RESUMEN
Adult retinal ganglion cells (RGCs) can regenerate their axons in vitro. Using proteomics, we discovered that the supernatants of cultured retinas contain isoforms of crystallins with crystallin beta b2 (crybb2) being clearly up-regulated in the regenerating retina. Immunohistochemistry revealed the expression of crybb within the retina, including in filopodial protrusions and axons of RGCs. Cloning and overexpression of crybb2 in RGCs and hippocampal neurons increased axonogenesis, which in turn could be blocked with antibodies against beta-crystallin. Conditioned medium from crybb2-transfected cell cultures also supported the growth of axons. Finally real time imaging of the uptake of green fluorescent protein-tagged crybb2 fusion protein showed that this protein becomes internalized. These data are the first to show that axonal regeneration is related to crybb2 movement. The results suggest that neuronal crystallins constitute a novel class of neurite-promoting factors that likely operate through an autocrine mechanism and that they could be used in neurodegenerative diseases.
Asunto(s)
Axones/fisiología , Cadena B de beta-Cristalina/fisiología , Secuencia de Aminoácidos , Animales , Callithrix , Técnicas de Cultivo de Célula , Línea Celular , Células Cultivadas , Medios de Cultivo , Electroforesis en Gel Bidimensional , Conos de Crecimiento/fisiología , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiología , Proteómica , Ratas , Ratas Sprague-Dawley , Retina/citología , Células Ganglionares de la Retina , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Cadena B de beta-Cristalina/químicaRESUMEN
The purpose of this study was to identify the gene expression profile of the regenerating retina in vitro. To achieve this goal, three experimental groups were studied: (1) an injury control group (OC-LI group) that underwent open crush (OC) of the optic nerve and lens injury (LI) in vivo; (2) an experimental group (OC-LI-R group) that comprised animals treated like those in the OC-LI group except that retinal axons were allowed to regenerate (R) in vitro; and (3) an experimental group (OC-LI-NR group) that comprised animals treated as those in the OC-LI group, except that the retinas were cultured in vitro with the retinal ganglion cell (RGC) layer facing upwards to prevent axonal regeneration (NR). Gene expression in each treatment group was compared to that of untreated controls. Immunohistochemistry was used to examine whether expression of differentially regulated genes also occurred at the protein level and to localize these proteins to the respective retinal cells. Genes that were regulated belonged to different functional categories such as antioxidants, antiapoptotic molecules, transcription factors, secreted signaling molecules, inflammation-related genes, and others. Comparison of changes in gene expression among the various treatment groups revealed a relatively small cohort of genes that was expressed in different subsets of cells only in the OC-LI-R group; these genes can be considered to be regeneration-specific. Our findings demonstrate that axonal regeneration of RGC involves an orchestrated response of all retinal neurons and glia, and could provide a platform for the development of therapeutic strategies for the regeneration of injured ganglion cells.
Asunto(s)
Regulación de la Expresión Génica/genética , Conos de Crecimiento/metabolismo , Regeneración Nerviosa/genética , Neuroglía/metabolismo , Traumatismos del Nervio Óptico/terapia , Células Ganglionares de la Retina/metabolismo , Animales , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Femenino , Perfilación de la Expresión Génica , Conos de Crecimiento/ultraestructura , Inmunohistoquímica , Cristalino/lesiones , Cristalino/fisiopatología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Nervio Óptico/citología , Nervio Óptico/metabolismo , Traumatismos del Nervio Óptico/genética , Traumatismos del Nervio Óptico/fisiopatología , Técnicas de Cultivo de Órganos , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Células Ganglionares de la Retina/citología , Activación Transcripcional/genética , Regulación hacia Arriba/genéticaRESUMEN
Synapses are specialized contact sites mediating communication between neurons. Synaptogenesis requires the specific assembly of protein clusters at both sides of the synaptic contact by mechanisms that are barely understood. We studied the synaptic targeting of multi-domain proteins of the ProSAP/Shank family thought to serve as master scaffolding molecules of the postsynaptic density. In contrast to Shank1, expression of green-fluorescent protein (GFP)-tagged ProSAP1/Shank2 and ProSAP2/Shank3 deletion constructs in hippocampal neurons revealed that their postsynaptic localization relies on the integrity of the C-termini. The shortest construct that was perfectly targeted to synaptic sites included the last 417 amino acids of ProSAP1/Shank2 and included the C-terminal sterile alpha motif (SAM) domain. Removal of 54 residues from the N-terminus of this construct resulted in a diffuse distribution in the cytoplasm. Altogether, our data delineate a hitherto unknown targeting signal in both ProSAP1/Shank2 and ProSAP2/Shank3 and provide evidence for an implication of these proteins and their close homologue, Shank1, in distinct molecular pathways.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Sinapsis/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Células Cultivadas , Proteínas Fluorescentes Verdes/genética , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Ratas , Proteínas Recombinantes de Fusión/genética , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
In a yeast two hybrid screen with the human isoform of Dendrin (KIAA0749), a putative modulator of the postsynaptic cytoskeleton, we isolated a cDNA coding for a novel protein, KIBRA, possessing two amino-terminal WW domains, an internal C2-like domain and a carboxy-terminal glutamic acid-rich stretch. Northern blot analysis revealed that the expression of KIBRA mRNA was predominately found in kidney and brain. In vitro interaction studies revealed that the first KIBRA WW domain binds specifically to PPxY motifs. Transient transfection of monkey kidney cells with constructs encoding Myc-tagged KIBRA displayed a cytoplasmic localization and a perinuclear enrichment of the protein.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Chlorocebus aethiops , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosfoproteínas , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Técnicas del Sistema de Dos HíbridosRESUMEN
Two isoforms of alpha-actinin 1 (ACTN1) known to be generated by tissue-specific alternative splicing of mutually exclusive exons have been described. Muscle cells express ACTN1 containing the smooth muscle exon (SM), while other (non-muscle) cells contain the non-muscle exon (NM). In this report, we describe the characterization of a novel ACTN1 isoform in adult rat brain in which both exons (NM + SM) are combined in the same transcript to give a brain-specific sequence domain (BS). Reverse transcriptase polymerase chain reaction (RT-PCR) demonstrated that expression of the BS exon was restricted to the brain. During development, weak expression of the BS exon was observed at early postnatal stages whereas in adult brain, it represented the predominant isoform of ACTN1. In situ hybridization analysis revealed that BS expression was highest in neurons of the hippocampus, cortex, and caudate putamen while the cerebellum and other subcortical structures showed only weak labeling.