Abstract
Lineage conversion of glia into induced neurons (iNs) by forced expression of defined transcription factors emerges as a promising strategy for regenerating new neurons for functional restoration. Proneural transcription factors, Ascl1 and Neurog2, have long been known to convert cortical astroglia to iNs. Moreover, they are phosphorylated at multiple serine-proline sites and their phospho-deficient mutant forms appear to exhibit greater neurogenic potential. However, whether phospho-deficient mutant forms of proneural factors lead to enhanced astroglia-to-neuron reprogramming and what the underlying molecular mechanisms are remain unclear. Hence, my thesis aims to delve into the molecular underpinnings of proneural transcription factor- and their phospho- deficient mutant forms-induced glia-to-neuron conversion, leveraging the well- established in vitro astroglia-to-neuron reprogramming model along with single-cell RNA sequencing, CUT&RUN technique, and bioinformatics tools.Firstly, a comparative analysis between Ascl1 and its phospho-deficient mutant Ascl1SA6-induced astroglia-to-neuron reprogramming was conducted. Pure cortical astroglia cultures prepared from P5 mice were transduced with lentiviral vectors encoding reprogramming factors, and cells were harvested for single-cell RNA sequencing at various time points post-induction. The use of RNA velocity revealed distinct reprogramming trajectories for Ascl1 and Ascl1SA6-expressing cells, which became transcriptionally different iN types. Surprisingly, unlike Ascl1-iNs, Ascl1SA6-iNs specifically upregulated non-cortical interneuron development genes and failed to induce Dlx family genes crucial for GABAergic interneuron development. Comparisons with the in vivo Ascl1 and Ascl1SA6-induced glia-to-neuron reprogramming single-cell dataset further revealed surprisingly similar transcriptomes of Ascl1-iNs and Ascl1SA6- iNs generated in vitro and in vivo, suggesting the importance of the identity of the reprogramming factor on establishing the iN identity.
Given such differences observed, CUT&RUN experiments were conducted to profile the binding of Ascl1 or Ascl1SA6 in astroglia. The binding of Ascl1 or Ascl1SA6 to target loci was directed by the sequence-specific bHLH DNA binding motif. Common Ascl1 and Ascl1SA6 binding sites were identified including known Ascl1 target gene loci, such as Hes6. Differences between Ascl1 and Ascl1SA6 binding were also observed. For example, Ascl1SA6, but not Ascl1, bound to regulatory regions of genes that were only up- regulated in Ascl1SA6-iNs, suggesting differential binding could potentially influence downstream gene activation and contribute to the establishment of iN identities in lineage conversion.
Moreover, the effect of another proneural transcription factor, Neurog2, and its phospho-deficient mutant Neurog2SA9, in astroglia-to-neuron reprogramming was explored. The difference in the neurogenic potential between Neurog2 and Neurog2SA9 was less pronounced as compared to between Ascl1 and Ascl1SA6. Bioinformatic tools identified dynamically regulated genes along the conversion trajectory, including genes involved in various signalling pathways and extracellular matrix organisation. The expression of one dynamically regulated gene, Qsox1, was confirmed using smFISH in an in vivo glia-to-neuron reprogramming paradigm. Qsox1 encodes for an oxidase for catalysing protein disulfide bond formation and its upregulation is reported to make cells susceptible to ferroptosis, a known bottleneck in lineage conversion. Therefore, the transient induction of Qsox1 may be critical in reprogramming.
Lastly, the commonalities and specificities of Ascl1, Neurog2 and their phospho-mutants in glia-to-neuron reprogramming were investigated using Seurat integration and gene specificity index correlation methods. Despite roles of Ascl1 and Neurog2 in directing GABAergic and glutamatergic neuron development, respectively, Ascl1-iNs surprisingly showed a relatively higher similarity to Neurog2-iNs than to Ascl1SA6-iNs. Additionally, common dynamically regulated genes along Ascl1, Ascl1SA6, Neurog2 or Neurog2SA9- induced reprogramming trajectory were identified.
Altogether, my work provided mechanistic insights into the proneural factor-induced astroglia-to-neuron lineage conversion and highlighted potential molecular players that might be targeted for improving reprogramming outcomes in future.
| Date of Award | 1 Dec 2024 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Benedikt Berninger (Supervisor) |