RESUMEN
BACKGROUND: Synapsins are abundant synaptic vesicle associated phosphoproteins that are involved in the fine regulation of neurotransmitter release. The Drosophila member of this protein family contains three conserved domains (A, C, and E) and is expressed in most or all synaptic terminals. Similar to mouse mutants, synapsin knock-out flies show no obvious structural defects but are disturbed in complex behaviour, notably learning and memory. RESULTS: We demonstrate that the N-terminal phosphorylation consensus motif RRxS that is conserved in all synapsins investigated so far, is modified in Drosophila by pre-mRNA editing. In mammals this motif represents the target site P1 of protein kinase A (PKA) and calcium/calmodulin dependent protein kinase I/IV. The result of this editing, by which RRFS is modified to RGFS, can be observed in cDNAs of larvae and adults and in both isolated heads and bodies. It is also seen in several newly collected wild-type strains and thus does not represent an adaptation to laboratory culture conditions. A likely editing site complementary sequence is found in a downstream intron indicating that the synapsin pre-mRNA can form a double-stranded RNA structure that is required for editing by the adenosine deaminase acting on RNA (ADAR) enzyme. A deletion in the Drosophila Adar gene generated by transposon remobilization prevents this modification, proving that the ADAR enzyme is responsible for the pre-mRNA editing described here. We also provide evidence for a likely function of synapsin editing in Drosophila. The N-terminal synapsin undeca-peptide containing the genomic motif (RRFS) represents an excellent substrate for in-vitro phosphorylation by bovine PKA while the edited peptide (RGFS) is not significantly phosphorylated. Thus pre-mRNA editing by ADAR could modulate the function of ubiquitously expressed synapsin in a cell-specific manner during development and adulthood. CONCLUSION: Similar to several other neuronal proteins of Drosophila, synapsin is modified by ADAR-mediated recoding at the pre-mRNA level. This editing likely reduces or abolishes synapsin phosphorylation by PKA. Since synapsin in Drosophila is required for various forms of behavioural plasticity, it will be fascinating to investigate the effect of this recoding on learning and memory.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Drosophila/metabolismo , Edición de ARN/fisiología , Precursores del ARN/metabolismo , Sinapsinas/metabolismo , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Secuencias de Aminoácidos/fisiología , Animales , Animales Modificados Genéticamente , Drosophila , Proteínas de Drosophila/genética , Expresión Génica/fisiología , Mutagénesis/fisiología , Fosforilación , Proteínas de Unión al ARN , Análisis de Secuencia de ADN/métodos , Sinapsinas/química , Sinapsinas/genéticaRESUMEN
Mouse AKR-2B cells express two forms of caspase-12: the full-length form coding for a protein of 47.8 kDa and a new splice variant of 40.2 kDa which is devoid of the CARD domain. In addition, three point mutations were disclosed: I/L-15, E/D-46 and P/L-105. A major portion of the two protein variants was found in the cytosol. Immunofluorescence studies showed an even distribution of caspase-12 within the cell, indicative for a cytoplasmatic localization. Transfection of AKR-2B cells with wild-type caspase-12 showed a colocalization of this protein with the endoplasmic reticulum (ER). Unlike mouse embryonal fibroblasts (MEF) which contain wild-type caspase-12, AKR-2B cells were largely resistant against treatment with the endoplasmatic reticulum stressing reagents brefeldin and tunicamycin. In AKR-2B cells, cytoplasmatic caspase-12 is bound to high molecular weight complexes of >1000 kDa [Cell Death Differ. 9 (2001) 125] and serum depletion leads to cleavage and detachment of caspase-12 from this high molecular weight complex. Cleavage of caspase-12 and -3 occurred almost simultaneously reaching a maximum 3-5 h after serum deprivation at which time also maximum apoptosis is found. Analysis of caspase-12 cleavage in vitro in comparison with fragmentation in vivo suggests that during death in AKR-2B cells induced by starvation, cleavage was brought about by caspase-3 at positions D24 and D94. Thus, mutated caspase-12 is differently integrated in signaling pathways of cell death and has lost its function as initiator caspase upon ER-stress. Instead, it is turned into a substrate of effector caspases. The implication of these findings in the pathological phenotype of ARK-2B mice is discussed.
Asunto(s)
Caspasas/genética , Caspasas/metabolismo , Citosol/enzimología , Retículo Endoplásmico/enzimología , Mutación Puntual , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Apoptosis , Western Blotting , Caspasas/química , Línea Celular , Medio de Cultivo Libre de Suero/farmacología , Citosol/metabolismo , Electroforesis en Gel de Agar , Retículo Endoplásmico/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente Directa , Ratones , Datos de Secuencia Molecular , Peso Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
Vertebrate synapsins are abundant synaptic vesicle phosphoproteins that have been proposed to fine-regulate neurotransmitter release by phosphorylation-dependent control of synaptic vesicle motility. However, the consequences of a total lack of all synapsin isoforms due to a knock-out of all three mouse synapsin genes have not yet been investigated. In Drosophila a single synapsin gene encodes several isoforms and is expressed in most synaptic terminals. Thus the targeted deletion of the synapsin gene of Drosophila eliminates the possibility of functional knock-out complementation by other isoforms. Unexpectedly, synapsin null mutant flies show no obvious defects in brain morphology, and no striking qualitative changes in behaviour are observed. Ultrastructural analysis of an identified 'model' synapse of the larval nerve muscle preparation revealed no difference between wild-type and mutant, and spontaneous or evoked excitatory junction potentials at this synapse were normal up to a stimulus frequency of 5 Hz. However, when several behavioural responses were analysed quantitatively, specific differences between mutant and wild-type flies are noted. Adult locomotor activity, optomotor responses at high pattern velocities, wing beat frequency, and visual pattern preference are modified. Synapsin mutant flies show faster habituation of an olfactory jump response, enhanced ethanol tolerance, and significant defects in learning and memory as measured using three different paradigms. Larval behavioural defects are described in a separate paper. We conclude that Drosophila synapsins play a significant role in nervous system function, which is subtle at the cellular level but manifests itself in complex behaviour.
