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Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Among the catalog of oxidative stress-responsive genes is the Lipocalin Apolipoprotein D (ApoD), an extracellular lipid binding protein endowed with antioxidant capacity. Within the nervous system, cell types in the defense frontline, such as astrocytes, secrete ApoD to help neurons cope with the challenge. The protecting role of ApoD is known from cellular to organism level, and many of its downstream effects, including optimization of autophagy upon neurodegeneration, have been described. However, we still cannot assign a cellular mechanism to ApoD gene that explains how this protection is accomplished. Here we perform a comprehensive analysis of ApoD intracellular traffic and demonstrate its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. By combining single-lysosome in vivo pH measurements with immunodetection, we demonstrate that ApoD is endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins. Our results uncover a previously unknown biological function of ApoD, member of the finely regulated and evolutionary conserved gene family of extracellular Lipocalins. They set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular functions, critical for the outcome of a wide variety of neurodegenerative diseases. These results open therapeutic opportunities by providing a route of entry and a repair mechanism for lysosomes in pathological situations. [ABSTRACT FROM AUTHOR]

The mammalian central nervous system (CNS) is unable to regenerate. In contrast, the CNS of fish, including the visual system, is able to regenerate after damage. Moreover, the fish visual system grows continuously throughout the life of the animal, and it is therefore an excellent model to analyze processes of myelination and re-myelination after an injury. Here we analyze Sox10+ oligodendrocytes in the goldfish retina and optic nerve in controls and after two kinds of injuries: cryolesion of the peripheral growing zone and crushing of the optic nerve. We also analyze changes in a major component of myelin, myelin basic protein (MBP), as a marker for myelinated axons. Our results show that Sox10+ oligodendrocytes are located in the retinal nerve fiber layer and along the whole length of the optic nerve. MBP was found to occupy a similar location, although its loose appearance in the retina differed from the highly organized MBP+ axon bundles in the optic nerve. After optic nerve crushing, the number of Sox10+ cells decreased in the crushed area and in the optic nerve head. Consistent with this, myelination was highly reduced in both areas. In contrast, after cryolesion we did not find changes in the Sox10+ population, although we did detect some MBP- degenerating areas. We show that these modifications in Sox10+ oligodendrocytes are consistent with their role in oligodendrocyte identity, maintenance and survival, and we propose the optic nerve head as an excellent area for research aimed at better understanding of de- and remyelination processes. [ABSTRACT FROM AUTHOR]

The Pax2 transcription factor plays a crucial role in axon-guidance and astrocyte differentiation in the optic nerve head (ONH) during vertebrate visual system development. However, little is known about its function during regeneration. The fish visual system is in continuous growth and can regenerate. Müller cells and astrocytes of the retina and ONH play an important role in these processes. We demonstrate that pax2a in goldfish is highly conserved and at least two pax2a transcripts are expressed in the optic nerve. Moreover, we show two different astrocyte populations in goldfish: Pax2+ astrocytes located in the ONH and S100+ astrocytes distributed throughout the retina and the ONH. After peripheral growth zone (PGZ) cryolesion, both Pax2+ and S100+ astrocytes have different responses. At 7 days after injury the number of Pax2+ cells is reduced and coincides with the absence of young axons. In contrast, there is an increase of S100+ astrocytes in the retina surrounding the ONH and S100+ processes in the ONH. At 15 days post injury, the PGZ starts to regenerate and the number of S100+ astrocytes increases in this region. Moreover, the regenerating axons reach the ONH and the pax2a gene expression levels and the number of Pax2+ cells increase. At the same time, S100+/GFAP+/GS+ astrocytes located in the posterior ONH react strongly. In the course of the regeneration, Müller cell vitreal processes surrounding the ONH are primarily disorganized and later increase in number. During the whole regenerative process we detect a source of Pax2+/ PCNA+ astrocytes surrounding the posterior ONH. We demonstrate that pax2a expression and the Pax2+ astrocyte population in the ONH are modified during the PGZ regeneration, suggesting that they could play an important role in this process. [ABSTRACT FROM AUTHOR]

CRB3 (Crumbs homologue 3), a member of the CRB protein family (homologous to the Drosophila Crumbs), is expressed in different epithelium-derived cell types in mammals, where it seems to be involved in regulating the establishment and stability of tight junctions and in ciliogenesis. This protein has been also detected in the retina, but little is known about its localization and function in this tissue. Our goal here was to perform an in-depth study of the presence of CRB3 protein in the mouse retina and to analyze its expression during photoreceptor ciliogenesis and the establishment of the plexiform retinal layers. Double immunofluorescence experiments for CRB3 and well-known markers for the different retinal cell types were performed to study the localization of the CRB3 protein. According to our results, CRB3 is present from postnatal day 0 (P0) until adulthood in the mouse retina. It is localized in the inner segments (IS) of photoreceptor cells, especially concentrated in the area where the connecting cilium is located, in their synaptic terminals in the outer plexiform layer (OPL), and in sub-populations of amacrine and bipolar cells in the inner plexiform layer (IPL).