More than 600 disorders afflict the nervous system. Common disorders such as stroke, epilepsy, Parkinson’s disease and autism are well-known. Many other neurological disorders are rare, known only to the patients and families affected, their doctors and scientists who look to rare disorders for clues to a general understanding of the brain as well as for treatments for specific diseases. Neurological disorders strike an estimated 50 million Americans each year, exacting an incalculable personal toll and an annual economic cost of hundreds of billions of dollars in medical expenses and lost productivity. There are many potential applications for using human embryonic stem (hES) cells to treat neurological diseases and injuries; however, a critical barrier to progress in the field is the ability to efficiently and reliably control neuronal differentiation from these cells. The main goal of this proposal is to define the gene regulatory mechanisms that control the acquisition of neuronal fate from hES cells. Longer term, we plan to produce small compounds (drugs) that greatly facilitate this process. Drugs that enhance neuron formation are likely to improve scientists’ ability to manipulate hES cells and create in vitro models for studying neurological diseases. Most importantly, drugs of this type may stimulate endogenous stem cells within adults to self-repair damaged areas of the brain. Because so little is known about how hES cells differentiate into neurons at the molecular level, this grant will focus on understanding how a single neuronal subtype is generated – motor neurons. Why motor neurons? Motor neuron diseases are a group of progressive neurological disorders that destroy cells that control essential muscle activity such as speaking, walking, breathing and swallowing. Eventually, the ability to control voluntary movement can be lost. Motor neuron diseases may be inherited or acquired, and they occur in all age groups. In adults, symptoms often appear after age 40. In children, particularly in inherited or familial forms of the disease, symptoms can be present at birth or appear before the child learns to walk. Is there a treatment? There is no cure or standard treatment for motor neuron diseases. Prognosis varies depending on the type of motor neuron disease and the age of onset; however, many types such as ALS and some forms of spinal muscular atrophy are typically fatal.The experiments in this proposal seek to understand mechanisms that will be directly applicable to hES cells and their use for treating motor neuron diseases. Moreover, the mechanisms controlly motor neuron formation are also likely to be relevant to many other neuronal subtypes. Therefore, these studies should provide essential and general insight into medically deploying strategies for converting hES cells into specific neuronal subtypes and thereby serve as a platform for treating a wide range of neurological diseases.
The long term goal of this research grant proposal is to understand and treat diseases and injuries of the nervous system using hES cells. Neurological disorders such as stroke, epilepsy, Parkinson’s disease and autism strike an estimated 5 million Californians each year, exacting an incalculable personal toll and an annual economic cost of billions of dollars in medical expenses and lost productivity. Thus, one benefit that will be derived from this area of research is the generation of specific tools and methods for reducing medical costs and increasing the quality of life and level of productivity of afflicted Californians. A second key benefit derived from this research grant proposal is the training of new scientists to serve as educators and researchers for the future, many in the burgeoning area of stem cell biology for which the State of California has emerged as a world’s leader. Finally, the discoveries derived from innovative and multidisciplinary research on hES cells described in this proposal, including the use of chemistry to create drug leads for regulating stem cell differentiation, are likely to lead to important new areas of intellectual property that are essential for creating high quality jobs in the biotechnology and pharmaceutical industries in California.
SYNOPSIS: This application seeks to define the regulatory mechanisms that control the differentiation of hES cells into motoneurons. The main goal of this proposal is to clarify the role in hESC motor neuron (MN) differentiation of lysine specific demethylase (LSD1) and SCP1, which suppress neuronal gene expression. Preliminary data show that dominant negative constructs of SCP1 promote neuronal differentiation. In Aim I, the PI will monitor expression of a battery of neuronal genes, MN-specific genes, transcription cofactor genes and other cell type-specific markers at 6 stages in the progression of hESCs into MNs in 8 hES lines. In addition, the PI will perform chromatin immunoprecipitation assays to monitor the regulatory changes that occur at key neuronal genes during MN differentiation. In Aim II, the PI will determine the effect on MN differentiation of LSD1 and SCP1 on hESCs using siRNA, dominant negative constructs and structure-based chemical inhibitors to target SCP1 and LSD1 in hES cells. Finally, these studies may identify drugs that affect hESC gene expression.
