We followed longitudinally the behavior and dopaminergic function of Göttingen minipigs exposed repetitively to MPTP with multi-tracer PET imaging. The MPTP paradigm was similar to that previously used in a large animal model which successfully induced parkinsonism15–20. Our data show that minipigs dosed with MPTP over a one month period demonstrate significant reductions in dopaminergic presynaptic function, observed up to one year later, along with cell death, even in the absence of sustained behavioral symptoms. Despite the significant reductions in tracer binding at all timepoints post-MPTP, we found spontaneous partial recovery of striatal [18F]-FDOPA Ki uptake when comparing data 1 month vs 9 months after the final MPTP injection. Even though the minipigs showed only mild and reversible behavioral changes after MPTP in the most aggressive phase, the administration protocol was effective and sufficient to induce in vivo dopamine storage and VMAT2 binding changes along with pathological nigral dopamine cell loss.
The MPTP model in non-human primates is still the gold-standard in PD animal lesion models, especially for trialing the efficacy of novel symptomatic medications1. However, there are well known limitations to MPTP administration, including spontaneous recovery from motor symptoms23,24 which is not observed in idiopathic PD patients. This limits its potential to investigate long-term efficacy of putative neuroprotective medications on motor symptoms. Studies have found that cell loss, however, is not statistically different between non-human primates that recover from MPTP exposure compared to those with stable motor deficits25 suggesting variable degrees of dopamine terminal adaptation to the nigral lesions are occurring. Indeed, it has been found that remaining TH + fibers can sprout and increase branching after partial lesion with MPTP in non-human primates26 and our data suggest an increase in aromatic L-amino acid decarboxylase (AADC) activity important for the intracellular retention of [18F]-FDOPA in remaining terminals at both 9 and 12 months post-MPTP. It is also known that the same MPTP dose can lead to mild to severe motor deficits in the same species. Previous studies of Göttingen minipigs administered MPTP have shown behavioral sensitivity and moderate parkinsonism, though they reported a non-significant tendency towards improved scores throughout the 6-month study period17,18. In contrast to their low 0.7-1 mg/kg daily SC injection (cumulative dose of 6.6 ± 1.4 mg/kg over a 22-day injection period), our initial dose of 1 mg/kg three times a week, was doubled in the final 2 weeks of injections due to the apparent lack of motor symptoms, resulting in a cumulative dose of 18 mg/kg. Previous studies in non-human primates administered cumulative doses of 1.6 mg/kg27 and 1.8 mg/kg as a progressive intoxication dose, and 3.2 mg/kg as an acute intoxication dose25. It is unclear why the MPTP dose in the current cohort of minipigs, which far exceeded the dose administered in previous large animal studies, was not sufficient to elicit moderate parkinsonian symptoms, or any stable behavioral impairment.
However, we speculate that our use of midazolam and (S)-ketamine anesthesia prior to MPTP-injections to increase safety of the staff handling MPTP might have protected the minipigs against extensive fiber loss. Midazolam and MPTP are both competitive inhibitors of various cytochrome P450 isozymes, that are involved in the neurotoxicity of MPTP28,29. Furthermore, a recent study found that (R)-ketamine attenuated MPTP-induced reduction in striatal dopamine transporter (DAT) and TH, while (S)-ketamine only had a small effect on DAT30. Therefore, the combination of midazolam (S)-ketamine may have reduced the dopamine terminal loss. Nevertheless, the dose was sufficient to induce 71% TH + area loss in the SN and significant reductions in PET markers of striatal presynaptic dopamine neurotransmission.
