Introduction The research project that I have conducted throughout my junior and senior year is entitled “The Effects of DMAB-Anabaseine on APPL Flies, a Model for the Treatment of Alzheimer’s”. This study was conducted to test the effect of DMAB-Anabaseine on APPL flies. These are flies that have been genetically mutated to develop early-onset Alzheimer’s disease (AD). DMAB-Anabaseine is the chemical I used to determine if cognitive behavior could be restored in the APPL flies. The concentrations used to test the APPL and wild type flies were 1uM, 10uM, 100uM, and 200uM. Eight fly vials were prepared for each concentration. Fifteen milliliters of treatment was poured into fifteen milliliters of the Drosophila media. After a week I tested both the regular wild type and the APPL flies using a positive phototaxis assay and a negative geotaxis assay to determine if the DMAB-Anabaseine restored functions to the APPL flies and had any effect on the normal controls. Alzheimer's is a disease of the brain that is characterized by impairment of memory, planning, perception, reasoning, and language. An increase in the production of a specific protein (beta–amyloid protein) in the brain leads to nerve cell death. As age increases, the risk of getting Alzheimer's increases. “Ten percent of people over 65 years of age and 50% of those over 85 years of age have Alzheimer's disease” (http://www.medicinenet.com/alzheimers_disease/page2.htm). If new treatments aren’t developed to decrease the chances of developing Alzheimer's disease, the number of people with Alzheimer's disease in the United States is expected to be 14 million by the year 2050 (http://www.medicinenet.com/alzheimers_disease/page2.htm). There are also genetic risk factors for Alzheimer's disease. “Most patients develop Alzheimer's disease after age 70. However, 2%–5% of patients develop the disease in their 40’s or 50’s. At least half of these early onset patients have Alzheimer’s by genetic mutations” (http://www.medicinenet.com/alzheimers_disease/page2.htm). The offspring of patients with early onset Alzheimer's disease from genetic mutations have a 50% chance of developing Alzheimer's disease as well. The onset of Alzheimer's disease is usually gradual and is slowly progressive. Memory problems that family members initially dismiss as "a normal part of aging" are, in retrospect, noted by the family to be the first stages of Alzheimer's disease. Memory issues, particularly short–term memory loss, are common beginning symptoms for the disease. For example, a person may forget to lock the door at night, or may have trouble remembering his/her medicine. In addition, early on a person may suffer mild personality changes, a lower activity level and withdrawal from social interactions. As the disease progresses, problems in thinking and in other cognitive functions develop. For example, the person may start to have troubles paying bills and may not understand what is being read to him/her. Additionally, disturbances in behavior will also be seen at this point. This includes agitation, irritability, and a decreased ability to dress appropriately. Later on in the progression of the disease, the person may become confused or disoriented about what day it is, and also may not remember simple information such as where they live. Sooner or later, the person may start to wander, become unable to engage in conversation, have erratic mood swings, become uncooperative, and lose bladder and bowel control. In late stages of the disease, people can become completely incapable of caring for themselves. Death usually follows, because illnesses take over the body during severely deteriorated states of health. Those who develop the disease later on in their life more commonly die from other illnesses (such as heart disease) rather than Alzheimer's disease. Symptoms of Alzheimer’s include memory loss, difficulty performing familiar tasks, problems with language, disorientation to time and place, poor or decreased judgment, problems with abstract thinking, misplacing things, changes in mood or behavior, changes in personality, loss of initiative (http://www.medicinenet.com/alzheimers_disease/page4.htm). Individuals who exhibit several of these symptoms should see a physician for a complete evaluation. DMAB-Anabaseine has been suggested as a possible treatment method for Alzheimer’s. However, little research has been conducted with this chemical. DMAB-Anabaseine is derived from the urine of a Nemertine worm. The chemical name of DMAB-Anabaseine is 4-[(5,6-Dihydro[2,3'-bipyridin]-3(4H)-ylidene)methyl]-N ,N-dimethylbenzenamine dihydrochloride and it is known to paralyze insects if the concentration of it is too strong. This chemical is currently being tested on rats before being considered for use in humans (Dr. William Kem, 2004). This chemical aims to eliminate the amyloid plaques located around the nerve cells in the brain so the path of the nerve impulse can be restored. The amyloid plaques found in Alzheimer's disease are made of beta amyloid. Beta amyloid is a protein fragment snipped