2. Universidad Cardenal Herrera-CEU

Connexins (Cx) are a family of transmembrane proteins that form gap junction intercellular channels that connect neighboring cells. These channels allow the passage of ions and other biomolecules smaller than 1 kDa, thereby synchronizing the cells both electrically and metabolically. Cxs are expressed in all retinal cell types and the diversity of Cx isoforms involved in the assembly of the channels provides a functional syncytium required for visual transduction. In this chapter, we summarize the status of current knowledge regarding Cx biology in retinal tissues and discuss how Cx dysfunction is associated with retinal disease pathophysiology. Although the contribution of Cx deficiency to retinal degeneration is not well understood, recent findings present Cx as a potential therapeutic target. Therefore, we will briefly discuss pharmacological approaches and gene therapies that are being explored to modulate Cx function and fight sight-threatening eye diseases.

Purpose: During lens fiber cell differentiation, organelles are removed in an ordered manner to ensure lens clarity. A critical step in this process is removal of the cell nucleus, but the mechanisms by which this occurs are unclear. In this study, we investigate the role of a cyclin-dependent kinase 1 (CDK1) regulatory loop in controlling lens fiber cell denucleation (LFCD). Methods: We examined lens differentiation histologically in two different vertebrate models. An embryonic chick lens culture system was used to test the role of CDK1, cell division cycle 25 (CDC25), WEE1, and PP2A in LFCD. Additionally, we used three mouse models that express high levels of the CDK inhibitor p27 to test whether increased p27 levels affect LFCD. Results: Using chick lens organ cultures, small-molecule inhibitors of CDK1 and CDC25 inhibit LFCD, while inhibiting the CDK1 inhibitory kinase WEE1 potentiates LFCD. Additionally, treatment with an inhibitor of PP2A, which indirectly inhibits CDK1 activity, also increased LFCD. Three different mouse models that express increased levels of p27 through different mechanisms show impaired LFCD. Conclusions: Here we define a conserved nonmitotic role for CDK1 and its upstream regulators in controlling LFCD. We find that CDK1 functionally interacts with WEE1, a nuclear kinase that inhibits CDK1 activity, and CDC25 activating phosphatases in cells where CDK1 activity must be exquisitely regulated to allow for LFCD. We also provide genetic evidence in multiple in vivo models that p27, a CDK1 inhibitor, inhibits lens growth and LFCD.

Inability to preserve proteostasis with age contributes to the gradual loss of function that characterizes old organisms. Defective autophagy, a component of the proteostasis network for delivery and degradation of intracellular materials in lysosomes, has been described in multiple old organisms, while a robust autophagy response has been linked to longevity. The molecular mechanisms responsible for defective autophagic function with age remain, for the most part, poorly characterized. In this work, we have identified differences between young and old cells in the intracellular trafficking of the vesicular compartments that participate in autophagy. Failure to reposition autophagosomes and lysosomes toward the perinuclear region with age reduces the efficiency of their fusion and the subsequent degradation of the sequestered cargo. Hepatocytes from old mice display lower association of two microtubule-based minus-end-directed motor proteins, the well-characterized dynein, and the less-studied KIFC3, with autophagosomes and lysosomes, respectively. Using genetic approaches to mimic the lower levels of KIFC3 observed in old cells, we confirmed that reduced content of this motor protein in fibroblasts leads to failed lysosomal repositioning and diminished autophagic flux. Our study connects defects in intracellular trafficking with insufficient autophagy in old organisms and identifies motor proteins as a novel target for future interventions aiming at correcting autophagic activity with anti-aging purposes.