HAL Id: hal-01927452
https://hal.archives-ouvertes.fr/hal-01927452
Submitted on 19 Nov 2018
HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
FoxO3a overexpression prevents both glycogen overload and autophagic buildup in Pompe disease
Julien Pichon, Lydie Lagalice, Johan Deniaud, Candice Babarit, Virginie Maurier, Laurence Dubreil, Thibaut Larcher, Eduard Ayuso, Carine Ciron,
Karl Rouger, et al.
To cite this version:
Julien Pichon, Lydie Lagalice, Johan Deniaud, Candice Babarit, Virginie Maurier, et al.. FoxO3a overexpression prevents both glycogen overload and autophagic buildup in Pompe disease. 26th annual Congress of the ESGCT, Oct 2018, Lausanne, Switzerland. Human Gene Therapy, 29 (12), 2018. �hal-01927452�
Background and Objectives
Fundings: This research was supported by the grant from the ministère de l’Enseignement supérieur et de la Recherche
Material and Methods
1- or 2- day-old
IV injection of PBS/AAV9-CMV-FoxO3a
(10
11vg/animal)
4 month-old
Euthanasia
Gaa
+/+: n = 5 male
Gaa
-/-: n = 5 male
Gaa
-/--FoxO3a: n = 5 male
•
Characterization of the glycogen overload: Periodic Acid Schiff (PAS) staining and glycogen assay
•
Exploration of the autophagic buildup: Immunolabelling of LC3 (autophagosomal marker) and
WGA (a lysosomal content marker); western blot against p62 (autophagic flux impairment marker)
•
Investigation of the morphological damages: Anisocytosis and centronucleation quantification
Pompe disease (glycogen storage disease type II) is a lysosomal storage disorder caused by the
mutation of acid α-glucosidase (Gaa), the unique enzyme degrading glycogen in glucose into
lysosomes. A
massive glycogen overload
is described in Pompe patients, mainly in skeletal and
cardiac muscles. Furthermore, severe impairment of autophagic flux has been described,
highlighted by
autophagic buildup
. Contrary to cardiac muscle,
no treatment currently allows
to cure efficiently and durably the skeletal muscle
. We have identified the transcription factor
Forkhead box O3 (FoxO3a) as a potential target
to alleviate skeletal muscle impairments
through its
key role on regulation of both glycogen homeostasis and autophagy
.
The objectives of the study were:
1/ to explore the preventive effect of FoxO3a overexpression on :
2/ to investigate the impact of FoxO3a overexpression on skeletal muscle remodeling
2/ FoxO3a overexpression prevents the glycogen overload in skeletal muscle
3/ FoxO3a overexpression prevents the autophagic buildup accumulation in skeletal muscle
4/ FoxO3a overexpression prevents
skeletal muscle remodeling
Gaa
+/+Gaa
-/-Gaa
-/-FoxO3a
Biceps femoris muscle
Take home messages
Scale bar: 50µm
Glycogen assay of Biceps femoris muscle extract. Statistical analysis : One-way ANOVA, Newman-Keuls post hoc test. **p<0.01, ***p<0,001. n=5 mice/group.
Quantification of total FoxO3a mRNA expression in Biceps femoris muscle by qPCR. Statistical analysis : One-way ANOVA, Newman-Keuls post hoc test. ***p<0,001. n=5 mice/group.
LC3 -im m unol abe lli ng
Representative LC3 (green) and dystrophin (red) immunolabelling of Biceps femoris muscle. Nuclei were counterstained using Hoechst. Scale bar: 25µm
Phagophore Lysosome Autophagolysosome Autophagosome LC3 Isolated membrane p62 Initiation
Elongation Docking and fusion Degradation
Autophagic flux markers
p62 GAPDH
Ga
a
+/+Ga
a
-/-Ga
a
-/-FoxO
3a
Representative western blot analysis of Biceps femoris muscle lysates using anti-p62 antibody. An anti-GAPDH antibody was used as a loading control. Statistical analysis : One-way ANOVA, Newman-Keuls post hoc test. ***p<0,001. n=5 mice/group.
