Ex Vivo Recording of Axonal Transport Dynamics on Postnatal Organotypic Cortical Slices

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Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization.

peer reviewedMacrophage polarization is a process whereby macrophages acquire distinct effector states (M1 or M2) to carry out multiple and sometimes opposite functions. We show here that translational reprogramming occurs during macrophage polarization and that this relies on the Elongator complex subunit Elp3, an enzyme that modifies the wobble uridine base U34 in cytosolic tRNAs. Elp3 expression is downregulated by classical M1-activating signals in myeloid cells, where it limits the production of pro-inflammatory cytokines via FoxO1 phosphorylation, and attenuates experimental colitis in mice. In contrast, alternative M2-activating signals upregulate Elp3 expression through a PI3K- and STAT6-dependent signaling pathway. The metabolic reprogramming linked to M2 macrophage polarization relies on Elp3 and the translation of multiple candidates, including the mitochondrial ribosome large subunit proteins Mrpl3, Mrpl13, and Mrpl47. By promoting translation of its activator Ric8b in a codon-dependent manner, Elp3 also regulates mTORC2 activation. Elp3 expression in myeloid cells further promotes Wnt-driven tumor initiation in the intestine by maintaining a pool of tumor-associated macrophages exhibiting M2 features. Collectively, our data establish a functional link between tRNA modifications, mTORC2 activation, and macrophage polarization

The Universal Periodic Review's look on human rights in Iran. Analysis of the three cycles of UPR dedicated to Iran

reservedLo scopo di questa tesi è quello di riuscire a comprendere, tramite un’accurata analisi, se il meccanismo di controllo > attuato dalle Nazioni Unite consista in un’azione meramente diplomatica, in cui l’utilizzo dei termini viene accuratamente calibrato per non sbilanciarsi troppo nelle raccomandazioni; o se, al contrario, metta realmente in rilievo le eventuali problematiche riscontrate all’interno di un determinato paese, che in questo specifico caso è l’Iran. Nel primo capitolo vengono analizzati tutti i report preparati dal paese e quelli preparati dall’Alto Commissariato per i diritti umani, e vengono confrontate tutte le raccomandazioni fatte all’Iran nell’arco dei tre cicli di UPR da parte di tutti i paesi membri delle Nazioni Unite. Nel secondo capitolo viene dato maggior rilievo alla problematica femminile e a quella delle minoranze religiose, entrambe categorie che si classificano nei cosiddetti gruppi vulnerabili. Infine, nel terzo capitolo viene dato maggiore spazio a quelli che sono stati i progressi e i passi indietro fatti nel corso della storia in Iran proprio in ambito di diritti delle donne, anche alla luce delle recenti proteste

Molecular regulation of local translation at the synapse: a novel role for Elongator?

Genetic variants of Elongator subunits (Ep1-Elp6) have been associated with neurological disorders characterized by synaptic deficits. This complex is expressed in the developing cerebral cortex where it controls the early steps of neurogenesis preceding synapse formation. Importantly, Elongator colocalizes with synaptic vesicles and is enriched at the pre-synaptic side of neuromuscular junction buttons. At the molecular level, Elongator promotes the modification of selected tRNAs by catalysing the first step reaction of the methoxy-carbonyl-methyl (mcm5) group addition to wobble uridines. Through this activity Elongator controls the fidelity and efficiency of translation. Whether Elongator modulates cortical synaptogenesis by regulating tRNAs modification at synapses, thereby local protein synthesis, has not been investigated yet. Given the important contribution of local translation to synaptogenesis, we therefore hypothesize that impaired tRNA modifications could be a common pathophysiological mechanism in Elongator-related disorders characterized by connectivity defects. Here, we show that Elongator is expressed at axonal growth cone, dendrites, and excitatory/inhibitory synapses. In addition, western blotting on synaptosomes isolated from postnatal cortices detected the expression of Elongator subunits, Alkbh8 and Ctu1, the three enzymes that act in a multi-step reaction to promote mcm5s2 group addition to selected tRNAs. In cortical neuronal cultures, we observed a decreased density of excitatory and inhibitory synapses in the absence of Elongator. Moreover, Elp3-depleted neurons display reduced puromycin labelling in their soma, along their dendrites and axons, suggesting reduced total protein synthesis. Interestingly, the PLA-Puromycin assay revealed a decreased number of newly synthesized PSD95, the major scaffolding protein in the excitatory postsynaptic density. Taken together, these preliminary data support a possible role for Elongator in local protein synthesis at synapses

Pietre al bivio. Marcatori territoriali e tracce stradali nel paesaggio resiliente fra Este e Padova

A re-examination of the archaeological evidences dated back from the 6th to the end of the 4th/beginning of the 3rd centuries bc, together with a specific focus towards the role of three significant funerary stones bearing Venetic inscriptions and the results of a UAV-derived analysis, has led to highlight the entanglement of the territorial and itinerary dynamics of pre-Roman and Roman periods in the area of the Venetia between Este and Padova

Molecular mechanisms of long-distance transport: a new role for local translation?

