FAK phosphorylation plays a central role in thrombin-induced RPE cell migration.

The migration of retinal pigment epithelial (RPE) cells is an important step in various pathologic conditions including subretinal neovascularization (SRN), proliferative vitreoretinopathy (PVR) and, importantly, as a consequence of retinal surgery. Therefore, the elucidation of the mechanisms underlying RPE trans-differentiation and migration is essential for devising effective treatments aimed to the prevention of these disorders. A common event in these pathologies is the alteration of the blood-retina barrier (BRB), which allows the interaction of RPE cells with thrombin, a pro-inflammatory protease contained in serum. Our previous work has demonstrated that thrombin induces RPE cell cytoskeletal remodeling and migration, hallmark processes in the development of PVR; however, the molecular mechanisms involved are still unclear. Cell migration requires the disassembly of focal adhesions induced by Focal Adhesion Kinase (FAK) phosphorylation, together with the formation of actin stress fibers. The aim of the present work was to identify thrombin-activated signaling pathways leading to FAK phosphorylation and to determine FAK participation in thrombin-induced RPE cell migration. Results demonstrate that the activation of PAR1 by thrombin induces FAK autophosphorylation at Y397 and the subsequent phosphorylation of Y576/577 within the activation loop. FAK phosphorylation was shown to be under the control of c/nPKC and PI3K/PKC-ζ, as well as by Rho/ROCK, since the inhibition of these pathways prevented thrombin-induced FAK phosphorylation and the consequent disassembly of focal adhesions, in parallel to FAK-dependent actin stress fiber formation and RPE cell migration. These findings demonstrate, for the first time, that thrombin stimulation of RPE cell transformation and migration are regulated by FAK tyrosine phosphorylation. Thus, targeting FAK phosphorylation may provide a strategical basis for PVR treatment.

Thioredoxin rod-derived cone viability factor protects against photooxidative retinal damage.

Rod-derived cone viability factor (RdCVF) is a trophic factor of the thioredoxins family that promotes the survival of cone photoreceptors. It is encoded by the nucleoredoxin-like gene 1 Nxnl1 which also encodes by alternative splicing a long form of RdCVF (RdCVFL), a thioredoxin enzyme that interacts with TAU. The known role of thioredoxins in the defense mechanism against oxidative damage led us to examine the retinal phenotype of the Nxnl1(-/-) mice exposed to photooxidative stress. Here we found that, in contrast to wild-type mice, the rod photoreceptors of Nxnl1(-/-) mice are more sensitive to light after exposure to 1700 or 2500 lx. The delivery of RdCVF by AAV to mice deficient of Nxnl1(-/-) protects rod photoreceptors from light damage. Interestingly, the RdCVF2L protein, encoded by the paralog gene Nxnl2, is able to reduce TAU phosphorylation, as does RdCVFL, but does not protect the rod from light damage. Our result shows that the Nxnl1 gene, through the thioredoxin RdCVFL, is part of an endogenous defense mechanism against photooxidative stress that is likely of great importance for human vision.

The disruption of the rod-derived cone viability gene leads to photoreceptor dysfunction and susceptibility to oxidative stress.

Rod-derived cone viability factor (RdCVF) is a thioredoxin-like protein, which has therapeutic potential for rod-cone dystrophies such as retinitis pigmentosa (RP). Cone loss in rodent models of RP is effectively reduced by RdCVF treatment. In this study, we investigate the physiological role of RdCVF in the retina by analyzing the phenotype of the mouse lacking the RdCVF gene, Nxnl1. Although the mice do not show an obvious developmental defect, an age-related reduction of both cone and rod function and a delay in the dark-adaptation of the retina are recorded by electroretinogram (ERG). This functional change is accompanied by a 17% reduction in cone density and a 20% reduction in thickness of the outer nuclear layer. The transcriptome of the retina reveals early changes in the expression of genes involved in programmed cell death, stress-response and redox-signaling, which is followed by a generalized injury response with increased microglial activation, GFAP, FGF2 and lipid peroxidation levels. Furthermore, cones of the mice lacking Nxnl1 are more sensitive to oxidative stress with a reduction of 65% in the cone flicker ERG amplitude measured under hyperoxic conditions. We show here that the RdCVF gene, in addition to therapeutic properties, has an essential role in photoreceptor maintenance and resistance to retinal oxidative stress.

Developmental expression of N-methyl-D-aspartate glutamate receptor 1 splice variants in the chick retina.

Glutamate is the major excitatory neurotransmitter in the vertebrate retina. The N-methyl-D-aspartate glutamate receptor (NMDAR) is assembled as a tetramer containing NR1 and NR2, and possibly NR3 subunits, NR1 being essential for the formation of the ion channel. The NMDAR1 (NR1) gene encodes for mRNAs that generate at least eight functional variants by alternative splicing of exon 5 (cassette N1), 21 (cassette C1), or 22 (cassettes C2 or C2'). NR1 splice variants were identified in the mature chick retina, and their variation during embryonic development (ED) was analyzed. NR1 was shown to lack N1 in early ED, shifting to N1-containing variants in the mature retina, which could contribute to explaining the distinct biochemical properties of retinal NMDARs compared with the CNS. Sequence analysis of C-terminal variants containing C1 and C2 cassettes suggests a membrane-targeting mechanism for avian NMDARs distinct from that in mammals. An NR1 variant containing a novel alternative C-terminal splice exon named C3 was found, which encodes six amino acids containing a predicted casein kinase II phosphorylation site. This new variant is expressed in the retina during a restricted period of ED, coincident with the generation of spontaneous calcium activity waves, which precedes synapse formation in the retina, suggesting its participation in this process.

Escherichia coli cells with mutations in the gene for adenylate cyclase (cya) exhibit a heat shock response.

Adenylate cyclase mutants of Escherichia coli showed the heat-shock response. The heat-shock response was studied in two different mutants and in different growth media, including rich and minimal media. These results are in disagreement with the proposal that the cya gene regulates the expression of the heat-shock genes.