Concerted regulation of skeletal muscle metabolism and contractile properties by the orphan nuclear receptor Nr2f6.

BACKGROUND: The maintenance of skeletal muscle plasticity upon changes in the environment, nutrient supply, and exercise depends on regulatory mechanisms that couple structural and metabolic adaptations. The mechanisms that interconnect both processes at the transcriptional level remain underexplored. Nr2f6, a nuclear receptor, regulates metabolism and cell differentiation in peripheral tissues. However, its role in the skeletal muscle is still elusive. Here, we aimed to investigate the effects of Nr2f6 modulation on muscle biology in vivo and in vitro. METHODS: Global RNA-seq was performed in Nr2f6 knockdown C2C12 myocytes (N = 4-5). Molecular and metabolic assays and proliferation experiments were performed using stable Nr2f6 knockdown and Nr2f6 overexpression C2C12 cell lines (N = 3-6). Nr2f6 content was evaluated in lipid overload models in vitro and in vivo (N = 3-6). In vivo experiments included Nr2f6 overexpression in mouse tibialis anterior muscle, followed by gene array transcriptomics and molecular assays (N = 4), ex vivo contractility experiments (N = 5), and histological analysis (N = 7). The conservation of Nr2f6 depletion effects was confirmed in primary skeletal muscle cells of humans and mice. RESULTS: Nr2f6 knockdown upregulated genes associated with muscle differentiation, metabolism, and contraction, while cell cycle-related genes were downregulated. In human skeletal muscle cells, Nr2f6 knockdown significantly increased the expression of myosin heavy chain genes (two-fold to three-fold) and siRNA-mediated depletion of Nr2f6 increased maximal C2C12 myocyte's lipid oxidative capacity by 75% and protected against lipid-induced cell death. Nr2f6 content decreased by 40% in lipid-overloaded myotubes and by 50% in the skeletal muscle of mice fed a high-fat diet. Nr2f6 overexpression in mice resulted in an atrophic and hypoplastic state, characterized by a significant reduction in muscle mass (15%) and myofibre content (18%), followed by an impairment (50%) in force production. These functional phenotypes were accompanied by the establishment of an inflammation-like molecular signature and a decrease in the expression of genes involved in muscle contractility and oxidative metabolism, which was associated with the repression of the uncoupling protein 3 (20%) and PGC-1α (30%) promoters activity following Nr2f6 overexpression in vitro. Additionally, Nr2f6 regulated core components of the cell division machinery, effectively decoupling muscle cell proliferation from differentiation. CONCLUSIONS: Our findings reveal a novel role for Nr2f6 as a molecular transducer that plays a crucial role in maintaining the balance between skeletal muscle contractile function and oxidative capacity. These results have significant implications for the development of potential therapeutic strategies for metabolic diseases and myopathies.

Identifying cellular markers of focal cortical dysplasia type II with cell-type deconvolution and single-cell signatures.

Focal cortical dysplasia (FCD) is a brain malformation that causes medically refractory epilepsy. FCD is classified into three categories based on structural and cellular abnormalities, with FCD type II being the most common and characterized by disrupted organization of the cortex and abnormal neuronal development. In this study, we employed cell-type deconvolution and single-cell signatures to analyze bulk RNA-seq from multiple transcriptomic studies, aiming to characterize the cellular composition of brain lesions in patients with FCD IIa and IIb subtypes. Our deconvolution analyses revealed specific cellular changes in FCD IIb, including neuronal loss and an increase in reactive astrocytes (astrogliosis) when compared to FCD IIa. Astrogliosis in FCD IIb was further supported by a gene signature analysis and histologically confirmed by glial fibrillary acidic protein (GFAP) immunostaining. Overall, our findings demonstrate that FCD II subtypes exhibit differential neuronal and glial compositions, with astrogliosis emerging as a hallmark of FCD IIb. These observations, validated in independent patient cohorts and confirmed using immunohistochemistry, offer novel insights into the involvement of glial cells in FCD type II pathophysiology and may contribute to the development of targeted therapies for this condition.

