RESUMEN

Neurodegenerative diseases have been linked to inflammation, but whether altered immunomodulation plays a causative role in neurodegeneration is not clear. We show that lack of cytokine interferon-ß (IFN-ß) signaling causes spontaneous neurodegeneration in the absence of neurodegenerative disease-causing mutant proteins. Mice lacking Ifnb function exhibited motor and cognitive learning impairments with accompanying α-synuclein-containing Lewy bodies in the brain, as well as a reduction in dopaminergic neurons and defective dopamine signaling in the nigrostriatal region. Lack of IFN-ß signaling caused defects in neuronal autophagy prior to α-synucleinopathy, which was associated with accumulation of senescent mitochondria. Recombinant IFN-ß promoted neurite growth and branching, autophagy flux, and α-synuclein degradation in neurons. In addition, lentiviral IFN-ß overexpression prevented dopaminergic neuron loss in a familial Parkinson's disease model. These results indicate a protective role for IFN-ß in neuronal homeostasis and validate Ifnb mutant mice as a model for sporadic Lewy body and Parkinson's disease dementia.

Asunto(s)

Interferón beta/metabolismo , Neuronas/metabolismo , Receptor de Interferón alfa y beta/metabolismo , Animales , Autofagia , Modelos Animales de Enfermedad , Terapia Genética , Interferón beta/genética , Interferón beta/uso terapéutico , Enfermedad por Cuerpos de Lewy/metabolismo , Enfermedad por Cuerpos de Lewy/patología , Ratones , Ratones Endogámicos C57BL , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/terapia , Receptor de Interferón alfa y beta/genética , Transducción de Señal , Transcriptoma , alfa-Sinucleína/metabolismo

Asunto(s)

Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuritas/fisiología , Isoformas de Proteínas/metabolismo , Transducción de Señal/fisiología , Animales , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Quinasa 2 de Adhesión Focal/genética , Quinasa 2 de Adhesión Focal/metabolismo , Humanos , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Moléculas de Adhesión de Célula Nerviosa/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Isoformas de Proteínas/genética , Proteína Quinasa C/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-fyn/genética , Proteínas Proto-Oncogénicas c-fyn/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo

Asunto(s)

Moléculas de Adhesión de Célula Nerviosa/biosíntesis , Isoformas de Proteínas/metabolismo , Empalme Alternativo , Transporte Biológico , Regulación del Desarrollo de la Expresión Génica , Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/genética , Isoformas de Proteínas/genética , Procesamiento Proteico-Postraduccional , Distribución Tisular

RESUMEN

The two major isoforms (180 kDa and 140 kDa) of the neural cell adhesion molecule (N-CAM) are crucially involved in neurogenesis and brain repair via activation of the mitogen-activated protein kinase (MAPK) cascade. Modification by glycosylation, and homophilic and heterophilic interactions regulate the function of N-CAM, but little is known about the interplay of these processes. In the neuron-like PC12 cell line, extracellular small acidic peptides have been shown to modulate the expression of N-CAM mRNA and protein and regulate its translocation to the plasma membrane. Among these peptides, a synthetic Ig-III-like short sequence (H2N-DDSDEEN-COOH), designated sSP, was particularly potent. In this study, we analyzed the cross-talk between nerve growth factor (NGF) and extracellular sSP in native and N-CAM-transfected PC12 cells to determine if these systems interact to modulate transduction pathways and regulate early steps of neurogenesis in vitro. Our results indicate that sSP accelerated the phosphorylation of extracellular regulated kinase-1 (ERK1) and -2 (ERK2) and promoted plasma membrane translocation of 180 kDa N-CAM. By stabilizing cell-cell contacts and promoting cell cluster formation, these events, which were mediated via a significant increase in intracellular Ca2+, regulated some of the early stages of the NGF-induced differentiation process.

