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

Intracerebral Administration of a Ligand-ASO Conjugate Selectively Reduces α-Synuclein Accumulation in Monoamine Neurons of Double Mutant Human A30P*A53T*α-Synuclein Transgenic Mice

α-Synuclein (α-Syn) protein is involved in the pathogenesis of Parkinson’s disease (PD). Point mutations and multiplications of the α-Syn, which encodes the SNCA gene, are correlated with early-onset PD, therefore the reduction in a-Syn synthesis could be a potential therapy for PD if delivered to the key affected neurons. Several experimental strategies for PD have been developed in recent years using oligonucleotide therapeutics. However, some of them have failed or even caused neuronal toxicity. One limiting step in the success of oligonucleotide-based therapeutics is their delivery to the brain compartment, and once there, to selected neuronal populations. Previously, we developed an indatraline-conjugated antisense oligonucleotide (IND-1233-ASO), that selectively reduces α-Syn synthesis in midbrain monoamine neurons of mice, and nonhuman primates. Here, we extended these observations using a transgenic male mouse strain carrying both A30P and A53T mutant human α-Syn (A30P*A53T*α-Syn). We found that A30P*A53T*α-Syn mice at 4–5 months of age showed 3.5-fold increases in human α-Syn expression in dopamine (DA) and norepinephrine (NE) neurons of the substantia nigra pars compacta (SNc) and locus coeruleus (LC), respectively, compared with mouse α-Syn levels. In parallel, transgenic mice exhibited altered nigrostriatal DA neurotransmission, motor alterations, and an anxiety-like phenotype. Intracerebroventricular IND-1233-ASO administration (100 µg/day, 28 days) prevented the α-Syn synthesis and accumulation in the SNc and LC, and recovered DA neurotransmission, although it did not reverse the behavioral phenotype. Therefore, the present therapeutic strategy based on a conjugated ASO could be used for the selective inhibition of α-Syn expression in PD-vulnerable monoamine neurons, showing the benefit of the optimization of ASO molecules as a disease modifying therapy for PD and related α-synucleinopathies.This study was supported by grants SAF2016-75797-R, PID2019-105136RB-100, Retos- Colaboración Subprogram RTC-2015-3309-1, Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (ERDF), UE; and CB/07/09/0034 Center for Networked Biomedical Research on Mental Health (CIBERSAM)

Human α-synuclein overexpression in mouse serotonin neurons triggers a depressive-like phenotype. Rescue by oligonucleotide therapy

Anxiety and depression affect 35–50% of patients with Parkinson’s disease (PD), often precede the onset of motor symptoms, and have a negative impact on their quality of life. Dysfunction of the serotonergic (5-HT) system, which regulates mood and emotional pathways, occurs during the premotor phase of PD and contributes to a variety of non-motor symptoms. Furthermore, α-synuclein (α-Syn) aggregates were identified in raphe nuclei in the early stages of the disease. However, there are very few animal models of PD-related neuropsychiatric disorders. Here, we develop a new mouse model of α-synucleinopathy in the 5-HT system that mimics prominent histopathological and neuropsychiatric features of human PD. We showed that adeno-associated virus (AAV5)-induced overexpression of wild-type human α-Syn (h-α-Syn) in raphe 5-HT neurons triggers progressive accumulation, phosphorylation, and aggregation of h-α-Syn protein in the 5-HT system. Specifically, AAV5-injected mice displayed axonal impairment in the output brain regions of raphe neurons, and deficits in brain-derived neurotrophic factor (BDNF) expression and 5-HT neurotransmission, resulting in a depressive-like phenotype. Intracerebroventricular treatment with an indatraline-conjugated antisense oligonucleotide (IND-ASO) for four weeks induced an effective and safe reduction of h-α-Syn synthesis in 5-HT neurons and its accumulation in the forebrain, alleviating early deficits of 5-HT function and improving the behavioural phenotype. Altogether, our findings show that α-synucleinopathy in 5-HT neurons negatively affects brain circuits that control mood and emotions, resembling the expression of neuropsychiatric symptoms occurring at the onset of PD. Early preservation of 5-HT function by reducing α-Syn synthesis/accumulation may alleviate PD-related depressive symptoms.We thank to the Coordenação de Aperfeiçoamento de Nivel Superior (CAPES-PDSE: 19/2016 88881.135527/2016-01), Brazil, for their financial support via a scholarship awarded to C.C.-S. We also thank JAE-Intro Program, CSIC (JAEINT_20_02116), Spain, for their financial support via a scholarship awarded to M.T.-L. This study was supported by grants SAF2016-75797-R, PID2019-105136RB-100, Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (ERDF), UE; and CB/07/09/0034 Center for Networked Biomedical Research on Mental Health (CIBERSAM)

