Date of Award
Doctor of Philosophy (PhD)
Tauopathies are a family of neurodegenerative disorders whose pathology include intracellular tau aggregates, synaptic dysfunction, and neuronal death. Advanced age is the strongest risk factor for developing a tauopathy, independent of a predisposing mutation, therefore the ability to study tauopathies in adult human neurons is vital to understand the age-associated changes which underly disease onset. Tau, the protein product of the gene Microtubule Associated Protein Tau (MAPT), is expressed highly in neurons. There are six tau isoforms whose splicing control and expression is both developmentally and tightly regulated, with only one isoform expressed in fetal brain and all six expressed in the adult brain. Alternative splicing occurs in both the N-terminal and C-terminal regions, varying between 0-2N domains or 3-4 microtubule binding domains (MBD), respectively. Tau isoforms are generally broken into two subgroups: 3R-tau and 4R-tau which represent the inclusion of either 3 or 4 MBDs, with the alternatively spliced MBD encoded by exon 10. Developmentally, the fetal brain expresses 3R-tau as the predominant isoform while the adult human brain expresses an approximately even ratio of 3R:4R. Importantly, imbalances in the isoform ratio, for instance with increased 4R-tau expression over 3R-tau, are sufficient to lead to pathogenic events leading to neurodegeneration. Currently, the molecular mechanisms controlling the tight regulation of 4R-expression remains largely unknown, and a human neuron-based platform that recapitulates 4R-tau expression of the endogenous MAPT as in the adult brain will be vital for studying tauopathies. Induced pluripotent stem cells (iPSCs) have been an instrumental tool to generate human neurons. However, evidence indicates that the induction of pluripotency in iPSCs reverts the cellular age to an embryonic state, thereby limiting the ability of iPSC-derived neurons to capture cellular events that occur in adult neurons. This is exemplified by recent studies showing that iPSC-derived neurons only produce the fetal isoform, 0N3R, limiting our ability to study the function and pathogenicity of adult tau isoforms, along with identifying the endogenous factors that regulate tau isoforms. Alternatively, direct conversion (reprogramming) of adult human fibroblasts to neurons bypasses the stem cell stage and propagates the age signature stored in original somatic cells to the reprogrammed neurons, offering the experimental benefit of modeling adult-onset neurodegenerative diseases. This transdifferentiation is directed by the ectopic expression of the brain-enriched microRNAs (miRNAs) miR-9/9* and miR-124 (miR-9/9*-124). When applied to adult fibroblasts, miR-9/9*-124 converts them to neurons (miNs) by first causing cell cycle exit and fibroblast identity erasure, followed by the neuronal program activation in sequence1. The resulting miNs maintain the epigenetic age of the starting fibroblasts, leading us to ask if they recapitulate adult tau isoform expression. Here, we reveal that miRNA-mediated direct reprogramming generates human neurons that express both 3R and 4R tau in the same ratio detected in adult human brains. RNA profiling by transcript assays and protein profiling by mass spectrometry demonstrates that adult human brain and miNs from adult fibroblasts have indistinguishable 4R tau profiles, which starkly contrasts with the low 4R tau expression in primary fetal human neurons and iPSC-Ns. The endogenous tau isoform regulation in miNs is sensitive to a point mutation within the splice site proximal to exon 10 in tauopathy patients resulting in reprogrammed neurons that showed increased 4R:3R tau ratio. Significantly, the increased level of 4R tau correlated with the formation of insoluble tau and seed-competent tau, reminiscent of that seen in human 4R tauopathies, and contrasting the lack of seed-competent tau seen in iPSC-Ns harboring the same patient mutation. Therefore, miRNA-mediated neuronal reprogramming provides a robust model for studying both the normal and abnormal biology of tau and exon 10 pathological mutations. We propose to use miNs as a model to dissect the adult protein and RNAs controlling tau isoform expression.
Chair and Committee
Andrew S. Yoo
Aaron DiAntonio, David Holtzman, Celeste Karch, Matthew Walter,
Capano, Lucia, "Dissecting Tau Isoform Control and Modeling Tauopathies in Directly-Reprogrammed Neurons" (2023). Arts & Sciences Electronic Theses and Dissertations. 2836.