Establishment of human Embryonic Stem Cell Models to Study the Impact of Alzheimer's Disease Mutant Genes on Neuronal Functions
Alzheimer’s disease (AD) afflicts over 5 million Americans and is characterized by deposits of insoluble protein aggregates (amyloid plaques) and neurofibillary tangles as well as massive neuronal loss in regions affecting learning and memory. Currently, the best model for studying AD and testing novel therapies is a transgenic mouse that shows some, but not all, of the pathologies associated with AD. Mouse and human cells do not respond in the same ways to deleterious cell signals, so there is a great need for a model in which AD pathology can be studied in human cells. One difficulty in advancing AD research is that it is hard to obtain and examine live non-dividing (post-mitotic) neurons from AD patients. Even if such neurons are obtained, it is impossible to amplify them for reproduction of the obtained data or for sharing those neurons with other laboratories. As long as there is a possibility that different mechanisms may underlie different AD cases, it is considerably disadvantageous to investigate neurons from an individual AD patient with an unidentified cause. Our study will be conducted to overcome these difficulties. With recent progress in differentiating embryonic stem cells (ESC) into post-mitotic neurons, construction of ES cells expressing familial Alzheimer’s Disease (FAD) genes would allow for the creation of post-mitotic neurons bearing AD pathology. In this proposal, we have the advantage of having grown multiple HUES and some NIH-approved human ESC (hESC)lines and being able to express foreign genes at high levels in hESCs using lentiviral infection, as well as our expertise in the molecular and cell biology of AD. We propose to fully characterize these ES cell models in the context of AD pathologies such as A? generation and tau hyperphosphorylation, neuronal susceptibility to various neural toxicities, gene profiling, and neuronal differentiation. Three key genes/proteins known to be involved in AD pathogenesis are APP (?-amyloid (A?) precursor protein, from which A? is derived, PS1 (presenilin1) and tau. All the mutations in APP and PS1 are autosomal dominant leading to early onset of AD; mutations in tau (associated with familial frontotemporal dementia) favor the formation of neurofibrillary tangles (NFT). In addition to their well-defined functions in causing AD pathological lesions, these three genes/proteins have also been known to play key physiological roles including those in neurogenesis, neural development and synaptic functions. Defects in neuronal functions such as neurogenesis and neuronal differentiation have been found in AD brains in association with pathological abnormalities such as amyloid plaques (consisting of A?) and neurofibrillary tangles (NFT, consisting of hyperphosphorylated tau) as well as neuronal death in selected brain regions. The established hES cell models would be ideally important for drug screening which should eventually be instrumental for development of effective AD therapies.
Statement of Benefit to California:
"DRAFT OF A DRAFT" California has a tradition of taking the lead in technology and medical breakthroughs, not only by invention and innovation, but also by following through with research, development, and commercialization of ideas. Californians have a tradition of encouraging an entrepreneurial spirit, making the state an attractive site to launch new and risky ventures. Our proposal takes advantage of California's greatest strengths: innovative scientific research and high tech expertise. We propose a collaboration that involves the best of cutting edge laboratory research with the kind of entrepreneurial spirit in computer technology that launched HP, Apple, Google, and Pixar. We propose to generate and analyze a large amount of information about stem cells, focusing first on the molecular signatures of these cells, and then adding information about their utility for scientific research and their efficacy in preclinical development and clinical trials. A partnership with experts in launching web-based databases will allow us to make these data accessible to scientists so that they can make intelligent choices about which of the hundreds of embryonic and somatic stem cell lines will be best for their needs. This will eliminate wasted resources by creating a shared information database, encourage collaboration, and immediately enable scientists from non-stem cell disciplines to plan and carry out their experiments in a sound scientific context. This project will be a unique opportunity for California to demonstrate the power of its large intellectual base, and will create a magnet for other researchers, inside and outside California, to contribute their own information and expertise, which will leverage the power of the California stem cell community to explore novel approaches. The proposed project will be a springboard to new commercial ventures, and attract investment in research and development. Ultimately, by encouraging sharing of information, the project will help maintain high standards of scientific research, and speed the development of clinical applications for stem cells that will benefit all Californians. ADD A SENTENCE ABOUT THE LARGER AGING POPULATION IN CA SO MORE AD - HUAXI TO ADD TO ABOVE
