the effect of pseudophosphorylated tau protein in the absence of fibrillar beta amyloid
DESCRIPTION
Alzheimer\'s Disease Research presented at Monmouth University in April, 2010TRANSCRIPT
The Effect of Pseudophosphorylated Tau Protein in the Absence of
Fibrillar Beta-Amyloid
By,Robert Dorfman
Purpose
Examine role tau proteins play in causing cell death attributed to Alzheimer’s disease (AD)
Find the cause of AD
BackgroundFatal brain disease
Most common cause of dementiaMassive brain shrinkageSixth leading cause of death in U.S.
Normal Alzheimer’s
AD Statistics5,000,000
Americans suffer30,000,000
Worldwide$180-Billion
(U.S.)No effective
treatment available
Tau ProteinsMicrotubule – associated
phosphoprotein (MAP)Microtubules make up
cytoskeleton of cellNormal tau
3 moles of phosphate/mole of protein
Hyperphosphorylated tau7-10 moles of
phosphate/mole of protein Courtesy of JYI.org
NEUROFIBRILLARY DEGENERATION
1 2 3 4 5 62 3 4 5
Courtesy of Alonso et al.
Pathogenesis of Alzheimer Disease
TauTangles
-AmyloidPlaques
ADAD
BACKGROUNDTau is Essential to Beta-Amyloid-
Induced NeurotoxicityMark 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
HYPOTHESIS
Pseudophosphorylated tau vectors, along with a mutation tau vector, are toxic for cells lacking fibrillar beta-amyloidToxicity measured by caspase-3
activationEnzyme that is essential for cell death
MATERIALS & METHODS
Preparation of E. coli4 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
METHODS
Isolate and Purify DNA Centrifuge and filter to obtain
plasmid DNA
UV spectrophotometryQuantify amount of DNA
Transient TransfectionInsert plasmid DNA into nucleus of kidney fibroblasts and make
cells express tau vectors
METHODSFixation
Freezes internal components of cells with gluteraldehyde
Permeabilization
Allows antibodies to be taken in
ImmunocytochemistryStaining of caspase-3
Confocal ImagingTook pictures and counted cells
Trial #1 - Normal Tau
DAPI Tau
Caspase-3 Overlay
Trial #1 - Mutated Tau
EGFP 8A
TauDAPI
Caspase-3 Overlay
Trial #1 - Pseudophosphorylated Normal
EGFP 6B
DAPI Tau
Caspase-3 Overlay
Trial #1 - Pseudophosphorylated Mutated
DAPI Tau
Caspase-3 Overlay
Trial #1 - Normal Tau
DAPI Tau
Caspase-3 Overlay
Trial #1 - Pseudophosphorylated Mutated
DAPI Tau
Caspase-3 Overlay
Trial #2 - Normal Tau
TauDAPI
Caspase-3 Overlay
Trial #2 - Mutated Tau
DAPI Tau
Caspase-3 Overlay
Trial #2 - Pseudophosphorylated Normal
TauDAPI
Caspase-3 Overlay
Trial #2 - Pseudophosphorylated Mutated
TauDAPI
Caspase-3 Overlay
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
Percentage of Caspase-3 Activation
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
SUMMARY OF RESULTS & CONCLUSIONS
Pseudophosphorylated tau vectors caused caspase-3 activation in cells lacking beta-amyloidIndependent of Beta-amyloidNon-mutated also toxic
Tau potentially responsible for cell death characteristics of AD Tangles present before plaques in AD
brainPossible cause of AD
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 neuronsMore trials
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. "Cytosolic Abnormally Hyperphosphorylated Tau But Not Paired Helical Filaments Sequester Normal MAPs and Inhibit Microtubule Assembly." 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. "Tau is essential to β-amyloid-induced neurotoxicity." Proceedings of the National Academy of Sciences of the United States of America 99.9 (30 Apr. 2002): 6364.
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 SchoolMy Mom and My GrandmaJSHS