Viral vector-mediated Cre recombinase expression in substantia nigra induces lesions of the nigrostriatal pathway associated with perturbations of dopamine-related behaviors and hallmarks of programmed cell death

This work is dedicated to the memory of Dr. Marie-Pascale Martres.International audienceCre/loxP recombination is a widely used approach to study gene function in vivo, using mice models expressing the Cre recombinase under the control of specific promoters or through viral delivery of Cre-expressing constructs. A profuse literature on transgenic mouse lines points out the deleterious effects of Cre expression in various cell types and tissues, presumably by acting on illegitimate loxP-like sites present in the genome. However, most studies reporting the consequences of Cre-lox gene invalidation often omit adequate controls to exclude the potential toxic effects of Cre, compromising the interpretation of data. In this study, we report the anatomical, neurochemical and behavioral consequences in mice of adeno-associated virus (AAV)-mediated Cre expression in the dopaminergic nuclei substantia nigra, at commonly used viral titers (3×109 genome copies/0.3 μL or 2×109 genome copies/0.6 μL). We found that injecting AAV-eGFP-Cre into the SN engendered drastic and reproducible modifications of behavior, with increased basal locomotor activity as well as impaired locomotor response to cocaine compared to AAV-eGFP-injected controls. Cre expression in the SN induced a massive decrease in neuronal populations of both pars compacta and pars reticulata, and dopamine depletion in the nigrostriatal pathway. This anatomical injury was associated with typical features of programmed cell death, including an increase in DNA break markers, evidence of apoptosis and disrupted macroautophagy. These observations underscore the need for careful control of Cre toxicity in the brain and the reassessment of previous studies. In addition, our findings suggest that Cre-mediated ablation may constitute an efficient tool to explore the function of specific cell populations and areas in the brain, and the impact of neurodegeneration in these populations. This article is protected by copyright. All rights reserved

Organic cation transporter 2 contributes to SSRI antidepressant efficacy by controlling tryptophan availability in the brain

Abstract Selective serotonin reuptake inhibitors (SSRI) are common first-line treatments for major depression. However, a significant number of depressed patients do not respond adequately to these pharmacological treatments. In the present preclinical study, we demonstrate that organic cation transporter 2 (OCT2), an atypical monoamine transporter, contributes to the effects of SSRI by regulating the routing of the essential amino acid tryptophan to the brain. Contrarily to wild-type mice, OCT2-invalidated mice failed to respond to prolonged fluoxetine treatment in a chronic depression model induced by corticosterone exposure recapitulating core symptoms of depression, i.e., anhedonia, social withdrawal, anxiety, and memory impairment. After corticosterone and fluoxetine treatment, the levels of tryptophan and its metabolites serotonin and kynurenine were decreased in the brain of OCT2 mutant mice compared to wild-type mice and reciprocally tryptophan and kynurenine levels were increased in mutants’ plasma. OCT2 was detected by immunofluorescence in several structures at the blood-cerebrospinal fluid (CSF) or brain-CSF interface. Tryptophan supplementation during fluoxetine treatment increased brain concentrations of tryptophan and, more discreetly, of 5-HT in wild-type and OCT2 mutant mice. Importantly, tryptophan supplementation improved the sensitivity to fluoxetine treatment of OCT2 mutant mice, impacting chiefly anhedonia and short-term memory. Western blot analysis showed that glycogen synthase kinase-3β (GSK3β) and mammalian/mechanistic target of rapamycin (mTOR) intracellular signaling was impaired in OCT2 mutant mice brain after corticosterone and fluoxetine treatment and, conversely, tryptophan supplementation recruited selectively the mTOR protein complex 2. This study provides the first evidence of the physiological relevance of OCT2-mediated tryptophan transport, and its biological consequences on serotonin homeostasis in the brain and SSRI efficacy

miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context

International audienceBackground: Duchenne (DMD) and Becker (BMD) muscular dystrophies are caused by mutations in the DMD gene coding for dystrophin, a protein being part of a large sarcolemmal protein scaffold that includes the neuronal nitric oxide synthase (nNOS). The nNOS was shown to play critical roles in a variety of muscle functions and alterations of its expression and location in dystrophic muscle fiber leads to an increase of the muscle fatigability. We previously revealed a decrease of nNOS expression in BMD patients all presenting a deletion of exons 45 to 55 in the DMD gene (BMDd45-55), impacting the nNOS binding site of dystrophin. Since several studies showed deregulation of microRNAs (miRNAs) in dystrophinopathies, we focused on miRNAs that could target nNOS in dystrophic context.Methods: By a screening of 617 miRNAs in BMDd45-55 muscular biopsies using TLDA and an in silico study to determine which one could target nNOS, we selected four miRNAs. In order to select those that targeted a sequence of 3′UTR of NOS1, we performed luciferase gene reporter assay in HEK393T cells. Finally, expression of candidate miRNAs was modulated in control and DMD human myoblasts (DMDd45-52) to study their ability to target nNOS.Results: TLDA assay and the in silico study allowed us to select four miRNAs overexpressed in muscle biopsies of BMDd45-55 compared to controls. Among them, only the overexpression of miR-31, miR-708, and miR-34c led to a decrease of luciferase activity in an NOS1-3′UTR-luciferase assay, confirming their interaction with the NOS1-3′UTR. The effect of these three miRNAs was investigated on control and DMDd45-52 myoblasts. First, we showed a decrease of nNOS expression when miR-708 or miR-34c were overexpressed in control myoblasts. We then confirmed that DMDd45-52 cells displayed an endogenous increased of miR-31, miR-708, and miR-34c and a decreased of nNOS expression, the same characteristics observed in BMDd45-55 biopsies. In DMDd45-52 cells, we demonstrated that the inhibition of miR-708 and miR-34c increased nNOS expression, confirming that both miRNAs can modulate nNOS expression in human myoblasts.Conclusion: These results strongly suggest that miR-708 and miR-34c, overexpressed in dystrophic context, are new actors involved in the regulation of nNOS expression in dystrophic muscle

miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context

Abstract Background Duchenne (DMD) and Becker (BMD) muscular dystrophies are caused by mutations in the DMD gene coding for dystrophin, a protein being part of a large sarcolemmal protein scaffold that includes the neuronal nitric oxide synthase (nNOS). The nNOS was shown to play critical roles in a variety of muscle functions and alterations of its expression and location in dystrophic muscle fiber leads to an increase of the muscle fatigability. We previously revealed a decrease of nNOS expression in BMD patients all presenting a deletion of exons 45 to 55 in the DMD gene (BMDd45-55), impacting the nNOS binding site of dystrophin. Since several studies showed deregulation of microRNAs (miRNAs) in dystrophinopathies, we focused on miRNAs that could target nNOS in dystrophic context. Methods By a screening of 617 miRNAs in BMDd45-55 muscular biopsies using TLDA and an in silico study to determine which one could target nNOS, we selected four miRNAs. In order to select those that targeted a sequence of 3′UTR of NOS1, we performed luciferase gene reporter assay in HEK393T cells. Finally, expression of candidate miRNAs was modulated in control and DMD human myoblasts (DMDd45-52) to study their ability to target nNOS. Results TLDA assay and the in silico study allowed us to select four miRNAs overexpressed in muscle biopsies of BMDd45-55 compared to controls. Among them, only the overexpression of miR-31, miR-708, and miR-34c led to a decrease of luciferase activity in an NOS1-3′UTR-luciferase assay, confirming their interaction with the NOS1-3′UTR. The effect of these three miRNAs was investigated on control and DMDd45-52 myoblasts. First, we showed a decrease of nNOS expression when miR-708 or miR-34c were overexpressed in control myoblasts. We then confirmed that DMDd45-52 cells displayed an endogenous increased of miR-31, miR-708, and miR-34c and a decreased of nNOS expression, the same characteristics observed in BMDd45-55 biopsies. In DMDd45-52 cells, we demonstrated that the inhibition of miR-708 and miR-34c increased nNOS expression, confirming that both miRNAs can modulate nNOS expression in human myoblasts. Conclusion These results strongly suggest that miR-708 and miR-34c, overexpressed in dystrophic context, are new actors involved in the regulation of nNOS expression in dystrophic muscle

Antidepressant efficacy of a selective organic cation transporter blocker in a mouse model of depression

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Additional file 3: of miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context

Table S1. Predictive candidate miRNA binding sites on the human NOS1 3’UTR (DOCX 12 kb

Additional file 1: of miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context

Supplementary methods. (DOCX 12 kb

Additional file 4: of miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context

Figure S1. Nuclear localization of nNOS in DMD muscular biopsy and in myoblasts. a) Control (ctrl) and DMD human muscular biopsy sections immunolabeled with anti-nNOS (red) antibody, nuclei with Dapi (blue), and imaged by confocal microscopy. Representative of 4 DMD patients. b) nNOS, GAPDH, and histone H3 (H3) immunoblots on cytoplasmic (CE) and nuclear (NE) protein extracts from control (ctrl) and DMDd45-52 myoblasts and total extract of control human muscular biopsy (ctrl biopsy). (TIFF 912 kb

Rescue of a Dystrophin-like Protein by Exon Skipping In Vivo Restores GABAA-receptor Clustering in the Hippocampus of the mdx Mouse

Dystrophin, the cytoskeletal protein whose defect is responsible for Duchenne muscular dystrophy (DMD), is normally expressed in both muscles and brain. Genetic loss of brain dystrophin in the mdx mouse model of DMD reduces the capacity for type A γ-aminobutyric acid (GABAA)-receptor clustering in central inhibitory synapses, which is thought to be a main molecular defect leading to brain and cognitive alterations in this syndrome. U7 small nuclear RNAs modified to encode antisense sequences and expressed from recombinant adeno-associated viral (rAAV) vectors have proven efficient after intramuscular injection to induce skipping of the mutated exon 23 and rescue expression of a functional dystrophin-like product in muscle tissues of mdx mice in vivo. Here, we report that intrahippocampal injection of a single dose of rAAV2/1-U7 can rescue substantial levels of brain dystrophin expression (15–25%) in mdx mice for months. This is sufficient to completely restore GABAA-receptor clustering in pyramidal and dendritic layers of CA1 hippocampus, suggesting exon-skipping strategies offer the prospect to investigate and correct both brain and muscle alterations in DMD. This provides new evidence that in the adult brain dystrophin is critical for the control of GABAA-receptor clustering, which may have an important role in activity-dependent synaptic plasticity in hippocampal circuits