RESUMEN
Galbeta1,3GalNAc and Galbeta1,4GIcNAc are the subterminal saccharide structures present on the CT carbohydrate antigen GalNAcbeta1,4[NeuAcalpha2,3]-Galbeta1-(3GalNAc or 4GIcNAc)-R, which is localized at the mammalian neuromuscular junction. Here we show that Galbeta1,3GalNAc, Galbeta1,4GIcNAc, and the CT carbohydrate antigen affect postsynaptic assembly in cultured muscle cells. Treatment of C2C12 myotubes with benzyl-O-alpha-GalNAc or neuraminidase increased peanut agglutinin (PNA) expression and AChR clustering. Induction of AChR clustering was blocked by PNA and by muscle agrin. Addition of Galbeta1,4GIcNAc or Galbeta1,3GalNAc increased AChR clustering in myotubes and muscle-specific kinase (MUSK) autophosphorylation in vitro, while NeuAcalpha2,3Galbeta1,4GIcNAc and Galbeta1,4GIc did not. Neural agrin activated MuSK in vitro if the lactosamine-containing mucin domain was present, and this activation was blocked in large part by Galbeta1,3GalNAc and Galbeta1,4GIcNAc. Agrin fragments and MuSK bound to these disaccharides with differing specificities. Overexpression of the CT carbohydrate antigen also increased AChR clustering and MuSK autophosphorylation in the presence of neural agrin. These data suggest a model in which different portions of the CT carbohydrate structure contribute to agrin-dependent signal transduction.
Asunto(s)
Agrina/metabolismo , Amino Azúcares/metabolismo , Gangliósidos/metabolismo , Músculo Esquelético/enzimología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Colinérgicos/metabolismo , Acetilgalactosamina/análogos & derivados , Acetilgalactosamina/farmacología , Agrina/análisis , Agrina/genética , Amino Azúcares/química , Animales , Antígenos de Carbohidratos Asociados a Tumores/química , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Compuestos de Bencilo/farmacología , Células COS , Línea Celular , ADN Complementario , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Gangliósidos/química , Gangliósidos/genética , Expresión Génica/fisiología , Humanos , Riñón/citología , Músculo Esquelético/química , Músculo Esquelético/citología , Neuraminidasa/farmacología , Oligopéptidos/genética , Aglutinina de Mani , Péptidos/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas Receptoras/análisis , Proteínas Tirosina Quinasas Receptoras/genética , TransfecciónRESUMEN
Adenoviral antisense constructs of the rat N-methyl-D-aspartate (NMDA) receptor subunit 1 (R1) were assessed for creating NMDAR1 knockouts in rat hippocampal CA1 regions in vivo. In situ hybridization analyses showed that virus-derived antisense transcripts were detected up to 5 weeks postinfection (p.i.). Although immunological methods failed to demonstrate a reduction of NMDA receptor protein, whole-cell recording showed that neurons in the transduced regions were deficient in NMDA receptor-mediated synaptic currents, but not alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor (AMPAR)-mediated synaptic currents. Thus, the data suggest that adenovirus technology can be used to locally knockout specific gene function for dissecting molecular mechanisms of synaptic plasticity.
Asunto(s)
Hipocampo/fisiología , Células Piramidales/fisiología , ARN sin Sentido/biosíntesis , Receptores de N-Metil-D-Aspartato/deficiencia , Receptores de N-Metil-D-Aspartato/genética , Sinapsis/fisiología , 2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Adenoviridae , Secuencia de Aminoácidos , Animales , Inmunohistoquímica , Hibridación in Situ , Potenciales de la Membrana/efectos de los fármacos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Ratas , Ratas Mutantes , Receptores AMPA/fisiología , Receptores de N-Metil-D-Aspartato/biosíntesis , Transcripción GenéticaRESUMEN
Replication-deficient adenoviral recombinants were assessed for in vivo transduction of rat hippocampal CA1 cells. Results show that efficient widespread transduction of CA1 in vivo was rapidly achievable and was sustained for more than 5 weeks. Assessment of electrophysiological properties in acute hippocampal slices showed that synaptic functioning and mechanisms involved in long-term potentiation (LTP) were preserved for minimally 5 weeks postinfection. Hence, adenovirus-mediated gene transfer in vivo promises to be a valuable tool for dissecting molecular mechanisms of synaptic plasticity, such as LTP and long-term depression (LTD).
