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RN1-00530-1: Molecular mechanisms of neural stem cell differentiation in the developing brain
Recommendation: Recommended for funding
Scientific Score: 77
First Year Funds Requested: $436,206
Total Funds Requested: $2,200,715
Public Abstract (provided by applicant)
One of the most exciting possibilities in stem cell biology is the potential to replace damaged or diseased neural tissues affected by neurodegenerative disorders. Stem-cell-derived neurons provide a potentially limitless supply of replacement cells to repair damaged or diseased neurons. Typically, only one or a very few types of neurons are affected in most neurodegenerative diseases, and simply transplanting stem cells directly into a degenerating or damaged brain will not guarantee that the stem cells will differentiate into the specific neurons types needed. In fact, they may instead cause tumor formation. Thus, we must learn how to guide stem cells, cultured in a laboratory, toward a specific differentiation pathway that will produce neurons of the specified type. These cells would then provide a safe, effective way to treat neurodegenerative diseases and central nervous system injuries.
Since there are hundreds or thousands of types of neurons in the cerebral cortex, functionally repairing damaged neurons in the cortex will require a detailed understanding of the mechanisms controlling differentiation, survival, and connectivity of specific neuronal subtypes. In this proposal, I propose to investigate the molecular mechanisms that guide the neural stem cells in developing embryonic brains to generate two specific types of neurons – corticospinal motor neurons (CSMNs) and corticothalamic projection neurons (CTNs).
Our first goal is to understand what regulates the development of CSMNs. CSMNs are clinically important neurons that degenerate in Amyotrophic Lateral Sclerosis (ALS), and are damaged in spinal cord injuries. With our current technology, replacing damaged CSMNs has been impossible, due largely to a lack of understanding of what signals regulate their development. Our second goal is to identify genes that direct the neural stem cells to generate the CTNs. Despite their essential importance in sensory processing and involvement in epilepsy, mechanisms governing the development of CTNs have not yet been revealed. CSMNs and CTNs express many identical genes, and are generated from common neural stem cells in the embryonic brains. Yet it is unclear how they are specified from common stem cells. Our third goal is to identify transcription factor codes that neural stem cells employ to specifically generate either CSMNs or CTNs.
Currently, there is no cure for neurodegenerative diseases. Understanding how CSMNs and CTNs are generated during development provides the opportunity to design procedures to direct the stem cells cultured in a laboratory to specifically produce CSMNs or CTNs, which can then be used to replaced damaged or diseased neurons, such as those affected by ALS, or spinal cord injuries.
Statement of Benefit to California (provided by applicant)
Neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), affect tens of thousands of Californians. There are no cures for these devastating diseases, nor effective treatments that consistently slow or stop them. The research proposed in this application may provide the basis for a novel, cost-effective, cell replacement therapy for ALS, thereby benefiting the State of California and its citizens.
Stem cells offer a potential renewable source of a wide range of cell types that could be used to replace damaged cells involved in neurodegenerative diseases or in spinal cord injuries. At present, transplanting stem cells directly into patients is problematic, because this approach may instead cause tumor growth. To support safe and effective cell transplants, it is important to differentiate stem cells prior to the therapy into the specific cell types affected by the diseases. Understanding how different types of neurons are generated during development provides an opportunity to develop new methods to guide the differentiation of stem cells into the proper neuron types.
In this application, we propose to uncover the mechanisms that regulate the neural stem cells in developing mouse brains to generate different neuronal types in the cerebral cortex, including the corticospinal motor neurons (CSMNs) and the corticothalamic neurons (CTNs). CSMNs are the neurons that degenerate in ALS and are affected in spinal cord injuries. Dysfunction of CTNs has been implicated in epilepsy. Understanding the mechanisms regulating neural stem cells to generate CSMNs and CTNs in vivo will help scientists and physicians to direct stems cells to produce CSMNs or CTNs to replace damaged neurons in patients with neurodegenerative conditions.
Review
SYNOPSIS: This project proposes to analyze the function of candidate genes in regulating the differentiation of embryonic neurons. The goal of the proposal is to understand cell fate decisions that regulate the formation of two related neural subtypes in the cortex: cortical spinal motor neurons (CSMNs) that reside in layer five and cortical thalamic neurons (CTNs) that reside in layer six. CSMNs are clinically important neurons that have been shown to degenerate in Amyotrophic Lateral Sclerosis (ALS) and are damaged in spinal cord injury. CTNs play important roles in sensory processing and their dysfunction has been implicated in epilepsy.
The applicant has identified candidate genes that, by virtue of their expression patterns and activities, are likely to contribute to cortical neuron subtype determination and to participate in the transcription factor codes that regulate cortical projection neuron fates. S/he proposes to analyze the functions of these genes in regulating the in vivo differentiation of embryonic neural progenitors of CSMNs or CTNs by analyzing gene expression and axonal projections in existing mutant mice, and by manipulating gene expression in neural progenitors in vivo using electroporation of siRNA or gain-of-function constructs. Collectively, these studies will enable researchers to explore the mechanisms of fate specification in cortical development.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: This proposal deals with a fundamental problem in stem cell-based therapies for neurological disease: how can cultured neural stem cells (NSCs) be instructed to differentiate into specific neural subtypes? The proposed research is likely to make a major contribution to our understanding of how subtypes of cortical projection neurons are determined. The issues dealt with in this proposal could have an important impact on the treatment of human neurodegenerative diseases, and they could be applied to human embryonic stem cell (hESC) differentiation protocols for stem cell therapy applications.
