The Morris K. Udall Center of Excellence for Parkinson’s Disease Research at the University of Kentucky

Director: Greg A. Gerhardt, Ph.D.

Title: Restoration of Dopamine Function in Parkinson’s Disease

Central Theme

The Udall Center at the University of Kentucky focuses on the use of growth factors to restore function to damaged dopamine neurons in Parkinson’s disease (PD). In particular, we focus on the use of nonhuman primate models of PD to lay the foundation for translational search and development of new therapies for the treatment of PD. Our current studies have focused on use of the glial cell line-derived neurotrophic factor (GDNF) to carry out three potential alterations to damaged or dying dopamine neurons: 1) restoration of function of the damaged cells, 2) slow the progression of dying dopamine neurons, and 3) augment the function of remaining dopamine cells. Our studies have involved the development of an infusion delivery system to directly deliver GDNF and/or other molecules to the brain of animal models or patients with PD. In addition, we are investigating a new peptide that may enhance dopamine neuron function, promote survival of dopamine neurons and may represent a new drug candidate for treatment of PD.

 

Center Structure

The Center consists of three research projects. Project 1 (Greg A. Gerhardt, Ph.D.) pursues quantitative pharmacological studies of the effects of chronic delivery of GDNF in rhesus monkeys. Project 2 (Zhiming Zhang, M.D.) utilizes behavioral and functional magnetic resonance imaging (fMRI) to evaluate GDNF actions in the monkey model of PD. Project 3 (Don M. Gash, Ph.D.) evaluates histopathological effects of GDNF delivery in these animals, including restoration of neural circuitry. These projects are supported by three resource Cores, including Administration (Gerhardt), Animal (Zhang) and Data Analysis (Richard J. Kryscio, Ph.D.).

 

Recent Significant Advances

Intracarotid injection of a low-dose of MPTP produces a stable, early stage model of Parkinson’s disease in middle-aged rhesus monkeys. An important issue raised in testing new neuroprotective/restorative treatments for Parkinson’s disease (PD) is the optimal stage in the disease process to initiate therapy. Current palliative treatments are effective in the early disease stages raising ethical concerns about substituting an experimental treatment for a proven therapy. Thus, a nonhuman primate model of the earlier stages of PD would be useful for assessing the benefits and risks of early intervention. We have endeavored to create a stable earlier disease MPTP rhesus monkey model by controlling for route of administration of MPTP (intracarotid infusion) and age of the animals (middle-aged, 16-19 years old). Using this approach, parkinsonian features emerged by two weeks and were stable by four-six weeks after MPTP treatment without evidence of either behavioral recovery or progressive functional decline throughout the 12-month behavioral evaluation. A subset of animals were utilized to quantify nigral dopamine cell loss, loss of striatal axons and striatal dopamine levels. A group of five severely-lesioned animals modeling the late-stage of PD was used for comparisons. Unbiased stereological cell counts of tyrosine hydroxylase-positive (TH+) neurons in the substantia nigra revealed a 68% neuronal loss in the early-stage PD animals vs. 87% cell loss in the late-stage PD animals. Also, TH+ fiber density analyzed using densitometry on the MPTP-lesioned caudate and putamen was ~25-30% that of the comparable contralateral regions in the early-stage PD animals, whereas TH+ fiber density in the caudate and putamen was only about 1% that of the comparable contralateral region in the late-stage animals. Consistent with these findings, dopamine, HVA and DOPAC tissue levels were significantly higher in the early vs. late-stage PD animals. Overall, our data support that, by controlling for age and route of administration, a low dose of MPTP can produce a stable, earlier stage model of PD in middle-aged rhesus monkeys. This is a major goal of our Udall Center that was achieved.

