In vivo18F-DOPA PET imaging identifies a dopaminergic deficit in a rat model with a G51D α-synuclein mutation

Parkinson’s disease (PD) is a neurodegenerative condition with several major hallmarks, including loss of substantia nigra neurons, reduction in striatal dopaminergic function, and formation of α-synuclein-rich Lewy bodies. Mutations in SNCA, encoding for α-synuclein, are a known cause of familial PD, and the G51D mutation causes a particularly aggressive form of the condition. CRISPR/Cas9 technology was used to introduce the G51D mutation into the endogenous rat SNCA gene. SNCA^G51D/+ and SNCA^G51D/G51D rats were born in Mendelian ratios and did not exhibit any severe behavourial defects. L-3,4-dihydroxy-6-¹⁸F-fluorophenylalanine (¹⁸F-DOPA) positron emission tomography (PET) imaging was used to investigate this novel rat model. Wild-type (WT), SNCA^G51D/+ and SNCA^G51D/G51D rats were characterized over the course of ageing (5, 11, and 16 months old) using ¹⁸F-DOPA PET imaging and kinetic modelling. We measured the influx rate constant (K_i) and effective distribution volume ratio (EDVR) of ¹⁸F-DOPA in the striatum relative to the cerebellum in WT, SNCA^G51D/+ and SNCA^G51D/G51D rats. A significant reduction in EDVR was observed in SNCA^G51D/G51D rats at 16 months of age indicative of increased dopamine turnover. Furthermore, we observed a significant asymmetry in EDVR between the left and right striatum in aged SNCA^G51D/G51D rats. The increased and asymmetric dopamine turnover observed in the striatum of aged SNCA^G51D/G51D rats reflects one aspect of prodromal PD, and suggests the presence of compensatory mechanisms. SNCA^G51D rats represent a novel genetic model of PD, and kinetic modelling of ¹⁸F-DOPA PET data has identified a highly relevant early disease phenotype.

Introduction

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.

Materials and methods

Point mutation in rat genome with CRISPR/Cas9

CRISPR/Cas9 constructs used were the gRNA_Cloning vector (a gift from George Church, Addgene plasmid # 41824¹; RRID:Addgene_41824) and humanized Cas9 nuclease vector (a gift from George Church, Addgene plasmid # 41815²; RRID:Addgene_41815) (Mali et al., 2013). The gRNA sequence was designed, synthesized, and inserted into the gRNA_Cloning vector into the AflII site. The gRNA sequence is: 5′-GTCGTTCATGGAGTGACAAC-3′. The gRNA and Cas9 vectors were each linearized with XhoI and in vitro transcribed using a MessageMAXT7 ARCA-Capped Message Transcription Kit (CELLSCRIPT, Madison, WI, USA). A 80-nt ssDNA mutant donor oligonucleotide with the desired 2-bp mutation (GGA to GAT) was synthesized. The sequence of the ssDNA donor is: 5′-CAATTCTTTTTTTAGGTTCCAAAACTAAGGAGGGAGTCGTTCATGatGTGACAACAGGTAAGCTCTGTTGTCTTTTATCC-3′. The gRNA, Cas9 mRNA, and ssDNA donor oligonucleotide were co-injected into male pronuclei of F344/Stm rat zygotes. Eleven (11) founder rats were screened by Sanger sequencing. Five (5) founders had mutations in exon 3 of SNCA, and one founder had the desired GGA to GAT (G51D) mutation (Supplementary Figure S1). This founder was mated to produce F1 progeny, and the mutation transmitted through the germ-line as determined by Sanger sequencing of genomic DNA extracted from ear notches (Figure 1A). Since the mutation introduced a new BspHI restriction site (Figure 1B), subsequent genotyping was performed by SNCA exon 3 PCR (forward primer 5′-TGGTGGCTGTTTGTCTTCTG-3′ and reverse primer 5′-TCCTCTGAAGACAATGGCTTTT-3′), a BspHI restriction enzyme digest, and agarose gel electrophoresis (Figure 1C).

FIGURE 1

Figure 1. Genotyping of SNCA^G51D rats. (A) Sanger sequencing of rat SNCA exon 3 of a WT and an SNCA^G51D/+ F1 rat revealed a heterozygous mutation (GGA/GAT) at codon 51. (B) The G51D mutation generated a new BspHI restriction site (TCATGA) in exon 3. (C) SNCA exon 3 PCR and BspHI restriction enzyme digest can reliably genotype WT, SNCA^G51D/+ and SNCA^G51D/G51D rats. (D) 44 neonatal (P2, P3) pups from five (5) SNCA^G51D/+ X SNCA^G51D/+ crosses were genotyped, and Mendelian ratios were observed.

