Presentation is loading. Please wait.

Presentation is loading. Please wait.

ACS nano.

Similar presentations

Presentation on theme: "ACS nano."— Presentation transcript:

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

Download ppt "ACS nano."

Similar presentations

Ads by Google