“Melatonin’s Effect on Learning and Memory in a Tauopathy Model of Alzheimer’s Disease in Drosophila melanogaster”
Sada Nichols-Worley, Precollegiate Research Student
Sophie Haugen, Precollegiate Research Student
STEM Fellowship, St. Mark’s School
Lindsey Lohwater, STEM Fellowship Teacher
Alzheimer’s Disease (AD) is a progressive neurodegenerative disease that causes devastating memory loss and cognitive decline in humans. There is no current cure for AD. Research studies show that oxidative stress is correlated to and possibly a cause of this neurodegeneration. Because antioxidants such as melatonin have been found to reduce oxidative stress, melatonin could alleviate neurodegeneration and serve as an effective dietary supplement for people with with AD. In this experiment, a tauopathy Drosophila melanogaster group that express human tau (MAPT) under gal4in neurons were used to model AD in humans. This study measured learning and memory of the Drosophila through an olfactory vortex learning assay in a t-maze. Groups of flies with and without melatonin supplementation were tested in the t-maze. Following experimentation and data collection, preliminary results from this study suggest, but do not confirm, that melatonin reduces memory loss and improves cognitive function in a Drosophila AD model. Further trials are needed to confirm the suggested results.
Alzheimer’s Disease (AD) is is a devastating disease that afflicts more than five million people in the United States and upwards of fifty million around the world. AD is the most common form of dementia, which is any type of serious degenerative memory loss and cognitive decline; over 70% of dementia cases are AD. AD primarily affects people older than sixty-five but the early-onset effects of the disease can appear ten to twenty years earlier. AD is a progressive disease, meaning it gradually worsens over time. The beginning stages of AD are characterized by mild memory loss and concentration problems. However, by the late stages of AD, individuals are afflicted with severe memory loss and impaired physical abilities, such as eating or walking. The affected population is increasing both in the United States and worldwide. AD is an age-related disease but may also be connected to environmental and lifestyle factors including diet, exercise, or lack thereof, and sleep. Each case and level of risk is dependent on the individual, but genetics also play a role.
The brain is made up of neurons, also known as nerve cells. In individuals with AD, these neurons are damaged or destroyed, causing the memory loss, cognitive decline, and other symptoms in afflicted individuals. Currently, the suspected causes of neuron damage are beta-amyloid plaques and neurofibrillary tangles made of hyperphosphorylated tau protein. Beta-amyloid is a protein that is typically removed from the brain by being transported across the blood-brain barrier . In AD, beta-amyloid clearance fails, so it accumulates as plaques. Neurofibrillary tangles are twisted microtubules. When tangled, microtubules prevent nutrients from being transported within neurons. Brain cells die. The brain also stops producing acetylcholine, which is a neurotransmitter responsible for memory. Microtubules are destabilized by hyperphosphorylation and the pathology induces loss of neuronal function and tau toxicity. Both cause neurological decline. There are greater than thirty phosphorylation sites in tau protein, but recent studies have concluded that thirteen play a role in phosphorylation in Alzheimer’s Disease .
The specific AD degeneration changes occur in the hippocampal formation and temporal lobes . “White matter” is also a characteristic of AD patients and can be seen from MRI examinations . Beta-amyloid accumulation and hyperphosphorylated tau protein are usually evident from a microscopic view. Unfortunately, these neurological changes and damage likely begin to happen years before symptoms of dementia and AD emerge. There have been studies to show that people with no cognitive degeneration symptoms also have these neurological changes and characteristics .
Figure 1. Structural and characteristic differences between healthy and AD neurons.
Oxidative stress is theorized to cause aging and is thought to be involved in the pathogenesis of AD . Oxidative stress results from an imbalance between reactive oxygen species (ROS) and the body’s antioxidant. ROS encompass free radicals and free radical mimics. Free radicals are molecules with an unpaired electron that desperately want to pair with another electron, thus making these molecules extremely reactive . Free radicals and free radical mimics can acquire another electron from virtually any biomolecule, and by doing so they damage these molecules . These ROS have been found to increase with aging in the brain, but also have been correlated with shorter lifespan and increased progression of AD . Oxidative stress was previously thought to be a consequence of the neurofibrillary tangles and beta-amyloid plaques in AD patients, but recently has been implicated as a cause of neurodegeneration rather than a side effect or correlation . The body has endogenous antioxidant defense mechanisms as well as antioxidants from the diet . However, as the ROS increase with age, the ROS begin to outnumber the antioxidants, thus raising oxidative stress. According to recent studies, oxidative stress has been determined to be one of the earliest markers of AD onset. Other conditions or diseases may cause higher oxidative stress including brain injury, diabetes mellitus, stroke, and other environmental or lifestyle factors such as smoking or lack of exercise . Formation of beta-amyloid plaques and fibrillary tangles may occur at a faster rate with oxidative stress, but it is not guaranteed that oxidative stress also directly causes the hyperphosphorylation of tau .
