneration and striatal deafferentation associated with the disease (Murphy and McKernan, 2022, Wong et al., 2019, Tofaris, 2022, Tagliaferro and Burke, 2016).Thus, in this study, we sought to determine if administration of this α-synuclein aggregator into the striatum of rats already overexpressing α-synuclein (induced by AAV vectors) would drive further α-synuclein pathology, and lead to nigrostriatal neurodegeneration and the precipitation of motor dysfunction.

Animals & Ethical Statement
Forty adult female Sprague-Dawley rats (Charles River, Margate, UK) were used in this research.All procedures involving the use of animals were completed in accordance with the European Union Directive 2010/63/EU and the Irish Statutory Instrument S.I.No. 543 of 2012; were sanctioned by the Animal Care and Research Ethics Committee (ACREC) of the University of Galway; and were carried out under Project and Individual Authorisations issued by the Irish Health Products Regulatory Authority.Animals were pair housed, on a 12:12 light:dark cycle, with 19-23 *C temperature and 40-70% humidity.Water and food were provided ad libitum over the course of the experiments except for 24 h before the Corridor Test was performed in which their food ration was restricted (to ensure they were driven to perform this food-motivated test).All behavioural testing and post-mortem analyses were completed by a researcher blinded to the treatment of the rats.

Experimental Design
Prior to any procedures, animals were habituated and baseline tested in the Stepping, Whisker and Corridor Tests before being randomly allocated to treatment groups.Rats then received unilateral intra-nigral injections of AAV vector expressing the human α-synuclein transgene (or a control AAV vector expressing GFP) followed 4 weeks later by intra-striatal injection of FN075 (or its vehicle as a control) to yield 4 final groups (Table 1).Animals were tested for motor defects in the Stepping, Whisker and Corridor Tests prior to AAV surgery as well as every 4 weeks post-AAV surgery.Animal sacrifice was carried out by transcardial perfusion-fixation under terminal pentobarbital anaesthesia for immunohistochemical analyses at 20 weeks post-AAV surgery.
Table 1.Groups used in this study to determine the effects of sequential intra-nigral administration of AAV-a-synuclein and intra-striatal FN075.After behavioural baselines were established, rats were randomly divided into 2 groups for intra-nigral infusion of AAV-a-synuclein or AAV-GFP.Four weeks later, they were randomly subdivided for intra-striatal infusion of FN075 or vehicle to yield 4 final groups.

Group
Virus injection FN075 injection n AAV Virus Production AAV2 recombinant genomes encoding wild-type human α-synuclein, or GFP, under the transcriptional control of the PGK1 (phosphoglycerate kinase) promoter were pseudotyped in serotype 6 capsids as previously described (Berger et al., 2015).In brief, viral particles were produced by co-transfection of HEK-293T cells with an adenovirus helper plasmid (pXX6-80), an AAV packaging plasmid carrying the rep2 and cap6 genes, and a plasmid encoding a recombinant AAV2 genome containing the transgene expression cassette.Seventytwo hours after transfection, viral particles were purified and concentrated from cell lysates and supernatants by ultracentrifugation on an iodixanol density step gradient, followed by dialysis against PBSMK buffer (0.5 mM MgCl 2 and 1.25 mM KCl in PBS).The concentration of vector stocks was estimated by real-time PCR and expressed as vector genomes per μL of concentrated stocks (vg/μL).On the day of surgery, the vectors were diluted in PBS with 0.01% Pluronic F-68 to the appropriate titer.

Surgery
All surgery was performed under aseptic conditions under isoflurane anaesthesia (5% in O 2 for induction, ˜2% in O 2 for maintenance) in a stereotaxic frame with the nose bar set at -2.3 mm.For the AAV surgery, all rats received a dual intra-nigral injection unilaterally in the substantia nigra (at the coordinates AP -4.8 & -5.8, ML +2.0 and DV -7.2).The AAV vector titre was 1.67 x 10 10 vg/μL for surgeries.Infusions were carried out at a rate of 0.5 μL/min with a total volume of 3 μL per site (2 sites per rat), and a further 5 min were then allowed for diffusion.For the FN075 surgery, all rats received four intra-striatal injections unilaterally into the striatum of FN075 (4 x 1.9 μg; 4 μl of 1 mM) or its vehicle (100 μM imidazole in PBS + 0.5% DMSO); at the coordinates AP +1.3, ML +2.7; AP +0.4,ML +3.1; AP-0.4,ML +4.3; AP -1.3, ML +4.7 (from bregma) and DV -5.0 below dura.Infusions were carried out at a rate of 1 μL/min with a total volume of 4 μL per site (4 sites per rat), and a further 2 min were then allowed for diffusion.

