acs nano. subventricular zone (svz) subgranular zone (sgz) rostral migratory stream (rms) neurogenic...
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ACS nano
Subventricular Zone (SVZ) Subgranular Zone (SGZ)
Rostral Migratory Stream (RMS)
Neurogenic Niches - SVZ
Neural Stem Cells (NSCs) display three cardinal features:
-proliferation-self renewal-multipotency
Santos et al., 2012, Integr Biol
In adult mammalian brain there are cells with stem cell properties, responsible for the formation of new neurons
Many factors regulate neurogenesis
(Maden et al. Nature 2002)
One developmental molecule of particular interest is retinoic acid (RA).
Enhancing neurogenesis in the injured brain is a promissing therapy
Retinoic Acid (RA)
Maden et al., 2007, Nat Rev Neurosci
- Regulates proliferation and differentiation of stem cells in the developing and adult brainSantos et al., 2012, Integr Biol
- Can improve age-related neuronal and cognitive lossCrandall et al. 2004
-- Can influence LTP, LTD and neurite and axonal outgrowth Chiang et al. 1998 & Corcoran and Maden et al 1999
A better understanding on how RA regulates postnatal neurogenesis may therefore offer regenerative strategies to treat brain injury or degeneration
Retinoic Acid
However, RA:
Is rapidly metabolized
Has poor water solubility
Requires a fine-tuning of concentration
Aim:
We propose an innovative approach to induce neuronal differentiation by using RA-loaded nanoparticles for the controlled release of RA.
Retinoic Acid
The use of nanoparticles (NPs) can be a powerful strategy to overcome these limitations
Neurosphere+EGF/FGF2
Doetsch et al 1999
SVZ cultures
RA delivery systemSince RA is rapidly metabolized by cells and has low solubility in aqueous solutions
DS/PEI RA-releasing nanoparticles (RA+-NPs) were used as a vehicle for intracellular delivery
dissociation
Methods
RA-NPs achieve a proneurogenic effect with a RA concentration 2500-fold lower than ∼the one with free RA
RA-NPs induce functional neuronal differentiation via nuclear RAR activation
Retinoic Acid-Loaded Nanoparticles
50
100
150
200 NanoparticlesRA in solution*** ***
***
[RA] nM
Neu
N-p
osi
tive
cel
ls(%
of
con
tro
l)
Maia & Santos et al., 2011, ACSnano
0
5
10
15
20
25** ##
% o
f N
eu
N-p
os
itiv
e c
ells
(% o
f to
tal c
ells
)
Contro
l
RA+ -N
Ps
RA+ -N
Ps
+ BM
S493
0
10
20
30
40
50
*** ###
% o
f n
eu
ron
al-
like
ce
lls(H
ist/
KC
l <0
.8)
Contro
l
RA+ -N
Ps
RA+ -N
Ps
+ BM
S493
Sox2
-/-
Sox2
-/+
Sox2
+/+
Nuclei Sox2 Dlx2
Merge
Stemness
Dissociation
Treatment
5day
s
5 days
Sphere countingdissociation
Secondary spheres countingSelf-renewal assay
24hrs-/- pro-commitment
+/+ pro-stemness
-/+ neutralSox-2, Dlx2Cell pair assay
Stemness
Control
-NPs
+
RA 4 nM
RAM
RA
10
Control
-NPs
+
RA 4 nM
RAM
RA
10
Control
-NPs
+
RA 4 nM
RAM
RA
10
0
20
40
60
80***
***
******
So
x-2
ce
ll p
air
s(%
of
tota
l pa
irs
)
Control
-NPs
+
RA Control
-NPs
+
RA
40
60
80
100
120
****
Nu
mb
er
of
ne
uro
sp
he
res
(% o
f C
on
tro
l)
Sox2+/+ Sox2+/- Sox2-/-
Self-renewal assay Cell pair assay
RA-NPs induce the commitment of SVZ NSCs
Phospho-JNK6hrs
RA-NP treatment increased the total length and the number of P-JNK positive ramifications on Tau positive axons
Control
-NPs
+
RA
0.0
0.5
1.0
1.5
2.0
2.5 **
Ra
mif
ica
tio
ns
/ne
uro
sp
he
re
Control
-NPs
+
RA
0
500
1000
1500*
To
tal r
am
ific
ati
on
len
gth
(
m)
/ ne
uro
sp
he
re
Signalling
Cross-linking
Sonication
Immunoprecipitation(H3K4me3)
Reverse cross linksPurify DNA
Quantitative PCRfor the promoter region of proneurogenic genes
Epigenetic regulation by histone methylation is thought to be
involved in long-term maintenance of certain regions of
the genome
Several genes are reported to become more expressed during
this process
Mash1 and Neurogenin1
qChIP
(Abcam protocols)
RA facilitates the expression of pro-neurogenic genes
Day 0
Plating of neurospheresIn differentiation conditions
(without EGF/FGF)
Day 7 Day 9 Day 11
qChIP
NP-RA exposure
Day 10
qChIP
Control
-NPs
1d
+
RA-N
Ps 2d
+
RAContro
l
-NPs
1d
+
RA-N
Ps 2d
+
RA
0
2
4
6 Mash1 Ngn1
**
*R
ec
ruit
me
nt
of
H3
K4
me
3(f
old
inc
rea
se
of
co
ntr
ol)
qChIP
Day 0
Plating of neurospheresIn differentiation conditions
(without EGF/FGF)
Day 7 Day 9 Day 12
RT-PCR
NP-RA exposure
RA-NPs promote a stronger expression of proneurogenic genes than solubilized RA
Control
-NPs
+
RA 4nM
RAM
RA
10 Contro
l
-NPs
+
RA 4nM
RAM
RA
10
0
2
4
6 Mash1 Ngn1
*
**
Ex
pre
ss
ion
of
mR
NA
(fo
ld in
cre
as
e o
f c
on
tro
l)
Mash1
Ngn1
RA+-NPs - +
- + RA+-NPs
qRT-PCR
RA-NPs induce the expression of proneurogenic genes in the SVZ neurogenic niche in vivo
In Vivo
Control
-NPs
3d
+
RA-N
Ps 4d
+
RA-N
Ps 7d
+
RA 4nM
RA 7
d
M R
A 7d
10
0
1
2
3 **
Ex
pre
ss
ion
of
mR
NA
(fo
ld in
cre
as
e o
f c
on
tro
l)
Control
-NPs
3d
+
RA-N
Ps 4d
+
RA-N
Ps 7d
+
RA 4nM
RA 7
d
M R
A 7d
10
0
1
2
3
**
*
Ex
pre
ss
ion
of
mR
NA
(fo
ld in
cre
as
e o
f c
on
tro
l)
In Vivo
LV
100µm
LV
SVZ
RA-NPs intracerebroventricular
injection
7 days
SVZ laser microdissection
LV: lateral ventricle
Our work presents a novel method to modulate the differentiation of SVZ cells involving the use of retinoic acid-releasing nanoparticles
The use of RA-NPs may open new perspectives for brain repair
• Nanoparticles proved to deliver efficiently RA within the cells and consequently promote neuronal differentiation
•Treated cultures presented a higher expression of neuronal markers and functional neuronal activity.
•Retinoic acid promotes de trimethylation of H3K4 upregulating the expression of pro-neurogenic genes
•Retinoic Acid activates JNK pathway
• We demonstrated successfully that a NP formulation could be used in vivo to control the differentiation of NSCs
•RA-NPs were more robust in maintaining the signature of gene expression both in vitro and in vivo than solubilized RA
Conclusions