1. Neuronal DEATH in Alzheimer’s Disease Molecular Events Linking Disease Pathologies During Neuronal Demise Jack Reifert FNS 10/30/09 100 billion 10 billion (10%) 200 million (0.2%)

2. Alzheimer’s Disease Quick Facts: Over 100 years since the first diagnosis Clues from inherited forms? Vulnerability of aged neurons? Mark P. Mattson et al., Nature Reviews Neuroscience V-7, P-286, 2006. Most common form of dementia - 4.5 to 4.7 million Americans Average duration from detectable symptoms to death = 8 years Chronic, age dependent disease (10% over 65, >40% over 85) Diagnosis only complete with detection of post-mortem pathologies

3. 2 Pathologies = 2 Protein Aggregates No Aggregates Tangles Plaques & Tangles Nature Medicine 10 , 1055 - 1063 (2004) AD FTDP PD HD ALS CJD

4. Amyloid Beta Plaques AICD Extracellular Domain Amyloid Precursor Protein (APP)   Amyloid-  Insoluble Fibers / Plaques Soluble Oligomers W. E. KLUNK AND C. A. MATHIS, UNIVERSITY OF PITTSBURGH

5. MAPT / Tau Tangles Cairns et al., Journal of Pathology V-204 P-438 2004 N C 441 1 45 74 103 Microtubule Binding Domains Alzheimer’s disease brain Assembled in-vitro from recombinant tau with  K280 FTDP-17 mutation Eckhard Mandelkow et al., Brain Pathology 17 (1) , 83–90 , January 2007 Calpain / Caspase Cleavage Sites

6. Amyloid Cascade Hypothesis APP Dysfunction FAD ? Plaques Tau Dysfunction Tangles FTDP-17 Cell Death LOF - MTs GOF - Aggregation Human Genetics: Biochemistry: Pathology: Other? Experimental Evidence: - A  treatment on wt mouse neurons in culture = cell death while A  treatment on tau -/- neurons = no cell death Rapoport et al. (2002). PNAS. - Tau -/- mice crossed with APP Alzheimer’s mouse model alleviates age . related memory deficits observed in the mouse models with tau present. Robberson et al. (2007). Science.

7. Killing Neurons with Amyloid Beta

8. Killing Neurons with Amyloid Beta Tau Tubulin T=0 4 Hr 8 Hr 24 Hr

9. Rapid Destruction of Tau

10. Tau Disappearance - A Closer Look 3min 48Hr Amyloid Beta 3min 48Hr Amyloid Beta

11. 5 Min 10 Min 15 Min 30 Min 1 Hour 2 Hour 150% 475% 325% 250% 225% 140% Time: % Calpain Activity: % Caspase Activity: 160% 150% 135% 120% 45% 40% N C 441 1 45 74 103 4 Hour 75% 20% Protease Cleavage of Tau - Low MW 52 kD 17 kD 25 341 421 Caspase Cleavage Sites 257 11 44 230 254 267 310 340 394 Calpain Cleavage Sites

12. Kinase Activity - Tau Phosphorylation APP Dysfunction FAD ? Plaques Tau Dysfunction Tangles FTDP-17 Cell Death LOF - MTs GOF - Aggregation Other? Neuronal DEATH after amyloid beta treatment ~ 50% @ 24 Hours Rapid protease activity on tau produces low MW fragments GSK3  CDK5

13. Signaling Summary

14. Tau Targeting Kinases pGSK(S9)

15. Suggested Kinase Activity ERK 1/2 Ras Stress! Cell Death Calpain p35 p25 CDK-5 GSK3 

16. Decreased Phosphorylation? 199/202 217 231 396 396/404 Tau-5 KO UN UN 3’ 10’ 20’ 40’ 1 2 4 8 24

17. How About Phospho-Tau?

18. De-Phosphorylation or Degradation? pTau 181 pTau 199/202 pTau 205 pTau 217 pTau 231 UN 10’ 20’ 40’ 1 2 4 8 24 Tau-1 = de-phospho selective (181-205) 3’ None!

19. Tau Hyperphosphorylation in AD! N C 441 1 45 74 103 257 11 44 230 254 267 310 340 394 Calpain Cleavage Sites pTau 262 pTau 400 pTau 396/404 pTau 413 25 341 421 Caspase Cleavage Sites PHF “Core”

20. Revisit The Model APP Dysfunction FAD ? Plaques Tau Dysfunction Tangles FTDP-17 Cell Death ERK AKT GSK3  CDK5

21. Future Directions: Characterize tau fragments by Mass Spec Determine relative toxicity of fragments alone Evaluate phosphorylation in relation to protease activity Culture experiments In-vitro aggregation assays In-vitro MT polymerization assays Evaluate importance of observed neuronal culture phenomenon in both mouse models and human condition

22. PanTau pTau 217 pTau 231 pTau 396

23. Happy Halloween 2009! Thanks: 1) Feinstein Lab Members / Stu 2) Committee Members : Zach Ma, Joel Rothmann, Les Wilson 3) Friends….. Funding: NIH (RO1 NS035010) California Dept. of Public Health - Alzheimer’s Research Foundation

28. ( a ) Senile plaques in neocortex of Alzheimer disease. © Lewy body in substantia nigra of Parkinson disease. ( d ) Intranuclear polyglutamine inclusion in neocortex of Huntington disease. ( e ) Ubiquitinylated inclusion in spinal cord motor neuron of ALS. ( f ) Protease-resistant PrP in cerebellum of CJD (panel f courtesy of Nigel Cairns). b ) NFTs in hippocampus of FTDP-17 (R406W mutation).

29. 5 Min 10 Min 15 Min 30 Min 1 Hour 2 Hour 150% 475% 325% 250% 225% 140% Time: % Calpain Activity: Tau GAPDH % Caspase Activity: 160% 150% 135% 120% 45% 40% 4 Hour 75% 20%

30. 3min 10min 20min 40min 1 Hour 2 Hours 4 Hours 8 Hours 24 Hours Time After A  Treatment % Relative Activity Survival Kinases Tau Kinases