Anti-parkinsonian actions of a GDNF propeptide.A hallmark of PD is the degeneration of dopamine neurons in the pars compacta of the substantia nigra. Neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), promote neuron survival, differentiation and maintenance, and have shown great promise for protection and restoration of damaged or dying dopamine neurons in animal models and in some PD therapy clinical trials. However, the delivery of neurotrophic factors like GDNF to the CNS is difficult due to their large size and poor bio-distribution, and their production is hampered by the difficulty of synthesis and structural modification. Small molecules with neurotrophic actions that are easy to synthesize and modify to improve bioavailability are needed. We now report the neurobiological actions of Dopamine Neuron Stimulating Peptide-11 (DNSP-11), an 11-mer peptide from the proGDNF domain. In vitro, DNSP-11 supports the survival of foetal mesencephalic neurons, increasing both the number of surviving cells and neuritic outgrowth analogous to native GDNF. Studies in MN9D cells show that DNSP-11 or GDNF protect against 6-OHDA-induced cell death significantly decreasing TUNEL-positive cells and levels of caspase-3 activity. In vivo, a single injection of DNSP-11 into the rat substantia nigra is rapidly taken up into neurons and residing in the cytoplasm, nucleus and neurites, with antibody staining disappearing after 24 hours in vivo. One injection of DNSP-11 into the normal adult rat substantia nigra increases resting levels of dopamine and its metabolites for up to 28 days. Of particular note, DNSP-11 significantly improves apomorphine-induced rotation behaviour, and increases dopamine and dopamine metabolite tissue levels in the substantia nigra in a rat model of PD. Preliminary data do not support that DNSP-11 binds to the GDNFRα1 receptor. Finally, pull down assays reveal that DNSP-11 is a binding partner to GAPDH, which has been implicated in the effects of anti-parkinsonian drugs and PD. Unlike GDNF, studies on dopamine cells in culture support that DNSP-11 inhibits the effects of mitochondrial toxins. Taken together, these data support that this small peptide shows similar neurotrophic actions as mature GDNF, thus making it a viable candidate for further development as a PD therapeutic. However, its neurotrophic actions may work through its direct actions on mitochondria.

 

Available Resources

• GLP primate facility for FDA accepted pre-IND or pre-IDE investigations.
• Young and aged rhesus monkey colony in AAALACI-accredited facility.
• Anatomical, functional and pharmacological MRI research facility (3T) for imaging measures in nonhuman primates and humans.
• Unilateral extensive and milder MPTP-induced primate models of Parkinson’s disease.
• High speed microelectrode array (MEA) recording facilities for in vivo measures of glutamate, dopamine, choline, acetycholine, lactate, glucose, GABA and NO in awake and anesthetized mice, rats and monkeys.
• Unilateral striatal and medial forebrain bundle 6-OHDA-induced rat models of PD.
• HPLC-EC facilities for measures of dopamine, norepinephrine, serotonin and their metabolites in brain tissues from mice, rat, monkeys and humans.
• Microdialysis recording facility for measures in mice, rats and monkeys.

 

Plans for the Coming Year

Project 1 will be focusing on the effects of intranigral and combined intraputamenal/intranigral GDNF in the “milder parkinsonian” Rhesus monkeys. In addition, they will carry out studies of the effects of GDNF on glutamate regulation in the striatum and substantia nigra of unilateral 6-OHDA lesioned rats and putamen of awake monkeys. In addition, they will continue studies of the use HPLC-EC and microdialysis methods to study DA storage, synthesis and metabolism in postmortem tissues from the caudate nucleus, putamen, globus pallidus and substantia nigra in animals treated with GDNF or GDNF propeptide (DNSP-11). Finally, they will continue studies of the in vivo effects of GNDF-related peptides on DA neurons in normal rats and rats that have been made parkinsonian with a unilateral 6-OHDA lesion.

Project 2 will continue to test the hypothesis that phMRI can be used to monitor therapeutic effects of PD and develop new imaging protocols to sensitively and reliably map functional changes associated with the dopaminergic neuronal system in the brain. In addition, they will finish studies of the behavioral effects of combined putamenal and nigral delivery of GDNF. Finally, they will work with projects 1 and 3 on new studies using DNSP-11 as a neurotrophic factor for damaged or dyeing dopamine neurons.