mTOR knockdown in the infralimbic cortex evokes a depressive-like state in mouse

Fast and sustained antidepressant effects of ketamine identified the mammalian target of rapamycin (mTOR) signaling pathway as the main modulator of its antidepressive effects. Thus, mTOR signaling has become integral for the preclinical evaluation of novel compounds to treat depression. However, causality between mTOR and depression has yet to be determined. To address this, we knocked down mTOR expression in mice using an acute intracerebral infusion of small interfering RNAs (siRNA) in the infralimbic (IL) or prelimbic (PrL) cortices of the medial prefrontal cortex (mPFC), and evaluated depressive- and anxious-like behaviors. mTOR knockdown in IL, but not PrL, cortex produced a robust depressive-like phenotype in mice, as assessed in the forced swimming test (FST) and the tail suspension test (TST). This phenotype was associated with significant reductions of mTOR mRNA and protein levels 48 h post-infusion. In parallel, decreased brain-derived neurotrophic factor (BDNF) expression was found bilaterally in both IL and PrL cortices along with a dysregulation of serotonin (5-HT) and glutamate (Glu) release in the dorsal raphe nucleus (DRN). Overall, our results demonstrate causality between mTOR expression in the IL cortex and depressive-like behaviors, but not in anxiety.Funding: This research was funded by grants of the Ministerio de Economía y Competitividad (SAF2011-25020 and SAF2015-67457-R MINECO); Ministerio de Ciencia, Innovación y Universidades (RTI2018-097534-B-I00); Ministerio de Ciencia e Innovación (PID2019-105136RB-100); and the European Regional Development Fund (ERDF), UE; Instituto de Salud Carlos III (PI19/00170), and Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM)

Selective siRNA-mediated suppression of astroglial glutamate transporters in infralimbic cortex induces depressive-like behaviors in mice

Trabajo presentado en el 29th ECNP Congress, celebrado en Viena, Austria, del 17 al 20 de septiembre de 2016Peer Reviewe

Antisense oligonucleotide reduction of human alpha-synuclein accumulation in dopamine and serotonin neurons prevents early dysfunctions in a mouse model of Parkinson's disease

Trabajo presentado en el Alpha-synuclein & Parkinson’s disease Conference: Lessons from the past 20 years, celebrado en la Universitat de Barcelona los días 11 y 12 de mayo de 2017Pathological changes in end-state of Parkinson’s disease (PD) are well characterized. However, there is an urgent need to identify early functional changes to develop new therapeutic strategies stopping the development of the illness. α-Synuclein is a protein accumulating in the brain of PD patients. We generated a mouse model overexpressing wild-type human-α-synuclein in dopamine-DA or serotonin-5-HT neurons of substantia nigra-(SNc) and raphe nuclei-(RN), respectively. These mice showed increased human-α-synuclein mRNA levels in the ipsilateral SNc and RN, without loss tyrosine hydroxylase-(TH)-positive or tryptophan hydroxylase- (TPH2)-positive neurons. Moreover, reduced DA and 5-HT release with development of αsynuclein-positive axonal swelling in striatum were found in these mice. In addition, AAV5 mice exhibited motor deficits at 8 weeks post-infection into SNc and depressive-like behaviors at 4 weeks post-infection into RN. Here, we evaluated whether reducing human-α-synuclein expression in the mesencephalic nuclei prevented early dysfunctions in the AAV5 model. We used a conjugated antisense oligonucleotide targeting intracellular human-α-synuclein (ASO1337) selectively in DA and 5-HT neurons. Mice overexpressing human-α-synuclein in the SNc/VTA or RN were treated intracerebroventricularly with vehicle, nonsense ASO sequence (ASO1227) or ASO1337 (30 or 100μg/day) during 28 days using osmotic minipumps implanted subcutaneously. ASO1337 reduced dose-dependent the human-α-synuclein mRNA levels (ASO1337 30μg/day: 60,21%±15.93; ASO1337 100μ/day: 46.71%±8.63, respect vehicle) whereas endogenous α-synuclein expression remained unaltered. Immunohistochemistry shows the reduction of human-α-synuclein protein density (vehicle: 245,56%±5.32: ASO1337 30μg/day: 177.7%±7.65; ASO1337 100μ/day: 173%±2.93, respect contralateral side). Furthermore, recovery of striatal DA release was achieved by reduction of human-α-synuclein expression in SNc/VTA. Our study indicated that the reduction of intracellular α-synuclein accumulation selectively in DA and 5-HT neurons using ASO molecules represents an optimal PD therapy.Spanish Ministry of Economy and Competitiveness, INNPACTO Subprogram IPT2012-1208-300000 and Retos-Colaboración Subprogram RTC-2014-2812-1; Instituto de Salud Carlos III PI13/01390 co-financed by the European Regional Development Fund “A way to build Europe”.Peer reviewe