Asunto(s)
Adenovirus Humanos , Hipocampo/fisiología , Neuronas/fisiología , Sinapsis/fisiología , beta-Galactosidasa/biosíntesis , Animales , Línea Celular , Estimulación Eléctrica , Genes Reporteros , Hipocampo/citología , Humanos , Técnicas In Vitro , Potenciación a Largo Plazo , Masculino , Plasticidad Neuronal , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/biosíntesis , TransfecciónRESUMEN
In order to investigate glucose transport at the blood-brain barrier (BBB), glucose transport properties were studied pharmacologically with a novel model system of inverted bovine brain cortical arteries. These vessels displayed glucose transport characteristics of facilitative glucose transporters (GLUT1-5) and of sodium- and energy-dependent glucose transporters (SGLT1-2). So far, glucose transport in the central nervous system (CNS) has only been shown to be achieved by facilitative transporters, in particular by GLUT1 at the blood-brain barrier and in glial cells and by GLUT3 in a subset of neurons. We report here that a SGLT-like transporter might partake in glucose transport at the bovine BBB, as indicated by immunocytochemical analysis and by Western immunoblot analysis of cultured bovine brain endothelial cells. A RNA protection assay revealed the presence of a SGLT-like gene fragment in rabbit cortex.
Asunto(s)
Proteínas Portadoras/metabolismo , Arterias Cerebrales/metabolismo , Corteza Cerebral/metabolismo , Metabolismo Energético/fisiología , Glicoproteínas de Membrana , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Sodio/metabolismo , Animales , Western Blotting , Bovinos , Células Cultivadas , Corteza Cerebral/irrigación sanguínea , Glucosa/metabolismo , Inmunohistoquímica , Técnicas In Vitro , Proteínas de Transporte de Monosacáridos/biosíntesis , ARN Mensajero/metabolismo , Conejos , Transportador 1 de Sodio-GlucosaRESUMEN
The deposition of amyloid in senile plaques and along the walls of the cerebral vasculature is a characteristic feature of Alzheimer disease. The peptide comprising the carboxyl-terminal 100 amino acids of the beta-amyloid precursor protein (beta APP) has been shown to aggregate into amyloid-like fibrils in vitro and to be neurotoxic, suggesting that this fragment may play a role in the etiology of Alzheimer disease. To address this question, we expressed this carboxyl-terminal 100-amino acid peptide of beta APP in transgenic mice under the control of the brain dystrophin promoter. We used an antibody to the principal component of amyloid, beta/A4, to demonstrate cell-body and neuropil accumulation of beta/A4 immunoreactivity in the brains of 4- and 6-month-old transgenic mice. Only light cytoplasmic staining with this antibody was visible in control mice. In addition, immunocytochemical analysis of the brains with an antibody to the carboxyl terminus of beta APP revealed abnormal aggregation of this epitope of beta APP within vesicular structures in the cytoplasm and in abnormal-appearing neurites in the CA2/3 region of the hippocampus in transgenic mice, similar to its aggregation in the cells of Alzheimer disease brains. Thioflavin S histochemistry suggested accumulations of amyloid in the cerebrovasculature of transgenic mice with the highest expression of the beta APP-C100 transgene. These observations suggest that expression of abnormal carboxyl-terminal subfragments of beta APP in vivo may cause amyloidogenesis and specific neuropathology.