Drosophila as an AD Model
Drosophila melanogaster (common fruit flies) are frequently used as a model for neurodegenerative diseases, and for AD in particular. Drosophila are easy to work with and can be crossbred to create different phenotype strains to model different diseases. Because they are invertebrates, less restrictions exist for how we can work with them. Furthermore, Drosophila are genetically similar to humans, sharing about 70% of disease-causing genes, and have a similar nervous system . Drosophila is an effective model for AD specifically because it has over 13,000 genes and similar pathologies and systems. Fruit flies have brains with 200,000 neurons and the equivalent of a vertebrate central nervous system; the fly brain and neurons function similarly to those of humans and other vertebrates .
Figure 2 (left). Human and Drosophila Nervous System
Figure 3 (right). Drosophila Neurons
In terms of AD, the best Drosophila models are Aβ-expressing or hyperphosphorylated tau/tauopathy. Both Aβ and tau are important for replicating neurodegenerative diseases and dementia in Drosophila: Aβ toxicity and Aβ-expressing flies model the accumulation of the plaques while tauopathy models are the neurofibrillary protein tangles of abnormally phosphorylated tau.
We are planning to use a tauopathy model by expressing wild-type and mutant human tau in the nervous system of Drosophila. Expressing forms of human tau causes replicative neurodegeneration similar to that of human subjects. Tau R406W- expressing transgenic flies undergo neurodegeneration by apoptotic cell death . The R406 mutation technically creates frontotemporal dementia but the effects and characteristics of those with the mutation resemble AD. It is an effective model for clinical AD because it causes late-onset symptoms and characteristics of the disease with rapidly progressing dementia . The most distinct characteristics of the Tau R406 mutation are neuronal loss, “abundant tau-positive inclusions”, and neurofibrillary tangles . The mutation generally does not cause the same Aβ accumulation as in human AD.
Role of Melatonin as an Antioxidant
As previously discussed, ROS is commonly believed to play a role in development of Alzheimer’s Disease. In recent years, studies have shown that oxidative stress may play more of a causal role than just being a correlation or effect of AD. Therefore, antioxidant therapy for treating AD is widely tested and practiced in an attempt to reduce ROS pathology and lower oxidative stress. Neurofibrillary tangles and tau phosphorylation are both suspected to be results of oxidative stress. Endogenous defense mechanisms inherently protect humans from ROS and free radical damage . There are several natural and active antioxidants in the body including vitamin C, E, sodium selenite, and melatonin . Dietary supplementary antioxidants are also very common and beneficial to balancing ROS and antioxidant mechanisms. While each antioxidant, endogenous or dietary, has specific benefits, some are more effective than others in reducing oxidative stress.
Melatonin is hormone and antioxidant naturally produced in the brain, but its production decreases with age . The most commonly-known benefits of melatonin include improving sleep, regulating circadian rhythms, and helping immunity, but it also decreases the rate of cognitive decline, especially in AD patients . Melatonin prevents Aβ accumulation and reduces tau hyperphosphorylation by protecting neuronal cells . Melatonin decreases with age and recent studies have shown that lower levels of melatonin in cerebrospinal fluid (CSF) are particularly evident in suspected AD patients . Thus, measuring levels of melatonin may help to determine the onset of early AD symptoms. Because of this, and because of melatonin’s ability to prevent and decrease the rate of cognitive decline, it is an effective antioxidant to use to test its effects on neurodegeneration.
Figure 4. Flow chart of melatonin and deterioration of immune system and cell function.
Melatonin decreases tau hyperphosphorylation by inhibiting various protein kinase activations and decreasing oxidative stress that results from these kinase activations . It has also been shown that decreasing melatonin levels in rats increases memory and cognitive impairment as well as tau phosphorylation . Melatonin is a naturally powerful and effective antioxidant but does have direct effects on improving conditions of AD, neurodegeneration, and overall oxidative stress.
Alzheimer’s Disease and Learning
Olfactory shock learning assays can be used to measure the progression of neurodegeneration. These assays test the memory and learning ability in Drosophila. Flies are exposed to a certain odor and presented with an electric shock; they are then exposed to a different odor associated with no shock. Afterwards, flies navigate through a T-maze where they can choose between the shock-paired odor and the non shock-paired odor . Flies are then tested immediately after being exposed to the odors and again thirty minutes to one hour later to test their memory.
