RESUMO
The cell adhesion molecule L1 (L1CAM, L1 in short) plays crucial roles during neural development, regeneration after injury, synapse formation, synaptic plasticity and tumor cell migration. L1 belongs to the immunoglobulin superfamily and comprises in its extracellular part six immunoglobulin (Ig)-like domains and five fibronectin type III homologous repeats (FNs). The second Ig-like domain has been validated for self- (so-called homophilic) binding between cells. Antibodies against this domain inhibit neuronal migration in vitro and in vivo. The fibronectin type III homologous repeats FN2 and FN3 bind small molecule agonistic L1 mimetics and contribute to signal transduction. FN3 has a stretch of 25 amino acids that can be triggered with a monoclonal antibody, or the L1 mimetics, to enhance neurite outgrowth and neuronal cell migration in vitro and in vivo. To correlate the structural features of these FNs with function, we determined a high-resolution crystal structure of a FN2FN3 fragment, which is functionally active in cerebellar granule cells and binds several mimetics. The structure illustrates that both domains are connected by a short linker sequence allowing a flexible and largely independent organization of both domains. This becomes further evident by comparing the X-ray crystal structure with models derived from Small-Angle X-ray Scattering (SAXS) data for FN2FN3 in solution. Based on the X-ray crystal structure, we identified five glycosylation sites which we believe are crucial for folding and stability of these domains. Our study signifies an advance in the understanding of structure-functional relationships of L1.
Assuntos
Fibronectinas , Molécula L1 de Adesão de Célula Nervosa , Fibronectinas/fisiologia , Raios X , Espalhamento a Baixo Ângulo , Difração de Raios X , Anticorpos Monoclonais , Adesão Celular/fisiologia , NeuritosRESUMO
Cell adhesion molecule L1 regulates multiple cell functions and L1 deficiency is linked to several neural diseases. Proteolytic processing generates functionally decisive L1 fragments, which are imported into the nucleus. By computational analysis, we found at L1's C-terminal end the chromo shadow domain-binding motif PxVxL, which directs the binding of nuclear proteins to the heterochromatin protein 1 (HP1) isoforms α, ß, and É£. By enzyme-linked immunosorbent assay, we show that the intracellular L1 domain binds to all HP1 isoforms. These interactions involve the HP1 chromo shadow domain and are mediated via the sequence 1158 KDET1161 in the intracellular domain of murine L1, but not by L1's C-terminal PxVxL motif. Immunoprecipitation using nuclear extracts from the brain and from cultured cerebellar and cortical neurons indicates that HP1 isoforms interact with a yet unknown nuclear L1 fragment of approximately 55 kDa (L1-55), which carries ubiquitin residues. Proximity ligation indicates a close association between L1-55 and the HP1 isoforms in neuronal nuclei. This association is reduced after the treatment of neurons with inhibitors of metalloproteases, ß-site of amyloid precursor protein cleaving enzyme (BACE1), or É£-secretase, suggesting that cleavage of full-length L1 by these proteases generates L1-55. Reduction of HP1α, -ß, or -É£ expression by siRNA decreases L1-dependent neurite outgrowth from cultured cortical neurons and decreases the L1-dependent migration of L1-transfected HEK293 cells in a scratch assay. These findings indicate that the interaction of the novel fragment L1-55 with HP1 isoforms in nuclei affects L1-dependent functions, such as neurite outgrowth and neuronal migration.
