Fine tune control of dopamine neurotransmission by alpha-synuclein: down- and over-expression models

Póster presentado en el IX Simposi de Neurobiologia Experimental, celebrado los días 22 y 23 de octubre de 2014 en Barcelona y organizado por la Societat Catalana de Biologia del Institut d'Estudis CatalansAlpha-synuclein protein (α-syn) accumulates in the brain of patients with Parkinson´s disease (PD) and leaves a degeneration of midbrain dopamine (DA) neurons. However, the normal function of α-syn on DA neurotransmission in vivo remains poorly understood. Here, we used two mouse models with a) reduced α-syn expression in the substantia nigra compacta (SNc) and ventral tegmental area (VTA) induced by antisense oligonucleotide molecule (ASO) and, b) modest α-syn over-expression in tyrosine hydroxylase (TH)-positive neurons in the absence of overt toxicity. ASO sequence against α-syn was conjugated to a cell-specific ligand, indatraline (monoamine transporter inhibitor), to promote its selective delivery into monoamine neurons after intranasal administration. Indatraline-α-syn-ASO conjugate (1233ASO) entered into midbrain DA cells followed by trafficking to deep endomembrane vesicles associated with Rab7 resulting in an efficient α-syn knockdown. Indeed, 4-day 1233ASO treatment (30µg/day) decreased α-syn mRNA and protein levels in SNc/VTA (84.1±1.7% and 57.7±7.8% of PBS-treated animals, respectively). Alpha-synuclein suppression displayed an enhancement striatal DA tone using intracerebral microdialysis. Local veratridine (50 µM) perfusion increased extracellular DA levels more efficient in 1233ASO-treated than PBS-treated mice. Similarly, nomifensine (1-10-50 µM) or amphetamine (1-10-100 µM) showed a marked doseeffect which phenotypic differences. Tetrabenazine (VMAT2 inhibitor, 100 µM) reduced striatal DA levels in 1233ASO-treated mice. This effect was lower than in control mice. Conversely, we found that over-expressed α-syn inhibits striatal DA release. Together, this evidence indicates a physiological role for a-syn as a >fine tune> modulator of nigroestriatal DA release and the effects depend on the a-syn expression levelsSpanish Ministery of Economy and Competitiveness, INNPACTO Subprogram IPT-2012-1208-300000; Instituto de Salud Carlos III (ISCIII) Grant PI13/01390. Some of these grants are co-financed by the European Regional Development Fund “A way to build Europe”Peer Reviewe

Therapeutic antidepressant potential of a conjugated siRNA silencing the serotonin transporter after intranasal administration

A Ferrés-Coy et al.Major depression brings about a heavy socio-economic burden worldwide due to its high prevalence and the low efficacy of antidepressant drugs, mostly inhibiting the serotonin transporter (SERT). As a result, similar to 80% of patients show recurrent or chronic depression, resulting in a poor quality of life and increased suicide risk. RNA interference (RNAi) strategies have been preliminarily used to evoke antidepressant-like responses in experimental animals. However, the main limitation for the medical use of RNAi is the extreme difficulty to deliver oligonucleotides to selected neurons/systems in the mammalian brain. Here we show that the intranasal administration of a sertraline-conjugated small interfering RNA (C-SERT-siRNA) silenced SERT expression/function and evoked fast antidepressant-like responses in mice. After crossing the permeable olfactory epithelium, the sertraline-conjugated-siRNA was internalized and transported to serotonin cell bodies by deep Rab-7-associated endomembrane vesicles. Seven-day C-SERT-siRNA evoked similar or more marked responses than 28-day fluoxetine treatment. Hence, C-SERT-siRNA (i) downregulated 5-HT1A-autoreceptors and facilitated forebrain serotonin neurotransmission, (ii) accelerated the proliferation of neuronal precursors and (iii) increased hippocampal complexity and plasticity. Further, short-term C-SERT-siRNA reversed depressive -like behaviors in corticosterone-treated mice. The present results show the feasibility of evoking antidepressant -like responses by selectively targeting neuronal populations with appropriate siRNA strategies, opening a way for further translational studies.This work was supported by grants from CDTI—Spanish Ministry of Science and Innovation—DENDRIA contribution, 'nLife all rights reserved' (to AB and FA); Instituto de Salud Carlos III PI10/00290 and PI13/01390 (to AB), PI/10/0123 (to JCL) and Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM); NARSAD Independent Investigator Grant from the Brain & Behavior Research Foundation Grant 20003 (to AB); Ministry of Economy and Competitiveness SAF2012-35183 (to FA) and SAF2011-25020 (to AP); and Generalitat de Catalunya, Secretaria d’Universitat i Recerca del Departament d’Economia i Coneixement (SGR2014) Catalan Government Grant 2009SGR220 (to FA). Some of these grants are co-financed by the European Regional Development Fund 'A way to build Europe'. AF-C is a recipient of a fellowship from Spanish Ministry of Education, Culture and Sport.Peer Reviewe

