1. Biophysical Chemistry Unit for High School Chemistry: Alzheimer’s Disease Study with Polydiacetylene Assay By: Sherry Finkel and Elizabeth Yates

2. Lesson 1: Background information on Beta-amyloid (Aβ) and Alzheimer’s Disease

3. Alzheimer’s disease is a neurodegenerative disease. – Most common form of dementia. – Causes severe memory loss and brain function. What causes memory loss? – Deposits within the brain cause damage to neurons. Neuritic plaques: A  – Located extracellularly (on the outside of the neuron) Neurofibrillary tangles: tau – Located intracellularly (on the inside of the neuron) Alzheimer’s Disease

4. Write down 3 differences you have observed between the healthy and Alzheimer's brain Researchers are interested in studying the proteins that cause the brain deposits so that we may find a cure.

5. Assignment for tomorrow: Read this article Fill out this log on a separate sheet of paper. We will define the words in class. Reading Log Words I did not know:Definitions:

6. In-Class Discussion What is our hypothesis about Aβ and Alzheimer’s Disease? Ideas on how can we test this hypothesis?

7. Lesson 2: Hypothesis Testing Making Polydiacetylene (PDA)

8. Formulating our hypothesis What have we learned about Alzheimer’s disease? – A  deposits on the brain damage the neuron. Therefore, causing memory loss. Types of A  aggregates: oligomers, protofibrils, fibrils, and plaques Let’s come up with our hypothesis using what we know! – We believe that… Certain Aβ aggregates play a larger role in Alzheimer’s disease than others. Great hypothesis, but what is a hypothesis we can test? – It must be more specific!

9. What are we missing? We must know what procedures are available to test our hypothesis. What are our ideas on how to test our hypothesis?

10. To test our hypothesis: use an assay A colorimetric ASSAY has been developed to test A  aggregate interaction with a model cell surface. What is an assay? – a standard test used in the sciences What do you mean by colorimetric? – See a visible color change when the desired reaction occurs.

11. What is our assay made of? Polydiacetylene (PDA) is a color indicator. – When the desired reaction occurs, it changes colors from blue to red. Color change can be measured quantitatively with a machine that records light absorbance measurements. – This process is called spectrophotometry. – *More information on Spectrophotometry *More information on Spectrophotometry The PDA molecule has a hydrophilic head and a hydrophobic tail. These features help PDA bind to lipid bilayers.

12. How does the assay work? PDA is mixed with lipid extract to make vesicles. These vesicles are brillant blue to start and will show a colorimetric response depending on the reaction.

13. Colorimetric Response Green: Aβ aggregate Grey: Lipid bilayer Blue: PDA Red: PDA after color change Aβ aggregates are added to the solution. The aggregates can respond it three ways: 1.They do not interact with the lipid bilayer, leaving the PDA blue. 2. They interact at the surface of the lipid bilayer, turning the PDA from blue to red. 3. They insert themselves into the bilayer, turning PDA from blue to purple.

14. Colorimetric Response is also known as %CR %CR is calculated using these formulas: PB = A 640 /(A 640 + A 500 ) %CR = (PB 0 -PB 1 )/PB 0 x 100% – PB is the ratio of blue reflected light to total reflected light – PB 0 is the control, meaning no Aβ is added to the PDA lipid vesicles. No color change should occur in the control. PB 1 is the sample with Aβ. Calculations

15. In-Class Discussion Read and re-read this lesson. In class, you will write a paragraph describing PDA and %CR in your own words This was a lot of information! We will make sense of it in class.

16. Lesson 3: Putting it Together

17. New Hypothesis New, testable hypothesis: We believe that… We will see a higher colorimetric response from certain aggregates of A  than others.

18. Aβ Fragments Fragments of Aβ are made of different amino acid sequences and sizes. For our assay, we will test: 7 fragments of the Aβ protein. Can you guess what this picture shows?

19. Atomic Force Microscope An Atomic Force Microscope Diagram. We will discuss how it works in class. The images from the last slide were taken using an AFM capable of measuring in nanometers. 1nm=0.000000001m

20. Aβ Aggregates A lipid bilayer was exposed to various Aβ fragments and observed to see if they formed aggregates. Any aggregates on the surface of the image appear in white or black patterns. These images can help us predict which fragment will show the greatest colorimetric response. How large is each image?

21. Make your prediction Which or fragment(s) do you think will have the highest colorimetric responses? Why? Write down your rationale (reasoning or justification)

22. Lesson 4: Methods

23. We will be performing a modified version of the research experiment from the Legleiter Lab at West Virginia University in class. We will make the following equipment substitutions: – PDA: Bromothymol Blue – Aβ: Unknown concentrations of Acetic Acid labeled as each Aβ fragment. Experiment modifications

24. Methods You and your group members will add 1mL of each “fragment” (unknown Acetic Acid dilution) to 9mL of PDA (Bromothymol Blue). You will allow the solution to sit for 5 minutes before using the spectrophotometer (review lesson 2 on using spectrophotometer). You will record your results in Microsoft Excel, where we will calculate %CR.

25. In-Class Tomorrow Remember to wear: Pants Close-toed shoes Hair tie for long hair

26. Lesson 5: Data Analysis

27. Legleiter Lab Aβ Data Legleiter Lab performed a similar experiment to us. What do you think? Write down your observations. Can you identify the positive and negative controls?

28. Legleiter Lab Aβ Data

29. Putting it Together What do the graph and image show you? What can you determine from the graphs Draw your conclusions from the Legleiter Lab data.

30. Supplemental Assignment: Statistics We know that Aβ 22-35 and Aβ 1-40 showed the greatest colorimetric responses. Can we really assume that these results will be repeatable? The only way we can assume is with statistics.

31. Innocent until Proven Guilty We must be 95% sure that our results will hold the same relationships if the experiment is repeated. We can only be sure if we perform a probability test, called a t-test, which gives us a probability value.

32. The Probability That Our Results are Not Repeatable… The Probability value (called a p-value) is the probability that our results cannot be repeated. (Read aloud 5 times until you memorize it!) So, the lower the p-value, the more likely your results are statistically significant. We are looking for a low p-value!

33. P<0.05 If p is less than 0.05, then there is less than a 5% (0.05x100) likelihood that our results are due to chance! There is a 95% chance that our results are repeatable! Small p-values=statistical significance.

34. Why p<0.05? We can never be 100% sure in science. We are willing to accept results that are highly probable in order to move science forward.

35. In-Class Discussion We will work on: – Statistics: t-tests analyzing p-values – Graphing Data analysis Different plots R 2 values