Asunto(s)

Señalización del Calcio/fisiología , Moléculas de Adhesión de Célula Nerviosa/biosíntesis , Oligopéptidos/farmacología , Animales , Señalización del Calcio/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Activación Enzimática , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacología , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Células PC12 , Estructura Terciaria de Proteína , Transporte de Proteínas , Ratas , Transfección

RESUMEN

The brain acid-soluble protein BASP1 (CAP-23, NAP-22) belongs to the family of growth-associated proteins, which also includes GAP-43, a protein recently shown to regulate neural cell adhesion molecule (NCAM)-mediated neurite outgrowth. Here, the effects of BASP1 overexpression were investigated in PC12E2 cells and primary hippocampal neurons. BASP1 overexpression stimulated neurite outgrowth in both cell types. The effects of BASP1 and trans-homophilic NCAM interactions were additive, and BASP1-induced neurite outgrowth was not inhibited by ectopic expression of cytoplasmic NCAM domains. Furthermore, inhibition of signaling via the fibroblast growth factor receptor, Src-family nonreceptor tyrosine kinases, protein kinase C, or GSK3beta, and expression of constructs of the cytoskeletal proteins spectrin and tau inhibited NCAM- but not BASP1-induced neurite outgrowth. Expression of BASP1 mutated at the serine-5 phosphorylation site stimulated neurite outgrowth to a degree comparable to that observed in response to overexpression of wild-type BASP1, whereas expression of BASP1 mutated at the myristoylation site at glycine-1 completely abrogated the stimulatory effects of the protein on neurite outgrowth. Finally, coexpression experiments with dominant negative and wild-type versions of GAP-43 and BASP1 demonstrated that the two proteins could substitute for each other with respect to induction of NCAM-independent neurite outgrowth, whereas BASP1 was unable to replace the stimulatory effect of GAP-43 on NCAM-mediated neurite outgrowth. These observations demonstrate that BASP1 and GAP-43 have overlapping, but not identical, functions in relation to neurite outgrowth and indicate that the main function of BASP1 is to regulate the organization and morphology of the plasma membrane.

Asunto(s)

Hipocampo/embriología , Hipocampo/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuritas/metabolismo , Proteínas Represoras/metabolismo , Animales , Membrana Celular/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Proteína GAP-43/metabolismo , Humanos , Immunoblotting , Inmunohistoquímica , Ratones , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Células PC12 , Ratas , Transducción de Señal/fisiología , Transfección

RESUMEN

The neural cell adhesion molecule (NCAM), and the growth-associated protein (GAP-43), play pivotal roles in neuronal development and plasticity and possess interdependent functions. However, the mechanisms underlying the functional association of GAP-43 and NCAM have not been elucidated. In this study we show that (over)expression of GAP-43 in PC12E2 cells and hippocampal neurons strongly potentiates neurite extension, both in the absence and in the presence of homophilic NCAM binding. This potentiation is crucially dependent on the membrane association of GAP-43. We demonstrate that phosphorylation of GAP-43 by protein kinase C (PKC) as well as by casein kinase II (CKII) is important for the NCAM-induced neurite outgrowth. Moreover, our results indicate that in the presence of GAP-43, NCAM-induced neurite outgrowth requires functional association of NCAM-180/spectrin/GAP-43, whereas in the absence of GAP-43, the NCAM-140/non-receptor tyrosine kinase (Fyn)-associated signaling pathway is pivotal. Thus, expression of GAP-43 presumably acts as a functional switch for NCAM-180-induced signaling. This suggests that under physiological conditions, spatial and/or temporal changes of the localization of GAP-43 and NCAM on the cell membrane may determine the predominant signaling mechanism triggered by homophilic NCAM binding: NCAM-180/spectrin-mediated modulation of the actin cytoskeleton, NCAM-140-mediated activation of Fyn, or both.

Asunto(s)

Proteína GAP-43/fisiología , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuritas/fisiología , Neuronas/citología , Animales , Células Cultivadas , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Fibroblastos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Ratones , Modelos Biológicos , Mutagénesis/fisiología , Moléculas de Adhesión de Célula Nerviosa/genética , Neuritas/efectos de los fármacos , Neuritas/ultraestructura , Ratas , Sinaptosomas/metabolismo , Transfección/métodos

RESUMEN

The neural cell adhesion molecule (NCAM) is pivotal in neural development, regeneration, and learning. Here we characterize two peptides, termed P1-B and P2, derived from the homophilic binding sites in the first two N-terminal immunoglobulin (Ig) modules of NCAM, with regard to their effects on neurite extension and adhesion. To evaluate how interference of these mimetic peptides with NCAM homophilic interactions in cis influences NCAM binding in trans, we employed a coculture system in which PC12-E2 cells were grown on monolayers of fibroblasts with or without NCAM expression and the rate of neurite outgrowth subsequently was analyzed. P2, but not P1-B, induced neurite outgrowth in the absence of NCAM binding in trans. When PC12-E2 cells were grown on monolayers of NCAM-expressing fibroblasts, the effect of both P1-B and P2 on neurite outgrowth was dependent on peptide concentrations. P1-B and P2 acted as conventional antagonists, agonists, and reverse agonists of NCAM at low, intermediate, and high peptide concentrations, respectively. The demonstrated in vitro triple pharmacological effect of mimetic peptides interfering with the NCAM homophilic cis binding will be valuable for the understanding of the actions of these mimetics in vivo.

