Funding opportunities

hES Differentiation in Lesch Nyhan Disease

Funding Type: 
Comprehensive Grant
Grant Number: 
Funds requested: 
$1 987 915
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
PUBLIC ABSTRACT. A number of human neurodegenerative disease are caused by degeneration or abnormal expression of vital signaling systems in the brain. Because some of these disorders result from complex interactions of many genes with still inadequately-defined environmental factors, some of our most powerful current methods of genetic analysis cannot be easily applied to understand how these diseases develop. However, one human disorder of the dopamine system results from the action of a single abnormal gene, making it an ideal model for studying how a single defective gene can do so much damage to one of the brain’s most important signaling systems. Lesch Nyhan Disease (LND) is a devastating metabolic and neurological disease of children that results from abnormalities in a single gene, HPRT, that regulates the production of purines – building blocks of DNA and RNA and vital elements of information flow in the brain. Children with this disease demonstrate mental retardation, uncontrollable movements, kidney damage and, most disturbingly, a bizarre and completely untreatable involuntary self-mutilatory behavior in which the children bite and amputate their finger tips, the corners of their mouth, etc. We do not understand at all the ways in which a body-wide defect in purine metabolism causes the abnormalities in brain dopamine function. We have long experience in studying the genetic mechanisms of this disorder, largely by characterizing the affected brain regions in a HPRT-deficient mouse model of the disease. We have found that nerve cells isolated from affected brain have reduced levels of dopamine and are unable to take up dopamine from their environment, just as occurs in human LND patients. We have interpreted that finding to suggest that HPRT deficiency probably leads to the dopamine deficit by affecting the expression of other genes whose role is to direct the normal development of the dopamine neurotransmitter system in and between brain neurons. We have approached that possibility by testing the levels of expression of virtually all 30,000 genes in affected HPRT-deficient mouse brain and by examining the proteins present in normal and HPRT-deficient tissue (proteomic analysis). We have discovered that a small number of genes are expressed abnormally in HPRT-deficient tissue and that some of the protein products of those genes are present in unusual forms or amounts. We are now working to determine the functional significance of these changes. We infer from these results that HPRT deficiency may cause a defect in the mechanisms by which human embryonic stem cells generate and differentiate into dopamine neurons and possibly other cells in the brain. We therefore wish to search for differences in the ways in which the genes of normal and HPRT-deficient human ES cells are expressed and how those differences may affect the proteins found in affected ES cells as they differentiate into dopamine neurons in culture.
Statement of Benefit to California: 
BENEFIT TO CALIFORNIA. Through the establishment of the California Institute of Regenerative Medicine, the State of California has taken a major step to fill a great gap in biomedical research funding in the United States in the area of stem cell biology. At the present time, investigators in the United States are seriously hampered by insufficient and restricted federal funding for human stem cell research and cannot pursue some of the most promising basic and clinical research directions in this vital new area of biomedicine. The present proposal describes studies intended to understand the ways in which human embryonic stem cells develop into the brain neurons involved in vital brain neurotransmitter systems that cause some of our society’s most troublesome neurological diseases. Hundred of thousands of people in California suffer from degenerative neurological disease and related movement disorders, producing enormous harm to the well being and productivity of our society and constituting a huge financial burden on the State of California. If our studies with a crucial model disease are fruitful, the improved knowledge of the dopamine pathways in health and disease may help to improve the lives of many people in our State and country. In addition, the boost provided by these kinds of studies to the academic research environment in California can make the biomedical effort in this State even more vibrant than it is now, particularly in neurosciences, a field in which California is already a world leader. Furthermore, knowledge derived from these studies also has the potential for wide application in existing and new commercial settings, thereby catalyzing the biotechnology and pharmaceutical efforts in California and strengthening their intellectual property base.
Review Summary: 
SYNOPSIS OF PROPOSAL: The investigator is planning to use human embryonic stem cells (hESCs)to better understand the molecular mechanisms responsible for the aberrant development of dopaminergic neurotransmitter pathways in Lesch Nyhan disease. Lesch Nyhan disease is a neurodevelopmental disorder caused by mutations in the gene encoding purine reutilization enzyme hypoxanthine guanine phosphoribosyltransferase (HPRT). This disease is characterized by mental retardation, severe metabolic and motor deficits and a compulsive self injury behavior. The neurological deficit associated with Lesch Nyhan disease includes a severe depletion of basal ganglia, depletion of dopamine levels and dopamine uptake and the demonstration of deficient dopamine levels and dopamine uptake in cultured primary mid-brain neuron cultures from knockout mice. These defects suggest abnormalities in a neurotransmitter dopamine pathway intrinsic to HPRT deficiency, and may involve a developmental defect in dopamine neurons. IMPACT AND SIGNIFICANCE:Lesch Nyhan disease is a rare metabolic defect with several neurologic consequences. There is no effective therapy for the disease, especially with regards to the neurologic complications. The use of stem cells and the methodologies in this proposal are routine. There is no original scientific hypothesis. The results of using stem cells in gene expression studies in this proposal will have modest scientific impact. The PI proposes to use hESCs to study the dysregulation of dopamine in the Lesch Nyhan syndrome. Although the gene responsible for the disease is known, how a problem with a enzyme for purine re-utilization (HPRT) produces the disease is not well understood. The applicant has studied murine HPRT-deficient ESC and now proposes to apply the tools he has developed from the mouse to hESCs under the assumption that purine metabolism abnormalities cause multiple genetic problems. Lesch Nyhan disease is an interesting model disease with clear evidence for involvement of the DA neuron system. These studies could serve as an example on how to study a monogenic disease using hESCs. However, there are several weaknesses (see below) that significantly reduce enthusiasm for this study in its current form. QUALITY OF THE RESEARCH PLAN: