Perinatal exposure to pesticides alters synaptic plasticity signaling and induces behavioral deficits associated with neurodevelopmental disorders.

Increasing evidence from animal and epidemiological studies indicates that perinatal exposure to pesticides cause developmental neurotoxicity and may increase the risk for psychiatric disorders such as autism and intellectual disability. However, the underlying pathogenic mechanisms remain largely elusive. This work was aimed at testing the hypothesis that developmental exposure to different classes of pesticides hijacks intracellular neuronal signaling contributing to synaptic and behavioral alterations associated with neurodevelopmental disorders (NDD). Low concentrations of organochlorine (dieldrin, endosulfan, and chlordane) and organophosphate (chlorpyrifos and its oxon metabolite) pesticides were chronically dosed ex vivo (organotypic rat hippocampal slices) or in vivo (perinatal exposure in rats), and then biochemical, electrophysiological, behavioral, and proteomic studies were performed. All the pesticides tested caused prolonged activation of MAPK/ERK pathway in a concentration-dependent manner. Additionally, some of them impaired metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD). In the case of the pesticide chlordane, the effect was attributed to chronic modulation of MAPK/ERK signaling. These synaptic alterations were reproduced following developmental in vivo exposure to chlordane and chlorpyrifos-oxon, and were also associated with prototypical behavioral phenotypes of NDD, including impaired motor development, increased anxiety, and social and memory deficits. Lastly, proteomic analysis revealed that these pesticides differentially regulate the expression of proteins in the hippocampus with pivotal roles in brain development and synaptic signaling, some of which are associated with NDD. Based on these results, we propose a novel mechanism of synaptic dysfunction, involving chronic overactivation of MAPK and impaired mGluR-LTD, shared by different pesticides which may have important implications for NDD.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by the Intertalentum Postdoctoral Program (Marie Curie cofund UAM-UE, EU project 713366) for V.B. and by grants from the Spanish Ministry of Science and Innovation (SAF2017-86983-R, PID2020-117651RB) and from the Spanish Ministry of Economy and Competitiveness (PCIN-2016– 095) for J.A.E.. V.B. was also recipient of the 2019 Eduardo Gallego postdoctoral fellowship from Fundación Francisco Cobos. M.I.C. was recipient of a postdoctoral fellowship from the Spanish Ministry of Economy (IJCI-2015–25507). E.LM. was recipient of a predoctoral fellowship from the Spanish Ministry of Science and Innovation (FPU18/02838).S

Functional specialization of different PI3K isoforms for the control of neuronal architecture, synaptic plasticity, and cognition

Neuronal connectivity and activity-dependent synaptic plasticity are fundamental properties that support brain function and cognitive performance. Phosphatidylinositol 3-kinase (PI3K) intracellular signaling controls multiple mechanisms mediating neuronal growth, synaptic structure, and plasticity. However, it is still unclear how these pleiotropic functions are integrated at molecular and cellular levels. To address this issue, we used neuron-specific virally delivered Cre expression to delete either p110α or p110β (the two major catalytic isoforms of type I PI3K) from the hippocampus of adult mice. We found that dendritic and postsynaptic structures are almost exclusively supported by p110α activity, whereas p110β controls neurotransmitter release and metabotropic glutamate receptor–dependent long-term depression at the presynaptic terminal. In addition to these separate functions, p110α and p110β jointly contribute to N-methyl-d-aspartate receptor–dependent postsynaptic long-term potentiation. This molecular and functional specialization is reflected in different proteomes controlled by each isoform and in distinct behavioral alterations for learning/memory and sociability in mice lacking p110α or p110β.This work was supported by the Spanish Ministry of Science and Innovation grants SAF2017-86983-R and PID2020-117651RB (to J.A.E.), Spanish Ministry of Science and Innovation grants SAF2017-89116R-P (FEDER/EU) and PID2020-116184RB (to M.G.), Carlos III Institute of Health-Fondo de Investigación Sanitaria grant PRB3 (IPT17/0019–ISCIII-SGEFI/ERDF, ProteoRed) and CIBERCV (to J.A.L.), Spanish Ministry of Economy postdoctoral contract IJCI-2015-25507 (to M.I.C.), Marie Curie cofund UAM-UE (EU project 713366) Intertalentum Postdoctoral Program (to V.B.), and Spanish Ministry of Science and Innovation predoctoral contracts (to C.S.-C., A.F.-R., and S.L.-G.). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN), and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033)