Overall, the proposal is well-written, the study plan is carefully considered and well-organized, and the experiments will produce interpretable results. The directions used by the Principal Investigator (PI) are creative ones. The experimental approach and concepts are fairly standard, and use existing mouse mutants and technologies. There is an impressive use of cutting-edge technology within the expertise of the PI, as the PI builds on studies that s/he performed during a post-doctoral fellowship. The applicant has paid attention to potential pitfalls and devised alternative approaches where possible. The work proposed will probably succeed and become a useful contribution to the literature in the fullness of time. The preliminary data and the quality of the PIs publications demonstrate that the PI can carry out the proposed studies, and provide reasonable evidence that the genes that s/he has chosen to study should be further pursued.
The major weakness of this proposal from the perspective of this particular granting mechanism is that the experimental approach is more solid than innovative: one does not sense any paradigm shift in the making. Apart from moderate innovation, the other drawback is the degree of relevance to the goals of CIRM, since the applicant is studying embryonic neural progenitors (not stem cells as stated).
The PI was involved in the characterization of the transcription factor to be studied, which is expressed in NSCs and subcortical projection neurons, including CSMNs. In addition, the PI generated null mice which have no corticospinal tract. The PI has preliminary data that show that the targeted transcription factor is required for the activation of a second transcription factor that is essential for CSMN development into postmitotic neurons, although confusingly electroporating the first transcription factor rescues development of CSMNs but does not restore expression of the second transcription factor.
One aim is to investigate downstream targets of the targeted transcription factor in NSCs using genome-wide screens. The planned experiments are feasible and worth doing, although there may be difficulties in determining the factors involved in CSMN development if post-transcriptional alterations play an important role. A question not addressed is whether the PI will identify direct versus indirect targets of the transcription factor. Also, when is the transcription factor first expressed in forebrain? Conditional ablation and analysis within a day would get closer to the question of direct targets.
Another aim is to determine the functions of three transcription factors that the PI found are expressed specifically in CTNs and/or their progenitors. In order to carry out these studies, the PI will study mice that carry mutations in these genes (these mice have been prepared in other labs). These experiments are complex, but should clarify the non-cell autonomous issue and the function of the three genes in regulating layer 6 CTN development.
The proposed experiments are time- and money-consuming, as only 1/8 of the embryos are the right genotype. A complication in analyzing at least one of these mutant mice strains is that the transcription factor in question is required for subplate development. It is possible that pioneering axons from subplate neurons guide corticothalamic projections. Thus, absence of corticothalamic axons in the null mice may be due to a defective subplate or to a cell-automomous defect in CTNs. To distinguish between these two possibilities, the PI will restore normal subplate neurons and pioneering axons by generating aggregation chimeras.
QUALIFICATIONS AND POTENTIAL OF THE PRINICIPAL INVESTIGATOR: An additional strength of the proposal is the PI. The candidate has a solid research record and has been well trained in genetics and neurobiology. Although the candidate’s publications are not great in number, they have been of excellent quality. The candidate is committed to the stem cell field and has the potential to become a leader in the field. S/he is likely to contribute original papers to the field of cortical development and has the potential to be a leader in the stem cell field.
The applicant received an undergraduate education at a top tier college abroad. S/he earned a Ph.D. at notable institution in New York, and conducted postdoctoral research at a premier institution in California studying the role of one of the transcription factors discussed in the proposal in cortical development. S/he has published several first-author papers, and two of these papers have appeared in very respectable journals. The applicant’s graduate work led to receipt of a prestigious thesis award. As a post-doc, the applicant published an important paper in a top tier journal relevant to the work proposed here. In 2006, the applicant joined his/her current department, and s/he has since recruited a post-doctoral fellow and graduate student to the lab.
The candidate has written a carefully-considered plan for career development. The milestones are realistic and achievable. S/he states that receiving a CIRM grant would allow him/her to turn much energy from grant writing to developing the proposed research program. In addition, should the applicant receive a CIRM New Faculty Award, the department has agreed to allow the applicant to buy one year of teaching relief, further enabling him/her to concentrate on generating the data necessary to obtain future federal funding. The applicant has a number of excellent mentors at top institutions within California who will guide him/her in cortical development and stem cell concepts, and the applicant’s postdoctoral advisor will continue to be supportive.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: The candidate’s department chair has provided a letter documenting the standard institutional commitments to junior faculty. In particular, the department has provided adequate space to the candidate and an opportunity to move into new space in 2007. All necessary core facilities are available for conduct of the research. Importantly, the applicant has access to a core microarray and transgenic mouse facility as well as a communal facility for FACS. The institutional commitment regarding the candidate’s start-up is not detailed, but the applicant will have one full year of teaching relief – apparently only if the present application is funded. The candidate’s appointment is tenure-track. At this institution, tenure is typically conferred within five to seven years of the initial appointment at the rank of Assistant Professor.
The scientific environment for stem cell research at this institution is good. The department anticipates “hiring 3-4 new stem cell researchers in the near future.” A stem cell research facility is due to begin construction in the Fall. The PI is a member of a CIRM stem cell research group that consists of over 10 individuals and organizes symposia, hosts outside speakers, etc. In addition, the candidate has a mentoring committee, and grants from junior faculty are reviewed by senior faculty. There are also collaborative interactions with individuals at other top-level research institutions in California.
DISCUSSION: Reviewers commented that this is a strong, well-trained candidate in a good environment for stem cell research. The proposal is a solid basic science project that is interesting if not particularly innovative, although it does make use of the latest technology for gain-of-function and loss-of-function analyses. The PI has a foothold in a field that not many people are in, although s/he is studying neural progenitors rather than stem cells. The topic of the proposal has great relevance to diseases (such as ALS) involving cortical spinal motor neurons or cortical thalamic neurons. Overall, this is a talented investigator studying an important field at a good institution.
The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:
- None