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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.

Degeneration of photoreceptors (rods and cones) results in blindness. As we rely almost entirely on our daytime vision mediated by the cones, it is the loss of these photoreceptors that results in legal blindness and poor quality of life. Cone dysfunction is usually observed due to two mechanisms: noncell-autonomous due to the secondary effect of rod death if the causative gene is specifically expressed in rods and cell autonomous, if the mutation is in a cone-specific gene. However, it is difficult to dissect cone autonomous effect of mutations in the genes that are expressed in both rods and cones. Here we report a property of murine cone photoreceptors, which is a cone-autonomous effect of the genetic perturbation of the retinitis pigmentosa 2 (Rp2) gene mutated in human X-linked RP. Constitutive loss of Rp2 results in abnormal extension of the cone outer segment (COS). This effect is phenocopied when the Rp2 gene is ablated specifically in cones but not when ablated in rods. Furthermore, the elongated COS exhibits abnormal ultrastructure with disorganized lamellae. Additionally, elongation of both the outer segment membrane and the microtubule cytoskeleton was observed in the absence of RP2. Taken together, our studies identify a cone morphological defect in retinal degeneration due to ablation of RP2 and will assist in understanding cone-autonomous responses during disease and develop targeted therapies.

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.

Overexpression and/or abnormal cleavage of amyloid precursor protein (APP) are linked to Alzheimer's disease (AD) development and progression. However, the molecular mechanisms regulating cellular levels of APP or its processing, and the physiological and pathological consequences of altered processing are not well understood. Here, using mouse and human cells, we found that neuronal damage induced by UV irradiation leads to specific APP, APLP1, and APLP2 decline by accelerating their secretase-dependent processing. Pharmacological inhibition of endosomal/lysosomal activity partially protects UV-induced APP processing implying contribution of the endosomal and/or lysosomal compartments in this process. We found that a biological consequence of UV-induced γ-secretase processing of APP is impairment of APP axonal transport. To probe the functional consequences of impaired APP axonal transport, we isolated and analyzed presumptive APP-containing axonal transport vesicles from mouse cortical synaptosomes using electron microscopy, biochemical, and mass spectrometry analyses. We identified a population of morphologically heterogeneous organelles that contains APP, the secretase machinery, molecular motors, and previously proposed and new residents of APP vesicles. These possible cargoes are enriched in proteins whose dysfunction could contribute to neuronal malfunction and diseases of the nervous system including AD. Together, these results suggest that damage-induced APP processing might impair APP axonal transport, which could result in failure of synaptic maintenance and neuronal dysfunction. Fil: Almenar Queralt, Angels. University of California at San Diego; Estados Unidos Fil: Falzone, Tomas Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of California at San Diego; Estados Unidos Fil: Shen, Zhouxin. University of California at San Diego; Estados Unidos Fil: Lillo, Concepción. University of California at San Diego; Estados Unidos Fil: Killian, Rhiannon L.. University of California at San Diego; Estados Unidos Fil: Arreola, Angela S.. University of California at San Diego; Estados Unidos Fil: Niederst, Emily D.. University of California at San Diego; Estados Unidos Fil: Ng, Kheng, S.. University of California at San Diego; Estados Unidos Fil: Kim, Sonia N.. University of California at San Diego; Estados Unidos Fil: Briggs, Steven P.. University of California at San Diego; Estados Unidos Fil: Williams, David S.. University of California at San Diego; Estados Unidos Fil: Goldstein, Lawrence S. B.. University of California at San Diego; Estados Unidos