Figure 5. Example of a t-maze learning assay for Drosophila .
In this AD tauopathy model, learning assays more accurately reflect neurodegeneration than measuring the amount of tau present. Tau aggregation does not necessarily cause neurodegeneration, and conversely, neurodegeneration can occur in the absence of tau aggregation. Because AD is progressive disease, markers of aging should also be measured.
The goal of this investigation is to determine whether melatonin supplementation can improve the learning ability and memory typically impaired by Alzheimer’s Disease using Drosophila models. Short- and long-term memory loss and reduced learning ability are caused by neurodegeneration that results from Alzheimer’s Disease.
The objectives of this investigation are:
If a tauopathy model of Alzheimer’s Disease in Drosophila melanogaster is raised on a melatonin-supplemented diet, then they will learn, perform, and maintain long-term memory more consistently as measured by a olfactory learning assay than Drosophila raised on a normal diet.
If a tauopathy model of Alzheimer’s Disease in Drosophila melanogaster eat a melatonin-supplemented diet, their learning and memory, as measured by an olfactory learning assay, will be no different than that of flies raised on a normal diet.
The following criteria need to be met to be included in this
The following criteria met will be grounds for exclusion in this
½ inch acrylic pieces: Left Arm, Right Arm, T-Maze Base
¼ inch acrylic pieces: T-Maze Elevator and T-Maze Wall
Creating Scent Flasks
Creating and Assembling Tubing
Creating Training Tubes
June 2017 — March 2018
June — August 2017: Conducted individual preliminary independent literature research under the direction of STEM Fellowship Teacher, Lindsey Lohwater. This research helped us to narrow in on a topic, but at that time we had not yet finalized our research plan and were researching more generally aging, Alzheimer’s Disease, and different potential models.
September — October 2017: Joined together as a partner project. Decided upon researching Alzheimer’s Disease in Drosophila. Completed backgrounded literature research and determined that we would use melatonin as a dietary supplement. Emailed and visited Dr. Farah Bardai of the Feany Lab to affirm our project plan and determine exactly which genotype of Drosophila to use to model Alzheimer’s Disease.
November 2017: Completed necessary forms to submit project to the SRC for approval. Continued developing protocol for project and began ordering necessary supplies. Received approval for project.
December 2017: Began making media for both experimental groups and ordered flies from Bloomington stock. Received flies and maintained them while finalizing protocol and assembling t-maze.
January-February 2018: Finished assembling t-maze. Carried out the research investigation by training flies and then testing them in the t-maze.
March 2018: Analyzed data and made conclusions on the investigation. Continuing to maintain Drosophila for future use and expansion of study. Poster designed, printed, and assembled, and final protocol completed. Entry into WRSF.
The following protocol is met by the investigators:
The primary independent variable in this experiment is diet. More specifically, the variable is the addition of the antioxidant melatonin to the typical cornmeal-based diet of Drosophila. Three different strains of flies are tested. One of which, the tauopathy strain, is the experimental strain and second variable (whether or not the flies have a tauopathy). Each genotype of flies is separated into groups that either receive no melatonin supplementation or receive melatonin supplementation at a dosage of 100 ug/ml (.43 mM) for a total of six groups of flies.
The controls for the experiment are flies receiving no melatonin and flies without a tauopathy. In addition to wild-type flies, a group of elav-gal4 flies is also used as a control to determine that the UAS-gal4 system and expression of gal4 in a wild-type fly does not affect learning and memory ability. Other elements of this study that are kept constant and controlled are the environment, training conditions, and training and testing protocol. All organisms tested were Drosophila melanogaster. All Drosophila are kept in an incubator set to 24º Celsius.
Table 8.1: Scent Choices and Performance Indices for Flies (All Groups)
Figure 8.1: Comparison Between Performance Indices for All Groups
Figure 8.2: Effect of Melatonin on Learning and Performance Index
The data collected for this investigation will be analyzed by:
Table 8.2: P-value per fly strain group
A half PI score is determined for each trial (one strain on one diet) with one of the scents as CS+. Another PI score is determined for the same strain and diet but with the other scent as CS+. The average of these half PI scores is the full PI score for the complete trial. An adjusted PI is determined by multiplying the half PI for one trial by the fraction representing how many flies were in that trial compared to the total. Then, the same is done for the other half of the trial (with opposite CS+ scent) and the two results are added together to yield the adjusted PI score.
