Proinsulin: From Hormonal Precursor to Neuroprotective Factor

In the last decade, non-canonical functions have been described for several molecules with hormone-like activities in different stages of vertebrate development. Since its purification in the 1960s, proinsulin has been one of the best described hormonal precursors, though it has been overwhelmingly studied in the context of insulin, the mature protein secreted by the pancreas. Beginning with our discovery of the presence and precise regulation of proinsulin mRNA in early neurulation and neurogenesis, we uncovered a role for proinsulin in cell survival in the developing nervous system. We subsequently demonstrated the ability of proinsulin to prevent pathological cell death and delay photoreceptor degeneration in a mouse model of retinitis pigmentosa. In this review, we focus on the evolution of proinsulin/insulin, beginning with insulin-like peptides expressed in mainly the neurosecretory cells of some invertebrates. We summarize findings related to the regulation of proinsulin expression during development and discuss the possible effects of proinsulin in neural cells or tissue, and its potential as a neuroprotective molecule

Bravo/Nr-CAM Is Closely Related to the Cell Adhesion Molecules L1 and Ng-CAM and Has a Similar Heterodimer Structure

Diverse cell-surface molecules of the nervous system play an important role in specifying cell interactions during development. Using a method designed to generate mAbs against neural surface molecules of defined molecular weight, we have previously reported on the surface protein, Bravo, found in the developing avian retinotectal system. Bravo is immunologically detected on developing optic fibers in the retina, but absent from distal regions of the same fibers in the tectum. We have isolated cDNA clones encompassing the entire coding region of Bravo, including clones containing five alternative sequences of cDNA. These putative alternatively spliced sequences encode stretches of polypeptide ranging in length from 10-93 amino acids and are predicted to be both extra- and intracellular. The deduced primary structure of Bravo reveals that, like the cell adhesion molecules (CAMs) chicken Ng- CAM and mouse L1, Bravo is composed of six Ig-like domains, five fibronectin type III repeats, a transmembrane domain, and a short cytoplasmic region. Recently, the cDNA sequence of a related molecule, Nr-CAM, was reported and its possible identity with Bravo discussed (Grumet, M., V. Mauro, M. P. Burgoon, G. E. Edelman, and B. A. Cunningham. 1991. J. Cell Biol. 113:1399-1412). Here we confirm this identity and moreover show that Bravo is found on Muller glial processes and end-feet in the developing retina. In contrast to the single polypeptide chain structure of Nr-CAM reported previously, we show that Bravo has a heterodimer structure composed of an alpha chain of M(r) 140/130 and a beta chain of 60-80 kD. As with L1 and Ng-CAM, the two chains of Bravo are generated from an intact polypeptide by cleavage at identical locations and conserved sites within all three molecules (Ser-Arg/Lys-Arg). The similar domain composition and heterodimer structure, as well as the 40% amino acid sequence identity of these molecules, defines them as an evolutionarily related subgroup of CAMs. The relationship of Bravo to molecules known to be involved in cell adhesion and process outgrowth, combined with its pattern of expression and numerous potential isoforms, suggests a complex role for this molecule in cell interactions during neural development

How autophagy is related to programmed cell death during the development of the nervous system

5 páginas, 3 figuras -- PAGS nros. 813-817Programmed cell death, together with proliferation and differentiation, is an essential process during the development of the nervous system. During neurogenesis, neurons and glia are generated in large numbers and, subsequently, they die in a process that depends on trophic signalling that refines the cytoarchitecture and connectivity of the nervous system. In addition, programmed cell death also affects proliferating neuroepithelial cells and recently differentiated neuroblasts. Autophagy is a lysosomal degradative pathway that allows the recycling of cell constituents, and seems to be able to play a dual role. It may serve to protect the cell by preventing the accumulation of deleterious products and organelles and supplying energy and amino acids. On the other hand, it has been considered a type of cell death. The role of autophagy during development is little characterized. The retina provides an excellent model system to study autophagy in the context of neural development, and to establish its relationship with proliferation, differentiation and cell death. In the present review, we summarize recent findings showing that autophagy contributes to the development of the nervous system by providing energy for cell corpse removal after physiological cell death, a process associated with retinal neurogenesisResearch in our laboratory is supported by grants from the Spanish Ministerio de Educación y Ciencia (BFU2006-00508 and BFU2006-26073-E to P.B. and SAF2007-66175 to E.J. de la R.). M.A.M. is an FPU (Formación de Profesorado Universitario) Fellow and P.B. is a Ramón y Cajal Fellow (both programmes are financed by the Ministerio de Educación y Ciencia)Peer reviewe

