Objective/Rationale:
We have generated two lines of human dopamine neurons with parkin mutations and two lines from normal people using stem cells derived from their skin cells. Several interesting differences were found between neurons with parkin mutations and normal neurons. Because these differences can potentially be used to discover novel drugs for Parkinson’s disease, we would like to generate additional lines of neurons with different parkin mutations and normal neurons to validate these important findings.
Project Description:
We will use viruses to introduce five genes (Oct4, Sox2, c-Myc, Klf4 and Nanog) to skin cells isolated from two normal subjects and four Parkinson’s disease patients with various parkin mutations. This process will produce induced pluripotent stem cells (iPSC) that can give rise to any types of cells in the human body, including dopamine neurons. Along with our existing lines of iPSCs, we will thus have six lines of human dopamine neurons with different parkin mutations and four lines of normal neurons. We will use this panel of dopamine neurons to examine dopamine release, uptake and metabolism. A number of biochemical and molecular biology techniques will be utilized to validate our preliminary findings on the deficiencies of dopamine neurons with parkin mutations, compared to normal neurons.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
The project aims to validate our discovery that parkin mutations result in dopamine-specific deficiencies in human dopamine neurons. Results from the study will allow us to screen for novel therapeutic targets for Parkinson’s disease using cells that are most severely affected by parkin mutations – the human midbrain dopaminergic neurons. Thus, the study may provide not only new targets for drug discovery research but also a unique and physiologically relevant platform to screen these new targets.
Anticipated Outcome:
We anticipate that by generating additional lines of iPSCs, we will be able to use the ten lines of human dopamine neurons (six with parkin mutations and four normal) to confirm our preliminary findings. The results will lay the foundation for drug discovery research based on the identified targets and also allow us to establish a screening platform using these unique resources.
Progress Report
We have generated induced pluripotent stem cells (iPSCs) from 6 PD patients with different parkin mutations and 4 normal subjects. The ten lines of iPSCs have been differentiated to midbrain dopaminergic (DA) neurons. Using some of the iPSC-derived midbrain DA neurons, we have found that parkin mutations significantly increase spontaneous dopamine release and reduce dopamine reuptake. This greatly affects the precision of dopamine’s action, which is critical for the brain to support locomotion. On the other hand, parkin mutations markedly increase oxidative stress because they significantly increase the expression of monoamine oxidases – enzymes that are responsible for dopamine oxidation. All these dopamine-specific phenotypes are significantly reversed by the normal parkin gene, but not its PD-linked mutant. This study not only reveals the unique and critical functions of parkin in human midbrain DA neurons, but also generates novel drug targets and a new platform for discovery of disease-modifying drugs for PD that may mimic the protective functions of parkin.
Presentations & Publications
Paper:
(1) H Jiang, Y Ren, EY Yuen, P Zhong, M Ghaedi, Z Hu, G Azabdaftari, K Nakaso, Z Yan, J Feng (2012). Parkin Controls Dopamine Utilization in Human Midbrain Dopaminergic Neurons Derived from Induced Pluripotent Stem Cells. Nature Communications, 3:668. DOI 10.1038/ncomms1669.
Abstract:
(1) H. Jiang, Y. Ren, E.Y. Yuen, P. Zhong, M. Ghaedi, Z. Hu, G. Azabdaftari, K. Nakaso, Z. Yan, J. Feng. Dopaminergic phenotypes in parkin-deficient human midbrain dopaminergic neurons derived from patient-specific induced pluripotent stem cells. Soc. Neurosci. Abst. 37: 552.06 (2011)
(2) H. Jiang, Y. Ren, E.Y. Yuen, P. Zhong, M. Ghaedi, Z. Hu, G. Azabdaftari, K. Nakaso, Z. Yan, J. Feng. Parkin Mutations Increase Oxidative Stress in Human Midbrain Dopaminergic Neurons Derived from Patient-specific iPS Cells. International Society for Stem Cell Research poster #3121 (2011).
Selected Presentations:
3/12: Third iPSC Consortia Meeting, National Institute of Neurological Disorders and Stroke, Washington, DC.
11/11: Life Sciences Summit 2011, New York, NY.
10/11: Public lecture preceding the Michael J. Fox Distinguished Speakers lecture, Buffalo, NY