In this proposal the investigator and collaborators will attempt to differentiate human embryonic stem cells to a dopamine phenotype and then to knock down the expression of HPRT in attempt to determine if this alters the dopaminergic phenotype with particular focus on gene expression profiles. It is not at all clear that usable data will be obtained from this approach for a variety of reasons; most importantly the reliable differentiation of human embryonic stem cells and the appropriate knock-down of the human gene have not been established. Furthermore, since this is a developmental disorder, it is not at all clear that the knock-down of a gene in embryonic stem cells will at all come close to recapitulating the more complex developmental changes that clearly occur in this disease as defined in literature and in the background of this grant. Pure embryonic stem cells and culture are unlikely to accurately recapitulate/mimic the complex synaptic and metabolic interactions between dopaminergic neurons and other cells. Also difficult to interpret will be effects of gene alterations that might be associated with HPRT deficiency-- which occurs not just in dopaminergic neurons- but surrounding interneurons and glia cells. Of course most importantly gene array studies have proven to have very limited utility in truly uncovering the biological underpinning of neurologic disease. The research plan requires better focus and detail. The aims described in the abstract do not really match the aims in the main application. The applicants proposed to use both hES derived NS cells and primary hNS cells differentiated with B49 medium. The applicants propose to both define novel differentiation factor for DA neuron development and find disease specific genes for Lesch Nyhan disease. No discussion on how any candidates will be selected from the screens and integrated in the overall study of Lesch Nyhan disease. The rescue studies are not well described, and it would seem essential to first understand the timing of rescue (e.g. by reintroducing the HPRT gene) to understand the critical period in the development of DA specific defects (HPRT rescue in adult DA neuron does not rescue phenotype). STRENGTHS: The greatest strengths are the investigator who has extensive experience in the HPRT models. The PI is a key expert in the field who has developed mouse models and studied the disease for more than a decade. A strong set of collaborators with critical expertise in many areas of the project has been assembled. An interesting disease model is proposed that could serve as a prototype CNS disorder to be studied using a hESC approach; HPRT is easier to model by homologous recombination than most other disorders (Y-chrom, alternative means of cell selection). WEAKNESSES: The weaknesses of the proposal are extensive. Gene arrays in hypothesis driven research are rarely as informative as proposed - although expectations may exist - many uses of these in past literature have been unremarkable and unrevealing, and few have revealed any reproducible fundamental information leading to understanding pathogenesis of disease. The limited studies proposed are not likely to give much insight into what may be happening in transplantation experiments. The inability to truly recapitulate the loss of function that is present from birth and throughout development using RNAi mediated knock-down of this gene in ES cells is considered a weakness. There is a real potential that multiple genes (dozens to hundreds) will be altered and the direction that one will have to follow to over express or under express these proteins seems, not just daunting but not a well described hypothesis. Another concern: the plans to do hybridization in a mouse based on human gene expression abnormalities. Some of the wild type mouse data alone would likely already be in the Allen brain atlas where thousands of genes have already been mapped out (at least normal expression patterns) and it would be foolish to repeat this with wild type mice again. The proposal is weak with regard to midbrain specification and DA neuron development. It is unlikely to provide novel insights into DA neuron development as proposed In general, the proposal is too diffuse of a study marked by poorly defined outcomes and decision points. TH will not read out midbrain DA neuron fate but only catecholaminergic neurons. There are some concerns regarding the overall hypothesis of the proposal. The PI states that the key defect in vivo seems to be the loss of DA axon terminals in the striatum. It is unclear how this phenotype will be modeled in the current proposal. The rescue studies are not well described with respect to selection of candidates or timing of reintroduction. There is no discussion of technical pitfalls with regard to promoter specific lentiviral vectors. Random integration into the genome often leads to stoichastic loss of or inappropriate expression that can interfere with analysis. The use of hNS differentiated into DA neurons using B49 conditioned medium is problematic. There is no evidence that such a protocol will lead to bona fide midbrain neurons rather than neural cells expressing TH. DISCUSSION: The strength of this proposal is the PI and the expertise in the disease and model. However, the PI has no experience in hESC differentiation into dopaminergic neurons, and there was concern about whether cells would migrate to the desired place. One reviewer felt that given previous work by the research team, the proposed studies would be feasible, but questioned why using hESC would be an added benefit. There was also a problem with experiments designed to create authentic, functional dopaminergic neurons with the proposed conditional mutant. The mis-expression or lack of expression of the promoter is a signficant complication, and others in the field have shown that this approach hasn't worked. At what stage of the rescue experiment will HPRT be added? Also, simply putting HPRT back in is not expected to rescue the dopaminergic phenotype. One reviewer stated that while the PI needs to get mid-brain dopaminergic neurons, it will not be possible with the protocols proposed. The PI has experience making primary mouse dopaminergic neurons, but not from mESC. Another reviewer was more enthusiastic, saying that the proposed study is of great interest because it is a template for a disease with a solid genetic basis and that the study of this in a human system is very important. The PI has already done the work in the mouse, and it is very reasonable to see if humans differ from mice in making these neurons. This is a risky project, but the risk is equal to others reviewed similarly. Recommendations to improve the proposal were offered. The study should be focused on Lesch Nyhan specific aspects of the hES model and less on study of dopaminergic neuron differentiation. A better description of how candidates will be selected and reintroduced should be included. hNS studies should be dropped as they introduce yet another model system / variable and do not really solve any of the major problems of the study. Moreover, there is no evidence that these cells will yield midbrain type dopaminergic neurons.

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