Mouse model of astroglial glutamate transporters knockdown in infralimbic cortex induces a depressive phenotype

Trabajo presentado en el X Simposi de Neurobiologia, celebrado en Barcelona, España, el 6 y 7 de octubre de 2016Emerging evidences suggest that dysregulations of glutamatergic neurotransmission in prefrontal cortex (PFC) are involved in the pathophysiology of depression. Astrocytes regulate excitatory neurotransmission by removing synaptic glutamate via GLAST and GLT-1 transporters. We hypothesized that reduced expression of GLAST and GLT-1 in mouse infralimbic-PFC would induce depressive-like behaviors due to disabled glutamate reuptake. We microinjected small interfering RNAs (siRNA) targeting GLAST or GLT-1unilaterally into prelimbic (PrL) or infralimbic (IL) cortices of mice and examined cellular and behavioral effects. Local GLAST-siRNA microinfusion in IL (4.2 nmol) reduced selectively GLAST mRNA and protein levels to ~80% of control mice 24h post-administration. GLAST knockdown mice exhibited depressive-like behaviors including anhedonia and, increased immobility time in the TST (125% of controls) and FST (128% of controls). Likewise, intra-IL GLT-1-siRNA infusion (4.2 nmol) reduced GLT-1 expression (~70% of controls) and mice showed a depressive-like phenotype in TST, FST and sucrose preference. Depressive-like IL symptoms were reverted by citalopram (10 mg/kg, i.p.). Conversely, intra-Prl microinfusion of both siRNAs did not affect behavioral responses despite of the reductions of GLAST and GLT-1-1 expression. Downregulation of GLAST and GLT-1 remained diminished for at least 3 day and recovered by the 7th day after acute infusion. Despite local veratridine infusion in the dorsal raphe nucleus (DRN) of control and knockdown mice evoked similar 5-HT release, reduced basal 5-HT levels in DRN were detected in IL GLAST-siRNA or GLT-1-siRNA treated mice. Local perfusion of GABAA receptor antagonist bicuculline (30 and 100¿M) increased DRN-5-HT release. This effect was more marked in knockdown than control mice, suggesting higher activation of GABA interneurons in siRNA-treated mice. Overall, these findings improve our understanding of the pathophysiology of depression, suggesting differences in connectivity between IL and PrL cortices with other cortical and subcortical regions and helping to identify novel targets in antidepressant drug development.Peer Reviewe

Regulation of nigrostriatal dopamine neurotransmission by gamma-synuclein: down- and over-expression mouse models

