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1.
Int J Mol Sci ; 22(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34445375

RESUMO

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.


Assuntos
Depressão/psicologia , Córtex Pré-Frontal/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Depressão/genética , Depressão/metabolismo , Modelos Animais de Doenças , Núcleo Dorsal da Rafe/metabolismo , Técnicas de Silenciamento de Genes , Ácido Glutâmico/metabolismo , Elevação dos Membros Posteriores , Masculino , Camundongos , Serotonina/metabolismo , Natação
2.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805843

RESUMO

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.


Assuntos
Glicoconjugados/genética , Oligonucleotídeos Antissenso/administração & dosagem , Doença de Parkinson/terapia , Mutação Puntual , alfa-Sinucleína/antagonistas & inibidores , alfa-Sinucleína/genética , Substituição de Aminoácidos , Animais , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Glicoconjugados/administração & dosagem , Glicoconjugados/metabolismo , Humanos , Indanos/administração & dosagem , Indanos/química , Indanos/metabolismo , Injeções Intraventriculares , Locus Cerúleo/metabolismo , Locus Cerúleo/patologia , Masculino , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Metilaminas/administração & dosagem , Metilaminas/química , Metilaminas/metabolismo , Camundongos , Camundongos Transgênicos , Norepinefrina/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Parte Compacta da Substância Negra/metabolismo , Parte Compacta da Substância Negra/patologia , Transmissão Sináptica , alfa-Sinucleína/metabolismo
3.
EBioMedicine ; 59: 102944, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32810825

RESUMO

BACKGROUND: Progressive neuronal death in monoaminergic nuclei and widespread accumulation of α-synuclein are neuropathological hallmarks of Parkinson's disease (PD). Given that α-synuclein may be an early mediator of the pathological cascade that ultimately leads to neurodegeneration, decreased α-synuclein synthesis will abate neurotoxicity if delivered to the key affected neurons. METHODS: We used a non-viral gene therapy based on a new indatraline-conjugated antisense oligonucleotide (IND-ASO) to disrupt the α-synuclein mRNA transcription selectively in monoamine neurons of a PD-like mouse model and elderly nonhuman primates. Molecular, cell biology, histological, neurochemical and behavioral assays were performed. FINDINGS: Intracerebroventricular and intranasal IND-ASO administration for four weeks in a mouse model with AAV-mediated wild-type human α-synuclein overexpression in dopamine neurons prevented the synthesis and accumulation of α-synuclein in the connected brain regions, improving dopamine neurotransmission. Likewise, the four-week IND-ASO treatment led to decreased levels of endogenous α-synuclein protein in the midbrain monoamine nuclei of nonhuman primates, which are affected early in PD. CONCLUSIONS: The inhibition of α-synuclein production in dopamine neurons and its accumulation in cortical/striatal projection areas may alleviate the early deficits of dopamine function, showing the high translational value of antisense oligonucleotides as a disease modifying therapy for PD and related synucleinopathies. FUNDING: Grants SAF2016-75797-R, RTC-2014-2812-1 and RTC-2015-3309-1, Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (ERDF), UE; Grant ID 9238, Michael J. Fox Foundation; and Centres for Networked Biomedical Research on Mental Health (CIBERSAM), and on Neurodegenerative Diseases (CIBERNED).


Assuntos
Neurônios/metabolismo , Oligonucleotídeos Antissenso/administração & dosagem , Doença de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Animais , Comportamento Animal , Modelos Animais de Doenças , Feminino , Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/genética , Haplorrinos , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Teste do Labirinto Aquático de Morris , Doença de Parkinson/diagnóstico , Doença de Parkinson/etiologia , Doença de Parkinson/terapia , Transmissão Sináptica , Resultado do Tratamento
4.
Glia ; 67(6): 1122-1137, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30635928

RESUMO

Elevation of energy metabolism and disturbance of astrocyte number/function in the ventral anterior cingulate cortex (vACC) contributes to the pathophysiology of major depressive disorder (MDD). Functional hyperactivity of vACC may result from reduced astrocytic glutamate uptake and increased neuronal excitation. Here we tested this hypothesis by knocking-down astrocytic glutamate transporter GLAST/GLT-1 expression in mouse infralimbic (IL, rodent equivalent of vACC) or prelimbic (PrL) cortices using RNAi strategies. Unilateral siRNA (small interfering RNA) microinfusion targeting GLAST or GLT-1 in mouse IL induced a moderate (20-30%) and long-lasting (7 days) decrease in their expression. Intra-IL GLAST-/GLT-1 siRNA microinfusion reduced the number of glial fibrillary acidic protein (GFAP)-positive and glutamine synthetase (GS)-positive astrocytes and evoked a depressive-like phenotype reversed by citalopram and ketamine. Intra-IL GLAST or GLT-1 knockdown markedly reduced serotonin (5-HT) release in the dorsal raphe nucleus (DR) and induced an overall reduction of brain-derived neurotrophic factor (BDNF) expression in ipsilateral and contralateral hemispheres. Egr-1 (early growth response protein-1) labeling suggests that both siRNAs enhance the GABAergic tone onto DR 5-HT neurons, leading to an overall decrease of 5-HT function, likely related to the widespread reduction on BDNF expression. Conversely, similar reductions of GLAST and GLT-1 expression in PrL did not induce a depressive-like phenotype. These results suggest that a focal glial change in IL translates into global change of brain activity by virtue of the descending projections from IL to DR and the subsequent attenuation of serotonergic function in forebrain, an effect perhaps related to the varied symptomatology of MDD.