Asunto(s)

Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Técnicas de Cocultivo , Prueba de Complementación Genética , Células L , Ratones , Modelos Moleculares , Imitación Molecular , Mutagénesis Sitio-Dirigida , Moléculas de Adhesión de Célula Nerviosa/genética , Neuritas/metabolismo , Neuritas/ultraestructura , Neuronas/citología , Neuronas/metabolismo , Células PC12 , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección

RESUMEN

The role of protein kinase C (PKC) isoforms in the neural cell adhesion molecule (NCAM)-mediated neurite outgrowth was tested using a co-culture system consisting of fibroblasts with or without NCAM expression upon which either primary cerebellar granular neurones (CGN) or pheochromocytoma (PC12-E2) cells were grown. The latter transiently expressed various PKC isoforms and domains derived from selected PKCs. PKC inhibitors of various specificity inhibited NCAM-stimulated neuritogenesis from CGN, indicating that PKC is involved in this process. Moreover, stimulation by the NCAM-mimetic peptide, C3d, elicited phosphorylation of PKC in CGN. Expression of kinase-deficient forms of PKCalpha, betaI and betaII blocked NCAM-mediated neurite extension, but had no effect on nerve growth factor (NGF)-mediated neurite outgrowth. Expression of two PKCepsilon constructs: (i) a fragment from PKCepsilon encompassing the pseudosubstrate, the C1a domain (including the actin-binding site, ABS), and parts of the V3 region, or (ii) the PKCepsilon-specific ABS blocked NCAM-mediated neurite extension in both cases. These two constructs also partially inhibited NGF-stimulated neuritogenesis indicating that PKCepsilon is a positive regulator of both NCAM- and NGF-mediated differentiation. We suggest that PKCepsilon is a common downstream mediator for several neuritogenic factors, whereas one or more conventional PKCs are specifically involved in NCAM-stimulated neurite outgrowth.

Asunto(s)

Aumento de la Célula , Moléculas de Adhesión de Célula Nerviosa/fisiología , Neuritas/fisiología , Proteína Quinasa C/fisiología , Animales , Aumento de la Célula/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Indoles/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/fisiología , Maleimidas/farmacología , Neuritas/efectos de los fármacos , Células PC12 , Proteína Quinasa C/antagonistas & inhibidores , Ratas

RESUMEN

The neural cell adhesion molecule, NCAM, is involved in multiple cis- and trans-homophilic interactions (NCAM binding to NCAM) thereby facilitating cell-cell adhesion through the formation of zipper-like NCAM-complexes. NCAM is also involved in heterophilic interactions with a number of proteins and extracellular matrix molecules. Some of these heterophilic interactions are mutually exclusive, and some interfere with or are dependent on homophilic NCAM interactions. Furthermore, both homo- and heterophilic interactions are modulated by posttranslational modifications of NCAM. Heterophilic NCAM-interactions initiate several intracellular signal transduction pathways ultimately leading to biological responses involving cellular differentiation, proliferation, migration and survival. Both homo- and heterophilic NCAM-interactions can be mimicked by synthetic peptides, which can induce NCAM-like signalling, and in vitro and in vivo studies suggest that such NCAM mimetics may be used for the treatment of neurodegenerative disorders.

