Estudio de la especificidad de vías de insulina/IGF-1 en los patrones de arborización y señalización mediante el uso de virus asociados a adenovirus

Objetivo: Las vías de señalización de insulina y del factor de crecimiento insulínico tipo I(IGF-1) están involucradas en el control de la arborización dendrítica, señalización y crecimientocelular. Varios estudios han demostrado que déficits en estas vías están relacionadoscon trastornos cognitivos, tales como autismo, y con un elevado riesgo de sufrir demencia.Con tal de elucidar el papel exacto que tiene la señalización de insulina cerebral en las funcionesneuronales importantes para los procesos cognitivos, pretendemos silenciar la expresiónde los substratos del receptor de insulina; IRS-1 e IRS-2, los cuales son dos componentesclave en la vía de señalización de Insulina-IGF-1. Diseño experimental y Métodos: Contal de estudiar el papel de IRS-1 e IRS-2 en la arborización dendrítica y en la plasticidadneuronal, generaremos varios virus adeno asociados (AAV) que sean específicos de neurona.Además, estas partículas víricas contendrán específicos shRNA para poder silenciar laexpresión de IRS-1 y/o IRS-2, junto con un gen chivato para comprobar la eficiencia de lainfección del virus. La generación de estas partículas la hemos realizado siguiendo el métodode recombinación Gateway, utilizando un promotor específico de neurona y un shRNAcontra IRS-1/IRS-2 junto con EGFP como gen chivato. Resultados y conclusiones: Hemossido capaces de generar una partícula vírica que consta de un promotor genérico potente(CMV) junto con el gen chivato EGFP. Hemos testado la eficiencia y actividad de esta partículavírica in vivo en varias regiones del cerebro de rata.Objective: The Insulin and insulin-like growth factor 1 (IGF-1) pathway are involved in thenormal control of dendritic arborisation, cell signalling and development. Several studieshave shown that deficits in these pathways are related to cognitive disorders, such as autism,and an increased risk of dementia. To elucidate the specific role of brain insulin signallingin neuronal functions that are relevant for cognitive processes we want to silence thegene expression of the Insulin Receptor Substrate 1, and 2, (IRS-1, IRS-2) two key componentsof the Insulin-IGF-1 pathway. Research Design and Methods: To study the roleof IRS-1 and IRS-2 in dendritic arborisation and neural plasticity we want to generate severaladeno-associated viruses (AAV) that are neuron-specific. Besides these virus willcontain a specific shRNA to silence both IRS-1or IRS-2 and a reporter gene in order to tracktheir infection efficiency. To do so, we have followed the Gateway method for viral particlesgeneration, with a neuron-specific promoter and a shRNA against IRS-1/IRS-2 along withthe enhanced green fluorescent protein (EGFP) as a reporter gene. Results and conclusions:We have been able to generate a viral particle that contains a strong generic promoter(CMV) along with a reporter gene (EGFP). We have tested the efficiency and activityof this viral particle in vivo in several regions of the rat brain

AAV delivery of shRNA against IRS1 in GABAergic neurons in rat hippocampus impairs spatial memory in females and male rats

