Postmitotic state of a cell as a challenge for regeneration: inner ear as a model

Supporting cells (SCs) of the mammalian inner ear are differentiated, postmitotic cells that hold promise as a platform in therapeutic interventions to replace lost sensory cells, the hair cells (HCs). However, SCs exhibit an age-dependent decline in their responsiveness to regenerative manipulations that aim to trigger cell cycle re-activation or to stimulate transdifferentiation into HCs. The aim of this PhD thesis project was to identify barriers restricting the therapeutic potential of auditory and vestibular SCs. Using viral-mediated ectopic expression of cyclin D1 (cD1) in organotypic cultures to force postmitotic SCs to re-enter the cell cycle, the efficiency of the cells to complete cell cycles was shown to decrease with maturation. Unscheduled cell cycle re-activation was found to be associated with accumulation of DNA double-strand breaks (DSBs), indicated by the upregulation of the serine 139 phosphorylated form of histone H2AX (γH2AX). By studying the dynamics of the DNA repair protein Rad51, the underlying reason for age-related restrictions in proliferative plasticity was shown to be delayed or inefficient DNA repair. Furthermore, delayed repair of DNA damage was found to lead to SC death. To study the possible involvement of DNA damage in stimulated SC-to-HC transdifferentiation, the pharmacological inhibitor of Notch signaling that triggers SC transdifferentiation into HCs was applied to inner ear explant cultures. It was shown that unlike forced cell cycle re-activation, stimulated transdifferentiation does not trigger DNA damage, suggesting that it might be a “safe” way generate HCs. p53 has been shown to antagonize cell proliferation and regenerative events in other contexts. Thus, it was investigated here whether this is the case also in the inner ear. Using a loss-of-function mutant mouse model, p53 was shown to be dispensable for inner ear development. By applying various growth-promoting manipulations on cochlear explants from these mutant mice in vitro, inactivation of p53 was shown to not confer regenerative potential to SCs. Excess levels of p53 that are generally associated with cellular stress response have been shown to direct cells to cell death. Thus, p53 levels are under a tight control. The significance of controlled levels of p53 in different developmental situations and in a tissue context is poorly understood. Thus, inner ear was used here as a model to study the consequences of p53 overexpression in various developmental contexts: proliferation; differentiation; and homeostasis. Mutant mouse models in which the interaction between p53 and its negative regulator Mouse double minute 2 (Mdm2) was abolished demonstrated that p53 accumulation is lethal to both proliferating HC, SC and neuronal progenitors, and quiescent, differentiating SCs and HCs. More thorough analysis that focused on SCs showed that their sensitivity to p53 decreases with postnatal maturation. The data presented here thus suggests that epigenetic signaling and maturation-related mechanisms that regulate chromatin conformation might limit p53´s pro-apoptotic functions in maturing SCs. This PhD work has revealed DNA damage signaling and inefficient DNA repair as important barriers restricting the proliferative potential of mammalian SCs. This work also demonstrates the importance of controlled levels of p53 for the survival of cells of the auditory organ. Furthermore, the data obtained from this work can be extrapolated to other postmitotic, differentiated cell types when evaluating their potential for regenerative therapies.