Parkinson’s disease (PD) is a common progressive neurodegenerative condition affecting ~1% of people over the age of 60 (Reeve et al., 2014). The condition results in deficits of motor function including abnormal posture and gait, tremor, and rigidity (Gelb et al., 1999; Lees et al., 2009). Characteristic neuropathological findings include the loss of neurons from the substantia nigra pars compacta (SNpc) which project axons to the striatum where dopamine (DA) is released (Bernheimer et al., 1973). The pathological hallmark of PD is the formation of Lewy bodies and Lewy neurites, complex intracellular inclusions abundant in α-synuclein protein (Gibb and Lees, 1988; Spillantini et al., 1997). Cases of familial PD have been shown to result from mutations in SNCA, encoding α-synuclein, and the G51D mutation (c.152 G > A) has been identified to cause an early onset and aggressive form of PD with associated dementia and multiple system atrophy (Kiely et al., 2013; Lesage et al., 2013).

It has been extensively reported that PD patients have a decreased number of tyrosine hydroxylase (TH) positive neurons in the striatum when compared to controls, and this occurs when the condition has significantly progressed (Huot et al., 2007; Kordower et al., 2013). However, prior to significant neuronal loss, earlier deficits in presynaptic dopaminergic function in the striatum of PD patients can be investigated using positron emission tomography (PET) and the radiotracer L-3,4-dihydroxy-6-¹⁸F-fluorophenylalanine (¹⁸F-DOPA) (Garnett et al., 1983; Nahmias et al., 1985). ¹⁸F-DOPA is metabolized in dopaminergic nerve terminals by the enzyme aromatic L-amino acid decarboxylase (AADC) to produce ¹⁸F-DA, which is incorporated into synaptic vesicles and then released into the synaptic cleft following neuronal stimulation (Firnau et al., 1987). In patients with PD, dopaminergic function in the striatum is impaired, and the influx rate constant (K_i) of ¹⁸F-DOPA is significantly decreased in the caudate and putamen (Brooks et al., 1990; Snow et al., 1993; Holthoff-Detto et al., 1997). Dopamine turnover can be measured with extended scanning time, and the effective distribution volume ratio (EDVR) of ¹⁸F-DOPA can be calculated (Sossi et al., 2001). A decreased EDVR in the striatum indicates an increase in dopamine turnover, and this was observed in newly diagnosed PD patients relative to healthy controls prior to a decrease in K_i (Sossi et al., 2002). An increase in dopamine turnover was also observed in asymptomatic LRRK2 mutation carriers before any evidence of changes in K_i (Sossi et al., 2010). This has been hypothesized to be a compensatory mechanism in early PD potentially due to upregulation of AADC decarboxylase activity (Lee et al., 2000; Adams et al., 2005). The rat is easily amenable to ¹⁸F-DOPA PET imaging and correlation with numbers of surviving dopaminergic neurons in the 6-hydroxy-dopamine (6-OHDA) lesion rat model of PD has been demonstrated (Kyono et al., 2011). Furthermore, in vivo dopaminergic compensation has been reported in this rat 6-OHDA lesion model of PD (Sossi et al., 2009). The MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) lesion model in monkeys have also identified a very close correlation with ¹⁸F-DOPA uptake in the striatum, and multiple measures of parkinsonism, include symptoms on the Kurlan scale, striatal dopamine content, and markers of dopaminergic neurons, such as tyrosine hydroxylase (Blesa et al., 2012). PET imaging with ¹⁸F-DOPA has also been applied to genetic rodent models of PD. Weaver mutant mice have a point mutation in the GIRK2 potassium channel and ¹⁸F-DOPA PET imaging non-invasively identified a dopaminergic deficit in the striatum and this deficit worsened with increasing age (Sharma and Ebadi, 2005; Sharma et al., 2006).

