In this application, we propose to identify small molecule compounds that can stimulate human embryonic stem cells to become dopamine-producing neurons. These neurons degenerate in Parkinson’s disease, and currently have very limited availability, thus hindering the cell replacement therapy for treating Parkinson’s disease. Our proposed research, if successful, will lead to the identification of small molecule compounds that can not only stimulate cultured human embryonic stem cells to become DA neurons, but may also stimulate endogenous brain stem cells to regenerate, since the small molecule compounds can be made readily available to the brain due to their ability to cross the blood-brain barrier. In addition, these small molecule compounds may serve as important research tools, which can tell us the fundamental biology of the human embryonic stem cells.
Statement of Benefit to California:
The proposed research will potentially lead to a cure for the devastating neurodegenerative, movement disorder, Parkinson’s disease. The proposed research will potentially provide important research tools to better understand hESCs. Such improved understanding of hESCs may lead to better treatments for a variety of diseases, in which a stem-cell based therapy could make a difference.
SYNOPSIS: The goal of this proposal is to identify small molecules that will stimulate hESCs to differentiate into dopaminergic (DA) neurons. Because of the large variability of DA neuron production of different batches of cultured ES cells, the PI plans to use a TH-GFP zebrafish line that he has established as an in vivo assay for a chemical screen of 5,000 small molecules. In aim 1, the PI will identify small molecules that affect the induction of DA neurons or their later differentiation and survival in zebrafish. The zebrafish are screened in a 96 well format under a fluorescent microscope. The chemically treated embryos will be observed under the fluorescent dissecting microscope at days 1, 2, and 3 post fertilization. The morphology and the state of GFP+ neurons will be examined. Those embryos, in which alterations of GFP patterns are observed, will be processed for TH immunolabeling to further validate the alterations of DA neurons. In aim 2, the PI will take the small molecule hits found in aim 1 and test them in hESC. INNOVATION AND SIGNIFICANCE: The reviewers found there to be a tremendous amount of innovation and significance in this proposal. At present, there are difficulties obtaining a large number of homogeneous dopaminergic neurons that are pure and free of xenobiotic products, such as feeder cells. In addition, the transcription factors and the pathways that are important in the differentiation and maintenance of these cells are poorly defined. Therefore, this proposal is significant since it may lead to knowledge and to the efficient generation of dopaminergic differentiated cells that may impact on the treatment of Parkinson's disease. The small molecules that may be identified in the planned studies may have a use in direct treatment of patients as they may cross the blood-brain-barrier and may slow down DA neuronal death, or they may stimulate regeneration and thus avoid transplantation into a delicate region of the brain. The proposal is very innovative since it uses zebrafish to carry out a chemical genetic screen. Although this approach has been used before for chemical screens in other conditions, it remains very novel. In fact, the PI was responsible for generating the TH-GFP transgenic zebrafish that make the proposal possible by allowing for the direct visualization of the state of DA neurons. The zebrafish provides a potential number of advantages over in vitro studies, especially since cells that differentiate in vitro may not maintain their differentiated state when transplanted, i.e., it may be valuable to screen for molecules that lead to differentiation of ESCs in an organism rather in vitro. STRENGTHS: This is an elegantly interwoven set of two experimental aims to glean small molecules able to induce DA neurogenesis in the zebrafish and later in a hESC culture setting. The availability of the transgenic TH-GFP zebrafish line that was generated by the PI is a potentially powerful tool. The approach that the PI is taking involving the use of the zebrafish with its consistent, small and easily quantifiable numbers of DA neurons to identify a molecule that can then be tested in hES cells is a valuable and clever screening approach. Zebrafish have been previously used to screen chemical libraries, i.e., there is precedence for this approach. The reviewer, however noted that these previous studies seem to have involved the rescue of transgenic mutant zebrafish. The use of an in vivo assay to look for small molecules important in the differentiation of DA neurons is clearly more physiologically relevant than an in vitro assay – as suggested by the fact that in vitro differentiation sometimes disappears after placement of cells within the CNS. Also, an ongoing NIH R01 in the PI’s lab looking at DA neuron development in the zebrafish is highly complementary to the studies proposed here. The UCSF SMDC is a valuable resource for the planned studies. The PI, an Assistant Professor in the Department of Biopharmaceutical Science at UCSF since 2000, is productive with publications in excellent journals on the topic of DA neurons in zebrafish as well as a publication related to the use of zebrafish in studies of toxins in Parkinson’s disease. Finally, the collaboration of Dr. Renee Reijo Pera, CoDirector, Program in Human Stem Cell Biology at UCSF, is a valuable one. WEAKNESSES: The weakest point of this proposal is the possibility that the PI’s fishing for active compounds that affect DA precursor gene expression and DA neuron generation is clearly risky and might not prove successful. For example, the drugs that affect DA neuron differentiation in the zebrafish may be species-specific and not function in hESC. Five thousand compounds is a relatively small library for screening. The zebrafish model is likely limiting for high thoughput screening - thus the focus on a five thousand compound library. If no hits are found in this library, there is a growing list of small molecule/drug libraries potentially available to screen in this elegant model so lack of hits in the proposed library might not be a daunting issue. The PI certainly has thought about the issue of no hits and proposed to try combinations of small molecules (simultaneously or sequentially) if the single small molecule attempts fail to translate from the zebrafish embryo to the hESC culture approach. There was a lack of clarity as to the endpoint and what constituted a positive hit. It appeared that the PI is screening for small molecules by examining the general morphology as well as the state of the GFP+ neurons. One reviewer was not quite certain what this meant. Does the PI expect that new DA neurons will form, that DA neurons will form earlier, that DA neurons will grow larger than normal, that DA neurons will have an increase in fluorescence? Are there examples where zebrafish neurons change their neurotransmitter characteristics? These are important questions that should have been more clearly explained in the proposal. Also, it is not clear how the PI will follow up with small molecules that have a negative effect on the DA-GFP. Might these also be of interest? Finally, aim 1 involving the zebrafish screen could be carried out with federal grant money and also was surprised that the PI only listed 10% effort on this proposal. DISCUSSION: A creative and important aspect of the proposal is the use of the transgeneic zebrafish line. There was discussion about how the screen will work, such as is it automated, is there quantitative fluorescence used, and what was the endpoint - what constitutes a "positive" hit. There was a discussion as to the timing of the exposure with respect to whether would be observing a direct or an indirect effect. Finally, the biotechnology aspect of the proposal was very attractive including the potential for industry interest in a successful outcome.