Funding opportunities

Stem cell regeneration of neural circuits in murine Huntington's disease models

Funding Type: 
New Faculty I
Grant Number: 
RN1-00547
Funds requested: 
$1 617 540
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Discovery of embryonic and tissue stem cells has provided renewed hope for the development of cell replacement therapies to treat neurodegenerative disorders. Currently, a number of stem cell-related strategies require preclinical evaluation in order to move such treatments to clinical trial. The present proposal will examine the therapeutic potential of endogenous and transplanted neural stem cells (NSCs) in a transgenic model of Huntington’s Disease (HD). HD is an adult-onset disorder involving specific loss of striatal and cortical neurons that is accompanied by progressive motor system abnormalities (e.g. uncontrollable ballistic movements or “chorea”) and an array of cognitive impairments. HD is caused by a dominant mutation in a single gene, huntingtin (htn), containing expanded CAG repeats and genomic insertion of the mutant htn induces pathology in animals similar (or identical) to that observed clinically. Importantly, transgenic mice exhibit nearly identical abnormalities to HD patients in terms of motor and cognitive deficits and neuronal loss in the striatum and cerebral cortex. Further, as the pathology of transgenic HD mice develops, adult NSCs undergo population expansion and their progeny are redirected toward the striatum and a parallel increase in adult NSCs occurs in HD patients. Thus, stem cell function is influenced in a similar fashion by the presence of mutant huntingtin in mouse and human brain. These factors make transgenic HD mouse models an ideal system for cell replacement experimentation, particularly for strategies using stem cells. Alternatives in cell replacement strategies has expanded immensely with the discovery and characterization of embryonic and adult stem cells. Our proposal provides a comparison of activation of endogenous NSCs and transplanted NSCs. Specificaly,endogenous NSCs will be stimulated in situ with factors that induce proliferation and survival of precursors. NSCs for transplantation will be derived from adult brain or from "neuralization" of embryonic stem stems, including human cells. Our laboratory is particularly well suited to conduct research of this nature because our combined cellular, systems neuroscience, and behavioral expertise will facilitate a thorough evaluation of stem cell therapeutics at the organism level necessary to move from preclinical studies to clinical trials.
Statement of Benefit to California: 
Nervous system disorders, ranging from idiopathic neurodegeration to trauma and stroke, account for an ever increasing portion of medical costs and for most of these syndromes there are few treatments and no cures. Cell replacement therapies offer a avenue of tremendous potential for the future of regenerative medicine. Clinical trials employing fetal tissue grafts for potential treatment of Parkinson's and Huntington's disease are under way and have yielded promising results. However, such potential treatments currently require aborted fetal tissue which provides both a moral obstacle and a limitation on the widespread employment of these approaches because of insufficient tissue sources. Discovery and characterization of embryonic and adult stem cells provide the potential opportunity for generating the large amounts of cells for widespred grafting because these cells (by definition) have extensive self-renewal capacity. Currently, many strategies for the use of stem cells require preclinical evaluation prior to the initiation of clinical trials. Our proposal will conduct preclinical trials using a mouse model of Huntington's disease to compare the therapeutic potential of activating endogenous neural stem cells, transplanting neural stem cells, and transplanting neuralized embryonic stem cells. The findings of these studies will have direct relevance to developing clinical trials employing stem cells in the treatment of Huntington's disease, as well as, other neurodegenerative disorders and traumatic brain injury. Accordingly, the proposed research will initiate steps towards fulfilling the hope that stem cells will be able to treat neurodegenerative disorders. Specifically, Huntington's disease afflicts several thousand individuals in California and there is no cure or treatment for this disease. It is a progressive disorder that robs the individual of motor, cognitive, and emotional control that produces tremendous distress for the afflicted and their families. As such, creating therapeutic avenues for the management of this condition will be of direct benefit to the citizens of California as well as reducing long-term health costs associated with Huntington's disease. Further, the proposed research will increase the diversity of stem cell research in the state and provide training and employment to future researchers.
