Role of RXR in Nurr1-induced Differentiation of Human ES Cells and in Parkinson's Disease

Funding Type: 
SEED Grant
Grant Number: 
RS1-00455
ICOC Funds Committed: 
$0
Disease Focus: 
Genetic Disorder
Muscular Dystrophy
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
Embryonic Stem Cell
Public Abstract: 
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.
Progress Report: 
  • Facioscapulohumeral muscular dystrophy (FSHD) is the third most common hereditary muscular dystrophy. There is no cure or treatment for this disease since the disease mechanism is not understood. We found evidence that FSHD is a “chromatin abnormality” disease, in which specific histone methylation and factor binding affecting gene silencing is lost at specific genomic regions. We hypothesize that the chromatin structure important for gene regulation is compromised in FSHD. Since we found that this chromatin structure is established in normal human embryonic stem (hES) cells, it is critical to examine how this process goes awry in FSHD during differentiation of hES cells into skeletal muscle cells. Therefore, our major goal is to establish hES cell lines from affected and normal embryos and to optimize a protocol to differentiate hES cells into skeletal muscle cells for comparative analyses. This is not only for our research, but also for use in the FSHD research community, which should help to understand the etiology and pathogenesis of FSHD as well as to possibly develop therapeutic strategies. During the past one year, we continued to optimize skeletal myoblast differentiation from hES cells by using several different protocols. While we were able to detect expression of muscle lineage-specific marker genes (i.e., Pax7 and MyoD) during ES cell differentiation, the expression level remained low and we continue to investigate different protocols for better gene induction and enrichment of the proper cell population by cell sorting. It is essential to attain a sufficiently high density of derived myoblasts in order to allow them to form myotubes, which is the basic fundamental element of skeletal muscle. The very recent publication (i.e., June, 2009) of a detailed protocol for derivation of skeletal muscle cells from hES cells should be of great assistance to us, as it highlights several key factors that are likely to be crucial for success. In addition, we have acquired with proper hSCRO approval the first batch of embryos of seven donated by an FSHD patient donor. Out of seven embryos, we have thawed five embryos initially and followed their growth in culture dishes. However, these embryos were frozen five years ago under suboptimal conditions, and failed to further develop in culture for us to derive any ES cells. We are in the process of thawing the remaining two embryos, although similar problems may persist. Presently, we have identified two additional sources of FSHD embryos. The acquisition process has been considerably prolonged due to recent changes in NIH regulations that necessitated the modification of the consenting form and process. However, with the help of our collaborator from Scripps, Dr. Jeanne Loring, we are in the process of obtaining five additional embryos and we have requested a “no cost” extension of this project in order to complete the derivation of hES cells from these embryos. During the past year, we published our initial characterization of the chromatin structure in FSHD patient cells by comparison with normal cells of different cell types, including hES cells. We detected differences in how one of the histone proteins is chemically modified in certain regions of the genomes in normal versus FSHD cells. This is important since histone proteins not only help to package DNA to form the basic element of chromatin, they also play a huge role in controlling gene expression. Since the effects of FSHD likely result from abnormal regulation of expression of certain genes, our work identified a relevant chromatin “marker” which is potentially useful for diagnostic purposes. In addition, our findings define a way to follow any changes in target gene regulation during the differentiation process (i.e., hES cells to myoblasts and myotubes) in normal and FSHD hES cells. The published manuscript was selected for “Faculty of 1000”, and we have already been asked to write a review on this subject. Furthermore, we have obtained NIH R01 funding for further high-throughput analysis of chromatin structure in FSHD. Taken together, with the support of the CIRM SEED grant, we are moving forward to understand the chromatin abnormality during cellular differentiation linked to FSHD pathogenesis.

© 2013 California Institute for Regenerative Medicine