Assuntos
Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Transtorno Depressivo Maior/metabolismo , Transportador 1 de Aminoácido Excitatório/deficiência , Transportador 2 de Aminoácido Excitatório/deficiência , Fenótipo , Animais , Transtorno Depressivo Maior/genética , Transportador 1 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/genética , Técnicas de Silenciamento de Genes/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
5.
Mol Neurobiol ; 56(4): 3038-3052, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30088175

RESUMO

Current pharmacological treatments for major depressive disorder (MDD) are severely compromised by both slow action and limited efficacy. RNAi strategies have been used to evoke antidepressant-like effects faster than classical drugs. Using small interfering RNA (siRNA), we herein show that TASK3 potassium channel knockdown in monoamine neurons induces antidepressant-like responses in mice. TASK3-siRNAs were conjugated to cell-specific ligands, sertraline (Ser) or reboxetine (Reb), to promote their selective accumulation in serotonin (5-HT) and norepinephrine (NE) neurons, respectively, after intranasal delivery. Following neuronal internalization of conjugated TASK3-siRNAs, reduced TASK3 mRNA and protein levels were found in the brainstem 5-HT and NE cell groups. Moreover, Ser-TASK3-siRNA induced robust antidepressant-like behaviors, enhanced the hippocampal plasticity, and potentiated the fluoxetine-induced increase on extracellular 5-HT. Similar responses, yet of lower magnitude, were detected for Reb-TASK3-siRNA. These findings provide substantial support for TASK3 as a potential target, and RNAi-based strategies as a novel therapeutic approach to treat MDD.


Assuntos
Depressão/tratamento farmacológico , Técnicas de Silenciamento de Genes , Neurônios/metabolismo , Canais de Potássio/metabolismo , Reboxetina/administração & dosagem , Sertralina/administração & dosagem , Administração Intranasal , Animais , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico , Comportamento Animal , Regulação para Baixo/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Norepinefrina/metabolismo , RNA Interferente Pequeno/metabolismo , Reboxetina/farmacologia , Neurônios Serotoninérgicos/efeitos dos fármacos , Neurônios Serotoninérgicos/metabolismo , Serotonina/metabolismo , Sertralina/farmacologia
6.
Mol Ther ; 26(6): 1552-1567, 2018 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-29628303

RESUMO

The possible implication of transcription factor EB (TFEB) as a therapeutic target in Parkinson's disease has gained momentum since it was discovered that TFEB controls lysosomal biogenesis and autophagy and that its activation might counteract lysosomal impairment and protein aggregation. However, the majority of putative direct targets of TFEB described to date is linked to a range of biological processes that are not related to the lysosomal-autophagic system. Here, we assessed the effect of overexpressing TFEB with an adeno-associated viral vector in mouse substantia nigra dopaminergic neurons. We demonstrate that TFEB overexpression drives a previously unknown bona fide neurotrophic effect, giving rise to cell growth, higher tyrosine hydroxylase levels, and increased dopamine release in the striatum. TFEB overexpression induces the activation of the mitogen-activated protein kinase 1/3 (MAPK1/3) and AKT pro-survival pathways, phosphorylation of mTORC1 effectors 4E-binding protein 1 (4E-BP1) and S6 kinase B1 (S6K1), and increased protein synthesis. We show that TFEB overexpression prevents dopaminergic cell loss and counteracts atrophy and the associated protein synthesis decline in the MPTP mouse model of Parkinson's disease. Our results suggest that increasing TFEB activity might prevent neuronal death and restore neuronal function in Parkinson's disease and other neurodegenerative diseases through different mechanisms.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/terapia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Modelos Animais de Doenças , Dopamina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doença de Parkinson/genética , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo
7.
Mol Ther ; 26(2): 550-567, 2018 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-29273501

RESUMO

Progressive neuronal death in brainstem nuclei and widespread accumulation of α-synuclein are neuropathological hallmarks of Parkinson's disease (PD). Reduction of α-synuclein levels is therefore a potential therapy for PD. However, because α-synuclein is essential for neuronal development and function, α-synuclein elimination would dramatically impact brain function. We previously developed conjugated small interfering RNA (siRNA) sequences that selectively target serotonin (5-HT) or norepinephrine (NE) neurons after intranasal administration. Here, we used this strategy to conjugate inhibitory oligonucleotides, siRNA and antisense oligonucleotide (ASO), with the triple monoamine reuptake inhibitor indatraline (IND), to selectively reduce α-synuclein expression in the brainstem monoamine nuclei of mice after intranasal delivery. Following internalization of the conjugated oligonucleotides in monoamine neurons, reduced levels of endogenous α-synuclein mRNA and protein were found in substantia nigra pars compacta (SNc), ventral tegmental area (VTA), dorsal raphe nucleus (DR), and locus coeruleus (LC). α-Synuclein knockdown by ∼20%-40% did not cause monoaminergic neurodegeneration and enhanced forebrain dopamine (DA) and 5-HT release. Conversely, a modest human α-synuclein overexpression in DA neurons markedly reduced striatal DA release. These results indicate that α-synuclein negatively regulates monoamine neurotransmission and set the stage for the testing of non-viral inhibitory oligonucleotides as disease-modifying agents in α-synuclein models of PD.


Assuntos
Neurônios Dopaminérgicos/metabolismo , Oligonucleotídeos/genética , alfa-Sinucleína/genética , Administração Intranasal , Animais , Células Cultivadas , Corpo Estriado/metabolismo , Dopamina/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Camundongos , Vias Neurais , Oligonucleotídeos/administração & dosagem , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/terapia , Prosencéfalo/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Serotonina/metabolismo , Transdução de Sinais , Substância Negra/metabolismo , Substância Negra/fisiopatologia , Transmissão Sináptica/genética
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