Asunto(s)
Precursor de Proteína beta-Amiloide/biosíntesis , Hipocampo/patología , Neuronas/patología , Proteínas Recombinantes de Fusión/biosíntesis , Precursor de Proteína beta-Amiloide/análisis , Precursor de Proteína beta-Amiloide/genética , Animales , Clonación Molecular , Cruzamientos Genéticos , Distrofina/genética , Femenino , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos , Ratones Transgénicos , Neuronas/citología , Regiones Promotoras Genéticas , Tractos Piramidales/citología , Tractos Piramidales/metabolismo , Tractos Piramidales/patología , Proteínas Recombinantes de Fusión/análisis , Valores de Referencia , Mapeo Restrictivo , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructuraRESUMEN
PC12 cells transfected with retroviral recombinants expressing the carboxyl-terminal 104 amino acids of the Alzheimer amyloid protein precursor (beta APP-C104) or PC12 cells transfected with the retroviral vector (DO) alone were transplanted into the brains of newborn mice. At 20 days after grafting, transplants could be detected in all of the mouse brains examined. At 4 months after transplantation, experimental animals exhibited significant cortical atrophy. Some also revealed immunoreactivity with Alz-50, an antibody that detects an Alzheimer disease-related protein, in the somatodendritic domain of neurons in the cortex surrounding the transplants. In addition, disorganization of the neuropil in the CA2/3 region of the hippocampus ipsilateral to the transplant was revealed by staining with an antibody to the carboxyl-terminal end of the amyloid protein precursor. A decrease in cell body immunoreactivity for this portion of the amyloid protein precursor was also detected with this antibody. Together, these results suggest that the carboxyl-terminal fragment of beta APP may cause specific neuropathology and neurodegeneration in vivo.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Corteza Cerebral/patología , Hipocampo/patología , Neuronas/patología , Trasplante Heterotópico/patología , Envejecimiento , Precursor de Proteína beta-Amiloide/genética , Animales , Animales Recién Nacidos , Atrofia , Corteza Cerebral/crecimiento & desarrollo , Vectores Genéticos , Hipocampo/crecimiento & desarrollo , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias/patología , Células PC12 , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Valores de Referencia , Transfección , Trasplante Heterotópico/fisiologíaRESUMEN
The plasma membrane Ca(2+)-pumping ATPase (Ca(2+)-ATPase) mRNAs are encoded on four different genes designated PMCA1-PMCA4. The primary transcripts from some of these genes are known to be alternately spliced in the region encoding the regulatory domains of the enzymes. The known alternately spliced forms of these Ca(2+)-ATPase mRNAs and a new spliced variant of PMCA4 (PMCA4b), presented here, represent at least nine different mRNAs encoding the Ca(2+)-ATPases. In this report, the examination of the tissue-specific distribution of these alternately spliced mRNAs using polymerase chain reaction amplification of cDNA coupled with Southern blotting revealed that each spliced variant had a unique tissue distribution. PMCA1b and PMCA4a were present in all tissues examined. PMCA1a, PMCA1b, and PMCA4b were expressed in excitable tissues, whereas PMCA1d was expressed only in muscle tissues. PMCA2 was found in liver, adrenal gland, spinal cord, and brain. PMCA3a was present in spinal cord, and PMCA3b in thymus, adrenal gland, spinal cord, and brain. The mRNA for a new spliced variant of PMCA4 (PMCA4b) was detected in this study. Complementary DNAs for this isoform were isolated and characterized from human and bovine brain. This alternately spliced form of the PMCA4 mRNA contained an exon inserted at the splice junction immediately following the sequence encoding the calmodulin-binding domain. As has also been shown for PMCA1a, this insertion produced a shift in the reading frame at the 3'-end of the PMCA4 mRNA that yielded a sequence encoding a Ca(2+)-ATPase lacking a large portion of the C-terminal regulatory domain. When the human PMCA4 gene spanning this region of variable exon splicing was sequenced, it confirmed the intron-exon boundaries where alternate splicing occurs to produce PMCA4a and PMCA4b.