The neuromuscular transmission of the SOD1 (G93A) mouse model of amyotrophic lateral sclerosis

Tese de mestrado, Neurociências, Faculdade de Medicina, Universidade de Lisboa, 2013A Esclerose Lateral Amiotrófica (ELA), uma das doenças do neurónio motor mais comum, caracteriza-se pela perda selectiva de neurónios motores do tracto corticoespinhal. Vários estudos sugerem que a degeneração inicia-se na porção distal do axónio com uma progressão retrógrada. Assim, o presente trabalho teve como objectivo avaliar a transmissão sináptica na junção neuromuscular dos animais SOD1(G93A), nos períodos correspondentes às fases pré-sintomática e sintomática da ELA. As experiências foram efectuadas em ratinhos transgénicos SOD1(G93A) e não transgénicos (WT), na fase pré-sintomática (4 a 6 semanas de idade) e fase sintomática (12 a 16 semanas de idade). Após o nascimento, os animais foram genotipados por polymerase chain reaction (PCR). Nas respectivas fases da doença, os animais foram testados no rotarod, e em seguida fizeram-se registos electrofisiologicos: potenciais de placa evocados (EPPs), potenciais de placa miniatura (MEPPs) e MEPPs gigantes (GMEPPs: MEPPs > 1mV). Os registos foram feitos em fibras musculares do diafragma inervado, paralisadas com μ-conotoxina GIIIB. O conteúdo quântico dos EPPs foi calculado através da razão entre a amplitude média dos EPPs e a amplitude média dos MEPPs. Na fase pré-sintomática da doença, os ratos SOD1(G93A) não exibiram alterações na função motora a 10 rpm. Relativamente à transmissão neuromuscular, estes animais apresentaram um aumento significativo da amplitude média dos EPPs e do conteúdo quântico dos EPPs, quando comparados com os animais WT, sugerindo uma maior eficiência da transmissão neuromuscular nos animais SOD1(G93A). Para além disso, o aumento significativo da frequência de GMEPPs, o que pela literatura parece estar associado a uma desregulação dos níveis intracelulares de Ca2+, e as alterações na amplitude e cinética dos MEPPs sugerem a existência de alterações ao nível da junção neuromuscular numa fase pré-sintomatica. Na fase sintomática, os animais SOD1(G93A) apresentaram um défice motor aos 10 rpm. Os registos electrofisiológicos revelaram a existência de dois grupos de junções neuromusculares nos ratos SOD1(G93A): SOD1a e SOD1b. O grupo SOD1a apresentou EPPs e MEPPs com amplitudes significativamente reduzidas bem como um rise-time dos MEPPs aumentado, quando comparado com os grupos SOD1b e WT, sugerindo um enfraquecimento da transmissão neuromuscular, nesse grupo. Pelo contrário, o grupo SOD1b apresentou uma transmissão neuromuscular semelhante tanto à dos animais SOD1(G93A) pré-sintomáticos, como também à dos WT com 12-14 semanas. Em conclusão, este trabalho mostra que a transmissão neuromuscular dos animais SOD1(G93A) encontra-se aumentada na fase pré-sintomática. Na fase sintomática, a presença de uma população mista de junções neuromusculares é consistente com os ciclos de desinervação/ re-inervação, já descritos noutros estudos. As alterações iniciais na transmissão neuromuscular dos animais SOD1(G93A) representam assim mais uma evidência que os mecanismos patológicos da ELA iniciam-se antes do aparecimento dos primeiros sintomas.Amyotrophic Lateral Sclerosis (ALS) is the most frequent adult-onset motor neuron disease and is characterized by a selective and progressive loss of motor neurons in the corticospinal tract. Growing evidence suggest that degeneration may begin at the distal axon proceeding in a dying-back pattern, increasing the need to focus on neuromuscular junction parameters. It seemed therefore of interest to investigate synaptic transmission at the neuromuscular junction (NMJ) in both pre- and symptomatic phases of the disease. Experiments were performed in SOD1(G93A) mice and in non-transgenic littermates (WT) with 4-6 and 12-14 weeks-old, corresponding respectively to pre- and symptomatic phases. After birth, mice were genotyped through polymerase chain reaction (PCR). At the respective age, mice were submitted rotarod, then low-frequency (0.5 Hz) evoked endplate potentials (EPPs), miniature endplate potentials (MEPPs) and giant MEPPs (GMEPPs: MEPPs >1mV) were recorded from innervated diaphragm muscle fibers, paralyzed with μ-conotoxin GIIIB. The quantal content of EPPs was estimated as the ratio between EPPs amplitude and MEPPs amplitude. In the pre-symptomatic phase, SOD1(G93A) mice did not present motor deficits on the rotarod at 10rpm. However, SOD1(G93A) mice exhibited a significant increase of the mean amplitude of EPPs together with an increase in the mean quantal content of EPPs, suggesting that more acetylcholine is being released into the synaptic cleft. Also, SOD1(G93A) mice presented a higher frequency of GMEPPs, suggestive of intracellular Ca2+ deregulation in nerve terminals. The observed increase in the mean amplitude of MEPPs and the decreased mean rise-time of MEPPs in SOD1(G93A) mice point as well to post-synaptic related changes. In symptomatic phase, SOD1(G93A) mice presented a lower motor balance and coordination. Electrophysiological data showed evidence for two NMJ groups in SOD1(G93A) mice: SOD1a and SOD1b. The SOD1a group presented both mean amplitude of EPPs and of MEPPs reduced. The mean rise-time of MEPPs was increased, when compared to WT and to SOD1b group, indicating an impairment in the neuromuscular transmission. In contrast, the neuromuscular transmission of SOD1b group was not different from age-matched WT or from the pre-symptomatic SOD1(G93A) mice. Altogether these results clearly show that the neuromuscular transmission of SOD1(G93A) mice is enhanced in the pre-symptomatic phase. In the symptomatic phase our results are consistent with the hypothesis that the diaphragm of SOD1(G93A) mice are undergoing cycles of denervation/re-innervation supported by the mixed population of neuromuscular junctions. These early changes in the neuromuscular transmission of SOD1(G93A) mice is a novel proof that the ALS associated events starts long before the symptoms appear