Review Summary: 
SYNOPSIS: This proposal seeks to identify the optimal cell source and conditions for cell replacement treatment of a Huntington’s Disease (HD) transgenic mouse. The PI will address the potential therapeutic value of NSC and ESC in HD in several ways. First, the PI will characterize the impact of growth and neurotrophic factors on long-term NSC function in normal and HD mouse brain. Next, the PI will determine the effectiveness of endogenous NSC and transplanted NSCs (including those derived from ESCs) with and without additional trophic factors on repairing circuits damaged in HD disease. The research is organized into four specific aims. Aim 1. Characterize NSCs in YAC128 mice. Human HD patients have increased NSCs and his research with the R6/2 transgenic mouse demonstrates not only an expansion in NSCs (both neurosphere-forming cells, “B” cells, and BrdU-retaining cells) in response to disease pathology but also precursor migration into the damaged striatum. Aim 2. Determine effectiveness of trophic factor-induced activation of endogeneous NSCs to repair neuodegernation. Characterize dose-response effects of trophic factors for the long-term tolerability and maximal striatal and cortical neurogenesis. The PI will initiate trophic factor treatments early, mid and late in the course of disease progression. Aim 3. Determine effectiveness of transplanted adult-derived NSC and progenitor cell types to repair neurodegernation. Transplants containing sorted or unsorted cells will be made into striatum and cortex of YAC128 to compare their effects on functional recovery. Aim 4. Determine effectiveness of transplanted “primitive” NSCs to repair neurodegeneration. Again, the influence of trophic factors to enhance repair following ESC transplantation will be assessed. Finally, in order to establish congruency between effects observed with mouse and human cells, parallel experiments will employ human ESCs to repair degeneration in mouse brain. STRENGTHS AND WEAKNESSES OF THE RESEARCH PLAN: This proposal will explore the feasibility of restoring neurons and neuronal circuitry in a mouse model of Huntington’s disease (HD). The hypothesis that is tested in this proposal is that cell replacement strategies will lead to behavioral recovery and an increase in lifespan in HD transgenic mice. The PI has preliminary data that demonstrate an increase in NSCs in R6/2 transgenic mice; similar increases have been found following excitotoxic damage to the striatum. The data suggest that a population of proliferative and undifferentiated cells are available for recruitment and would be stimulated to replace degenerating neurons given the proper conditions. The studies are largely applications of previously carried out experiments on a different model of HD. The PI has previously studied the R6/2 transgenic mouse but now will switch to the YAC128 mouse, which has a more comparable time course and pathology to HD in humans. One has to assume that his preliminary data on the R6/2 mouse will be applicable to the YAC128, and his first specific aim will detail this. The PI proposes to explore both endogenous and exogenous repair. The results could have important implications for HD, and possibly to other neurodegenerative diseases and injury. However, the focus is rather narrow, since it is mainly applicable to one disease. In specific aim 1, the PI will characterize the same parameters in the YAC128 mouse as was done in the R6/2 mouse. The PI chose the YAC128 mouse because it has a longer duration of disease and exhibits a window of therapeutic opportunity. These studies will examine whether there are increased numbers of subpendymal neurosphere-forming cells in the YAC128 mouse compared to controls. The PI has found that particular growth factors lead to striatal neurogenesis. In specific aim 2, YAC128 mice will be treated by infusion of growth factors (EGF, bFGF, TGF, and erythropoietin) into the lateral vectricles by osmotic minipump attached to a 30-guage cannula for varying times to determine the effect on: the number of B cell and BrdU-retaining cells in the subependyma, the striatal and cortical cytoarchitecture, and neurochemical responsiveness. The most effective trophic factor protocol will then be used on the YAC128 mice (since functional neurogenesis takes a period of several weeks) at 3, 8 and 12 months. These mice will be examined with respect to the number of NSCs and also with a battery of behavioral tests and neurochemical assessments. In addition to the intraventricular administration of growth factors, the PI will produce novel formulations of small molecules that cross the blood-brain barrier to allow for systemic adminstration. In the third specific aim, the PI will determine the effect of transplanting sorted and unsorted adult NSCs on the functional recovery of YAC128 mice. The mice will be evaluated as in specific aim 2. In some cases, trophic factor administration will be used to increase survival and the incorporation of grafts. In the last specific aim, the PI will determine the effect of transplanting primitive NSCs (which are derived from mouse or human ESCs) to repair neurodegeneration; the cells will be treated (e.g., sorting vs. unsorting, trophic factor treated) per