GRF2 Is Crucial for Cone Photoreceptor Viability and Ribbon Synapse Formation in the Mouse Retina.

Using constitutive GRF1/2 knockout mice, we showed previously that GRF2 is a key regulator of nuclear migration in retinal cone photoreceptors. To evaluate the functional relevance of that cellular process for two putative targets of the GEF activity of GRF2 (RAC1 and CDC42), here we compared the structural and functional retinal phenotypes resulting from conditional targeting of RAC1 or CDC42 in the cone photoreceptors of constitutive GRF2 and GRF2 mice. We observed that single RAC1 disruption did not cause any obvious morphological or physiological changes in the retinas of GRF2 mice, and did not modify either the phenotypic alterations previously described in the retinal photoreceptor layer of GRF2 mice.

Scribble basal polarity acquisition in RPE cells and its mislocalization in a pathological AMD-like model.

Apicobasal polarity is a hallmark of retinal pigment epithelium cells and is required to perform their functions; however, the precise roles of the different proteins that execute polarity are still poorly understood. Here, we have studied the expression and location of Scribble, the core member of the polarity basal protein complex in epithelial-derived cells, in human and mouse RPE cells in both control and pathological conditions. We found that Scribble specifically localizes at the basolateral membrane of mouse and human RPE cells.

Insulin receptor activation by proinsulin preserves synapses and vision in retinitis pigmentosa.

Synaptic loss, neuronal death, and circuit remodeling are common features of central nervous system neurodegenerative disorders. Retinitis pigmentosa (RP), the leading cause of inherited blindness, is a group of retinal dystrophies characterized by photoreceptor dysfunction and death. The insulin receptor, a key controller of metabolism, also regulates neuronal survival and synaptic formation, maintenance, and activity.

Evolutionary Origin of Insulin-Degrading Enzyme and Its Subcellular Localization and Secretion Mechanism: A Study in Microglial Cells.

The insulin-degrading enzyme (IDE) is a zinc-dependent metalloendopeptidase that belongs to the M16A metalloprotease family. IDE is markedly expressed in the brain, where it is particularly relevant due to its in vitro amyloid beta (Aβ)-degrading activity. The subcellular localization of IDE, a paramount aspect to understand how this enzyme can perform its proteolytic functions in vivo, remains highly controversial.

Impairment of Autophagic Flux Participates in the Antitumor Effects of TAT-Cx43 in Glioblastoma Stem Cells.

Autophagy is a physiological process by which various damaged or non-essential cytosolic components are recycled, contributing to cell survival under stress conditions. In cancer, autophagy can have antitumor or protumor effects depending on the developmental stage. Here, we use Western blotting, immunochemistry, and transmission electron microscopy to demonstrate that the antitumor peptide TAT-Cx43, a c-Src inhibitor, blocks autophagic flux in glioblastoma stem cells (GSCs) under basal and nutrient-deprived conditions.

Key Role for CRB2 in the Maintenance of Apicobasal Polarity in Retinal Pigment Epithelial Cells.

Apicobasal polarity is essential for epithelial cell function, yet the roles of different proteins in its completion is not fully understood. Here, we have studied the role of the polarity protein, CRB2, in human retinal pigment epithelial (RPE) cells during polarization , and in mature murine RPE cells . After establishing a simplified protocol for the culture of human fetal RPE cells, we studied the temporal sequence of the expression and localization of polarity and cell junction proteins during polarization in these epithelial cells.

Targeting metabolic plasticity in glioma stem cells in vitro and in vivo through specific inhibition of c-Src by TAT-Cx43.

Background: Glioblastoma is the most aggressive primary brain tumour and has a very poor prognosis. Inhibition of c-Src activity in glioblastoma stem cells (GSCs, responsible for glioblastoma lethality) and primary glioblastoma cells by the peptide TAT-Cx43 reduces tumorigenicity, and boosts survival in preclinical models. Because c-Src can modulate cell metabolism and several reports revealed poor clinical efficacy of various antitumoral drugs due to metabolic rewiring in cancer cells, here we explored the inhibition of advantageous GSC metabolic plasticity by the c-Src inhibitor TAT-Cx43.

Sfrp1 deficiency makes retinal photoreceptors prone to degeneration.

Millions of individuals worldwide suffer from impaired vision, a condition with multiple origins that often impinge upon the light sensing cells of the retina, the photoreceptors, affecting their integrity. The molecular components contributing to this integrity are however not yet fully understood. Here we have asked whether Secreted Frizzled Related Protein 1 (SFRP1) may be one of such factors.

Extracellular Vesicles Secreted by Astroglial Cells Transport Apolipoprotein D to Neurons and Mediate Neuronal Survival Upon Oxidative Stress.

Extracellular vesicle (EV)-mediated glia-to-neuron communication has been recognized in a growing number of physiological and pathological situations. They transport complex sets of molecules that can be beneficial or detrimental for the receiving cell. As in other areas of biology, their analysis is revolutionizing the field of neuroscience, since fundamental signaling processes are being re-evaluated, and applications for neurodegenerative disease therapies have emerged.

Expression and localization of the polarity protein CRB2 in adult mouse brain: a comparison with the CRB1 mutant mouse model.