Junctional instability in neuroepithelium and network hyperexcitability in a focal cortical dysplasia human model.

Focal cortical dysplasia is a highly epileptogenic cortical malformation with few treatment options. Here, we generated human cortical organoids from patients with focal cortical dysplasia type II. Using this human model, we mimicked some focal cortical dysplasia hallmarks, such as impaired cell proliferation, the presence of dysmorphic neurons and balloon cells, and neuronal network hyperexcitability. Furthermore, we observed alterations in the adherens junctions zonula occludens-1 and partitioning defective 3, reduced polarization of the actin cytoskeleton, and fewer synaptic puncta. Focal cortical dysplasia cortical organoids showed downregulation of the small GTPase RHOA, a finding that was confirmed in brain tissue resected from these patients. Functionally, both spontaneous and optogenetically-evoked electrical activity revealed hyperexcitability and enhanced network connectivity in focal cortical dysplasia organoids. Taken together, our findings suggest a ventricular zone instability in tissue cohesion of neuroepithelial cells, leading to a maturational arrest of progenitors or newborn neurons, which may predispose to cellular and functional immaturity and compromise the formation of neural networks in focal cortical dysplasia.

Clusters of SARS-CoV-2 Lineage B.1.1.7 Infection after Vaccination with Adenovirus-Vectored and Inactivated Vaccines.

A SARS-CoV-2 B.1.1.7 variant of concern (VOC) has been associated with increased transmissibility, hospitalization, and mortality. This study aimed to explore the factors associated with B.1.1.7 VOC infection in the context of vaccination. On March 2021, we detected SARS-CoV-2 RNA in nasopharyngeal samples from 14 of 22 individuals vaccinated with a single-dose of ChAdOx1 (outbreak A, n = 26), and 22 of 42 of individuals with two doses of the CoronaVac vaccine (outbreak B, n = 52) for breakthrough infection rates for ChAdOx1 of 63.6% and 52.4% for CoronaVac. The outbreaks were caused by two independent clusters of the B.1.1.7 VOC. The serum of PCR-positive symptomatic SARS-CoV-2-infected individuals had ~1.8-3.4-fold more neutralizing capacity against B.1.1.7 compared to the serum of asymptomatic individuals. These data based on exploratory analysis suggest that the B.1.1.7 variant can infect individuals partially immunized with a single dose of an adenovirus-vectored vaccine or fully immunized with two doses of an inactivated vaccine, although the vaccines were able to reduce the risk of severe disease and death caused by this VOC, even in the elderly.

Dysregulation of NEUROG2 plays a key role in focal cortical dysplasia.

OBJECTIVE: Focal cortical dysplasias (FCDs) are an important cause of drug-resistant epilepsy. In this work, we aimed to investigate whether abnormal gene regulation, mediated by microRNA, could be involved in FCD type II. METHODS: We used total RNA from the brain tissue of 16 patients with FCD type II and 28 controls. MicroRNA expression was initially assessed by microarray. Quantitative polymerase chain reaction, in situ hybridization, luciferase reporter assays, and deep sequencing for genes in the mTOR pathway were performed to validate and further explore our initial study. RESULTS: hsa-let-7f (p = 0.039), hsa-miR-31 (p = 0.0078), and hsa-miR34a (p = 0.021) were downregulated in FCD type II, whereas a transcription factor involved in neuronal and glial fate specification, NEUROG2 (p < 0.05), was upregulated. We also found that the RND2 gene, a NEUROG2-target, is upregulated (p < 0.001). In vitro experiments showed that hsa-miR-34a downregulates NEUROG2 by binding to its 5'-untranslated region. Moreover, we observed strong nuclear expression of NEUROG2 in balloon cells and dysmorphic neurons and found that 28.5% of our patients presented brain somatic mutations in genes of the mTOR pathway. INTERPRETATION: Our findings suggest a new molecular mechanism, in which NEUROG2 has a pivotal and central role in the pathogenesis of FCD type II. In this way, we found that the downregulation of hsa-miR-34a leads to upregulation of NEUROG2, and consequently to overexpression of the RND2 gene. These findings indicate that a faulty coupling in neuronal differentiation and migration mechanisms may explain the presence of aberrant cells and complete dyslamination in FCD type II. Ann Neurol 2018;83:623-635.