Animal husbandry

The breeding and maintenance of wild-type (WT), SNCA^G51D/+ and SNCA^G51D/G51D rats, and the in vivo experiments conducted were approved by the UK Home Office under project licence PC6C08D7D. WT, SNCA^G51D/+ and SNCA^G51D/G51D rats were analyzed over the course of ageing at 5, 11, and 16 months using ¹⁸F-DOPA PET imaging and no rats were re-used for repeat scanning (n = 4 per genotype per age-group, for a total of 36 rats).

PET imaging

¹⁸F-DOPA was produced using a multi-step nucleophilic fluorination pathway, and used the TRACERlab MX synthesiser and cassette with the nucleophilic precursor to ¹⁸F-DOPA (ABX Advanced Biochemical Compounds, PEDP-0062-H), with the final product formulated in PBS (Martin et al., 2013).

Imaging was conducted under general anesthesia and used Isoflurane in 0.6 L/min O₂ and 0.4 L/min N₂O. 10 mg/kg Carbidopa (Sigma-Aldrich, C1335), then 10 mg/kg Entacapone (Sigma-Aldrich, SML-0654) were injected intravenously to prevent the peripheral metabolism of ¹⁸F-DOPA by AADC and catechol-O-methyltransferase (COMT), respectively (Walker et al., 2013a). Thirty minutes after injection of Carbidopa and Entacapone, 18.5 +/− 7.1 MBq (mean +/− SD) of ¹⁸F-DOPA was injected as a bolus via the tail vein. Activity in the empty syringe was measured, with the activity injected calculated and decay corrected. Dynamic PET imaging lasted 2 h.

Images were obtained using the nanoScan PET/CT scanner (Mediso Medical Imaging Systems, Budapest, Hungary), with PET/CT data acquired using Nucline™ v2.01 acquisition software. Prior to radiotracer injection a scout view CT image was acquired. Dynamic PET imaging commenced upon injection of ¹⁸F-DOPA and used a coincidence mode 1–5 and coincidence time window of 5 ns. After PET imaging, CT data was acquired (trajectory semi-circular, maximum field of view, 480 projections, 55 kVp and 300 ms, binning 1:4) and used for attenuation correction of PET data.

The PET data that was reconstructed and extended from the olfactory bulbs to the caudal border of the heart. Reconstruction was 3D, dynamic and used the TeraTomo3D reconstruction method. Coincidence mode was 1–3, voxel size 0.4 mm × 0.4 mm × 0.4 mm, used 4 iterations and 6 subsets, and was corrected for scatter, attenuation and randoms. Data was reconstructed into frames comprising 6 frames of 30 s, 3 frames of 60 s, 2 frames of 120 s and 22 frames of 300 s. All PET imaging data is freely accessible.³

Image analysis

Images were analyzed using PMOD 3.409 software (PMOD Technologies LLC) and a hand-drawn template. Volumes of interest (VOIs) comprised the left and right striatum, whole striatum, and the cerebellum which was a reference region for non-specific uptake. The same VOI template was used for all rats and were only moved into position over the respective anatomical areas. PET images for display purposes and to aid with VOI drawing were obtained by averaging over 0–120 min of emission data then a 1 mm × 1 mm × 1 mm Gaussian filter was applied. Standardized uptake value (SUV) images were produced using rat body weight and the activity injected. Similarly, SUV TACs were calculated as follow SUV (g/mL) = activity concentration in the target VOI (kBq/mL)/[decay corrected amount of ¹⁸F-DOPA injected (MBq)/weight of the rat (kg)].