A p-value was determined per two full trials of each type of fly. (3 groups). All wild-type flies = one p-value. All elav-gal4 flies = one p-value. All tauopathy flies = one p-value.
Statistics for all groups:
= Melatonin adjusted PI for each group || p = Mean ||
Positive z-value → (1 - (% of z))
Negative z-value → (% of z)
Statistics for wild-type group:
.4149 > .05 therefore: we fail to reject the null hypothesis. The test used an alpha value of .05 for significance. The p-value is .4149, which is greater than the a-value. We do not have enough significance to reject the null hypothesis.
Statistics for elav-gal4 group:
.18658 > .05 therefore: we fail to reject the null hypothesis. The test used an alpha value of .05 for significance. The p-value is .18658, which is greater than the a-value. We do not have enough significance to reject the null hypothesis.
Statistics for tauopathy group:
.00002 < .05 therefore: we reject the null hypothesis. The test used an alpha value of .05 for significance. The p-value is approximately zero, which is less than the a-value. We reject the null hypothesis. We have made simplifying assumptions and recognize the variability in sample size of individual trials.
Results from the learning assays were condensed into a Performance Index (PI) that encompasses how many flies chose correctly and incorrectly in the t-maze. As can be seen in Figure 2, melatonin supplementation improved the performance of all types of flies, ranging from an improvement of 0.018 for wild-type flies to 0.877 for tauopathy flies. These results suggest that there is a correlation between melatonin supplementation and reduced memory and learning loss. The results for the tauopathy flies are especially important because they suggest that melatonin supplementation can reduce memory and learning loss, likely through reducing oxidative stress and neurodegeneration, in an AD model.
Wild-type flies receiving melatonin supplementation showed a small, if not insignificant, difference in learning assay performance to those not receiving melatonin supplementation. This would suggest that melatonin supplementation does not have any effect on learning and memory in the standard, healthy brain. The elav-gal4 and tauopathy flies receiving melatonin supplementation, however, showed a much more significant improvement as compared to those not receiving supplements.
As expected, tauopathy flies both with and without melatonin supplementation performed worse on the learning assay than did elav-gal4 or wild-type flies (Figure 3). This worsened performance was expected due to the neurodegeneration that occurs in a tauopathy. Even though melatonin supplementation did improve learning and memory of tauopathy flies, we did not expect that melatonin supplementation would reverse or fully stop the progression of neurodegeneration. Elav-gal4 flies on a normal diet performed worse than wild-type flies on a normal diet, but elav-gal4 flies on a melatonin-supplemented diet performed better than wild-type flies on a melatonin-supplemented diet. We, therefore, cannot conclude whether or not elav-gal4 changes melatonin’s effect on learning and memory. Furthermore, results shown in Figures 3 and 4 confirm that the scent chosen to be the CS+/CS- has no effect on how well flies perform in the learning assay.
Through statistical analysis of the different fly groups, the standard deviation value for the control wild-type fly group was .0884. The standard deviation value for the control elav-gal4 group was .2425, and the value for the tauopathy group was .0661. Through completing a Z-test, a p-value was determined for all three fly groups according to the standard deviation to either reject or fail to reject the null hypothesis. The p-values for the wild-type and elav-gal4 were both greater than the set alpha-value of .05, indicating that the data lacks support against the null hypothesis. The p-value for the tauopathy flies was less than .05 as it was ~.0002, approximately zero, indicating that this data rejects the null hypothesis and suggests to support the hypothesis.
As we conducted only half of one trial for tauopathy on normal diet and only one trial for melatonin-supplemented tauopathy flies, we acknowledge that our population is too small to make significant conclusions. The adjusted PI values and the p-value for tauopathy flies would be more varied with one more trial.
To continue this project, first we would perform more trials of this same experiment to determine whether our results would still be significant for tauopathy flies. Following these trials, if they showed the same promising results as our first set of data, we would expand our project in three main ways. Firstly, we would test the flies not just right after training but also an hour after training to test their long-term memory in addition to learning ability and short-term memory. Furthermore, we would test flies not just once in their lifetimes but multiple times over the span of their life (days 1, 5, 10, 15, and 20). Doing so would allow us to see how the flies’ learning and memory capabilities decay as they age, and how the effect of melatonin supplements may differ with age. Finally, we would further divide the flies receiving melatonin supplementation into groups receiving melatonin supplementation from the beginning of their lives and groups not receiving melatonin until the adult stages of their lives. We could then determine whether the age (and level of neurodegeneration) at which melatonin supplementation is introduced to one’s diet changes melatonin’s effect. We could also advance the t-maze learning assay to include two t-mazes and make further modifications to the training cycles.
