Summary

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. SNCAG51D/+ and SNCAG51D/G51D rats were born in Mendelian ratios and did not exhibit any severe behavourial defects. L-3,4-dihydroxy-6-18Ffluorophenylalanine (18F-DOPA) positron emission tomography (PET) imaging was used to investigate this novel rat model. Wild-type (WT), SNCAG51D/+ and SNCAG51D/ 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 (Ki ) and effective distribution volume ratio (EDVR) of 18F-DOPA in the striatum relative to the cerebellum in WT, SNCAG51D/+ and SNCAG51D/G51D rats. A significant reduction in EDVR was observed in SNCAG51D/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 SNCAG51D/G51D rats. The increased and asymmetric dopamine turnover observed in the striatum of aged SNCAG51D/G51D rats reflects one aspect of prodromal PD, and suggests the presence of compensatory mechanisms. SNCAG51D rats represent a novel genetic model of PD, and kinetic modelling of 18F-DOPA PET data has identified a highly relevant early disease phenotype.

Results from nanoScan® PET/CT

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. Images were obtained using the nanoScan PET/CT scanner (Mediso Medical Imaging Systems), with PET/ CT data acquired using Nucline™ v2.01 acquisition software. A homogenous solution of 18F-FDG was used in a National Electrical Manufacturers Association (NEMA) NU-4 mouse image quality (IQ) phantom. Imaging data was acquired and reconstructed using variations of several parameters (Figure 1). 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). 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 5ns. After PET imaging, CT data was acquired (trajectory semi-circular, maximum field of view, 480 projections, 55 kVp and 300ms, binning 1:4) and used for attenuation correction of PET data. The PET data 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 0.4mm×0.4mm×0.4mm, used 4 iterations and 6 subsets, and was corrected for scatter, attenuation and randoms. Data was reconstructed into frames comprising 6 frames of 30s, 3 frames of 60s, 2 frames of 120s and 22 frames of 300s.

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 0–120min of emission data then a 1mm×1mm×1mm Gaussian filter was applied. Standardized uptake value (SUV) images were produced using rat body weight and the activity injected. Similarly, SUV Time activity curves (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)].

• 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, and dopamine receptor binding in rat cerebellum is similar to background.
• 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.
  
  

Figure 1. PET IQ Phantom data. The phantom was filled with 3.8 MBq of 18F-FDG solution. (A) Images of the central chamber after reconstructing the PET data using different methods varying the resolution (fast/normal/fine) and the number of iterations and subsets used for reconstruction. (B) Effect of the reconstruction method on the % standard deviation (% SD) in image uniformity. (C) Images of air- and water-filled inserts of the phantom. (D,E) Effect of spillover ratio (SOR) of activity into air (D) and water (E). (F) Images of rods of differing diameters (1mm – 5mm) in the phantom. (G,H) Recovery coefficients (RC) for 2 mm rods (G), and 3 mm rods (H).

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).

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

WT, SNCAG51D/+ and SNCAG51D/G51D rats were subjected to 18FDOPA 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, SNCAG51D/+ and SNCAG51D/G51D rats (Figure 3A), and plotted to show the specific uptake of 18F-DOPA into the striatum compared with the cerebellum. Kinetic modelling used the Patlak reference tissue model and Logan reference tissue model to determine the Ki of 18F-DOPA in the striatum and the distribution volume ratio (DVR) of 18FDOPA in the striatum relative to the cerebellum, respectively. The Patlak reference tissue model is a graphical analysis method that models the irreversible uptake of radiotracers. The Logan reference tissue model analyzed reversible tracer uptake. effective DVR (EDVR) can be used to estimate the effective dopamine turnover. The Logan reference tissue model was also used to determine the EDVR of 18F-DOPA which involved subtracting the TAC (kBq/mL) for the cerebellum from the TACs for the striatum before analysis. Differences in EDVR between left and right striatum were also investigated by calculating asymmetry in EDVR which = (EDVR contralateral − EDVR ipsilateral)/EDVR contralateral.

• The mean Ki values of 18F-DOPA in 5–16 month old SNCAG51D/+ and SNCAG51D/G51D rats compared with age-matched WT rats were not significantly different (Figure 3B).
• 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 3C,D).
• 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, the results indicate an increase in mean dopamine turnover in 16 month SNCAG51D/G51D rats compared with age-matched WT rats.

Since early Parkinson’s often presents with an asymmetric dopaminergic deficit, they investigated this phenotype in their rat model.

• Interestingly, the mean EDVR of 18F-DOPA in the left and right striatum of 5 month old and 16 month old SNCAG51D/G51D rats were significantly different, but this was not observed in age-matched SNCAG51D/+ and WT rats (Figure  3E).
• The EDVR asymmetry in SNCAG51D/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.

Figure 3. Kinetic modelling of 18F-DOPA PET data from WT, SNCAG51D/+ and SNCAG51D/G51D rats at 5, 11, and 16 months of age. (A) Standardized uptake value (SUV) time activity curves (TACs) for all rat genotypes—WT, SNCAG51D/+, and SNCAG51D/G51D rats, n = 4 per genotype per age-group. m-months old, Str.-Striatum, Cer.-Cerebellum. (B) The mean Ki in the striatum of 5–16 month old WT, SNCAG51D/+ and SNCAG51D/G51D rats. (C) There was a significant decrease in mean DVR of 18F-DOPA in 16 month old SNCAG51D/G51D rats when 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 SNCAG51D/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 results obtained by in vivo PET/CT imaging demonstrated that SNCAG51D/G51D rats show a significant increase in dopamine turnover in the striatum at 16months 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. SNCAG51D rats represent an interesting model of early PD pathophysiology, and provide a tractable platform for investigating additional genetic or environmental triggers of Parkinson’s.

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