RESUMEN
The ability of the nervous system to undergo long-term plasticity is based on changes in cellular and synaptic proteomes. While many studies have explored dynamic alterations in neuronal proteomes during plasticity, there has been less attention paid to the astrocytic counterpart. Indeed, progress in identifying cell type-specific proteomes is limited owing to technical difficulties. Here, we present a cell type-specific metabolic tagging technique for a mammalian coculture model based on the bioorthogonal amino acid azidonorleucine and the mutated Mus musculus methionyl-tRNA synthetaseL274G enabling azidonorleucine introduction into de novo synthesized proteins. Azidonorleucine incorporation resulted in cell type-specific protein labeling and retained neuronal or astrocytic cell viability. Furthermore, we were able to label astrocytic de novo synthesized proteins and identified both Connexin-43 and 60S ribosomal protein L10a upregulated upon treatment with Brain-derived neurotrophic factor in astrocytes of a neuron-glia coculture. Taken together, we demonstrate the successful dissociation of astrocytic from neuronal proteomes by cell type-specific metabolic labeling offering new possibilities for the analyses of cell type-specific proteome dynamics.
Asunto(s)
Astrocitos/metabolismo , Alanina/análogos & derivados , Alanina/química , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Azidas/análisis , Azidas/química , Factor Neurotrófico Derivado del Encéfalo/farmacología , Técnicas de Cocultivo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Metionina-ARNt Ligasa/genética , Metionina-ARNt Ligasa/metabolismo , Neuroglía/citología , Norleucina/análogos & derivados , Norleucina/análisis , Norleucina/química , Biosíntesis de Proteínas , Proteínas/química , Proteínas/metabolismo , Proteoma , Proteómica/métodos , Ratas WistarRESUMEN
BACKGROUND/AIMS: The trefoil factor family (TFF) peptide TFF3 is typically secreted by mucous epithelia, but is also expressed in the immune system and the brain. It was the aim of this study to determine the cerebral cell types which express Tff3. METHODS: Primary cultures from rat embryonic or neonatal cerebral cortex and hippocampus, respectively, were studied by means of RT-PCR and immunofluorescence. Moreover, Tff3 expression was localized by immunocytochemistry in sections of adult rat cerebellum. RESULTS: Tff3 transcripts were detectable in neural cultures of both the cortex and the hippocampus as well as in glial cell-enriched cultures. Tff3 peptide co-localized with Map2 indicating an expression in neurons in vitro. The neuronal expression was confirmed by immunofluorescence studies of adult rat cerebellum. Furthermore, Tff3 peptide showed also a clear co-localization with Iba-1 in vitro typical of activated microglial cells. CONCLUSION: The neuronal expression of Tff3 is in line with a function of a typical neuropeptide influencing, e.g., fear, memory, depression and motoric skills. The expression in activated microglial cells, which is demonstrated here for the first time, points towards a possible function for Tff3 in immune reactions in the CNS. This opens a plethora of additional possible functions for Tff3 including synaptic plasticity and cognition as well as during neuroinflammatory diseases and psychiatric disorders.
Asunto(s)
Corteza Cerebral/metabolismo , Regulación de la Expresión Génica , Neuronas/metabolismo , Neuropéptidos/biosíntesis , Animales , Hipocampo/metabolismo , Microglía/metabolismo , Neuropéptidos/metabolismo , Ratas , Factor Trefoil-3RESUMEN
The proper organization of the presynaptic cytomatrix at the active zone is essential for reliable neurotransmitter release from neurons. Despite of the virtual stability of this tightly interconnected proteinaceous network it becomes increasingly clear that regulated dynamic changes of its composition play an important role in the processes of synaptic plasticity. Bassoon, a core component of the presynaptic cytomatrix, is a key player in structural organization and functional regulation of presynaptic release sites. It is one of the most highly phosphorylated synaptic proteins. Nevertheless, to date our knowledge about functions mediated by any one of the identified phosphorylation sites of Bassoon is sparse. In this study, we have identified an interaction of Bassoon with the small adaptor protein 14-3-3, which depends on phosphorylation of the 14-3-3 binding motif of Bassoon. In vitro phosphorylation assays indicate that phosphorylation of the critical Ser-2845 residue of Bassoon can be mediated by a member of the 90-kDa ribosomal S6 protein kinase family. Elimination of Ser-2845 from the 14-3-3 binding motif results in a significant decrease of Bassoon's molecular exchange rates at synapses of living rat neurons. We propose that the phosphorylation-induced 14-3-3 binding to Bassoon modulates its anchoring to the presynaptic cytomatrix. This regulation mechanism might participate in molecular and structural presynaptic remodeling during synaptic plasticity.
Asunto(s)
Proteínas 14-3-3/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sinapsis/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Ratones , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Plasticidad Neuronal , Neuronas/citología , Neuronas/metabolismo , Fosforilación , Ratas , Proteínas Quinasas S6 Ribosómicas/metabolismo , Sinapsis/fisiologíaRESUMEN
Synaptic transmission relies on effective and accurate compensatory endocytosis. F-BAR proteins may serve as membrane curvature sensors and/or inducers and thereby support membrane remodelling processes; yet, their in vivo functions urgently await disclosure. We demonstrate that the F-BAR protein syndapin I is crucial for proper brain function. Syndapin I knockout (KO) mice suffer from seizures, a phenotype consistent with excessive hippocampal network activity. Loss of syndapin I causes defects in presynaptic membrane trafficking processes, which are especially evident under high-capacity retrieval conditions, accumulation of endocytic intermediates, loss of synaptic vesicle (SV) size control, impaired activity-dependent SV retrieval and defective synaptic activity. Detailed molecular analyses demonstrate that syndapin I plays an important role in the recruitment of all dynamin isoforms, central players in vesicle fission reactions, to the membrane. Consistently, syndapin I KO mice share phenotypes with dynamin I KO mice, whereas their seizure phenotype is very reminiscent of fitful mice expressing a mutant dynamin. Thus, syndapin I acts as pivotal membrane anchoring factor for dynamins during regeneration of SVs.