Quantification of LC3+-vacuolated fibers (left) and of the LC3+-buildup size (right) in Biceps
femoris muscle. Statistical analysis : Mann-Whitney test, *p<0,05. n=5 mice/group.
Gaa
+/+Gaa
-/-Gaa
-/-FoxO3a
Analysis of the minimum Feret diameter (MinFeret; shortest distance between two parallel tangents of the muscle fiber edges) of Biceps
femoris muscle. Statistical analysis: Two-way ANOVA, Newman-Keuls
post hoc test. *p<0.05. n=5 mice/group.
1/ Validation of FoxO3a overexpression
Ø
Prevention of the autophagic buildup
Gaa
+/+Gaa
-/-Gaa
-/-FoxO3a
L ys os om al c ont ent im m unol abe lli ng
Representative lysosomal content immunolabelling using WGA (green) of Biceps femoris muscle. Edges of muscle fibers were marked using white line dots for Gaa-/- and Gaa
-/-FoxO3a. Scale bar: 10µm.
Quantification of lysosomal content (vesicles WGA+) size in Biceps femoris muscle.
Statistical analysis : Mann-Whitney test, **p<0,01. n=5 mice/group.
Ø
Prevention of the lysosome overload
Ø
Prevention of the autophagic flux impairments
• FoxO3a overexpression prevents both the glycogen overload and the autophagic
flux impairment in skeletal muscle of Pompe disease.
• FoxO3a overexpression prevents the skeletal muscle remodeling occurring in
Pompe disease.
• FoxO3a overexpression could exhibit a therapeutic relevance to efficiently
correct skeletal muscle in Pompe disease, in combination with gene therapy
inducing overexpression of GAA or enzyme replacement therapy (ERT).
v
Experimental design
v
Analyses performed
PA
S
s
ta
ini
ng
Gaa+/+ Gaa-/- Gaa-/- FoxO3a 0 2 4 6 8 10
Re
la
tiv
e
to
ta
l F
ox
O
3a
m
RNA
ex
pr
es
si
on
(f
ol
d/
cont
rol
)
***
***
Gaa+/+ Gaa-/- Gaa-/- FoxO3a
0 50 100 150
Gl
yc
og
en
c
on
te
nt
(%
G
A
A
-/-u
ntr
ea
te
d)
***
**
***
Gaa-/- Gaa-/- FoxO3a 0 10 20 30 40 LC 3 + v ac uo la te d m us cl e fi be rs / T ota l m us cl e fi be rs ( % )
*
Gaa-/- Gaa-/- FoxO3a 0 1 2 3 LC 3 + b u ild u p a re a / M u sc le fib e rs a re a ( A U )
*
Gaa+/+ Gaa-/- Gaa-/- FoxO3a
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 p62/ G A P D H ( A U )
***
***
Gaa-/- Gaa-/- FoxO3a
0 2 4 6 8 10 12 Ly sos om al c ont ent ar ea / Mu sc le fi be r are a (% ) **
Gaa+/+ Gaa-/- Gaa-/- FoxO3a 25 30 35 40
Mi
nF
ere
t o
f mu
sc
le
fib
er
s (
µm)
*
*
Gaa+/+ Gaa-/- Gaa-/- FoxO3a 0 5 10 15 20 25 30 35
Ce
nt
ro
nu
cle
at
ed
fib
er
s
(%
)
***
***
*
Ø
Prevention of atrophy
Ø
Increase of regenerative fibers
Quantification of centronucleated muscle fibers in Biceps femoris muscle. Statistical analysis: One-way ANOVA, Newman-Keuls post hoc test. *p<0.05, ***p<0.001. n=5 mice/group.
Conclusion
Autophagosomes
Lysosomes
Glycogen
