Corey A. Shafer

andNicholas M.

Kanaan

CELLULAR TOXICITY OF MUTANT FORMS OF THE

TAU PROTEIN

LET’S DIVE DEEP INTO THE BRAIN

Alzheimer's disease (AD) and tauopathiesNeural degeneration

memory and cognitionGenetic mutations in the tau gene (MAPT) Paperclip fold Amino acids 2-18 of tau

phosphatase activating domain (PAD) activation of Protein Phosphatase 1 (PP1) and Glycogen Synthase

Kinase 3 (GSK3) inhibits anterograde fast axonal transport (FAT)

THE BASICS

ROLE OF PAD IN FAT DYSFUNCTION

Point mutation Located at the beginning of PAD which activates the

PP1/GSK3 signaling cascadeKnown to reduce tau’s ability to stabilize microtubulesDisrupts anterograde transport

THE LANGUAGE OF R5L

R5LOriginal Amino Acid:Arginine

Mutated Amino

Acid:Leucine

Position:5th Amino

Acid

Does the R5L mutation in tau cause toxicity to cells?

QUESTION

STEP 1: RECOMBINANT TAU PROTEINS

Mutagenesis

Mini-prep

Gel Electrophores

is / DNA Sequencing

Maxi-prep

STEP 2: CELLTITER-GLO ASSAY

Transfection

Change Media

GloMax Multi-Detection System

Plate Cells

HEK-293 cells transfection:•Mock (control)•Ht40 (wild-type tau)•R5L mutation

STEP 3: ELISA

Capture Antibodi

es

Detection

Antibodies

Washing

Spectrophotometer

Capture Antibodies: •Tau5: total tau•TOC1: PAD exposed•TNT1: oligomers present

Detection Antibodies:•R1

STEP 4: MICROSCOPY

Nikon A1+ Confocal Microscopy System

TNT1

DAPI

TOC1

DAP1: Stains live and fixed cells

CELLTITER-GLO ASSAY

* True (error free) value

ELISA

ELISA

Tau 5 TNT1 TOC1

MICROSCOPY

ht40

R5L

TNT1 TOC1 Merge + DAPI

What we know…Alzheimer’s disease affects roughly 5.2 million Americans each

Economic, social and healthcare burden

No effective treatments

What we learned…Better understanding of what might cause tauopathies

May lead to viable and effective therapeutic treatments

R5L was toxic to cells and exhibited known pathogenic changesWT-tau appeared to be equally as toxic to cells PAD exposure and oligomer formation present lead to toxicityFuture studies are required to determine the underlying molecular mechanisms of R5L tau-mediated cell toxicity

CONCLUSION

These findings would not have existed without the help of:•my mentor, Dr. Nicholas Kanaan, who took me into his lab and provided me with this unique research experience•the members of the Kanaan lab that guided me in my research methods (especially Dr. Benjamin Combs and Tessa Grabinski)•my teachers, Mrs. Leigh Eriks and Mrs. Jessica Malecki who have inspired my love of science•my mother, Ms. Shawn Shafer who always believed in me•my fellow research students who have supported me throughout the entire process

AKNOWLEDGEMENTS

Kanaan NM, Collier TJ, Marchionini DM, McGuire SO, Fleming MF, Sortwell CE. (2006) Exogenous erythropoietin provides neuroprotection of grafted dopamine neurons in a rodent model of Parkinson's disease. Brain Res. 2006 Jan. 12;1068(1):221-9

Kannan NM, Morfini GA, LaPointe NE, Pigino GF, Patterson KR, Song Y, Andreadis A, Fu Y, Brady ST, Binder LI (2011) Pathogenic Forms of Tau Inhibit Kinesin-Dependent Axonal Transport through a Mechanism Involving Activation of Axonal Phototransferases. The Journal of Neuroscience. 31(27):9858-9868.

Kannan NM, Pigino GF, Brady ST, Lazarov O, Binder LI, Morfini GA (2012) Axonal Degeneration in Alzheimer's Disease: When Signaling Abnormalities Meet the Axonal Transport System. Experimental Neurology. 246(2013) 44-53.

Morfini G, et. Al (2009) Axonal Transport Defects in Neurodegenerative Diseases. The Journal of Neuroscience. 29(41):12776 –12786

Ward SM, Himmelstein DS, Lancia JK, Binder LI (2012) Tau oligomers and tau toxicity in neurodegenerative disease. Biochemical Society Transactions. 40(4):667-71.

REFERENCES