1. Cotinine: Potential Therapeutic agent in Alzheimer’s Disease Sharmila Thiagarajan C1 Bio-tech

2. What is AD?  AD is a type of dementia that causes problems with memory, behavior, and thinking  The neuron cells start degrading causing the brain to shrivel up  It’s a progressive disease that worsens over time  It’s the fourth leading cause of death behind heart disease, cancer, and stroke  There is no cure yet, but fortunately AD is researched by many  Notable people that died of AD are Ronald Reagan and Winston Churchill

3. History  Alzheimer’s Disease was discovered in 1906 when Dr. Alois Alzheimer noticed changes in the brain of his patient, Auguste. After she died he examined her brain and found what is now known as the signs of AD. Dr. Alzheimer Auguste Deter

4. Statistics  There are 3 million new cases of AD per year in the United States  25 million people suffer from different types of dementia today  One new case is diagnosed every 7 seconds  The number of cases might double in the United States and triple in both India and China over the next three decades

5. Comparing Stages of Alzheimer’s From the healthy brain to the severe AD brain one can see that the brain starts shrinking and parts of the brain deteriorates. Since AD is first affected in the hippocampus, (the red circles) the severe AD picture shows a hole where the hippocampus should be and unfortunately that’s where the memory and nervous systems are located. That’s why many AD patients have trouble with remembering things. 1. 2. 3.

6. Amyloid Plaque  There are two lesions that cause AD  The first lesion is a plaque which is basically deposits of the protein beta- amyloid accumulated in the spaces between nerve cells in the hippocampus

7. Pathways When looking at the pathway of the plaque, one can see there are two directions: one direction resulted in a normal product and the other harmful product. Heading to the right (pink side) leads to amyloid plaque, also freed by the APP protein, which travels through the beta secretase and attaches to the other amyloid proteins. The correct pathway which is towards the left (blue side) shows the large protein called APP. It frees the beta- amyloid which than goes through the alpha secretase which is then cleared in the body.

8. Tau Tangles  The second lesion is called tau tangles also known as neurofibrillary tangles  Tau tangles are deposits of the protein tau that accumulate inside of nerve cells in the hippocampus  When neurons communicate with each other they send signals from the soma to the synapse through the neuron’s skeleton which is made up of microtubules that are stabilized by normal tau protein. In AD, tau protein becomes defective from the microtubules which ultimately breaks the skeleton. Picture on the right is the neuron’s skeleton when the tau protein is defective and breaks the skeleton Microtubules Tau protein

9. Differences in Neurons When looking closely at the neurons one can see the tangles accumulating in the AD picture The neurons are then unable to send signals to each other, which ultimately kills the neuron.

10. Alzheimer’s disease equation A β a changes tau changes cell death and dementia = Alzheimer’s disease Researcher John Hardy published an article about the relationship between both lesions. He derived the equation (right) stating: 1. If amyloid plaque increased then some of the plaque would enter the cell 2. The tau protein would phosphorylate and aggregate 3.Causing the neuron to die which would indicate AD.

11. Cotinine- Therapeutic Agent to AD  Cotinine is a natural product from tobacco leaves  Studies have shown that cotinine consumed by mice showed a decrease in amyloid plaque in the brain  An anagram of nicotine, cotinine does not have the negative effects of addiction like nicotine does

12. Research Question  What effect would cotinine have on mice that develop amyloid plaque and neurofibrillary tangles?

13. Inductive Reasoning Plaques only (Known to decrease plaques from previous studies) Plaques and tangles Hypothesis: Cotinine should decrease plaques which will ultimately decrease tangles process and slow down neuronal death As researched in article “Cotinine halts the advance of Alzheimer's disease-like pathology and associated depressive-like behavior in Tg6799 mice” it is shown that plaque is decreased by the presence of cotinine yet the effect cotinine on the tau tangles is unknown. By understanding the information, one can hypothesize that cotinine, if given to mice with both tau tangles and plaque, would decrease plaque levels leading to decreased tau tangles formation ultimately slowing down neuronal death. Cotinine

14. Pre-Existing Mouse Models  1. Normal mouse – control  2. Mouse with only plaque - control  3. Mouse with tau tangles and plaque Using Pre Existing Mouse Models, three types of mice will be used for this experiment. A normal mouse which is the control. A mouse that only develops plaque(is sold by Jacksons Lab) which will also be a control. And a mouse that develops tau tangles and plaque will give an answer to the research question.

