Corey A. Shafer and Nicholas M. Kanaan CELLULAR TOXICITY OF MUTANT FORMS OF THE TAU PROTEIN

LET’S DIVE DEEP INTO THE BRAIN

 Alzheimer's disease (AD) and tauopathies  Neural degeneration  memory and cognition  Genetic 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 cascade  Known to reduce tau’s ability to stabilize microtubules  Disrupts anterograde transport THE LANGUAGE OF R5L R5LR5L Original Amino Acid: Arginine Mutated Amino Acid: Leucine Position: 5 th Amino Acid

Does the R5L mutation in tau cause toxicity to cells? QUESTION

STEP 1: RECOMBINANT TAU PROTEINS Mutagenesis Mini-prep Gel Electrophoresis / 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 Antibodies 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

Tau 5TNT1 TOC1

MICROSCOPY ht40 R5L TNT1 TOC1Merge + 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 changes  WT-tau appeared to be equally as toxic to cells  PAD exposure and oligomer formation present lead to toxicity  Future 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 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):  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)  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): REFERENCES