Generation of oligodendrocytes and characterisation of their role in axonal support

Domingues Pedro, Liliana (2023): Generation of oligodendrocytes and characterisation of their role in axonal support. Dissertation, LMU München: Graduate School of Systemic Neurosciences (GSN)

Abstract

Glial cells, comprising of oligodendrocytes, astrocytes and microglia, have come a long way from their simplistic description as nerve glue. Today, we know that neuron-glia interactions are a fundamental aspect of neuronal function and brain homeostasis. Oligodendrocytes (OLGs) are the myelinating glia of the central nervous system forming myelin sheaths that enwrap axons. These cells are involved in axonal maintenance and survival, neuronal circuitry adaptation and immunomodulation. However, many aspects of oligodendrocyte physiology and pathology are still not completely understood. Factors that have contributed to the knowledge gaps associated with these cells include the complicated and inefficient protocols for the isolation of primary cells, the lack of defined stage specific markers and of adequate transgenic tools that would allow the manipulation of these cells in vitro. In this thesis I conducted a comparative study to evaluate the differentiation efficiency of OLGs derived from human induced pluripotent stem cells (iPSCs) via the ectopic expression of oligodendrocyte transcription factors. I aimed to provide benchmarking criteria for the reproducibility and robustness of iPSC derived OLGs (iOLGs) protocols. I observed that iPSCs overexpressing solely SOX10 differentiated, at low yields, into O4 and MBP expressing iOLGs, while overexpression of a combination of three transcription factors, SOX10, OLIG2 and NKX6.2 (SON) lead to a higher differentiation efficiency. By including a purification step I significantly improved oligodendrocyte differentiation yields and reduced the population heterogeneity. The gene expression profile of SON-induced iOLGs confirmed the expression of oligodendrocyte differentiation markers. Furthermore, using a co-culture platform with iPSC-derived neurons, astrocytes and microglia we showed that these cells can migrate within the culture and form myelin-like structures in vitro. In this thesis I also aimed to address the wider knowledge gap of the mechanism of axonal support by myelinating oligodendrocytes. Oligodendrocytes secrete ferritin heavy chain (FTH1) protein, which may be internalised by neighbouring neurons and act as an antioxidant defence system by storing and detoxifying the excess of neuronal intracellular iron. Interestingly, Fth1 mRNA is among the three most highly abundant transcripts found in purified myelin, despite not behaving like a myelin-resident protein. Given the importance of FTH1 protein for neuronal protection it is reasonable to expect that the targeted transport and local translation of Fth1 mRNA could effectively provide a way for oligodendrocytes to rapidly respond to external stimuli. As such I aimed to characterise the nature of the Fth1 transcript in oligodendrocytes. Fth1 mRNA shows the characteristic granular distribution along the distal processes, reminiscent of other locally repressed mRNAs in both mouse and human oligodendrocytes. Moreover, Fth1 mRNA is not associated with processing bodies or stress granules in mature oligodendrocytes, instead it appears to be a unique type of cytoplasmic RNA. To identify the RNA-binding proteins that promote Fth1 mRNA localization or translation repression, I developed a proteomics approach to selectively isolate native RNA-protein complexes. I identified 19 potential protein candidates that could be associated with Fth1 mRNA translocation and/or translation repression in oligodendrocytes. In conclusion, our study is the first step to establish a standardised method for oligodendrocyte differentiation via ectopic transcription factor expression. Indeed, continuous improvement of the established protocols will allow the development of reproducible and cost-effective human iPSC-derived oligodendrocyte models and ultimately facilitate the utilisation of the iPSC technology to study the mechanisms and pathways of human OLG migration and myelination. In addition, our study also suggests the existence of a previously unknown RNA trafficking mechanism for Fth1 mRNA and local protein translation in myelin. We also identified potential Fth1 mRNA binding proteins that could provide additional insights into the impact of neuronal cues in the regulation of FTH1 protein expression and secretion by oligodendrocytes.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Oligodendrocytes, iPSC, Stem cells, Ferritin, mRNA, axonal support, local mRNA translation
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Graduate School of Systemic Neurosciences (GSN)
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 17. Februar 2023
• 1. Bericht­erstatter:in: Simons, Mikael