Restoration of β-Hexosaminidase A deficiency through the use of molecular chaperones

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First Advisor

Gregory J. Long

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Scholarship Domain(s)

Scholarship of Discovery


Tay-Sachs disease (TSD, also known as GM2-gangliosidosis) is an autosomal-recessive neurodegenerative lysosomal storage disease caused by a mutation in the HEX A gene which codes for the lysosomal enzyme β-hexosaminidase A (Hex A)7,8. For patients with TSD, GM2-gangliosides cannot be properly broken down, and, as a result, accumulate in their neurons, causing severe neurological complications5.

Currently, there is no cure for TSD. Although all past treatment options have been ineffective, the novel pharmacological chaperone therapy has shown promise as a means for effective treatment by improving the folding and transportation of Hex A. Here, pharmacological chaperone therapy for TSD was investigated using both chemical chaperones and pharmaceutical chaperones. This study set out to reduce the amount of GM2 gangliosides in cells by increasing Hex A activity. To stop the accumulation of GM2 gangliosides, only about 10 percent of enzyme activity is actually needed2.

This study set out to ameliorate Hex A activity in TSD cells and decrease the GM2 ganglioside buildup in a TSD derived cell line, GM00502, using four factors. Each of these factors; DMSO, glutamic acid, Pyrimethamine, and a decrease in temperature, have been shown to improve Hex A activity in in vitro cell culture models of TSD3,4. Pyrimethamine is currently the leading pharmacological chaperone for treating TSD and is currently in its Phase I and II clinical trials1.

It is important to note that the fate of mutated Hex A enzymes depends on their specific mutation. Of the 74 mutations for Hex A, three mutations (1278ins4, 1421+1G→C, and Gly269Ser) account for 98 percent of all TSD cases8. The human kidney fibroblast cell line, GM00502, used in this study contained two of these mutations (1278ins4 and 1421+1G→C)6. The effects of the molecular chaperones outlined in this project have yet to be tested on a cell line with both of these major mutations. I hypothesized that temperature reduction, DMSO, glutamic acid, and pyrimethamine would rescue Hex A activity in the TSD derived human kidney fibroblast cell line GM00502.

A study in cytotoxicity was conducted to find the optimum concentration of DMSO, glutamic acid, and Pyrimethamine to treat both GM00502 and HEK293 (control) human kidney fibroblasts. After confirming proposed treatment concentrations were not cytotoxic, a gradient of three concentrations of each factor was then used in the subsequent testing phase. Differences in GM2 ganglioside levels can be quantified through the use of LysoTracker DND-26 staining and fluorescence microscopy. GM00502 TSD derived cells were shown to contain an increased accumulation of GM2 gangliosides when compared to the unafflicted HEK293 control cells. After treating GM00502 cells with DMSO, glutamic acid, Pyrimethamine, and a temperature reduction it was found that all treatments were able to reduce the overall GM2 ganglioside level. Likewise, each treatment was also able to further reduce the GM2 ganglioside levels in HEK293 cells. Of the four factors tested, 250 mM glutamic acid was shown to be the most effective in reducing lysosomal accumulation in HEK293 cells whereas 1.5 ug/mL pyrimethamine was most effective in GM00502 cells.

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Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

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