Metagenomic analysis shows the presence of bacteria related to free-living forms of sulfur-Oxidizing Chemolithoautotrophic Symbionts in the rhizosphere of the seagrass Zostera marina

Seagrasses play an important role as ecosystem engineers; they provide shelter to many animals and improve water quality by filtering out nutrients and by controlling pathogens. Moreover, their rhizosphere promotes a myriad of microbial interactions and processes, which are dominated by microorganisms involved in the sulfur cycle. This study provides a detailed insight into the metabolic sulfur pathways in the rhizobiome of the seagrass Zostera marina, a dominant seagrass species across the temperate northern hemisphere. Shotgun metagenomic sequencing revealed the relative dominance of Gamma- and Deltaproteobacteria, and comparative analysis of sulfur genes identified a higher abundance of genes related to sulfur oxidation than sulfate reduction. We retrieved four high-quality draft genomes that are closely related to the gill symbiont of the clam Solemya velum, which suggests the presence of putative free-living forms of symbiotic bacteria. These are potentially highly versatile chemolithoautotrophic bacteria, able to alternate their metabolism between parallel pathways of sulfide oxidation (via sqr and fcc), nitrate reduction (denitrification or DNRA) and carbon fixation (via CBB or TCA cycle), depending on the environmental availability of sulfide. Our results support the hypothesis that seagrass meadows might function as a source of symbionts for invertebrates that inhabit within or around seagrass meadows. While providing ideal conditions for the proliferation of these free-living forms of symbionts, seagrasses would benefit from their genetic versatility, which contributes to sulfide detoxification and ammonium production, the seagrasses' preferred nitrogen source.European Union ERC 322551 European Science Foundation ConGenOmics program 6349 Fundacao para a Ciencia e Tecnologia (FCT) SFRH/BPD/63/03/2009 SFRH/BPD/107878/2015info:eu-repo/semantics/publishedVersio

Molecular Taxonomic Profiling of Bacterial Communities in a Gilthead Seabream (Sparus aurata) Hatchery