This is a pre-print of an article published in Brain Structure and Function. The final authenticated version is available online at: https://doi.org/10.1007/s00429-020-02155-xBrain insulin resistance is a major factor leading to impaired cognitive function and it is considered as the onset of Alzheimer´s disease. Insulin resistance is intimately linked to inflammatory conditions, many studies have revealed how pro-inflammatory cytokines lead to insulin resistance, by inhibiting IRS1 function. Thus, the dysfunction of insulin signaling is concomitant with inflammatory biomarkers. However, the specific effect of IRS1 impaired function in otherwise healthy brain has not been dissected out. So, we decided in our study, to study the specific role of IRS1 in the hippocampus, in the absence of comorbidities. To that end, shRNA against rat and human IRS1 was designed and tested in cultured HEK cells to evaluate mRNA levels and specificity. The best candidate sequence was encapsulated in an AAV vector (strain DJ8) under the control of the cytomegalovirus promoter and together with the green fluorescent protein gene as a reporter. AAV-CMV-shIRS1-EGFP and control AAV-CMV-EGFP were inoculated into the dorsal hippocampus of female and male Wistar rats. One month later, animals undertook a battery of behavioral paradigms evaluating spatial and social memory and anxiety. Our results suggest that females displayed increased susceptibility to AAV-shIRS1 in the novel recognition object paradigm; whereas both females and males show impaired performance in the T maze when infected with AAV-shIRS1 compared to control. Anxiety parameters were not affected by AAV-shIRS1 infection. We observed specific fluorescence within the hilum of the dentate gyrus, in immuno-characterized parvalbumin and somatostatin neurons. AAV DJ8 did not enter astrocytes. Intense green fibers were found in the fornix, mammillary bodies, and in the medial septum indicating that hippocampal efferent had been efficiently targeted by the AAV DJ8 infection. We observed that AAV-shIRS1 reduced significantly synaptophysin labeling in hippocampal-septal projections compared to controls. These results support that, small alterations in the insulin/IGF1 pathway in specific hippocampal circuitries can underlie alterations in synaptic plasticity and affect behavior, in the absence of inflammatory condition

Examination of gammarid transcriptomes reveals a widespread occurrence of key metabolic genes from epibiont bdelloid rotifers in freshwater species

Previous data revealed the unexpected presence of genes encoding for long-chain polyunsaturated fatty acid (LC-PUFA) biosynthetic enzymes in transcriptomes from freshwater gammarids but not in marine species, even though closely related species were compared. This study aimed to clarify the origin and occurrence of selected LC-PUFA biosynthesis gene markers across all published gammarid transcriptomes. Through systematic searches, we confirmed the widespread occurrence of sequences from seven elongases and desaturases involved in LC-PUFA biosynthesis, in transcriptomes from freshwater gammarids but not marine species, and clarified that such occurrence is independent from the gammarid species and geographical origin. The phylogenetic analysis established that the retrieved elongase and desaturase sequences were closely related to bdelloid rotifers, confirming that multiple transcriptomes from freshwater gammarids contain contaminating rotifers’ genetic material. Using the Adineta steineri genome, we investigated the genomic location and exon–intron organization of the elongase and desaturase genes, establishing they are all genome-anchored and, importantly, identifying instances of horizontal gene transfer. Finally, we provide compelling evidence demonstrating Bdelloidea desaturases and elongases enable these organisms to perform all the reactions for de novo biosynthesis of PUFA and, from them, LC-PUFA, an advantageous trait when considering the low abundance of these essential nutrients in freshwater environments

Abscisic Acid Supplementation Rescues High Fat Diet-Induced Alterations in Hippocampal Inflammation and IRSs Expression

Accumulated evidence indicates that neuroinflammation induces insulin resistance in the brain. Moreover, both processes are intimately linked to neurodegenerative disorders, including Alzheimer’s disease. Potential mechanisms underlying insulin resistance include serine phosphorylation of the insulin receptor substrate (IRS) or insulin receptor (IR) misallocation. However, only a few studies have focused on IRS expression in the brain and its modulation in neuroinflammatory processes. This study used the high-fat diet (HFD) model of neuroinflammation to study the alterations of IR, an insulin-like growth factor receptor (IGF1R) and IRS expressions in the hippocampus. We observed that HFD effectively reduced mRNA and protein IRS2 expression. In contrast, a HFD induced the upregulation of the IRS1 mRNA levels, but did not alter an IR and IGF1R expression. As expected, we observed that a HFD increased hippocampal tumor necrosis factor alpha (TNFα) and amyloid precursor protein (APP) levels while reducing brain-derived neurotrophic factor (BDNF) expression and neurogenesis. Interestingly, we found that TNFα correlated positively with IRS1 and negatively with IRS2, whereas APP levels correlated positively only with IRS1 but not IRS2. These results indicate that IRS1 and IRS2 hippocampal expression can be affected differently by HFD-induced neuroinflammation. In addition, we aimed to establish whether abscisic acid (ABA) can rescue hippocampal IRS1 and IRS2 expression, as we had previously shown that ABA supplementation prevents memory impairments and improves neuroinflammation induced by a HFD. In this study, ABA restored HFD-induced hippocampal alterations, including IRS1 and IRS2 expression, TNFα, APP, and BDNF levels and neurogenesis. In conclusion, this study highlights different regulations of hippocampal IRS1 and IRS2 expression using a HFD, indicating the important differences of these scaffolding proteins, and strongly supports ABA therapeutic effects

