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Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016., Analyses of RasGRF1 knockout (GRF1-KO), GRF2-KO and the GRF1/2 double-KO mouse retinas, revealed specific alterations in cone photoreceptors in the GRF2-KO and GRF1/2-DKO. These alterations consist in the specific accumulation of misplaced “ectopic” nuclei in the photoreceptors segments area. The pathological displacement of cone nuclei occurred postnatally and peaked between postnatal day 11 (P11) and P15 coinciding with the so called “late migratory phase” of cone nuclei. The late migratory phase is a physiological process whereby cone nuclei move towards the outer limiting membrane (OLM) where they reach their final position. In the GRF2-KO and GRF1/2-DKO retinas this movement is deregulated and cone nuclei are located closer to the OLM and in some regions of the retina even trespass it. Using detailed inmunocytochemical analyses we have identified disruption of the OLM and accumulation of activated, phosphorylated forms of PAK, MLC2 and VASP, molecules known to participate in nuclear migration and cytoskeletal reorganization, in cone photoreceptors of GRF2-KO and GRF1/2-DKO retinas. Electroretinographic recordings show specific impairment of cone photoreceptor cells in GRF1-KO and GRF1/2-DKO retinas. These data support a critical role of GRF2 in cone nuclear migration and proper retinal development and functioning.

Resumen del trabajo presentado al XXXXVIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Valencia del 7 al 10 de septiembre de 2015., During nervous system development secretion must be tightly controlled meanwhile neurons differentiate projecting axons and dendrites to their specific targets. Neurotrophins regulate, among other functions, differentiation and regulated secretion in the nervous system. However, the molecular mechanisms underlying the temporal coordination of differentiation and secretion by neurotrophins are unknown. Here we describe the involvement of ARMS/Kidins220, a downstream protein of Trk neurotrophin receptors, acting through Synembryn-B and Trio in the regulated secretion mediated by neurotrophins. We observed that PC12 differentiation and regulated secretion are temporally controlled since non-differentiated or differentiated neurons display a poor or strong regulated secretion in response to neurotrophins, respectively. Interestingly, high levels of ARMS/Kidins220 and Synembryn-B are required for differentiation, when regulated secretion is minimal, whereas a strong downregulation of both proteins occurred once the cells are differentiated, when regulated secretion is maximal. Overexpression or downregulation of ARMS/Kidins220 and Synembryn-B levels in non-differentiated PC12 cells blocks or potentiates NGF-mediated secretion, respectively. Similarly, secretion of BDNF in cortical neurons in response to NT-3 or NT-4 augments with a concomitant downregulation of ARMS/Kidins220 and Synembryn-B protein levels. In addition, knockdown of ARMS/Kidins220 and Synembryn-B potentiated further BDNF evoked secretion. Finally, downregulation of ARMS/Kidins220 protein in vivo enhanced BDNF secretion from cortex in response to depolarization, NT-3 or NT-4 and a signifi cant accumulation of BDNF in the striatum coming from the cortex and hippocampus.

Mutations in the MYO7A gene cause a deaf-blindness disorder, known as Usher syndrome 1B. In the retina, the majority of MYO7A is in the retinal pigmented epithelium (RPE), where many of the reactions of the visual retinoid cycle take place. We have observed that the retinas of Myo7a-mutant mice are resistant to acute light damage. In exploring the basis of this resistance, we found that Myo7a-mutant mice have lower levels of RPE65, the RPE isomerase that has a key role in the retinoid cycle. We show for the first time that RPE65 normally undergoes a light-dependent translocation to become more concentrated in the central region of the RPE cells. This translocation requires MYO7A, so that, in Myo7a-mutant mice, RPE65 is partly mislocalized in the light. RPE65 is degraded more quickly in Myo7a-mutant mice, perhaps due to its mislocalization, providing a plausible explanation for its lower levels. Following a 50-60% photobleach, Myo7a-mutant retinas exhibited increased all-trans-retinyl ester levels during the initial stages of dark recovery, consistent with a deficiency in RPE65 activity. Lastly, MYO7A and RPE65 were co-immunoprecipitated from RPE cell lysate by antibodies against either of the proteins, and the two proteins were partly colocalized, suggesting a direct or indirect interaction. Together, the results support a role for MYO7A in the translocation of RPE65, illustrating the involvement of a molecular motor in the spatiotemporal organization of the retinoid cycle in vision.