Biology

Melatonin’s Effect on Learning and Memory in a Tauopathy Model of Alzheimer’s Disease in Drosophila melanogaster

Investigators:

Sada Nichols-Worley, Precollegiate Research Student

Sophie Haugen, Precollegiate Research Student

STEM Fellowship, St. Mark’s School

Adult Sponsor:        

Lindsey Lohwater, STEM Fellowship Teacher

Investigation Schema

  1. Introduction
  1. Abstract
  2. Background and Rationale
  1. Goal and Objectives
  1. Goal
  2. Objectives
  1. Hypothesis Statements
  1. Hypothesis
  2. Null Hypothesis
  1. Selection Criteria
  1. Inclusion criteria
  2. Exclusion criteria
  1. Materials
  1. Test Materials
  1. Methods
  1. Procedural Steps
  2. Calendar of Events
  3. Safety Considerations
  1. Experimental variables and controls
  1. Variables
  2. Controls
  1. Data Collection and Analysis
  1. Data collection
  2. Data analysis
  1. Discussion
  2. Further Directions of Research
  3. Quality Control and Assurance
  4. Data Handling and Record Keeping
  5. Publication of Results
  6. Budget
  7. References

  1. Introduction
  1. Abstract

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.

  1. Background and Rationale

Overview

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 [9]. 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 [3].

        

The specific AD degeneration changes occur in the hippocampal formation and temporal lobes [9]. “White matter” is also a characteristic of AD patients and can be seen from MRI examinations [9]. 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 [9].

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 [2]. 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 [2]. Free radicals and free radical mimics can acquire another electron from virtually any biomolecule, and by doing so they damage these molecules [2]. 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 [2]. 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 [2]. The body has endogenous antioxidant defense mechanisms as well as antioxidants from the diet [2]. 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 [3]. 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 [3].

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 [5]. 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 [5].

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.

Tauopathy Model

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 [1]. 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 [10]. The most distinct characteristics of the Tau R406 mutation are neuronal loss, “abundant tau-positive inclusions”, and neurofibrillary tangles [10]. 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 [2]. There are several natural and active antioxidants in the body including vitamin C, E, sodium selenite, and melatonin [2]. 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 [7]. 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 [7]. Melatonin prevents Aβ accumulation and reduces tau hyperphosphorylation by protecting neuronal cells [7]. 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 [7]. 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 [7]. It has also been shown that decreasing melatonin levels in rats increases memory and cognitive impairment as well as tau phosphorylation [7]. 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 [4]. Flies are then tested immediately after being exposed to the odors and again thirty minutes to one hour later to test their memory.

jove-90-50107-0.jpg

Figure 5. Example of a t-maze learning assay for Drosophila [4].

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.

  1. Goal and Objectives
  1. Goal

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.

  1. Objectives

The objectives of this investigation are:

  1. Hypothesis Statements
  1. [H1] Hypothesis Statement

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.

  1. [H2] Null Hypothesis Statement

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.

  1. Selection criteria
  1. Inclusion Criteria

 The following criteria need to be met to be included in this

 investigation:

  1. Exclusion Criteria

 The following criteria met will be grounds for exclusion in this

  investigation:

  1. Materials
  1. Test Materials

  1. Methods
  1. Procedural Steps

Making Media

  1. Collect the materials necessary for making media. This includes distilled water, yeast, soy flour, yellow cornmeal, agar, light corn syrup, propionic acid, tegosept, Melatonin, a hot plate, a large beaker, a scale, and Erlenmeyer flasks and stoppers.
  2. Using the scale, measure contents of ingredients. Measure 1 liter of distilled water, 17.3g yeast, 10g soy flour, 73.1g yellow cornmeal, 5.8g agar, 77 ml light corn syrup, 4.8 ml propionic acid, 1g tegosept. Tegosept and propionic acid are anti-molding agents that prevent the solution from molding.
  3. If making control food, then no melatonin should be added. If making experimental food, add .025g of melatonin to the beaker. This can be added at any time.
  4. Measure half of the distilled water into the beaker. Place this on a hot plate and bring to a boil. Once it boils, add half of the yellow cornmeal. Stir this constantly to avoid clumping. While stirring, slowly add rest of yellow cornmeal. When the mixture is smooth and combined, add the remainder of the distilled water, still stirring. Slowly add the soy flour, agar, and yeast to the water and cornmeal mixture, still stirring. When well combined, add the 77 ml of light corn syrup to the beaker.
  5. Once the mixture has reached a boil, add the 1 g of Tegosept to the beaker.
  6. When it stops boiling, keep the mixture slowly mixing and still warm so that it does not harden. When it appears to be fully combined and homogenous, remove from heat and pipet the 4.8 ml propionic acid to the beaker and stir. Allow to cool to 70ºC.
  7. Pour media into vials, about 2 inches high in each.
  8. Allow media to cool overnight at room temperature in the fly room.

Making Scents

  1. Mix the scents in a 1:1000 ratio with mineral oil. Drosophila are repelled by strong scents, so the scents should be diluted to this ratio. Label one beaker “MCH” and one beaker “OCT”. Pipette 200 ml of mineral oil into each beaker. Pipette 200uL of each scent into in each respective beaker.
  2. Parafilm each beaker to seal and prevent any exposure or further dilution. These scents will be used for olfactory learning assays later in experimentation.

Maintaining Flies

  1. Fly vials should be flipped every 1 to 2 generations or earlier if a vial becomes overcrowded (40+ flies) or the media in a vial begins to dry out. This will ensure that the flies maintain a general state of well-being. In this study, we will flip adult flies into new vials as soon as they repopulate in order to distinguish between generations to organize by diet.
  2. To flip flies from one vial to another, first tap the flies in the original vial down 4 to 5 times on a table to ensure that the flies stay on the bottom of the vial and do not escape. Then, quickly flip the original vial of flies over the new vial of flies. Tap both vials down until all flies in the original vial are in the new vial. Make sure not to tap too hardly or the media from the original vial may fall, crushing the flies and any eggs or larvae. Finally, plug the two vials with stoppers.
  3. The flies should be maintained at a constant temperature in the incubator. In this investigation, the flies will be kept at 24ºC.

Assembling T-maze

  1. Gather necessary pieces of t-maze for construction. Crate files in software such as Autodesk Inventor or access pre-cut acrylic pieces to assemble.
  2. The base and walls include:

                ½ inch acrylic pieces: Left Arm, Right Arm, T-Maze Base

¼ inch acrylic pieces: T-Maze Elevator and T-Maze Wall

  1. Attach Left Arm of T-Maze to the Base about ¼ inch away from the center. Align Right Arm with Left Arm so that the two pieces are touching, and attach with a clamp or removable hold.
  2. Insert elevator between Left and Right Arms so that large holes are aligned.

Creating Scent Flasks

  1. Obtain four 125 ml glass Erlenmeyer flasks that will hold the odors for training and testing.
  2. Put a two-hole size five rubber stopper in the flask. Put a bent 1 mL plastic pipette in one hole of the rubber stopped. Bend with a heat gun. Cut a straight pipette, the same length as the bent pipette, and put this pipette in the other hole of the rubber stopped. Ensure that pipettes fit snugly in the holes.
  3. Prior to beginning a training and testing cycle for a group of flies, add 20 mL of each respective odor into its own flask. Label which flask is which with corresponding tape colors. One flask of each odor will be used for testing and one for each for testing (total of 2 flasks per odor).

Creating and Assembling Tubing

  1. Obtain ¼” thick OD polyethylene tubing, an air pump, and quick connects.
  2. Attach OD polyethylene tubing to the air pump and use a cap and sleeve to attach it to a needle valve which will control air flow and air pressure. Attach another cap to the other side of the tube and valve.
  3. This tube will connect to a straight or angle quickconnect. The straight quickconnect attaches to another tube that attaches to a training flask. Training flasks will be kept near the valve tube and air pump.
  4. The tube connected to an angle quickconnect will attach it to the two T-maze testing flasks. The angle quickconnect will have one other segment of polyethylene tubing attached to a T connect.
  5. T connect will have two tubes attached on either side, which will go left and right into one testing flask.
  6. Each testing flask will have a two-hole rubber stopper, similarly to the scent flasks, in which one hole will contain a straight pipette and the other will contain the bent pipette. Each pipette tip is attached to a segment of polyethylene tubing of equal length.