[18F]-FDOPA PET has previously been used to assess decline in striatal dopamine terminal function in MPTP-induced parkinsonism and its response to fetal mesencephalic dopaminergic allografts in pigs17 and humans31. The previous transplant study reported motor symptoms and a stable 60% loss of dopa decarboxylase activity 3- and 6-months after MPTP in the non-grafted minipigs. We show here that asymptomatic minipigs demonstrate a clear and persistent reduction in [18F]-FDOPA uptake even when behaviorally intact but, for the first time, we also demonstrate a significant recovery at 9 months after MPTP intoxication, which was also sustained at 12 months post-MPTP. The observed decline in striatal [18F]-FDOPA uptake of MPTP-lesioned pigs is consistent with changes reported in MPTP-exposed non-human primates32 and both idiopathic and MPTP-exposed PD patients33. Franke et al. also suggested that mild pathological changes were reversible after cessation of MPTP administration in marmosets34.
We have previously shown reductions in striatal [11C]-DTBZ binding in minipig models of parkinsonism induced by acute and chronic proteasome inhibition11,14, and preformed a-synuclein fibrils35 or adeno-associated viral (AAV) vector induced a-syn overexpression in rodents36. However, this is the first study of VMAT2 binding in MPTP-injected minipigs. Radio-labeled DTBZ binds with nanomolar-affinity to the VMAT2 transporter protein of synaptic vesicles in monoaminergic neurons37 and can non-invasively track presynaptic dopamine terminal density in the striatum38. It was once thought that DTBZ binding was insensitive to pharmacological challenges39, however, it is now accepted that binding may be altered by changes in intravesicular concentrations of stored dopamine40–42. Selective loss of VMAT2 is one of the first measurable effects of aging and active PD in human, and aging and MPTP exposure in non-human primates22,43 and precedes the appearance of clinical features. VMAT2 reductions in striatum and SN correlate with disease severity of PD patients44. Here we show 55–60% decreases in VMAT2 availability in MPTP-treated minipigs in the absence of overt motor features of parkinsonism. Unlike [18F]-FDOPA, at the time of the final imaging sessions there was no significant recovery in [11C]-DTBZ binding. Previous studies have found 35–46% loss of VMAT2 in striatal regions of asymptomatic non-human primates 2 months after onset of MPTP exposure which progressed to a 69–78% loss by 10 months43. The different results are likely attributable to the fact that the investigators continued to administer MPTP throughout the entire non-human primate study, whereas the scans conducted in minipig were done at specific times after the final MPTP administration in our subchronic protocol.
As [18F]-FDOPA Ki reflects blood-brain barrier transport, AADC activity and vesicular storage, whereas [11C]-DTBZ BPND only reflects synaptic monoamine vesicle availability, the main differences between the uptake and binding should be reflected by the decarboxylation. Using [18F]-FDOPA, [11C]-DTBZ and [11C]-methylphenidate (labeling DAT) PET, Lee at al. have found that [18F]-FDOPA uptake was reduced to a lesser extent than [11C]-DTBZ and [11C]-methylphenidate in PD basal ganglia45. This difference in uptake and binding was argued to reflect up-regulation of AADC activity in the parkinsonian striatum as a plastic mechanism to increase availability of dopamine to postsynaptic dopamine receptors45. Our functional recovery in [18F]-FDOPA uptake at 9 and 12 months post-MPTP might therefore be explained by AADC activity being more prone to up-regulation following dopamine denervation than VMAT2 due to dynamic regulation and compensatory mechanisms46,47. Furthermore, the significant correlation of striatal [18F]-FDOPA uptake and [11C]-DTBZ binding only at the 9 month timepoint supports the differential behavior of the two PET tracers across time after MPTP lesioning in minipigs. We show the temporal stability of [11C]-DTBZ as a marker of terminal damage that correlates with TH + area. This makes the case for using [11C]-DTBZ when planning trials of neuroprotective agents in MPTP-lesioned animals.
In conclusion, we show here for the first time the temporal evolution of two markers of presynaptic dopamine function in a large animal model, the Göttingen minipig, as an alternate to the non-human primate, in response to MPTP. Our asymptomatic to mildly symptomatic animals showed similar radioligand changes observed in behaviorally impaired animal models as well as in human PD. The recovery of [18F]-FDOPA binding 9 and 12 months after the final dose of MPTP suggests that caution should be taken when planning investigations of therapeutic interventions, which may be confounded by spontaneous recovery of the model.