Genetic mouse and rat models of PD have been generated using different approaches with variable success. Although the majority of transgenic and knock-out/knock-in models are in the mouse, there have been several reported rat models. One transgenic rat model has been produced with a human SNCA construct with two PD mutations (A30P and A53T) under control of the rat TH promoter, and the major deficits reported in this model were olfactory (Lelan et al., 2011). A bacterial artificial chromosome (BAC) transgenic rat model expressing human SNCA with the E46K mutation identified a trend for decreased immunostaining for TH in the striatum and α-synuclein aggregates were identified in neuronal processes in the striatum (Cannon et al., 2013). A spontaneous non-coding SNCA mutation in a Sprague–Dawley rat colony was found to significantly increase SNCA expression in the brain, caused accumulation of nitrosylated and phosphorylated α-synuclein protein, and lead to a significant impairment of dopamine release in the striatum (Stoica et al., 2012; Guatteo et al., 2017). The most representative genetic rat model of PD to date used a human BAC transgene to over-express WT human SNCA (BAC-hSNCA) (Nuber et al., 2013). Transgenic rats were observed to have significantly decreased expression of TH in the striatum as well as significantly decreased striatal dopamine levels in aged BAC-hSNCA rats compared with healthy controls (Nuber et al., 2013). Furthermore, neuritic α-synuclein pathology was identified in the striatum of aged transgenic rats and significant motor deficits were observed from 12 months of age (Nuber et al., 2013). A subsequent study found that BAC-hSNCA rats have a significant early-onset (4 months) serotonergic deficit leading to anxiety-like phenotypes prior to the onset of motor deficits (Kohl et al., 2016).

To date the majority of genetic rat models of PD have been generated by the random insertion of a transgene or the knock-out of a PD-related gene (Creed and Goldberg, 2018). Replicating the precise single amino acid substitution observed in PD kindreds has become possible in the rat with Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome engineering (Yoshimi et al., 2014). Here, we used CRISPR/Cas9 and a donor oligonucleotide in rat zygotes to mutate glycine-51 to aspartic acid (G51D) in rat SNCA exon 3 to produce a novel SNCA^G51D rat model of PD. Striatal dopamine metabolism and turnover were investigated in SNCA^G51D/+ and SNCA^G51D/G51D rats over the course of ageing using ¹⁸F-DOPA PET imaging and kinetic modelling.

The aim of the study was to characterize a novel SNCA^G51D rat model of Parkinson’s disease using ¹⁸F-DOPA PET imaging. Experiments were conducted over an ageing time-course, since phenotypes were anticipated to worsen with time. In patients with PD, striatal dopaminergic function decreases prior to degeneration of nerve terminals (Morrish et al., 1995, 1998; Nurmi et al., 2001).

¹⁸F-DOPA PET imaging data indicated no significant differences in mean K_i of ¹⁸F-DOPA in the striatum of ageing SNCA^G51D/+ and SNCA^G51D/G51D rats compared with age-matched WT rats (Figure 4B). However, there was a significant decreased mean DVR and EDVR of ¹⁸F-DOPA in the striatum of 16 month old SNCA^G51D/G51D rats compared with age-matched WT rats (Figures 4C,D), which indicates increased dopamine turnover in the aged SNCA^G51D/G51D rats.

In early PD the EDVR of ¹⁸F-DOPA in the striatum relative to the cerebellum is significantly decreased compared with healthy controls, which indicates increased effective dopamine turnover and this is likely due to a compensatory change in response to striatal dysfunction (Sossi et al., 2002). This data is supported by results from ¹⁸F-DOPA PET imaging studies of asymptomatic MPTP lesioned monkeys which have also implicated increased effective dopamine turnover as a compensatory mechanism (Doudet et al., 1998). The significantly decreased mean EDVR in 16 month old SNCA^G51D/G51D rats may indicate a compensatory change in dopaminergic function in the striatum of SNCA^G51D/G51D rats following putative striatal dysregulation.

