Investigation of Multipotent Neural Progenitor Cell to Oligodendrocyte Differentiation including the Transcriptional Activator Protein Pur-alpha by Quantitative Proteomics

Abstract

Oligodendrocytes are neuroglial cells, localized in the white matter of the central nervous system. They develop from neural stem cells (NSCs), which produce, among others, migrating and proliferating oligodendrocyte precursor cells (OPCs), the progenitors of mature oligodendrocytes. These cells in turn are responsible for the synthesis and maintenance of myelin sheaths of axons to enable fast saltatory signal transduction. Their importance for proper brain function in adults becomes evident in cases where injury-mediated or disease-mediated demyelination of axons occurs, like in multiple sclerosis, where patients display severe neurological deficits. As a consequence, formerly quiescent adult OPCs migrate to the lesion, where they proliferate and differentiate into oligodendrocytes to allow for remyelination of axons. The extent of remyelination however differs between patients and even fails, especially in patients suffering from chronic disorders. On that account, the understanding of molecular mechanisms underlying the differentiation process from neural progenitor cells (NPCs) to oligodendrocytes is crucial to develop appropriate therapeutic strategies.<br /> In the first and second part of this thesis, quantitative large scale mass spectrometry approaches were used to analyze alterations in the oligodendroglial proteome during the in-vitro differentiation of murine neurospheres (mainly consisting of NPCs) to oligospheres (mainly consisting of OPCs) and of rat OPCs to oligodendrocytes. Moreover, cells were analyzed at multiple time points during both processes to allow for the generation of protein kinetics, enabling a much more precised analysis of changes in protein levels compared to approaches with only two time points. In addition, this allows for the determination of early and late upregulated proteins. <br /> Analysis of five biological replicates addressing the differentiation from NPCs to OPCs resulted in the identification of 932 significantly upregulated and 1,184 downregulated proteins. The shift from NPCs to cells committed to the oligodendroglial lineage could be shown by both, immunocytochemistry staining and mass spectrometric analyses. Accordingly, the number of cells expressing the OPC marker chondroitin sulfate proteoglycan NG2 increased and multiple proteins associated with oligodendroglia were upregulated, while proteins involved in the regulation of the mitotic cell cycle were downregulated. Further potential proteins involved in the differentiation process were identified, such as the transcriptional activator protein Pur-alpha (PURA), the transcriptional modulator prohibitin (PHB) and the histone modifier NAD-dependent protein deacetylase sirtuin-2 (SIRT2).<br /> In four biological replicates conducted of the OPC to oligodendrocyte differentiation process 402 significantly upregulated and 485 downregulated proteins were identified. The successful differentiation could be confirmed by increasing numbers of cells expressing the oligodendrocyte marker myelin basic protein (MBP) and by ascending expression levels of proteins synthesizing lipids for myelin sheaths, myelin specific proteins and cytoskeleton organizing proteins. It further became apparent that expression levels of some proteins were altered instantly after exposition to differentiation medium, whereas expression levels of other proteins were altered later on. Early upregulated or downregulated proteins in the case of differentiation inhibitors, represented potential candidates responsible for the induction of OPC differentiation, including the peptidyl arginine deiminase, type II (PADI2) or the inositol 1,4,5-trisphosphate receptor type 2 (ITPR2). <br /> In the third part, the during NSC to oligodendrocyte differentiation continuously upregulated transcriptional activator protein Pur-alpha was particularly investigated regarding its degradation and interaction partners. It binds to purine-rich parts of single-stranded DNA and RNA, thereby regulating DNA replication and transcription of various genes. Pur-alpha further plays an essential role in synapse formation and development of dendrites, explainable by its function as transporting protein for mRNA to sites of translation in dendrites. Accordingly, Pur-alpha is crucial for proper brain development during early post-natal stages. <br /> In contrary to protein levels, no significant differences in Pur-alpha RNA levels were observed between neurospheres and oligospheres. Several ubiquitination sites were identified within Puralpha and the protein possesses multiple anaphase promoting complex/cyclosome (APC/C) recognition sites. We thus assumed that the elevated expression levels of Pur-alpha in oligospheres compared to neurospheres are due to decreased ubqiuitination and thus degradation. Besides, its function as regulator of proliferation regulating proteins, like E2F1, indicated Puralpha as a potential factor triggering the differentiation to oligospheres. However, we could not confirm this hypothesis used on in vitro degradation and ubiquitination assays. Further conducted interaction partner analyses using two different approaches significantly identified a few subunits of the proteasome complex and one component of the APC/C. Nonetheless, multiple promising Pur-alpha interaction partners were detected, including the La-related protein 1 (LARP1), the polyadenylate-binding protein 1 (PABPC1) or the ATP-dependent RNA helicase DDX3X. Furthermore, the serine protease HTRA1, together with Pur-alpha, might promote the differentiation from NPCs to OPCs. To finally confirm a direct interaction between Pur-alpha and these proteins additional experiments are required.