RESUMO
The primary cilium, hereafter cilium, is an antenna-like organelle that modulates intracellular responses, including autophagy, a lysosomal degradation process essential for cell homeostasis. Dysfunction of the cilium is associated with impairment of autophagy and diseases known as "ciliopathies". The discovery of autophagy-related proteins at the base of the cilium suggests its potential role in coordinating autophagy initiation in response to physiopathological stimuli. One of these proteins, beclin-1 (BECN1), it which is necessary for autophagosome biogenesis. Additionally, polycystin-2 (PKD2), a calcium channel enriched at the cilium, is required and sufficient to induce autophagy in renal and cancer cells. We previously demonstrated that PKD2 and BECN1 form a protein complex at the endoplasmic reticulum in non-ciliated cells, where it initiates autophagy, but whether this protein complex is present at the cilium remains unknown. Anorexigenic pro-opiomelanocortin (POMC) neurons are ciliated cells that require autophagy to maintain intracellular homeostasis. POMC neurons are sensitive to metabolic changes, modulating signaling pathways crucial for controlling food intake. Exposure to the saturated fatty acid palmitic acid (PA) reduces ciliogenesis and inhibits autophagy in these cells. Here, we show that PKD2 and BECN1 form a protein complex in N43/5 cells, an in vitro model of POMC neurons, and that both PKD2 and BECN1 locate at the cilium. In addition, our data show that the cilium is required for PKD2-BECN1 protein complex formation and that PA disrupts the PKD2-BECN1 complex, suppressing autophagy. Our findings provide new insights into the mechanisms by which the cilium controls autophagy in hypothalamic neuronal cells.
Assuntos
Autofagia , Proteína Beclina-1 , Cílios , Hipotálamo , Neurônios , Canais de Cátion TRPP , Animais , Camundongos , Proteína Beclina-1/metabolismo , Cílios/metabolismo , Hipotálamo/metabolismo , Hipotálamo/citologia , Neurônios/metabolismo , Canais de Cátion TRPP/metabolismo , Canais de Cátion TRPP/genéticaRESUMO
OBJECTIVE: Mucociliary transport function in the airway mucosa is essential for maintaining a clean mucosal surface. This function is impaired in upper and lower airway diseases. Nasal polyps are a noticeable pathological feature that develop in some of the patients with chronic rhinosinusitis. Like ordinary nasal mucosae, nasal polyps have a ciliated pseudostratified epithelium with vigorous ciliary beating. We measured ex vivo Mucociliary Transport Velocity (MCTV) and Ciliary Beat Frequency (CBF) and explored the expressions of Planar Cell Polarity (PCP) proteins in nasal polyps in comparison with turbinate mucosae. METHODS: Inferior turbinates and nasal polyps were surgically collected from patients with chronic rhinosinusitis. Ex vivo MCTV and CBF were measured using a high-speed digital imaging system. Expressions of PCP proteins were explored by fluorescence immunohistochemistry and quantitative RT-PCR. RESULTS: The MCTV of nasal polyps was significantly lower than that of the turbinates (7.43⯱â¯2.01 vs. 14.56⯱â¯2.09⯵m/s; pâ¯=â¯0.0361), whereas CBF did not differ between the two tissues. The MCTV vector was pointed to the posteroinferior direction in all turbinates with an average inclination angle of 41.0 degrees. Immunohistochemical expressions of Dishevelled-1, Dishevelled-3, Frizzled3, Frizzled6, Prickle2 and Vangl2 were lower in the nasal polyps than in the turbinates. Confocal laser scanning microscopy showed that Frizzled3 was localized along the cell junction on the apical surface. The expression levels of mRNAs for Dishevelled-1, Dishevelled-3 and Frizzled3 in the nasal polyps were also decreased in comparison with the turbinates. CONCLUSION: These results indicate that muco ciliary transport in nasal polyps is impaired although vigorous ciliary beating is maintained, and that the impairment may be caused by a decrease in Dishevelled/Frizzled proteins and resultant PCP disarrangement. LEVEL OF EVIDENCE: Level 3.
