Spatiotemporal regulation of GSK3β levels by miRNA-26a controls axon development in cortical neurons

© 2020. Published by The Company of Biologists Ltd. Both the establishment of neuronal polarity and axonal growth are crucial steps in the development of the nervous system. The local translation of mRNAs in the axon provides precise regulation of protein expression, and is now known to participate in axon development, pathfinding and synaptic formation and function. We have investigated the role of miR-26a in early stage mouse primary cortical neuron development. We show that micro-RNA-26a-5p (miR-26a) is highly expressed in neuronal cultures, and regulates both neuronal polarity and axon growth. Using compartmentalised microfluidic neuronal cultures, we identified a local role for miR-26a in the axon, where the repression of local synthesis of GSK3β controls axon development and growth. Removal of this repression in the axon triggers local translation of GSK3β protein and subsequent transport to the soma, where it can impact axonal growth. These results demonstrate how the axonal miR-26a can regulate local protein translation in the axon to facilitate retrograde communication to the soma and amplify neuronal responses, in a mechanism that influences axon development

Distinct small non-coding RNA landscape in the axons and released extracellular vesicles of developing primary cortical neurons and the axoplasm of adult nerves

Neurons have highlighted the needs for decentralized gene expression and specific RNA function in somato-dendritic and axonal compartments, as well as in intercellular communication via extracellular vesicles (EVs). Despite advances in miRNA biology, the identity and regulatory capacity of other small non-coding RNAs (sncRNAs) in neuronal models and local subdomains has been largely unexplored. We identified a highly complex and differentially localized content of sncRNAs in axons and EVs during early neuronal development of cortical primary neurons and in adult axons in vivo. This content goes far beyond miRNAs and includes most known sncRNAs and precisely processed fragments from tRNAs, sno/snRNAs, Y RNAs and vtRNAs. Although miRNAs are the major sncRNA biotype in whole-cell samples, their relative abundance is significantly decreased in axons and neuronal EVs, where specific tRNA fragments (tRFs and tRHs/tiRNAs) mainly derived from tRNAs Gly-GCC, Val-CAC and Val-AAC predominate. Notably, although 5ʹ-tRHs compose the great majority of tRNA-derived fragments observed in vitro, a shift to 3ʹ-tRNAs is observed in mature axons in vivo. The existence of these complex sncRNA populations that are specific to distinct neuronal subdomains and selectively incorporated into EVs, equip neurons with key molecular tools for spatiotemporal functional control and cell-to-cell communication

Mitochondrial impairment activates the Wallerian pathway through depletion of NMNAT2 leading to SARM1-dependent axon degeneration.

Wallerian degeneration of physically injured axons involves a well-defined molecular pathway linking loss of axonal survival factor NMNAT2 to activation of pro-degenerative protein SARM1. Manipulating the pathway through these proteins led to the identification of non-axotomy insults causing axon degeneration by a Wallerian-like mechanism, including several involving mitochondrial impairment. Mitochondrial dysfunction is heavily implicated in Parkinson's disease, Charcot-Marie-Tooth disease, hereditary spastic paraplegia and other axonal disorders. However, whether and how mitochondrial impairment activates Wallerian degeneration has remained unclear. Here, we show that disruption of mitochondrial membrane potential leads to axonal NMNAT2 depletion in mouse sympathetic neurons, increasing the substrate-to-product ratio (NMN/NAD) of this NAD-synthesising enzyme, a metabolic fingerprint of Wallerian degeneration. The mechanism appears to involve both impaired NMNAT2 synthesis and reduced axonal transport. Expression of WLDS and Sarm1 deletion both protect axons after mitochondrial uncoupling. Blocking the pathway also confers neuroprotection and increases the lifespan of flies with Pink1 loss-of-function mutation, which causes severe mitochondrial defects. These data indicate that mitochondrial impairment replicates all the major steps of Wallerian degeneration, placing it upstream of NMNAT2 loss, with the potential to contribute to axon pathology in mitochondrial disorders

Local axonal translation: regulation of neuronal polarity, axon development and survival

