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| author | Karina Patricia Guaman |
| title |
Evaluating the Effects of Methylene Blue on the Growth of Axons and the Health of Primary Neuronal Cell Cultures; Implications in Alzheimer Disease Research
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| abstract | Alzheimer disease (AD) is a neurodegenerative illness that causes a gradual decline in memory and significantly affects cognitive
functioning. One compound that may aid AD patients is methylene blue (MB). MB is an inexpensive dye that has been known to cross the blood-brain barrier (Stack et al.
2014; Jiang et al. 2015) and has been safely used in humans (Naylor et al. 1986). Previously, a derivative of MB, Rember, was utilized in a phase II clinical drug
trial, where individuals who were administered MB demonstrated less of a cognitive decline in comparison to the placebo group. While the exact mechanism of action
of MB in relation to AD remains unknown, it is possible that MB may be working as an anti-tau aggregation drug (Hosokawa et al. 2012) or an energy-enhancing compound
(Shen et al. 2013). With this in mind, the overarching goal of this study was to provide insight into MB's effects in an in vitro primary neuronal cell culture
using embryonic rat neurons, while placing particular emphasis on the growth of axons. We first assessed whether nanomolar (nM) concentrations of MB produced neurotoxic
effects in primary neurons. Results indicated that doses up to 200 nM MB had no neurotoxic effects. Next, we observed tau, a protein that drives the formation of axons
(Wang and Liu 2008), in neurons treated with the same previous doses of MB, and we detected various growth rates of neurons in vitro. As part of this aim, we
raised the question if MB interacts with the axonal protein tau, would those interactions alter the development of axons? Based on our findings, there was no inhibition
of axonal growth, and 100 nM MB led to a significant increase in axonal lengths. With this enhancement property of MB eluded, axonal lengths were distributed into
frequencies, and we observed an increase in long axons and a decrease in short axons with 100 nM MB. This effect was more pronounced with a 24-hour MB stimulation as
opposed to a 48-hour MB stimulation. We also categorized axonal lengths into long axons (>75 microns), and we determined that there was a significant difference
between axonal lengths and doses with both 24-hour and 48-hour exposure periods. An increasing impact of dose was seen with doses up to 100 nM MB. Because axon
outgrowth is an energy intensive function, we examined ATP levels as a possible explanation for the enhancement property of 100 nM MB on axon lengths. We detected
a significant increase in ATP levels with a 48-hour exposure to MB but not with a 24-hour exposure. In a pilot study, we analyzed potential neuroprotective effects
of MB against glutamate excitotoxicity. Little to no attenuation was seen with MB. While we did not directly test whether MB became bound to phosphorylated tau,
because axon outgrowth was not inhibited, tau's function in elongating the axon did not appear to be disrupted. This study has implications for future AD research
by highlighting MB’s ability to serve as a cognitive enhancing drug.
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| school | The College of Liberal Arts, Drew University |
| degree | B.A. (2017) |
| advisor | Dr. Roger Knowles |
| committee | Dr. Joanna Miller
Dr. Minjoon Kouh |
| full text | KPGuaman.pdf |
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