Assuntos
Movimento Celular , Homólogo 5 da Proteína Cromobox/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neuritos/metabolismo , Motivos de Aminoácidos , Animais , Homólogo 5 da Proteína Cromobox/genética , Feminino , Masculino , Camundongos , Camundongos Mutantes , Molécula L1 de Adesão de Célula Nervosa/genética , Domínios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Abnormal functions of the cell adhesion molecule L1 are linked to several neural diseases. Proteolytic L1 fragments were reported to interact with nuclear and mitochondrial proteins to regulate events in the developing and the adult nervous system. Recently, we identified a 55 kDa L1 fragment (L1-55) that interacts with methyl CpG binding protein 2 (MeCP2) and heterochromatin protein 1 (HP1) via the KDET motif. We now show that L1-55 also interacts with histone H1.4 (HistH1e) via this motif. Moreover, we show that this motif binds to NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2), splicing factor proline/glutamine-rich (SFPQ), the non-POU domain containing octamer-binding protein (NonO), paraspeckle component 1 (PSPC1), WD-repeat protein 5 (WDR5), heat shock cognate protein 71 kDa (Hsc70), and synaptotagmin 1 (SYT1). Furthermore, applications of HistH1e, NDUFV2, SFPQ, NonO, PSPC1, WDR5, Hsc70, or SYT1 siRNAs or a cell-penetrating KDET-carrying peptide decrease L1-dependent neurite outgrowth and the survival of cultured neurons. These findings indicate that L1's KDET motif binds to an unexpectedly large number of molecules that are essential for nervous system-related functions, such as neurite outgrowth and neuronal survival. In summary, L1 interacts with cytoplasmic, nuclear and mitochondrial proteins to regulate development and, in adults, the formation, maintenance, and flexibility of neural functions.
Assuntos
Proteínas Mitocondriais , Molécula L1 de Adesão de Célula Nervosa , Citoplasma/metabolismo , Citosol/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Molécula L1 de Adesão de Célula Nervosa/química , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Humanos , Camundongos , AnimaisRESUMO
The cell adhesion molecule L1 is essential not only for neural development, but also for synaptic functions and regeneration after trauma in adulthood. Abnormalities in L1 functions cause developmental and degenerative disorders. L1's functions critically depend on proteolysis which underlies dynamic cell interactions and signal transduction. We showed that a 70 kDa fragment (L1-70) supports mitochondrial functions and gene transcription. To gain further insights into L1-70's functions, we investigated several binding partners. Here we show that L1-70 interacts with topoisomerase 1 (TOP1), peroxisome proliferator-activated receptor γ (PPARγ) and NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2). TOP1, PPARγ and NDUFV2 siRNAs reduced L1-dependent neurite outgrowth, and the topoisomerase inhibitors topotecan and irinotecan inhibited L1-dependent neurite outgrowth, neuronal survival and migration. In cultured neurons, L1 siRNA reduces the expression levels of the long autism genes neurexin-1 (Nrxn1) and neuroligin-1 (Nlgn1) and of the mitochondrially encoded gene NADH:ubiquinone oxidoreductase core subunit 2 (ND2). In mutant mice lacking L1-70, Nrxn1 and Nlgn1, but not ND2, mRNA levels are reduced. Since L1-70's interactions with TOP1, PPARγ and NDUFV2 contribute to the expression of two essential long autism genes and regulate important neuronal functions, we propose that L1 may not only ameliorate neurological problems, but also psychiatric dysfunctions.
Assuntos
Molécula L1 de Adesão de Célula Nervosa , Animais , Camundongos , Complexo I de Transporte de Elétrons/metabolismo , Flavoproteínas/metabolismo , Expressão Gênica , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Ubiquinona/metabolismo , DNA Topoisomerases Tipo I/metabolismoRESUMO
The neural cell adhesion molecule L1 (also called L1CAM or CD171) functions not only in cell migration, but also in cell survival, differentiation, myelination, neurite outgrowth, and signaling during nervous system development and in adults. The proteolytic cleavage of L1 in its extracellular domain generates soluble fragments which are shed into the extracellular space and transmembrane fragments that are internalized into the cell and transported to various organelles to regulate cellular functions. To identify novel intracellular interaction partners of L1, we searched for protein-protein interaction motifs and found two potential microtubule-associated protein 1 light-chain 3 (LC3)-interacting region (LIR) motifs within L1, one in its extracellular domain and one in its intracellular domain. By ELISA, immunoprecipitation, and proximity ligation assay using L1 mutant mice lacking the 70 kDa L1 fragment (L1-70), we showed that L1-70 interacts with LC3 via the extracellular LIR motif in the fourth fibronectin type III domain, but not by the motif in the intracellular domain. The disruption of the L1-LC3 interaction reduces L1-mediated neurite outgrowth and neuronal survival.
