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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). Sí

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). Sí

De novo protein synthesis is required for synapse modifications underlying stable memory encoding. Yet neurons are highly compartmentalized cells and how protein synthesis can be regulated at the synapse level is unknown. Here, we characterize neuronal signaling complexes formed by the postsynaptic scaffold GIT1, the mechanistic target of rapamycin (mTOR) kinase, and Raptor that couple synaptic stimuli to mTOR-dependent protein synthesis; and identify NMDA receptors containing GluN3A subunits as key negative regulators of GIT1 binding to mTOR. Disruption of GIT1/mTOR complexes by enhancing GluN3A expression or silencing GIT1 inhibits synaptic mTOR activation and restricts the mTOR-dependent translation of specific activity-regulated mRNAs. Conversely, GluN3A removal enables complex formation, potentiates mTOR-dependent protein synthesis, and facilitates the consolidation of associative and spatial memories in mice. The memory enhancement becomes evident with light or spaced training, can be achieved by selectively deleting GluN3A from excitatory neurons during adulthood, and does not compromise other aspects of cognition such as memory flexibility or extinction. Our findings provide mechanistic insight into synaptic translational control and reveal a potentially selective target for cognitive enhancement, Ramon y Cajal program RYC2014-15784, RETOS-MINECO SAF2016-76565-R, ERANET-Neuron JTC 2019 ISCIII AC19/00077 FEDER funds (to R.A.); RETOS-MINECO SAF2017-87928-R (to A.B.); an NIH grant (NS76637) and UTHSC College of Medicine funds (to S.J.T.); and NARSAD Independent Investigator Award and grants from the MINECO (CSD2008-00005, SAF2013-48983R, SAF2016-80895-R), Generalitat Valenciana (PROMETEO 2019/020)(to I.P.O.) and Severo-Ochoa Excellence Awards (SEV-2013-0317, SEV-2017-0723)

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.

El pdf del artículo es la versión post-print., The organochlorine chemicals endosulfan, dieldrin, and γ-hexachlorocyclohexane (lindane) are persistent pesticides to which people are exposed mainly via diet. Their antagonism of the γ-aminobutyric acid-A (GABAA) receptor makes them convulsants. They are also endocrine disruptors because of their interaction with the estrogen receptor (ER). Here, we study the effects of dieldrin, endosulfan, and lindane on ERs in primary cultures of cortical neurons (CN) and cerebellar granule cells (CGC). All the compounds tested inhibited the binding of [3H]-estradiol to the ER in both CN and CGC, with dieldrin in CGC showing the highest affinity. We also determined the effects of the pesticides on protein kinase B (Akt) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation. Dieldrin and endosulfan increased Akt phosphorylation in CN, which was inhibited by the ERb antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-α]pyrimidin-3-yl]phenol. Instead, Akt and ERK1/2 phosphorylation induced by dieldrin in CGC was mediated by multiple activation of ERa, ERb, and G protein- coupled receptor 30. Lindane did not activate these pathways, but it inhibited estradiol-mediated Akt and ERK1/2 activation. In CN, all the chemicals activated ERK1/2 through a mechanism involving GABAA and glutamate receptors. Long-term exposure to these pesticides reduced the levels of ERα, but not of ERβ. Moreover, extracts of CN treated with endosulfan, dieldrin, or lindane induced cell proliferation in MCF-7 human breast cancer-derived cells, whereas only extracts of CGC treated with dieldrin induced MCF-7 cell proliferation. Overall, the observed alterations on ER-mediated signaling and ER levels in neurons might contribute to the neurotoxicity of these organochlorine pesticides. © The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved., This research was supported by grants from Ministry of Health (FIS 061212, FIS10/0453), CIBERESP (AA08-001), Generalitat de Catalunya (2009/SGR/214) and from Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía (P09-CTS-5488).