Assuntos
Pólipos Nasais , Sinusite , Humanos , Pólipos Nasais/metabolismo , Depuração Mucociliar , Cílios/metabolismo , Cílios/patologia , Mucosa Nasal/metabolismo , Sinusite/metabolismoRESUMO
BACKGROUND: Supratentorial RELA fusion (ST-RELA) ependymomas (EPNs) are resistant tumors without an approved chemotherapeutic treatment. Unfortunately, the molecular mechanisms that lead to chemoresistance traits of ST-RELA remain elusive. The aim of this study was to assess RELA fusion-dependent signaling modules, specifically the role of the Hedgehog (Hh) pathway as a novel targetable vulnerability in ST-RELA. METHODS: Gene expression was analyzed in EPN from patient cohorts, by microarray, RNA-seq, qRT-PCR, and scRNA-seq. Inhibitors against Smoothened (SMO) (Sonidegib) and Aurora kinase A (AURKA) (Alisertib) were evaluated. Protein expression, primary cilia formation, and drug effects were assessed by immunoblot, immunofluorescence, and immunohistochemistry. RESULTS: Hh components were selectively overexpressed in EPNs induced by the RELA fusion. Single-cell analysis showed that the Hh signature was primarily confined to undifferentiated, stem-like cell subpopulations. Sonidegib exhibited potent growth-inhibitory effects on ST-RELA cells, suggesting a key role in active Hh signaling; importantly, the effect of Sonidegib was reversed by primary cilia loss. We, thus, tested the effect of AURKA inhibition by Alisertib, to induce cilia stabilization/reassembly. Strikingly, Alisertib rescued ciliogenesis and synergized with Sonidegib in killing ST-RELA cells. Using a xenograft model, we show that cilia loss is a mechanism for acquiring resistance to the inhibitory effect of Sonidegib. However, Alisertib fails to rescue cilia and highlights the need for other strategies to promote cilia reassembly, for treating ST-RELA tumors. CONCLUSION: Our study reveals a crucial role for the Hh pathway in ST-RELA tumor growth, and suggests that rescue of primary cilia represents a vulnerability of the ST-RELA EPNs.
Assuntos
Ependimoma , Neoplasias Supratentoriais , Humanos , Proteínas Hedgehog , Cílios/metabolismo , Cílios/patologia , Aurora Quinase A/genética , Ependimoma/patologia , Neoplasias Supratentoriais/patologia , Fator de Transcrição RelARESUMO
Palmitic acid (PA) is significantly increased in the hypothalamus of mice, when fed chronically with a high-fat diet (HFD). PA impairs insulin signaling in hypothalamic neurons, by a mechanism dependent on autophagy, a process of lysosomal-mediated degradation of cytoplasmic material. In addition, previous work shows a crosstalk between autophagy and the primary cilium (hereafter cilium), an antenna-like structure on the cell surface that acts as a signaling platform for the cell. Ciliopathies, human diseases characterized by cilia dysfunction, manifest, type 2 diabetes, among other features, suggesting a role of the cilium in insulin signaling. Cilium depletion in hypothalamic pro-opiomelanocortin (POMC) neurons triggers obesity and insulin resistance in mice, the same phenotype as mice deficient in autophagy in POMC neurons. Here we investigated the effect of chronic consumption of HFD on cilia; and our results indicate that chronic feeding with HFD reduces the percentage of cilia in hypothalamic POMC neurons. This effect may be due to an increased amount of PA, as treatment with this saturated fatty acid in vitro reduces the percentage of ciliated cells and cilia length in hypothalamic neurons. Importantly, the same effect of cilia depletion was obtained following chemical and genetic inhibition of autophagy, indicating autophagy is required for ciliogenesis. We further demonstrate a role for the cilium in insulin sensitivity, as cilium loss in hypothalamic neuronal cells disrupts insulin signaling and insulin-dependent glucose uptake, an effect that correlates with the ciliary localization of the insulin receptor (IR). Consistently, increased percentage of ciliated hypothalamic neuronal cells promotes insulin signaling, even when cells are exposed to PA. Altogether, our results indicate that, in hypothalamic neurons, impairment of autophagy, either by PA exposure, chemical or genetic manipulation, cause cilia loss that impairs insulin sensitivity.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Animais , Autofagia , Cílios/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Hipotálamo/metabolismo , Insulina/metabolismo , Resistência à Insulina/genética , Camundongos , Neurônios/metabolismo , Ácido Palmítico/metabolismo , Ácido Palmítico/farmacologia , Pró-Opiomelanocortina/metabolismo , Pró-Opiomelanocortina/farmacologiaRESUMO
CCDC28B (coiled-coil domain-containing protein 28B) was identified as a modifier in the ciliopathy Bardet-Biedl syndrome (BBS). Our previous work in cells and zebrafish showed that CCDC28B plays a role regulating cilia length in a mechanism that is not completely understood. Here we report the generation of a Ccdc28b mutant mouse using CRISPR/Cas9 (Ccdc28b mut). Depletion of CCDC28B resulted in a mild phenotype. Ccdc28b mut animals i) do not present clear structural cilia affectation, although we did observe mild defects in cilia density and cilia length in some tissues, ii) reproduce normally, and iii) do not develop retinal degeneration or obesity, two hallmark features of reported BBS murine models. In contrast, Ccdc28b mut mice did show clear social interaction defects as well as stereotypical behaviors. This finding is indeed relevant regarding CCDC28B as a modifier of BBS since behavioral phenotypes have been documented in BBS. Overall, this work reports a novel mouse model that will be key to continue evaluating genetic interactions in BBS, deciphering the contribution of CCDC28B to modulate the presentation of BBS phenotypes. In addition, our data underscores a novel link between CCDC28B and behavioral defects, providing a novel opportunity to further our understanding of the genetic, cellular, and molecular basis of these complex phenotypes.