RESUMO
L1 syndrome is a rare developmental disorder characterized by hydrocephalus of varying severity, intellectual deficits, spasticity of the legs, and adducted thumbs. Therapy is limited to symptomatic relief. Numerous gene mutations in the L1 cell adhesion molecule (L1CAM, hereafter abbreviated L1) were identified in L1 syndrome patients, and those affecting the extracellular domain of this transmembrane type 1 glycoprotein show the most severe phenotypes. Previously analyzed rodent models of the L1 syndrome focused on L1-deficient animals or mouse mutants with abrogated cell surface expression of L1, making it difficult to test L1 function-triggering mimetic compounds with potential therapeutic value. To overcome this impasse, we generated a novel L1 syndrome mouse with a mutation of aspartic acid at position 201 in the extracellular part of L1 (p.D201N, hereafter termed L1-201) that displays a cell surface-exposed L1 accessible to the L1 mimetics. Behavioral assessment revealed an increased neurological deficit score and increased locomotor activity in male L1-201 mice carrying the mutation on the X-chromosome. Histological analyses of L1-201 mice showed features of the L1 syndrome, including enlarged ventricles and reduced size of the corpus callosum. Expression levels of L1-201 protein as well as extent of cell surface biotinylation and immunofluorescence labelling of cultured cerebellar neurons were normal. Importantly, treatment of these cultures with the L1 mimetic compounds duloxetine, crotamiton, and trimebutine rescued impaired cell migration and survival as well as neuritogenesis. Altogether, the novel L1 syndrome mouse model provides a first experimental proof-of-principle for the potential therapeutic value of L1 mimetic compounds.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/tratamento farmacológico , Deficiência Intelectual/tratamento farmacológico , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Peptidomiméticos/uso terapêutico , Paraplegia Espástica Hereditária/tratamento farmacológico , Animais , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Cerebelo/patologia , Ventrículos Cerebrais/metabolismo , Ventrículos Cerebrais/patologia , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Cloridrato de Duloxetina/farmacologia , Cloridrato de Duloxetina/uso terapêutico , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Locomoção , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Molécula L1 de Adesão de Célula Nervosa/genética , Neurogênese , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Peptidomiméticos/farmacologia , Fenótipo , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/patologia , Toluidinas/farmacologia , Toluidinas/uso terapêutico , Trimebutina/farmacologia , Trimebutina/uso terapêuticoRESUMO
Cell adhesion molecule L1 regulates multiple cell functions, and L1 deficiency is linked to several neural diseases. Recently, we have identified methyl CpG binding protein 2 (MeCP2) as a potential binding partner of the intracellular L1 domain. By ELISA we show here that L1's intracellular domain binds directly to MeCP2 via the sequence motif KDET. Proximity ligation assay with cultured cerebellar and cortical neurons suggests a close association between L1 and MeCP2 in nuclei of neurons. Immunoprecipitation using MeCP2 antibodies and nuclear mouse brain extracts indicates that MeCP2 interacts with an L1 fragment of ~55 kDa (L1-55). Proximity ligation assay indicates that metalloproteases, ß-site of amyloid precursor protein cleaving enzyme (BACE1) and É£-secretase, are involved in the generation of L1-55. Reduction in MeCP2 expression by siRNA decreases L1-dependent neurite outgrowth from cultured cortical neurons as well as the migration of L1-expressing HEK293 cells. Moreover, L1 siRNA, MeCP2 siRNA, or a cell-penetrating KDET-containing L1 peptide leads to reduced levels of myocyte enhancer factor 2C (Mef2c) mRNA and protein in cortical neurons, suggesting that the MeCP2/L1 interaction regulates Mef2c expression. Altogether, the present findings indicate that the interaction of the novel fragment L1-55 with MeCP2 affects L1-dependent functions, such as neurite outgrowth and neuronal migration.