Selective suppression of α-Synuclein in monoaminergic neurons of mice by intranasal delivery of targeted small interfering RNA or antisense oligonucleotides: Potential therapy for Parkinson's disease

Póster presentado en: ACNP (American College of Neuropsychopharmacology) 52nd Annual Conference, celebrada del 8 al 12 de diciembre de 2013 en Hollywood, Florida (Estados Unidos)Abstract publicado en: Neuropsychopharmacology 38:S419-S420 (2013). ISSN: 0893-133X. eISSN: 1740-634X. DOI:10.1038/npp.2013.280α-Synuclein (α-Syn) appears to play a crucial role in the pathogenesis of several neurodegenerative disorders including Parkinson's disease (PD). The brains of Parkinson patients typically contain insoluble intracellular protein inclusions called Lewy bodies. Increased neuronal α-Syn levels represent a major component of Lewy bodies and therefore, the suppression of α-Syn expression provides a valid therapeutic target for PD. The goal of this study was to assess the ability of various small interfering RNA (siRNA) and antisense oligonucleotide (ASO) sequences directed against α-Syn to downregulate endogenous or overexpressed α-Syn mRNA levels in BE-M17 neuroblastoma cells. Moreover, we evaluated the feasibility of reducing α-Syn expression selectively in PD-vulnerable brain areas including substantia nigra pars compacta (SNc), ventral tegmental area (VTA), locus coeruleus (LC) and dorsal raphe nucleus (DR) of mice after the internalization of conjugated siRNA/ASO molecules into monoamine neurons following intranasal administration. Conclusions: These results set the stage for the testing of these molecules as potential disease-modifying agents in neurotoxin-based and genetic models of PD linked to pathogenic increases in α-Syn. In this study we have characterized conjugated siRNA and ASO molecules that actively reduce endogenous α-Syn expression in vivo using the intranasal route to deliver directly siRNA/ASO into the brainPeer Reviewe

Human pluripotent stem cell-derived neurons are functionally mature in vitro and integrate into the mouse striatum following transplantation

