Background. Human embryonic stem (hES) cells hold promise for replacement therapies. One of the promising targets for such therapy is Parkinson’s disease (PD). However, successful application of hES cell-based therapy depends on our ability to produce a large number of dopaminergic (DA) neurons and to control their survival once these neurons are transplanted into patients. This largely depends on our understanding of cellular components and pathways governing the proliferation, differentiation and survival of hES cells. Recent studies show that a protein called Nurr1 and its closely related protein Nur77 play a critical role in mouse ES cell differentiation, maturation, and survival. Introduction of Nurr1 into mouse ES cells induces their differentiation. However, the efficiency of generating DA neurons by Nurr1 is low and Nurr1-induced DA neurons are functionally immature, suggesting the necessity of additional factor(s). Nurr1 and Nur77 bind to RXR, a receptor protein for vitamin A-derived compounds and certain fatty acids. Interestingly, docosahexaenoic acid (DHA), which is enriched in brain and promotes the survival of newborn DA neurons, acts as a RXR ligand. Furthermore, synthetic RXR ligands enhance the differentiation of Nurr1-expressing cells and increase the survival of DA neuron. Recently, we discovered a novel Nur77-mediated survival/death pathway, in which Nur77 acts in the nucleus to confer cell survival, whereas its migration to mitochondria results in cell death. In addition, we showed that the survival and death activities of Nur77 are controlled by RXR and its ligands (such as DHA). Hypothesis: We hypothesize that RXR and its ligands are required for efficient hES cell differentiation, maturation and survival through their interaction with Nurr1 and Nur77 proteins. Objective: The objectives of this proposal are to establish an effective and reliable protocol for generating DA neurons from hES cells and to identify novel agents for the prevention and treatment of PD. We propose three specific aims: 1). To analyze the role of RXR and ligands in Nurr1-induced hES cell differentiation. Nurr1 and RXR proteins will be introduced into hES cells, which will be differentiated in the presence or absence of RXR ligands. 2). We will employ computer-based approaches to design and identify DHA analogs, which will be evaluated for their ability to modulate RXR activities, to induce hES cell differentiation, and to promote the survival of hES-derived neurons. 3). To evaluate hES cells in animals. The hES-derived DA neurons will be evaluated in a mouse PD model by transplantation. Two most effective DHA analogs will be also evaluated for their PD preventive effects. Results from these studies will not only enhance our understanding of the molecular pathways governing the differentiation of hES cells but may also lead to the identification of effective RXR-based agents for prevention and treatment of PD.
Statement of Benefit to California:
Parkinson’s disease (PD) is a progressive disorder of central nervous system, which occurs when a group of brain cells called dopamine (DA) neurons begin to malfunction and die. According to the National Institutes of Health, PD affects at least 500,000 people in the United States and some 50,000 new cases are diagnosed each year, a number that is expected to rise as the population ages. Parkinson's disease affects both men and women almost equally. People of every race, economic class, and ethnicity can get PD. Although age is a clear risk factor, the commonly used herbicide, paraquat, which is structurally similar to the neurotoxic chemical MPTP, is known to be able to induce parkinsonism. Thus, there is an increased PD mortality in California, as California uses approximately a quarter of all pesticides in the US. According to the National Parkinson Foundation, PD patients spend an average of $2,500 a year for medications. Thus, there is of great interest to Californian residents for the development of new agents for the prevention and treatment of human PD. Current therapies, including the administration of L-dopa and dopamine receptor agonists and on deep-brain stimulation in the subthalamic nucleus, are effective only for some symptoms. They are associated with side effects and do not stop the progression of the disease. Recent progress shows that PD is a prime candidate for treatment by stem cell transplantation. If cells with properties of DA neurons can be generated in vitro from human embryonic stem (hES) cells, they can be used to replace damaged brain cells in PD patients. Thus, hES cells hold promise for generating an unlimited supply of brain cells for PD therapy. In fact, the potential to use ES cells to replace damaged cells has already been demonstrated in animal. However, little is known whether human ES cells can be successfully instructed to brain cells with therapeutic value. Our proposed studies aim at the development of new approach for facilitating the generation of DA neurons from hES cells by genetic manipulation consisting of the specific activation of Nurr1, Nur77, and RXR proteins, which are known to regulate the development, maturation and survival of DA neurons. Results from these studies may lead to improved generation of DA neurons for PD therapy. Our proposed studies of identifying RXR-binding DHA analogs may also lead to the identification of new DHA-based agents for the prevention and treatment of PD. Thus, California residents who suffering PD will benefit from this research. Development of novel preventive and therapeutic strategies and agents will certainly stimulate economic growth in California.
The proposed study intends to examine the roles of Nurr1 and Nurr77 in the differentiation and survival of DA neurons that are derived from human ESCs. In particular, the PI will analyze whether the cell survival effect of Nurr1 and/or Nurr77 is mediated by RXR/Nurr1 interactions, i.e., dimerization of RXR/Nurr1 elicited by RXR ligand binding will prevent Nurr1 translocation from nucleus to mitochondria thus preventing cell death. SIGNIFICANCE AND INNOVATION: This system could very well be used to evaluate RXR-binding analogs for their potential to promote DA neuron survival. Therefore, the proposal is quite novel and bears application potential. STRENGTHS: The study is based on the PI’s experience in molecular biology of RXR and its ligands. It will first establish a DA neuron differentiation culture from human ESCs. By altering the Nurr1 expression in human ESCs using lentiviral vectors, the PI hopes to differentiate human ESCs to DA neurons. Using this system, the PI will evaluate the effect of RXR ligands and their analogs on the survival of DA neurons. The function of the ESC-derived DA neurons will be examined in the MPTP-induced mouse model. The experimental design is generally logical and clear. WEAKNESSES: The PI will introduce Nurr1-GFP and RXRa-GFP into human ESCs. It is not clear whether the PI intends to establish ESC lines. If so, it could take a couple of years to build a dozen of lines (in three different parental human ESC lines). There is no mention what promoters will be used to drive the expression of Nurr1 and RXRa. Many ubiquitous promoters are severely suppressed in human ESCs and/or down-regulated along neural differentiation, which may well jeopardize the proposed study. DA neuron differentiation is generally facilitated by FGF8 and sonic hedgehog. In the flowcharts (Figs. 1, 2), the PI will use FGF2 instead. It is not clear whether it reflects the lack of knowledge in the field or that the PI intends to avoid FGF8 and SHH in order to analyze the effect of Nurr1 and RXRa. The function and maturation of DA neurons in vivo will be assessed by transplanting the human ESC-derived DA neurons into the MPTP-treated mouse model and evaluating the behaviors and histology 5 weeks later. This timeline in general fits the maturation process of mouse cells but is far off the scale of human DA neuronal maturation. This again reflects the lack of attention being paid to literatures. It would be ideal to have collaborators to help ESC differentiation and PD transplantation.