Kinetic modelling used the Patlak reference tissue model and Logan reference tissue model to determine the K_i of ¹⁸F-DOPA in the striatum and the distribution volume ratio (DVR) of ¹⁸F-DOPA in the striatum relative to the cerebellum, respectively (Logan, 2000). The cerebellum is used as a reference brain region since dopamine receptor binding in rat cerebellum is much lower than in striatum and equivalent to background (Kuhar et al., 1978), and PET imaging in humans demonstrated the cerebellum is devoid of any dopaminergic signals (Wong et al., 1986). Furthermore, PD patients do not exhibit any Lewy pathology in the cerebellum (Devi et al., 2008). The Patlak reference tissue model used 60 min of data and a t* of 10 min in accordance with previous studies in Sprague Dawley rats (Kyono et al., 2011). This is a graphical analysis method that models the irreversible uptake of radiotracers (Patlak and Blasberg, 1985). In contrast, reversible tracer uptake was analyzed with the Logan reference tissue model using 120 min of data and t* of 30 min, which were parameters optimized from our WT F344 rat data and these differ from other parameters used to analyze PET data from Sprague Dawley rats (Walker et al., 2013a). The Logan reference tissue model was also used to determine the effective DVR (EDVR) of ¹⁸F-DOPA which involved subtracting the TAC (kBq/mL) for the cerebellum from the TACs for the striatum before analysis (Walker et al., 2013a). EDVR can be used to estimate the effective dopamine turnover (Sossi et al., 2002). Differences in EDVR between left and right striatum were also investigated by calculating asymmetry in EDVR which = (EDVR contralateral − EDVR ipsilateral)/EDVR contralateral (Walker et al., 2013b). All K_i, DVR, EDVR, and asymmetry EDVR data for each individual rat is supplied in Supplementary Tables 1, 2.

Statistical analysis

¹⁸F-DOPA PET data from WT, SNCA^G51D/+, and SNCA^G51D/G51D rats are presented where appropriate as the mean +/− standard deviation. K_i, DVR, and EDVR results from age-matched WT, SNCA^G51D/+ and SNCA^G51D/G51D rats were analyzed using a 1-way ANOVA with Tukey’s multiple comparison test. Left–right EDVR asymmetry results were investigated for statistical significance using a paired t test. Although the sample size is small for each experimental variable (n = 4 per genotype per time-point), quantile-quantile plots of the K_i, DVR, and EDVR data-sets indicated the data is normally distributed.

Results

Generation of the SNCA^G51D rat model

The G51D mutation in PD patients is due to a single nucleotide change (c.152 G > A) that mutates the Gly-51 codon (GGT) to GAT, encoding for aspartic acid. The rat SNCA Gly-51 codon is GGA, which requires a 2-nucleotide mutation to produce the Asp-51 codon GAT. Cas9 mRNA, gRNA and ssDNA donor oligonucleotide with GAT codon were co-injected into F344/Stm rat zygotes. Eleven founder rats were screened by Sanger sequencing and 5 F0 rats had mutations in SNCA, but only one founder had the desired GGA to GAT (G51D) mutation in exon 3 of SNCA (Supplementary Figure S1). This F0 founder rat was bred, and F1 animals with the G51D mutation were identified by Sanger sequencing confirming the mutation could transmit through the germ-line (Figure 1A). All subsequent progeny from the sequence-confirmed F1 rats were PCR-genotyped taking advantage of a new BspHI restriction site introduced by the G51D mutation (Figures 1B,C). Heterozygous SNCA^G51D/+ matings produced progeny in Mendelian ratios suggesting the mutation did not reduce embryonic or fetal viability (Figure 1D).

Optimization of ¹⁸F-DOPA PET imaging in Fischer 344 rats

The methods used for the reconstruction of in vivo PET imaging experiments were optimized since the rat striatum is a small structure and the optimal acquisition and reconstruction of PET data is scanner dependent. A homogenous solution of ¹⁸F-FDG was used in a National Electrical Manufacturers Association (NEMA) NU-4 mouse image quality (IQ) phantom (Harteveld et al., 2011). Imaging data was acquired and reconstructed using variations of several parameters (Supplementary Figure S2). The most promising reconstruction scenario used iterative methods and employed 4 iterations and 6 subsets (4i6S) at a normal resolution and a coincidence mode of 1–3. The in vivo imaging and kinetic modelling methods were optimized using data obtained from preliminary experiments using two WT F344 rats (Figure 2). Time activity curves (TACs) and standardized uptake values (SUV) were calculated and plotted for striatum and cerebellum (Figures 2B,C). The cerebellum was used as a reference region since it lacks AADC expression (Baker et al., 1991), and dopamine receptor binding in rat cerebellum is similar to background (Kuhar et al., 1978). Plotting the SUV ratio (SUVr) of the striatum and cerebellum for both rats showed the data was consistent and that the activity reached a pseudo-equilibrium between 50 and 85 min (Figure 2D). This data indicated that methods used for kinetic modelling in recent PET imaging studies of models of PD were optimal (Kyono et al., 2011). Kinetic modeling using Patlak and Logan graphical analysis used data collected over 60 and 180 min, respectively (Figure 3).

