The adult brain contains a pool of stem cells, termed adult neural stem cells, that could be used for regenerative purposes in diseases that affect the nervous system. The goal of this proposal is to understand the mechanisms that promote the maintenance of adult neural stem cells as an organism ages. Understanding the factors that maintain the pool of adult neural stem cells should open new avenues to prevent age-dependent decline in brain functions and to use these cells for therapeutic purposes in neurological and neurodegenerative diseases, such as Alzheimer’s or Parkinson’s diseases.
Our general strategy is to use genes that play a central role in organismal aging as we have recently discovered that two of these genes, Foxo and Sirt1, have profound effects on the maintenance and self-renewal of adult neural stem cells. We propose to use these genes as a molecular handle to understand the mechanisms of maintenance of neural stem cells. Harnessing the regenerative power of stem cells by acting on genes that govern aging will provide a novel angle to identify stem cell therapeutics for neurological and neurodegenerative diseases, most of which are age-dependent.
As the population of the State of California ages, neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease affect increasing numbers of patients. There are no efficient treatments of cures for these diseases. In addition to the devastating effects of neurodegenerative diseases on the patients and their relatives, the cost of caring for California’s Alzheimer patients—about $22.4 billion in 2000—has been estimated to triple by 2040 due to the aging of the baby-boomer’s generation.
Stem cells from the brain, or neural stem cells, hold the promise of treatments and cures for these neurodegenerative diseases. One therapeutic strategy will be to replace degenerating cells in patients with stem cells. Another approach would be to identify strategy to better maintain the pool of neural stem cell with age. Both approaches will only be possible when the mechanisms controlling the maintenance of these stem cells and their capacity to produce their functional progeny are better understood in young and old individuals.
We propose to study the mode of action in neural stem cells of two genes, Foxo and Sirt, that are known to play major roles to extend lifespan in a variety of species. These genes are major targets for the development of stem cell therapeutic strategies that will benefit a wide range of patients suffering from age-dependent neurodegenerative disorders.
The development of effective replacement therapies in neurodegenerative diseases will be a benefit for the rapidly aging population of California; it will also alleviate the financial burden that these age-related disorders create for the State of California.
SYNOPSIS: The Foxo3 transcription factor and its binding partner Sirt1 (a histone deacetylase) have been shown to extend the lifespan of genetically accessible invertebrate model organisms and (perhaps) also mice. Several recent studies indicate that Foxo-dependent signaling is required for long-term regenerative potential of the adult hematopoietic stem cell (HSC) compartment through regulation of HSC response to physiologic oxidative stress, quiescence, and survival. Foxo proteins are negatively regulated by direct phosphorylation by AKT and positively regulated by the PTEN phosphatase which blocks AKT signaling. The activity state of Foxo3 is also regulated by acetylation. Dr. Brunet’s work established that in response to oxidative stress stimuli, the histone deacetylase Sirt1 binds to and deacetylates Foxo3 tipping the balance of Foxo3 function towards growth arrest and survival.
The goal of this application is to define the role of Foxo3 and Sirt1 in neural progenitors. The applicant hypothesizes that Foxo3 and Sirt1 act to maintain the pool of functional neural stem cells (NSC) during aging by regulating a specific network of genes. The applicant reasons that depletion of NSC may contribute to cognitive changes associated with normal or pathological aging. Thus identifying ways to reactivate the Foxo3/ Sirt1 network to sustain the pool of NSC in the adult brain could have practical overtones for human mental health. Her study plan has three specific aims:
1) Assess the role of Foxo3 and Sirt1 in NSC as a function of age. Both gain of function and loss of function studies are proposed towards this end. For loss of function genetic studies in vitro, the applicant will use 1) lentiviral RNAi technology, 2) NSC derived from Foxo3 and Sirt1 knockout animals, and 3) small molecule inhibitors of Sirt1.
2) Define the mechanisms of action of Foxo3 and Sirt1 (i.e. their genetic targets). In preliminary studies, the applicant has identified two potential Foxo3 transcriptional targets. Using contemporary deep sequencing (ChIP Seq) technology, she hopes to identify additional Foxo3 and Sirt1 targets in NSC, and thus describe the global program whereby Foxo3 and Sirt1 moderate lifespan of adult stem cells.
3) Explore the importance of Foxo3 and Sirt1 in human stem cells. The applicant speculates that quantitative differences in the lifespan of humans and rodents may translate into qualitative differences in the molecular mechanisms that sustain the lifespan of human NSC. In collaboration with Drs. Theo Palmer and Julie Baker at Stanford, she will derive human NSC lines out of human embryonic stem (ES) cell cultures. Armed with these human NSC lines, she will basically repeat the in vitro studies on the function of Foxo3 and Sirt1 described for specific aims one and two.
STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: The proposal clearly addresses important facets of human aging and neurodegenerative disease. The idea that genes that regulate longevity may also regulate the long term renewal potential of adult stem cells is a recent and very interesting concept, and the applicant has been a major contributor to this idea. Although depletion of neural progenitor cells is probably only one of the multiple underlying causes of age-related dementias and neurodegenerative disease states in humans, the research proposed here has the potential to improve the standard of care for these problems in the fullness of time. The idea is that identifying ways to reactivate the Foxo3-Sirt1 network in aged NSC could maintain the pool of NSC for regenerative purposes. In this respect, the proposal is innovative and of high significance to CIRM.
