YADONG HUANG, MD, PhD

		Defining the Isoform-specfific Effects of Apolipoprotein E on the Development of IPS Cells into Functional Neurons in Vitro and in Vivo GOALS: We propose to determine the effects of different forms of apoE on the development of induced pluripotent stem (iPS) cells into functional neurons. In Aim 1, iPS cells will be generated from skin cells of adult knock-in (KI) mice expressing different forms of human apoE and in humans with different apoE genotypes. In Aim 2, the development of the iPS cells into functional neurons in culture and in mouse brains will be compared. In Aim 3, the effects of different forms of apoE on the functional recovery of mice with acute brain injury treated with iPS cell-derived neural stem cells (NSCs) will be assessed.
RATIONALE AND SIGNIFICANCE: The central nervous system (CNS) has limited ability to regenerate and recover after injury. For this reason, recovery from acute and chronic neurological diseases, such as stroke and Alzheimer's disease (AD), is often incomplete and disability results. Embryonic stem cells have great promise for treating or curing neurological diseases, but their therapeutic use is limited by ethical concerns and by rejection reactions after allogenic transplantation. The generation of iPS cells from somatic cells offers a way to potentially circumvent the ethical issues and to generate patient- and disease-specific stem cells for future therapy. In the CNS, apoE plays important roles in lipid homeostasis and in neuronal maintenance. However, apoE2, apoE3, and apoE4 differ in their ability to accomplish these tasks. ApoE4, the major genetic risk factor for AD, is associated with poor clinical outcome and more rapid progression or greater severity of head trauma, stroke, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis-all potential targets of stem cell therapy. This proposal builds on three novel findings in human apoE-KI mice. (1) NSCs express apoE. (2) ApoE plays a role in cell-fate determination (neuron vs astrocyte) of NSCs. (3) ApoE4 impairs the neuronal development of NSCs. Thus, we hypothesize that transplantation of iPS cells derived from apoE4 carriers (~20% of the general population and ~50% of AD patients) might not be beneficial or even detrimental for patients with neurological diseases. We propose in vitro and in vivo studies to assess the effects of different forms of apoE on the development of iPS cells into functional neurons and on the functional recovery of mice with acute brain injury treated with iPS cell-derived NSCs. These studies will shed light on the regulation of neuronal development of iPS cells and help to “optimize” future iPS cell therapy for neurological diseases. SPECIFIC AIMS: Aim 1. To establish adult mouse and human iPS cell lines with different apoE genotypes. Aim 2. To determine the isoform-specific effects of apoE on the development of iPS cells into functional neurons in culture and in mouse brains. Aim 3. To assess the isoform-specific effects of apoE on the functional recovery of mice with acute (stroke) brain injury treated with iPS cell-derived NSCs.   Study the Pathophysiology of Frontotemporal Dementia in Induced Pluripotent Stem (iPS) Cells and their Derived Neurons RATIONALE AND SIGNIFICANCE: The discovery that induced pluripotent stem (iPS) cells can be generated from somatic cells has provided a way to circumvent the ethical issues of using embryonic stem cells and to generate patient- and disease-specific stem cells for mechanistic studies, drug screening, and therapy. A number of disease- and patient-specific iPS cell lines have been established, including those from patients with amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Parkinson's disease (PD), Alzheimer's disease (AD), frontotemporal dementia (FTD), and Huntington's disease (HD). Many of those iPS cell lines recapitulate in cultures the detrimental effects of the related mutations observed in vivo in humans, demonstrating the usefulness of patient- and disease-specific iPS cells in mechanistic studies and potentially also in drug screening. Furthermore, correction of genetic mutations in disease-specific iPS cells can rescue phenotypes in mouse models of human diseases, such as sickle cell anemia. However, one major difficulty for mechanistic and drug-screening studies with iPS cells is the variation among iPS cell clones with the same mutation. To address this issue, we used homologous recombination mediated by zinc finger nuclease (ZFN) to establish a protocol for correcting gene mutations in iPS cells or neural stem cells (NSCs). ZFN technology is a powerful tool for generating pairs of iPS cells, one with and one without the tau mutation, from the same parental iPS cell clone for better control in mechanistic and drug-screening studies. In the future, iPS cells with corrected tau mutations might also be used for stem cell therapy. Our goal is to correct the mutation in multiple iPS cell lines from FTD patients with different mutations in tau, progranulin, or other genes to generate isogenic pairs of iPS cells for mechanistic and drug screening studies. We will then explore the effects of those mutations on gene and microRNA expression profiles and on neuronal development and/or degeneration in cultures and determine whether correcting the mutation rescues their detrimental effects. SPECIFIC AIMS: Aim 1. Generate iPS cells from dermal fibroblasts with or without FTD-related gene mutations. Aim 2. Correct the mutations in iPS cells or NSCs using ZFN-mediated gene editing technology. Aim 3. Compare gene and microRNA expression profiles and neuronal differentiation of iPS cells and NSCs carrying the mutant or corrected FTD-related genes.

 
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