The architecture and mechanical properties of tissues are essential for their function. Mechanical homeostasis maintains mechanical properties within an optimal range, but is challenged by remodelling processes, including cell turnover during ageing. Postmitotic tissues, as in the brain and the retina, cannot proliferate and employ structural remodelling instead of proliferation to adapt to cell loss. The retinal pigment epithelium (RPE) is a paradigmatic postmitotic tissue that ensures photoreceptor longevity via the daily internalization of photoreceptor outer segments and experiences structural remodelling in ageing. In this work, I developed an in vitro model that recapitulates the age-related cell density reduction the human RPE experiences in vivo during ageing via apoptosis-generated density reduction (AgeD). AgeD relies on the monolayer-scale, on-demand activation of the recombinant FKBP/casp8 fusion protein in mature hiPSC-derived RPE (iRPE) on hydrogels. The model recapitulates the age-related structural remodelling of the human RPE with reduced cell height, shortened apical microvilli and changes in the actin cytoskeleton. Functionally, AgeD-monolayers perform photoreceptor fragment phagocytosis differently by engulfing a lower number of particles of a larger size. The age-associated RPE structural phenotype presents a significant biomechanical shift on the tissue level, evidenced by tissue stiffening and enhanced elastic properties, and altered apical mechanics evidenced by decreased Brillouin shift. AgeD-monolayers exhibit higher force at cell-cell junctions, demonstrated by vinculin and phospho-myosin light chain accumulation. Transcriptional profiling revealed global changes of the expression of actin-associated genes, including increased expression of branched actin nucleator subunits, as well as decreased expression of formins and ezrin. Pharmacological inhibition of actin nucleators during phagocytosis revealed that Arp2/3 inhibition rescued the number of internalized particles in AgeD-monolayers, while formin inhibition in control iRPE led to larger average size of internalised fragments. This result suggests altered actin cytoskeleton plasticity in AgeD-iRPE during phagocytosis. Besides tissue architecture, the here presented model may recapitulate other aspects of RPE ageing, such as alterations in the genomic integrity and epigenetics. Additionally, the AgeD-iRPE condition shows increased transcriptional levels of basement-membrane related components and decreased chromatin compaction, in agreement with in vivo observations from old mice presented in this work. The murine RPE characterization reveals further that the RPE far-peripheral region undergoes the most pronounced structural remodelling from all retinal regions. Insights from our group’s previous work on the different mechanical status of this region agree with the differential modulatory functions of different laminin isoforms laminin-511 and laminin-332 observed in the AgeD model, where laminin-511 renders the monolayers with higher sensitivity to mechanical cues, while laminin-332 exhibits a protective role in terms of mechanics and function. Altogether, this work demonstrates that the age-associated tissue architecture of the postmitotic RPE correlates with the establishment of a new mechanical homeostasis. In this state, actin-related processes are leveraged towards structural reinforcement to preserve tissue integrity decreasing apical plasticity needed on-demand for the execution of phagocytosis. This work delivers novel insights into postmitotic mechanobiology and the age-related changes of retinal homeostasis and opens the door to future studies to address remaining questions on postmitotic mechano-epigenetics or the impact of the extracellular matrix.
Teodora Piskova (Wed,) studied this question.