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