from a protein called amyloid precursor protein. These fragments then are mixed together with other molecules, neurons, and non-nerve cells. In Alzheimer’s disease, plaques develop in the hippocampus, a deep structure located in the brain that helps to encode memories, and in other areas of the cerebral cortex that are used for thinking and making decisions (http://www.webbooks.com/eLibrary/Medicine/Neurological/Alzheimer_Amyloid.htm). The early-onset Alzheimer’s that occurs between the ages of 30 and 60 has also been demonstrated from the result of mutations of the genes involved in the production of beta amyloid. For this reason, beta amyloid is an important target in the development of new drugs against Alzheimer’s disease. However, as time passes by, Alzheimer’s damages the nerve cells more in humans. Thus, the earlier the symptoms are seen the better the results will be. A model for Alzheimer’s in flies has been developed in order to further understand the disease and possible treatment methods. These flies are genetically mutated by affecting the APPL gene, otherwise known as the β amyloid protein precursor (http://flybase.org/reports/FBti0051787.html). In addition, their mutations create amyloid plaques that are seen in Alzheimer’s patients. These flies exhibit a higher reaction time to light, they are less phototaxic. You can also see that these flies were much slower then the regular wild type flies when compared to the wild type flies. These flies also have a slower negative geotaxis meaning they rise up to the top of the fly vial at a slower rate. The APPL flies have early onset Alzheimer’s that is homologous to the beginning state of Alzheimer’s patients. Many previous studies regarding Alzheimer’s have been studied in conjunction with rats and monkeys. For example, the article “Hydroxy Metabolites of the Alzheimer’s Drug Candidate 3[(2, 4-Dimethoxy) Benzylidene]-Anabaseine Dihydrochloride (GTS-21): Their Molecular Properties, Interactions with Brian Nicotinic Receptors, and Brain Penetration” by Dr. William Kem describes the Alzheimer’s drug’s function. According to Kem talks the drug activates the a7 receptor, increasing cognitive behavior (Dr. William Kem, 2004). In the article “A Study of the Occurrence of Anabaseine in Paranemertes and Other Nemertines” by William Kem, he explains how the DMAB-Anabaseine was derived from organism of the Nemertine phylum’s toxin in order to understand its function better. Along with that, he also talks about how DMAB-Anabaseine became of great interest to him when working with the chemical (Dr. William Kem, 1971). Lastly, an article Dr. William Kem wrote and sent to me was “Biochemistry of Nemertine Toxins.” Here in this article, he goes into description of the chemical in its coloration, pH, and element composition. It was hypothesized that administration of DMAB-Anabaseine would increase cognition as measured by the response to a negative geotaxis and positive phototaxis by APPL and wild type flies. Materials and Methods The flies used in the experiment were APPL type flies and regular wild type flies. The APPL flies were obtained from Harvard Medical School which maintains a fly bank of mutants many of which are homologous for human diseases. The APPL flies are genetically mutated to develop early onset Alzheimer’s disease. The regular wild type flies were used as a control to measure any effects on the flies after treatment. The fly vials used were 3.2cm in width and 10.1cm in height. Sponge tops were used to cover the top of the fly vial and were 3.5cm in width and 3.7cm in height. The media used was from Carolina (WF-17-3120) named Formula 4-24 Instant Drosophila Media. Each vial was prepared with 15mL of the media and 15mL of water or different dilutions of the DMAB-Anabaseine solution. All flies were stored at room temperature and received about 12 hours of light. The chemical used to test on the flies, DMAB-Anabaseine, was obtained from William Kem of the University of Florida at Gainesville. He sent a 45.6 mg sample of DMAB-Anabaseine. DMAB-Anabaseine has a molecular weight of 364. The concentrations of DMAB-Anabaseine prepared were 1uM, 10uM, 100uM, and 200uM. The anabaseine was administered by using different concentrations of the chemical in the media instead of water. Fifteen mL of the media and 15mL of the anabaseine solution were mixed in together. Eight fly vials were prepped for each concentration; four for the wild type flies and four for the APPL flies. Then, after the media looked dry enough to transfer flies, the flies were inoculated with fly nap. After sorting the flies by gender, 6 males and 6 females, were put into each fly vial. This was done with both the wild type and APPL flies. After a week or so, observations were made to see if there was larva in the media or pupa on the vial wall. When this event took place all the adult flies from the fly vial were removed. The wild type flies reproduced faster then the APPL flies so they were tested first to use time efficiently. The negative geotaxis was preformed by placing approximately 15 to 20 