Behavioural Testing
To assess the impact of the intra-nigral AAV α-synuclein infusion combined with intra-striatal FN075 infusion on motor function, the rats were assessed for motor impairments using the Stepping Test of forelimb akinesia (Olsson et al., 1995), the Whisker Test of sensorimotor integration (Schallert et al., 2000) and the Corridor Test of sensorimotor neglect (Fitzsimmons et al., 2006, Dowd et al., 2005).In the Stepping Test, rats were held with two hands restraining both hindlimbs and one forelimb, they were then guided at a steady pace (90 cm in ˜5 s) across a table surface.This was performed in both the backhand and forehand directions on the ipsilateral and contralateral sides, counting the number of adjusting steps made by the free forelimb, and data are expressed as the number of contralateral steps taken (mean of both directions).In the Whisker Test, rats were held with two hands restraining both hindlimbs and one forelimb, on the side of the unrestrained limb their whiskers were brushed against the side of a table 10 times.This was performed on the ipsilateral and contralateral sides, where the number of vibrissae-elicited forelimb placings was recorded, and data are expressed as the number of contralateral placings made.In the Corridor Test, pots containing CocoPops® at regular intervals evenly spaced on both sides of a long corridor was set up, rats were placed in the corridor and allowed to freely retrieve the CocoPops®.Once a total of 20 retrievals were made or 5 min elapsed, the trial was deemed complete.The number of retrievals made from both the ipsilateral and contralateral sides was counted, and data are expressed as the % contralateral retrievals made.

Euthanasia and Tissue Processing
Animals were euthanised 20 weeks after AAV surgery via transcardial perfusion fixation under terminal pentobarbital anaesthesia (50 mg/kg).Brains were post-fixed in 4% paraformaldehyde for 24 h before being cryoprotected in a 30% sucrose solution with 0.1% sodium azide.Using a freezing sledge microtome (Bright, Cambridgeshire, UK), 30μm serial coronal sections were collected in a series of 6.

Immunohistochemistry
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.Tissue was then blocked to prevent non-specific antibody binding by incubation in a solution containing 3% normal horse serum or normal goat serum (depending on the host species of the secondary antibody) in tris-buffered saline (TBS) with 0.2% Triton X-100 for 1 hour at room temperature.Sections were then allowed to incubate overnight with the primary antibody (Mouse anti-tyrosine hydroxylase, 1:1000, Millipore MAB318; Mouse anti-human-α-synuclein, 1:10,000, Millipore 36-008; Rabbit anti-phospho-α-synuclein (S129), 1:5000, Abcam ab51253; Rabbit anti-α-synuclein-aggregate-specific, 1:8000, Abcam ab209538) which was diluted in 1% serum in TBS with 0.2% Triton X-100.The second day the appropriate biotinylated secondary antibody (Horse anti-mouse, 1:200, Vector BA-2001; Goat anti-rabbit, 1:200, Jackson ImmunoResearch 111-065-144) with 1% serum was allowed to incubate with the sections for 3 hours.Sections were subsequently incubated for a further 2 hours with a streptavidin-biotin-horseradish peroxidase solution (Vector PK-4000).A 0.5% diaminobenzidine tetrahydrochloride (DAB) (Sigma D5637) solution in TBS containing 0.3 μL/mL of hydrogen peroxide was used to develop the tissue staining.Sections were mounted onto gelatin-coated slides, dehydrated in a series of ascending alcohols concentrations, cleared in xylene and coverslipped with DPX mountant.

Image Analysis
Immunostained sections images were taken using a VS120 Virtual Slide Microscope (Olympus, Southend-on-Sea, UK).Manual counts and density image analyses were performed using ImageJ software (ImageJ v1.53k,National Institute of Health, Bethesda, MD, USA).Automatic counting image analysis was achieved using Qupath softwear (QuPath v0.4.4,Cancer Research & Cell Biology, Queen's University, Belfast, Northern Ireland).In an unbiased manner three images were selected along the rostrocaudal axis of the striatum or nigra based on their distance from bregma (striatal AP coordinates: +0.7, +1.0, +1.2 mm; nigral AP coordinates -5.6, -5.8, -6.04 mm).The optical density of the staining of α-synuclein in the substantia nigra and the striatum, and of tyrosine hydroxylase in the striatum was measured using ImageJ software.To do so, both the intact side and the lesioned side the mean grey value was measured.By applying the conversion formula in ImageJ (optical density = log 10 (255/mean grey value)), these were then converted to optical densities, this was then expressed as a percentage of the intact side.To quantify the number of tyrosine hydroxylase immunopositive cell bodies in the substantia nigra, manual counts were conducted on both the ipsilateral and contralateral sides according to distinct boundaries.Specifically, immune-positive cells in the substantia nigra pars reticulata, pars lateralis and pars compacta were counted and immunepositive cells in the ventral tegmental area (VTA) were excluded.Cell counts data were expressed as a percentage of the intact side.The number of pS129-α-synuclein-positive accumulations in the substantia nigra were also counted on the side of the brain ipsilateral to the lesion surgery.Data were expressed as the average number of pS129-α-synuclein-positive accumulations in the three sections analysed.To ascertain the number of pS129-α-synuclein-positive accumulations in the striatum, QuPaths pixel classifier feature was used to create a threshold.Once the threshold was set, the annotation was saved pS129-α-synuclein and split objects was selected to give the number of annotations found within the striatum.Staining vectors were set on each image prior to running the programme to create the annotations on the lesioned side of the brain.Data were expressed as the average number of pS129-α-synuclein-positive accumulations in the three sections analysed.To assess the number of α-synuclein aggregates in the substantia nigra, analysis was conducted using QuPath's built-in "cell detection".Staining vectors were first set on each image for consistency and the same cell detection parameters used throughout the project.This give the number of aggregates detected in the ipsilateral side.Data were expressed as the average number of aggregated α-synuclein-positive accumulations in the three sections analysed.

