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Date of Award
Doctor of Philosophy (PhD)
In Huntington’s disease (HD), expansion of CAG codons within the huntingtin gene (HTT) leads to the aberrant formation of protein aggregates and the differential degeneration of striatal medium spiny neurons (MSNs). Modeling HD using patient-specific MSNs has been challenging, as neurons differentiated from induced pluripotent stem cells yield few MSNs that are free of mutant HTT aggregates, and lack overt phenotypes. Alternatively, human fibroblasts can be directly converted, bypassing pluripotency, into a heterogeneous population of neurons upon the ectopic expression of brain-enriched microRNAs (miRNA), miR-9/9* and miR-124 (miR-9/9*-124). It remained unknown, nonetheless, whether miRNA-mediated neuronal conversion could yield a homogeneous population of a discrete neuronal subtype, such as MSNs. In my thesis work, I have found that co-expression of miR-9/9* and miR-124 with transcription factors enriched in the developing striatum, BCL11B (also known as CTIP2), DLX1, DLX2, MYT1L, can guide the conversion of human postnatal and adult fibroblasts into an enriched population of cells analogous to striatal medium spiny neurons (MSNs). When transplanted in the mouse brain, converted cells were functional, synaptically active, exhibited passive membrane properties indistinguishable from native MSNs, extended projections to correct target regions, and persisted in situ for over 6 months. We hypothesized, that MSNs generated from patient fibroblasts through microRNA-based neuronal conversion, which we have shown to retain age signatures of donor fibroblasts, could offer advantages in modeling HD in tissue culture. Within this thesis I show that directly converted patient MSNs consistently exhibited mutant HTT (mHTT) aggregates, mHTT-dependent DNA damage, mitochondrial dysfunction, and spontaneous degeneration over time in culture. We further provide evidence that erasure of age stored in starting fibroblasts through the induction of pluripotency and neuronal conversion of pre-symptomatic HD patients’ fibroblasts results in differential manifestation of cellular phenotypes associated with HD, highlighting the importance of age in modeling late-onset neurological disorders. While the applicability of induced pluripotent stem cells (iPSCs) for the development of stem cell-based therapies and modeling of developmental processes remains unequivocal, our findings address many of the challenges faced by modeling late-onset diseases using neurons differentiated from HD iPSCs.
Chair and Committee
Andrew S. Yoo
Aaron DiAntonio, Sergej Djuranovic, Hiroko Yano, Luis Batista,
Victor, Matheus B., "Modeling Huntington's Disease With Neurons Directly Converted from Patient Fibroblasts" (2017). Arts & Sciences Electronic Theses and Dissertations. 1185.
Available for download on Wednesday, December 15, 2117