2012 — 2017 |
Yoo, Andrew |
DP2Activity Code Description: To support highly innovative research projects by new investigators in all areas of biomedical and behavioral research. |
Microrna and Neural Factor-Mediated Direct Reprogramming of Cell Fates
DESCRIPTION (Provided by the applicant) Abstract: The ability to obtain neurons directly from the patients with neurological disorders will offer opportunities to study pathogenesis from the affected cells and develop novel therapeutic approaches. In hereditary neurological diseases, patient-specific neurons reprogrammed from non-neuronal cell types will harbor the same genetic mutation, thus offering valuable tools to study cell-autonomous pathology. For the disorders of somatic neurodegeneration, the isogenic, induced neurons may be used for cell replacement-based therapies. Most of current approaches towards deriving human individual-specific neurons have focused on transforming a differentiated cell type (for instance, dermal fibroblasts) to a pluripotent state and further differentiating them into neurons. This method requires forced expression of tumorigenic transcription factors that are highly expressed in embryonic stem cells. Moreover, differentiation of multipotent neural progenitors into specific subtypes of neurons is a difficult process to control. I have devised an alternative strategy of non-invasively obtaining neurons from adult human skin cells by converting their cell fate without going through pluripotent state (thus not requiring the expression of tumorigenic genes) and directly into post-mitotic neurons (direct reprogramming). I recently discovered that neuron-specific microRNAs (miR-9/9* and miR-124) could promote the switching of a non-neuronal cell fate into neurons when ectopically expressed with as few as one neural factor. The reprogramming efficiency was higher with more neural factors, and I devised a protocol in which miR-9/9* and -124 with neural factors, NeuroD2, ASCL1 and Myt1l generated neurons with cortical neuron-like characteristics. In this proposal, I will develop strategies to directly reprogram non-neuronal cells into various subtype-specific neurons. In developing tissue culture models of neurological diseases, it is important to obtain the type of neurons affected by the disease. Because the miR-9/9* and miR-124 are expressed pan-neuronally, I hypothesize that neuronal reprogramming can be customized for different subtypes of neurons by using transcription factors specific for the desired cell type in the background of miR-9/9*-124 expression. I will extend these studies to apply the direct reprogramming method in vivo from non-neuronal cells and hope to gain insights into possible restoration of the neuronal function lost in animal models of neuronal injury. Public Health Relevance: The ability to non-invasively obtain neurons from human individuals with neurological disorders will offer novel directions towards disease modeling and cell replacement-based therapeutic approaches. Here, we prose to devise methods to directly reprogram non-neuronal cell fates into subtype-specific neurons and develop in vivo application of this method in animal models of neuronal injury.
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0.915 |
2017 |
Yoo, Andrew |
RF1Activity Code Description: To support a discrete, specific, circumscribed project to be performed by the named investigator(s) in an area representing specific interest and competencies based on the mission of the agency, using standard peer review criteria. This is the multi-year funded equivalent of the R01 but can be used also for multi-year funding of other research project grants such as R03, R21 as appropriate. |
Modeling Neuronal Aging and Alzheimer's Disease in Human Neurons Directly Converted From Fibroblasts
ABSTRACT Late-onset Alzheimer?s disease (AD) is the most common form of late-onset neurodegenerative diseases. Because aging is a major risk factor in developing AD, dissection of molecular events that occur in the neurons of elderly individuals will provide important insights into the pathogenesis of late-onset AD (LOAD). Previous studies using post-mortem brain samples from cognitively normal human individuals demonstrated age- associated changes in gene expression. However, these molecular alterations are difficult to examine at a cellular level due to the unattainability of live human neurons cultured from elderly individuals. As such, the ability to obtain human neurons that reflect the late stage of human lifespan will provide a powerful experimental platform to investigate cellular properties of aged neurons. Recent advances in cell-fate reprogramming technologies allow the generation of human neurons from easily obtainable somatic cells such as fibroblasts. One commonly used method relies on the induction of pluripotent stem cells (iPSCs) from fibroblasts, which are sequentially differentiated into neurons. However, recent studies reported that the pluripotency induction erased the age signature stored in the original fibroblasts and consequently, iPSC- derived neurons resembled the neurons of an embryonic stage. Alternatively, we and other groups have shown that fibroblasts can be directly converted to neurons by ectopically expressing neurogenic genetic factors. In particular, we demonstrated that ectopic expression of neuronal microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124) in human adult fibroblasts led to the adoption of a neuronal state that can be guided to specific neuronal subtypes with neural transcription factors (TFs). Here, we propose to use our established miRNA-TF- based conversion protocol to generate human cortical neurons, a neuronal type largely affected in AD, from fibroblast donors across the age spectrum, and obtain cortical neurons that represent donors? ages as a cellular model of aging. The premise of maintaining the age of original fibroblasts in converted neurons is supported by our recent study demonstrating the retention of multiple age-associated marks including epigenetic, mRNA, miRNA and cellular signatures in converted human striatal neurons. In order to determine the consistency of age maintenance in cortical neurons, we will characterize an array of age marks in converted human cortical neurons from multiple age groups, and test if neurons derived from old individuals and LOAD patients would manifest AD-associated cellular phenotypes in comparison to neurons from young individuals. In addition, by leveraging our ability to control subtype specificity during neuronal conversion, we will address if different types of neurons (cortical, striatal and motor neurons) would differentially display AD phenotypes. We will also use converted human cortical neurons to identify genes whose altered expression underlies the age-related cellular phenotypes. Successful completion of our research aims will pave the way to model aging in human neurons.
