<p>Expansion in the glutamine encoding CAG triplet-repeat in the Huntingtin (HTT) gene causes Huntingtons disease (HD). The susceptibility, age of onset, and severity of HD is modified by ill-defined genetic and/or environmental factors. We have previously reported that expression of mutant HTT is associated with impaired Mn accumulation in a striatal cell line and mouse models of HD. Recognizing the regulatory relationship between HTT protein and metal transporters, I hypothesized that mutant HTT alters regulation of cellular Mn by aberrant interactions with the normal cellular Mn transport system.
<p>To identify the mechanism, I have developed and validated a high-throughput fluorescence-quenching based assay (cellular fura-2 manganese extraction assay - CFMEA) to measure Mn transport kinetics and storage properties in cultured striatal cells and rodent tissues. I tested the hypothesis that expression of mutant HTT impairs Mn accumulation by (i) modulating Mn uptake, (ii) intracellular trafficking dynamics and accumulation, and (iii) efflux transport mechanisms. I demonstrate that expression of mutant HTT alters instantaneous Mn uptake kinetics. I observed a significant decrease in net Mn storage capacity in mutant compared to wild-type striatal cells, despite the similar net Mn efflux rates between both genotypes.
<p>To dissect the metal transporter system(s) underlying altered mutant Mn homeostasis, I examined several known Mn transporters and failed to observe their role in mutant Mn uptake deficits. However, I demonstrate that the putative Mn transporter, Huntingtin interacting protein (HIP14), protein levels is significantly decreased in mutant compared to wild-type striatal cells. Furthermore, I observed a wild-type specific down regulation of HIP14 protein levels in response to excess Mn, which is consistent with a role of HIP14 in regulating Mn transport in the striatal cells. In addition, I demonstrate that Mn uptake in the striatal cells is substantially inhibited by the two highest affinity cations (Mg2+ and Sr2+) for the HIP14 transporter and its specific inhibitor (Ca2+). Finally, I show a functional relationship between the previously reported differential Mn induced cytotoxicity between wild-type and mutant HTT striatal cells and the cellular Mn pools that contribute to oxidative injury.
<p>In conclusion, I have developed a novel high throughput assay for assessing Mn transport dynamics and my findings suggest that the Mn accumulation deficit in mutant cells is due to impairment in Mn uptake and storage capabilities and not alteration in Mn efflux. In addition, mutant HTT impairment of HIP14 transporter function points to its responsibility for disruptions of Mn homeostasis and this may contribute to environmental modulation in HD.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-09122011-143041 |
| Date | 13 September 2011 |
| Creators | Kwakye, Gunnar Francis |
| Contributors | Kevin Currie, Judy Aschner, Aaron Bowman, Michael Aschner |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-09122011-143041/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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