Marina E. Emborg

Age-Related Declines in Nigral Neuronal Function Correlate With Motor Impairments in Rhesus Monkeys

Although the role of dopamine dysfunction is well established in Parkinsons disease, the effect of nigrostriatal degeneration on motor performance during normal aging is less well understood. In this study, aged rhesus monkeys (25–27 years old) displayed significant impairments relative to young (3–5 years old) cohorts in motor function as assessed on a fine motor task and home cage activity. Additionally, the clinical motor function of aged monkeys was impaired relative to young monkeys as assessed on a clinical rating scale. Unbiased stereologic measurements of the substantia nigra revealed a significant age‐related loss of tyrosine hydroxylase‐immunoreactive (TH‐ir; 50.3%) and dopamine transporter‐immunoreactive (DAT‐ir; 33.2%) nigral neurons. The monkeys performance on the fine motor task and on the clinical rating scale was correlated with TH‐ir neuronal counts. The number of DAT‐ir nigral neurons was correlated with activity and clinical rating scale scores. Our results suggest that age‐related motor impairments in nonhuman primates are associated with spontaneous decreases in TH‐ir and DAT‐ir nigral cells. The correlation of motor deficits with the loss of TH‐ir and DAT‐ir nigral neurons suggests that aged nonhuman primates may provide a useful model for mimicking changes seen in human aging and early Parkinsons disease. J. Comp. Neurol. 401:253–265, 1998.

Lentiviral Gene Transfer to the Nonhuman Primate Brain

Lentiviral vectors infect quiescent cells and allow for the delivery of genes to discrete brain regions. The present study assessed whether stable lentiviral gene transduction can be achieved in the monkey nigrostriatal system. Three young adult Rhesus monkeys received injections of a lentiviral vector encoding for the marker gene beta galatosidase (beta Gal). On one side of the brain, each monkey received multiple lentivirus injections into the caudate and putamen. On the opposite side, each animal received a single injection aimed at the substantia nigra. The first two monkeys were sacrificed 1 month postinjection, while the third monkey was sacrificed 3 months postinjection. Robust incorporation of the beta Gal gene was seen in the striatum of all three monkeys. Stereological counts revealed that 930,218; 1,192,359; and 1,501,217 cells in the striatum were beta Gal positive in monkeys 1 (n = 2) and 3 (n = 1) months later, respectively. Only the third monkey had an injection placed directly into the substantia nigra and 187,308 beta Gal-positive cells were identified in this animal. The injections induced only minor perivascular cuffing and there was no apparent inflammatory response resulting from the lentivirus injections. Double label experiments revealed that between 80 and 87% of the beta Gal-positive cells were neurons. These data indicate that robust transduction of striatal and nigral cells can occur in the nonhuman primate brain for up to 3 months. Studies are now ongoing testing the ability of lentivirus encoding for dopaminergic trophic factors to augment the nigrostriatal system in nonhuman primate models of Parkinsons disease.

Subthalamic Glutamic Acid Decarboxylase Gene Therapy: Changes in Motor Function and Cortical Metabolism

Parkinsons disease (PD) is associated with increased excitatory activity within the subthalamic nucleus (STN). We sought to inhibit STN output in hemiparkinsonian macaques by transfection with adeno-associated virus (AAV) containing the gene for glutamic acid decarboxylase (GAD). In total, 13 macaques were rendered hemiparkinsonian by right intracarotid 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine injection. Seven animals were injected with AAV-GAD into the right STN, and six received an AAV gene for green fluorescent protein (GFP). Videotaped motor ratings were performed in a masked fashion on a weekly basis over a 55-week period. At 56 weeks, the animals were scanned with 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET). Histological examination was performed at the end of the study. No adverse events were observed after STN gene therapy. We found that the clinical rating scores for the two treatment groups had different patterns of change over time (group × time interaction, P<0.001). On FDG PET, the GAD animals exhibited an increase in glucose utilization in the right motor cortex relative to GFP controls (P<0.001). Metabolism in this region correlated with clinical ratings at end point (P<0.01). Histology confirmed GAD expression in treated animals. These findings suggest that STN AAV-GAD is well tolerated and potentially effective in a primate model of PD. The changes in motor cortical glucose utilization observed after gene therapy are consistent with the modulation of metabolic brain networks associated with this disorder.