Project 3 will continue studies on the proGDNF trophic molecule DNSP-11 into the putamen/ substantia nigra of milder unilateral MPTP-treated monkeys. Studies in the coming year will address the distribution and actions of proGDNF DNSP-11 and GDNF on regeneration, functional recovery, potential neurogenesis or gliogenesis in the nigrostriatal dopaminergic system conjunction with Projects 1 and 2.

 

Selected Recent Publications

Gash DM, Chen Y and Gerhardt GA. Neurotrophic factors and Parkinson’s disease. In: Parkinson’s Disease and Related Disorders Handbook of Clinical Neurology, 3^rd Ed. WC Koller and E Melamed, Editors. 2007.

Gash DM, Rutland K, Hudson JL, Sullivan PG, Bing G, Cass WA, Pandya JD, Liu M, Choi D-Y, Hunter RL, Gerhardt GA, Smith CD, Slevin JT and Prince TS. Trichloroethylene: Parkinsonism and Complex I Mitochondrial Neurotoxicity. Ann Neurol Feb; 63(2):184-92, 2008. PMID: 18157908

Luan L, Ding F, Ai Y, Andersen A, Hardy P, Forman E, Gerhardt GA, Gash DM, Grondin R and Zhang Z. Pharmacological MRI (phMRI) monitoring of treatment in hemiparkinsonian rhesus monkeys. Cell Transplantation, 17(4):417-25, 2008. [Epub ahead of print] PMCID: PMC2605704

Grondin R, Zhang Z, Ai Y, Ding F, Walton AA, Surgener SP, Gerhardt GA and Gash DM. Intraputamenal infusion of exogenous neuturin protein restores motor and dopaminergic function in the globus pallidus of MPTP-lesioned rhesus monkeys. Cell Transplantation 17(4):373-81, 2008. PMCID: PMC2680490

Ding F, Luan L, Ai Y, Walton A, Gerhardt GA, Gash DM, Grondin R and Zhang Z. Development of a stable, early stage unilateral model of Parkinson's disease in middle-aged rhesus monkeys. Exp Neuron 212(2):431-9, 2008. [Epub ahead of print] PMCID: PMC2527750

Salvatore MF, Gerhardt GA and Stanford JA. Bilateral effects of unilateral GDNF administration on dopamine- and GABA-regulating proteins in the rat nigrostriatal system. Exp. Neurology, (in press; May), 2009. PMID: 19460370

Grondin R, Zhang Z, Ai Y, Slevin J, Young AB, Gash DM and Gerhardt GA. Direct Delivery of GDNF in the Non-Human Primate and Human Parkinsonian Brain: Success and Road Blocks, In: CNS Regeneration: Basic Science and Clinical Advances. Tuszynski and Kordower, Editors, Academic Press, pp. 223-244, 2008.

Gash DM, Gerhardt GA and Slevin JT. “GDNF (including Neurturin).” Review Chapter in Encyclopedia of Movement Disorders; Editors Katie Kompoliti, Leonard Verhagen, publisher Elsevier; expected publication date of 2009.

 

Public Health Statement

The Udall Center at the University of Kentucky focuses on the use of new drugs to restore function to dopamine neurons, which are damaged and dying in patients with PD. Our studies represent a paradigm shift in the treatment of PD. Instead of treating the symptoms, which are well treated in the earlier stages of PD, we are focusing on trying to repair the brain and/or slow down the progression of the disease. Our current studies have focused on use of the glial cell line-derived neurotrophic factor (GDNF) to carry out three potential alterations to the damaged brain: 1) restore function to damaged brain cells, 2) slow the progression of dying cells, and 3) augment the function of remaining cells to improve the improve the motor function in patients with PD. In addition, we have discovered a new drug, DNSP-11 that may be as good as or better than GDNF for treating dying dopamine cells in PD.