The Amyloid Hypothesis of Alzheimer Pathogenesis

1. APP Self-assembly Amyloid-b production tau neuronal cell death clinical AD The Amyloid Hypothesis of Alzheimer Pathogenesis

2. SWAN Proteins Wong & Wu APP: Chromosomal Location APP Genetic locus on Chr. 21q21.3 APP is a single copy gene. It exists as one copy per haploid genome.

3. SWAN Proteins Wong & Wu APP is a gene that encodes a complexity of proteins • A single locus: single copy per haploid genome • Multiple splice variant mRNAs • Multiple protein isoforms expressed • Some isoforms are ubiquitous, others neuron specific or T cell-specific • Variety of conserved functional domains throughout length of protein • Isoform differences in major domains • APP interacts with multiple proteins • Mutations throughout protein associated with familial Alzheimer Disease • Cleavage products generated by α-, β- and γ-secretase are involved in both normal and pathogenic protein processing.

4. APP Gene encodes 10 Isoforms produced by Alternative Splice Variants There are three major isoforms: There are seven and probably more additional isoforms. SWAN Proteins Wong & Wu

5. Caspase Zn+/Cu+ Kunitz Hep Hep TM Hep Goα Conserved Functional Protein Domains in APP Suggest Functional Differences between Protein Isoforms SWAN Proteins Wong & Wu

6. SWAN Proteins Wong & Wu APP does more than produce Aβ… but production of Aβ is the critical step for AD.

7. SWAN Proteins Wong & Wu Intracellular Processing of APP Generates a Variety of Cleavage Products APP is Cleaved into the following 12 chains:1- Soluble APP-α     2- Soluble APP-β3- C994- Beta-amyloid protein 425- Beta-amyloid protein 406- C837- P3(42): α-secretase and γ-secretase product8- P3(40): α-secretase and γ-secretase product9- Gamma-secretase C-terminal fragment 59Alternative name(s):            γ-CTF(59)            Amyloid intracellular domain 59              Short name=AICD-59 or AID(59)10- Gamma-secretase C-terminal fragment 57Alternative name(s):            γ-CTF(57)            Amyloid intracellular domain 57              Short name=AICD-57 or AID(57)11- Gamma-secretase C-terminal fragment 50Alternative name(s):            γ-CTF(50)            Amyloid intracellular domain 50              Short name=AICD-50 or AID(50)12- C31 http://journals.prous.com/journals/dnp/20061907/html/dn190411/images/fig1.gif

8. SWAN Proteins Wong & Wu Intracellular APP trafficking

9. APP interacts with many proteins http://string.embl.de/newstring_cgi/show_network_section.pl?taskId=TuwI5V8me0SR&use_java=no&network_flavor=evidence SWAN Proteins Wong & Wu

10. Amyloid Plaques b Amyloid-b (Ab40 vs Ab42) g Genetic Mutations in the Gene for APP Can Cause Alzheimer’s Disease APP

11. b-Secretase: Carries out the first cut that produces the N-terminus of Ab Wolfe. Sci. Am., 2006

12. Co-crystal structure of b-secretase with an inhibitor: coordination of hydroxyl group with the catalytic aspartates Hong et al. Science, 2000, 290, 150-3

13. The Complexities of b-Secretase (b-site APP cleaving enzyme; BACE1) BACE1 splice isoformsAlternative splicing within exons 3 and 4 BACE2 Homolog of BACE1; also cleaves APP. BACE1 substrates APP, neuregulin, ST6Gal 1, Nav1 b2, others Processing of BACE1 Signal sequence and prodomain removal, glycosylation Intracellular localization of BACE1 ER, Golgi, cell surface, endosomes Tissue distribution of BACE1 Ubiquitous but most highly in the brain

14. The Complexities of a-Secretase a-Secretases Metalloproteases ADAM-10, -17, and -9 Where they cut within APP Within the Ab sequence, thereby precluding Ab formation Other substrates These are major sheddases; many other membrane protein substrates How they are regulated ADAM-10 is constitutive, while ADAM-17 is inducible Why they may be important therapeutically Activity can be stimulated by agonists for certain muscarinic receptor subtypes

15. g-Secretase: Performs the second cut that produces the C-terminus of Ab Wolfe. Sci. Am., 2006

16. CTF NTF b Ab D D g e AICD lumen/extracellular Presenilin APP cytosol Wolfe et al. Nature1999, 398, 513. Li et al. Nature2000, 405, 689. Esler et al. Nature Cell Biology2000, 2, 428.