Dieldrin is an endocrine disruptor that accumulates in mammalian adipose tissue and brain. It induces convulsions due to its antagonism of the γ-aminobutyric acid A receptor (GABA AR). We have previously reported that long-term exposure to dieldrin causes the internalization of the N-methyl-D-aspartate receptor (NMDAR) as a result of persistent GABAAR inhibition. Because the neurosteroids 17β-estradiol (E2) and allopregnanolone are known to modulate the function and trafficking of GABA AR and NMDAR, we examined the effects of E2 and allopregnanolone on dieldrin-induced GABA AR inhibition, NMDAR internalization, and neuronal death in cortical neurons. We found that 1 nM E2 increased the membrane expression of NR1/NR2B receptors and postsynaptic density 95 but did not induce their physical association. In contrast, 10 nM E2 had no effect on these proteins but reduced NR2A membrane expression. We also found that exposure to 60 nM dieldrin for 6 d in vitro caused the internalization of NR1 and NR2B but not NR2A. Treatment with either 1 nM E2 or 10 οM allopregnanolone prevented the dieldrin-induced reduction in membrane levels of the NR1/NR2B receptors. Furthermore, prolonged exposure to 200 nM dieldrin down-regulated the expression of NR2A; this was inhibited only by allopregnanolone. Although both hormones restored NMDAR function, as measured by the NMDA-induced rise in intracellular calcium, allopregnanolone (but not E2) reversed the inhibition of GABA AR and neuronal death caused by prolonged exposure to dieldrin. Our results indicate that allopregnanolone protects cortical neurons against the neurotoxicity caused by long-term exposure to dieldrin by maintaining GABA AR and NMDAR functionality. Copyright © 2012 by The Endocrine Society., This work was supported by the Ministry of Science and Innovation (FIS 10/0453) (to CS); Generalitat de Catalunya (2009/SGR/214) (to CS); the Agence Nationale pour la Recherche Grants ANR-07-NEURO-026-03 and ANR-09-BLAN-0267 (to VP); the European Society for Pediatric Endocrinology (ESPE) Research Unit Grant 2009-2011 coordinated by Dr. Sabine Heger (Children’s Hospital Bult, Hannover, Germany) (to V. P.).

El pdf es la versión post-print., Glutamate is the mayor excitatory neurotransmitter in vertebrate nervous system. It has a crucial role in most brain functions under physiological conditions through the activation of both ionotropic and metabotropic glutamate receptors. In addition, extracellular glutamate concentration is tightly regulated through different excitatory amino acid transporters (EAAT). Glutamate neurotransmission is also involved in the neurotoxic effects of many environmental chemicals and drugs. Furthermore, homeostatic changes in glutamate neurotransmission appear in response to prolonged block / enhancement of electrical activity. Here we describe different approaches to evaluate alterations in glutamate neurotransmission regarding glutamate receptors and glutamate transporters by using primary cultures of neurons and of astrocytes. The methods are based on the increased fluorescence of calcium-sensitive probes in response to glutamate agonists, on radioligand binding to glutamate receptors and transport sites and on inmunocytochemistry visualization of glutamate receptors., This work was supported by grant PI 06/1212 (Spanish Ministry of Health) and SGR 2009/SGR/214 (Generalitat de Catalunya, Spain).

Trabajo presentado en el XII International Congress of Toxicology, celebrado en Barcelona, España, del 19 al 23 de julio de 2010, The organochlorine chemicals endosulfan, dieldrin, and lindane are persistent pesticides, and endosulfan is frequently found in fruits and vegetables, contributing to diet exposure. These compounds are convulsants due to their antagonism on the GABAA receptor. They are also endocrine disruptors because of their interaction with the estrogen receptor (ER) in reporter cell lines. The aim of this study was to investigate the effects of dieldrin, endosulfan, and γ-hexachlorocyclohexane (γ-HCH, lindane) and its isomer β-HCH on ER binding as well as on Akt and MAPK pathways in primary cultures of either cortical neurons (CN) or cerebellar granule cells (CGC) and also in SH-SY5Y neuroblastoma cells. We observed that all the tested compounds, except β-HCH isomer, inhibited [3H]-17β-Estradiol binding in both primary cultures but with different IC50s, with dieldrin in CGC showing the highest potency. In addition, dieldrin and endosulfan enhanced Akt and ERK1/2 phosphorylation, whereas lindane inhibited the activation of both proteins mediated by estradiol. We further analyzed whether these pesticides could altered the intracellular estrogenic charge of the neuronal cultures. For this purpose we used the E-Screen assay, in which MCF-7 human breast cancer-derived cells were incubated with the intracellular content of the neuronal cultures that were previously exposed to these pollutants for a short- and long-term period of time. Prolonged exposure to the three compounds enhanced the basal estrogenic activity of CN whereas only exposure to dieldrin increases that of CGC. On the other hand, we have observed that long-term exposure to dieldrin reduced the levels of ER-alpha, but not ER-beta, in both primary cultures. In summary, we demonstrated that the three organochlorine pesticides tested are endocrine disruptors in neuronal cultures., Supported by FIS PI061212 and CIBERESP (AA08_001).

