Valerie Joers

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.

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.

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.

Response of aged parkinsonian monkeys to in vivo gene transfer of GDNF.

This study assessed the potential for functional and anatomical recovery of the diseased aged primate nigrostriatal system, in response to trophic factor gene transfer. Aged rhesus monkeys received a single intracarotid infusion of MPTP, followed one week later by MRI-guided stereotaxic intrastriatal and intranigral injections of lentiviral vectors encoding for glial derived neurotrophic factor (lenti-GDNF) or beta-galactosidase (lenti-LacZ). Functional analysis revealed that the lenti-GDNF, but not lenti-LacZ treated monkeys displayed behavioral improvements that were associated with increased fluorodopa uptake in the striatum ipsilateral to lenti-GDNF treatment. GDNF ELISA of striatal brain samples confirmed increased GDNF expression in lenti-GDNF treated aged animals that correlated with functional improvements and preserved nigrostriatal dopaminergic markers. Our results indicate that the aged primate brain challenged by MPTP administration has the potential to respond to trophic factor delivery and that the degree of neuroprotection depends on GDNF levels.

A monoclonal antibody-GDNF fusion protein is not neuroprotective and is associated with proliferative pancreatic lesions in parkinsonian monkeys

Glial cell line derived neurotrophic factor (GDNF) is a neurotrophic factor that has neuroprotective effects in animal models of Parkinson’s disease (PD) and has been proposed as a PD therapy. GDNF does not cross the blood brain barrier (BBB), and requires direct intracerebral delivery to be effective. Trojan horse technology, in which GDNF is coupled to a monoclonal antibody (mAb) against the human insulin receptor (HIR), has been proposed to allow GDNF BBB transport (ArmaGen Technologies Inc.). In this study we tested the feasibility of HIRMAb-GDNF to induce neuroprotection in parkinsonian monkeys, as well as its tolerability and safety. Adult rhesus macaques were assessed throughout the study with a clinical rating scale, a computerized fine motor skills task and general health evaluations. Following baseline measurements, the animals received a unilateral intracarotid artery MPTP injection. Seven days later the animals were evaluated, matched according to disability and blindly assigned to receive twice a week iv. treatments (vehicle, 1 or 5 mg/kg HIRmAb-GDNF) for a period of three months. HIRmAb-GDNF did not improve parkinsonian motor symptoms and induced a dose-dependent hypersensitivity reaction. Quantification of dopaminergic striatal optical density and stereological nigral cell counts did not demonstrate differences between treatment groups. Focal pancreatic acinar to ductular metaplasia (ADM) was noted in four of seven animals treated with 1 mg/kg HIRmAb-GDNF; two of four with ADM also had focal pancreatic intraepithelial neoplasia 1B (PanIN-1B) lesions. Minimal to mild, focal to multifocal, nonsuppurative myocarditis was noted in all animals in the 5 mg/kg treatment group. Our results demonstrate that HIRmAb-GDNF dosing in a monkey model of PD is not an effective neuroprotective strategy and may present serious health risks that should be considered when planning future use of the IR antibody as a carrier, or of any systemic treatment of a GDNF-containing molecule.

Intracerebral Transplantation of Differentiated Human Embryonic Stem Cells to Hemiparkinsonian Monkeys

To explore stem cell therapy for Parkinsons disease (PD), three adult rhesus monkeys were first rendered hemiparkinsonian by unilateral intracarotid 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) infusion. Five months postinfusion, they were given MRI-guided stereotaxic intrastriatal and intranigral injections of green fluorescent protein (GFP)-labeled cultures of dopaminergic neurons derived from human embryonic stem cells (DA-hES cells). The animals were immunosuppressed using daily oral cyclosporine (CsA). Three months later, viable grafts were observed at the injection sites in one animal, while no obvious grafts were present in the other two monkeys. The surviving grafts contained numerous GFP-positive cells that were positively labeled for nestin and MAP2 but not for glial fibrillary acidic protein (GFAP), NeuN, or tyrosine hydroxylase (TH). The grafted areas in all animals showed dense staining for GFAP, CD68, and CD45. These results indicated that xenografts of human stem cell derivatives in CsA-suppressed rhesus brain were mostly rejected. Our study suggests that immunological issues are obstacles for preclinical evaluation of hES cells and that improved immunosuppression paradigms and/or alternative cell sources that do not elicit immune rejection are needed for long-term preclinical studies.

