1. ACS nano

2. Neurogenic Niches - SVZ
In adult mammalian brain there are cells with stem cell properties, responsible for the formation of new neurons
Subventricular Zone (SVZ)
Subgranular Zone (SGZ)
Rostral Migratory Stream (RMS)
Santos et al., 2012, Integr Biol
Neural Stem Cells (NSCs) display three cardinal features:
-proliferation
-self renewal
-multipotency

3. Retinoic Acid (RA) 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

4. Retinoic Acid
- Regulates proliferation and differentiation of stem cells in the developing and adult brain
Santos et al., 2012, Integr Biol
- Can improve age-related neuronal and cognitive loss
Crandall et al. 2004
-- Can influence LTP, LTD and neurite and axonal outgrowth
Chiang et al & Corcoran and Maden et al 1999
Maden et al., 2007, Nat Rev Neurosci
A better understanding on how RA regulates postnatal neurogenesis may therefore offer regenerative strategies to treat brain injury or degeneration

5. However, RA: Aim: Retinoic Acid Is rapidly metabolized
Has poor water solubility
Requires a fine-tuning of concentration
The use of nanoparticles (NPs) can be a powerful strategy to overcome these limitations
Aim:
We propose an innovative approach to induce neuronal differentiation by using RA-loaded nanoparticles for the controlled release of RA.

6. Methods SVZ cultures dissociation RA delivery system
Neurosphere
+EGF/FGF2
Doetsch et al 1999
dissociation
RA delivery system
Since 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

7. Retinoic Acid-Loaded Nanoparticles
Control
RA+-NPs
+ BMS493
Control
RA+-NPs
+ BMS493
RA-NPs induce functional neuronal differentiation via nuclear RAR activation
Maia & Santos et al., 2011, ACSnano
RA-NPs achieve a proneurogenic effect with a RA concentration ∼2500-fold lower than the one with free RA

8. Stemness Sphere counting dissociation Secondary spheres counting
Self-renewal assay
5 days
Treatment
5days
Dissociation
-/- pro-commitment
+/+ pro-stemness
-/+ neutral
24hrs
Sox-2, Dlx2
Cell pair assay
Merge
Nuclei
Sox2
Dlx2
Sox2 -/-
Sox2 -/+
Sox2 +/+

9. Stemness Self-renewal assay Cell pair assay Sox2+/+ Sox2+/- Sox2-/-
RA-NPs induce the commitment of SVZ NSCs

10. Signalling Phospho-JNK 6hrs
RA-NP treatment increased the total length and the number of P-JNK positive ramifications on Tau positive axons

11. for the promoter region of proneurogenic genes
qChIP
Epigenetic regulation by histone methylation is thought to be involved in long-term maintenance of certain regions of the genome
Cross-linking
Sonication
Immunoprecipitation
(H3K4me3)
Several genes are reported to become more expressed during this process
Mash1 and Neurogenin1
Reverse cross links
Purify DNA
Quantitative PCR
for the promoter region of proneurogenic genes
(Abcam protocols)

12. qChIP RA facilitates the expression of pro-neurogenic genes Day 0
Plating of neurospheres
In differentiation conditions
(without EGF/FGF)
Day 7
Day 9
Day 11
qChIP
NP-RA exposure
Day 10
RA facilitates the expression of pro-neurogenic genes

13. qRT-PCR
Day 0
Plating of neurospheres
In differentiation conditions
(without EGF/FGF)
Day 7
Day 9
Day 12
RT-PCR
NP-RA exposure
Mash1
RA+-NPs
Ngn1
RA+-NPs
RA-NPs promote a stronger expression of proneurogenic genes than solubilized RA

14. In Vivo
RA-NPs induce the expression of proneurogenic genes in the SVZ neurogenic niche in vivo

15. RA-NPs intracerebroventricular injection
In Vivo
RA-NPs intracerebroventricular injection
7 days
SVZ laser microdissection
100µm LV
SVZ LV
LV: lateral ventricle

16. The use of RA-NPs may open new perspectives for brain repair
Conclusions
Our work presents a novel method to modulate the differentiation of SVZ cells involving the use of retinoic acid-releasing nanoparticles
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
The use of RA-NPs may open new perspectives for brain repair