Asunto(s)
Conducta Animal/fisiología , Drosophila/fisiología , Sinapsinas/deficiencia , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Potenciales de Acción/fisiología , Animales , Animales Modificados Genéticamente , Conducta Animal/efectos de los fármacos , Western Blotting/métodos , Depresores del Sistema Nervioso Central/farmacología , Clonación Molecular/métodos , Condicionamiento Operante/fisiología , Análisis Mutacional de ADN , Drosophila/genética , Estimulación Eléctrica/métodos , Etanol/farmacología , Potenciales Postsinápticos Excitadores/genética , Inmunohistoquímica/métodos , Técnicas de Inmunoadsorción , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Microscopía Electrónica , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Mutagénesis/fisiología , Unión Neuromuscular/genética , Unión Neuromuscular/metabolismo , Unión Neuromuscular/fisiología , Desempeño Psicomotor/fisiología , Conducta Sexual/efectos de los fármacos , Conducta Sexual/fisiología , Sinapsis/metabolismo , Sinapsis/ultraestructura , Sinapsinas/genética , Sinapsinas/fisiología , Vesículas Sinápticas/genética , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructura , Factores de Tiempo , Distribución Tisular , Percepción Visual/genética , Percepción Visual/fisiología , Caminata/fisiología , Alas de Animales/fisiologíaRESUMEN
PURPOSE: To investigate stimulatory effects of PDGF-AA, PDGF-AB, PDGF-BB, bFGF, IL-1beta, TGF-beta1 and TGF-beta2 on the proliferation and myofibroblast transformation of cultured human Tenon's capsule fibroblasts and to characterize expression of PDGF- and TGF-beta-receptors in these cells. METHODS: To determine cell proliferation, cell number of 2nd passage cultured human Tenon's capsule fibroblasts was measured before and after addition of growth factors using a computer-based cell counter system. Immunoblotting was used to detect and quantitate alpha-smooth-muscle actin (alpha-SMA) expression. Expression of PDGF- and TGF-beta-receptor mRNA was detected by RT-PCR, expression of the corresponding protein was demonstrated using Western blot. RESULTS: A significant increase in proliferation (p < or = 0.05) was detected after exogenous stimulation with PDGF-AA (10 ng/ml and 100 ng/ml), PDGF-AB (10 ng/ml and 100 ng/ml), PDGF-BB (10 ng/ml and 100 ng/ml), bFGF (100 ng/ml), IL-1beta (1 ng/ml and 10 ng/ml), TGF-beta1 (0.5 ng/ml) and TGF-beta2 (0.5 ng/ml). Both TGF-beta1 and TGF-beta2 stimulated expression of alpha-SMA in a dose dependent manner with peak activity at a concentration of 50 ng/ml (TGF-beta1) and 500 ng/ml (TGF-beta2). Protein and mRNA of PDGF-receptor type alpha and type beta and TGF-beta-receptors type I, II and III are expressed in cultured human Tenon's capsule fibroblasts. CONCLUSIONS: The present investigation strongly supports the hypothesis that PDGF-isoforms are major stimulators of proliferation of Tenon's capsule fibroblasts after glaucoma filtering surgery while TGF-beta-isoforms are essential for the transformation of Tenon's capsule fibroblasts into myofibroblasts.
Asunto(s)
Células del Tejido Conectivo/efectos de los fármacos , Ojo/citología , Ojo/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Sustancias de Crecimiento/farmacología , Actinas/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Células del Tejido Conectivo/citología , Fibroblastos/citología , Humanos , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismoRESUMEN
Treatment of AKR-2B fibroblasts with anisomycin (10 microM) led to a rapid disintegration of the cells (t1/2 = 5 h) which was complete after 24 h. Cell death was associated with typical hallmarks of apoptosis like membrane blebbing, exposure of phophatidylserine on the cell surface, nuclear condensation and specific cleavage of rRNA. However, there was no dissipation of the mitochondrial potential and no intranucleosomal fragmentation. By affinity labeling with YVK(-bio)D.aomk in combination with immunostaining against activated caspase-3 analyzed by 2-D gel electrophoresis it was shown that caspase-3 is the dominant executioner caspase. Gel filtration experiments of cytosolic extract analyzed by Western blotting revealed the formation of high-molecular-weight complexes of caspase-3 (600 kDa and 250 kDa, respectively), but there was no complex formation of Apaf-1. Anisomycin treatment led to a strong activation of the stress kinases p38 kinases and the jun kinases, that was not sufficient for the activation of caspase-3 which required much higher concentrations. By using the selective inhibitors SB 203580 for p38 kinases and SP 600125 for c-jun kinases, respectively, it is shown that activation of these kinases is not necessary for cell death induced by anisomycin in AKR-2B cells. Furthermore, we disclose the activation of caspase-12 in AKR-2B cells following the addition of anisomycin. Caspase-12 zymogen present as a cytosolic complex (> 600 kDa) is activated by anisomycin leading to an uncomplexed cleaved enzyme. Since anisomycin treatment did neither lead to stress of the endoplasmic reticulum nor to a breakdown of intracellular Ca(2+)-stores, alternative pathways involved in the activation of caspases are discussed.