flies into an empty fly vial. For each test the fly vials were labeled on the bottom with the number of the fly culture and the type of fly. The empty test vials couldn’t get mixed up because different colored sponge tops were used. For each dosage the negative geotaxis was begun by doing a live transfer and then sealing the fly vial with the sponge top. Then, the fly vial was tapped down causing all the flies to fall to the bottom and the timer was started. When the first five flies reached the top then the timer was stopped and the ascent time was recorded. This assay was performed 4 times for each dosage and strain. The distance the flies had to travel was 6.5 cm for each trial. The positive phototaxis assay required the use of a light box created from a plastic container with dimensions of 36 cm in length, 26 cm in width, and 17.5 cm in height. The inside of the fly box was sprayed with black with black spray paint to create a dark environment. Fly vials were placed at either ends of the box. One fly vial was closer to a 12W 4V light at one end of the box. This light was the primary source of light at one end of the box and was used to lure the flies. The mountings were made by taking two wood blocks that were 11.5cm in length, 6 cm in width, and 2 cm high. These two blocks were attached to the box with two screws. Then, wood trim was glued on either side of the block to the exact fly vial width with wood glue. I repeated this process for the other block. Fly vials rested on mountings placed in the box in order to raise the positioning of the fly vials so the door could open. The door was made out of five pieces of plexi glass. The plexi glass used had a width of .2 cm. Two pieces were 19 cm long and 11.5 cm high, two were 4 cm long and 9.5 cm high, and the last piece of plexi glass was 6 cm long and 19.5 cm high. The cuts were made using a glass cutter to make sure the cuts were clean and the two 19 cm pieces had a hole made dead center of the plexi glass of the size of the fly vial. Now, the two 19cm long pieces were placed back to back, but before this could be done the two 4 cm pieces were placed on either side of the 6cm piece. After that, a mark was made where the two 4 cm pieces were located when pressed against the 6 cm piece. The pieces were then glued to one of the 19 cm pieces to make sure that adjustments can be made to the holes if necessary on the second 19 cm piece. This was all then held in place by two black binder clips to make sure the layers of plexi glass wouldn’t move. The phototaxis assay was performed by first letting the flies sit in the dark for five minutes with a lid placed on top of the box. Then the light was turned on for 15 seconds. Next, the box was tapped down and the passage way was then opened for 15 seconds. After the fifteen seconds the passage way was closed and the data was then recorded by counting the number of flies found in the second fly vial over the total number of flies. This invention allowed me to determine if treatment restored APPL flies cognitive behavior; the positive phototaxis response. Results The purpose of this experiment was to see if the DMAB-Anabaseine would have any effect on regular Wild type flies and on the APPL flies, a model for Alzheimer’s. I hypothesized that the DMAB-Anabaseine would have a positive effect on the APPL flies, improving cognitive behavior. The DMAB-Anabaseine was proven by Dr. William Kem to have positive effects on monkeys and on rats by improving their cognitive behavior and response rate; however I wanted to test to see if this is true with another species of animals, flies. The flies were tested using the positive phototaxis assay (Graph #2) using the light box and the negative geotaxis assay (Graph #1) using the fly vials. Negative Geotaxis Assay The negative geotaxis assay showed that for the 0uM dosage, it took the regular flies an average of 3.90 seconds to reach to top with the 4 trials for each fly vial. For the APPL flies, it took them an average of 8.99 seconds to reach the top. For the 1uM dosage the time it took the flies to reach the top has decreased dramatically. The regular flies took an average of 1.91 seconds to reach the top. The APPL flies had an average of the 1.90 seconds to reach the top. In comparison, the 1uM APPL flies negative geotaxis response was the exact same time for the regular wild type flies. With this dosage, the anabaseine improved response time for the negative geotaxis. At the 10uM dosage, the response times for the regular flies was 6.40 seconds and it took the APPL flies 4.76 seconds. In Graph #1 it can been seen that the 10uM dosage didn’t have as great an effect on the flies as the 1uM, but the time was still faster for the APPL flies when compared to both strains without treatment (0uM) Finally, for the 100uM dosage there wasn’t as great as an improvement in response time for regular flies or the APPL flies, but it was better than the 10uM dosage and the control results. The lowest standard deviations were found for the control and for the 1uM dosage. This shows that the data wasn’t