Evaluation of a New Digital Automated Glycemic Pattern Detection Tool

Background: Blood glucose meters are reliable devices for data collection, providing electronic logs of historical data easier to interpret than handwritten logbooks. Automated tools to analyze these data are necessary to facilitate glucose pattern detection and support treatment adjustment. These tools emerge in a broad variety in a more or less nonevaluated manner. The aim of this study was to compare eDetecta, a new automated pattern detection tool, to nonautomated pattern analysis in terms of time investment, data interpretation, and clinical utility, with the overarching goal to identify early in development and implementation of tool areas of improvement and potential safety risks. Methods: Multicenter web-based evaluation in which 37 endocrinologists were asked to assess glycemic patterns of 4 real reports (2 continuous subcutaneous insulin infusion [CSII] and 2 multiple daily injection [MDI]). Endocrinologist and eDetecta analyses were compared on time spent to analyze each report and agreement on the presence or absence of defined patterns. Results: eDetecta module markedly reduced the time taken to analyze each case on the basis of the emminens eConecta reports (CSII: 18 min; MDI: 12.5), compared to the automatic eDetecta analysis. Agreement between endocrinologists and eDetecta varied depending on the patterns, with high level of agreement in patterns of glycemic variability. Further analysis of low level of agreement led to identifying areas where algorithms used could be improved to optimize trend pattern identification. Conclusion: eDetecta was a useful tool for glycemic pattern detection, helping clinicians to reduce time required to review emminens eConecta glycemic reports. No safety risks were identified during the study

Rosiglitazone-induced CD36 up-regulation resolves inflammation by PPARγ and 5-LO-dependent pathways.

PPARγ-achieved neuroprotection in experimental stroke has been explained by the inhibition of inflammatory genes, an action in which 5-LO, Alox5, is involved. In addition, PPARγ is known to promote the expression of CD36, a scavenger receptor that binds lipoproteins and mediates bacterial recognition and also phagocytosis. As phagocytic clearance of neutrophils is a requisite for resolution of the inflammatory response, PPARγ-induced CD36 expression might help to limit inflammatory tissue injury in stroke, an effect in which 5-LO might also be involved. Homogenates, sections, and cellular suspensions were prepared from brains of WT and Alox5(-/-) mice exposed to distal pMCAO. BMMs were obtained from Lys-M Cre(+) PPARγ(f/f) and Lys-M Cre(-) PPARγ(f/f) mice. Stereological counting of double-immunofluorescence-labeled brain sections and FACS analysis of cell suspensions was performed. In vivo and in vitro phagocytosis of neutrophils by microglia/macrophages was analyzed. PPARγ activation with RSG induced CD36 expression in resident microglia. This process was mediated by the 5-LO gene, which is induced in neurons by PPARγ activation and at least by one of its products--LXA4--which induced CD36 independently of PPARγ. Moreover, CD36 expression helped resolution of inflammation through phagocytosis, concomitantly to neuroprotection. Based on these findings, in addition to a direct modulation by PPARγ, we propose in brain a paracrine model by which products generated by neuronal 5-LO, such as LXA4, increase the microglial expression of CD36 and promote tissue repair in pathologies with an inflammatory component, such as stroke.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness CSD2010-00045 (to M.A.M.) SAF2009-08145 and SAF2012-33216 (to M.A.M.), SAF2011-23354 (toI.L.), SAF2009-07466 and SAF2012-31483 (to M.R.), from Fondo Europeo de Desarrollo Regional (FEDER) “Instituto de Salud Carlos III” RETICS RD12/0014/0003 (to I.L. and from the local govern-ment of Madrid S2010/BMD-2336 (to M.A.M.) and S2010/BMD-2349 (to I.L.). I.B. and M.I.C. are fellows of the Spanish Ministry ofEconomy and Competitiveness. The authors thank Tamara Atanesand Roberto Cañadas for their technical assistance.S

Astrocytic p38α MAPK drives NMDA receptor-dependent long-term depression and modulates long-term memory.