Assuntos
Molécula L1 de Adesão de Célula Nervosa , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Células HEK293 , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Molécula L1 de Adesão de Célula Nervosa/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
Adhesion molecules regulate cell proliferation, migration, survival, neuritogenesis, synapse formation and synaptic plasticity during the nervous system's development and in the adult. Among such molecules, the neural cell adhesion molecule L1 contributes to these functions during development, and in synapse formation, synaptic plasticity and regeneration after trauma. Proteolytic cleavage of L1 by different proteases is essential for these functions. A proteolytic fragment of 70 kDa (abbreviated L1-70) comprising part of the extracellular domain and the transmembrane and intracellular domains was shown to interact with mitochondrial proteins and is suggested to be involved in mitochondrial functions. To further determine the role of L1-70 in mitochondria, we generated two lines of gene-edited mice expressing full-length L1, but no or only low levels of L1-70. We showed that in the absence of L1-70, mitochondria in cultured cerebellar neurons move more retrogradely and exhibit reduced mitochondrial membrane potential, impaired Complex I activity and lower ATP levels compared to wild-type littermates. Neither neuronal migration, neuronal survival nor neuritogenesis in these mutants were stimulated with a function-triggering L1 antibody or with small agonistic L1 mimetics. These results suggest that L1-70 is important for mitochondrial homeostasis and that its absence contributes to the L1 syndrome phenotypes.
Assuntos
Molécula L1 de Adesão de Célula Nervosa , Paraplegia Espástica Hereditária , Animais , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neuritos/metabolismo , Neurogênese/genética , Neurônios/metabolismo , Paraplegia Espástica Hereditária/metabolismoRESUMO
The important functions of cell adhesion molecule L1 in the nervous system depend on diverse proteolytic enzymes which generate different L1 fragments. It has been reported that cleavage in the third fibronectin type III (FNIII) homologous domain generates the fragments L1-80 and L1-140, while cleavage in the first FNIII domain yields the fragments L1-70 and L1-135. These results raised questions concerning the L1 cleavage sites. We thus generated gene-edited mice expressing L1 with mutations of the cleavage sites either in the first or third FNIII domain. By immunoprecipitations and immunoblot analyses using brain homogenates and different L1 antibodies, we show that L1-70 and L1-135 are generated in wild-type mice, but not or only to a low extent in L1 mutant mice. L1-80 and L1-140 were not detected in wild-type or mutant mice. Mass spectrometry confirmed the results from immunoprecipitations and immunoblot analyses. Based on these observations, we propose that L1-70 and L1-135 are the predominant fragments in the mouse nervous system and that the third FNIII domain is decisive for generating these fragments. Treatment of cultured cerebellar neurons with trypsin or plasmin, which were both proposed to generate L1-80 and L1-140 by cleaving in the third FNIII domain, showed by immunoprecipitations and immunoblot analyses that both proteases lead to the generation of L1-70 and L1-135, but not L1-80 and L1-140. We discuss previous observations on the basis of our new results and propose a novel view on the molecular features that render previous and present observations compatible.
Assuntos
Encéfalo/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neurônios/metabolismo , Proteólise , Animais , Camundongos , Camundongos MutantesRESUMO
The human natural killer (HNK-1) carbohydrate plays important roles during nervous system development, regeneration after trauma and synaptic plasticity. Four proteins have been identified as receptors for HNK-1: the laminin adhesion molecule, high-mobility group box 1 and 2 (also called amphoterin) and cadherin 2 (also called N-cadherin). Because of HNK-1's importance, we asked whether additional receptors for HNK-1 exist and whether the four identified proteins share any similarity in their primary structures. A set of 40,000 sequences homologous to the known HNK-1 receptors was selected and used for large-scale sequence alignments and motif searches. Although there are conserved regions and highly conserved sites within each of these protein families, there was no sequence similarity or conserved sequence motifs found to be shared by all families. Since HNK-1 receptors have not been compared regarding binding constants and since it is not known whether the sulfated or non-sulfated part of HKN-1 represents the structurally crucial ligand, the receptors are more heterogeneous in primary structure than anticipated, possibly involving different receptor or ligand regions. We thus conclude that the primary protein structure may not be the sole determinant for a bona fide HNK-1 receptor, rendering receptor structure more complex than originally assumed.