Human Pluripotent Stem Cells (hPSCs) are a powerful tool for modelling human development. In recent years, hPSCSs have become central in cell-based therapies for neurodegenerative diseases given their potential to replace affected neurons. However, directing hPSCs into specific neuronal types is complex and requires an accurate protocol that mimics endogenous neuronal development. Here we describe step-by-step a novel and fast feeder-free neuronal differentiation protocol to direct hPSCs onto mature forebrain neurons in 37 days in vitro (DIV). The protocol is grounded on a combination of specific morphogens, trophic and growth factors, ions, neurotransmitters and extracellular matrix elements. An induced hPSC line (Ctr-Q33) and an embryonic hPSC line (GEN-Q18) were used to reinforce the potential of the protocol. Neuronal activity was analysed by single-cell calcium imaging. At 8 DIV, we obtained a homogeneous population of hPSCs-derived neuroectodermal progenitors which self-arranged in bi-dimensional neural tube-like structures. At 16 DIV, we generated hPSC-derived neural progenitors (NPCs) with mostly subpallial identity along with a subpopulation of pallial NPCs. Terminal in vitro neuronal differentiation was confirmed by the expression of microtubule associated protein 2b (Map2b) by almost 100% of hPSC-derived neurons and the expression of specific-striatal neuronal markers including GABA, CTIP2 and DARPP-32. HPSC-derived neurons showed mature and functional phenotypes as they expressed synaptic markers, voltage-gated ion channels and neurotransmitter receptors. Neurons displayed diverse spontaneous activity patterns that were classified into three major groups, namely 'high', 'intermediate' and 'low' firing neurons. Finally, transplantation experiments in vivo showed that highly relevant, committed NPCs survived within mouse striatum for at least 3 months. NPCs embodied host environmental cues and differentiated into striatal medium size spiny neurons (MSNs), which successfully integrated into the endogenous circuitry without the appearance of any teratoma symptom. Altogether, present findings demonstrate the potential of this in vitro human neuronal differentiation protocol, which will bring new opportunities for the study of human neurodevelopment and neurodegeneration, and will open new avenues in cell-based therapies, cutting-edge pharmacological studies and toxicology

Recuperació de les dones en situació de violència masclista de parella : descripció i instrumentació

Podeu consultar la versió en castellà actualitzada de l'any 2016 a: http://hdl.handle.net/11703/106307Aquesta publicació presenta els resultats d'una recerca aplicada, patrocinada per l'Ajuntament de Barcelona, dirigida per la Universitat de Barcelona i realitzada amb la participació de l'Equip d'Atenció a les Dones de la Direcció del Programa de la Dona de l'Ajuntament de Barcelona i gestionat per la Fundació Salut i Comunitat

Recuperación de las mujeres en situación de violencia machista de pareja : descripción e instrumentación

Podeu consultar la versió en català de l'any 2013 a: http://hdl.handle.net/11703/91078Aquesta publicació presenta els resultats d'una recerca aplicada, patrocinada per l'Ajuntament de Barcelona, dirigida per la Universitat de Barcelona i realitzada amb la participació de l'Equip d'Atenció a les Dones de la Direcció del Programa de la Dona de l'Ajuntament de Barcelona i gestionat per la Fundació Salut i Comunitat

Soluble mutant huntingtin drives early human pathogenesis in Huntington’s disease

Huntington's disease (HD) is an incurable inherited brain disorder characterised by massive degeneration of striatal neurons, which correlates with abnormal accumulation of misfolded mutant huntingtin (mHTT) protein. Research on HD has been hampered by the inability to study early dysfunction and progressive degeneration of human striatal neurons in vivo. To investigate human pathogenesis in a physiologically relevant context, we transplanted human pluripotent stem cell-derived neural progenitor cells (hNPCs) from control and HD patients into the striatum of new-born mice. Most hNPCs differentiated into striatal neurons that projected to their target areas and established synaptic connexions within the host basal ganglia circuitry. Remarkably, HD human striatal neurons first developed soluble forms of mHTT, which primarily targeted endoplasmic reticulum, mitochondria and nuclear membrane to cause structural alterations. Furthermore, HD human cells secreted extracellular vesicles containing mHTT monomers and oligomers, which were internalised by non-mutated mouse striatal neurons triggering cell death. We conclude that interaction of mHTT soluble forms with key cellular organelles initially drives disease progression in HD patients and their transmission through exosomes contributes to spread the disease in a non-cell autonomous manner. Graphical abstract

GABA released from cultured cortical neurons influences the modulation of t-[35S]butylbicyclophosphorothionate binding at the GABAA receptor: Effects of thymol