MicroRNA hsa-miR-134 is a circulating biomarker for mesial temporal lobe epilepsy.

Epilepsy is misdiagnosed in up to 25% of patients, leading to serious and long-lasting consequences. Recently, circulating microRNAs have emerged as potential biomarkers in a number of clinical scenarios. The purpose of this study was to identify and to validate circulating microRNAs that could be used as biomarkers in the diagnosis of epilepsy. Quantitative real-time PCR was used to measure plasma levels of three candidate microRNAs in two phases of study: an initial discovery phase with 14 patients with mesial temporal lobe epilepsy (MTLE), 13 with focal cortical dysplasia (FCD) and 16 controls; and a validation cohort constituted of an independent cohort of 65 patients with MTLE and 83 controls. We found hsa-miR-134 downregulated in patients with MTLE (p = 0.018) but not in patients with FCD, when compared to controls. Furthermore, hsa-miR-134 expression could be used to discriminate MTLE patients with an area under the curve (AUC) of 0.75. To further assess the robustness of hsa-miR-134 as a biomarker for MTLE, we studied an independent cohort of 65 patients with MTLE, 27 of whom MTLE patients were responsive to pharmacotherapy, and 38 patients were pharmacoresistant and 83 controls. We confirmed that hsa-miR-134 was significantly downregulated in the plasma of patients with MTLE when compared with controls (p < 0.001). In addition, hsa-miR-134 identified patients with MTLE regardless of their response to pharmacotherapy or the presence of MRI signs of hippocampal sclerosis. We revealed that decreased expression of hsa-miR-134 could be a potential non-invasive biomarker to support the diagnosis of patients with MTLE.

Ciliary neurotrophic factor fused to a protein transduction domain retains full neuroprotective activity in the absence of cytokine-like side effects.

Ciliary neurotrophic factor (CNTF) is a multifunctional cytokine that can regulate the survival and differentiation of many types of developing and adult neurons. CNTF prevents the degeneration of motor neurons after axotomy and in mouse mutant progressive motor neuronopathy, which has encouraged trials of CNTF for human motor neuron disease. Given systemically, however, CNTF causes severe side effects, including cachexia and a marked immune response, which has limited its clinical application. The present work describes a novel approach for administering recombinant human CNTF (rhCNTF) while conserving neurotrophic activity and avoiding deleterious side effects. rhCNTF was fused to a protein transduction domain derived from the human immunodeficiency virus-1 TAT (transactivator) protein. The resulting fusion protein (TAT-CNTF) crosses the plasma membrane within minutes and displays a nuclear localization. TAT-CNTF was equipotent to rhCNTF in supporting the survival of cultured chicken embryo dorsal root ganglion neurons. Local or subcutaneous administration of TAT-CNTF, like rhCNTF rescued motor neurons from death in neonatal rats subjected to sciatic nerve transection. In contrast to subcutaneous rhCNTF, which caused a 20-30% decrease in body weight in neonatal rats between postnatal days 2 and 7 together with a considerable fat mobilization in brown adipose tissue, TAT-CNTF lacked such side effects. Together, these results indicate that rhCNTF fused with the protein transduction domain/TAT retains neurotrophic activity in the absence of CNTFs cytokine-like side effects and may be a promising candidate for the treatment of motor neuron and other neurodegenerative diseases.