Assuntos
Antígenos CD57/metabolismo , Caderinas/metabolismo , Proteína HMGB1/metabolismo , Proteína HMGB2/metabolismo , Laminina/metabolismo , Oligossacarídeos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Antígenos CD57/química , Caderinas/química , Proteína HMGB1/química , Proteína HMGB2/química , Humanos , Laminina/química , Ligantes , Regeneração Nervosa/fisiologia , Plasticidade Neuronal/fisiologia , Oligossacarídeos/química , Ligação Proteica , Domínios ProteicosRESUMO
The cell adhesion molecule L1 (also known as L1CAM) plays important roles in the mammalian nervous system under physiological and pathological conditions. We have previously reported that proteolytic cleavage of L1 by myelin basic protein leads to the generation of a 70â kDa transmembrane L1 fragment (L1-70) that promotes neuronal migration and neuritogenesis. Here, we provide evidence that L1-70 is imported from the cytoplasm into mitochondria. Genetic ablation of L1, inhibition of mitochondrial import of L1-70 or prevention of myelin basic protein-mediated generation of L1-70 all lead to reduced mitochondrial complex I activity, and impaired mitochondrial membrane potential, fusion, fission and motility, as well as increased retrograde transport. We identified NADH dehydrogenase ubiquinone flavoprotein 2 as a binding partner for L1, suggesting that L1-70 interacts with this complex I subunit to regulate complex I activity. The results of our study provide insights into novel functions of L1 in mitochondrial metabolism and cellular dynamics. These functions are likely to ameliorate the consequences of acute nervous system injuries and chronic neurodegenerative diseases.
Assuntos
Mitocôndrias/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Encéfalo/metabolismo , Citoplasma/metabolismo , Feminino , Masculino , Camundongos , Transporte ProteicoRESUMO
Glycosaminoglycans such as chondroitin sulfate (CS) and dermatan sulfate (DS) are long chains of repeating disaccharide units, covalently linked to core proteins to form proteoglycans. Proteoglycans can be cell membrane-bound or are part of the extracellular matrix. They are important in a wide range of biologic processes, including development, synaptic plasticity, and regeneration after injury, as well as modulation of growth factor signaling, cell migration, survival, and proliferation. Synthesis of CS and DS in the Golgi apparatus is mediated by sulfotransferases that modify sugar chains through transfer of sulfate groups to specific positions on the sugar moieties. To clarify the functions of CS and DS during nervous system regeneration, we studied the effect of chondroitin 4- O-sulfotransferase-1/carbohydrate sulfotransferase-11 (C4ST-1/Chst-11) and dermatan 4- O-sulfotransferase-1/Chst-14 (D4ST-1/Chst-14) down-regulation on spinal cord regeneration in larval and adult zebrafish. In our study, knockdown of C4ST1/Chst-11 accelerated regeneration after spinal cord injury in larval and adult zebrafish and knockdown of D4ST1/Chst-14 did not alter regenerative capacity. From these and previous observations, we drew the conclusion that different CS and DS expression patterns can be growth permitting, growth inhibiting, or neutral for regrowing or sprouting axons, depending on the tissue environment of a particular animal species.-Sahu, S., Li, R., Loers, G., Schachner, M. Knockdown of chondroitin-4-sulfotransferase-1, but not of dermatan-4-sulfotransferase-1, accelerates regeneration of zebrafish after spinal cord injury.