Who will record data: Sophie Haugen and Sada Nichols-Worley
Who will be supervising data collection: Lindsey Lohwater and Ken Wells
Where will data be recorded: Google and Excel Spreadsheets and laboratory notebooks
When will data be recorded: Between February 13, 2018 and March 1, 2018
Where will data be collected: St. Mark’s School Biotechnology Lab Room 285, “Fly Room”
At this time, the investigators will not be publishing their results of this study.
2 (24 oz)
Bob’s Red Mill
1 (16 oz)
Light Corn Syrup
1 (16 fl oz)
Bloomington Drosophila Stock Center
Bloomington Drosophila Stock Center
Bloomington Drosophila Stock Center
1 (250 mL)
1 (250 mL)
1 (500 mL)
 Dias-Santagata, D., Fulga, T. A., Duttaroy, A., & Feany, M. B. (2007).
Oxidative stress mediates tau-induced neurodegeneration in Drosophila.
Journal of Clinical Investigation. https://doi.org/10.1172/JCI28769
 Aliev, G., Obrenovich, M. E., Reddy, V. P., Shenk, J. C., Moreira, P. I.,
Nunomura, A., … Perry, G. (2008). Antioxidant Therapy in Alzheimer’s
Disease: Theory and Practice. Mini Reviews in Medicinal Chemistry, 8(13),
 Liu, Z., Li, T., Li, P., Wei, N., Zhao, Z., Liang, H., … Wei, J. (2015). The
Ambiguous Relationship of Oxidative Stress, Tau Hyperphosphorylation,
and Autophagy Dysfunction in Alzheimer’s Disease. Oxidative Medicine
and Cellular Longevity, 2015, 352723. http://doi.org/10.1155/2015/352723
 Malik, B. R., Hodge, J. J. Drosophila Adult Olfactory Shock Learning. J. Vis.
Exp. (90), e50107, https://doi.org/10.3791/50107 (2014).
 Moloney, A., Sattelle, D. B., Lomas, D. A., & Crowther, D. C. (2010).
Alzheimer’s disease: insights from Drosophila melanogaster models.
Trends in Biochemical Sciences, 35(4), 228–235.
 Gistelinck, M., & Lambert, J.-C. (2012). Drosophila Models of Tauopathies:
What Have We Learned? International Journal of Alzheimer's Disease.
 Lin, L., Huang, Q.-X., Yang, S.-S., Chu, J., Wang, J.-Z., & Tian, Q. (2013).
Melatonin in Alzheimer’s Disease. International Journal of Molecular
Sciences, 14(7), 14575–14593. http://doi.org/10.3390/ijms140714575
 AH, B., & N, P. (1993). Targeted gene expression as a means of altering cell
fates and generating dominant phenotypes. NCBI. Abstract retrieved from
 Tarasoff-Conway, J. M., Carare, R. O., Osorio, R. S., Glodzik, L., Butler, T.,
Fieremans, E., … de Leon, M. J. (2015). Clearance systems in the
brain—implications for Alzheimer disease. Nature Reviews. Neurology,
11(8), 457–470. http://doi.org/10.1038/nrneurol.2015.119
 Mutations MAPT R406W. (n.d.). Retrieved November 2, 2017, from Alzforum
Networking for a Cure website:
Background & Rationale Figures:
[Figure 1] Komaroff, A., Dr. (2015, June 25). Plaques and tangles [Image].
[Figure 2] [Organisation of the nervous system into brain in the head,
ventral/spinal cord in the trunk and segmental nerves]. (2015, June). Retrieved from https://droso4schools.wordpress.com/organs/
[Figure 3] N, S.-S. (2007, January 28). [File:Neurons used for studies on
neuronal growth at different stages of Drosophila development]. Retrieved from https://commons.wikimedia.org/wiki/File:Neurons_used_for_studies_on_neuronal_growth_at_different_stages_of_Drosophila_development.jpg
[Figure 4] Espino, J. (2012, December). Schematic diagram depicting beneficial
effects of melatonin on age-associated deterioration of immune system. [Image]. Retrieved from https://www.researchgate.net/figure/235368827_fig1_Schematic-diagram-depicting-beneficial-effects-of-melatonin-on-age-associated
[Figure 5] Malik, B. R., Hodge, J. J. Drosophila Adult Olfactory Shock Learning. J.
Vis. Exp. (90), e50107, https://doi.org/10.3791/50107 (2014).