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      Genomic Instability Associated with p53 Knockdown in the Generation of Huntington’s Disease Human Induced Pluripotent Stem Cells

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          Abstract

          Alterations in DNA damage response and repair have been observed in Huntington’s disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis, while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX, indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus, increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown.

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          Suppression of induced pluripotent stem cell generation by the p53-p21 pathway.

          Induced pluripotent stem (iPS) cells can be generated from somatic cells by the introduction of Oct3/4 (also known as Pou5f1), Sox2, Klf4 and c-Myc, in mouse and in human. The efficiency of this process, however, is low. Pluripotency can be induced without c-Myc, but with even lower efficiency. A p53 (also known as TP53 in humans and Trp53 in mice) short-interfering RNA (siRNA) was recently shown to promote human iPS cell generation, but the specificity and mechanisms remain to be determined. Here we report that up to 10% of transduced mouse embryonic fibroblasts lacking p53 became iPS cells, even without the Myc retrovirus. The p53 deletion also promoted the induction of integration-free mouse iPS cells with plasmid transfection. Furthermore, in the p53-null background, iPS cells were generated from terminally differentiated T lymphocytes. The suppression of p53 also increased the efficiency of human iPS cell generation. DNA microarray analyses identified 34 p53-regulated genes that are common in mouse and human fibroblasts. Functional analyses of these genes demonstrate that the p53-p21 pathway serves as a barrier not only in tumorigenicity, but also in iPS cell generation.
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            Dominant phenotypes produced by the HD mutation in STHdh(Q111) striatal cells.

            Lengthening a glutamine tract in huntingtin confers a dominant attribute that initiates degeneration of striatal neurons in Huntington's disease (HD). To identify pathways that are candidates for the mutant protein's abnormal function, we compared striatal cell lines established from wild-type and Hdh(Q111) knock-in embryos. Alternate versions of full-length huntingtin, distinguished by epitope accessibility, were localized to different sets of nuclear and perinuclear organelles involved in RNA biogenesis and membrane trafficking. However, mutant STHdh(Q111) cells also exhibited additional forms of the full-length mutant protein and displayed dominant phenotypes that did not mirror phenotypes caused by either huntingtin deficiency or excess. These phenotypes indicate a disruption of striatal cell homeostasis by the mutant protein, via a mechanism that is separate from its normal activity. They also support specific stress pathways, including elevated p53, endoplasmic reticulum stress response and hypoxia, as potential players in HD.
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              Oxidative damage and metabolic dysfunction in Huntington's disease: selective vulnerability of the basal ganglia.

              The etiology of the selective neuronal death that occurs in Huntington's disease (HD) is unknown. Several lines of evidence implicate the involvement of energetic defects and oxidative damage in the disease process, including a recent study that demonstrated an interaction between huntingtin protein and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using spectrophotometric assays in postmortem brain tissue, we found evidence of impaired oxidative phosphorylation enzyme activities restricted to the basal ganglia in HD brain, while enzyme activities were unaltered in three regions relatively spared by HD pathology (frontal cortex, parietal cortex, and cerebellum). Citrate synthase-corrected complex II-III activity was markedly reduced in both HD caudate (-29%) and putamen (-67%), and complex IV activity was reduced in HD putamen (-62%). Complex I and GAPDH activities were unaltered in all regions examined. We also measured levels of the oxidative damage product 8-hydroxydeoxyguanosine (OH8dG) in nuclear DNA, and superoxide dismutase (SOD) activity. OH8dG levels were significantly increased in HD caudate. Cytosolic SOD activity was slightly reduced in HD parietal cortex and cerebellum, whereas particulate SOD activity was unaltered in these regions. These results further support a role for metabolic dysfunction and oxidative damage in the pathogenesis of HD.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                16 March 2016
                2016
                : 11
                : 3
                : e0150372
                Affiliations
                [1 ]Department of Neurology, Vanderbilt University Medical Center, Nashville, TN, 37240, United States of America
                [2 ]Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN, 37232, United States of America
                [3 ]Genetics Associates Inc., Nashville, TN, 37203, United States of America
                [4 ]Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY, 10461, United States of America
                [5 ]Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN, 37240, United States of America
                [6 ]Center in Molecular Toxicology, Vanderbilt University, Nashville, TN, 37232, United States of America
                University of Minnesota Medical School, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: AMT MDN ABB. Performed the experiments: AMT MDN RC AAA KKK BH MRB. Analyzed the data: AMT MRB MDN KCE ABB. Contributed reagents/materials/analysis tools: RC KCE. Wrote the paper: AMT MDN AAA MA KCE ABB.

                Article
                PONE-D-15-19601
                10.1371/journal.pone.0150372
                4794230
                26982737
                d88fd902-228e-4b8e-b652-254dcb156c8c
                © 2016 Tidball et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 7 May 2015
                : 13 February 2016
                Page count
                Figures: 5, Tables: 3, Pages: 16
                Funding
                This study was supported by the National Institute of Environmental Health Sciences: ES016931 (ABB) and ES010563 (ABB/MA); the National Institute of Neurological Disorders and Stroke: F31 NS077632 (AMT) as well as additional support from the Vanderbilt Medical Scientist Training Program funded by the National Institute of General Medical Sciences: T32 GM07347 (KKK).
                Categories
                Research Article
                Biology and life sciences
                Genetics
                DNA
                DNA damage
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA damage
                Medicine and Health Sciences
                Clinical Genetics
                Genetic Diseases
                Autosomal Dominant Diseases
                Huntington Disease
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                Neurology
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