The transcription factor p53 contributes to the regulation of neurogenesis and oligodendrogenesis in embryonic neural progenitor cells by fine-tuning intracellular ROS levels

Abstract

The transcription factor p53 has recognized roles in cell cycle arrest, apoptosis, DNA damage response and cellular redox regulation. In situ hybridization has demonstrated high expression level of p53 mRNA in all embryonic mouse tissues up to the embryonic day 10.5, after which p53 expression becomes more restricted being profound only in the germinal zones of the developing brain, such as in the ventricular (VZ) and sub-ventricular zone (SVZ) and in the ganglionic eminence (GE) of the developing brain. Nevertheless, in spite of the fact that 16-24% of p53-/- mouse embryos die from defective neural tube closure in the midbrain-hindbrain boundary, which suggests a role for p53 in brain development, the conventional view is that p53 is dispensable during CNS development.

In this work I have set out to explore explanations for p53 expression in the germinal zones of the embryonic telencephalon. Since a recent work attributed a regulatory function to reactive oxygen species (ROS) in the normal homeostasis of neural stem cells (NSC) and since some of the target genes of p53 possess antioxidant properties, I set out in this dissertation work to explore the hypothesis that p53 regulates the abundance of intracellular ROS in NSCs and thereby their proliferation/differentiation decisions in the embryonic and postnatal germinal zones. To study this, I have used embryonic mouse NPC cultures and telencephalic samples of various embryonic and postnatal developmental stages, conducted various drug treatments and applied methods such as ex utero electroporation, viral transduction, Affymetrix gene expression analysis, immunocytochemistry, immunohistochemistry, flow cytometry, immunoblotting and real time PCR.

The findings point to the importance of ROS in the regulation of embryonic neural progenitor cell homeostasis and imply a role for p53 in the fine-tuning of progenitor cell ROS levels and the concomitant DNA damage, and proliferation versus neurogenesis/oligodendrogenesis decisions. Indeed, elevation in ROS in embryonic p53-/- neural progenitor cells may provide a compensatory mechanism, which prevents overproliferation inducing instead neurogenesis. The study presents novel neurogenic genes, whose expression is responsive to cellular redox status and shows that in the absence of p53 PI3K-Akt signalling is elevated. P53 contributes to redox regulation at least in part by regulating expression of gene(s) with antioxidant properties and by counteracting PI3K-Akt signalling. Furthermore, elevation in ROS is not only a characteristic of embryonic p53-/- telencephalons, since the SVZ and hippocampus of the adult p53-/- mice, the neurogenic regions of the adult brain, exhibit an increase in ROS compared to the wild type animals.

In conclusion, this work casts light to the somewhat enigmatic role of p53 in the germinal zones of the developing telencephalon. The findings of the study imply that p53 has a role in redox regulation not only in the embryonic but also in the adult brain. Moreover, though in the embryonic brain elevation in ROS seems to compensate the absence of p53 and to induce on one hand neurogenesis and to impair on the other hand oligodendrogenesis, in the adult brain an increase in ROS is a potential source of neurological damage, which would deserve investigations.

Eine der spannendsten Fragen der Entwicklungsbiologie ist, wie sich das Gehirn entwickelt. Zahlreiche verschiedene Transkriptionfaktoren regulieren die Entwicklung des Gehirns. In der vorliegenden Dissertation habe ich untersucht, welche Rolle/Rollen der Transkriptionfaktor p53 in diesem Prozess haben könnte. Trotz dass p53 üppige Expression auf den germinalen Regionen des embryonalen Telencephalons hat, ist der Konsensus, dass p53 nicht notwendig für die normale Entwicklung des Gehirns ist.

In der vorliegenden Arbeit wird gezeigt, dass die in vitro kultivierte p53-/- embryonalen neuronalen Stammzellen, p53-/- embryonalen Telencephalonen, die SVZ und der Hippocampus des adulten Gehirns erhöhte ROS haben. ROS scheinen den Mechanismus darzustellen, den die p53-/- neuronalen Stammzellen verwenden, um die Überproliferation zu vermeiden. Erhöhte ROS stehen in Zusammenhang mit neuronaler Genexpression und haben deswegen auf die Neurogenese eine positive Wirkung, aber verschlechtern die Oligodendrogenese, weil die Stammzellen von Oligodendrozyten empfindlich gegenüber erhöhte ROS sind. ROS schädigen auch DNA. Der positive Einfluss, den p53 auf ROS-Homöostase hat, basiert auf der Wirkung p53 auf die Expression von einigen Antioxidantgenen und negativen Regulatoren der Akt-mTOR Signaltransduktion hat.

Kurz gesagt, die vorliegende Dissertation schlägt vor, dass p53 aktiv in embryonalen Telenecephalonen ist und dort auf positive Weise am Gleichgewichtzustand der neuronalen Stammzellen mitwirkt. Zudem können erhöhte ROS ein Mittel darstellen, das die normale Entwicklung auf p53-/- Telenecephalonen ermöglicht, aber später, in Erwachsenen Neurodegeneration verursachen kann.