Acquisition of cell polarization is essential for the performance of crucial functions, like a successful secretion and appropriate cell signaling in many tissues, and it depends on the correct functioning of polarity proteins, including the Crumbs complex. The CRB proteins, CRB1, CRB2 and CRB3, identified in mammals, are expressed in epithelial-derived tissues like brain, kidney and retina. CRB2 has a ubiquitous expression and has been detected in embryonic brain tissue; but currently there is no data regarding its localization in the adult brain.

Myelin extracellular leaflet compaction requires apolipoprotein D membrane management to optimize lysosomal-dependent recycling and glycocalyx removal.

To compact the extracellular sides of myelin, an important transition must take place: from membrane sliding, while building the wraps, to membrane adhesion and water exclusion. Removal of the negatively charged glycocalyx becomes the limiting factor in such transition. What is required to initiate this membrane-zipping process? Knocking-out the Lipocalin Apolipoprotein D (ApoD), essential for lysosomal functional integrity in glial cells, results in a specific defect in myelin extracellular leaflet compaction in peripheral and central nervous system, which results in reduced conduction velocity and suboptimal behavioral outputs: motor learning is compromised.

Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress.

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.

Sox10 Expression in Goldfish Retina and Optic Nerve Head in Controls and after the Application of Two Different Lesion Paradigms.

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.

ARMS/Kidins220 and synembryn-B levels regulate NGF-mediated secretion.

Proper development of the nervous system requires a temporally and spatially orchestrated set of events including differentiation, synapse formation and neurotransmission. Nerve growth factor (NGF) acting through the TrkA neurotrophin receptor (also known as NTRK1) regulates many of these events. However, the molecular mechanisms responsible for NGF-regulated secretion are not completely understood.

RASGRF2 controls nuclear migration in postnatal retinal cone photoreceptors.

Detailed immunocytochemical analyses comparing wild-type (WT), GRF1-knockout (KO), GRF2-KO and GRF1/2 double-knockout (DKO) mouse retinas uncovered the specific accumulation of misplaced, 'ectopic' cone photoreceptor nuclei in the photoreceptor segment (PS) area of retinas from GRF2-KO and GRF1/2-DKO, but not of WT or GRF1-KO mice. Localization of ectopic nuclei in the PS area of GRF2-depleted retinas occurred postnatally and peaked between postnatal day (P)11 and P15. Mechanistically, the generation of this phenotype involved disruption of the outer limiting membrane and intrusion into the PS layer by cone nuclei displaying significant perinuclear accumulation of signaling molecules known to participate in nuclear migration and cytoskeletal reorganization, such as PAR3, PAR6 and activated, phosphorylated forms of PAK, MLC2 and VASP.

CRB2 completes a fully expressed Crumbs complex in the Retinal Pigment Epithelium.

The CRB proteins CRB1, CRB2 and CRB3 are members of the cell polarity complex Crumbs in mammals that together with Scribble and Par complexes stablish the polarity of a variety of cell types. Although many members of the Crumbs complex proteins are expressed in the retinal pigment epithelium (RPE), and even though the mRNA of CRB2 has been detected in ARPE-19 cells and in the RPE/Choroid, to date no CRB protein has yet been found in this tissue. To investigate this possibility, we generated an antibody that specifically recognize the mouse CRB2 protein, and we demonstrate the expression of CRB2 in mouse RPE.

Loss of retinitis pigmentosa 2 (RP2) protein affects cone photoreceptor sensory cilium elongation in mice.

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.

Microtubule motors transport phagosomes in the RPE, and lack of KLC1 leads to AMD-like pathogenesis.

The degradation of phagosomes, derived from the ingestion of photoreceptor outer segment (POS) disk membranes, is a major role of the retinal pigment epithelium (RPE). Here, POS phagosomes were observed to associate with myosin-7a, and then kinesin-1, as they moved from the apical region of the RPE. Live-cell imaging showed that the phagosomes moved bidirectionally along microtubules in RPE cells, with kinesin-1 light chain 1 (KLC1) remaining associated in both directions and during pauses.

UV irradiation accelerates amyloid precursor protein (APP) processing and disrupts APP axonal transport.

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.

Immunocytochemical evidence of the localization of the Crumbs homologue 3 protein (CRB3) in the developing and mature mouse retina.

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.

Numb/Numbl-Opo antagonism controls retinal epithelium morphogenesis by regulating integrin endocytosis.

Polarized trafficking of adhesion receptors plays a pivotal role in controlling cellular behavior during morphogenesis. Particularly, clathrin-dependent endocytosis of integrins has long been acknowledged as essential for cell migration. However, little is known about the contribution of integrin trafficking to epithelial tissue morphogenesis.

Elevated PGC-1α activity sustains mitochondrial biogenesis and muscle function without extending survival in a mouse model of inherited ALS.

The transcriptional coactivator PGC-1α induces multiple effects on muscle, including increased mitochondrial mass and activity. Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, adult-onset neurodegenerative disorder characterized by selective loss of motor neurons and skeletal muscle degeneration. An early event is thought to be denervation-induced muscle atrophy accompanied by alterations in mitochondrial activity and morphology within muscle.

Characterization of Pax2 expression in the goldfish optic nerve head during retina regeneration.

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.

The Usher 1B protein, MYO7A, is required for normal localization and function of the visual retinoid cycle enzyme, RPE65.

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.