Topologically Restricted Appearance in the Developing Chick Retinotectal System of Bravo, a Neural Surface Protein: Experimental Modulation by Environmental Cues

A novel neural surface protein, Bravo, shows a pattern of topological restriction in the embryonic chick retinotectal system. Bravo is present on the developing optic fibers in the retina; however, retinal axons in the tectum do not display Bravo. The appearance of Bravo in vitro is modulated by environmental cues. Axons growing out from retinal explants on retinal basal lamina, their natural substrate, express Bravo, whereas such axons growing on collagen do not. Retinal explants provide a valuable system to characterize the mechanism of Bravo restriction, as well as the cellular signals controlling it. Bravo was identified with monoclonal antibodies from a collection generated against exposed molecules isolated by using a selective cell surface biotinylation procedure. The NH2-terminal sequence of Bravo shows similarity with L1, a neural surface molecule which is a member of the immunoglobulin superfamily. This possible relationship to L1, together with its restricted appearance, suggests an involvement of Bravo in axonal growth and guidance

Muerte neural temprana: un proceso inadvertido en el desarrollo del sistema nervioso.

15 p.-7 fig.[EN]During the development of the vertebrate nervous system, multiple physiological processes are involved in the generation of its complex cytoarchitecture and functionality. Among them, programmed cell death has been recognized as a key process that affects connecting neurons. By contrast, there is limited information available regarding the cell death that affects neuroepithelial cells, and recently born neurons and glia, hindering the comprehensive understanding of neural development. We have demonstrated that exquisitely regulated PCD occurs during early stages of neural development such as neurulation and neurogenesis. We have characterized how survival signals from proteins like proinsulin/insulin, c-Raf, and HSC70 counteract caspase-dependent apoptosis, which affects neuroepithelial cells proliferation and the generation of retinal ganglion cells. Furthermore, the characterization of these physiological signals during retinal neurogenesis has the potential to provide new therapeutic tools to attenuate retinal neurodegeneration.[ES]Durante el desarrollo del sistema nervioso de vertebrados, múltiples procesos fisiológicos participan en la generación de su compleja arquitectura celular y funcionalidad. Entre ellos, la muerte celular programada que afecta a neuronas de conexión está reconocido como un proceso fundamental. Por otro lado, hay escasa información disponible acerca de la muerte celular que afecta a células neuroepiteliales y a neuronas y glía recién nacidas, lo que impide que tengamos una noción completa sobre el desarrollo neural. Los estudios de nuestro laboratorio han demostrado que la muerte celular programada se encuentra finamente regulada y ocurre en etapas tan tempranas del desarrollo como la neurulación o la neurogénesis. Hemos caracterizado el papel que moléculas de supervivencia, como la proinsulina/insulina, c-Raf o HSC70, desempeñan bloqueando la apoptosis dependiente de caspasas, proceso que afecta a células neuroepiteliales proliferativas, así como a la generación de las células ganglionares de la retina. Es más, la caracterización de estas señales fisiológicas originadas durante la neurogénesis de la retina nos ha proporcionado una nueva herramienta terapéutica potencial para el tratamiento y atenuación de las neurodegeneraciones retinianas.Research in the laboratory is funded by grants-in-aid from the Spanish Ministerio de Educación y Ciencia (SAF2007-66175-C02-01 to EJdlR, BFU2007-61055/BMC to FdP, and BFU2006-00508 to PB).Peer reviewe

Científicas pioneras en el vivero del CIB

La saga de investigadoras del siglo XX que llegaron a tener un muy merecido prestigio y voz propia en el área de biomedicina en nuestro país, tiene su máximo referente en Margarita Salas. Pero coetáneas con ella, o algunos años mayores, un grupo de científicas florecieron en el Centro de Investigaciones Biológicas (CIB) del Consejo Superior de Investigaciones Científicas (CSIC). Fueron pioneras en sus respectivas especialidades: la Biología Molecular, la Endocrinología Molecular o la Bioquímica y el Metabolismo, entre otras. Este breve repaso a sus trayectorias quiere rendir tributo tanto a aquellas que tuvieron un reconocimiento general, en algunos casos a nivel internacional, como a algunas menos conocidas pero que contribuyeron definitivamente a impulsar la menguada ciencia española de la segunda mitad del siglo pasado