Axonal microtubules (MT) regulate long-distance transport of cargos to peripheral sub-compartments. Among those cargoes, mRNAs and their associated RNA-binding proteins are transported as membraneless ribonucleoprotein (RNP) granules that, together with ribosomes, can hitchhike on fast-moving membrane-bound organelles for their transport along MTs and subsequent translation. Importantly, components of the translation machinery including ribosomes, tRNAs and the translation elongation factor 1a (eEF1a) have also been identified at the surface of distinct organelle subtypes suggesting that some organelles may serve as platforms for mRNA translation. Accordingly, axonal late endosomes provide sites for local protein synthesis contributing to the maintenance of the local mitochondria proteome. However, while active protein translation on fast-moving organelles has only been described in the fungi Ustilago maydis, the role for a functional coordination between organelle transport and local translation remains poorly understood in neuronal cells. In order to characterize the population of transcripts actively translated on moving organelles, we use a transgenic mouse model that co-expresses fluorescently-labelled motile organelles and the RiboTag system. By combining sequential centrifugation and fluorescent flow-cytometry, we isolate motile organelles whose associated ribosome-bound mRNAs will be further characterized via translatome studies. We further established an in vitro model of neuron-like cells that undergo axonal transport dynamics and local translation events, and that expresses the SunTag system to probe the translation dynamics of selected candidate mRNAs. This work will therefore combine translatome analysis of sorted motile vesicles with imaging of translation dynamics to elucidate the functional contribution of motile organelles to the axonal proteome, thus revealing new insights into the regulation of local translation in neurons

Molecular mechanisms of long-distance transport: a new role for local translation?

Axonal microtubules (MT) regulate long-distance transport of cargos to peripheral sub-compartments. Among those cargoes, mRNAs and their associated RNA-binding proteins are transported as membraneless ribonucleoprotein (RNP) granules that, together with ribosomes, can hitchhike on fast-moving membrane-bound organelles for their transport along MTs and subsequent translation. Deep-sequencing studies have characterized the axonal translatome of several cellular subtypes, unravelling developmental translation programs that support compartment-specific biological processes. Importantly, components of the translation machinery including ribosomes, tRNAs and the translation elongation factor 1a (eEF1a) have also been identified at the surface of distinct organelle subtypes suggesting that some organelles may serve as platforms for mRNA translation. Accordingly, axonal late endosomes provide sites for local protein synthesis contributing to the maintenance of the local mitochondria proteome. However, while active protein translation on fast-moving organelles has only been described in the fungi Ustilago maydis, the role for a functional coordination between organelle transport and local translation remains poorly understood in neuronal cells. In order to characterize the population of transcripts actively translated on moving organelles, we use a transgenic mouse model that co-expresses fluorescently-labelled motile organelles and the RiboTag system. By combining sequential centrifugation and fluorescent flow-cytometry, we isolate motile organelles whose associated ribosome-bound mRNAs will be further characterized via translatome studies. We further established an in vitro model of neuron-like cells that undergo axonal transport dynamics and local translation events, and that expresses the SunTag system to probe the translation dynamics of selected candidate mRNAs. This work will therefore combine translatome analysis of sorted motile vesicles with imaging of translation dynamics to elucidate the functional contribution of motile organelles to the axonal proteome, thus revealing new insights into the regulation of local translation in neurons

Molecular Analysis of Axonal Transport Dynamics upon Modulation of Microtubule Acetylation.

Axonal transport is used by neurons to distribute mRNAs, proteins, and organelles to their peripheral compartments in order to sustain their structural and functional integrity. Cargoes are transported along the microtubule (MT) network whose post-translational modifications influence transport dynamics. Here, we describe methods to modulate MT acetylation and record its impact on axonal transport in cultured mouse cortical projection neurons as well as in motoneurons of Drosophila melanogaster third-instar larvae. Specifically, we provide a step-by step procedure to reduce the level of MT acetylation and to record and analyze the transport of dye-labeled organelles in projection neuron axons cultured in microfluidic chambers. In addition, we describe the method to record and analyze GFP-tagged mitochondria transport along the motoneuron axons of transgenic Drosophila melanogaster third-instar larvae