Creating Training Tubes

  1. Drill a ¼ hole in the bottom of a 50 mL test tube. Cut off the top of the tube above 50 mL mark line. Cut again below the 5 mL line mark but save the piece to reattach.
  2. Cut a small piece of mesh so that it covers the bottom of the test tube completely right below the cut at 5 mL and tape mesh pieces on the side of the test tube.
  3. Tape the large test tube to the small drilled bottom side of the test tube so that the drilled hole is to the outside.
  4. Obtain two size six rubber stoppers for the training tubes and fill both holes with mesh to serve as a cap during training that will prevent flies from flying through the holes of the rubber stopper.

Training Flies

  1. When flies enter the adult stage, at about 3-5 days old, they are mature and ready for training and testing. In the case of this study, it is optimal to test the flies between 7-10 days old because this tauopathy model begins experiencing neurodegeneration at this stage.
  2. Obtain a vial of adult flies that are all on the same control media or dosage of melatonin.
  3. Flip the flies into a testing tube, which consists of a 50 mL tube with mesh blocking the drilled hole at the bottom. Attach testing tube to the polyethylene tubing connected to the air pump and air valve.
  4. Turn on the air pump and let the chosen CS- scent to be piped through the tubing for 30 seconds so that the flies gain exposure to it and associate it with no negative stimulus. Turn off air pump. Wait 5 minutes. Repeat this step with the CS+ being piped through the tubing into the training tube containing flies.
  5. Immediately following the 30 seconds of CS+ piping, lightly tap flies on a vortexer for 15 seconds. Preferably tap on vortexer while CS+ scent is being piped through. Vortexing is traumatic and aversive. The flies will associate the CS+ scent/negative stimuli with this vortexing experience. Wait 5 minutes.
  6. Repeat step (e.) with CS+ a total of three times so that flies are sure to associate the stimulus with the vortex experience.

Testing Flies

  1. Immediately following the last 5 minute break after 3 vortex training cycles, turn on the air pump and load the flies into the top arm of the T-maze for testing. Turn the T-maze on its side so that the elevator is parallel to the ground. This will make transition from training tube to T-maze more effective.
  2. Tap the training tube down and quickly remove the rubber stopper, still tapping to ensure no flies escape. Flip the tube upside down so that the top opening is aligned with the open top arm of the T-maze. Tap the bottom of the training tube or bump the t-maze down so that flies fall and fly into the containment area between the elevator and left and right walls.
  3. Slide the elevator down so that the flies are contained in the middle of the two ports (loading and testing/arm ports).
  4. Keeping the flies in the middle containment area, turn on the air pump and allow the scents to run through the tubing into the two arm ports of the t-maze where two more tubes are attached. Ensure that the liquid scent at the bottom of each flask is bubbling and flowing evenly. Allow the scents to fill the arm ports and tubes for about 15 seconds so that the odors already fill the space by the time flies are exposed.
  5. Lower the elevator so that the flies are between the two arm ports/tubes of the T-maze testing site. Allow the flies to make a decision for 45 seconds, then raise the elevator so that they have to choose either the left or right port. Remove the tubes from the arm ports and cap with rubber stoppers filled with mesh or foam stoppers.
  6. Count the flies in the tubes by eye or by anesthetizing and counting with a paintbrush. Counting method depends on sample size.
  7. If there are any flies remaining in the elevator containment area or original vial, they can be lowered to the ports again and tested for more data. Return all flies to original food vial and save to test later in life. Keep flies alive in control conditions.
  8. Conduct the same testing procedure with a different group of the same strain of flies but reverse which odor is CS+/vortex-paired to account for all/any differences between the two odors themselves.

