Supervisors info:
Ευθυμιόπουλος Σπύρος, Καθηγητής Φυσιολογίας Ζώων και Ανθρώπου – Νευροβιολογίας, Τμήμα Βιολογίας, ΕΚΠΑ
Τσιτσιλώνη Ουρανία, Καθηγήτρια Ανοσολογίας, Τμήμα Βιολογίας, ΕΚΠΑ
Παπαζαφείρη Παναγιώτα, Αναπληρώτρια Καθηγήτρια Φυσιολογίας Ζώων, Τμήμα Βιολογίας, ΕΚΠΑ
Summary:
Direct reprogramming involves the trans-differentiation of human somatic cells into induced neurons without the generation of induced pluripotent stem cells. These induced neurons can be used for the in vitro study of neurodegenerative diseases pathogenesis (disease-in-a-dish model) as well as for the discovery of new prognostic, diagnostic or therapeutic biomarkers for these diseases. Moreover, the replacement of lost neurons due to trauma, neurodegeneration or stroke by directly reprogramming resident glial cells into induced neurons holds great promise as an alternative therapeutic approach to brain transplantation. To this end, reprogramming of astrocytes to induced neurons has been well studied during the last years both in vitro and in vivo using combinations of transcription factors (TFs), chemical cocktails and miRNAs. These molecules can change the cell fate after inducing changes both at transcriptional or epigenetic level of the cell. There has been a great deal of studies indicating that directly reprogrammed induced neurons acquire specific neuronal subtypes, while they can be functional on an in vivo system. However, the underlying mechanisms that dictate the reprogramming process have been poorly addressed so far. In our group, se have investigated the role of the brain enriched miRNA, miR-124 and the small neurogenic molecule ISX9 in inducing neuronal reprogramming of cortical mouse astrocytes, focusing on the characterization of the transcriptional networks that instruct the process during in vitro reprogramming. Molecular characterization of early induced-neurons with RNA-seq has revealed that the major TFs upregulated early during the reprogramming process, namely Neurogenin2, NeuroD1, Tbr2 and Gsx2, are implicated in forebrain development,. However, the transcriptomic analysis also revealed a set of highly upregulated genes attributed to the addition of ISX9. These genes are related to midbrain development and maturation of dopaminergic neurons (Lmx1b, En1, Foxa1, Girk2 and DAT), proposing the existence of an alternative route the induced-neuronal cells are potent to undergo following ISX9 addition in the reprogramming coctail, other than obtaining a forebrain neuronal identity. Moreover, this analysis has also revealed that Shh as well as Gli1, which are major mediators of ventral brain identity, were downregulated after the addition of miR-124 alone, while they were upregulated after the addition of miR-124+ISX9. These findings prompted us to modify our protocol of direct reprogramming in order to create midbrain dopaminergic neurons. Firstly, we conformed with real time RT-PCR the levels of the chosen genes and then we added Shh and Fgf8 to our previous protocol, in order to enhance dopaminergic neurons’production pathways. To this end the major aim of this study was to elucidate the role of Shh and Fgf8 in reinforcing the latent midbrain dopaminergic program of reprogrammed cells by real time RT-PCR, immunofluorescence and electrophysiological analysis. Our results indicate that addition of Shh along with Fgf8 enhances the maturation and morphological complexity of induced-neurons. Furthermore, the addition of these two factors seems to repress markers that relate with immature dopaminergic neurons’ identity, such as Lmx1b while, it enhances the expression of mature dopaminergic markers, such as DAT. Importantly, it represses the cortical fate, as revealed by the downregulation of cortical markers, such as Tbr1 and Tbr2. As far as the electrophysiological analysis is concerned, our results indicate that addition of Shh and Fgf8 in miR124/ISX9 reprogramming cocktail, boosts the formation of electrophysiogically active induced neurons-that can respond to external stimuli and participate to neuronal networks in vitro.