Assuntos
Condroitina/metabolismo , Traumatismos da Medula Espinal/genética , Sulfotransferases/metabolismo , Animais , Técnicas de Silenciamento de Genes , Sulfotransferases/genética , Peixe-ZebraRESUMO
Because of the importance of the HNK-1 carbohydrate for preferential motor reinnervation after injury of the femoral nerve in mammals, we screened NIH Clinical Collection 1 and 2 Libraries and a Natural Product library comprising small organic compounds for identification of pharmacologically useful reagents. The reason for this attempt was to obviate the difficult chemical synthesis of the HNK-1 carbohydrate and its isolation from natural sources, with the hope to render such compounds clinically useful. We identified six compounds that enhanced neurite outgrowth from cultured spinal motor neurons at nM concentrations and increased their neurite diameter, but not their neurite branch points. Axons of dorsal root ganglion neurons did not respond to these compounds, a feature that is in agreement with their biological role after injury. We refer to the positive functions of some of these compounds in animal models of injury and delineate the intracellular signaling responses elicited by application of compounds to cultured murine central nervous system neurons. Altogether, these results point to the potential of the HNK-1 carbohydrate mimetics in clinically-oriented settings.
Assuntos
Antígenos CD57/análogos & derivados , Gânglios Espinais/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Compostos Orgânicos/farmacologia , Animais , Células Cultivadas , Embrião de Mamíferos , Feminino , Gânglios Espinais/citologia , Masculino , Camundongos , Neurônios Motores/citologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológicoRESUMO
The immunoglobulin superfamily adhesion molecule close homolog of L1 (CHL1) plays important roles during nervous system development. Here, we identified the hedgehog receptor patched-1 (PTCH1) as a novel CHL1-binding protein and showed that CHL1 interacts with the first extracellular loop of PTCH1 via its extracellular domain. Colocalization and co-immunoprecipitation of CHL1 with PTCH1 suggest an association of CHL1 with this major component of the hedgehog signaling pathway. The trans-interaction of CHL1 with PTCH1 promotes neuronal survival in cultures of dissociated cerebellar granule cells and of organotypic cerebellar slices. An inhibitor of the PTCH1-regulated hedgehog signal transducer, smoothened (SMO), and inhibitors of RhoA and Rho-associated kinase (ROCK) 1 and 2 prevent CHL1-dependent survival of cultured cerebellar granule cells and survival of cerebellar granule and Purkinje cells in organotypic cultures. In histological sections from 10- and 14-day-old CHL1-deficient mice, enhanced apoptosis of granule, but not Purkinje, cells was observed. The results of the present study indicate that CHL1 triggers PTCH1-, SMO-, RhoA- and ROCK-dependent signal transduction pathways to promote neuronal survival after cessation of the major morphogenetic events during mouse cerebellar development.
Assuntos
Apoptose , Moléculas de Adesão Celular/metabolismo , Receptor Patched-1/metabolismo , Células de Purkinje/metabolismo , Transdução de Sinais , Animais , Moléculas de Adesão Celular/genética , Camundongos , Camundongos Knockout , Receptor Patched-1/genéticaRESUMO
Here we present a designer's approach to building cellular neuronal networks based on a biocompatible negative photoresist with embedded coaxial feedthroughs made of semiconductor microtubes. The diameter of the microtubes is tailored and adjusted to the diameter of cerebellum axons having a diameter of 2-3 µm. The microtubes as well as the SU-8 layer serve as a topographical cue to the axons. Apart from the topographical guidance, we also employ chemical guidance cues enhancing neuron growth at designed spots. Therefore, the amino acid poly-l-lysine is printed in droplets of pl volume in the front of the tube entrances. Our artificial neuronal network has an extremely high yield of 85% of the somas settled at the desired locations. We complete this by basic patch-clamp measurements on single cells within the neuronal network.
RESUMO
Polysialic acid (PSA) is a large, negatively charged, linear homopolymer of alpha2-8-linked sialic acid residues. It is generated by two polysialyltransferases and attached to N- and/or O-linked glycans, and its main carrier is the neural cell adhesion molecule (NCAM). PSA controls the development and regeneration of the nervous system by enhancing cell migration, axon pathfinding, synaptic targeting, synaptic plasticity, by regulating the differentiation of progenitor cells and by modulating cell-cell and cell-matrix adhesions. In the adult, PSA plays a role in the immune system, and PSA mimetics promote functional recovery after nervous system injury. In search for novel small molecule mimetics of PSA that are applicable for therapy, we identified idarubicin, an antineoplastic anthracycline, and irinotecan, an antineoplastic agent of the topoisomerase I inhibitor class, as PSA mimetics using a competition enzyme-linked immunosorbent assay. Idarubicin and irinotecan compete with the PSA-mimicking peptide and colominic acid, the bacterial analog of PSA, for binding to the PSA-specific monoclonal antibody 735. Idarubicin and irinotecan stimulate neurite outgrowth and survival of cultured cerebellar neurons after oxidative stress via protein kinase C and Erk1/2 in a similar manner as colominic acid, whereas Fyn, casein kinase II and the phosphatase and tensin homolog are only involved in idarubicin and irinotecan-stimulated neurite outgrowth. These novel results show that the structure and function of PSA can be mimicked by the small organic compounds irinotecan and idarubicin which trigger the same signaling cascades as PSA, thus introducing the possibility of retargeting these drugs to treat nervous system injuries.