17. Proposed Mechanism of g-Secretase Esler et al. PNAS, 2002, 99, 2720-7

18. lumen/extracellular lumen/extracellular Presenilin SPP S2,b SP S4, g D D D D signal peptide SPP S3, e cytosol NCT Aph-1 cytosol Pen-2 CTF 1999 2003 NTF lumen/extracellular lumen/extracellular S1P Spitz S2P Rhomboid S H Rhomboid HEXXH S2P LDG SREBP Reg cytosol cytosol 2001 1997 Wolfe and Kopan. Science2004

19. g-Secretase: A Complex and Unusual Protease NCT Aph-1 Pen-2 CTF NTF Takasugi et al. Nature2003 Kimberly et al. PNAS2003 Edbauer et al. Nat Cell Biol2003 1:1:1:1 Stoichiometry: Sato et al. JBC 2007

20. The size of the g-secretase complex is ~230 kDa by scanning transmission electron microscopy Osenkowski et al. JMB 2009.

21. CryoEM Structure of g-Secretase Osenkowski et al. JMB 2009.

22. The Complexities of g-Secretase PS mutations Over 100 FAD-associated mutations PS1 vs PS2 PS1 is the major player, both in AD and biology Other processing events at PS Caspase cleavage gives short CTF Aph1 isoforms Aph1-a and Aph1-b; Aph1-a1 and Aph1-a2 Nicastrin glycosylation 16 glycosylation sites that occur in transit from the ER Intracellular localization ER, Golgi, cell surface, endosomes, mitochondria

23. CTF NTF S2 Np3 D D S4 S3 NICD lumen/extracellular Presenilin Notch cytosol DeStrooper et al. Nature1999, 398, 518.

24. Ihara Model of Processive Proteolysis of APP APP Ab38/40 Ab45/46 Ab42/43 Ab48/49 b g’ g z e AICD

25. When Tau Goes Bad Wolfe. Sci. Am., 2006

26. SWAN Proteins Wong Tau: Chromosomal Location MAPT Genetic locus on Chr. 17q21.1 Source: MAPT Mapviewer (NCBI) WT: MAPT is a single copy gene. It exists as one copy per haploid genome. Disease: MAPT gene mutations have been associated with several neurodegenerative disorders such as Alzheimer's disease, Pick's disease, frontotemporal dementia, cortico-basal degeneration and progressive supranuclear palsy.

27. SWAN Proteins Wong Tau is a gene that encodes a complexity of proteins • A single locus: single copy per haploid genome • Multiple splice variant mRNAs • Mulitiple protein isoforms expressed • tau is not only present in neurons but is also clearly present in oligodendroglia and astrocytes • Variety of conserved functional domains throughout length of protein • Isoform differences in major domains • Tau interacts with multiple proteins • ~40 FTDP-associated mutations

28. Alternative Splicing of Tau in FTDP-17 Wolfe. JBC 2009

29. Stem Loop Structure and Exon 10 Splicing Wolfe. JBC 2009

30. Tau protein is highly phosphorylated: Phosphorylation state appears to be critical to disease SWAN Proteins Wong

31. Acknowledgments Wolfe Lab Proteases William Esler Taylor Kimberly Chittaranjan Das Frédéric Bihel Anna Kornilova Patrick Fraering Pam Osenkowski Toru Sato Sarav Narayanan Omar Quintero-Monzon Morgan Martin Marty Fernandez RNA Christine Donahue Yang Liu Eleanor Peacey Karen Mowrer Jean-François Fisette Collaborators Proteases Dennis Selkoe Rafi Kopan Bart De Strooper Todd Golde Huilin Li RNA Ken Kosik Jane Wu Eriks Rosners Marcie Glicksman Gabriele Varani LEAD Corinne Augelli-Szafran Han-Xu Wei Dai Lu Jing Zhang Yongli Gu Funding National Institutes of Health Alzheimer’s Association ISOA/ADDF