Unraveling the potential of marine invertebrates as a source of omega-3 long-chain polyunsaturated fatty acids for aquaculture

Trabajo presentado en Aquaculture Europe, celebrado en Rimini (Italia), del 27 al 30 de septiembre de 2022

Malformations of the craniocervical junction (chiari type I and syringomyelia: classification, diagnosis and treatment)

Chiari disease (or malformation) is in general a congenital condition characterized by an anatomic defect of the base of the skull, in which the cerebellum and brain stem herniate through the foramen magnum into the cervical spinal canal. The onset of Chiari syndrome symptoms usually occurs in the second or third decade (age 25 to 45 years). Symptoms may vary between periods of exacerbation and remission. The diagnosis of Chiari type I malformation in patients with or without symptoms is established with neuroimaging techniques. The most effective therapy for patients with Chiari type I malformation/syringomyelia is surgical decompression of the foramen magnum, however there are non-surgical therapy to relieve neurophatic pain: either pharmacological and non-pharmacological. Pharmacological therapy use drugs that act on different components of pain. Non-pharmacological therapies are primarly based on spinal or peripheral electrical stimulation

CIBERER : Spanish national network for research on rare diseases: A highly productive collaborative initiative

Altres ajuts: Instituto de Salud Carlos III (ISCIII); Ministerio de Ciencia e Innovación.CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research

How diet and temperature affect fatty acid biosynthesis in gammarids fed sidestream sources

Trabajo presentado en la International Conference & Exposition Aquaculture Europe, celebrada en Funchal, Maderia (Portugal) del 04 al 07 de octubre de 2021.[Introduction]: The fast and remarkable growth of global aquaculture in the recent years has created new economic and ecological challenges. With regard to fish farming, such challenges have been mostly linked to guarantee the supply of raw materials used for feed formulation in order to reduce the current usage of finite resources such as fishmeal and fish oil. Gammarids, particularly their biomasses, have prompted interest as alternative sources for feed formulation due to their nutritional profile. Recent studies have reported the ability of gammarids to grow on a wide range of sidestreams while accumulating relatively high levels of long-chain (C20-24) polyunsaturated fatty acids (LC-PUFA), physiologically essential compounds for normal growth and development of vertebrates. Diet and temperature have been suggested to modulate the abundance of LC-PUFA in aquatic invertebrates. Here, we aimed to elucidate the effects of three diets (the seaweed Fucus sp., carrot leaves, coconut flesh) and four different temperatures (5, 10, 15, and 20°C) on LC-PUFA profiles of Gammarus locusta, a marine gammarid with capacity for trophic upgrading when grown in low-LC-PUFA diets. [Results]: FA analysis of diets showed that coconut is rich in saturated fatty acids (SFA) in comparison to Fucus sp. and carrot leaves. Regarding LC-PUFA, Fucus sp. showed moderate levels of arachidonic acid (ARA, 11.96%) and eicosapentaenoic acid (EPA, 4.81%), and lower levels of docosahexaenoic acid (DHA). Both coconut and carrot leaves diets lack LC-PUFA. FA analysis of G. locusta showed that temperature did not significantly influenced the composition of gammarids irrespectively of the diet. However, the G. locusta FA profiles varied with diets. Statistical analysis revealed that diet is the leading factor accounting for the G. locusta FA profiles (p < 0.05). Interestingly, gammarids fed on Fucus sp. showed the highest content of total lipids among all dietary treatments, and a fairly similar FA profile to that of wild type specimens. Furthermore, G. locusta fed on carrot leaves and coconut showed levels of DHA similar to those fed on Fucus sp. diet. [Discussion]: The study demonstrates that diet is the main modulator of FA composition in G. locusta when compared to temperature. Our analyses also revealed the presence of DHA, albeit in small proportions, in gammarids fed on either coconut or carrot leaves, regardless of the presence of this LC-PUFA in these sidestreams. These results suggest that G. locusta might have some endogenous capacity enabling the production of certain LC-PUFA from precursors present in diets devoid of these essential compounds. Therefore, gammarids can contribute to trophic upgrading of aquatic food webs by the production of physiologically essential FA such as EPA and DHA from primary sources. Thus, it is fundamental to fine tune the culturing conditions for LC-PUFA production in G. locusta regarding the optimisation of such biosynthetic mechanisms. We conclude that cultured gammarids fed on either Fucus sp. or carrot leaves are promising candidates culturing marine gammarids from which high nutritional value biomasses can be processed for their use as ingredients for aquafeeds.This research was partially supported by the ERA-NET BlueBio COFUND Project SIDESTREAM [Grant ID 68], co-funded through national funds provided by Agencia Estatal de Investigación [PCI2020-111960] and Federal Ministry of Education and Research and the German Federal Ministry for Research and Education (BMBF/FKZ 161B0950B). Further funding was obtained through the project IMPROMEGA of the Ministry of Science, Innovation and Universities, Spanish Government (RTI2018-095119-B-100, MCIU/AEI/FEDER/UE)

