Your SlideShare is downloading. ×
The Effect Of Pseudophosphorylated Tau Protein In The Absence Of Fibrillar Beta Amyloid
Upcoming SlideShare
Loading in...5
×

Thanks for flagging this SlideShare!

Oops! An error has occurred.

×

Saving this for later?

Get the SlideShare app to save on your phone or tablet. Read anywhere, anytime - even offline.

Text the download link to your phone

Standard text messaging rates apply

The Effect Of Pseudophosphorylated Tau Protein In The Absence Of Fibrillar Beta Amyloid

661
views

Published on

Alzheimer\'s Disease Research presented at Monmouth University in April, 2010

Alzheimer\'s Disease Research presented at Monmouth University in April, 2010


0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total Views
661
On Slideshare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
5
Comments
0
Likes
0
Embeds 0
No embeds

Report content
Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
No notes for slide

Transcript

  • 1. The Effect of Pseudophosphorylated Tau Protein in the Absence of Fibrillar Beta-Amyloid By, Robert Dorfman
  • 2. Purpose
    • Examine role tau proteins play in causing cell death attributed to Alzheimer’s disease (AD)
    • Find the cause of AD
  • 3. Background
    • Fatal brain disease
      • Most common cause of dementia
      • Massive brain shrinkage
      • Sixth leading cause of death in U.S.
    Normal Alzheimer’s
  • 4. AD Statistics
    • 5,000,000 Americans suffer
      • 30,000,000 Worldwide
    • $180-Billion (U.S.)
    • No effective treatment available
  • 5. Tau Proteins
    • Microtubule – associated phosphoprotein (MAP)
      • Microtubules make up cytoskeleton of cell
    • Normal tau
      • 3 moles of phosphate/mole of protein
    • Hyperphosphorylated tau
      • 7-10 moles of phosphate/mole of protein
    Courtesy of JYI.org
  • 6. NEUROFIBRILLARY DEGENERATION 1 2 3 4 5 6 2 3 4 5 Courtesy of Alonso et al.
  • 7. Pathogenesis of Alzheimer Disease AD Tau Tangles  -Amyloid Plaques
  • 8. BACKGROUND
    • Tau is Essential to Beta-Amyloid-Induced Neurotoxicity
        • Mark Rapoport & Adriana Ferreira
      • Neurons depleted of tau did not degenerate in presence of A-beta
      • Cells did degenerate with re-expression of hyperphosphorylated tau
      • According to the Alzheimer’s Association , neurofibrillary tangles appear in AD brain before amyloid-beta plaques
  • 9. HYPOTHESIS
    • Pseudophosphorylated tau vectors, along with a mutation tau vector, are toxic for cells lacking fibrillar beta-amyloid
      • Toxicity measured by caspase-3 activation
        • Enzyme that is essential for cell death
  • 10. MATERIALS & METHODS
    • Preparation of E. coli
      • 4 E. coli samples containing different tau vectors
    EGFP WT normal tau, control EGFP R406W mutated tau, control EGFP 6B Pseudophosphorylated normal tau, variable EGFP 8A Pseudophosphorylated mutated tau, variable
  • 11. METHODS
    • Isolate and Purify DNA
    • Centrifuge and filter to obtain plasmid DNA
    • UV spectrophotometry
    • Quantify amount of DNA
    • Transient Transfection
    • Insert plasmid DNA into nucleus of kidney fibroblasts and make cells express tau vectors
  • 12. METHODS
    • Fixation
    • Freezes internal components of cells with gluteraldehyde
    • Permeabilization
    • Allows antibodies to be taken in
    • Immunocytochemistry
    • Staining of caspase-3
    • Confocal Imaging
    • Took pictures and counted cells
  • 13. Trial #1 - Normal Tau DAPI Tau Caspase-3 Overlay
  • 14. Trial #1 - Mutated Tau EGFP 8A Tau DAPI Caspase-3 Overlay
  • 15. Trial #1 - Pseudophosphorylated Normal EGFP 6B DAPI Tau Caspase-3 Overlay
  • 16. Trial #1 - Pseudophosphorylated Mutated DAPI Tau Caspase-3 Overlay
  • 17. Trial #1 - Normal Tau DAPI Tau Caspase-3 Overlay
  • 18. Trial #1 - Pseudophosphorylated Mutated DAPI Tau Caspase-3 Overlay
  • 19. Trial #2 - Normal Tau Tau DAPI Caspase-3 Overlay
  • 20. Trial #2 - Mutated Tau DAPI Tau Caspase-3 Overlay
  • 21. Trial #2 - Pseudophosphorylated Normal Tau DAPI Caspase-3 Overlay
  • 22. Trial #2 - Pseudophosphorylated Mutated Tau DAPI Caspase-3 Overlay
  • 23. Percentage of Caspase-3 Activation Trial #1 - % Caspase-3 Activation Trial #2 - % Caspase-3 Activation Pseudo Mutated 85.2 92 Mutated 66 52.3 Normal 0 16.9 Pseudo Normal 79 90
  • 24. Percentage of Caspase-3 Activation
  • 25. Statistical Analysis
    • Two-Proportion Z test
    • Compared caspase-3 activated cell amount in treatment vs. Normal WT tau
    • All values significant at a p-value of 0.05
    Trial #1 – P - Values Trial #2 – P - Values Normal vs. Mutated 8.159 x 10 -9 9.614 × 10 -11 Normal vs. Pseudo Normal 5.900 × 10 -12 0 Normal vs. Pseudo Mutated 0 0
  • 26. SUMMARY OF RESULTS & CONCLUSIONS
    • Pseudophosphorylated tau vectors caused caspase-3 activation in cells lacking beta-amyloid
      • Independent of Beta-amyloid
      • Non-mutated also toxic
    • Tau potentially responsible for cell death characteristics of AD
      • Tangles present before plaques in AD brain
      • Possible cause of AD
  • 27. Possible Expansions
    • More emphasis on Tau hypothesis in AD research
    • Future medications should focus on stopping or reversing hyperphosphorylation of tau
    • Future plans include:
      • Use human hippocampal neurons
      • More trials
  • 28. SOURCES
    • Alonso, Alejandra del C., B. Li, I. Grundke-Iqbal, and K. Iqbal. "Mechanism of Tau-Induced Neurodegeneration in Alzheimer Disease and Related Tauopathies." Current Alzheimer Research 5.4 (Oct. 2008): 375-384.
    • “ Alzheimer’s Facts and Figures”. Alzheimer’s Association . Alzheimer’s Association. 3 Dec. 2008. < http://www.alz.org alzheimers_disease_facts_figures.asp >.
    • Iqbal, Khalid, Alejandra del C. Alonso, and Inge Grundke-Iqbal. &quot;Cytosolic Abnormally Hyperphosphorylated Tau But Not Paired Helical Filaments Sequester Normal MAPs and Inhibit Microtubule Assembly.&quot; Journal of Alzheimer's Disease 14.4 (Dec. 2008): 365-370.
    • Rapoport, Mark, Hana N. Dawson, Lester I. Binder, Michael P. Vitek, and Adriana Ferreira. &quot;Tau is essential to β-amyloid-induced neurotoxicity.&quot; Proceedings of the National Academy of Sciences of the United States of America 99.9 (30 Apr. 2002): 6364.
  • 29. Acknowledgements:
    • Professor Alejandra del Carmen Alonso of the CUNY: College of Staten Island, Mentor
    • Christopher Corbo of the Center for Developmental Neuroscience
    • Susan Seigel, Mentor at Freehold High School
    • Michael Ramdeen of Freehold High School
    • My Mom and My Grandma
    • JSHS