As wild fish stocks decline worldwide, land-based fish rearing is likely to be of increasing relevance to feeding future human generations. Little is known about the structure and role of microbial communities in fish aquaculture, particularly at larval developmental stages where the fish microbiome develops and host animals are most susceptible to disease. We employed next-generation sequencing (NGS) of 16S rRNA gene reads amplified from total community DNA to reveal the structure of bacterial communities in a gilthead seabream (Sparus aurata) larviculture system. Early-(2 days after hatching) and late-stage (34 days after hatching) fish larvae presented remarkably divergent bacterial consortia, with the genera Pseudoalteromonas, Marinomonas, Acinetobacter, and Acidocella (besides several unclassified Alphaproteobacteria) dominating the former, and Actinobacillus, Streptococcus, Massilia, Paracoccus, and Pseudomonas being prevalent in the latter. A significant reduction in rearing-water bacterial diversity was observed during the larviculture trial, characterized by higher abundance of the Cryomorphaceae family (Bacteroidetes), known to populate microniches with high organic load, in late-stage rearing water in comparison with early-stage rearing-water. Furthermore, we observed the recruitment, into host tissues, of several bacterial phylotypes-including putative pathogens as well as mutualists-that were detected at negligible densities in rearing-water or in the live feed (i.e., rotifers and artemia). These results suggest that, besides host-driven selective forces, both the live feed and the surrounding rearing environment contribute to shaping the microbiome of farmed gilthead sea-bream larvae, and that a differential establishment of host-associated bacteria takes place during larval development.for ScienPortuguese Foundation ce and Technology [PTDC/MAR/112792/2009, UID/Multi/04326/2013, UID/BIO/04565/2013]; Programa Operacional Regional de Lisboa [007317]info:eu-repo/semantics/publishedVersio

Molecular exploration of bacterial communities associated with azooxanthellate gorgonians in the coast of Algarve, south Portugal

O presente trabalho avaliou as associações bacterianas entre três espécies de gorgónias (Eunicella verrucosa, E. gazella e Leptogorgia sarmentosa) relativamente ao seu perfil comunitário, diversidade, abundância e identificação filogenética de isolados seleccionados, e estabeleceu comparações entre os referidos perfis e as comunidades bacterianas presentes no meio circundante (isto é, água e sedimento). As associações microbianas entre tecidos saudáveis e doentes de E. verrucosa foram também comparadas utilizando os mesmos parâmetros. O perfil comunitário bacteriano foi realizado com a técnica Gel de Electroforese de Gradiente Desnaturante (DGGE) e revelou associações distintas entre as gorgónias e as amostras ambientais. As amostras de E. verrucosa doentes juntamente com água e sedimento representaram as associações mais diversas. Foi detectada uma especificidade no perfil bacteriano comunitário entre as três espécies estudadas, que revelou ser distinta nos tecidos necróticos. A quantificação bacteriana foi efectuada através de microscopia de epifluorescência e de plaqueamento. Os tecidos doentes de E. verrucosa apresentaram a maior contagem bacteriana tanto por epifluorescência como por plaqueamento [3.99x1010 cél/ml (Número Total de Células – NTC) e 1.8x106 UFC/ml (Unidades Formadoras de Colónias), respectivamente], seguidos pelas amostras de tecidos saudáveis da mesma espécie (2.29x1010 cél/ml e 3.5x105 UFC/ml, respectivamente). Os resultados obtidos das contagens por epifluorescência de E. gazella foram na ordem das 2.09x1010 cél/ml e via plaqueamento na ordem das 2.3x105 UFC/ml, enquanto que para L. sarmentosa foram de 2.42x1010 cél/ml e 1.3x105 UFC/ml, respectivamente. Foram encontradas diferenças significativas nas contagens de plaqueamento entre todas as amostras excepto entre as amostras de E. gazella e L. sarmentosa, e L. sarmentosa e água, e nas contagens por epifluorescência as diferenças significativas observadas foram entre água e todas as gorgónias (p<0.05), segundo o teste estatístico Mann-Whitney. Foram efectuadas análises filogenéticas a partir de isolados seleccionados utilizando o método de Máxima Verosimilhança com o modelo de substituição GTR+G+I. As culturas de tecidos de E. verrucosa revelaram a dominância de membros da classe Gammaproteobacteria (Vibrio sp., Pseudoalteromonas sp. e Shewanella sp., tendo sido os últimos dois géneros detectados apenas em tecidos doentes), embora também tenha sido determinada a presença de membros das classes Alphaproteobacteria (Ruegeria sp.) e Actinobacteria (Micrococcus sp.). O estudo presente identificou perfis comunitários bacterianos específicos entre as espécies estudadas, que diferem do ambiente circundante tanto em diversidade como abundância. Identificou também diferenças entre a diversidade comunitária bacteriana, estrutura e abundância em tecidos doentes e saudáveis de Eunicella verrucosa