Both the establishment of neuronal polarity and axonal growth are critical steps in the development of the nervous system, allowing neurons to fulfil their functional role, transmitting and receiving electrical signals. The local translation of mRNAs in the axon provides fine regulation of protein expression, and is now known to participate in axon development, homeostasis and degeneration. In this context, microRNAs play a fundamental role in the spatiotemporal regulation of axonal translation and, by doing so, can regulate almost every aspect nervous system development, physiology and disease. This thesis focuses on elucidating the mechanisms by which local protein translation in the axon can regulates axon development and survival. I show how axonal protein synthesis contributes in supplying the needs of the axon and maintaining its homeostasis. Repression of protein translation restricted to the axonal compartment of microfluidic chambers triggers axon degeneration in mouse sensory neurons. Moreover, I identified four microRNAs as potential candidate regulators of axon degeneration pathways. I then investigated the role of a single microRNA, miR-26a, in early stage primary cortical neuron development. I show that miR-26a is highly expressed in neuronal cultures and regulates both neuronal polarity and axon growth. Specifically, inhibition of miR-26a reduces the number of polarised neurons, whilst its over-expression produces the opposite phenotype and increases the number of neurons with multiple axon-like processes via the targeting of GSK3β. Using compartmentalised microfluidic neuronal cultures, I also identified a local role for miR-26a in the axon, where the repression of local synthesis of GSK3β controls axon development and growth. Removal of this repression in the axon triggers local translation of GSK3β protein and subsequent transport to the soma, where it can impact axonal growth mechanism. These results demonstrate how the axonal miR-26a can regulate local protein translation in the axon to facilitate retrograde communication to the soma and amplify neuronal responses, in a mechanism that influences axon development

Local axonal translation: regulation of neuronal polarity, axon development and survival

Both the establishment of neuronal polarity and axonal growth are critical steps in the development of the nervous system, allowing neurons to fulfil their functional role, transmitting and receiving electrical signals. The local translation of mRNAs in the axon provides fine regulation of protein expression, and is now known to participate in axon development, homeostasis and degeneration. In this context, microRNAs play a fundamental role in the spatiotemporal regulation of axonal translation and, by doing so, can regulate almost every aspect nervous system development, physiology and disease. This thesis focuses on elucidating the mechanisms by which local protein translation in the axon can regulates axon development and survival. I show how axonal protein synthesis contributes in supplying the needs of the axon and maintaining its homeostasis. Repression of protein translation restricted to the axonal compartment of microfluidic chambers triggers axon degeneration in mouse sensory neurons. Moreover, I identified four microRNAs as potential candidate regulators of axon degeneration pathways. I then investigated the role of a single microRNA, miR-26a, in early stage primary cortical neuron development. I show that miR-26a is highly expressed in neuronal cultures and regulates both neuronal polarity and axon growth. Specifically, inhibition of miR-26a reduces the number of polarised neurons, whilst its over-expression produces the opposite phenotype and increases the number of neurons with multiple axon-like processes via the targeting of GSK3β. Using compartmentalised microfluidic neuronal cultures, I also identified a local role for miR-26a in the axon, where the repression of local synthesis of GSK3β controls axon development and growth. Removal of this repression in the axon triggers local translation of GSK3β protein and subsequent transport to the soma, where it can impact axonal growth mechanism. These results demonstrate how the axonal miR-26a can regulate local protein translation in the axon to facilitate retrograde communication to the soma and amplify neuronal responses, in a mechanism that influences axon development

How urbanization affects multiple dimensions of biodiversity in tropical butterfly assemblages

We evaluated how the taxonomic, phylogenetic and functional diversities of butterflies and their community-weighted traits are affected by urbanization in the southeastern Brazilian Atlantic Forest. For this purpose, a dataset of Nymphalidae species distributed across 15 urban, semiurban, and rural fragments was analyzed. Urbanization was defined by a set of environmental variables. Furthermore, the total area of each fragment was also considered in the analyses but did not influence the results, in which disturbance level and patch connectivity drove the environmental variation across the urban matrix. Species diversity increased towards the more connected fragments, while phylogenetic and functional diversity did not vary in relation to urbanization. A high forewing:hindwing ratio and the frequency of tiger-like wings were positively related to the urban fragments, while a low forewing:hindwing ratio and iridescent wings were related to the semiurban and rural fragments. The suitability of highly interconnected rural habitats for the maintenance of butterfly diversity was corroborated as expected. Nonetheless, our results also showed that semiurban fragments preserved the ecologically relevant traits of butterflies related to forested habitats, expressed in butterfly groups possibly linked with dispersal capability to avoid predation. Careful management of semiurban fragments and urban landscaping, including highly structured and native vegetation outside urban parks, may increase the functional and taxonomic diversities or at least maintain the current levels of functionality in the urban matrix. Thus, it is possible to preserve the biological diversity of native fauna and flora and recover relevant ecosystem services, ensuring the conservation of Neotropical urban center283621638CNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESP – Fundação de Amparo à Pesquisa Do Estado De São PauloNSF - National Science Foundation303834/2015-3; 307886/2015-8; 563332 / 2010-7; 465610 / 2014-52011 / 50225-3; 2012 / 50260-6DEB-1256742Não temNão temINCT - Instituto Nacional de Ciência e TecnologiaFAPEG - Fundação de Amparo a Pesquisa do Estado de Goiá