The PPAR-γ agonist pioglitazone modulates inflammation and induces neuroprotection in parkinsonian monkeys

BackgroundActivation of the peroxisome proliferator-activated receptor gamma (PPAR-γ) has been proposed as a possible neuroprotective strategy to slow down the progression of early Parkinsons disease (PD). Here we report preclinical data on the use of the PPAR-γ agonist pioglitazone (Actos®; Takeda Pharmaceuticals Ltd.) in a paradigm resembling early PD in nonhuman primates.MethodsRhesus monkeys that were trained to perform a battery of behavioral tests received a single intracarotid arterial injection of 20 ml of saline containing 3 mg of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Twenty-four hours later the monkeys were assessed using a clinical rating scale, matched accordingly to disability, randomly assigned to one of three groups [placebo (n = 5), 2.5 (n = 6) or 5 (n = 5) mg/kg of pioglitazone] and their treatments started. Three months after daily oral dosing, the animals were necropsied.ResultsWe observed significant improvements in clinical rating score (P = 0.02) in the animals treated with 5 mg/kg compared to placebo. Behavioral recovery was associated with preservation of nigrostriatal dopaminergic markers, observed as higher tyrosine hydroxylase (TH) putaminal optical density (P = 0.011), higher stereological cell counts of TH-ir (P = 0.02) and vesicular monoamine transporter-2 (VMAT-2)-ir nigral neurons (P = 0.006). Stereological cell counts of Nissl stained nigral neurons confirmed neuroprotection (P = 0.017). Pioglitazone-treated monkeys also showed a dose-dependent modulation of CD68-ir inflammatory cells, that was significantly decreased for 5 mg/kg treated animals compared to placebo (P = 0.018). A separate experiment to assess CSF penetration of pioglitazone revealed that 5 mg/kg p.o. induced consistently higher levels than 2.5 mg/kg and 7.5 mg/kg. p.o.ConclusionsOur results indicate that oral administration of pioglitazone is neuroprotective when administered early after inducing a parkinsonian syndrome in rhesus monkeys and supports the concept that PPAR-γ is a viable target against neurodegeneration.

Contributions of non-human primates to neuroscience research

Non-human primates have a small but important role in basic and translational biomedical research, owing to similarities with human beings in physiology, cognitive capabilities, neuroanatomy, social complexity, reproduction, and development. Although non-human primates have contributed to many areas of biomedical research, we review here their unique contributions to work in neuroscience, and focus on four domains: Alzheimers disease, neuroAIDS, Parkinsons disease, and stress. Our discussion includes, for example, the role of non-human primates in development of new treatments (eg, stem cells, gene transfer) before phase I clinical trials in patients; basic research on disease pathogenesis; and understanding neurobehavioural outcomes resulting from genotype-environment interactions.

Specification of Midbrain Dopamine Neurons from Primate Pluripotent Stem Cells

By sequentially applying sonic hedgehog (C25II) and CHIR99021 (GSK3β inhibitor) to induce the midbrain floor plate (FP) progenitors and fibroblast growth factor 8 (FGF8) to promote dopaminergic differentiation in a chemically defined medium, we have established a robust system for the generation of midbrain dopamine (DA) neurons from human and rhesus monkey embryonic stem cells and induced pluripotent stem cells (PSCs). We found that CHIR99021 specifies diencephalon to hind brain fates in a concentration‐dependent manner and only a narrow concentration range of CHIR99021 at a particular window is necessary to induce the midbrain FP progenitors, expressing Corin, En1, FoxA2, and Lmx1a. FGF8 enhances the dopaminergic fate of the progenitors, thus generating DA neurons with midbrain characteristics, including expression of tyrosine hydroxylase, Lmx1a/b, FoxA2, FoxP1, Nurr1, and En1 as well as typical electrophysiological properties. More than half of these DA neurons expressed A9 DA neuron markers Girk2 and ALDH1a1. The new strategy will allow generation of enriched populations of functional midbrain DA neurons from both human and monkey PSCs for disease modeling, drug testing, and potential cell therapy. STEM CELLS2012;30:1655–1663

Induced pluripotent stem cell-derived neural cells survive and mature in the nonhuman primate brain.

The generation of induced pluripotent stem cells (iPSCs) opens up the possibility for personalized cell therapy. Here, we show that transplanted autologous rhesus monkey iPSC-derived neural progenitors survive for up to 6 months and differentiate into neurons, astrocytes, and myelinating oligodendrocytes in the brains of MPTP-induced hemiparkinsonian rhesus monkeys with a minimal presence of inflammatory cells and reactive glia. This finding represents a significant step toward personalized regenerative therapies.

Distribution of high affinity choline transporter immunoreactivity in the primate central nervous system.

