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Alzheimer's Statistics

Alzheimer’s disease (AD) is the 6th leading cause of death in the U.S.
5.8 million Americans are currently living with AD
By 2050, it is estimated that this number will rise above 14 million

https://www.alz.org/alzheimers-dementia/facts-figures

https://www.sciencedirect.com/science/article/pii/S1552526019300317

Figure 1: Percentage of Americans diagnosed with AD based on age group (2019)

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Proteins and Tangles

Two proteins: Beta-amyloid and tau
AD: the buildup of intraneuronal clumps of tau protein
Tau protein mutates causing the aggregation of clumps

Leads to destroyed neurons and neurodegenerative diseases

Figure 2: Diagram of Alzheimer's cell with buildup of beta-amyloid plaques, neurofibrillary tangles, and clumps formed by tau

https://www.pinterest.com/pin/80994493277529754/

Figure 2

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Drosophila Models

Drosophila are more easily examined and dissected, in comparison to traditional rodent models
High reproduction rates allow researchers to use greater sample sizes
Drosophila models are successful at displaying buildup of neurodegenerative proteins

Drosophila clearly demonstrate the negative results of tauopathy and beta-amyloid accumulation in an easily obtained invertebrate model.

(Passarella, 2018)

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Olfactory Testing

This model system provides understanding of molecular and cellular mechanisms involved in learning and memory processes
Linking odors with a reward system generates olfactory association; this pairing can be used to test and analyze Drosophila performance

The use of Drosophila in aversive olfactory training allows for large Drosophila populations during testing.

(Jiang, 2016)

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Lithium Testing

Lithium based medications are currently used to treat patients with bipolar depression
Unsure whether lithium will help those suffering from neurodegenerative disorders by combating tau aggregation and its negative effects

Lithium failed to break up beta-amyloid plaques, but was somewhat effective for dissembling tau protein in transgenic rodent models.

(Caccamo, 2007)

Lithium and memantine in rodent models effectively reduced neuroinflammation and destroyed beta-amyloid plaques; effects on tau not recorded.

(Budni, 2017)

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Gap and Hypothesis

Gap in Literature:
Lack of research on the effects of lithium on tau protein and its impact on memory and behavior
No previous research done with various concentrations of lithium in Drosophila
Unsure which concentration of lithium is most effective at restoring factors lost to neurodegeneration

Hypothesis:

The use of higher levels of lithium chloride in Drosophila models will demonstrate a positive effect by allowing for increased memory and function in transgenic tau flies.

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Tubes and T-Maze

Figure 3:

Displays the odor tubes used for reward training; purple tubes are for BA; blue tubes are used with MCH.

Figure 4:

Example of approximately 100 flies inside T-maze; flies are dumped through funnel at the top and are free to crawl to whichever side they prefer.

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Odor Bubblers

Figure 5:

Demonstrates the set-up of the odor tubes; BA in the bubbler on the left and MCH on the right. Air emitted by the bubbling odors travels down tube and to respective side of the T-maze.

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Odor Testing (for training purposes)

Odors created:

750mL plain mineral oil
Mixed with desired amount of BA or MCH

Flies placed in vials the day before and starved overnight;

3 vials for MCH, 3 for BA, and 2 for both odors

Tested odor preference with BA on one side of T-maze and MCH on the other;

Flies showed no preference

Same procedure done to test BA avoidance;

Continued testing amounts until flies avoided both odors

MCH avoidance tested by placing flies in T-maze for 3 min. trial;

Record number of flies on either side

Amounts tested:

700uL, 750uL, 800uL BA
300uL, 350uL, 400uL MCH

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Reward-Based Training

Concentrations of lithium chloride solution:

-1mM, 15mM, 50mM

Flies put into 16 vials; every 4 have different lithium

Exposed to both odors for 3 minutes in darkness;

Number of flies on either side of T-maze was recorded

Flies starved in their vials overnight;

Odor solutions made with concentrations found through odor testing

Flies associated one odor with a reward & one without;

Placed in T-maze with both odors flowing

Flies exposed to one odor in tube lined with filter paper soaked in sucrose (reward);

