ALLN induces accumulation of novel amyloid precursor
protein (APP) fragments
Haizhi Wang1,2, Nianli Sang1, Can Zhang3, Ramesh Raghupathi2, Rudoph E. Tanzi3, Aleister Saunders1,2,4
1
Department of Biology, College of Arts and Sciences, Drexel University; 2 Department of Neurobiology
and Anatomy, College of Medicine, Drexel University; 3 Harvard University Massachusetts General Hospital,
Boston; 4 Department of Biochemistry and Molecular Biology, College of Medicine, Drexel University.
Figure 1. Summary of known cleavage sites in APP 1,2,3,4.
Figure 2. ALLN treatment induces accumulation of novel APP CTFs.
10 kD
A, schematic representation of APP695-Myc construct and epitopes of C1/6.1 and
9B11. B, naive HEK293 or HEK293 cells transiently over-expressing APP695-Myc were
treated with Vehicle, 5 μM L685,485 or 5 μM ALLN for 24 hrs before collection. Top, anti Myc
antibody (9B11); bottom, anti APP antibody (C1/6.1). Notice the presence of η-CTF-Myc(30 kD,
red double arrowhead).
C1/6.1,
6E10
G12A 9B11
Aβ TM AICD
Ectodomain
Myc
APP-FL
58
AL
LN
85
L6
Ve
hi
cle
LN
AL
,4
8
,45
ox
Proteasome activity,
% of vehicle
32
G1
M
icl
Ve
h
G1
Ep
cle
i
h
Ve
actin
A
B
MDL28170
ci le
h
e
V
20
μM
40
μM
C
N,
L
AL μM
5
**
**
2,
3
1
G
M μM
20
x,
p
E μM
1
APP-FL
100 kD
APP-FL
100 kD
120
100
80
60
40
20
0
15 kD
10 kD
η-CTF-Myc(30 kD)
η-CTF
15 kD-CTF-Myc(20 kD)
β-CTF-Myc
α-CTF-Myc
AICD-Myc
AICD
actin
References:
1.Vassar, R., et al, 1999. 2. Simons, M., 1996. 3. Weidemann, A., et al, 2002. 4. Zhao, G., et al, 2005
5. Meng, L., et al, 1999.
actin
F
h
e
V
ci le
Talin-FL
50 kD
Talin-cleaved
37 kD
actin
1
5
*
η-CTF
25 kD
20 kD
10 kD
37 kD
G
APP-FL
N
Pepstatin A
Cathepsin G inhibitor I
e
l
M μM μM
M
M
M
c
ci le LN
M
M
i
N
μ
μ
μ
μ
μ 0
h LL
μ 0
h
0 40
L
e
e
1
5
20 40
1
5
2
A
A
V
V
APP-FL
15 kD
15 kD-CTF
β-CTF
α-CTF
Cathepsin L inhibitor II
e
l
c
i
μM μM
μM μM eh
1
5
1 5 V
APP-FL
100 kD
15 kD
25 kD
α-CTF 20 kD
25 kD-CTF
15 kD
15 kD-CTF
β-CTF
α-CTF
AICD
10 kD
actin
10 kD
37 kD
actin
15 kD-CTF
β-CTF
α-CTF
25 kD-CTF
MK-0822
L
L
A
η-CTF
actin
actin
37 kD
μM
APP-FL
15 kD-CTF
β-CTF
α-CTF
15 kD
scrambled Calpastatin
A
25 kD
20 kD
25 kD
20 kD
37 kD
Ve
10 kD
CA-074Me
μM
40
N
L
L
100 kD
α-CTF
10 kD
250 kD
37 kD
100 kD
APP-FL
15 kD-CTF
β-CTF
α-CTF
20
cl
i
h
100 kD
8
η-CTF
25 kD
20 kD
E
25 kD
20 kD
e
D
45
β-CTF-Myc
α-CTF-Myc
AICD-Myc
β-CTF
α-CTF
15 kD-CTF
β-CTF
α-CTF
85
,
η-CTF-Myc(30 kD)
15kD-CTF
η-CTF
120
100
80
60
40
20
0
A, MDL28170 inhibits cathepsin and calpain activitiy. Naive HEK293 cells were treated with MDL28170 for 24 hrs before collection. Note the accumulation
of η-CTFs. B, we then utilize a selective calpain inhibitor, Calpastatin peptide, which is a synthetic peptide corresponding to the active domain of
calpastatin (endogneous inhibitor of calpain). Cells were treated with either calpastatin or scrambled peptide for 24 hrs before collection. No changes in
CTFs are observed. Calpain inhibition is confirmed by a sifinificant decrease in cleaved talin, a known substrate of calpain (B, C). D, Treatment with a general
cathepsin inhibitor (E-64D) induced accumulation of APP-CTFs, including η-CTF. Further pharmacologic studies with selective cathepsin inhibitors reveals
that inhibition of cathepsin L is likely responsible for the accumulation of novel APP fragments(E-H).