Assuntos
Camptotecina/análogos & derivados , Idarubicina/farmacologia , Crescimento Neuronal/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Proteína Quinase C/metabolismo , Ácidos Siálicos/farmacologia , Animais , Camptotecina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Irinotecano , Moléculas de Adesão de Célula Nervosa/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/citologia , Ratos Sprague-Dawley , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
The serotonergic system plays important roles in multiple functions of the nervous system and its malfunctioning leads to neurological and psychiatric disorders. Here, we show that the cell adhesion molecule close homolog of L1 (CHL1), which has been linked to mental disorders, binds to a peptide stretch in the third intracellular loop of the serotonin 2c (5-HT2c) receptor through its intracellular domain. Moreover, we provide evidence that CHL1 deficiency in mice leads to 5-HT2c-receptor-related reduction in locomotor activity and reactivity to novelty, and that CHL1 regulates signaling pathways triggered by constitutively active isoforms of the 5-HT2c receptor. Furthermore, we found that the 5-HT2c receptor and CHL1 colocalize in striatal and hippocampal GABAergic neurons, and that 5-HT2c receptor phosphorylation and its association with phosphatase and tensin homolog (PTEN) and ß-arrestin 2 is regulated by CHL1. Our results demonstrate that CHL1 regulates signal transduction pathways through constitutively active 5-HT2c receptor isoforms, thereby altering 5-HT2c receptor functions and implicating CHL1 as a new modulator of the serotonergic system.
Assuntos
Comportamento Animal/fisiologia , Moléculas de Adesão Celular/metabolismo , Neurônios GABAérgicos/metabolismo , Receptor 5-HT2C de Serotonina/metabolismo , Transdução de Sinais/fisiologia , Animais , Moléculas de Adesão Celular/genética , Corpo Estriado/citologia , Corpo Estriado/metabolismo , Neurônios GABAérgicos/citologia , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Camundongos Knockout , Receptor 5-HT2C de Serotonina/genéticaRESUMO
Prion protein (PrP) protects neural cells against oxidative stress, hypoxia, ischemia, and hypoglycemia. In the present study we confirm that cultured PrP-deficient neurons are more sensitive to oxidative stress than wild-type neurons and present the novel findings that wild-type, but not PrP-deficient astrocytes protect wild-type cerebellar neurons against oxidative stress and that exosomes released from stressed wild-type, but not from stressed PrP-deficient astrocytes reduce neuronal cell death induced by oxidative stress. We show that neuroprotection by exosomes of stressed astrocytes depends on exosomal PrP but not on neuronal PrP and that astrocyte-derived exosomal PrP enters into neurons, suggesting neuronal uptake of astrocyte-derived exosomes. Upon exposure of wild-type astrocytes to hypoxic or ischemic conditions PrP levels in exosomes were increased. By mass spectrometry and Western blot analysis, we detected increased levels of 37/67 kDa laminin receptor, apolipoprotein E and the ribosomal proteins S3 and P0, and decreased levels of clusterin/apolipoprotein J in exosomes from wild-type astrocytes exposed to oxygen/glucose deprivation relative to exosomes from astrocytes maintained under normoxic conditions. The levels of these proteins were not altered in exosomes from stressed PrP-deficient astrocytes relative to unstressed PrP-deficient astrocytes. These results indicate that PrP in astrocytes is a sensor for oxidative stress and mediates beneficial cellular responses, e.g. release of exosomes carrying PrP and other molecules, resulting in improved survival of neurons under hypoxic and ischemic conditions.
