Environmental toxins have been implicated in the etiology of Parkinson's disease. Recent findings of defects in the ubiquitin-proteasome system in hereditary and sporadic forms of the illness suggest that environmental proteasome inhibitors are candidate PD-inducing toxins. Here, we systemically injected six doses of naturally occurring (epoxomicin) or synthetic (Z-lle-Glu(OtBu)-Ala-Leu-al [PSI]) proteasome inhibitors into adult rats over a period of 2 weeks. After a latency of 1 to 2 weeks, animals developed progressive parkinsonism with bradykinesia, rigidity, tremor, and an abnormal posture, which improved with apomorphine treatment. Positron emission tomography demonstrated reduced carbon-11-labeled 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) binding to dopaminergic nerve terminals in the striatum, indicative of degeneration of the nigrostriatal pathway. Postmortem analyses showed striatal dopamine depletion and dopaminergic cell death with apoptosis and inflammation in the substantia nigra pars compacta. In addition, neurodegeneration occurred in the locus coeruleus, dorsal motor nucleus of the vagus, and the nucleus basalis of Meynert. At neurodegenerative sites, intracytoplasmic, eosinophilic, alpha-synuclein/ubiquitin-containing, inclusions resembling Lewy bodies were present in some of the remaining neurons. This animal model induced by proteasome inhibitors closely recapitulates key features of PD and may be valuable in studying etiopathogenic mechanisms and putative neuroprotective therapies for the illness.

The metabolic activation resulting from direct dopaminergic stimulation can be detected using auto-radiography, positron emission tomography (PET) or, potentially, fMRI techniques. To establish the validity of the latter possibility, we have performed a number of experiments. We measured the regional selectivity of two different dopaminergic ligands: the dopamine release compound D-amphetamine and the dopamine transporter antagonist 2 beta-carbomethoxy-3 beta-(4-fluoropheny) tropane (CFT). Both compounds led to increased signal intensity in gradient echo images in regions of the brain with high dopamine receptor density (frontal cortex, striatum, cingulate cortex > > parietal cortex). Lesioning the animals with unilaterally administered 6-hydroxydopamine (6-OHDA) led to ablation of the phMRI response on the ipsilateral side; control measurements of rCBV and rCBF using bolus injections of Gd-DTPA showed that the baseline rCBV and rCBF values were intact on the lesioned side. The time course of the BOLD signal changes paralleled the changes observed by microdialysis measurements of dopamine release in the striatum for both amphetamine and CFT; peaking at 20-40 min after injection and returning to baseline at about 70-90 min. Signal changes were not correlated with either heart rate, blood pressure or pCO2. Measurement of PET binding in the same animals showed an excellent correlation with the phMRI data when compared by either measurements of the number of pixels activated or percent signal change in a given region. The time course for the behavioral measurements of rotation in the 6-OHDA lesioned animals correlated with the phMRI. These experiments demonstrate that phMRI will become a valuable, noninvasive tool for investigation of neurotransmitter activity in vivo.

Summary Autologous transplantation of patient-specific iPSC-derived neurons is a potential clinical approach for treatment of neurological disease. Preclinical demonstration of long-term efficacy, feasibility and safety of iPSC-derived dopamine neurons in non human primate models will be an important step in clinical development of cell therapy. Here, we analyzed cynomolgus monkey (CM) iPSC-derived midbrain dopamine neurons for up to 2 years following autologous transplantation in a Parkinson's disease (PD) model. In one animal, with the most successful protocol, we found that unilateral engraftment of CM-iPSCs could provide a gradual onset of functional motor improvement contralateral to the side of dopamine neuron transplantation, and increased motor activity, without a need for immunosuppression. Post-mortem analyses demonstrated robust survival of midbrain-like dopaminergic neurons and extensive outgrowth into the transplanted putamen. Our proof of concept findings support further development of autologous iPSC-derived cell transplantation for treatment of PD.