E-64D, μM
15 kD
APP
(C1/6.1)
e
32
e
37 kD
L6
AL
L
N
e
icl
Ve
h
,4
L68
5
AL
L
N
e
icl
Ve
h
APP-FL-Myc
Myc
(9B11)
25 kD
20 kD
15 kD-CTF 15 kD
β-CTF
α-CTF
AICD
10 kD
actin
37 kD
10 kD
C
APP-FL
APP-Myc transient
58
Naive
15 kD-CTF
Figure 7. Cathepsin inhibition mediates the accumulation of η-CTF.
Figure 3. ALLN induces same changes in exogenous APP.
A
η-CTF
15 kD
15 kD-CTF
β-CTF
100 kD
APP-FL
Talin-cleaved / Talin-FL
% of Vehicle
15 kD
B
at
in
25 kD
20 kD
η-CTF
10 kD
Vehicle ALLN Triton X-100
25 kD
20 kD
η-CTF
η-CTF
15 kD
20 kD-CTF
18 kD-CTF
15 kD-CTF 10 kD
β-CTF
α-CTF
AICD
η-CTF
20 kD-CTF
18 kD-CTF
15 kD-CTF
β-CTF
α-CTF
AICD
A
15 kD
st
long exposure
p=0.734
Ca
lp
a
long exposure
actin
15 kD-CTF
β-CTF
α-CTF
25 kD
AICD
20 kD
A
N
L
L
25 kD
20 kD
ed
15 kD-CTF
β-CTF
α-CTF
AICD
100 kD
50 kD
N
L
L
**
ra
m
bl
15 kD
η-CTF
120
100
80
60
40
20
0
sc
η-CTF
A
AL
25 kD
20 kD
10 kD
APP-FL
100 kD
h
e
V
Naive HEK293 cells were treated with vehicle, 5 μM ALLN, 20 μM MG132(A) or 1 μM epoxomicin (Epox, a selective proteasome inhibitor, B)5 for 6 hrs before
collection and subject to western analysis with anti APP antibody. Notice accumulation of η-CTF can be observed in ALLN and MG132 treated cells, but not
in Epox treated cells. This suggests that proteasome inhibition is not responsible for η-CTF accumulation, and therefore, these η-CTFs are unlikely
ubiquitinated forms of canonical CTFs. C, Cells were treated with above proteasome inhibitor as indicated for 6 hrs before lysis. In vitro 20S proteasome
activity were measured. Efficient proteasome inhibition is achieved after MG132 and Epox treatment.
icl
actin
B
CHX (long exposure)
e
le
l
c
c
i
i
N
h
L
h
e
L
e
V
A
V
Figure 6. Accumulation of η-CTF is not due to proteasome inhibition.