Effects of diet and temperature on the fatty acid composition of the gammarid Gammarus locusta fed alternative terrestrial feeds

© 2022 Ribes-Navarro, AlbertsHubatsch, Monroig, Hontoria and Navarro.The fast and remarkable growth of global aquaculture in recent years has created new challenges, such as guaranteeing a sustainable supply of raw materials used for aquafeed formulation. Gammarids are low-trophic crustaceans with an increasing interest in aquaculture due to their high nutritional profiles and their capacity to grow under high-density conditions. Moreover, gammarids have the ability to thrive on a wide range of sidestreams while accumulating relatively high levels of long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA). In the present study, juveniles of the marine gammarid Gammarus locusta were cultured at four different temperatures (5°C, 10°C, 15°C, and 20°C) for 21 days and fed three diets, including the seaweed Fucus sp. as control, and carrot leaves and coconut flesh representing two agri-food industry sidestreams. Our results indicate that both the survival and biomass of G. locusta were highly affected by diet, with coconut showing the lowest growth performance. The temperature had no effect on biomass, although high temperature (20°C) resulted in a decrease in survival. The effects of temperature on the gammarid fatty acids were not evident, with diet being the main modulator of the profiles. Furthermore, the results also reveal that the Fucus sp. diet was associated with relatively high percentages of n-3 and n-6 LC-PUFA. Interestingly, essential LC-PUFA such as eicosapentaenoic (20:5n-3, EPA) and docosahexaenoic (22:6n-3, DHA) acids were detected in gammarids fed on either Fucus sp. or any of the sidestreams irrespectively of their presence in the diets. These results suggest an ability of G. locusta for LC-PUFA biosynthesis (trophic upgrading) and/or retention, making this species a promising candidate for the production of high-value ingredients for aquafeeds.This research was supported by the ERA-NET BlueBio COFUND Project SIDESTREAM [Grant ID 68], cofunded through national funds provided by the Agencia Española de Investigación, Spain, grant no. PCI2020-111960/MCIN/AEI/10.13039/501100011033 and the EU NextGenerationEU/ PRTR, and the Funding by the German Federal Ministry of Education and Research (BMBF), FKZ161B0950B. Additional funding was received through the project IMPROMEGA Agencia Española de Investigación, Spain, grant no. RTI2018- 095119-B-100, MCIU/AEI/FEDER/UE/MCIN/AEI/10.13039/ 501100011033/and FEDER “A way to make Europe.”Peer reviewe

Freshwater gammarids as novel omega-3 long-chain polyunsaturated fatty acid sources for aquaculture: investigating the de novo biosynthesis capacity

Trabajo presentado en el International Symposium on Fish Nutrition and Feeding - "Towards precision fish nutrition and feeding (ISFNF2022)", celebrado en Sorrento (Italia) del 05 al 09 de junio de 2022