A mouse monoclonal antibody (clone 62‐2E8) raised against a human recombinant high‐affinity choline transporter (CHT)‐glutathione‐S‐transferase fusion protein was used to determine the distribution of immunoreactive profiles containing this protein in the monkey central nervous system (CNS). Within the monkey telencephalon, CHT‐immunoreactive perikarya were found in the striatum, nucleus accumbens, medial septum, vertical and horizontal limb nuclei of the diagonal band, nucleus basalis complex, and the bed nucleus of the stria terminalis. Dense fiber staining was observed within the islands of Calleja, olfactory tubercle, hippocampal complex, amygdala; moderate to light fiber staining was seen in iso‐ and limbic cortices. CHT‐containing fibers were also present in sensory and limbic thalamic nuclei, preoptic and hypothalamic areas, and the floccular lobe of the cerebellum. In the brainstem, CHT‐immunoreactive profiles were observed in the pedunculopontine and dorsolateral tegmental nuclei, the Edinger‐Westphal, oculomotor, trochlear, trigeminal, abducens, facial, ambiguus, dorsal vagal motor, and hypoglossal nuclei. In the spinal cord, CHT‐immunoreactive ventral horn motoneurons were seen in close apposition to intensely immunoreactive C‐terminals at the level of the cervical spinal cord. CHT immunostaining revealed a similar distribution of labeled profiles in the aged human brain and spinal cord. Dual fluorescent confocal microscopy revealed that the majority of CHT immunoreactive neurons contained the specific cholinergic marker, choline acetyltransferase, at all levels of the monkey CNS. The present observations indicate that the present CHT antibody labels cholinergic structures within the primate CNS and provides an additional marker for the investigation of cholinergic neuronal function in aging and disease. J. Comp. Neurol. 463:341–357, 2003.

GDNF-secreting human neural progenitor cells increase tyrosine hydroxylase and VMAT2 expression in MPTP-treated cynomolgus monkeys

Human neural progenitor cells (hNPCs) have been proposed as a potential source of cells for ex vivo gene therapy. In this pilot study, three 5-year-old female cynomolgus monkeys received a single intracarotid infusion of MPTP, followed 1 week later by MRI-guided stereotaxic intrastriatal and intranigral injections of male hNPCs transgenic for GDNF. Immunosupression with oral cyclosporine (30–40 mg/kg) began 48 h before hNPC transplants and continued throughout the study. We monitored the animals using a clinical rating scale (CRS). Three months postsurgery, we euthanized the animals by transcardiac perfusion, then retrieved and processed their brains for morphological analysis. Our findings include the following. 1) hNPCs survived and produced GDNF in all animals 3 months postsurgery. 2) hNPCs remained in the areas of injection as observed by GDNF immunostaining and in situ hybridization for the human Y chromosome. 3) A “halo” of GDNF expression was observed diffusing from the center of the graft out into the surrounding area. 4) We observed increased TH- and VMAT2-positive fibers in areas of GDNF delivery in two of the three animals. The two animals with TH- and VMAT2-positive fibers also showed reductions in their CRS scores. 5) Some GFAP-positive perivascular cuffing was found in transplanted areas. 6) General blood chemistry and necropsies did not reveal any abnormalities. Therefore, we conclude that hNPCs releasing GDNF may be a possible alternative for intracerebral trophic factor delivery in Parkinsons disease.

6-[18F]Fluoro-l-m-tyrosine: metabolism, positron emission tomography kinetics, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine lesions in primates

The tracer 6-[18F]fluoro-L-m-tyrosine (FMT) was studied with regard to its biochemistry and kinetics, as well as its utility in evaluating brain dopaminergic function in primates before and after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment using positron emission tomography (PET). Plasma analysis of FMT and its F18-labeled metabolites 6-fluoro-3-hydroxyphenylacetic acid (FPAC) and 6-fluoro-3-hydroxyphenylethylamine (FMA) during PET scanning enabled kinetic analysis of FMT uptake. A separate study examined brain FMT metabolism in MPTP-naive monkeys euthanized 60 or 120 min after FMT injection. Almost 60% of total plasma F-18 activity was associated with FPAC and FMA 120 min after FMT injection. The FMT signal accumulated preferentially in dopaminergic areas such as caudate and putamen. This bilateral FMT signal was disrupted after unilateral intracarotid artery (ICA) MPTP infusion which reduced ipsilateral striatal activity. A three compartment three kinetic rate constant model for FMT uptake revealed reduced FMT decarboxylation (k3) in ipsilateral caudate and putamen after unilateral MPTP although a further decrease was not evident after intravenous MPTP. FPAC was the major F-18 species in all brain regions except in cerebellum where FMT was predominant 60 min post-mortem. FPAC was most concentrated in dopaminergic areas whereas lower levels occurred in areas containing few dopamine terminals. These data demonstrate preferential FMT metabolism and F-18 retention in dopaminergic tissue and support the use of FMT to evaluate normal and abnormal dopaminergic function.