DNA Repair Deficiency in Huntington's Disease Fibroblasts and Induced Pluripotent Stem Cells

<p>Mutant huntingtin protein (mhtt)– the protein responsible for cellular dysfunction in Huntington’s disease (HD) –is a product of an expanded trinucleotide repeat (TNR) cytosine-adenine-guanine (CAG) sequence in exon 1 of the <em>huntingtin</em> <em>(HTT)</em> gene. The pathology of HD has been extensively researched; however, the mechanism by which the disease-causing TNR expansions occur in somatic cells remains elusive. Interestingly, HD has often been referred to a ‘DNA repair disease’, even though DNA repair dysfunction <em>in situ</em> has not been identified. We hypothesized that presence of the mhtt protein affects the expression of DNA repair genes used to address DNA repair, ultimately affecting genome stability, thus providing a possible mechanism for TRN instability. Using quantitative polymerase chain reaction (qPCR) gene arrays for 84 DNA repair genes, we identified 18 DNA repair genes with decreased fold changes between 2- and 3- fold, as well as 11 genes down regulated greater than 3- fold in one HD fibroblast sample relative to a wild-type sample. To ensure our results were not limited to the samples tested, we then increased our number of HD samples and investigated gene expression of <em>APEX1, BRCA1, RPA1, </em>and<em> RPA3</em> using sensitive TaqMan Gene Expression assays. Further, immunocytochemistry (ICC) analysis validated expression deficiencies at the protein level. These data identify down-regulated genes necessary to maintain stability in the genome of multiple HD affected fibroblast lines. Our data infers that the presence of the toxic mutant huntingtin (mHtt) protein is involved in the DNA repair gene inhibition.</p> <p>The mutant huntingtin protein (mHtt) produced in HD exhibits a partial gain-of-function in that the hydrophobic, expanded polyglutamine region at the N-termini aggregates to unintended targets, such as transcription factors and histone modifiers. To identify the broad pathway regulating gene expression down-regulation, we investigated epigenetic regulatory mechanisms. Rapid revival of selected DNA repair expression was observed in response to pharmacological hypomethylation treatment, but not to histone modification treatments. This identifies differential methylation patterns occur as a result of mHtt presence. Using capillary electrophoresis fragment analysis to characterize <em>HTT</em> TNR gene expansions, our data reveals that intermittent 5-azacytidine treatments induced <em>HTT</em> gene stability over 4 population doublings, elucidating methylation patterning involvement in TNR instability.</p> <p>Furthermore, induced pluripotent stem cells (iPSCs) undergo global epigenetic changes relative to its native cell type that include methylation patterning changes. Upon reprogramming of HD-affected fibroblasts into a pluripotent state we revealed that gene expression was recovered to wild-type levels and was maintained through 20 population doublings. As well, iPSC-HD lines show contraction-biased instability, opposite to expansion-biased instability in native fibroblast cell types. Differentiation of iPSC-HD lines into mesenchymal-like cells (MLCs) further revealed that APEX1 expression remained static, while others retreated to pre-iPSC expression levels. Interestingly, HD-iPSC derived MLCs showed that TNR regions maintained stability in the <em>HTT</em> gene pathogenic region, showing no changes in (CAG) repeats. These findings demonstrate that DNA repair gene expression in HD fibroblasts is altered, thus providing insight into the mechanism in which TNR instability persists, ultimately leading to genetic anticipation. This also identifies possible biomarkers that can be used to monitor disease progression and therapeutic treatment success. This study also provides evidence that TNR instability is pharmacologically alterable. This is the first evidence that a DNA repair gene deficiency is present in cells affected by Huntington’s disease. More so, our data suggests that mechanisms involved in pluripotency has a protective effect on the pathogenic TNR region of the <em>HTT</em> gene.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biological Sciences

Year dc:date.available

2015

Author dc:creator

• Mollica, Peter Anthony

Contributors dc:contributor

• Christopher J. Osgood
• Roy C. Ogle
• Lesley H. Greene

• 5-azacytidine
• DNA repair
• Huntington's disease
• Induced pluripotent stem cells
• Neurodegeneration
• Trinucleotide repeat instability
• Cell and Developmental Biology
• Cell Biology
• Developmental Biology
• Molecular Biology

Statement dc:rights

• <p>In Copyright. URI: <a href="http://rightsstatements.org/vocab/InC/1.0/">http://rightsstatements.org/vocab/InC/1.0/</a> This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).</p>

Identifier

9781339386508

OAI identifier oai:identifier

oai:digitalcommons.odu.edu:biology_etds-1006