Sonic Hedgehog Pathway Components Implicated in APP Metabolism
Gabriel Horwitz, Michal Sharoni, Anna Vorobyeva, Aleister Saunders
Drexel University, Biology Department
300
50
shRNA 2
Percentage knockdown of GLi3 versus
Scrambled
shRNA 3
shRNA 4
shRNA 5
CTFs/FL
Percentage Change versus scrambled
shRNA 5
shRNA 4
shRNA 3
shRNA 2
shRNA 1
SHRNA 1
SHRNA 2
SHRNA 3
SHRNA 4
SHRNA 5
CTF/actin
CONTROL
SHRNA 1
SHRNA 2
SHRNA 3
shRNA's
SHRNA 4
Figure 3. HeLa cells were plated and grown to 80% confluence in DMEM 10%
FBS. Cells were transfected with 5 shRNA’s to knockdown Gli3 expression and
incubated for 24 hours. Antibody c-terminal anti APP c1/6.1 was used to detect
full-length APP and APP-CTFs. Graphs show no significant affect on CTF
accumulation as a result of the knockdown.
SHRNA 5
•
Down-regulation of Gli3 seems to have an affect of full length APP and
should be clarified.
•
Our lab has previously shown that knockdown of Gli1 and Gli2 in HeLa cells
do not exhibit any changes in APP-CTF accumulation and therefore we
have experimented with Gli3.
•
Preliminary data from our laboratory shows that pharmacological inhibition
of Patch1 leads to an accumulation in APP-CTFs. Therefore, we would like
to investigate Patch1 and its relationship to APP metabolism.
shRNA 5
Figure 3. Knockdown of Gli3 Shows No Affect on
APP Metabolism
The data showing no significant change CTF accumulation with molecular
down-regulation of Gli3 seems to indicate Gli3 is not involved in APP
metabolism. Further experiments are needed to verify these results.
shRNA 4
•
shRNA 3
Figure 2. HeLa cells were plated and grown to 80% confluence in DMEM 10% FBS. Cells were
simultaneously transfected with overexpression of myc-Gli3 and 5 shRNA’s for knockdown and
incubated for 24 hours. Antibody c-myc (by Roche) was used to detect myc-Gli3 (around 180
kD). Graph shows expression of Gli3-myc decreased with knockdown mediated by shRNA’s.
Control
Mammalian Shh Signaling Pathway
100
CONTROL
shRNA 2
•
150
FL/actin
Myc-Gli3
shRNA 1
•
•
•
•
200
Control
Figure 2. Successful Knockdown of Gli3
(www.Vanderbilt.edu)
Alzheimer’s disease (AD) is the most common form of dementia for which
there is no cure
Age is the greatest risk factor
Two pathways of APP proteolysis, non-amyloidogenic and amyloidogenic.
Amyloidogenic pathway produces Aβ
Our lab identified components of the Sonic hedgehog (Shh) signaling
pathway as regulators of APP metabolism
Here we show the effects of modulating two Shh components, Gli3 and
Ptch1
250
0
Background & Significance
•
PERCENTAGE CHANGE VERSUS SCRAMBLED
Figure 1. Plasmids containing both myc-Gli3 and myc-Patch1 were purified using miniprep and maxi-prep protocols. DNA electrophoresis showed myc-Ptch1 and myc-Gli3,
both with base plasmid pCS2, migrated to the correct size.
shRNA 1
Alzheimer’s disease (AD) is a neurodegenerative disease characterized by
memory loss and neuronal death. AD is initiated by a sequential proteolytic
cleavage of amyloid precursor protein (APP), leading to a formation of Aβ
fragments as plaques. Strong evidence suggests, Aβ plays a role in AD
pathogenesis. This would indicate any modulation in APP metabolism could
lead to a change in Aβ levels and affect AD initiation. Certain modulators of
APP metabolism have been introduced, however there has yet to be a
successful therapy developed. Previous data from our lab , utilizing in vitro and
in vivo models, demonstrated that inhibition of Shh pathway via Cyclopamine
results in altered APP processing and Aβ generation. It is known that
Cyclopamine acts as a Sonic hedgehog (Shh) signaling pathway inhibitor. The
major players in the Shh pathway are sonic hedgehog peptide (shh),
Smoothened (Smo), patched1 (Ptch1) and Gli1, 2 and 3 transcription factors.
In this research I monitored changes in APP metabolism through downregulation of Gli3 and Ptch1 by following changes in APP c-terminal fragment
(CTF) accumulation. Our study is the first to indicate a connection between
Shh signaling pathway and APP metabolism and could lead to development of
novel therapeutic target.
Figure 1. Purification of myc-Gli3 and myc-Patch1
Control
Abstract
FL APP
Conclusions & Future Direction
ACTIN
Figure 1. Shh binds Patched (Ptch1), allowing Smoothened to migrate into cell.
Gli transcription factors are then activated by Smo. These factors either induce
or repress transcription within the nucleus. In the absence of Shh, Patch will
inhibit Smo activities.
Acknowledgements
APP-CTF
Thanks to Dr. Aleister Saunders, Dr. Michal Sharoni and Dr. Anna Vorobyeva