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

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.

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.

SWAN Proteins Wong & Wu 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 IsoformHepZn + /Cu + KunitzHep βαγGoαGoαCasp Isoform APP770Isoform APP770 P Isoform APP751Isoform APP751 P Isoform APP695Isoform APP695 P Isoform L-APP677Isoform L-APP677 P Isoform L-APP696Isoform L-APP696 P Isoform L-APP733Isoform L-APP733 P Isoform APP714Isoform APP714 P Isoform APP305Isoform APP305 P Isoform L-APP752Isoform L-APP752 P Isoform APP639Isoform APP639 P TM Kunitz Hep Zn + /Cu + Hep GoαGoα Caspase Conserved Functional Protein Domains in APP Suggest Functional Differences between Protein Isoforms

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

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-β 3- C99 4- Beta-amyloid protein Beta-amyloid protein C83 7- P3(42): α-secretase and γ-secretase product 8- P3(40): α-secretase and γ-secretase product 9- Gamma-secretase C-terminal fragment 59 Alternative name(s): γ-CTF(59) Amyloid intracellular domain 59 Short name=AICD-59 or AID(59) 10- Gamma-secretase C-terminal fragment 57 Alternative name(s): γ-CTF(57) Amyloid intracellular domain 57 Short name=AICD-57 or AID(57) 11- Gamma-secretase C-terminal fragment 50 Alternative name(s): γ-CTF(50) Amyloid intracellular domain 50 Short name=AICD-50 or AID(50) 12- C31

SWAN Proteins Wong & Wu Intracellular APP trafficking

SWAN Proteins Wong & Wu APP interacts with many proteins avor=evidence APP Binds UniProtAPP Binds UniProt APBA1Q02410HSD17B10Q99714 APBB1O00213MAPK8IP 1 Q9UQF2 Apbb1P46933Mapk8ip1Q9WVI9-1 APBB2Q92870MAPTP10636 Apbb3O35827MAPTP APLP1P51693PRNPP04156 APLP2Q06481PSEN1P49768 APOA1P02647PSEN2P49810 CHRNA7P36544SHC1P29353 Chrna7Q05941SHC3Q92529 CNTN3Q9P232Slc5a7Q8BGY9 CNTN4Q8IWV2TGFB1P01137P FLOT1O75955TGFB2P61812 HOMER2Q9NSB8TP53BP2Q13625 HOMER3Q9NSC5

  APP Amyloid-  (A  40 vs A  42) Amyloid Plaques Genetic Mutations in the Gene for APP Can Cause Alzheimer’s Disease

 -Secretase: Carries out the first cut that produces the N-terminus of A  Wolfe. Sci. Am., 2006

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

BACE1 splice isoforms Alternative splicing within exons 3 and 4 BACE2 Homolog of BACE1; also cleaves APP. BACE1 substrates APP, neuregulin, ST6Gal 1, Nav1  2, 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 The Complexities of  -Secretase (  -site APP cleaving enzyme; BACE1)

 -Secretases Metalloproteases ADAM-10, -17, and -9 Where they cut within APP Within the A  sequence, thereby precluding A  formation 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 The Complexities of  -Secretase Other substrates These are major sheddases; many other membrane protein substrates

 -Secretase: Performs the second cut that produces the C-terminus of A  Wolfe. Sci. Am., 2006

  D D lumen/extracellular cytosol Presenilin  NTF CTF AA AICD APP Wolfe et al. Nature 1999, 398, 513. Li et al. Nature 2000, 405, 689. Esler et al. Nature Cell Biology 2000, 2, 428.

Proposed Mechanism of  -Secretase Esler et al. PNAS, 2002, 99,

SREBP Reg S1P S2P lumen/extracellular cytosol S2P SPP SP lumen/extracellular cytosol signal peptide SPP S4  S2,  D D lumen/extracellular cytosol Aph-1 NCT CTF NTF Pen-2 Presenilin S3,  Spitz Rhomboid S H lumen/extracellular cytosol Rhomboid Wolfe and Kopan. Science 2004 D D HEXXH LDG

Aph-1 NCT NTF Pen-2  -Secretase: A Complex and Unusual Protease Takasugi et al. Nature 2003 Kimberly et al. PNAS 2003 Edbauer et al. Nat Cell Biol :1:1:1 Stoichiometry: Sato et al. JBC 2007 CTF

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

CryoEM Structure of  -Secretase Osenkowski et al. JMB 2009.

PS mutations Over 100 FAD-associated mutations PS1 vs PS2 PS1 is the major player, both in AD and biology Aph1 isoforms Aph1-  and Aph1-  ph1-  and Aph1-  2 Nicastrin glycosylation 16 glycosylation sites that occur in transit from the ER Other processing events at PS Caspase cleavage gives short CTF Intracellular localization ER, Golgi, cell surface, endosomes, mitochondria The Complexities of  -Secretase

S4 S2 D D lumen/extracellular cytosol Presenilin S3 NTF CTF Np3 NICD Notch DeStrooper et al. Nature 1999, 398, 518.

APP  AICD   A  48/49  A  45/46A  42/43 ’’ A  38/40 Ihara Model of Processive Proteolysis of APP

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

SWAN Proteins Wong Tau: Chromosomal Location MAPT Genetic locus on Chr. 17q21.1 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. Source: MAPT Mapviewer (NCBI)MAPT Mapviewer

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

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

Stem Loop Structure and Exon 10 Splicing Wolfe. JBC 2009

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

Acknowledgments Funding National Institutes of Health Alzheimer’s Association ISOA/ADDF 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