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

RNAi-mediated serotonin transporter suppression rapidly increases serotonergic neurotransmission and hippocampal neurogenesis

Current antidepressants, which inhibit the serotonin transporter (SERT), display limited efficacy and slow onset of action. Here, we show that partial reduction of SERT expression by small interference RNA (SERT-siRNA) decreased immobility in the tail suspension test, displaying an antidepressant potential. Moreover, short-term SERT-siRNA treatment modified mouse brain variables considered to be key markers of antidepressant action: reduced expression and function of 5-HT(1A)-autoreceptors, elevated extracellular serotonin in forebrain and increased neurogenesis and expression of plasticity-related genes (BDNF, VEGF, Arc) in hippocampus. Remarkably, these effects occurred much earlier and were of greater magnitude than those evoked by long-term fluoxetine treatment. These findings highlight the critical role of SERT in serotonergic function and show that the reduction of SERT expression regulates serotonergic neurotransmission more potently than pharmacological blockade of SERT. The use of siRNA-targeting genes in serotonin neurons (SERT, 5-HT(1A)-autoreceptor) may be a novel therapeutic strategy to develop fast-acting antidepressants

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

Reduced alfa-MSH underlies hypothalamic ER-stress-induced hepatic gluconeogenesis

Alterations in ER homeostasis have been implicated in the pathophysiology of obesity and type-2 diabetes (T2D). Acute ER stress induction in the hypothalamus produces glucose metabolism perturbations. However, the neurobiological basis linking hypothalamic ER stress with abnormal glucose metabolism remains unknown. Here, we report that genetic and induced models of hypothalamic ER stress are associated with alterations in systemic glucose homeostasis due to increased gluconeogenesis (GNG) independent of body weight changes. Defective alpha melanocyte-stimulating hormone (α-MSH) production underlies this metabolic phenotype, as pharmacological strategies aimed at rescuing hypothalamic α-MSH content reversed this phenotype at metabolic and molecular level. Collectively, our results posit defective α-MSH processing as a fundamental mediator of enhanced GNG in the context of hypothalamic ER stress and establish α-MSH deficiency in proopiomelanocortin (POMC) neurons as a potential contributor to the pathophysiology of T2D

Modulación Post-Transcripcional de Genes Expresados en Neuronas Serotoninérgicas de Ratón como Nuevas Dianas Terapéuticas en Depresión

[spa] La tesis doctoral titulada “Modulación post-transcripcional de genes expresados en neuronas serotoninérgicas de ratón como nuevas dianas terapéuticas en depresión” ha sido realizada en el Instituto de Investigaciones Biomédicas de Barcelona (CSIC) bajo la supervisión del Dr. Francesc Artigas y la Dra. Analía Bortolozzi. Dicha tesis fue defendida el día 16 del mes de diciembre del año 2016. Se ha trabajado con la hipótesis de que los niveles de expresión del autoreceptor 5-HT1A, el transportador de serotonina (SERT) y el canal de potasio TASK3 en las neuronas serotoninérgicas podrían determinar la vulnerabilidad al estrés y por ende, la susceptibilidad a la depresión y/o respuesta antidepresiva en modelos murinos. El objetivo principal ha sido determinar las consecuencias moleculares, histológicas, neuroquímicas y comportamentales asociadas a la modulación post-transcripcional selectiva de los genes del receptor 5- HT1A, el transportador SERT o del canal de potasio TASK3 en neuronas monoaminérgicas de ratón. La metodología utilizada se basa bien en la aplicación local de moléculas de siRNAs contra el receptor 5- HT1A (1A-siRNA), el transportador SERT (SERT-siRNA) o el canal de potasio TASK3 (TASK3-siRNA) directamente en el área cerebral de interés utilizando técnicas estereotáxicas, o bien en la administración por vía intranasal de estos siRNAs conjugados con sertralina o reboxetina para para favorecer el silenciamiento celular selectivo en neuronas serotoninérgicas o noradrenérgicas, respectivamente. Los resultados obtenidos han posibilitado la publicación de 4 artículos originales más un quinto en preparación y han permitido establecer las siguientes conclusiones: * La modulación post-transcripcional del receptor 5-HT1A de las neuronas serotoninérgicas evoca efectos antidepresivos en ratones, probablemente debido a la mayor capacidad de las neuronas serotoninérgicas para liberar 5-HT en condiciones de estrés. * La disminución de la expresión del SERT en ratones produce un efecto antidepresivo más rápido y potente que el que se obtiene con el SSRI fluoxetina. Además, el knockdown del SERT consigue revertir de modo más rápido el comportamiento de tipo depresivo del modelo de depresión asociado al consumo crónico de corticosterona en comparación al tratamiento con fluoxetina. * La reducción de la expresión del canal de potasio TASK3 en neuronas serotoninérgicas produce efectos antidepresivos en ratón, demostrando el potencial de TASK3 como una nueva diana para el tratamiento de la depresión. Además, el knockdown de TASK3 en neuronas noradrenérgicas, aunque de menor magnitud que el knockdown en neuronas serotoninérgicas, también produce efectos antidepresivos. * Finalmente, la ruta de administración intranasal utilizada para silenciar el receptor 5-HT1A, el SERT o el canal de potasio TASK3 mediante siRNAs, dota los estudios de gran valor translacional, representando nuevas posibilidades terapéuticas que mejoran las principales limitaciones de los antidepresivos actuales: baja eficacia y acción retardada.[eng] It has been hypothesized that expression levels of 5-HT1A-autoreceptor, serotonin transporter (SERT) and potassium channel TASK3 in serotonergic neurons could determine vulnerability to stress and hence susceptibility to depression and/or antidepressant response in murine models. The main objective has been to determine the molecular, histological, neurochemical and behavioral consequences associated with selective post-transcriptional modulation of 5-HT1A receptor, SERT transporter or TASK3 potassium channel genes in mouse monoaminergic neurons. The methodology used is based on either the local application of siRNA molecules against 5-HT1A receptor (1A-siRNA), SERT transporter (SERT-siRNA) or potassium channel TASK3 (TASK3-siRNA) directly into the brain area of interest using stereotactic techniques, or the intranasal administration of these siRNAs conjugated with sertraline or reboxetine to promote selective cell silencing in serotonergic or noradrenergic neurons, respectively. The results obtained have allowed to establish the following conclusions: * Post-transcriptional modulation of 5-HT1A receptor in serotonergic neurons evokes antidepressant effects in mice, probably because of the increased ability of serotonergic neurons to release 5-HT under stress conditions. * Decreased expression of SERT in mice produces a more rapid and potent antidepressant effect than that obtained with SSRI fluoxetine. In addition, SERT's knockdown is able to revert the depressive- like behavior of a depression mouse model associated with chronic corticosterone consumption faster than fluoxetine treatment. * The reduction of TASK3 potassium channel expression in serotonergic neurons produces antidepressant-like effects in mice, demonstrating the potential of TASK3 as a new target for the treatment of depression. In addition, TASK3 knockdown in noradrenergic neurons, although of smaller magnitude than the knockdown in serotoninergic neurons, also produces antidepressant- like effects. * Finally, the intranasal route of administration used to silence 5-HT1A receptor, SERT or TASK3 potassium channel through siRNAs, provides studies of great translational value, representing new therapeutic possibilities that improve the main limitations of current antidepressants: low efficacy and delayed onset of action