The experiments proposed play off of a now well-established conceptual paradigm of Foxo transcription factors and Sirt deacetylase proteins as master regulators of cellular aging. However, the use of NSC in this context is novel, and especially the proposal to investigate the regulation of miRNA is significant. The study plan exploits state of the art technologies and bioassays but there are no novel approaches at work. Critical reagents, technologies and collaborators are in place for all the studies proposed.
The experiments in Aims 1 and 2 (analysis of Foxo3 and Sirt1 function in NSC in vitro and in vivo using mouse models) are logical and straightforward, whereas the ones in Aim 3 (analysis of Foxo3 and Sirt1 function in vitro in human NSC, derived form human ES cells) are hard to judge. For example, it is not clear what cells will be used. Nevertheless, a study of human NSC is of importance and provides stronger relevance to the mission of CIRM. The preliminary data supports the central premise of the studies, to wit that Foxo3 and Sirt1 enhance the lifespan of NSC. Although the work on the genetic targets of Foxo3 and Sirt1 in rodent and human NSC seems unlikely to go beyond the descriptive level in the five year time frame of this grant, this was not considered a major concern.
The applicant acknowledges potential pitfalls in the work proposed and where possible describes alternative approaches. For example, functional redundancy of Foxo3 with other members of the Foxo protein family is a potential problem. To address the issue of functional redundancy and compensation, the applicant has recently produced mice with a null allele of Foxo6, the other Foxo transcription factor that is highly expressed in brain.
Several recommendations were made with regard to prioritizing or improving certain experiments. For instance, the applicant’s proposal to examine NSC maintenance in mice in which the Foxo3 and Sirt1 genes are conditionally deleted in adult NSC is listed as a long-term goal. However, one reviewer felt that these are critical experiments that should be performed earlier rather than later. Since the applicant is new to in vivo studies, this may be the reason for the hesitance to start these experiments now. Another reviewer acknowledged that the applicant has demonstrated the ability to properly perform microarray-based miRNA assays. However, (s)he suggests that new technologies to amplify and analyze miRNA could be employed, because the amount of miRNA could be minimal when isolating it from subsets of cells exhibiting certain phenotypes. Since neurospheres are known to consist of heterogeneous cell populations, isolation of a certain phenotype of cells, e.g. NSC, or a different type of culture method (monolayer culture) may be necessary. Also, factors added to the serum free culture media might affect the results, and stress from gene transfection may cause spontaneous differentiation of the cells. Thus, careful selection of control experiments should be considered.
QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Brunet is clearly a very talented scientist, and her potential to become a leader in the stem cell field and make seminal contributions is outstanding. Dr. Brunet has been productive at every stage of her career development to this point. She generated a number of first author research publications at both the predoctoral and postdoctoral levels - and these papers appeared in top-tier scientific journals. Dr. Brunet has expertise in Foxo research, and several of her papers address the genetic control of longevity.
Dr. Brunet was appointed to the faculty at Stanford University Medical School in 2004, and her first publications as an independent investigator are already appearing in high-quality journals. She has already secured R01 grant support from the National Institutes of Health, together with several smaller grants from private foundations.
Dr. Brunet has also presented a carefully considered plan for career development. Several official mentors, including Greg Barsh, and collaborators have been listed. She will meet with these individuals together with the department chair at least once a year to discuss progress and plans. The scientific environment at Stanford University is outstanding.
INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: An enthusiastic letter from Dr. Brunet's Department Chair details a generous commitment of space (1,355 sq. ft) and resources ($700,000 start up package). The Department supports 40% of Dr. Brunet’s salary. It has also assigned an official mentor to Dr. Brunet to guide her and advise her through the early years of her career development. The institutional track record of Stanford University for developing the careers of its junior faculty is outstanding, and Stanford has made a major commitment to increase stem cell research and biomedical engineering.
DISCUSSION: Overall, there was great enthusiasm for this proposal, since the applicant is outstanding, and so is the institutional environment. One reviewer stated that this application was one of the three best proposals in his/her pile. The application itself was considered a great proposal with a great research plan, and with all critical elements in place. However, the innovation level of this proposal was considered a little low; it is “not a never before seen” application. Aim 1 was described as being conventional. Aim 2 is mechanistic, and utilizes elegant ChIP technology. Aim 3 was described as less well thought out (derive human NSC from human ES cells and repeat aims 1 and 2), but also as the most interesting aim, since it intends to test the hypothesis that depletion of NSC may contribute to neurological changes associated with aging. This aim is based on the general idea that humans live longer than mice due to molecular differences, and consequently differences in lifespan, in their adult stem cells. It was acknowledged that it is challenging to study aging. One suggestion for improvement of Aim 3 was to include in the analysis NSC derived from mouse ES cells to compare to those derived from human ES cells. It was also mentioned that neurospheres contain a heterogeneous population of cells, which may create problems, and that Foxo function has already been well studied in HSC. Notwithstanding these minor suggestions, overall the reviewers agreed that this is great application from an excellent candidate with a stellar track record and great collaborators.