Induced pluripotent stem cells from patients with ataxia-telangiectasia

Nayler, Samuel (2014). Induced pluripotent stem cells from patients with ataxia-telangiectasia PhD Thesis, School of Medicine, The University of Queensland. doi:10.14264/uql.2015.23

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Author Nayler, Samuel
Thesis Title Induced pluripotent stem cells from patients with ataxia-telangiectasia
School, Centre or Institute School of Medicine
Institution The University of Queensland
DOI 10.14264/uql.2015.23
Publication date 2014-12-19
Thesis type PhD Thesis
Open Access Status Other
Supervisor Ernst Wolvetang
Martin Lavin
Sergei Kozlov
Total pages 324
Language eng
Subjects 0601 Biochemistry and Cell Biology
Formatted abstract
A-T (Ataxia-Telangiectasia) is a multi-systemic, rare genetic disorder hallmarked by predisposition to cancer, immune deficiency and most notably progressive neurodegeneration [1]. A-T results from mutation of the ATM gene (11q22.3). ATM is a protein kinase belonging to the phosphatidylinositol 3-kinase-like family [2]. ATM exerts control over genomic integrity by recognizing and responding to DNA damage, through phosphorylation of substrates involved in cell cycle control and DNA damage repair [1]. The ATM protein is involved with numerous cellular processes and as such, A-T represents a paradigm for neurodegenerative disorders, as well as cancer.

Experimental models of ATM function have been restricted to systems of limited context to understanding ATM biology and its potential role in development and tissue formation. Animal models have failed to faithfully recapitulate the full spectrum of A-T symptoms including the neurodegenerative aspect, which remains poorly understood. Existing human cellular models (such as fibroblasts and lymphoblastoid cell lines from patients) allow analysis of ATM in limited contextual space. Little is known about the involvement of ATM or its downstream substrates have in the mechanisms that exist to direct and protect development within the embryo. Perturbed regulation of DNA damage at critical temporal junctures within the developing embryo could lead to the progressive degenerative characteristics observed in this disorder. Further to this, accumulating evidence points to involvement of ATM in areas outside of its canonical role of orchestrating the DNA damage response including; meiosis [3], proteasome-mediated protein degradation [4], mitochondrial function [5], insulin resistance and glucose metabolism [5, 6], modulation of synaptic functions in neurons [7], vesicle trafficking [8], pentose-5 pathway signaling [9], HDAC4 localization [10] and as a sensor and responder to oxidative stress [11, 12]. Emerging evidence also suggests that ATM has tissue specific functionality and is required during development [13, 14].

hESCs (human embryonic stem cells) constitute a powerful tool for modeling of development and disease. To date only one report describes the effect of ATM knockout in hESCs by BAC-mediated transgenesis [15]. The authors show that ATM knockout results in cells that display hypersensitivy to IR (ionizing radiation) and show lack of G2M cell cycle arrest after DNA damage, recapitulating aspects of the phenotype seen in existing cellular A-T models and demonstrating that ATM is a critical responder to DNA damage in this context. A recent technology to emerge from Japan has made it possible to reprogram terminally differentiated somatic cells, such as fibroblasts, into cells which resemble hESCs, in terms of self-renewal and their ability to generate cells of all three germ layers [16] and are so named induced pluripotent stem cells (iPSCs).

To date, studies on A-T have failed to answer the fundamental question of why certain cell types are seemingly more affected than others. As access to pluripotent stem cells becomes mainstream, it becomes possible, to a degree, to recreate and study in vitro processes giving rise to various cell types, including neurons, using directed differentiation protocols. We hypothesized that ATM deficient cells may be difficult or impossible to reprogram without intervention or assistance, and that this difficulty may be at least in part due excessive levels of reprogramming-induced or existing DNA damage, to the poor growth characteristics of A-T cells or the inability to participate in certain pathways necessary for reprogramming. We show that it was indeed possible to generate iPSCs from A-T patients albeit at reduced efficiency. This thesis describes the first generation and characterization of bona fide iPS cells from patients with A-T. Additionally we use this model system to explore the role and functionality of ATM in an embryonic setting, showing that ATM signaling is vitally required for the maintenance of cell cycle control and DNA fidelity after DNA damage. We have defined the transcriptional landscape of pluripotent stem cells from patients with A-T and point to novel findings regarding oxidative phosphorylation pathways. Further to this, we have used this model system to explore the role of ATM in neurogenesis and neuronal activity. We provide evidence that fosters support for the theory that A-T involves aspects of mitochondrial dysfunction and explore whether calcium trafficking is defective in neurons generated from patient iPSCs. Importantly we have illustrated the proof of concept that genetic manipulation of neuronal cells is possible by delivery of full length ATM, which also restored a functional DNA damage response.

Finally, we utilized a neuronal differentiation protocol to generate neural progenitors characteristic of the developing cerebellum, and describe the transcriptome of these cells in the absence of ATM. These data present unique and novel insights into the developing A-T brain and recapitulate many of the existing findings regarding the molecular pathways that may underpin the neurodegeneration in A-T. We speculate that this dataset will be a useful tool in understanding the growth requirements required for further expansion and study of these cell types in vitro, which could be harnessed to identify and screen drugs useful for the treatment of A-T patients.
Keyword iPS
Induced pluripotent stem cells
Pluripotency
DNA damage
Cell cycle
ATM
Ataxia-Telangiectasia
Neurodegeneration
Disease model
Cerebellar neurogenesis

Document type: Thesis
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Created: Fri, 28 Nov 2014, 04:42:41 EST by Samuel Nayler on behalf of Scholarly Communication and Digitisation Service