Ve
h
AL
LN
e
icl
Ve
h
50 kD
B
A
LN
AL
APP-FL
APP-FL
CHX
e
l
c
i
100 kD
e
icl
Ve
h
LN
AL
Ve
hic
le
Naive HEK293 cells were treated with 5 μM ALLN or vehicle for 24 hrs before collection, and
subject to western analysis probing with different anti APP antibody(C1/6.1, 6E10 and G12A,
epitopes shown in Fig 3A). ALLN treatment induced accumulation of canonical α-CTF and
β-CTF, as well as APP intracellular domain (AICD). Interestingly, ALLN treatment also induce
accumulation of several hitherto undocumented APP-CTF: a CTF of 25 kD(red arrowhead,
C1/6.1, 6E10 and G12A), and a CTF of 15 kD(blue arrow, C1/6.1, 6E10 and G12A). The 15 kD-CTF
could potentially be the δ-CTF, previously reported in hippocampal neurons2. Here, we named
C1/6.1
G12A
the CTF of 25 kD as η-CTF. Longer
exposure reveal two more additional
CTFs: 18 kD and 20 kD (orange
arrowhead).
6E10
100 kD
85
50
M
46
LDH cytotoxicity assay
40 42
LN
16
L6
LN
AL
Figure 5. ALLN induced changes are independent of cytotoxicity and protein synthesis.
A, naive HEK293 cells were treated with vehicle, 5 μM ALLN or 2%
N-terminus ~~GLTNIKTEEISEVKMDAEFRHDSGYEVHHQKLVFFAEDVGSNK GAIIGLMVGGVVI ATVIVITLVML KKKQ~~C-terminus Triton X- 100 for 24 hrs before LDH in the medium were measured.
B, naive HEK293 cells were treated with vehicle or 5 μM ALLN with
or without cycloheximide(CHX, 50 μg/ml) for 4 hrs before collection.
δ
β
γ ζ ε
α
1
Ve
hi
cle
,4
85
L6
Ve
hi
cle
LN
AL
Ve
hi
cle
LN
AL
Ve
hi
cle
58
Figure 4. ALLN induced accumulation of novel APP-CTFs can be observed across different cell types.
Abstract:
Naive HeLa cells, Bovine brain microvasculature endothelial cells (BBMEC), H4 neuroglioma and SH-SY5Y human neuroblastoma cells stably
Alzheimer’s disease (AD) is a progressive neurodegenerative
expressing APP, were treated with vehicle, 5 μM L685,485 or 5 μM ALLN for 24 hrs before collection.
HEK293
HeLa
H4/APP
SH-SY5Y/APP
BBMEC
disease characterized by the deposition of amyloid β peptide(Aβ). Aβ
is a proteolytic product of amyloid precursor protein(APP). In this
study, we investigate whether protein degradation plays a role in the
processing of APP. We found that inhibiting protein degradation with
APP-FL
100 kD
ALLN induces the accumulation of novel APP fragments. This effect is
independent of cytotoxicity and protein synthesis. We further
50 kD
showed that inhibition of cathepsin, and not calpain or proteasome
mediates the accumulation of novel APP fragments. Our data
25 kD-CTF
25 kD
suggests that APP undergoes alternative processing which generates
20 kD
10 kD
the novel fragments; these fragments undergoes rapid clearance/
15 kD
15 kD-CTF
degradation via cathepsin (most likely cathepsin L) under
physiological conditions.
H
100 kD
25 kD
20 kD
15 kD
10 kD
37 kD
cathepsin L inhibitor III
ci le N
L
h
μM
μM
L
e
5
1
A
V
APP-FL
25 kD-CTF
15 kD-CTF
β-CTF
α-CTF
AICD
actin
Conclusion: By inhibiting protein degradation, we observed an accumulation of novel APP fragments.
Here, we name the 25 kD-CTF as η-CTF. Our data suggests that this novel CTF is an alternative
processing product of APP, and it is not observed under physiological conditions due to rapid
processing/clearance by protein degradation system, most likely cathepsin L.