SYNOPSIS: Exogenous, Alzheimer's disease-associated genes APP, PS1, and tau will be studied in hES cells to create a better model (compared to current transgenic mouse models) of human disease using several lines (including ones generated by Doug Melton) to study how neuronal functions will be affected. Four specific aims are proposed that: 1. Will engineer stable hES cells expressing functional APP, PS1, and tau genes, individually or together (as in the generation of the triple transgenic mice); 2. Examine the production of A beta and hyperphosphorylation of tau and testing if these cells exhibit an altered sensitivity to stress/excitotoxicity associated with A beta and NFT pathologies; 3. Understand the effects of these AD-associated genes on the growth and differentiation of hES cells (phenotyping and whole genome gene expression profiling); and 4. Examining how these AD related genes affect neuronal differentiation (neuronal markers and synaptic functions). SIGNIFICANCE AND INNOVATIONS: The proposed study, in principle, has the potential to yield cell lines and reagents that would be invaluable to the study of the pathophysiology of Alzheimer's Disease (AD). The approach of introducing the known "bad genes" associated with AD into human ES cells and then differentiating them into neuronal cultures to provide an in vitro model system to gain insight into the pathologic process associated with AD seems highly innovative. STRENGTHS: There is a need to have highly reproducible cellular models of AD and other neurodegenerative diseases, and hES cells expressing AD genes offer such a model. The PI has extensive expertise in the genes and their products that contribute to the pathogenesis of AD; the collaborator on this project, Dr. Loring, has extensive experience with ES cells, therefore this is a good team to be able to accomplish these studies. WEAKNESSES: Reviewer one: There is too little description of how the studies will be conducted and data analyzed; for example, defining normal or abnormal neuronal differentiation and synaptic function is only alluded to through what will be stained in the immunofluorescence studies and quantitative PCR for expression of particular gene markers. This is an overambitious proposal with 4 specific aims that will certainly require much more than the requested time to complete. Each one of these aims is time consuming and requires quality control and cell homogeneity testing and confirmation before being able to ascertain the effects of THREE DIFFERENT GENES on different populations of heterogeneous cultures of hES-derived cells. The “Potential Pitfalls and Alternative Methods” section is too short and is cryptic, and open-ended. For example, in one of the few sentences of this section, the following disquieting statement is presented: “….it is possible that the mutations may not affect neural development under our differentiation protocols. In this case, we will switch the goal to study their sensitivity to stress after their differentiation into neuronal cells. Reviewer two: Aim 1: No preliminary data to show that the lentiviral vectors transduce the human ES cells -- the preliminary data provided were in neuronal stem cells, which doesn't really support this part of the proposed research. Also, the investigators provide no details regarding the lentiviral vector genome design -- what promoter/enhancer will be used to express the AD genes? It appears to be a ubiquitous promoter, since many of their studies are based on study of the impact of the expression of the AD genes in the undifferentiated culture. It might be interesting to also include a neuronal-specific promoter, since normally these are very tissue-specific in their expression patterns. Finally, the method they plan to use to select for the transduced cells is based solely on FACS sorting, without the benefit of any selectable marker. Generally, long-term stable expression is not as easily achieved with transduction schemes based on selection solely on FACS sorting rather than biochemical selection methods. Apparently, none of the vectors are made. Aims 2 and 3: The investigator plans to do a very detailed assessment of the impact of expression of AD-associated genes on gene expression using microarray technology and RT-PCR. In principle, this sounds like a good idea, however, the proposal appears to indicate that the studies will be performed in the undifferentiated human ES cultures that have been transduced to express the AD-associated genes, rather than derivative differentiated neurons. Since the pathology of AD is exclusively found in the CNS, one reviewer doesn't understand why the proposal is to look at the undifferentiated cultures, rather than neuronal cultures. This is potentially an interesting and important proposal. In light of space constraints, the applicant is advised to spend less space and effort toward reviewing the AD literature and more to experimental design, outcomes, pitfalls, and follow-up studies. DISCUSSION: There were no further comments other than those of the reiewers.