351. Inhibition of Thyroid Hormone Receptors in the Neural Stem Cells of Newborn and Adult Mice In Vivo: Consequences on Target Gene Expression
Authors: Zahra Hassani, Gladys Alfama, Jean-Christophe François, Carinne Giovannangeli, Barbara A. Demeneix
Journal: Molecular Therapy (2006)

The discovery of adult neural stem cells (NSC) in the mammalian brain a few years ago raised the possibility of new therapeutic perspectives for the treatment of genetic or inherited neurological disorders. However, little is known about the fundamental biology of this NSC population, and functional analysis of target genes has to be done to better understand the mechanisms underlying their proliferation or differentiation pathways. The tightly localised position of these neural stem cells in the subependymal area lining the lateral ventricles in the brain make them accessible to gene transfer by stereotaxic injections in the lateral ventricles. Knocking down the expression of a target gene by RNA interference (RNAi) in the NSC population of adult mice brains could lead to the determination of the function of a given protein in its in vivo cellular context. After having demonstrated that RNAi technology can be efficient in vivo by stereotaxic injection of small interfering RNAs (siRNAs) in the lateral ventricles of newborn mice (Hassani et al., 2005), we now address the question of whether small hairpin RNAs (shRNAs) could lead to the specific inhibition of a target gene in the same conditions. The advantage in the use of shRNAs is that we actually know that linear polyethylenimine (L-PEI) is able to specifically deliver plasmids to NSC and neural progenitors located in the subventricular zone of adult mice brains (Lemkine et al., 2002) after stereotaxic injections in the lateral ventricles. However, PEI is not an efficient vector for the delivery of siRNAs in the brain in vivo (Hassani et al., 2005). On this basis we injected plasmids carrying small hairpin RNAs complexed by L-PEI to the lateral ventricles of newborn mice brains in order to specifically inhibit the expression of a target gene. Our first observation was that shRNAs under the control of a H1- or a CMV-promoter are not efficient for the inhibition of the luciferase target gene in vivo. Surprisingly, we found that an hybrid CMV-H1 promoter directing the expression of the shRNA gave significantly better inhibition of the target gene than a H1- or CMV- promoter alone. As thyroid hormone tri-iodothyronin (T3) is necessary for proliferation of NSC in vivo (Lemkine et al., 2005), we sought to identify the genes involved in T3-mediated regulation of NSC proliferation. Two genes potentially implicated in NSC proliferation were tested and found to respond to T3 in the newborn mouse brain: CyclinD1 and Sox2. We found that cyclinD1 expression was significantly decreased after T3 treatment of hypothyroid animals, and that this repression involved Thyroid hormone Receptor alpha1 (TRa1). Sox2 is a transcription factor that is a major player in embryonic neurogenesis. Its expression is limited to NSC in the adult. Sox2 was found to be activated by T3 in newborn mice brains. We now aim to determine whether Sox2 is also regul ated by T3 via TRa1 or via another TR, and we will assess this question by inhibiting TRa1 with specific shRNAs.