Intraoperative intracerebral MRI-guided navigation for accurate targeting in nonhuman primates.

During in vivo intracerebral infusions, the ability to perform accurate targeting towards a 3D-specific point allows control of the anatomical variable and identification of the effects of variations in other factors. Intraoperative MRI navigation systems are currently being used in the clinic, yet their use in nonhuman primates and MRI monitoring of intracerebral infusions has not been reported. In this study rhesus monkeys were placed in a MRI-compatible stereotaxic frame. T1 MRIs in the three planes were obtained in a 3.0T GE scanner to identify the target and plan the trajectory to ventral postcommisural putamen. A craniotomy was performed under sterile surgical conditions at the trajectory entry point. A modified MRI-compatible trajectory guide base (Medtronic Inc.) was secured above the cranial opening and the alignment stem applied. Scans were taken to define the position of the alignment stem. When the projection of the catheter in the three planes matched the desired trajectory to the target, the base was locked in position. A catheter replaced the alignment stem and was slowly introduced to the final target structure. Additional scans were performed to confirm trajectory and during the infusion of a solution of gadoteridol (ProHance, Bracco Diagnostics; 2 mM/L) and bromophenol blue (0.16 mg/ml) in saline. Monitoring of the pressure in the infusion lines was performed using pressure monitoring and infusion pump controller system (Engineering Resources Group Inc.) in combination with a MRI-compatible infusion pump (Harvard). MRI during infusion confirmed successful targeting and matched postmortem visualization of bromophenol blue. Assessment of the accuracy of the targeting revealed an overall 3D mean ± SD distance error of 1.2 ± 0.6 mm and angular distance error of 0.9 ± 0.5 mm. Our results in nonhuman primates confirm the accuracy of intraoperative MRI intracerebral navigation combined with an adaptable, pivot point-based targeting system and validates its use for preclinical intracerebral procedures.

Cardiac Sympathetic Denervation in 6-OHDA-Treated Nonhuman Primates

Cardiac sympathetic neurodegeneration and dysautonomia affect patients with sporadic and familial Parkinsons disease (PD) and are currently proposed as prodromal signs of PD. We have recently developed a nonhuman primate model of cardiac dysautonomia by iv 6-hydroxydopamine (6-OHDA). Our in vivo findings included decreased cardiac uptake of a sympathetic radioligand and circulating catecholamines; here we report the postmortem characterization of the model. Ten adult rhesus monkeys (5–17 yrs old) were used in this study. Five animals received 6-OHDA (50 mg/kg iv) and five were age-matched controls. Three months post-neurotoxin the animals were euthanized; hearts and adrenal glands were processed for immunohistochemistry. Quantification of immunoreactivity (ir) of stainings was performed by an investigator blind to the treatment group using NIH ImageJ software (for cardiac bundles and adrenals, area above threshold and optical density) and MBF StereoInvestigator (for cardiac fibers, area fraction fractionator probe). Sympathetic cardiac nerve bundle analysis and fiber area density showed a significant reduction in global cardiac tyrosine hydroxylase-ir (TH; catecholaminergic marker) in 6-OHDA animals compared to controls. Quantification of protein gene protein 9.5 (pan-neuronal marker) positive cardiac fibers showed a significant deficit in 6-OHDA monkeys compared to controls and correlated with TH-ir fiber area. Semi-quantitative evaluation of human leukocyte antigen-ir (inflammatory marker) and nitrotyrosine-ir (oxidative stress marker) did not show significant changes 3 months post-neurotoxin. Cardiac nerve bundle α-synuclein-ir (presynaptic protein) was reduced (trend) in 6-OHDA treated monkeys; insoluble proteinase-K resistant α-synuclein (typical of PD pathology) was not observed. In the adrenal medulla, 6-OHDA monkeys had significantly reduced TH-ir and aminoacid decarboxylase-ir. Our results confirm that systemic 6-OHDA dosing to nonhuman primates induces cardiac sympathetic neurodegeneration and loss of catecholaminergic enzymes in the adrenal medulla, and suggests that this model can be used as a platform to evaluate disease-modifying strategies aiming to induce peripheral neuroprotection.