Asunto(s)

Moléculas de Adhesión de Célula Nerviosa/fisiología , Transducción de Señal/fisiología , Animales , Humanos , Isomerismo , Modelos Moleculares , Moléculas de Adhesión de Célula Nerviosa/química , Procesamiento Proteico-Postraduccional

RESUMEN

The neural cell adhesion molecule, NCAM, mediates Ca(2+)-independent cell-cell and cell-substratum adhesion via homophilic (NCAM-NCAM) and heterophilic (NCAM-non-NCAM molecules) binding. NCAM plays a key role in neural development, regeneration, and synaptic plasticity, including learning and memory consolidation. The crystal structure of a fragment comprising the three N-terminal Ig modules of rat NCAM has been determined to 2.0 A resolution. Based on crystallographic data and biological experiments we present a novel model for NCAM homophilic binding. The Ig1 and Ig2 modules mediate dimerization of NCAM molecules situated on the same cell surface (cis interactions), whereas the Ig3 module mediates interactions between NCAM molecules expressed on the surface of opposing cells (trans interactions) through simultaneous binding to the Ig1 and Ig2 modules. This arrangement results in two perpendicular zippers forming a double zipper-like NCAM adhesion complex.

Asunto(s)

Anticuerpos/inmunología , Anticuerpos/fisiología , Adhesión Celular/fisiología , Moléculas de Adhesión de Célula Nerviosa/inmunología , Cristalografía por Rayos X , Dimerización , Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/fisiología , Neuritas/fisiología

RESUMEN

The neural cell adhesion molecule, NCAM, is known to stimulate neurite outgrowth from primary neurones and PC12 cells presumably through signalling pathways involving the fibroblast growth factor receptor (FGFR), protein kinase A (PKA), protein kinase C (PKC), the Ras-mitogen activated protein kinase (MAPK) pathway and an increase in intracellular Ca2+ levels. Stimulation of neurones with the synthetic NCAM-ligand, C3, induces neurite outgrowth through signalling pathways similar to the pathways activated through physiological, homophilic NCAM-stimulation. We present here data indicating that phosphatidylinositol 3-kinase (PI3K) is required for NCAM-mediated neurite outgrowth from PC12-E2 cells and from cerebellar and dopaminergic neurones in primary culture, and that the thr/ser kinase Akt/protein kinase B (PKB) is phosphorylated downstream of PI3K after stimulation with C3. Moreover, we present data indicating a survival-promoting effect of NCAM-stimulation by C3 on cerebellar and dopaminergic neurones induced to undergo apoptosis. This protective effect of C3 included an inhibition of both DNA-fragmentation and caspase-3 activation. The survival-promoting effect of NCAM-stimulation was also shown to be dependent on PI3K.

Asunto(s)

Diferenciación Celular/fisiología , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Moléculas de Adhesión Celular Neuronal/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Humanos , Etiquetado Corte-Fin in Situ , Ligandos , Ratones , Datos de Secuencia Molecular , Moléculas de Adhesión de Célula Nerviosa/farmacología , Neuritas/efectos de los fármacos , Neuronas/citología , Neuronas/efectos de los fármacos , Péptidos/farmacología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología

RESUMEN

Cell migration is required during development of the nervous system. The regulatory mechanisms for this process, however, are poorly elucidated. We show here that expression of or exposure to the neural cell adhesion molecule (NCAM) strongly affected the motile behaviour of glioma cells independently of homophilic NCAM interactions. Expression of the transmembrane 140 kDa isoform of NCAM (NCAM-140) caused a significant reduction in cellular motility, probably through interference with factors regulating cellular attachment, as NCAM-140-expressing cells exhibited a decreased attachment to a fibronectin substratum compared with NCAM-negative cells. Ectopic expression of the cytoplasmic part of NCAM-140 also inhibited cell motility, presumably via the non-receptor tyrosine kinase p59(fyn) with which NCAM-140 interacts. Furthermore, we showed that the extracellular part of NCAM acted as a paracrine inhibitor of NCAM-negative cell locomotion through a heterophilic interaction with a cell-surface receptor. As we showed that the two N-terminal immunoglobulin modules of NCAM, which are known to bind to heparin, were responsible for this inhibition, we presume that this receptor is a heparan sulfate proteoglycan. A model for the inhibitory effect of NCAM is proposed, which involves competition between NCAM and extracellular components for the binding to membrane-associated heparan sulfate proteoglycan.

Asunto(s)

Adhesión Celular/fisiología , Membrana Celular/metabolismo , Movimiento Celular/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteoglicanos de Heparán Sulfato/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Animales , Sitios de Unión/genética , Membrana Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Citoplasma/genética , Citoplasma/metabolismo , Proteínas de la Matriz Extracelular/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Glioma , Humanos , Integrinas/metabolismo , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/farmacología , Isoformas de Proteínas/genética , Estructura Terciaria de Proteína/genética , Ratas , Células Tumorales Cultivadas