Sampling performance of bait traps in high Andean fruit-feeding butterflies

Studies on model organisms such as butterflies are useful tools for conservation decision-making. However, in tropical ecosystems with an intrinsic high diversity a full understanding of biotic communities is difficult to obtain. Bait trap samplings have traditionally been used for community appraisals related to ecological and conservation issues. Nonetheless, in the Andes Mountains, there is little knowledge related to the effectiveness of bait traps for butterfly sampling. In this study, we tested the success of fermented fruits and rotten fish baits for butterfly sampling in four land-cover types (páramo, cloud forests, mixed, and pasture) in the upper Rio Chico basin of the northern Central Cordillera of the Colombian Andes. A butterfly survey was conducted between 2011 and 2014, in an elevation range of 2650 to 3300 masl, within a total of 132 field days. Three sampling units for each land cover were established with four standard Van Someren-Rydon traps (VSR) per sampling unit. Traps were baited alterna-tively with fermented fruits and carrion (rotten fish). All 57 recorded species were captured using rotten fish, while approximately 65% (37 species) were collected from fermented fruit. Moreover, species richness was higher in all sampled land covers using rotten fish bait, but the dominant species in the land covers differed between baits. The rotten fish bait proved to be highly effective for butterfly sampling in páramo and cloud forest, although the combination of traps baited with fermented banana and rotten fish, allowed the collection of data suitable for comparison among all studied land cover

Treatment of benign endobronchial tumors: when, how, and why. Insights, experiences, and interventional pulmonology strategies

Benign endobronchial tumors are rare clinical entities characterized by considerable variability in etiology and clinical presentation. The authors report four cases of endobronchial hamartomas treated and followed up from 2018 to 2023. Three of these cases, with identical endobronchial localization in the right lower lobe, were radically treated in flexible bronchoscopy with the only use of biopsy forceps. Another case with a different localization in the left main bronchus was treated with a laser through rigid bronchoscopy. In addition, the authors outline the main interventional pulmonological strategies for the treatment of benign tumors with endobronchial growth based on the existing literature

Distinct small non-coding RNA landscape in the axons and released extracellular vesicles of developing primary cortical neurons and the axoplasm of adult nerves

Neurons have highlighted the needs for decentralized gene expression and specific RNA function in somato-dendritic and axonal compartments, as well as in intercellular communication via extracellular vesicles (EVs). Despite advances in miRNA biology, the identity and regulatory capacity of other small non-coding RNAs (sncRNAs) in neuronal models and local subdomains has been largely unexplored. We identified a highly complex and differentially localized content of sncRNAs in axons and EVs during early neuronal development of cortical primary neurons and in adult axons invivo. This content goes far beyond miRNAs and includes most known sncRNAs and precisely processed fragments from tRNAs, sno/snRNAs, Y RNAs and vtRNAs. Although miRNAs are the major sncRNA biotype in whole-cell samples, their relative abundance is significantly decreased in axons and neuronal EVs, where specific tRNA fragments (tRFs and tRHs/tiRNAs) mainly derived from tRNAs Gly-GCC, Val-CAC and Val-AAC predominate. Notably, although 5ʹ-tRHs compose the great majority of tRNA-derived fragments observed invitro, a shift to 3ʹ-tRNAs is observed in mature axons invivo. The existence of these complex sncRNA populations that are specific to distinct neuronal subdomains and selectively incorporated into EVs, equip neurons with key molecular tools for spatiotemporal functional control and cell-to-cell communication