Modulación post-transcripcional de genes expresados en neuronas serotonérgicas de ratón como nuevas dianas terapéuticas en depresión

Peer Reviewe

Reduced α-MSH Underlies Hypothalamic ER-Stress-Induced Hepatic Gluconeogenesis

Marc Schneeberger et al.© 2015 The Authors. Alterations in ER homeostasis have been implicated in the pathophysiology of obesity and type-2 diabetes (T2D). Acute ER stress induction in the hypothalamus produces glucose metabolism perturbations. However, the neurobiological basis linking hypothalamic ER stress with abnormal glucose metabolism remains unknown. Here, we report that genetic and induced models of hypothalamic ER stress are associated with alterations in systemic glucose homeostasis due to increased gluconeogenesis (GNG) independent of body weight changes. Defective alpha melanocyte-stimulating hormone (α-MSH) production underlies this metabolic phenotype, as pharmacological strategies aimed at rescuing hypothalamic α-MSH content reversed this phenotype at metabolic and molecular level. Collectively, our results posit defective α-MSH processing as a fundamental mediator of enhanced GNG in the context of hypothalamic ER stress and establish α-MSH deficiency in proopiomelanocortin (POMC) neurons as a potential contributor to the pathophysiology of T2D.This work has been supported by grants PI10/01074 (to M.C.), PI13/01604 (to M.C.), and PI13/01390 (to A.B.; Plan Estatal de I+D+I 2013-2016) cofunded by ISCIII-Subdirección General de Investigación y Fomento de la Investigación el Fondo Europeo de Desarrollo Regional (FEDER); RecerCaixa 2010ACUP_00275 (to M.C.); Generalitat de Catalunya 2014SGR659 (to R.G.) and 2014SGR48 (to A.Z.); Ministerio de Ciencia y Competitividad SAF2013-40987R (to A.Z.); Marie Curie People Cofund Fellowship, Seventh Framework Programme of the European Commission grant 267248:DIATRAIN (to A.G.G.-V.); and co-funding from Fundaçao para a Ciencia e a Tecnologia (FCT) and FEDER through COMPETE programme for grants REDE/1506/REM/2005 (National Mass Spectrometry Network), RECI/QEQQFI/0168/2012 (UC-NMR Center), EXCL/DTP-PIC/0069/2012, and structural funding for the Center for Neurosciences (PEst-C/SAU/LA0001/2011; to J.G.J. and B.M.). M.S. is a recipient of an undergraduate grant from the UB. A.F.-C. is a recipient of a fellowship from Spanish Ministry of Education, Culture and Sport. M.C. is a recipient of a Miguel Servet contract (MICINN-ISCIII; CP09/00233). P.M.G.-R. is a recipient of a Ramon y Cajal contract (MICINN; RYC-2009-05158). A.Z. is a recipient of an ICREA Acadèmia (Generalitat de Catalunya). This work was carried out in part at the Esther Koplowitz CentrePeer Reviewe

RNAi-mediated serotonin transporter suppression rapidly increases serotonergic neurotransmission and hippocampal neurogenesis

Póster presentado en el 2013 ECNP Workshop on Neuropsychopharmacology for Young Scientists in Euope, celebrado del 7 al 10 de marzo de 2013 en Nice (Francia)Abstract publicado en: European Neuropsychopharmacology 23(Suppl. 1): S6 (2013). ISSN: 0924-977X. e-ISSN: 1873-7862. DOI: 10.1016/S0924-977X(13)70100-7Peer Reviewe

Therapeutic potential of RNAi for the treatment of major depressive disorder

Trabajo presentado en el 7th Mediterranean Neuroscience Conference, celebrado en Marrakech (Marruecos), del 23 al 27 de junio de 2019Major depressive disorder (MDD) is a severe psychiatric syndrome with very high socioeconomic impact worldwide, due to three main factors: i) its high prevalence, ii) the emergence of depressive episodes during active periods of life, and iii) the slow clinical action and limited efficacy of standard antidepressant treatments, based on the pharmacological blockade of serotonin (5-HT) transporter (SERT)