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Deciphering the mRNP Code for Successful Glia to Neuron Reprogramming

Subject Area General Genetics and Functional Genome Biology
Developmental Biology
Cell Biology
Term from 2019 to 2024
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 427451793
 
Final Report Year 2024

Final Report Abstract

In this collaborative project we took advantage of a novel experimental approach using Neurogenin 2, allowing to convert glial cells into functional primary neurons. This enables us to investigate the role of key RNA-binding proteins (RBPs), e.g. the RBP Pumilio2 and the double-stranded RBP Staufen2 in the successful astrocyte-to-neuron conversion. In brief, we focused on unraveling the roles of both RBPs in neurogenic mRNA localization and translation during this process. For this task, we down-regulated either Stau2 or Pum2 by lentiviral shRNA transduction of cultured glial cells and found that the absence of functional Pum2 caused a significant decrease in the observed reprograming efficiency. Further experiments then revealed a specific role of Pum2 in the late phase of the cell fate conversion process, when the early transcriptional changes associated with neuronal fate have already been implemented. Interestingly, the absence of Pum2 also profoundly impacted the underlying cell morphology. In addition to cells with normal astrocyte morphology and cells with typical neuronal morphology, we found a third category of cells being enriched under these conditions: cells with an aberrant morphology, showing small size, shriveled shape with uneven surface, and/or the presence of abnormal cell protrusions. Importantly, these (mostly non-reprogrammed) cells did not show typical signs of neurons based on Doublecortin-immunostainings (a typical neuronal marker) but rather atypical glial-like features exhibiting unusual glia morphology. We consider these results to be very exciting, as we identified by single cell RNA-based trajectory analysis a massively enriched new cell population in the context of adult neurogenesis in the hippocampus using our published Pum2-deficient mouse model. We termed this new population of cells transient state cells (TSCs) as it is found in between glial/neural stem cells and neurons in the mentioned trajectory analysis. In parallel to these in cellulo experiments, we have performed extensive molecular analysis to understand the physiological contributions of both RBPs in the brain. Whereas Stau2 is predominantly responsible for localizing a special set of mRNAs to specific compartments in cells, Pum2 is primarily regulating (local) translation. The identification of a new set of RNA targets in our reprogrammed neurons allowed us to look for changes in either RNA localization or subsequently in translation in those reprogrammed neurons. Future experiments will therefore have to unravel the exact contribution of Pum2 in this process, most likely in translational control of specific mRNAs that we have identified in those reprogrammed neurons. Based on our results from this project, we aim at combining two distinct biological processes, e.g. astrocyte-to-neuron reprogramming (studied in this project) and neurogenesis in the mammalian hippocampus (in a parallel project) with the fascinating outlook of studying direct neurogenesis under physiological conditions.

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