Sos1 disruption impairs cellular proliferation and viability through an increase in mitochondrial oxidative stress in primary MEFs.

Using a 4-hydroxytamoxifen (4OHT)-inducible, conditional Sos1-null mutation, we analyzed wild-type (WT), single Sos1-KO, Sos2-KO and double Sos1/2 KO primary mouse embryonic fibroblasts (MEF) with an aim at evaluating the functional specificity or redundancy of the Sos1 and Sos2 alleles at the cellular level. The 4OHT-induced Sos1-KO and Sos1/2-DKO MEFs exhibited distinct flat morphology, enlarged cell perimeter and altered cytoskeletal organization that were not observed in the WT and Sos2-KO counterparts. The Sos1-KO and Sos1/2-DKO MEFs also displayed significant accumulation, in comparison with WT and Sos2-KO MEFs, of cytoplasmic vesicular bodies identified as autophagosomes containing degraded mitochondria by means of electron microscopy and specific markers. Cellular proliferation and migration were impaired in Sos1-KO and Sos1/2-DKO MEFs in comparison with WT and Sos2-KO MEFs, whereas cell adhesion was only impaired upon depletion of both Sos isoforms. RasGTP formation was practically absent in Sos1/2-DKO MEFs as compared with the other genotypes and extracellular signal-regulated kinase phosphorylation showed only significant reduction after combined Sos1/2 depletion. Consistent with a mitophagic phenotype, in vivo labeling with specific fluorophores uncovered increased levels of oxidative stress (elevated intracellular reactive oxygen species and mitochondrial superoxide and loss of mitochondrial membrane potential) in the Sos1-KO and the Sos1/2-DKO cells as compared with Sos2-KO and WT MEFs. Interestingly, treatment of the MEF cultures with antioxidants corrected the altered phenotypes of Sos1-KO and Sos1/2-DKO MEFs by restoring their altered perimeter size and proliferative rate to levels similar to those of WT and Sos2-KO MEFs. Our data uncover a direct mechanistic link between Sos1 and control of intracellular oxidative stress, and demonstrate functional prevalence of Sos1 over Sos2 with regards to cellular proliferation and viability.

Detection of macroglial cells in the fish optic nerve by intracellular injection in fixed tissue / Detección de células macrogliales en el nervio óptico de peces mediante inyección intracelular en tejido fijado

In this study we made intracellular injections of Lucifer Yellow fluorochrome into macroglial cells, astrocytes and oligodendrocytes of the fixed optic nerve of tench (Tinca tinca). From their three-dimensional morphology, we identified oligodendrocytes and at least four different types of astrocytes, both in the central zones of the nerve and in that forming part of the glia limitans. Moreover, we have identified and described groups of associated astrocytes (AU)

En este estudio realizamos inyecciones intracelulares del fluorocromo Amarillo Lucifer en las células macrogliales, astrocitos y oligodendrocitos del nervio óptico fijado de la tenca (Tinca tinca). En base a su morfología tridimensional, pudimos identificar oligodendrocitos y al menos cuatro tipos distintos de astrocitos, tanto en las zonas centrales del nervio como en la zona que forma parte de la glia limitans. Adicionalmente, identificamos y describimos grupos de astrositos asociados (AU)

Non-neuronal cells involved in the degeneration and regeneration of the fish retina.

In the present work we show that during the degenerative process occurring after the cryo-elimination of the tench peripheral growing zone many non-neuronal cell types in addition to the resident microglial cells, appear within the affected areas. Some of them are normally found in the retina, such as the retinal pigmented epithelium cells and others originate from extra-retinal tissues. We identified these as granular leukocytes and macrophages. The microglial cells and macrophages, those resident in the sub-retinal space, and the invasive ones, act as phagocytes. The analysis of the injured retina following lesion shows that the invasive macrophages, arising from the scleral extra-retinal tissues, penetrate the neural retina, and migrate from the scleral to the vitreal portion. In contrast those coming from the vitreal extra-retinal tissues migrate in the opposite direction. Moreover, the retinal pigmented epithelium cells present remarkable modifications in their morphology and distribution and enter the neural retina, where they disrupt the surrounding tissue. We have also observed that this cryo-lesion causes an inflammation mediated by a type of granular leukocyte, denominated heterophils which penetrate the neural retina and probably come from the blood supply. Our results suggest that, during the first days after the lesion, the participation of diverse non-neuronal cells removing cell debris from the damaged zone should create a favourable environment allowing the regeneration of the neural retina.

Growing and regenerating axons in the visual system of teleosts are recognized with the antibody RT97.

We have analyzed the immunolabeling with the antibody RT97, a good marker for ganglion cell axons in several species, in the normal and regenerating visual pathways of teleosts. We have demonstrated that RT97 antibody recognizes several proteins in the tench visual system tissues (105, 115, 160, 200, 325 and 335 kDa approximately). By using immunoprecipitation and Western blot we have found that after crushing the optic nerve the immunoreactivity to anti RT97 increased markedly in the optic nerve. In immunohistochemical analysis we also found a different pattern of labeling in normal and regenerating visual pathways. In normal tench RT97 is a good marker for the horizontal cells in the retina, for growing ganglion cell axons which run along the optic nerve from the retina to the optic tectum and of the axon terminals in the stratum opticum and stratum fibrosum and griseum superficiale in the optic tectum. After optic nerve crush, no immunohistochemistry modifications were observed in the retina. However, in accordance with Western blot experiments, in the optic nerve intensely stained groups of regenerating axons appeared progressively throughout the optic nerve as far as the optic tectum. We conclude that the antibody RT97 is an excellent marker of growing and regenerating axons of the optic nerve of fish.

