Inhaltszusammenfassung

In vivo glia-to-neuron conversion mediated by transcription factors is emerging as a possible cell replacement strategy to respecify brain resident cells into lost neuronal cell types and to reinstate the function of disrupted circuitries in the presence of injury, neuropsychiatric disorders or other insults. Different factors, such as the absence or presence of injury, the cell type of origin and the choice of transcription factors influence the ability to convert cortical glia into neurons ...In vivo glia-to-neuron conversion mediated by transcription factors is emerging as a possible cell replacement strategy to respecify brain resident cells into lost neuronal cell types and to reinstate the function of disrupted circuitries in the presence of injury, neuropsychiatric disorders or other insults. Different factors, such as the absence or presence of injury, the cell type of origin and the choice of transcription factors influence the ability to convert cortical glia into neurons in vivo. However, the role that post-translational modification of transcription factors or the intrinsic heterogeneity of the starting cell population have in this process is largely unknown. In this thesis, I show that the ability of the proneural transcription factor ASCL1 to direct cell fate-switch in proliferative postnatal cortical glia is increased by the lack of protein phosphorylation. I found that, in postnatal cortical astrocytes cultures transduced with retroviruses encoding for three forms of ASCL1 differing in their phosphorylation states, the induction of a phospho-deficient mutant leads to increased reprogramming efficiency compared to the wild type protein or a phospho-mimetic mutant. However, independently on its phosphorylation state, ASCL1 is not able to efficiently convert postnatal cortical glia into neurons in vivo but rather decreases the proportion of astroglia favouring generation of oligodendroglia. The exogenous expression of both phospho-mutants leads to an increase in mature oligodendrocytes, consistent with a role of Ascl1 in the differentiation of oligodendrocyte progenitors. Moreover, I demonstrated that co-transduction of Bcl2 with Ascl1 successfully induces glia-to-neuron conversion. Remarkably, when associated with Bcl2, both phospho-mutants showed a greater neurogenic ability compared to the wild type protein and the phospho-deficient mutant drove the formation of a higher proportion of NeuN-expressing neurons. In summary, while the phosphorylation state of ASCL1 does not seem to promote cell fate-switch per se, it influences the maturation level of reprogrammed postnatal cortical glia and the conversion efficiency in association with BCL2. Furthermore, I describe an adaptation of a clonal labelling system, called StarTrack, for in vivo cortical astrocyte-to-neuron conversion and, eventually, clonal analysis of induced neurons. Here, I illustrate the results obtained from preliminary experiments in which the tamoxifen-activated proneural factors ASCL1ERT2 and NEUROG2ERT2 are induced in GFAP-expressing postnatal cortical astrocytes. These experiments revealed the limitations of the system, such as insufficient expression levels of the plasmids expressing Ascl1ERT2 and Neurog2ERT2 and the failure to induce formation of DCX-positive neurons to date. After identification of the pitfalls of the system, I describe a possible strategy to circumvent them. In the future, coupling tamoxifen-inducible activation of forced neurogenesis in astrocytes with clonal labelling of converted cells will permit the identification of the glial clones from which induced neurons are generated and, conversely, identification of the astrocytic populations that expressed the proneural factors but failed to reprogram. Together, understanding the mechanisms favouring the execution of neurogenic programs instructed by transcription factors and the reasons, why some glial subpopulations fail to erase their original fate and switch lineage, will help developing strategies to improve cell fate conversion for regenerative medicine.» weiterlesen» einklappen

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DDC Sachgruppe:
Biowissenschaften, Biologie