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.
Aging is a major risk factor for developing Alzheimer's disease. The possibility of producing human neurons from skin cells that remember the age of the individual they came from offers unprecedented promise for learning about cellular events that occur in aged human neurons. The major aim of our proposal is to use our established methods to generate human neurons of elderly individuals by directly converting their skin cells and use these neurons to model aging and cellular phenotypes associated with Alzheimer's disease.
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