Data_Sheet_1_Metagenomic Analysis Shows the Presence of Bacteria Related to Free-Living Forms of Sulfur-Oxidizing Chemolithoautotrophic Symbionts in the Rhizosphere of the Seagrass Zostera marina.PDF

<p>Seagrasses play an important role as ecosystem engineers; they provide shelter to many animals and improve water quality by filtering out nutrients and by controlling pathogens. Moreover, their rhizosphere promotes a myriad of microbial interactions and processes, which are dominated by microorganisms involved in the sulfur cycle. This study provides a detailed insight into the metabolic sulfur pathways in the rhizobiome of the seagrass Zostera marina, a dominant seagrass species across the temperate northern hemisphere. Shotgun metagenomic sequencing revealed the relative dominance of Gamma- and Deltaproteobacteria, and comparative analysis of sulfur genes identified a higher abundance of genes related to sulfur oxidation than sulfate reduction. We retrieved four high-quality draft genomes that are closely related to the gill symbiont of the clam Solemya velum, which suggests the presence of putative free-living forms of symbiotic bacteria. These are potentially highly versatile chemolithoautotrophic bacteria, able to alternate their metabolism between parallel pathways of sulfide oxidation (via sqr and fcc), nitrate reduction (denitrification or DNRA) and carbon fixation (via CBB or TCA cycle), depending on the environmental availability of sulfide. Our results support the hypothesis that seagrass meadows might function as a source of symbionts for invertebrates that inhabit within or around seagrass meadows. While providing ideal conditions for the proliferation of these free-living forms of symbionts, seagrasses would benefit from their genetic versatility, which contributes to sulfide detoxification and ammonium production, the seagrasses' preferred nitrogen source.</p

Phenolic chemistry of the seagrass Zostera noltei Hornem. Part 1: First evidence of three infraspecific flavonoid chemotypes in three distinctive geographical regions

The flavonoid content of Zostera noltei leaves was investigated over a broad spatial scale using chromatographic and spectroscopic techniques (HPLC-DAD, LC/MS and NMR). Samples were collected at fifteen localities covering Mediterranean Sea and NE Atlantic coast, and representative of three types of coastal ecosystems: mesotidal bays, coastal lagoons, and open-sea. Three geographically distinct flavonoid chemotypes were identified on the basis of their respective major compound. One is characterized by apigenin 7-sulfate (Eastern part of Gulf of Cadiz), one by diosmetin 7-sulfate (French Atlantic coast and Mediterranean Sea), and the third contained similar quantities of the above two compounds (Mauritania and South Portugal). Our results show that metabolomic profiling using a combination of analytical techniques is a tool of choice to characterize chemical phenotype accurately. This work emphasizes for the first time the spatial variability in the flavonoid chemistry of Z. noltei throughout Atlantic and Mediterranean range, and constitutes the first report of chemical races in the Zosteraceae family. This infraspecific chemical differentiation should be considered when dealing with the role of Z. noltei in coastal ecosystems or in the selection of the best population donor for Z. noltei beds restoration. Combined with molecular identification, phenolic fingerprinting might be helpful to elucidate the evolutionary history of Z. noltei

Deep ocean prokaryotic communities are remarkably malleable when facing long-term starvation

International audienceThe bathypelagic ocean is one of the largest ecosystems on Earth and sustains half of the ocean's microbial activity. This microbial activity strongly relies on surface-derived particles, but there is growing evidence that the carbon released through solubilization of these particles may not be sufficient to meet the energy demands of deep ocean prokaryotes. To explore how bathypelagic prokaryotes respond to the absence of external inputs of carbon, we followed the long-term (1 year) dynamics of an enclosed community. Despite the lack of external energy supply, we observed a continuous succession of active prokaryotic phylotypes, which was driven by recruitment of taxa from the seed bank (i.e., initially rare operational taxonomic units [OTUs]). A single OTU belonging to Marine Group I of Thaumarchaeota, which was originally rare, dominated the microbial community for similar to 4 months and played a fundamental role in this succession likely by introducing new organic carbon through chemolithoautotrophy. This carbon presumably produced a priming effect, because after the decline of Thaumarchaeota, the diversity and metabolic potential of the community increased back to the levels present at the start of the experiment. Our study demonstrates the profound versatility of deep microbial communities when facing organic carbon deprivation