El pdf del artículo es la versión pre-print.Thymol is a monoterpene that specifically interacts with synaptic neural functions in neuronal GABA-operated Cl(-) channels. Here we explore the effects of thymol, and propofol as positive control, on t-[(35)S]butylbicyclophosphorothionate ([(35)S]TBPS) binding in primary cultures of cortical neurons. The study includes a meaningful analysis of the effect of various exposure buffers, and their correlation with GABA released from cells, chloride influx through the GABA(A) receptor and GABA transporter activity. Cell viability was also determined. Thymol and propofol inhibited the binding of [(35)S]TBPS to cells exposed to Tris-citrate-NaCl buffer whereas a biphasic effect was observed in HEPES solution. The different effects of the two buffers analysed are due to the higher capacity of Tris-citrate-NaCl buffer to induce the release of endogenous GABA facilitating the binding of [(35)S]TBPS to its recognition site at the GABA(A) receptor. Released GABA in the presence of this buffer was inhibited by the neuronal GABA transporter inhibitor SKF 100330-A. Tris-citrate-NaCl buffer also induced a chloride influx, which was reverted by picrotoxinin. TBPS binding in living cells is facilitated by GABA released from the cells, which in turn activates basal GABA(A) receptor activity. The results deepen on the allosteric mechanism of thymol as positive modulator of the GABA(A) receptor. Furthermore, we corroborate [(35)S]TBPS binding as an important test to verify the capacity of drugs to act on and recognize a site at the GABA(A) receptor.This study was supported by grants PI061212 and 2005-SGR-00826 (Ministry of Health and Generalitat de Catalunya, respectively, Spain), SECYT-UNC (Argentina) and CAEN-ISN. DAG is a member of CONICET (Argentina) and was recipient of a post-doctoral fellowship from the Fundación Carolina (Spain). IV was recipient of a CSIC Fellowship in the I3P program, co-financed with European Social Funds.Peer reviewe

Long-term exposure to dieldrin prevents glutamate toxicity by reducing NMDA receptor and mGLUR5 functionality in primary cultures of cortical neurons.

Trabajo presentado en el 12th Biennial Meeting of the International Neurotoxicology Association, celebrado en Jerusalén (Israel) del 07 al 12 de julio de 2009.[Purpose]: Dieldrin is a previously used pesticide that accumulates in the adipose tissue and the brain of mammals. Despite it is known that an acute exposure to dieldrin evokes convulsions, due to its antagonism on the GABAA receptor, 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 in cerebellar granule cells. In the present work we use primary cultures of cortical neurons, mainly composed of GABAergic neurons, to support these observations in a different neuronal population and to further deep into the mechanisms involved in the toxic action of this pesticide. [Methods]: Cell viability: MTT and LDH assays. Functionality of the glutamate receptors (GluR): by measuring the [Ca2+]i with the fluorescence probe Fluo-3AM. Levels and localization of GluR: Western Blot and immunocytochemistry. [Results]: We have observed that the long-term (2 and 6DIV) exposure to a subcitotoxic concentration (60nM) of dieldrin prevents the excitotoxicity and reduces the [Ca2+]i increase caused by glutamate. However only after 6 DIV of treatment, dieldrin was able to reduce NMDAR and type-I metabotropic GluR functionalities. The NMDAR immunolabelling revealed that it undergoes internalization after 6 DIV of exposure to dieldrin. Whereas mGLUR1 expression remained unaffected, mGLUR5 was downregulated after the longer treatment. In contrast no changes were observed neither on receptor functionality nor on the receptor levels after 2 DIV of exposure to dieldrin. However double immunostaining for NMDA and mGLUR5 showed that these receptors loosed colocalization on the cell membrane in cells treated with dieldrin either for 2 and 6 DIV. [Conclusions]: We confirmed that the permanent blockade of the GABAA receptor by this persistent pesticide triggers adaptative neuronal changes consisting on the reduction of the glutamatergic transmission. Moreover it seems that the presence of mGLUR5 plays crucial role on the glutamate excitotoxicity in cortical neurons. Altough these results could attribute protective effects to this pollutant in a excitotoxic model, it seems properly that, through interfering with the glutamatergic transmission, dieldrin might impair memory and learning and could also alter behaviour.Supported by FIS PI 06121

Reduction of glutamatergic neurotransmission by prolonged exposure to dieldrin involves NMDA receptor internalization and metabotropic glutamate receptor 5 downregulation

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.Peer Reviewe