Apoptosis in the trabecular meshwork of glaucomatous patients

We established and validated an in toto method to perform TdT-mediated dUTP nick end labeling to study apoptosis in human trabecular meshwork tissue obtained during trabeculectomy in glaucoma patients. In specimens from patients with primary open-angle glaucoma and primary angle-closure glaucoma, we detected a tendency for more apoptotic cells to accumulate in patients with primary open-angle glaucoma. The utility of this method to study apoptosis in the trabecular meshwork is discussed, as well as its application as a tool in biologic samples

A GSK-3b modulator delays photoreceptor cell death and preserves visual function in the rd10 mouse model of retinitis pigmentosa

1 p.Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal dystrophies that lead to blindness. Photoreceptor cell death, reactive gliosis and retinal inammation are common features in animal models of the disease. The enzyme Glycogen Synthase Kinase-3 Beta (GSK-3b) is involved in inammatory processes associated to diverse neurodegenerative pathologies. The aim of our study is to test in the rd10 mouse whether the GSK-3b inhibitor VP3.15 (a small heterocyclic molecule) is a potential therapeutic treatment for RP.SAF2013-41059-RPeer reviewe

Intravitreal injection of proinsulin-loaded microspheres delays photoreceptor cell death and vision loss in the rd10 mouse model of retinitis pigmentosa

9 p.-6 fig.PURPOSE. The induction of proinsulin expression by transgenesis or intramuscular gene therapy has been shown previously to retard retinal degeneration in mouse and rat models of retinitis pigmentosa (RP), a group of inherited conditions that result in visual impairment. We investigated whether intraocular treatment with biodegradable poly (lactic-co-glycolic) acid microspheres (PLGA-MS) loaded with proinsulin has cellular and functional neuroprotective effects in the retinaMETHODS. Experiments were performed using the Pde6brd10 mouse model of RP. Methionylated human recombinant proinsulin (hPI) was formulated in PLGA-MS, which were administered by intravitreal injection on postnatal days (P) 14 to 15. Retinal neuroprotection was assessed at P25 by electroretinography, and by evaluating outer nuclear layer (ONL) cellular preservation. The attenuation of photoreceptor cell death by hPI was determined by TUNEL assay in cultured P22 retinas, as well as Akt phosphorylation by immunoblottingRESULTS. We successfully formulated hPI PLGA-MS to deliver the active molecule for several weeks in vitro. The amplitude of b-cone and mixed b-waves in electroretinographic recording was significantly higher in eyes injected with hPI-PLGA-MS compared to control eyes.Treatment with hPI-PLGA-MS attenuated photoreceptor cell loss, as revealed by comparing ONL thickness and the number of cell rows in this layer in treated versus untreated retinas. Finally, hPI prevented photoreceptor cell death and increased AktThr308 phosphorylation in organotypic cultured retinas.CONCLUSIONS. Retinal degeneration in the rd10 mouse was slowed by a single intravitreal injection of hPI-PLGA-MS. Human recombinant proinsulin elicited a rapid and effective neuroprotective effect when administered in biodegradable microspheres, which may constitute a future potentially feasible delivery method for proinsulin-based treatment of RP.Supported by Grants from the Spanish Ministerio de Ciencia e Innovacion (MICINN) and Spanish Ministerio de EconomÍa y Competitividad (MINECO), SAF2010-21879 (EJdlR and PdlV), SAF2013-41059-R (FdP and EJdlR), and technical personnel support from CIBERDEM, ISCIII, Madrid, SpainPeer reviewe

Visual decline in aged mice is delayed by proinsulin

1 p.Visual decline is normally associated to the aging process.We search for cellular and molecular processes associated to normal aging. In addition, we have previously shown that human proinsulin (hPi) delays vision loss, as determined by electroretinography (ERG),and retinal degeneration, as determined by photoreceptor counting,in the rd10 mouse and the P23H rat models of Retinitis Pigmentosa.Our aim is to reveal additional potential benets of a hPi-based treatment in the aged retina.CONSOLIDER CSD2010-00045 SpainPeer reviewe