The Amyloid Hypothesis
of Alzheimer Pathogenesis
APP
Amyloid-b
production
Self-assembly
tau
neuronal
cell death
clinical AD
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
Conserved Functional Protein Domains in APP Suggest
Functional Differences between Protein Isoforms
Caspase
Zn+/Cu+
Hep
Kunitz
Hep
Hep
Goα
TM
Hep
Zn+/Cu+
Kunitz
Hep
Hep
Hep
β
α
γ
G oα
Casp
Isoform APP770 P05067-1
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Isoform APP751 P05067-8
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Isoform APP695 P05067-4
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Isoform L-APP677 P050673
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Isoform L-APP696 P050675
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Isoform L-APP733 P050677
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Isoform APP714 P05067-6
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Isoform APP305 P05067-2
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Isoform L-APP752 P050679
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Isoform APP639 P05067-10
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Isoform
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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 42
5- Beta-amyloid protein 40
6- 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)
http://journals.prous.com/journals/dnp/20061907/html/dn190411/images/fig1.gif
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
http://string.embl.de/newstring_cgi/show_network_section.pl?taskId=TuwI5V8me0SR&use_java=no&network_fl
avor=evidence
SWAN Proteins Wong & Wu
APP
Binds
UniProt
APP
Binds
UniProt
APBA1
Q02410
HSD17B10
Q99714
APBB1
O00213
MAPK8IP
1
Q9UQF2
Apbb1
P46933
Mapk8ip1
Q9WVI9-1
APBB2
Q92870
MAPT
P10636
Apbb3
O35827
MAPT
P10636-8
APLP1
P51693
PRNP
P04156
APLP2
Q06481
PSEN1
P49768
APOA1
P02647
PSEN2
P49810
CHRNA7
P36544
SHC1
P29353
Chrna7
Q05941
SHC3
Q92529
CNTN3
Q9P232
Slc5a7
Q8BGY9
CNTN4
Q8IWV2
TGFB1
P011371
FLOT1
O75955
TGFB2
P61812
HOMER2
Q9NSB8
TP53BP2
Q13625
HOMER3
Q9NSC5
Genetic Mutations in the Gene for APP
Can Cause Alzheimer’s Disease
APP
Amyloid
Plaques
b
Amyloid-b (Ab40 vs Ab42)
g
b-Secretase: Carries out the first cut
that produces the N-terminus of Ab
Wolfe. Sci. Am., 2006
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
The Complexities of b-Secretase
(b-site APP cleaving enzyme; BACE1)
BACE1 splice isoforms
Alternative 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
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
g-Secretase: Performs the second cut
that produces the C-terminus of Ab
Wolfe. Sci. Am., 2006
lumen/extracellular
Presenilin
APP
CTF
NTF
b
Ab
D
g
D
e
cytosol
O
Ph
OH
O
N
H
Ph
NH
O
O
NH
AICD
NH-Bt
Ph
O
Br H O
N
O
F F H O
N
N
H OH O
N
H
OBt
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 g-Secretase
Esler et al. PNAS, 2002, 99, 2720-7
lumen/extracellular
lumen/extracellular
Presenilin
SPP
S2, b
S4, g
D
D
D
SPP
S3, e
signal peptide
D
SP
cytosol
NCT
Aph-1
Pen-2
cytosol
CTF
1999
2003
NTF
lumen/extracellular
lumen/extracellular
S1P
S2P
Spitz
Rhomboid
S
H
Rhomboid
HEXXH
S2P
cytosol
cytosol
1997
2001
Wolfe and Kopan. Science 2004
g-Secretase: A Complex and Unusual Protease
NCT
Aph-1
Pen-2
CTF
NTF
Takasugi et al. Nature 2003
Kimberly et al. PNAS 2003
Edbauer et al. Nat Cell Biol 2003
1:1:1:1 Stoichiometry: Sato et al. JBC 2007
The size of the g-secretase complex is ~230 kDa
by scanning transmission electron microscopy
Osenkowski et al. JMB 2009.
CryoEM Structure of g-Secretase
Osenkowski et al. JMB 2009.
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
lumen/extracellular
Presenilin
Notch
CTF
NTF
S2
Np3
D
D
S4
S3
cytosol
NICD
DeStrooper et al. Nature 1999, 398, 518.
APP
Ihara Model of Processive Proteolysis of APP
Ab45/46
Ab48/49
Ab42/43
b
e
g
z
AICD
g’
Ab38/40
When Tau
Goes Bad
Wolfe. Sci. Am., 2006
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.
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
SWAN Proteins Wong
Alternative Splicing of Tau in FTDP-17
Wolfe. JBC 2009
Stem Loop Structure and Exon 10 Splicing
Wolfe. JBC 2009
Tau protein is highly phosphorylated: Phosphorylation state
appears to be critical to disease
SWAN Proteins Wong
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
Acknowledgments
Collaborators
LEAD
Corinne Augelli-Szafran
Han-Xu Wei
Dai Lu
Jing Zhang
Yongli Gu
Funding
National Institutes of Health
Alzheimer’s Association
ISOA/ADDF
Proteases
Dennis Selkoe
Rafi Kopan
Bart De Strooper
Todd Golde
Huilin Li
RNA
Ken Kosik
Jane Wu
Eriks Rosners
Marcie Glicksman
Gabriele Varani