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•We studied how Efnb2 haploinsufficiency could affect cochlear gap junction channels.•Cx30 homomeric channels associate with ephrin-B2 into caveolae membrane domains.•Cx30 channels ...mostly interact with ephrin-B2 in the peripheral regions of the GJPs.•Efnb2 haploinsufficiency results in disassembly and endocytosis of Cx30 GJPs.•Ephrin-B2 reverse signalling promotes the clathrin-dependent endocytosis of Cx30 GJPs.
Chromosome 13q deletions encompassing EFNB2, which encodes the transmembrane protein ephrin-B2, are likely to cause syndromic forms of sensorineural hearing loss of unclear origin. Thus, unravelling the pathogenic mechanisms could help to improve therapeutic strategies. In the cochlea, adjacent non-sensory epithelial cells are connected via gap junction channels, the activity of which is critical to maintain cochlear homeostasis. Here we show that ephrin-B2 promotes the assembly of connexin 30 (Cx30) gap junction plaques (GJPs) between adjacent non-sensory Deiters’ cells. An in situ proximity ligation assay revealed that ephrin-B2 preferentially interacts with Cx30 in the periphery of the GJPs, i.e. where newly synthesized connexin hemichannels accrue to the GJP. Moreover, we observed that heterozygous mice encoding an Efnb2 null allele display excessive clathrin-mediated internalization of Cx30 GJPs in early postnatal stages. Finally, an in vitro organotypic assay revealed that ectopic activation of ephrin-B2 reverse signalling promotes the internalization of Cx30 GJPs. These data argue in favor of a cell-autonomous, Eph receptor-independent role of ephrin-B2 in the assembly of Cx30 GJPs. According to recent observations, early GJP degradation could certainly play a role in the pathogenic process leading to progressive sensorineural hearing loss due to Efnb2/EFNB2 haploinsufficiency.
Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial lung disease of unknown aetiology which leads rapidly to death. As diagnosis of IPF is complex, we aimed to characterise ...microRNA (miRNA) content of exosomes from sputum of patients with IPF. Using miRNA quantitative PCR array, we found a substantial dysregulation of sputum exosomal miRNA levels between patients with IPF and healthy subjects and identified a unique signature of three miRNAs. Interestingly, we found a negative correlation between miR-142-3p and diffusing capacity of the lungs for carbon monoxide/alveolar volume. This is the first characterisation of miRNA content of sputum-derived exosomes in IPF that identified promising biomarkers for diagnosis and disease severity.
Peripartum cardiomyopathy (PPCM) is a life-threatening pregnancy-associated cardiomyopathy in previously healthy women. Although PPCM is driven in part by the 16-kDa N-terminal prolactin fragment ...(16K PRL), the underlying molecular mechanisms are poorly understood. We found that 16K PRL induced microRNA-146a (miR-146a) expression in ECs, which attenuated angiogenesis through downregulation of NRAS. 16K PRL stimulated the release of miR-146a-loaded exosomes from ECs. The exosomes were absorbed by cardiomyocytes, increasing miR-146a levels, which resulted in a subsequent decrease in metabolic activity and decreased expression of Erbb4, Notch1, and Irak1. Mice with cardiomyocyte-restricted Stat3 knockout (CKO mice) exhibited a PPCM-like phenotype and displayed increased cardiac miR-146a expression with coincident downregulation of Erbb4, Nras, Notch1, and Irak1. Blocking miR-146a with locked nucleic acids or antago-miRs attenuated PPCM in CKO mice without interrupting full-length prolactin signaling, as indicated by normal nursing activities. Finally, miR-146a was elevated in the plasma and hearts of PPCM patients, but not in patients with dilated cardiomyopathy. These results demonstrate that miR-146a is a downstream-mediator of 16K PRL that could potentially serve as a biomarker and therapeutic target for PPCM.
Hereditary hearing loss affects about 1 per 1000 children. Mutations in GJB2, which encodes the connexin 26 protein (Cx26) involved in cochlear homeostasis, are found in about 50% of patients with ...autosomal recessive non-syndromic hearing loss. Deciphering the trafficking pathway of cochlear Cx26 in situ should represent an advance in understanding the pathogenic significance of many of these mutations. Connexins trafficking and delivery to lipid raft-associated gap junction plaques usually requires successively microtubule and actin networks. Here we show that cochlear Cx26 exhibits an unusual trafficking pathway. We observed that Cx26 assembly occurs in non-lipid raft membrane domains and that junctional plaques are devoid of actin and associated zonula occludens proteins. Using cytoskeleton-disrupting drugs in organotypic culture, we found that cochlear Cx26 gap junction assembly requires microtubules but not actin filaments. Altogether, our data provide an unexpected insight into Cx26 trafficking pathway and gap junction assembly in the cochlea.
•Cx26 gap junctions are devoid of cortical actin and associated ZOs in the cochlea.•Cochlear Cx26 assembles into non-lipid raft gap junction plaques.•Cochlear Cx26 gap junction assembly requires microtubules but not the actin network.
The cerebral cortex contains layers of neurons sequentially generated by distinct lineage-related progenitors. At the onset of corticogenesis, the first-born progenitors are apical progenitors (APs), ...whose asymmetric division gives birth directly to neurons. Later, they switch to indirect neurogenesis by generating intermediate progenitors (IPs), which give rise to projection neurons of all cortical layers. While a direct lineage relationship between APs and IPs has been established, the molecular mechanism that controls their transition remains elusive. Here we show that interfering with codon translation speed triggers ER stress and the unfolded protein response (UPR), further impairing the generation of IPs and leading to microcephaly. Moreover, we demonstrate that a progressive downregulation of UPR in cortical progenitors acts as a physiological signal to amplify IPs and promotes indirect neurogenesis. Thus, our findings reveal a contribution of UPR to cell fate acquisition during mammalian brain development.