Exposed to odor for 2 minutes

Flies exposed to other odor in tube with filter paper soaked in water (no reward);

Exposed to odor for 2 minutes

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Analysis

For each vial tested, %’s of successful and unsuccessful flies calculated

Successful – Crawled toward odor associated with reward
Unsuccessful – Crawled toward odor associated with water

Descriptive analysis - mean % of successful and unsuccessful flies for each lithium concentration
ANOVA tests - compare the p-values of %’s of successful and unsuccessful flies for control and experimental groups

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Results – Control

Unless flies have abnormal aggregations of tau protein, they demonstrate no significant effects due to lithium treatment
Lithium is unable to perform memory and behavior rescuing effects on flies without symptoms of AD
P-value = 0.67 (not significant)

Figure 6: Represents the % of wildtype flies that crawled away from the aversive odor; similar results for flies that did and did not receive lithium

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Results – Experimental

Tau flies did not demonstrate significant success rates after receiving lithium treatment
Flies receiving treatment had a success rate only ~6% higher than flies receiving water
P-value = 0.23 (not significant)

Figure 7: Displays the % of tau flies that were successful in going towards the odor associated with the reward

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Results – Comparison

Both tau and wildtype flies portray insignificant effects after receiving lithium treatment
Results between experimental and control flies varied less than 0.3% for each lithium concentration
The % of flies that were attracted to the odor associated with the reward was almost identical across all groups

Figure 8: Graph compares the % of successful flies for wildtype and tau groups; similar results for flies receiving water & flies receiving lithium

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Discussion

Alteration of behaviors and memory occur due to the destructive effects of tau
In Drosophila models, tau has failed to show increased toxicity in cells leading to impairment

Lithium chloride did not have any affect on flies with tauopathy

Flies with and without neurodegenerative protein buildup are neither harmed nor helped by this quantity of lithium chloride solution

Could likely be due to human error during experimentation

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Conclusion and Future Research

Conclusion:

In this study, the use of Drosophila to model tau accumulation was not effective, as they were not appropriately inhibited by the aggregation of the protein
Lithium testing on tau aggregation could serve as a pathway to the development of lithium-centered treatments, but should not be tested in Drosophila models

Future Research:

Lithium testing on tau buildup can be conducted in rodent models to view impact of the treatment in mammals
Further investigation must be done to confirm lithium treatment as effective Alzheimer’s medication

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Acknowledgments

I thank my research mentor for her guidance and expertise throughout my experimentation process
Thank you also to my science research teacher and family for their continuous support

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References

Alzheimer's Association. (2018). Facts and Figures. Retrieved from https://www.alz.org/alzheimers-dementia/factsfigures?utm_source=google&utm_medium=paidsearch&utm_campaign=google_grants&utm_content=alzheimers&gclid=EAIaIQobChMIluXPo5-j5AIV4f_jBx2H_AX8EAAYASAAEgLMB_D_BwE
Alzheimer’s Association. (2019). 2019 Alzheimers disease facts and figures. Alzheimers & Dementia, 15(3), 321–387. doi: 10.1016/j.jalz.2019.01.010
Budni, J., Feijó, D., Batista-Silva, H., Garcez, M., Mina, F., Belletini-Santos, T., & Quevedo, J. (2017). Lithium and memantine improve spatial memory impairment and neuroinflammation induced by β-amyloid 1-42 oligomers in rats. Neurobiology of Learning and Memory, 141, 84–92. doi: 10.1016/j.nlm.2017.03.017
Caccamo, A., Oddo, S., Tran, L. X., & Laferla, F. M. (2007). Lithium Reduces Tau Phosphorylation but Not Aβ or Working Memory Deficits in a Transgenic Model with Both Plaques and Tangles. The American Journal of Pathology, 170(5), 1669–1675. doi: 10.2353/ajpath.2007.061178
Passarella, D., & Goedert, M. (2018). Beta-sheet assembly of Tau and neurodegeneration in Drosophila melanogaster. Neurobiology of Aging, 72, 98–105. doi: 10.1016/j.neurobiolaging.2018.07.022