Analyzing Data

  1. The performance index (PI) is calculated from data once it is collected. The PI can be obtained by subtracting the amount of flies that chose incorrectly (chose CS+) from the amount of flies that chose correctly (chose CS-) and divide this number by the total number of flies in said trial. This formula is: (# CS-  - # CS+)/(total #). This PI value is known as the half PI score.
  2. The total PI score is calculated by averaging the half PI values for two trials of the same group of flies, with the scent being the CS+ in one trial being the CS- in the other.
  3. Adjusted PI accounts for variation in sample size between the trials accounted for in the same total PI. The adjusted PI can be found by multiplying the half PI for one trial by the fraction representing how many flies were in that trial compared to the total. Then, do the same for the other trial and add the two results together to get the adjusted PI.
  4. Determine the standard deviation of each strain of flies (all wild-type, all elav-gal4, all tauopathy in each respective food group with respective standard deviation value). Perform a Z-test on each strain to obtain a p-value. A p-value will reject or fail to reject the null hypothesis of the study.

  1. Calendar of Events 

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.

  1. Safety Considerations

                The following protocol is met by the investigators:

  1. Experimental Variables and Controls
  1. Variables

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.

  1. Controls

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.

  1. Data Collection and Analysis
  1. Data collection  

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

  1. Data analysis

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:

Actual: .5

= .0883

.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:

Actual: .529

= .2425

.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:

Actual: .21

= .0661

.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.

  1. Discussion

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.

  1. Future Directions of Research

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.

  1. Quality Control and Assurance

  1. Data Handling and Record Keeping

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”

  1. Publication of Results

At this time, the investigators will not be publishing their results of this study.

  1. Budget

Item

Brand

Quantity

Cost/Item

Total Cost

Cornmeal

Quaker

2 (24 oz)

$1.69

$3.38

Soy Flour

Bob’s Red Mill

1 (16 oz)

$3.99

$3.99

Light Corn Syrup

Karo

1 (16 fl oz)

$2.69

$2.69

Melatonin

Sigma Aldrich

1 (250mg)

$37.50

$37.50

Wild-type flies

Bloomington Drosophila Stock Center

2

$18.00

$36.00

Elav-gal4 flies

Bloomington Drosophila Stock Center

3

$18.00

$54.00

Tauopathy flies

Bloomington Drosophila Stock Center

4

$7.50

$30.00

MCH [Scent]

Sigma Aldrich

1 (250 mL)

$44.10

$44.10

OCT [Scent]

Sigma Aldrich

1 (250 mL)

$40.00

$40.00

Paraffin Oil

Carolina Biological

1 (500 mL)

$9.35

$9.35

Polyethylene tubing

Watts

100 feet

$16.38

$16.38

Total:

$281.39

  1. References

[1] 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  

[2] 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),

1395–1406. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2921812/ 

[3] 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 

[4] Malik, B. R., Hodge, J. J. Drosophila Adult Olfactory Shock Learning. J. Vis.

Exp. (90), e50107, https://doi.org/10.3791/50107 (2014).  

[5] 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.

http://doi.org/10.1016/j.tibs.2009.11.004  

[6] Gistelinck, M., & Lambert, J.-C. (2012). Drosophila Models of Tauopathies:

What Have We Learned? International Journal of Alzheimer's Disease.

             http://dx.doi.org/10.1155/2012/970980   

[7] 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 

[8] AH, B., & N, P. (1993). Targeted gene expression as a means of altering cell

fates and generating dominant phenotypes. NCBI. Abstract retrieved from

https://www.ncbi.nlm.nih.gov/pubmed/8223268    

[9] 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

[10] Mutations MAPT R406W. (n.d.). Retrieved November 2, 2017, from Alzforum

Networking for a Cure website:

http://www.alzforum.org/mutation/mapt-r406w

Background & Rationale Figures:

[Figure 1] Komaroff, A., Dr. (2015, June 25). Plaques and tangles [Image].

Retrieved from

https://www.askdoctork.com/how-does-alzheimers-wreak-so-much-havoc-in-the-brain-201506258011   

[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).