Dieldrin was previously used as a pesticide. Although its use has been discontinued, humans are still exposed to it due to its high environmental persistence and because it accumulates in the adipose tissue of animals. Acute exposure to dieldrin provokes convulsions due to its antagonism on the gamma-aminobutyric acid-A (GABAA) receptor. However, little is known about the effects of low chronic exposure to this pollutant. In the present work, we use primary cultures of cortical neurons to study the mechanisms involved in the toxic action of dieldrin. We found that 2 and 6 days in vitro (DIV) exposure to a subcytotoxic concentration (60nM) of dieldrin reduced the increase in intracellular calcium concentration ([Ca2+]i) and the excitotoxicity caused by glutamate. Exposure to dieldrin for 6 DIV induced N-methyl-D-aspartate receptor (NMDAR) internalization and reduced metabotropic glutamate receptor 5 (mGLUR5) levels. Double immunostaining for NMDAR and mGLUR5 showed that these receptors lose colocalization on the cell membrane in neurons treated with dieldrin. No changes were observed in receptor functionalities or receptor levels after 2 DIV of exposure to dieldrin. However, the increase in [Ca2+]i induced by coactivation of NMDAR and mGLUR5 was significantly reduced. Thus, a functional interaction between the two receptors seems to play an important role in glutamate-induced excitotoxicity. We confirm that permanent blockade of the GABAA receptor by this persistent pesticide triggers adaptive neuronal changes consisting of a reduction of glutamatergic neurotransmission. This might explain the cognitive and learning deficits observed in animals after chronic treatment with dieldrin. © The Author 2009. Published by Oxford University Press on behalf of the Society of Toxicology., This work was funded by the Instituto de Salud Carlos III, Ministry of Health, Spain (grant number PI 061212); Generalitat de Catalunya (grant number 2005/SGR/00826). V.B. is recipient of a predoctoral fellowship from Institut d’Investigacions Biomèdiques August Pi i Sunyer.

Trabajo presentado en el XII International Congress of Toxicology, celebrado en Barcelona, España, del 19 al 23 de julio de 2010, Dieldrin is a previously used pesticide that accumulates in the adipose tissue as well as in the brain of mammals. This organochlorine has convulsant activity because it blocks the GABAA receptor. However, little is known about the effects of a chronic exposure to this pollutant. We have previously reported that long-term exposure to dieldrin reduces the number of functional NMDA receptors (NMDARs) in neuronal cultures. On the other hand, estradiol increases synaptogenesis and promotes the delivery of NMDAR to the cell membrane. The aims of this study are: (i) elucidate which subunits of NMDAR are affected by dieldrin exposure and (ii) evaluate the effects of estradiol against dieldrin-induced NMDAR internalization. Long-term (6DIV) exposure to a subcitotoxic concentration (60 nM) of dieldrin caused the internalization of NR1 and NR2B, but not NR2A, subunits of NMDAR. Treatment with 1 nM (but not 10 nM) 17β-estradiol (E2) for 2DIV rescued dieldrin-induced decrease of NR1 and NR2B on the cell membrane but not their internalization, suggesting different mechanisms of action between the two compounds regarding NMDAR trafficking. E2 also restored NMDAR function, as measured by NMDA-induced [Ca2+]i increase. Furthermore, prolonged exposure to a higher concentration (200 nM) of dieldrin also reduced cell surface expression of NR2A, but this effect was not prevented by E2. In addition, we showed here by immunoprecipitation on membrane extraction that PSD-95 is highly physically associated with NR2A-containing (but not NR2B-containing) NMDARs in cortical neurons and moreover that this association is lost after prolonged exposure to 200 nM dieldrin. In the present study, we confirmed that the permanent blockade of the GABAA receptor by this persistent pesticide triggers adaptive neuronal changes consisting on NMDAR internalization, that might explain the learning and cognitive deficits observed in animals after chronic treatment with dieldrin. In addition, estradiol may exert beneficial effects against dieldrin-induced neurotoxicity., Supported by FIS PI061212.