NMDA receptor-dependent long-term depression (LTD) in the hippocampus is a well-known form of synaptic plasticity that has been linked to different cognitive functions. The core mechanism for this form of plasticity is thought to be entirely neuronal. However, we now demonstrate that astrocytic activity drives LTD at CA3-CA1 synapses. We have found that LTD induction enhances astrocyte-to-neuron communication mediated by glutamate, and that Ca2+ signaling and SNARE-dependent vesicular release from the astrocyte are required for LTD expression. In addition, using optogenetic techniques, we show that low-frequency astrocytic activation, in the absence of presynaptic activity, is sufficient to induce postsynaptic AMPA receptor removal and LTD expression. Using cell-type-specific gene deletion, we show that astrocytic p38α MAPK is required for the increased astrocytic glutamate release and astrocyte-to-neuron communication during low-frequency stimulation. Accordingly, removal of astrocytic (but not neuronal) p38α abolishes LTD expression. Finally, this mechanism modulates long-term memory in vivo.post-print5316 K

Ipsilesional Hippocampal GABA Is Elevated and Correlates With Cognitive Impairment and Maladaptive Neurogenesis After Cortical Stroke in Mice.

BACKGROUND Cognitive dysfunction is a frequent stroke sequela, but its pathogenesis and treatment remain unresolved. Involvement of aberrant hippocampal neurogenesis and maladaptive circuitry remodeling has been proposed, but their mechanisms are unknown. Our aim was to evaluate potential underlying molecular/cellular events implicated. METHODS Stroke was induced by permanent occlusion of the middle cerebral artery occlusion in 2-month-old C57BL/6 male mice. Hippocampal metabolites/neurotransmitters were analyzed longitudinally by in vivo magnetic resonance spectroscopy. Cognitive function was evaluated with the contextual fear conditioning test. Microglia, astrocytes, neuroblasts, interneurons, γ-aminobutyric acid (GABA), and c-fos were analyzed by immunofluorescence. RESULTS Approximately 50% of mice exhibited progressive post-middle cerebral artery occlusion cognitive impairment. Notably, immature hippocampal neurons in the impaired group displayed more severe aberrant phenotypes than those from the nonimpaired group. Using magnetic resonance spectroscopy, significant bilateral changes in hippocampal metabolites, such as myo-inositol or N-acetylaspartic acid, were found that correlated, respectively, with numbers of glia and immature neuroblasts in the ischemic group. Importantly, some metabolites were specifically altered in the ipsilateral hippocampus suggesting its involvement in aberrant hippocampal neurogenesis and remodeling processes. Specifically, middle cerebral artery occlusion animals with higher hippocampal GABA levels displayed worse cognitive outcome. Implication of GABA in this setting was supported by the amelioration of ischemia-induced memory deficits and aberrant hippocampal neurogenesis after blocking pharmacologically GABAergic neurotransmission, an intervention which was ineffective when neurogenesis was inhibited. These data suggest that GABA exerts its detrimental effect, at least partly, by affecting morphology and integration of newborn neurons into the hippocampal circuits. CONCLUSIONS Hippocampal GABAergic neurotransmission could be considered a novel diagnostic and therapeutic target for poststroke cognitive impairment.S

An insertional mutagenesis programme with an enhancer trap for the identification and tagging of genes involved in abiotic stress tolerance in the tomato wild-related species Solanum pennellii