Nonuniform cardiac denervation observed by 11C-meta-hydroxyephedrine PET in 6-OHDA-treated monkeys.

Parkinsons disease presents nonmotor complications such as autonomic dysfunction that do not respond to traditional anti-parkinsonian therapies. The lack of established preclinical monkey models of Parkinsons disease with cardiac dysfunction hampers development and testing of new treatments to alleviate or prevent this feature. This study aimed to assess the feasibility of developing a model of cardiac dysautonomia in nonhuman primates and preclinical evaluations tools. Five rhesus monkeys received intravenous injections of 6-hydroxydopamine (total dose: 50 mg/kg). The animals were evaluated before and after with a battery of tests, including positron emission tomography with the norepinephrine analog 11C-meta-hydroxyephedrine. Imaging 1 week after neurotoxin treatment revealed nearly complete loss of specific radioligand uptake. Partial progressive recovery of cardiac uptake found between 1 and 10 weeks remained stable between 10 and 14 weeks. In all five animals, examination of the pattern of uptake (using Logan plot analysis to create distribution volume maps) revealed a persistent region-specific significant loss in the inferior wall of the left ventricle at 10 (P<0.001) and 14 weeks (P<0.01) relative to the anterior wall. Blood levels of dopamine, norepinephrine (P<0.05), epinephrine, and 3,4-dihydroxyphenylacetic acid (P<0.01) were notably decreased after 6-hydroxydopamine at all time points. These results demonstrate that systemic injection of 6-hydroxydopamine in nonhuman primates creates a nonuniform but reproducible pattern of cardiac denervation as well as a persistent loss of circulating catecholamines, supporting the use of this method to further develop a monkey model of cardiac dysautonomia.

Titer and Product Affect the Distribution of Gene Expression after Intraputaminal Convection-Enhanced Delivery

Background: The efficacy and safety of intracerebral gene therapy for brain disorders like Parkinsons disease depends on the appropriate distribution of gene expression. Objectives: To assess whether the distribution of gene expression is affected by vector titer and protein type. Methods: Four adult macaque monkeys seronegative for adeno-associated virus 5 (AAV5) received a 30-µl inoculation of a high- or a low-titer suspension of AAV5 encoding glial cell line-derived neurotrophic factor (GDNF) or green fluorescent protein (GFP) in the right and left ventral postcommissural putamen. The inoculations were conducted using convection-enhanced delivery and intraoperative MRI (IMRI). Results: IMRI confirmed targeting and infusion cloud irradiation from the catheter tip into the surrounding area. A postmortem analysis 6 weeks after surgery revealed GFP and GDNF expression ipsilateral to the injection site that had a titer-dependent distribution. GFP and GDNF expression was also observed in fibers in the substantia nigra (SN) pars reticulata (pr), demonstrating anterograde transport. Few GFP-positive neurons were present in the SN pars compacta (pc), possibly by direct retrograde transport of the vector. GDNF was present in many neurons of the SNpc and SNpr. Conclusions: After controlling for target and infusate volume, the intracerebral distribution of the gene product was affected by the vector titer and product biology.