Trabajo presentado en el Alpha-synuclein & Parkinson’s disease Conference: Lessons from the past 20 years, celebrado en la Universitat de Barcelona los días 11 y 12 de mayo de 2017Synucleins are small proteins in vertebrates, especially abundant in neurons and presynaptic terminals. Mutations in α-synuclein gene have been reported in inherited forms of Parkinson’s disease, but far less is known about β- and γsynucleins, which have been implicated in some neurodegenerative diseases and synucleinopathies. Here, we examined the distribution and cellular localization of γ-synuclein in the mouse brain using in situ hybridization and immunohistochemistry procedures, and evaluated its functional role in nigrostriatal dopamine (DA) neurotransmission in vivo. We used two different models with: 1) down-regulated γ-synuclein expression in DA neurons of substantia nigra compacta/ventral tegmental area (SNc/VTA) by small interfering RNA molecules (siRNA), or 2) over-expressed γ-synuclein in SNc/VTA DA neurons using AAV10-CMV-mouse-γ-synuclein vector. In C57BL/6J control mice, abundant levels of γ-synuclein mRNA were found in monoaminergic nuclei (SNc/VTA, raphe nuclei and locus coeruleus). Double in situ hybridization and immunohistochemistry showed a specific colocalization of γ-synuclein in TH-positive DA neurons. Unilateral infusion of three siRNA sequences targeting γ-synuclein decreased its mRNA levels in DA neurons of SNc (50% of aCSF-treated mice 24h post-infusion). Furthermore, γ-synuclein suppression displayed an enhanced striatal DA tone using intracerebral microdialysis, which is reflected in higher extracellular DA levels in siRNAtreated mice during local veratridine, nomifensine and amphetamine administrations and lower levels with quinpirole; meanwhile, γ-synuclein overexpression led to lower levels during nomifensine administration. In conclusion, these results confirm that γ-synuclein is a negative regulator of DA neurotransmission and suggest that siRNA-induced γ-synuclein suppression in midbrain dopaminergic neurons may lead to new therapies for synucleinopathies.Peer reviewe

Murine model of depression: astroglial glutamate transporter knockdown in infralimbic cortex induces a depressive phenotype

Trabajo presentado en el Neuroscience 2017, celebrado en Washington del 11 al 15 de noviembre de 2017Emerging evidence suggests that dysregulations of glutamatergic neurotransmission in prefrontal cortex are involved in the pathophysiology of depression. Astrocytes regulate excitatory neurotransmission by removing synaptic glutamate via GLAST and GLT-1 transporters. We demonstrated that unilateral knockdown of GLAST and GLT-1 in mouse infralimbic (IL) cortex induced by the local micro-infusion of small interfering RNAs (siRNA) targeting either transporter evoked a depressive-like phenotype. Conversely, intra-prelimbic (Prl) cortex microinfusion of both siRNAs did not affect behavioral responses despite comparable reductions of GLAST and GLT-1-1 expression in both PFC subdivisions. Downregulation of GLAST and GLT-1 remained diminished for at least 3 day and recovered 7 days post-administration. The depressive-like phenotype induced by reducing GLT-1 and GLAST expression in IL may be due to a hypoactive serotonergic (5-HT) system since it was reversed by the antidepressant drug citalopram. Likewise, microdialysis experiments revealed that siRNA-treated mice exhibited a reduced basal 5-HT release in the dorsal raphe nucleus (DRN) compared with controls, and a similar effect of veratridine in both experimental groups, indicating a similar intracellular 5-HT content. Further, local infusion of the GABAA receptor antagonist bicuculline increased DRN-5-HT release more markedly in siRNA-treated mice, suggesting a greater inhibition of 5-HT neurons via GABAA receptors. However, local downregulation of both transporters in PrL did not alter baseline 5-HT in DRN not the effect of bicuculline on 5-HT release. Taken together, these observations suggest that 1) an alteration of astroglial glutamate uptake in ventral PFC regions (IL) evokes downstream changes in 5-HT function, associated to a depressive-like phenotype in mice, and 2) the regional specificity of the effect suggests a differential functional connectivity between PrL/IL and DRN 5-HT neurons. In addition, this potential new model of MDD shows several physio pathological markers observed in patients: reduced expression of GFAP, a smaller number of astrocytes in mPFC, as well as a decreased BDNF expression in mPFC and the hippocampal formation, which is highly and significantly correlated with the degree of glutamate transporter knockdown in IL, indicating a direct relationship between both processes. Overall, these findings improve our understanding of the pathophysiology of depression and could help to identify novel targets in antidepressant drug development, since this potential new MDD model mimics many of the functional and molecular alterations observed in MDD patients.Peer reviewe