Salinity and drought have a huge impact on agriculture since there are few areas free of these abiotic stresses and the problem continues to increase. In tomato, the most important horticultural crop worldwide, there are accessions of wild-related species with a high degree of tolerance to salinity and drought. Thus, the finding of insertional mutants with other tolerance levels could lead to the identification and tagging of key genes responsible for abiotic stress tolerance. To this end, we are performing an insertional mutagenesis programme with an enhancer trap in the tomato wild-related species Solanum pennellii. First, we developed an efficient transformation method which has allowed us to generate more than 2,000 T-DNA lines. Next, the collection of S. pennelli T0 lines has been screened in saline or drought conditions and several presumptive mutants have been selected for their salt and drought sensitivity. Moreover, T-DNA lines with expression of the reporter uidA gene in specific organs, such as vascular bundles, trichomes and stomata, which may play key roles in processes related to abiotic stress tolerance, have been identified. Finally, the growth of T-DNA lines in control conditions allowed us the identification of different development mutants. Taking into account that progenies from the lines are being obtained and that the collection of T-DNA lines is going to enlarge progressively due to the high transformation efficiency achieved, there are great possibilities for identifying key genes involved in different tolerance mechanisms to salinity and drought

Astrocytic p38 MAPK drives NMDA receptor-dependent long-term depression and modulates long-term memory

NMDA receptor-dependent long-term depression (LTD) in the hippocampus is a well-known form of synaptic plasticity that has been linked to different cognitive functions. The core mechanism for this form of plasticity is thought to be entirely neuronal. However, we now demonstrate that astrocytic activity drives LTD at CA3-CA1 synapses. We have found that LTD induction enhances astrocyte-to-neuron communication mediated by glutamate, and that Ca signaling and SNARE-dependent vesicular release from the astrocyte are required for LTD expression. In addition, using optogenetic techniques, we show that low-frequency astrocytic activation, in the absence of presynaptic activity, is sufficient to induce postsynaptic AMPA receptor removal and LTD expression. Using cell-type-specific gene deletion, we show that astrocytic p38α MAPK is required for the increased astrocytic glutamate release and astrocyte-to-neuron communication during low-frequency stimulation. Accordingly, removal of astrocytic (but not neuronal) p38α abolishes LTD expression. Finally, this mechanism modulates long-term memory in vivo.We thank the personnel at the fluorescence microscopy facility of the CBMSO (SMOC) for their expert technical assistance. We also thank Alfonso Araque, Carlos Dotti, Liset Menéndez de la Prida, Manuel Valero, Sara Mederos and Gertrudis Perea for expert advice and critical reading of the manuscript, and Simon Arthur (University of Dundee, UK) for the p38α knockout mice. We thank Godwin K. Dogbevia, Artur Luzgin, and Maria Calleja for technical help with molecular biology, virus purifications and characterization of AAV2/1-PGFAP-TeTxLC-2A-mKO. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness to J.A.E. (SAF2015-72988-EXP, PCIN-2016-095 and SAF2017-86983-R), to M.N. (SAF2014-58598-JIN; RYC-2016-20414), to M.I.C. (IJCI-2015-25507), and to J.A.E. and A.R.N. (CSD2010-0045). M.N. was also funded from BBVA Foundation and L'Oreal Unesco “For Women in Science”

TLR4-Binding DNA Aptamers Show a Protective Effect against Acute Stroke in Animal Models.

Since Toll-like receptor 4 (TLR4) mediates brain damage after stroke, development of TLR4 antagonists is a promising therapeutic strategy for this disease. Our aim was to generate TLR4-blocking DNA aptamers to be used for stroke treatment. From a random oligonucleotide pool, we identified two aptamers (ApTLR#1R, ApTLR#4F) with high affinity for human TLR4 by systematic evolution of ligands by exponential enrichment (SELEX). Optimized truncated forms (ApTLR#1RT, ApTLR#4FT) were obtained. Our data demonstrate specific binding of both aptamers to human TLR4 as well as a TLR4 antagonistic effect. ApTLR#4F and ApTLR#4FT showed a long-lasting protective effect against brain injury induced by middle cerebral artery occlusion (MCAO), an effect that was absent in TLR4-deficient mice. Similar effects were obtained in other MCAO models, including in rat. Additionally, efficacy of ApTLR#4FT in a model of brain ischemia-reperfusion in rat supports the use of this aptamer in patients undergoing artery recanalization induced by pharmacological or mechanical interventions. The absence of major toxicology aspects and the good safety profile of the aptamers further encourage their future clinical positioning for stroke therapy and possibly other diseases in which TLR4 plays a deleterious role