A comparative study of protein kinase C-like immunoreactive cells in the retina.

The present study is a morphological and quantitative analysis of protein kinase C-like immunoreactive (PKC-L ir) bipolar cells in the retinas of five different vertebrate species (chicken, tench, zebrafish, goldfish and rat). The morphology of PKC-L-ir bipolar cell axon terminals in fish differs significantly from those of chicken and rat retinas. Fish have bulky terminals whereas chicken and rat have their terminals in the form of small knob-shaped branches. In tench and goldfish, PKC-L-ir bipolar cells gradually decrease in size from the medial (i.e., in tench: mean +/- SD soma area of 30.09 +/- 5.98 microm2) to the peripheral (i.e., in tench: 19.93 +/- 1.73 microm2) retinal regions. This is not observed in chicken, rat or zebrafish where there is more homogeneity in s oma and axon terminal sizes between different retinal regions. Except in chicken, cell density increases from the central (i.e., in tench: mean +/- SD 1795.88 +/- 242.35 cells/mm2) to the peripheral (i.e., in tench: 4295.41 +/- 279.23 cells/mm2) retina. This study provides data that show relevant differences in the PKC-L-ir bipolar morphology and density among birds, fish and mammals. Moreover, these structural variations could mean not only differences in the cellular physiology, but also in the patterns of development and maintenance of the retina in each species.

Enzyme histochemical identification of microglial cells in the retina of a fish (Tinca tinca).

Histochemistry for nucleoside diphosphatase was used to study the microglial cells in the adult tench retina. An abundant population of microglial cells was located in the vascular membrane, nerve fibre layer, inner and outer plexiform layers and scattered cells were observed in the inner nuclear layer. Rounded and amoeboid cells could be seen close to the vessel in the vascular membrane, bipolar cells in the nerve fibre layer and ramified cells in the rest of the layers. Several microglial forms could correspond to developing cells. The pattern of distribution was similar to that described in other vertebrates, but with several differences, such as the presence of microglial cells in the vascular membrane and inner nuclear layer and the overlap of processes in the plexiform layers.

Response of microglial cells after a cryolesion in the peripheral proliferative retina of tench.

We studied the glial response after inducing a lesion in the zone of the peripheral retina of tench, where there is proliferative neuroepithelium. In the retina and optic nerve, the microglial response was analysed with tomato lectin and the macroglial response with antibodies against GFAP and S-100. In lesioned retinas, there was a temporal-spatial distribution pattern of microglia. One day after lesion, primitive ramified cells appeared in the nerve fibre layer. These cells appeared progressively from the vitreal to the scleral layers until day 7 when cells appeared in all layers, with the exception of the outer plexiform layer. From this point, labelling decreased. In the optic nerve, 3 days after lesion, an increase in the number of microglial cells was observed, first in the nerve folds and from day 15 in specific areas of the optic nerve. In the central retina, in the optic nerve head and within the optic nerve itself, the appearance of microglial cells, after the lesion, near the blood vessels, could indicate a vascular origin of microglia, as has been proposed by many authors. However, we cannot discount the idea that some of the reactive microglial cells arise by proliferation of the microglia existing in the normal state. Using GFAP and S-100 antibodies, no important changes in the retina were observed, however in the optic nerve there was response to the lesion. Thus, the macroglial cells appeared to be involved in reorganisation of the optic nerve axons after lesion.

Ultrastructural organization of the optic nerve of the tench (Cyprinidae, Teleostei).

Different parts of the tench optic nerve--the intraocular and intraorbital segments, the chiasm, and the post-chiasmatic segment--were studied using light and electron microscopy. From the head of the optic nerve, a zone of continuous growth constituted by the younger non-myelinated ganglion axons can be differentiated from a mature zone where almost all the axons are myelinated. The transition from one zone to the other is progressive. The area containing only non-myelinated axons is very restricted, and the presence of myelinated and non-myelinated axons in the same fascicle is frequent. In the head of the optic nerve, the growing zone surrounds the central artery. In the intraorbital segment, where the optic nerve is organized as a folded ribbon, the growing edge is surrounded by other mature folds. In the chiasm and in the post-chiasmatic segment of the optic nerve, the organization as a folded ribbon disappears and the youngest axons are situated on the periphery. In the growing zones, the immature astrocytes predominate; in the transition zones, oligodendrocytes, in different stages of maturity, begin to appear. In the mature zone, almost all the glial cells are differentiated, although immature cells can be found. The microglial cells are not abundant and are of the ramified type. Moreover, in contrast to the descriptions of other teleosts, the tench optic nerve is profusely supplied with blood vessels throughout its length.