results of the other specific aims. The PI has experience in culturing of adult NSCs. The work with ESCs will take advantage of guidance from the UCSB Laboratory for Stem Cell Research at UCSB. There were several strengths to this proposal. Overall, the experimental plan appears thorough and complete. Only occasional parts of the description are “dropped” in an unannounced manner, e.g. the use of minocycline. In many ways, this proposal builds and is based on previous studies and findings of others. Concerns were expressed with the proposal, including one major question: what will the PI do if there is no increase in NSC in this model of HD--that is, if the preliminary observations do not hold in the new model? There were some concerns with Aim 2 of the proposal. The PI should use a genetic approach to mark and follow cells to striatum.There was also concern about the small molecule studies in Aim 2, which will be in collaboration with Dr. Bruce Lipshutz of the Department of Chemistry and Biochemistry; this aspect of the study is not detailed and no letter from Dr. Lipshutz is provided. In Aim 3, one reviewer questioned, how will transplanted cells be identified since the transgenic carries gfp under control of the GFAP promoter (gfp-GFAP) rather than an independent promoter? In Aim 4, one reviewer commented that the PI likely needs to sort neural cells so no tumors form. One reviewer's enthusiasm for the project would have been bolstered with more innovative ideas and more risk-taking. A minor criticism of the proposal was that there were a number of typographical errors. One reviewer would discourage the applicant in the future from for using such small font as he does in the legends of the figures- they are almost unreadable. QUALIFICATIONS AND POTENTIAL OF THE PRINCIPAL INVESTIGATOR: Dr. Kippin received his PhD at Concordia University in 2000, followed by a post-doctoral fellowship at University of Toronto (2000-2004) and then at the Medical University of South Carolina (2003-2004). Dr. Kippin has been Assistant Professor in the Department of Psychology of the Psychology and Neuroscience Research Institute at UCSB since July 2004. Dr. Kippin states he has 14 years of experience in behavioral neuroscience and one year in cell and molecular biology. Dr. Kippin’s publication record is quite good. Since 2003, Dr. Kippin has published 13 papers with him as first, shared first author, or last author (including, notably, three J. Neuroscience papers and one Genes and Development paper). There are three publications in 2007, and 2 in 2006 (including one paper on HD). One reviewer commented that these publications are largely descriptive. He is presently the recipient of a NASAD grant entitled "antipsychotic drug regulation of NSCs and neurogenesis in the adult mammalian brain" and an Alcoholic Beverage Medical Research Foundation award on "alcohol and adult NSC function." The PI has training in HD and behavioral and molecular studies. Probably his training of greatest relevance to his current project was in Derek Von der Kooy’s lab. In reading his Career developmental Plans, one might get the impression that he is primarily interested in behavioral aspects of neuroscience and in the systems he studies, and that the neural stem cell component is somewhat secondary. He did not detail any plan in regard to mentoring and only an apparent collaboration with the Clegg lab at UCSB on human ESCs. INSTITUTIONAL COMMITMENT TO PRINCIPAL INVESTIGATOR: Dr. Kippin has apparently been very successful at UCSB since his starting there in 2004, and his Department Chair expects him to be tenured in one to two years. This is stated in a letter from his chair. Dr. Kippin is said to have received a "generous start-up package," although details are not provided. A senior mentor has been assigned to assist him in his career development. He presently has 750 sq. ft. of wetbench and behavioral testing space. It appears as if they have many of the Core’s needed to do his work and he has the appropriate equipment in his lab. There are core facilities for FACS, confocal microscopy, animal care, genomics and proteomics. UCSB has a growing interest in stem cell research and have been quite successful in achieving CIRM funding to date, all of which should help him. There is a shared Laboratory for Stem Cell Biology used by 13 UCSB PIs. A new shared lab is under construction through funding from CIRM. Funding for a future Director for a Stem Cell Center has been obtained and there are plans for recruitment for 3 additional PIs. DISCUSSION: Comments from the reviewers centered on the fact that the proposal is not very risk-taking, and might move the field forward only a little bit. The reviewers sensed a strong commitment to and self-described identity of the PI in behavioral neuroscience. There was some debate about the commitment of the investigator to stem-cell science, citing the following: the only component of the research plan to involve stem cells was via collaboration, and there was no mentoring plan in place for stem cell biology. Two reviewers commented on the difficulty of reading the proposal because of the typographical errors and the small font size.
Conflicts: 

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