Nestin regulation in the embryonic and adult CNS

Sammanfattning: The developing mammalian central nervous system (CNS), i.e. the brain and spinal cord, is generated from neuroectodermal stem/progenitor cells lining the neural tube. The CNS stem/progenitor cells proliferate and migrate to give rise to the three principal cell types in the developing CNS: neurons, astrocytes and oligodendrocytes. It has recently been established that the adult CNS also contains small populations of CNS stem/progenitor cells, which can be cultured in vitro and differentiate to neurons and glial cells. CNS stem/progenitor cells have received a lot of attention, because of the prospect of using these cells for transplantation and treatment of degenerative disorders in the CNS such as Parkinson's disease. The overall aim of this thesis has been to gain insight into the regulation of the nestin gene, which is expressed in CNS stem/progenitor cells both in the embryonic and adult CNS, and is a commonly used marker for these cells. We first investigated the relevance of nestin as a marker for CNS injury in animals and we found that reexpression of endogenous nestin was both rapid and longlasting in reactive astrocytes. Based on the nestin expression pattern at various time points after injury, we postulated that these cells may be derived from proliferative stem/progenitor cells that migrate away from the central canal towards the site of injury. In order to study in vivo how the nestin gene is regulated we generated transgenic mice carrying different putative nestin enhancer elements (regulatory regions) coupled to the reporter gene lacZ (encoding beta-galactosidase). The second intron of the human nestin gene was cloned and a 374-bp long regulatory region in the second intron is sufficient to direct gene expression to stem/progenitor cells, including early neural crest cells, in the developing CNS. A 120-bp long region in the 374-bp element is absolutely required for the pan-neural CNS expression, as deletion of the 120-bp region resulted in the retention of expression only in a small region in the developing mid/forebrain and at rhombomere boundaries. The 120-bp region contains putative binding sites for nuclear hormone receptors and we demonstrated binding of several nuclear receptors (TR, RAR, RXR and COUP-TF) to these motifs. Furthermore we analyzed the elements required for regulation of nestin expression in the adult CNS. A 714-bp region from the second intron of the human nestin gene, which is sufficient to control embryonic expression, did not yield expression in the neurogenic regions of the adult brain and spinal cord. In contrast, a larger nestin enhancer, encompassing 5 kb of rat nestin upstream sequences and the complete nestin gene, was able to induce lacZ expression in the ventricular wall of the brain and around the central canal of the spinal cord. Similarly, increased lacZ expression in response to brain or spinal cord injury was only seen with the larger rat nestin enhancer. The 714-bp element was however sufficient to control lacZ expression in adult CNS stem/progenitor cells and astrocytes cultured in vitro. In addition, an adenovirus containing the complete second intron of the human nestin gene linked to the lacZ gene was able to control expression in a nestin-positive CNS tumor cell line. These data show that the enhancer elements for adult CNS expression are complex, and that enhancer elements sufficient for embryonic expression only control expression in cultured adult CNS cells, but not in the adult neurogenic regions in vivo. In conclusion, data in this thesis provide new insight into how the nestin gene is regulated in embryonic and adult CNS, in response to CNS injury and tumor. The characterised nestin enhancer has become an important tool to express other genes of interest, both in transgenic mice and in adenoviral vectors, to direct expression to CNS stem/progenitor and tumor cells in vivo, for possible future therapeutic applications.