Historically, there has been no good way to isolate DA neurons from other cells of the midbrain. Thus, missing DA neurons have been replaced by mixed cell populations following transplantation of embryonic midbrain tissue in animal models of disease and in Parkinson's patients. Although, in many cases, these transplants have provided long-term benefit, the presence of unwanted cells, such as glia, non-DAergic neurons, or even excessive numbers of DA neurons, has produced serious side effects, and in rare cases, even death. Discovering ways in which to segregate DA neurons from other cell types poses a significant challenge, but a necessary next step. In the present proposal, our plan is to take advantage of several new advances in the laboratory; including the recent cloning of 11kb human tyrosine hydroxylase gene promoter (hTH). This sequence accurately targets the expression of the reporter, green fluorescent protein (GFP) to DA neurons of the mammalian CNS. Because GFP can be directly visualized in live fetal DA neurons, this approach allows enrichment via flourescent activated cell sorting (FACS) for study in vivo and in vitro. Moreover, it is possible to adapt these purification methods to mouse stem and human progenitor cells using a lentiviral vector to transduce cells with the hTH-GFP transgene. Following their DA differentiation and FACS sorting, our goal is to study purified populations of engineered stem/progenitor-derived DA neurons in culture or after transplantation into the Parkinsonian rat. These models offer us a unique opportunity to determine the ideal number of DA neurons needed as well as the optimal conditions which contribute to their survival and growth following transplantation. Graft function will be assessed in live animals via behavioral testing and in vivo microdialysis which will be correlated with biochemical and anatomical (at the light and electron microscopic levels) changes following sacrifice. This work will hopefully lay the foundation for the development of therapeutic treatments for Parkinson's and other diseases involving compromised DA systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
2R01NS032519-11A1
Application #
6818730
Study Section
Special Emphasis Panel (ZRG1-CNNT (01))
Program Officer
Murphy, Diane
Project Start
1994-08-01
Project End
2008-06-03
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
11
Fiscal Year
2004
Total Cost
$324,675
Indirect Cost
Name
Thomas Jefferson University
Department
Neurology
Type
Schools of Medicine
DUNS #
053284659
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
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