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•The UPR is a homeostat regulating the specification of cortical stem cells•Elp3 loss in cortical stem cells triggers UPR by decreasing codon translation rates•Gradual UPR suppression promotes the switch from direct to indirect neurogenesis
Laguesse, Creppe et al. demonstrate that the unfolded protein response (UPR) contributes to neurogenesis in the developing cerebral cortex. Depletion of the Elongator complex component Elp3 triggers the UPR through interference with codon translation speed. UPR activation impairs the balance between direct and indirect neurogenesis, leading to premature neuron generation.
A deficiency in pejvakin, a protein of unknown function, causes a strikingly heterogeneous form of human deafness. Pejvakin-deficient (Pjvk−/−) mice also exhibit variable auditory phenotypes. ...Correlation between their hearing thresholds and the number of pups per cage suggest a possible harmful effect of pup vocalizations. Direct sound or electrical stimulation show that the cochlear sensory hair cells and auditory pathway neurons of Pjvk−/− mice and patients are exceptionally vulnerable to sound. Subcellular analysis revealed that pejvakin is associated with peroxisomes and required for their oxidative-stress-induced proliferation. Pjvk−/− cochleas display features of marked oxidative stress and impaired antioxidant defenses, and peroxisomes in Pjvk−/− hair cells show structural abnormalities after the onset of hearing. Noise exposure rapidly upregulates Pjvk cochlear transcription in wild-type mice and triggers peroxisome proliferation in hair cells and primary auditory neurons. Our results reveal that the antioxidant activity of peroxisomes protects the auditory system against noise-induced damage.
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•Pejvakin-deficient mice and humans are hypervulnerable to sound exposure•Oxidative stress induces a pejvakin-dependent proliferation of peroxisomes•Peroxisome proliferation contributes to the physiological response to sound exposure•Pjvk gene transfer can rescue auditory dysfunction in Pjvk−/− mice
Hypervulnerability to sound exposure in pejvakin-deficient mice results from impaired adaptive proliferation of peroxisomes in response to the oxidative stress. In wild-type mice, loud sounds elicit adaptive peroxisomal proliferation.
The development of hearing in mammals requires the formation and maturation of a highly organized and specialized epithelium known as the organ of Corti. This epithelium contains two types of cells, ...the sensory cells, which are the true receptors of auditory information, and the surrounding supporting cells, which are composed of a highly developed cytoskeleton essential to the architecture of the mature organ of Corti. The supporting cells are the only mammalian cells reported to contain the unusual 15-protofilament microtubules. In this paper, we show that 15-protofilament microtubules appear between the second and fourth day after birth in the pillar cells of the organ of Corti in mice. We also show that contrary to what has been described in the nematode worm Caenorhabiditis. elegans, microtubule acetylation is not essential for the formation of 15-protofilament microtubules in mice but is required for fine-tuning of their diameter.Key words: Acetylation, cytoskeleton, microtubule, inner ear, supporting cells
Genetic loss-of-function studies on development, cancer and somatic cell reprogramming have suggested that the group of macroH2A histone variants might function through stabilizing the differentiated ...state by a yet unknown mechanism. Here, we present results demonstrating that macroH2A variants have a major function in maintaining nuclear organization and heterochromatin architecture. Specifically, we find that a substantial amount of macroH2A is associated with heterochromatic repeat sequences. We further identify macroH2A on sites of interstitial heterochromatin decorated by histone H3 trimethylated on K9 (H3K9me3). Loss of macroH2A leads to major defects in nuclear organization, including reduced nuclear circularity, disruption of nucleoli and a global loss of dense heterochromatin. Domains formed by DNA repeat sequences are disorganized, expanded and fragmented, and mildly re-expressed when depleted of macroH2A. At the molecular level, we find that macroH2A is required for the interaction of repeat sequences with the nucleostructural protein lamin B1. Taken together, our results argue that a major function of macroH2A histone variants is to link nucleosome composition to higher-order chromatin architecture.
Propranolol, a widely used non-selective beta-adrenergic receptor blocker, was recently shown to display anticancer properties. Its potential to synergize with certain drugs has been also outlined. ...However, it is necessary to take into account all the properties of propranolol to select a drug that could be efficiently combined with. Propranolol was reported to block the late phase of autophagy. Hence, we hypothesized that in condition enhancing autophagy flux, cancer cells should be especially sensitive to propranolol. 2DG, a glycolysis inhibitor, is an anti-tumor agent having limited effect in monotherapy notably due to induction of pro-survival autophagy. Here, we report that treatment of cancer cells with propranolol in combination with the glycolysis inhibitor 2DG induced a massive accumulation of autophagosome due to autophagy blockade. The propranolol +2DG treatment efficiently prevents prostate cancer cell proliferation, induces cell apoptosis, alters mitochondrial morphology, inhibits mitochondrial bioenergetics and aggravates ER stress in vitro and also suppresses tumor growth in vivo. Our study underlines for the first time the interest to take advantage of the ability of propranolol to inhibit autophagy to design new anti-cancer therapies.
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