1. Treating Alzheimer’s Disease with Nattokinase
Jason Li
2015 COSMOS
Cluster 1: Biotechnology

2. Alzheimer’s Disease (AD)
Neurodegenerative disease
It is not a normal phenomenon
Most common form of dementia
Leads to impairment of memory and cognition
Causes loss of connections among and death of neuron cells.

3. Causes of Alzheimer’s-Tau Protein
Hyper-phosphorylation of tau protein inside neuron cells
Normal tau proteins stabilizes microtubules
When hyper-phosphorylated, Tau proteins start clumping together and forming tangles instead of doing their normal job.
Blocks transportation of essential supplies inside the cell

4. Causes of Alzheimer’s-Amyloid Plaques
They are also known as beta-amyloids (Aβ)
Located in synapse of neuron cells
Misfolded proteins that tend to clump together.
Insoluble
Occupy synapses and lead to death of neuron cells.

5. Tau Protein or Plaques? Which one causes Alzheimer’s more?
There is no certain answer
Both contribute and lead to eventually death of neuron cells.

6. Nattokinase
Extracellular enzyme secreted by bacterium Bacillus subtilis natto found in the food natto
Natto: fermented soybeans that are more commonly eaten in Asia, especially Japan.
Despite its name, it is a protease.
A previous research has shown that Nattokinase can cleave samples of beta-amyloid but not from the brain
Another research has shown that Nattokinase can be taken orally by rats and enter their brain tissues.

7. Researchable Question
What effects would nattokinase have on the level of beta amyloids and the overall gene expression in a mouse’s brain tissues with Alzheimer’s?

8. Significance of Experiment
Further progress in alleviating the disease
Prove the potential of food treatment
Natto would be a convenient food to eat
Because there is no clear understanding of the relationship between amyloid plaques and tau protein, if amyloid can be reduced with nattokinase, then effects of reducing the plaques on the disease in general can be observed

9. Breakdown of Experiment
1. 2. feeding of nattokinase 3. extract brain tissue 4.thioflavin t binding assay to detect beta amyloid 5. mRNA sequencing to test gene expression

10. Thioflavin T Binding Assay-Background Info
Dye-binding assay to diagnose amyloid fibrils ex vivo or in vitro
Thioflavin T: a benzothiazole dye
The dye exhibits enhanced fluorescence when binding to amyloid fibrils

11. RNA-Seq Background Info
Detects gene expression of mRNA in the whole brain
Converting mRNA into cDNA libraries for further analysis through sequencing.
Advantages over other tests like microarrays and hybridization: faster and cheaper, more data output.
Sequencing only the exons in RNA transcripts, the RNA responsible for producing protein products.

12. Groups of Mice Group 1: Negative control group: healthy mice
Group 2: Positive control group 2: AD mice fed with water only
Group 3: AD mice treated with low dose of nattokinase
Group 4: AD mice treated with high dose of nattokinase

13. Hypothesis
In the brain tissue, addition of nattokinase reduces amyloid plaques and influences some gene expressions in the brain.
Predicted level of amyloid plaques among the groups:
Group 2 (positive control with untreated AD) > Group 3 (treated with low dose of nattokinase) > Group 4 (treated with high dose of nattokinase) > Group 1 (negative control without AD).

14. Step 1: Oral Administration of Nattokinase
Obtain mouse models
Purchase nattokinase from a dietary supplement market in USA.
Feed the experimental group with nattokinase suspended in water orally for 45 days daily

15. Step 2: Purification of βA
Animals fasted overnight
Brain extraction
Purification of protein:
Homogenizing brain tissue
Ultracentrifuging or chromatography

16. 3. Thioflavin T Binding Assay
Get isolated sample of Aβ
Incubate sample with dye added
Screen for fluorescence using screening machine

17. Step 4. RNA- Seq
Necessary materials can be purchased from Illumina, a prominent biotechnology company
Isolating all RNA: centrifuging and diluting lipids and proteins
Purifying mRNA (there is a lot more rRNA than mRNA)
Synthesize cDNA using reverse transcriptase
Fragmentation of cDNA
Addition of adapters to fragments for primers to bind in sequencing process
Sequencing of cDNA fragments
Analysis of sequence

18. 5. Analysis of mRNA sequence
Assemble expressed gene sequences using overlapping sequences of fragments
Identifying all expressed genes using Blast
Comparison of treated and untreated

19. Challenges of the Experiment
Keep in mind: the brain is complex
Thioflavin T assay: might be biased by the presence of exogenous factors.
RNA-Seq: difficulty of interpreting the data

20. Further Areas of Research
Switching from experiments for mice to humans
The exact mechanism by which amyloid plaques and tau protein cause neuronal damage
Gene expression of abnormal proteins in AD
Gene therapy might be possible to be developed

21. Bibliography
"Alzheimer's Disease & Dementia | Alzheimer's Association." Alzheimer's Disease & Dementia | Alzheimer's Association. Alzheimer's Association, n.d. Web. 28 July "Tau Protein." Wikipedia. Wikimedia Foundation, n.d. Web. 29 July Murphy, M. Paul, and Harry LeVine. "Alzheimer’s Disease and the β-Amyloid Peptide." Journal of Alzheimer's Disease : JAD. U.S. National Library of Medicine, n.d. Web. 29 July "Amyloid-Degrading Ability of Nattokinase from Bacillus Subtilis Natto."Journal of Agricultural and Food Chemistry. National Taiwan University, n.d. Web. 29 July "Serrapeptase and Nattokinase Intervention for Relieving Alzheimer’s Disease Pathophysiology in Rat Model." Human and Experimental Toxicology. N.p., n.d. Web. July Jameson, LaramieP., Nicholas W. Smith, and Sergei V. Dzyuba. "Dye-Binding Assays for Evaluation of the Effects of Small Molecule Inhibitors on Amyloid (Aβ) Self-Assembly." ACS Chemical Neuroscience. American Chemical Society, n.d. Web. 29 July Jameson, LaramieP., Nicholas W. Smith, and Sergei V. Dzyuba. "Dye-Binding Assays for Evaluation of the Effects of Small Molecule Inhibitors on Amyloid (Aβ) Self-Assembly." ACS Chemical Neuroscience. American Chemical Society, n.d. Web. 30 July Walsh, Dominic M., Eva Thulin, Aedín M. Minogue, Niklas Gustavsson, Eric Pang, David B. Teplow, and Sara Linse. "A Facile Method for Expression and Purification of the Alzheimer’s Disease-associated Amyloid β-peptide." The Febs Journal. Blackwell Publishing Ltd, n.d. Web. 30 July 2015.

22. Image Credits