largely spread out from the mean showing consistency for this experiment. The 100uM dilution also had a fairly low standard deviation as did the 10uM. Positive Phototaxis Assay For the positive phototaxis assay, the data was collected by counting the number of flies found in the fly vial closest to the light over the total number of flies used in the assay. Using percentages, you will see that as the number gets higher, the higher the number of flies went to the fly vial closest to the light (Graph #2). With the control, 50.55% of the regular wild type flies went to the vial closest to the light. For the APPL flies, only 38.84% of the flies did so. For the 1uM, 50.31% of the wild type went to the light as the control wild type flies, however, 66.87% of the APPL flies went to the light. Twice as many flies went to the light when compared to the untreated APPL flies. For the 10uM 63.02% of the wild type went to the light as did 50.00% of the APPL flies. The data shows an increase in cognitive function of response for the positive phototaxis of the regular flies, but for the APPL flies the cognitive function of response dropped slightly when compared to the control flies and 1uM flies. Response to the positive phototaxis for the 100uM dosage was 39.41%, a big decrease for the wild type flies, and the APPL flies had 82.64% of the flies go to the light. This was the highest percent response to the positive phototaxis for the APPL when you look at all the other dilutions. The hypothesis was that the cognitive behavior of the APPL flies would be restored. The data showed that treatment by oral administration of DMAB-Anabaseine had restored function in the APPL flies almost to the level of the normal wild type flies for both the negative geotaxis and positive phototaxis. Discussion and Conclusion After organizing the data, you can clearly see that the best dosages used that restored taxis response was the 1uM and the 100uM of DMAB-Anabaseine for both the negative geotaxis and positive phototaxis assay. This dosage had the highest response rate in seconds for the negative geotaxis and has the highest percent responders for the positive phototaxis assay. Along with that, the 1uM and 100uM also had the lowest standard deviation. For this project concentrations of 1uM, 10uM, 100uM, and 200uM, but the 200uM was too strong. It was so strong that the larva didn’t hatch from their pupa stage so there was no data collected for this dilution. This happened for both the APPL flies and regular wild type flies. This wasn’t the only limit to this project. No matter what the sources of error could have been, I tried to stay as consistent as possible with the measuring, and data collection. For the project, I hypothesized that the DMAB-Anabaseine would have positive effects on the flies. Anabaseine helped in increasing cognitive behavior in the APPL flies and in the regular wild type flies. This is based on the negative geotaxis and positive phototaxis results. For future research, I would use another apparatus to test for other improvements in cognitive function. A larger variety in the range of the dilutions of DMAB-Anabaseine would be better to present for the project because the viewer can see which dilutions are better than/worse than others. In conclusion, DMAB-Anabaseine which has shown promise as a treatment for Alzheimer’s disease in other animal models has, as a result of this study, shown to be effective in restoring function in another model for Alzheimer’s disease, the APPL flies. Bibliography (1996). Alzheimer's disease. Retrieved September 26, 2006, from MedicineNet.com Web site: http://www.medicinenet.com/alzheimers_disease/article.htm (2007). Alzheimer's disease. Retrieved November 1, 2006, from Wikipedia Web site: http://en.wikipedia.org/wiki/Alzheimer's_disease (1994). DMAB-Anabaseine dihydrochloride. Retrieved November 9, 2007, from Tocris Web site: http://www.tocris.com/products/DMAB-anabaseine.php (2007, Nov 1). DmelPBac{WH}Applf04460 Insertion . Retrieved March 3, 2007, from FlyBase Web site: http://flybase.org/reports/FBti0051787.html (1995). Fruit Flies Yield Clues To Clinical Heterogeneity Of Early-onset Alzheimer's Disease. Retrieved December 20, 2006, from ScienceDaily Web site: http://www.sciencedaily.com/releases/2006/05/060523072751.htm Kem, William (2004).Hydroxy Metabolites of the Alzheimer’s Drug Candidate 3[(2, 4- Dimethoxy) Benzylidene]-Anabaseine Dihydrochloride (GTS-21): Their Molecular Properties, Interactions with Brian Nicotinic Receptors, and Brain Penetration. Kem, William (1973).Marine Pharmacognosy. Biochemistry of Nemertine Toxins. Chapter 2, 37-81. (2007, Sep 11). The Beta Amyloid. Retrieved January 11, 2007 Web site: http://www.webbooks.com/eLibrary/Medicine/Neurological/ Alzheimer_Amyloid.htm Kem, William (1971).Toxicon. A Study of the Occurrence of Anabaseine in Paranemertes and Other Nemertines. Volume 9, 23-32. Crowther, Damian (2005). What flies can tell us about Alzheimer’s. Retrieved December 7, 2006, from The Royal Society Web site: http://www.royalsoc.ac.uk/exhibit.asp?id=3618&tip=1