In PD the K_i of ¹⁸F-DOPA in the striatum is significantly decreased compared with healthy controls (Brooks et al., 1990; Burn et al., 1994; Holthoff-Detto et al., 1997; Rinne et al., 2000), with K_i in the putamen reaching 57%–80% of normal levels before symptoms of PD develop (Morrish et al., 1995, 1998; Nurmi et al., 2001). The lack of change in mean K_i in the striatum of SNCA^G51D/+ and SNCA^G51D/G51D rats suggests any potential phenotypes are being sufficiently compensated for in this model and reflects what is observed in patients with prodromal PD. In chemical lesion models of PD, TH has been found to be significantly increased in the striatum post lesioning, a compensatory mechanism in response to nerve damage, which either results in increased TH protein expression or involves morphological changes such as the expansion of nerve terminals (Blanchard et al., 1995; Bezard and Gross, 1998). Compensatory changes in dopaminergic terminals have also been shown to involve the upregulation of AADC activity and the downregulation of DATs (Lee et al., 2000; Adams et al., 2005). In 6-OHDA lesion models of PD, PET imaging studies have identified a complex relationship between the k_ref of ¹⁸F-DOPA and the binding potential of ¹¹C-DTBZ (denervation severity) (Walker et al., 2013a).

Results measuring asymmetry in the EDVR of ¹⁸F-DOPA showed decreased mean EDVR in the left striatum when compared to the right in 5 out of 6 groups of rats, with greatest asymmetry in 16 month old SNCA^G51D/G51D rats (Figure 4E). Reasons that may explain the predilection for the left side could be due to the unique anatomy of the blood vessel supply to the brain in F344 rats which may result in non-uniform perfusion in this strain of rat (Iwasaki et al., 1995).

SNCA^G51D rats were generated using CRISPR/Cas9 technology to model the G51D α-synuclein mutation in humans. The G51D model has good construct validity, and mutant α-synuclein is expressed from the endogenous rat locus. SNCA^G51D rats have the potential to model the widespread neurological abnormalities found in PD and may be a more representative model of PD than the focal 6-OHDA lesion model, which has previously been studied using ¹⁸F-DOPA PET imaging (Kyono et al., 2011; Walker et al., 2013a). Since rodent models of human disease are almost always less severe, we expected the SNCA^G51D rats to have phenotypes similar to early stages of the disease (Petrucci et al., 2016). SNCA^G51D may exhibit a subtle phenotype in rats due to protective factors such as β-synuclein which have been shown to the ameliorate PD-like phenotypes in mice (Hashimoto et al., 2001; Fan et al., 2006). Furthermore, rodents may lack key triggers or cellular components necessary for exhibiting a full PD phenotype. Differences in the SNCA sequence between rodents and humans may explain some of the difficulties in modelling genetic PD, since rodents, indeed most vertebrates, encode a threonine at position 53 which is a cause of PD in humans, and human A53T mice have demonstrated exaggerated motor deficits and α-synuclein pathology following removal of endogenous mouse α-synuclein (Cabin et al., 2005). The SNCA^G51D rat model can be used to investigate sensitivity to potential environmental causes of Parkinson’s. Indeed, the E46K α-synuclein BAC transgenic rat model exhibited a significantly increased sensitivity to rotenone (Cannon et al., 2013).

SNCA^G51D/G51D rats show a significant increase in dopamine turnover in the striatum at 16 months of age, but not a significant decrease in Ki – dopamine synthesis and storage. These findings mimic one component of the early stages of Parkinson’s, and may reflect the compensatory changes in the dopaminergic system observed in humans. SNCA^G51D rats represent an interesting model of early PD pathophysiology, and provide a tractable platform for investigating additional genetic or environmental triggers of Parkinson’s.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

The animal study was reviewed and approved by UK Home Office under project licence PC6C08D7D.

VM, AT, and TK devised the study. TM (Kyoto University) generated the SNCA^G51D rat model. KD (University of Edinburgh) established genotyping of SNCA^G51D rats, maintained the rat colony, and conducted preliminary studies. TW and CL (University of Edinburgh) synthesized the ¹⁸F-DOPA radiotracer, and CA-C (University of Edinburgh) provided assistance during in vivo ¹⁸F-DOPA PET imaging experiments. VM and AT designed and conducted PET experiments and data analysis. VM and TK wrote the manuscript and AT edited the manuscript. All authors contributed to the article and approved the submitted version.

This work was funded by a Carnegie Trust PhD Scholarship to VM, Parkinson’s UK Senior Fellowship to TK, and a Wellcome Trust ISSF award to TK and AT.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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