Abstract
After completion of embryonic corticogenesis, there is no evidence of generation of new neurons in the cerebral cortex. Therefore, upon injury or neurodegenerative processes, neuronal loss cannot be restored naturally. Several experimental strategies to revert this situation have been developed and improved during the past years, including transplantation of neuronal precursor cells, mobilization of neuronal precursor cells and direct lineage reprogramming. The latter has emerged as one of the more prominent strategies for brain repair, and it consists in the conversion of non-neuronal cells into neurons by forced expression of transcription factors. In vivo work has demonstrated that forced expression of proneural transcription factors involved in neurogenesis during embryonic corticogenesis, such as Neurogenin 2 (Neurog2), can convert reactive glial cells located in the mouse cerebral cortex after acute injury. However, glia-to-neuron conversion without prior injury is not efficient in the adult mouse cerebral cortex, as a proliferative state of the starter cells seems to be needed for a successful conversion.In this thesis, in order to study the conversion of glial cells into induced neurons (iNs) in a largely injury- free environment, I took advantage of a recently developed experimental model in which proliferative glial-cell precursors resident in the postnatal mouse cerebral cortex are specifically targeted with retroviruses (RVs) encoding for the reprogramming factors of interest. The postnatal reprogramming paradigm could also allow the modification of dysfunctional circuits in models of neurodevelopmental disorders prior to adulthood in the future.
Using the novel postnatal reprogramming paradigm, I overexpressed Neurog2 in dividing glial cells resident in the mouse postnatal cerebral cortex and I found that it could convert a small proportion of them into DCX-positive and NeuN-positive cells (referred to as induced neurons, iNs). Lineage tracing experiments using a mGFAP;EGFP mouse line to trace cells of astrocytic origin showed that a substantial proportion of iNs were generated from astrocytes, supporting the authenticity of glia-to- neuron conversion. Furthermore, I could show that co-expression of Bcl2 with Neurog2 drastically improved the conversion of glial cells into iNs. I also studied the role of post-translational modifications of Neurog2 on its reprogramming capability by overexpressing a phosphosite mutant form of Neurog2, Neurog2SA9, in which phosphorylation in serine-proline sites is blocked. I observed that Neurog2SA9 alone exerted a higher reprogramming effect than Neurog2, although this effect was less clear when reprogramming was facilitated by Bcl2.
I also observed evidence of hallmarks of glutamatergic neuron specification of iNs. Even though RVs target proliferative glia that may be assumed to be more plastic, iNs appear to be hypothrophic by all parameters assessed. In an attempt to improve reprogramming outcome, a neural circuit mechanism known to operate in cortical neuronal maturation was activated. This did not have an overt effect on reprogramming or improve the hypothrophic state of iNs.
Altogether, I showed that it is possible to convert astroglial cells into iNs in the postnatal mouse cerebral cortex by co-expressing Neurog2 and Neurog2SA9 with Bcl2. iNs pass through a DCX-positive stage, like what is observed in physiological neurogenesis, reaching a glutamatergic identity. However, despite these promising results, future work should be directed towards improving specification and maturation of iNs.
| Date of Award | 1 Oct 2023 |
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| Original language | English |
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| Supervisor | Benedikt Berninger (Supervisor) & Beatriz Rico (Supervisor) |