SIGNIFICANCE AND INNOVATION: This proposal’s goal is to clarify the key transcription programs that regulate gene expression and development in hESCs, and are critical in the differentiation of hESCs into motor neurons. The hypothesis that LSD1 and SCP1 are important in the differentiation of hESCs into MNs is a novel one. The approaches that are used in the proposal are contemporary. The findings made as a result of studies in this proposal may be important in the better design of protocols to efficiently generate MNs from hESCs, in the identification of molecular signatures that correspond to motor neurons at different stages of differentiation, and in the design of drugs that can facilitate the growth and differentiation of MNs and can be used in hESC-derived cultures and the treatment of MN disease and spinal cord injury. In addition, understanding the role of these transcription factors in MN development may be a guide in developing drug targets for therapy of MN diseases. Improved methods to create a renewable source of MNs provides cells that could be used for transplantation in patients with MN disease or spinal cord injury or could be used in in vitro models for these disorders.
Overall, this is an exciting proposal that has the potential to reveal mechanisms by which motoneurons develop from hES cells. The proposal also has the potential to bring about pharmaceuticals to impede or enhance the development of motoneurons. The proposal is supported enthusiastically.
STRENGHTS: There are many strengths in this proposal. This productive PI brings expertise of the development of embryonic motoneurons in vivo to the current study, and he is an expert in the identification of gene expression programs used during the development of MNs in the spinal cord. The Pfaff laboratory has proven to be a leader in the study of motoneuron development in vivo and it is anticipated that there will be key results made from the current series of experiments. Also, the environment of the Salk is a rich one with respect to expertise in stem cells, and the collaboration of the PI with the Noel lab is valuable, with the latter having key expertise in synthetic chemistry and who is expected to generate new compounds. This collaboration brings state of the art technology and is likely to yield good information on the regulatory changes that occur at specific neuronal genes during motoneuron differentiation.
It is highly appropriate to study gene expression during the differentiation of hESCs into MNs in order to provide useful information with respect to the efficient generation of MNs. The preliminary data presented by the PI are of interest and suggest that SCP1 and LSD1 are likely to play important roles with respect to differentiation of hESCs into MNs. Also exciting are the recent structural studies of SCP1; this information will be helpful in designing targets for designing small molecules. The proposal follows logically from these exciting findings.
Caveats in the proposal are considered and addressed. For example, the applicants have taken into account the lower proportion of motoneurons compared to other cell types, and the heterogeneous populations at early stages of ES cell specification, and have proposed appropriate methods to enrich for motoneurons.
WEAKNESSES: The principal applicant has little experience working with hES cells. However, ample expertise exists in the Salk Institute and his track record indicates that he will quickly establish the knowledge and technology to study these cells in his laboratory. One reviewer also felt that the PI does not adequately and clearly state potential difficulties in some of the experiments and alternative directions that could be taken.
With respect to the experimental strategies employed, the first specific aim is very descriptive involving the assay of many genes and gene products under many conditions at many times in many different ESCs. The resulting gene expression profiles may yield extensive data sets that may be hard to follow. One is concerned that it will be difficult for the PI to generate clear correlations. While the applicants will focus on genes grouped into specific categories, several of these categories are products or markers of particular types of cells that are formed (e.g. oligodendrocyte factors) which may not guide results of what regulates the differentiation of the cells. However, it is reassuring that there is some focus on known genes (more interesting might be genes not currently implicated) that regulate neuronal differentiation based on knowledge from the developing embryonic motoneuron system in vivo. In addition, the inclusion of many genes may be important in identifying key ones.
DISCUSSION: Reviewers were very enthusiastic about this proposal, with one reviewer stating that this was the best s/he reviewed. The PI is an expert in spinal motor neurons who has been very productive, and while he has no experience with hESC, it is considered a benefit to bring his expertise to this field. A recent Science paper shows that SCP1inhibits neuronal gene expression in non-neuronal cells and that dominant negative constructs of SCP-1 lead to neuronal differentiation. The applicant also has a paper in press on the crystal structure of SCP1 which will allow the design of small molecule inhibitors. The main drawback is that aim 1 is a slightly overambitious cataloging experiment looking at multiple stages in multiple lines, and it may be hard to interpret the involvement of the additional genes that are found.