FIGURE 2

Figure 2. Representative PET-CT images and Standardized uptake value (SUV) time activity curves (TACs) for wild-type (WT) rats. (A) PET-CT images of two WT rats are shown in coronal and transverse planes. ¹⁸F-DOPA PET data averaged over frames 1–33 and smoothed using a 1 mm × 1 mm × 1 mm Gaussian filter. HU-Hounsfield Units. Specific activity (B), SUV TAC data (C), and SUV ratio (SUVr) data (D) for the specific uptake of ¹⁸F-DOPA into the striatum relative to the cerebellum is shown for two WT rats. The dashed box indicates the phase of pseudo-equilibrium (50–85 min).

FIGURE 3

Figure 3. Patlak and Logan analysis for WT rats. (A) Patlak graphical analysis of 60 min of data (t* of 10 min) from the whole striatum of a WT rat relative to cerebellum. (B) Logan graphical analysis of 180 min of data (t* of 130 min) from the whole striatum of a WT rat.

¹⁸F-DOPA PET imaging reveals a deficit in dopamine turnover

WT, SNCA^G51D/+ and SNCA^G51D/G51D rats were subjected to ¹⁸F-DOPA PET at 5, 11, and 16 months of age using the conditions optimized above. A total of 36 rats were scanned with 4 rats per genotype per age-group, and no rats were re-scanned for longitudinal studies. Data was analyzed to determine SUV TACs from WT, SNCA^G51D/+ and SNCA^G51D/G51D rats (Figure 4A), and plotted to show the specific uptake of ¹⁸F-DOPA into the striatum compared with the cerebellum.

FIGURE 4

Figure 4. Kinetic modelling of ¹⁸F-DOPA PET data from WT, SNCA^G51D/+ and SNCA^G51D/G51D rats at 5, 11, and 16 months of age. (A) Standardized uptake value (SUV) time activity curves (TACs) for all rat genotypes—WT, SNCA^G51D/+, and SNCA^G51D/G51D rats, n = 4 per genotype per age-group. m-months old, Str.-Striatum, Cer.-Cerebellum. (B) The mean K_i in the striatum of 5–16 month old WT, SNCA^G51D/+ and SNCA^G51D/G51D rats. (C) There was a significant decrease in mean DVR of ¹⁸F-DOPA in 16 month old SNCA^G51D/G51D rats when compared with age-matched WT rats (*p < 0.05), and (D) also a significant decrease in EDVR of ¹⁸F-DOPA in 16 months SNCA^G51D/G51D rats compared with age-matched WT rats (*p < 0.05). (E) Mean left–right asymmetry in EDVR was significantly different in 5 month old and 16 month old SNCA^G51D/G51D rats. Data shows the mean, standard deviation and individual data-points. n = 4 per genotype per age-group. Paired t test. *p < 0.05 L-left striatum, R-right striatum, m-months old.

The mean K_i values of ¹⁸F-DOPA in 5–16 month old SNCA^G51D/+ and SNCA^G51D/G51D rats compared with age-matched WT rats were not significantly different (Figure 4B). The mean DVR and EDVR of ¹⁸F-DOPA in the striatum relative to the cerebellum was significantly decreased in 16 month old rats compared to WT rats, but these differences were not observed at 5 and 11 months of age (Figures 4C,D) (Supplementary Table 1). The EDVR of ¹⁸F-DOPA is the ratio of the distribution volumes of ¹⁸F-DOPA in the specific and precursor compartments reduced by the factor k₂/(k₂ + k₃), and since EDVR is estimated to be the inverse of effective dopamine turnover (Sossi et al., 2002), the results indicate an increase in mean dopamine turnover in 16 month SNCA^G51D/G51D rats compared with age-matched WT rats. Since early Parkinson’s often presents with an asymmetric dopaminergic deficit (Djaldetti et al., 2006), we investigated this phenotype in our rat model. Interestingly, the mean EDVR of ¹⁸F-DOPA in the left and right striatum of 5 month old and 16 month old SNCA^G51D/G51D rats were significantly different, but this was not observed in age-matched SNCA^G51D/+ and WT rats (Figure 4E). The EDVR asymmetry in SNCA^G51D/G51D rats was between −0.4 and 0.3, while the normal range determined for Sprague Dawley rats is between −0.1 and 1.0 (Walker et al., 2013b).

Discussion

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).

Conclusion

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.

Data availability statement

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.

Ethics statement

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

Author contributions

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.

Funding

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.

Acknowledgments

The authors would like to thank Yayoi Kunihiro for assistance in generation of the SNCA^G51D rat model.

Conflict of interest

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.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnins.2023.1095761/full#supplementary-material

Footnotes

1. ^http://n2t.net/addgene:41824

2. ^http://n2t.net/addgene:41815

3. ^https://datashare.ed.ac.uk/handle/10283/4014

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