Assuntos
Astrócitos/metabolismo , Morte Celular/fisiologia , Exossomos/metabolismo , Hipóxia/metabolismo , Proteínas Priônicas/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismoRESUMO
Polysialic acid (PSA), a large, linear glycan composed of 8 to over 100 α2,8-linked sialic acid residues, modulates development of the nervous system by enhancing cell migration, axon pathfinding, and synaptic targeting and by regulating differentiation of progenitor cells. PSA also functions in developing and adult immune systems and is a signature of many cancers. In this study we identified vinorelbine, a semi-synthetic third generation vinca alkaloid, and epirubicin, an anthracycline and 4'-epimer of doxorubicin, as PSA mimetics. Similar to PSA, vinorelbine and epirubicin bind to the PSA-specific monoclonal antibody 735 and compete with the bacterial analog of PSA, colominic acid in binding to monoclonal antibody 735. Vinorelbine and epirubicin stimulate neurite outgrowth of cerebellar neurons via the neural cell adhesion molecule, via myristoylated alanine-rich C kinase substrate, and via fibroblast growth factor receptor, signaling through Erk pathways. Furthermore, the two compounds enhance process formation of Schwann cells and migration of cerebellar neurons in culture, and reduce migration of astrocytes after injury. These novel results show that the structure and function of PSA can be mimicked by the small organic compounds vinorelbine and epirubicin, thus raising the possibility to re-target drugs used in treatment of cancers to nervous system repair. Vinorelbine and epirubicin, identified as PSA mimetics, enhance, like PSA, neuronal migration, neuritogenesis, and formation of Schwann cell processes, and reduce astrocytic migration. Ablating NCAM, inhibiting fibroblast growth factor (FGFR) receptor, or adding the effector domain of myristoylated alanine-rich C kinase substrate (MARCKS) minimize the vinorelbine and epirubicin effects, indicating that they are true PSA mimetics triggering PSA-mediated functions.
Assuntos
Movimento Celular/efeitos dos fármacos , Epirubicina/farmacologia , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ácidos Siálicos/farmacologia , Vimblastina/análogos & derivados , Animais , Movimento Celular/fisiologia , Células Cultivadas , Epirubicina/química , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroglia/fisiologia , Neurônios/fisiologia , Estrutura Terciária de Proteína , Ácidos Siálicos/química , Vimblastina/química , Vimblastina/farmacologia , VinorelbinaRESUMO
The neural cell adhesion molecule (NCAM) plays important functional roles in development of the nervous system. We investigated the influence of a constitutive ablation of NCAM on the outcome of spinal cord injury. Transgenic mice lacking NCAM (NCAM-/-) were subjected to severe compression injury of the lower thoracic spinal cord using wild-type (NCAM+/+) littermates as controls. According to the single-frame motion analysis, the NCAM-/- mice showed reduced locomotor recovery in comparison to control mice at 3 and 6 weeks after injury, indicating an overall positive impact of NCAM on recovery after injury. Also the Basso Mouse Scale score was lower in NCAM-/- mice at 3 weeks after injury, whereas at 6 weeks after injury the difference between genotypes was not statistically significant. Worse locomotor function was associated with decreased monoaminergic and cholinergic innervation of the spinal cord caudal to the injury site and decreased axonal regrowth/sprouting at the site of injury. Astrocytic scar formation at the injury site, as assessed by immunohistology for glial fibrillary acidic protein at and around the lesion site was increased in NCAM-/- compared with NCAM+/+ mice. Migration of cultured monolayer astrocytes from NCAM-/- mice was reduced as assayed by scratch wounding. Numbers of Iba-1 immunopositive microglia were not different between genotypes. We conclude that constitutive NCAM deletion in young adult mice reduces recovery after spinal cord injury, validating the hypothesized beneficial role of this molecule in recovery after injury.