Jason Li
2015 COSMOS
Cluster 1: Biotechnology
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.
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
Causes of Alzheimer’s-Amyloid
Plaques
 They are also known as
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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.
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.
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.
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?
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
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
Thioflavin T Binding AssayBackground 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
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.
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
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).
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
Step 2: Purification of βA
 Animals fasted overnight
 Brain extraction
 Purification of protein:
 Homogenizing brain tissue
 Ultracentrifuging or chromatography
3. Thioflavin T Binding Assay
 Get isolated sample of Aβ
 Incubate sample with dye added
 Screen for fluorescence using screening machine
Step 4. RNA- Seq
 Necessary materials can be purchased from Illumina, a
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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
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
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
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
Bibliography
"Alzheimer's Disease & Dementia | Alzheimer's Association." Alzheimer's Disease & Dementia |
Alzheimer's Association. Alzheimer's Association, n.d. Web. 28 July 2015.
"Tau Protein." Wikipedia. Wikimedia Foundation, n.d. Web. 29 July 2015.
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 2015.
"Amyloid-Degrading Ability of Nattokinase from Bacillus Subtilis Natto."Journal of Agricultural and Food
Chemistry. National Taiwan University, n.d. Web. 29 July 2015.
"Serrapeptase and Nattokinase Intervention for Relieving Alzheimer’s Disease Pathophysiology in Rat
Model." Human and Experimental Toxicology. N.p., n.d. Web. July 2013.
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 2015.
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 2015.
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.
Image Credits
 http://www.webmd.com/alzheimers/ss/slideshow-alzheimers
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overview
http://flipper.diff.org/app/items/info/5017
http://www.uccs.edu/biology-educationalresources/alzheimers-disease.html
http://www.oksfood.com/sushi/nattomaki_hosomaki.html
https://en.wikipedia.org/wiki/Wood_mouse#/media/File:Apode
mus_sylvaticus_bosmuis.jpg
http://www.homeremedycentral.com/en/supplements/aminoacid/nattokinase.html
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503347/
http://www.assay-protocol.com/biochemistry/proteinfibrils/amyloid-beta