Statistical Analysis
GraphPad Prism software (Version 9.1.2.) was utilised to carry out the statistical analyses.Data were first tested for normality using Shapiro-Wilk's test and homogeneity of variance using Brown-Forsythe's test.All data were confirmed to be parametric and were consequently expressed as mean ± standard error of the mean and analysed by ANOVA.Behavioural data were analysed using a two-way ANOVA with repeated-measures (with within-subject factor of time and between-subject factor of group) with post hoc Tukey analyses.Histological data were analysed using one-way ANOVA (with between-subject factor of group) with post hoc Tukey analyses.Results were deemed significant if p < 0.05.Throughout the results, the main outcome from the ANOVA is given in the text while the outcome of any post hoc analysis is shown in the relevant figure and explained in the corresponding legend.

ΦΝ075 διδ νοτ πρεςιπιτατε μοτορ ιμπαιρμεντ ιν τηε ΑΑ῞-α-σψνυςλειν μοδελ
One critical aspect of Parkinson's disease is the progressive emergence of motor impairments.In unilateral models of the disease, this manifests as deficits on contralateral side of the body to the site injected, while the ipsilateral side of the body remains unaffected.Hence, the animals were subjected to a series of lateralised motor tests to investigate whether the single or combined Parkinsonian insults could provoke motor impairment specifically on the contralateral side.However, relative to the Control group, neither AAV-α-synuclein nor FN075, either alone or in sequence, induced any significant contralateral motor deficit in any of the tests (Fig. 1; Stepping: Group x Time, F (15, 180) = 0.66, p> 0.05; Whisker: Group x Time, F (15, 180) = 0.84, p > 0.05); Corridor Group x Time,F (15, 180) = 0.73, p > 0.05).
Neither unilateral administration of AAV-α-synuclein nor FN075, alone or sequentially, was sufficient to induce contralateral motor deficits in the Stepping, Whisker or Corridor tests.Data are represented as mean ± SEM with n = 10 animals per group, and were analysed by two-way ANOVA with repeated measures.Infusion days are represented by dashed lines.

ΦΝ075 διδ νοτ ενηανςε πατηολογιςαλ α-σψνυςλειν εξπρεσσιον ιν τηε ΑΑ῞-α-σψνυςλειν μοδελ
Having established that AAV-α-synuclein led to human α-synuclein overexpression in the nigrostriatal pathway, we explored what forms the protein took, and whether or not pathological forms were exacerbated by FN075 administration.Thus, we performed further immunohistochemical staining specific for pS129α-synuclein.Immunostaining revealed a significant increase in the number of nigral cell bodies in which pathological pS129-α-synuclein had accumulated in the groups which received AAV-α-synuclein (Fig. 3a; Group, F (3,36) =15.10, p < 0.05).However, there was no build-up of cellular pS129-α-synuclein in the substantia nigra as a result of FN075, either alone or in combination with AAV-α-synuclein.Similarly when assessing the number of pS129-α-synuclein aggregations found in the striatum on the side of the injection, there was a significant increase in the groups receiving the AAV-α-synuclein vector (Fig. 3b; Group,F (3,35) =10.75, p < 0.05), yet FN075 did not have any additive effect on the amount of staining.Finally, we carried out an immunohistochemical stain for aggregate specific α-synuclein which showed a significant increase in fibrillar α-synuclein in the substantia nigra in both groups receiving AAV-α-synuclein (Fig. 4; Group, F (3,35) =7.56, p < 0.05).However, again there was no added pathology induced by the sequential exposure to FN075.

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 FN075templated 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 Posted on 3 May 2024 -The copyright holder is the author/funder.All rights reserved.No reuse without permission.-https://doi.org/10.22541/au.171475235.57509359/v1-This is a preprint and has not been peer-reviewed.Data may be preliminary.
In summary, this approach to modelling Parkinson's disease, involving the combination of nigral AAVα-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.