Regionally selective knockdown of astroglial glutamate transporters in infralimbic cortex induces a depressive phenotype in mice.

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.

Selective α-Synuclein Knockdown in Monoamine Neurons by Intranasal Oligonucleotide Delivery: Potential Therapy for Parkinson's Disease.

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.

Anti-α-synuclein ASO delivered to monoamine neurons prevents α-synuclein accumulation in a Parkinson's disease-like mouse model and in monkeys.

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).

Selective Knockdown of TASK3 Potassium Channel in Monoamine Neurons: a New Therapeutic Approach for Depression.

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.

Inversions and adaptation to the plant toxin ouabain shape DNA sequence variation within and between chromosomal inversions of Drosophila subobscura.

Adaptation is defined as an evolutionary process allowing organisms to succeed in certain habitats or conditions. Chromosomal inversions have the potential to be key in the adaptation processes, since they can contribute to the maintenance of favoured combinations of adaptive alleles through reduced recombination between individuals carrying different inversions. We have analysed six genes (Pif1A, Abi, Sqd, Yrt, Atpα and Fmr1), located inside and outside three inversions of the O chromosome in European populations of Drosophila subobscura. Genetic differentiation was significant between inversions despite extensive recombination inside inverted regions, irrespective of gene distance to the inversion breakpoints. Surprisingly, the highest level of genetic differentiation between arrangements was found for the Atpα gene, which is located outside the O1 and O7 inversions. Two derived unrelated arrangements (O3+4+1 and O3+4+7) are nearly fixed for several amino acid substitutions at the Atpα gene that have been described to confer resistance in other species to the cardenolide ouabain, a plant toxin capable of blocking ATPases. Similarities in the Atpα variants, conferring ouabain resistance in both arrangements, may be the result of convergent substitution and be favoured in response to selective pressures presumably related to the presence of plants containing ouabain in the geographic locations where both inversions are present.

Reduced α-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.

Acute 5-HT1A autoreceptor knockdown increases antidepressant responses and serotonin release in stressful conditions.

RATIONALE: Identifying the etiological factors in anxiety and depression is critical to develop more efficacious therapies. The inhibitory serotonin(1A) receptors (5-HT(1A)R) located on 5-HT neurons (autoreceptors) limit antidepressant responses and their expression may be increased in treatment-resistant depressed patients. OBJECTIVES: Recently, we reported that intranasal administration of modified small interference RNA (siRNA) molecules targeting 5-HT(1A)R in serotonergic neurons evoked antidepressant-like effects. Here we extended this finding using marketed siRNAs against 5-HT(1A)R (1A-siRNA) to reduce directly the 5-HT(1A) autoreceptor expression and evaluate its biological consequences under basal conditions and in response to stressful situations. METHODS: Adult mice were locally infused with vehicle, nonsense siRNA, and 1A-siRNA into dorsal raphe nucleus (DR). 5-HT(1A)R knockout mice (1A-KO) were also used. Histological approaches, in vivo microdialysis, and stress-related behaviors were performed to assess the effects of 5-HT(1A) autoreceptor knockdown. RESULTS: Intra-DR 1A-siRNA infusion selectively reduced 5-HT(1A)R mRNA and binding levels and canceled 8-OH-DPAT-induced hypothermia. Basal extracellular 5-HT in medial prefrontal cortex (mPFC) did not differ among treatments. However, 1A-siRNA-treated mice displayed less immobility in the tail suspension and forced swim tests, as did 1A-KO mice. This was accompanied by a greater increase in prefrontal 5-HT release during tail suspension test. Moreover, intra-DR 1A-siRNA infusion augmented the increase of extracellular 5-HT in mPFC evoked by fluoxetine, up to the level in 1A-KO mice. CONCLUSION: Together with our previous report, the present results indicate that acute suppression of 5-HT(1A) autoreceptor expression evokes robust antidepressant-like effects, likely mediated by an increased capacity of serotonergic neurons to release 5-HT in stressful conditions.