Contributions
Abstract: P421
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - Genetics /Epigenetics and Pharmacogenetics
Background: NPCs are characterized by their ability to differentiate towards neurons and glia and thus may provide a valuable tool in stem cell-based therapies for neurodegenerative diseases. However, the inflammatory environment of the CNS seems to affect important biological processes of endogenous or transplanted NPCs. Epigenetic modifications constitute a major intracellular mechanism that controls gene expression in response to extracellular cues. In this context, we aimed to investigate the effects of cytokines, the main inflammatory mediators, on expression levels of differentiation-related genes and also on DNA methylation patterns of the corresponding promoter regions.
Methods: NPCs from postnatal C57bl/6J mice were cultured as free-floating neurospheres. Cells were treated with pro- or anti- inflammatory cytokines (IFNγ, TNFα and TGFβ) or vehicle, as control. Total RNA was isolated and reversely transcribed into cDNA. Real time PCR was used to evaluate mRNA expression levels of neural and glial genes. Also, genomic DNA was isolated from each group and was treated with bisulfite. PCR was used for the amplification of selected gene promoter regions. Finally, PCR products were cloned into pCR2.1 plasmids and analyzed with Sanger sequencing.
Results: IFNγ increased the expression of Neurogenin1 (Ngn1) (p< 0.05, 3.96-fold), Olig2 (p< 0.05, 2.5-fold), Tubb3 (p< 0.0001, 4.6-fold). TNFα induced expression of NeuroD1 (p< 0.005, 2.8-fold), Ngn1
(p< 0.005, 6.4-fold), Math1 (p< 0.05, 2.75-fold) and DCX (p< 0.0001, 3.5-fold). TGFβ increased mRNA levels of NeuroD1 (p< 0.005, 3.2-fold), Ngn1 (p< 0.05, 2-fold), Math1 (p< 0.005, 6.8-fold), Olig2 (p< 0.005, 2.9-fold) and reduced DCX (p< 0.005, 0.4-fold), NG2 (p< 0.005, 0.17-fold) and S100b (p< 0.005, 0.22-fold). Ngn1 (total methylation: ctrl-5%, IFNγ-4.2%, TNFα-3.05%, TGFβ-4.1%) and Tubb3 (total methylation: ctrl-88.3%, IFNγ- 92.3%) presented differential methylation pattern between cytokine-treated groups.
Conclusions: Pro-inflammatory cytokines seem to induce neuronal differentiation of NPCs, while TGFβ represses both neuronal and glial differentiation. Our results indicate that the induction of neural gene expression could be regulated via DNA methylation.
Disclosure: This research has been co-financed by the European Union (European Social Fund - ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: Heracleitus II. Investing in knowledge society through the European Social Fund.
Conflicts of interest: none
Kyriaki Nefeli Poulatsidou: Nothing to disclosure
Roza Lagoudaki: Nothing to disclosure
Olga Touloumi: Nothing to disclosure
Evangelia Kesidou: Nothing to disclosure
Elisavet Chartomatsidou: Nothing to disclosure
Maria Grigoriou: Nothing to disclosure
Katerina Chlichlia: Nothing to disclosure
Nikolaos Grigoriadis: Nothing to disclosure
Abstract: P421
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - Genetics /Epigenetics and Pharmacogenetics
Background: NPCs are characterized by their ability to differentiate towards neurons and glia and thus may provide a valuable tool in stem cell-based therapies for neurodegenerative diseases. However, the inflammatory environment of the CNS seems to affect important biological processes of endogenous or transplanted NPCs. Epigenetic modifications constitute a major intracellular mechanism that controls gene expression in response to extracellular cues. In this context, we aimed to investigate the effects of cytokines, the main inflammatory mediators, on expression levels of differentiation-related genes and also on DNA methylation patterns of the corresponding promoter regions.
Methods: NPCs from postnatal C57bl/6J mice were cultured as free-floating neurospheres. Cells were treated with pro- or anti- inflammatory cytokines (IFNγ, TNFα and TGFβ) or vehicle, as control. Total RNA was isolated and reversely transcribed into cDNA. Real time PCR was used to evaluate mRNA expression levels of neural and glial genes. Also, genomic DNA was isolated from each group and was treated with bisulfite. PCR was used for the amplification of selected gene promoter regions. Finally, PCR products were cloned into pCR2.1 plasmids and analyzed with Sanger sequencing.
Results: IFNγ increased the expression of Neurogenin1 (Ngn1) (p< 0.05, 3.96-fold), Olig2 (p< 0.05, 2.5-fold), Tubb3 (p< 0.0001, 4.6-fold). TNFα induced expression of NeuroD1 (p< 0.005, 2.8-fold), Ngn1
(p< 0.005, 6.4-fold), Math1 (p< 0.05, 2.75-fold) and DCX (p< 0.0001, 3.5-fold). TGFβ increased mRNA levels of NeuroD1 (p< 0.005, 3.2-fold), Ngn1 (p< 0.05, 2-fold), Math1 (p< 0.005, 6.8-fold), Olig2 (p< 0.005, 2.9-fold) and reduced DCX (p< 0.005, 0.4-fold), NG2 (p< 0.005, 0.17-fold) and S100b (p< 0.005, 0.22-fold). Ngn1 (total methylation: ctrl-5%, IFNγ-4.2%, TNFα-3.05%, TGFβ-4.1%) and Tubb3 (total methylation: ctrl-88.3%, IFNγ- 92.3%) presented differential methylation pattern between cytokine-treated groups.
Conclusions: Pro-inflammatory cytokines seem to induce neuronal differentiation of NPCs, while TGFβ represses both neuronal and glial differentiation. Our results indicate that the induction of neural gene expression could be regulated via DNA methylation.
Disclosure: This research has been co-financed by the European Union (European Social Fund - ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) - Research Funding Program: Heracleitus II. Investing in knowledge society through the European Social Fund.
Conflicts of interest: none
Kyriaki Nefeli Poulatsidou: Nothing to disclosure
Roza Lagoudaki: Nothing to disclosure
Olga Touloumi: Nothing to disclosure
Evangelia Kesidou: Nothing to disclosure
Elisavet Chartomatsidou: Nothing to disclosure
Maria Grigoriou: Nothing to disclosure
Katerina Chlichlia: Nothing to disclosure
Nikolaos Grigoriadis: Nothing to disclosure