Molecular dissection of pericyte-to-neuron reprogramming reveals cellular identity safeguarding mechanisms

Lohrer, Benjamin (2022): Molecular dissection of pericyte-to-neuron reprogramming reveals cellular identity safeguarding mechanisms. Dissertation, LMU München: Medizinische Fakultät

Abstract

Neurodegenerative diseases, strokes, and injuries affect millions of people worldwide and current treatment options are insufficient. Since death of neurons in the brain is a common feature of all these disorders, a potential therapy could replace the lost neurons by newly generated ones to restore brain function. Natural adult neurogenesis in humans has been proven inadequate to deal with a major loss of brain cells. Therefore, for many years, transplantation of fetal tissue or stem cell-derived neural progenitors have been the focus of investigations regarding new treatments. More recently, new methods and insights have rendered brain-resident cells a promising means of an alternative therapeutic approach. While cellular identity was believed to be irreversible once differentiated for a long time, this view has changed gradually over the last decades. Among other cells, it has been shown for human brain pericytes that retroviral expression of the transcription factors (TFs) Ascl1 and Sox2 (AS) is sufficient to generate functional induced neurons (iNs) by direct reprogramming, and that this process is accompanied by a neural stem cell (NSC)-like state. While it is clear now that even a terminal cellular identity can be changed, the exact mechanisms remain elusive. Therefore, in this study we aimed at (i) identifying barriers and molecular mechanisms involved in cellular identity conversion from somatic cells into induced neurons, (ii) improving the efficiency of pericyte-to-neuron reprogramming, and (iii) directing the reprogramming process towards the desired cell types. By single cell RNA sequencing, we generated a high-resolution dataset of cells during pericyte-to-iN conversion. Using RNA velocity analysis, we were able to predict the progression of cells towards the neuronal fate and could identify blocker and facilitator genes that obstruct or enable cells to pass past a designated decision point. Among the facilitator genes, we identified several chromatin remodelers and cytoskeleton genes, and revealed a temporal heterogeneity regarding their expression pattern. Interestingly, we show that the blocker genes are part of a cellular identity safeguarding mechanism triggered by AS reprogramming. We demonstrate that the metabolic transition from glycolysis to oxidative phosphorylation is an essential barrier cells must overcome to transit from a pericyte towards a neuronal identity. Our findings suggest that any failure to meet metabolic requirements results in cells being either unable to change their identity or adopting a confused fate. To impact on the NSC-like state, we used either modulation of NOTCH signaling or TGF-β signaling by inhibition of the γ-secretase or dual SMAD inhibition, respectively, via small molecules. Strikingly, both treatments counteracted pericyte identity safeguarding mechanisms and significantly lowered reprogramming barriers. Consequently, our results show a strong increase in the number of generated iNs. Interestingly, we demonstrate that TGF-β signaling inhibition is more potent in lowering these metabolic barriers than NOTCH signaling inhibition, re-routing cells onto an entirely different route towards neurons. Additionally, TGF-β signaling inhibition almost completely suppresses the generation of undesired off-target cells without a clear identity, likely due to antioxidant regulon activity, which supports the metabolic transition. Remarkably, we illustrate that despite different treatments, iNs are transcriptionally similar and that both neuronal subtypes can be mapped to developing human brain regions. Finally, we used a different approach and reprogrammed pericytes into TUBB3+ cells using Neurog2/Sox2 (NS). We show that NS generated cells have a distinct transcriptomic identity from AS generated ones: While they are more likely to lose their original identity, the NS-generated iNs exhibit more progenitor-like properties, pointing at the different reprogramming capacities of proneural TFs. Altogether, this thesis emphasizes not only that cellular identity even in terminally differentiated cells can still be altered without returning to a pluripotent state. It further illustrates several previously unknown mechanisms during direct pericyte-to-iN reprogramming and opens new ways to improve its efficiency. Every new insight into cross-lineage cellular identity conversion paves the way for future neuronal replacement therapies.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 28. November 2022
• 1. Bericht­erstatter:in: Karow, Marisa