15. Making Cotinine nano-particles Cotinine poly(lactic-co-glycolic acid Stir at 1200 rpm 1% polyvinyl alcohol With Emulsion Stir at13,000rpm Freeze- Dry Cotinine nano-particles

16. Making Cotinine nano-particles  A mixture containing cotinine (10 mg cotinine in 1 ml double distilled water), methanol (1 ml), and PLGA (100 mg dissolved in 8 ml dichloromethane) need to be stirred at 1200 rpm to form an emulsion, which will be added to a rapidly stirred 1% v/v aqueous solution of polyvinyl alcohol (8 ml). The resulting suspension will be stirred at room temperature for 2–3 h ensuring complete evaporation of dichloromethane and centrifuged at 13,000 rpm for 15 min at 4 1C. The supernatant will be removed and the particles will be washed twice with water, followed by freeze-drying to obtain cotinine encapsulated PLGA nanoparticles.

17. Experiment 1. Mice need to be kept under a12:12 hour light and dark cycle with access to food and water (2 mice for each type to test one with cotinine and one without) 2. After one month inject cotinine nanoparticles for six months daily 4. After seven months all mice will be euthanized 3. Every month all mice will get tau PET and amyloid PET scan

18. PET Scan  Stands for positron emission tomography  It is a imaging test  It uses radioactive tracers in a special dye specific for tau or amyloid plaque  These tracers are injected into a vein which are then absorbed by organs and tissues

19. Expected Results- Mouse 1 (normal) Amyloid plaque Month 7 Amyloid plaque PET scan with and without cotinine Expected results: in Mouse1 with cotinine and without cotinine in the amyloid plaque PET scan would be identical.

20. Expected Results- Mouse 1(normal) Tau protein PET scan with and without cotinine In Mouse 1 for the tau PET scan, both with and without cotinine would also be identical.

21. Expected Results- Mouse 2 (plaque only) with cotinine Amyloid plaque PET scan with cotinine Amyloid plaque PET scan without cotinine So this test would confirm the article about cotinine decreasing amyloid plaque levels when comparing both scans.

22. Expected Results-Mouse 2 (plaque only) Tau protein PET scan with and without cotinine Since this mouse does not develop tau so both PET scans for with and without cotinine would look identical.

23. Expected Results- Mouse 3 (tangles and plaque) Amyloid plaque PET scan with cotinine Amyloid plaque PET scan without cotinine So this test would also confirm the article about cotinine decreasing amyloid plaque levels when comparing both scans.

24. Expected Results- Mouse 3 (plaques and tangles) Tau protein PET scan with cotinine Tau protein PET scan without cotinine If hypothesis is correct as well as John Hardy’s research about the relationship between the tau and the plaque than the mouse with the cotinine would significantly have less tau protein than the same mouse without the cotinine

25. Summary  Mouse 1- No plaque or tau  Mouse 2- Significant decrease in plaque and no tau  Mouse 3- Significant decrease in plaque and tau Mouse 3 is expected to show a decrease in the tau tangles as a direct result of the cotinine-decreased amyloid plaque levels. It then follows that there would be less neuronal death and possibly diminished AD symptoms.

26. Further Questions  How would cotinine affect tau tangles?  Future Experiment: Get a mouse model that develops only tau to test the effect of cotinine  How effective is cotinine delivered by nanoparticles compared with oral cotinine?  Future Experiment: Get two mouse model and see how much cotinine is in the brain through both techniques.

27. Works Cited  Hardy, John. "The Relationship between Amyloid and Tau." - Springer. Journal of Neuroscience, 01 May 2003. Web. 28 July 2015.  Patel, Sagar. "Cotinine Halts the Advance of Alzheimer's Disease-like Pathology and Associated Depressive-like B..." Frontiers. University of South Florida, June-July 2014. Web. 28 July 2015.  Barreto, George E. "Beneficial Effects of Nicotine, Cotinine and Its Metabolites as Potential Agents for Parkinson's..." Frontiers. Mo2015.rsani Medical College, Jan.-Feb. 2015. Web.  Lonskaya, Irina. "Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, 13 Dec. 2013. Web. 28 July 2015  Bradt, Steve. "Researchers Create Colorful “Brainbow” Images of the Nervous System." Harvard Gazette. Harvard University, 31 Oct. 2007. Web. 28 July 2015.  Yang, Sarah. "Lifelong Brain-stimulating Habits Linked to Lower Alzheimer's Protein Levels." Berkeley News. University of Berkeley, Jan.-Feb. 2012. Web. 28 July 2015.