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0.915 |
2018 — 2021 |
Yoo, Andrew |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanistic Insights Into Neurodegeneration in Huntington's Disease Using Patient-Derived Neurons Through Direct Conversion of Fibroblasts
Huntington?s disease (HD) is an inherited adult-onset neurodegenerative disorder caused by an abnormal expansion of CAG codons in the huntingtin (HTT) gene. HD is characterized by the aggregation of mutant HTT (mHTT) protein and selective degeneration of striatal medium spiny neurons (MSNs). Modeling HD using patient-derived neurons has been challenging mainly due to the lack of experimental approaches to obtain adult neurons from HD patients. Our previous work demonstrated that human MSNs could be generated with high efficiency and specificity from adult skin fibroblasts through direct cell fate conversion (reprogramming) using microRNAs and transcription factors. Importantly, the converted human MSNs resembled the neurons of human adults, an important feature for modeling late-onset diseases. However, the utility of directly converted MSNs as a cellular model of adult-onset HD remained to be determined. Recently, our preliminary work demonstrated that MSNs could be generated from directly converting fibroblasts of HD patients (HD-MSNs), and the resulting HD-MSNs manifested key hallmarks of HD pathology such as mHTT aggregates, DNA damage, and spontaneous cell death in culture. In the current grant, we propose to use HD-MSNs as a cellular model of HD and define genetic factors that alleviate the neuronal death of HD-MSNs. In Aim 1, we focus on SP9, a transcription factor that we found to be significantly downregulated in HD-MSNs in comparison to control MSNs from healthy individuals (Ctrl-MSNs). Interestingly, SP9 has been reported to be required for the maintenance and survival of MSNs, and we discovered that enforcing SP9 expression in HD-MSNs protected the cells from spontaneous cell death. To define the neuroprotective role of SP9 in HD-MSNs, we will identify direct target genes of SP9 and reveal genes integral to SP9?s function to promote HD-MSN survival. In Aim 2, we will investigate the function a primate-specific microRNA, miR-663b as a neuroprotective miRNA in HD- MSNs. Our preliminary work indicated that miR-663b protected MSNs from oxidative stress-induced neurodegeneration. Given the link between oxidative cellular stress and neurodegeneration in HD-MSNs, we will test if increasing the miR-663b level in HD-MSNs would confer a neuroprotection and identify direct target genes of miR-663b to delineate the function of miR-663b in HD-MSNs. In Aim 3, we will identify genetic pathways responsible for differential vulnerability to neuronal death between MSNs at different stages of disease progression. We found that HD-MSNs generated from fibroblasts sampled before the onset of clinical symptoms (pre-HD-MSNs) displayed significantly lower degrees of DNA damage and cell death in comparison to HD-MSNs derived after the onset of clinical symptoms. We will conduct transcriptome analysis to identify differentially expressed genes between pre-HD-MSNs and symptomatic HD-MSNs and identify differentially expressed genes responsible for the differential vulnerability to neuronal death. Overall, results from the current proposal will provide insights to neuronal death in HD using patient-derived neurons.
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0.915 |