Assuntos
Síndrome de Bardet-Biedl , Degeneração Retiniana , Animais , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/metabolismo , Cílios/metabolismo , Camundongos , Fenótipo , Degeneração Retiniana/genética , Peixe-Zebra/genéticaRESUMO
Airway mucociliary clearance (MCC) is the main mechanism of lung defense keeping airways free of infection and mucus obstruction. Airway surface liquid volume, ciliary beating, and mucus are central for proper MCC and critically regulated by sodium absorption and anion secretion. Impaired MCC is a key feature of muco-obstructive diseases. The calcium-activated potassium channel KCa.3.1, encoded by Kcnn4, participates in ion secretion, and studies showed that its activation increases Na+ absorption in airway epithelia, suggesting that KCa3.1-induced hyperpolarization was sufficient to drive Na+ absorption. However, its role in airway epithelium is not fully understood. We aimed to elucidate the role of KCa3.1 in MCC using a genetically engineered mouse. KCa3.1 inhibition reduced Na+ absorption in mouse and human airway epithelium. Furthermore, the genetic deletion of Kcnn4 enhanced cilia beating frequency and MCC ex vivo and in vivo. Kcnn4 silencing in the Scnn1b-transgenic mouse (Scnn1btg/+), a model of muco-obstructive lung disease triggered by increased epithelial Na+ absorption, improved MCC, reduced Na+ absorption, and did not change the amount of mucus but did reduce mucus adhesion, neutrophil infiltration, and emphysema. Our data support that KCa3.1 inhibition attenuated muco-obstructive disease in the Scnn1btg/+ mice. K+ channel modulation may be a therapeutic strategy to treat muco-obstructive lung diseases.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Pneumopatias Obstrutivas/etiologia , Depuração Mucociliar/fisiologia , Animais , Cálcio/metabolismo , Células Cultivadas , Cílios/efeitos dos fármacos , Cílios/metabolismo , Modelos Animais de Doenças , Epitélio/metabolismo , Feminino , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Pulmão/fisiopatologia , Pneumopatias Obstrutivas/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Depuração Mucociliar/efeitos dos fármacos , Sódio/metabolismoRESUMO
Mycoplasma hyopneumoniae is the most costly pathogen for swine production. Although several studies have focused on the host-bacterium association, little is known about the changes in gene expression of swine cells upon infection. To improve our understanding of this interaction, we infected swine epithelial NPTr cells with M. hyopneumoniae strain J to identify differentially expressed mRNAs and miRNAs. The levels of 1,268 genes and 170 miRNAs were significantly modified post-infection. Up-regulated mRNAs were enriched in genes related to redox homeostasis and antioxidant defense, known to be regulated by the transcription factor NRF2 in related species. Down-regulated mRNAs were enriched in genes associated with cytoskeleton and ciliary functions. Bioinformatic analyses suggested a correlation between changes in miRNA and mRNA levels, since we detected down-regulation of miRNAs predicted to target antioxidant genes and up-regulation of miRNAs targeting ciliary and cytoskeleton genes. Interestingly, most down-regulated miRNAs were detected in exosome-like vesicles suggesting that M. hyopneumoniae infection induced a modification of the composition of NPTr-released vesicles. Taken together, our data indicate that M. hyopneumoniae elicits an antioxidant response induced by NRF2 in infected cells. In addition, we propose that ciliostasis caused by this pathogen is partially explained by the down-regulation of ciliary genes.
Assuntos
Antioxidantes/metabolismo , Proteínas de Bactérias/metabolismo , Cílios/genética , Células Epiteliais/metabolismo , Mycoplasma hyopneumoniae/genética , Mycoplasma hyopneumoniae/metabolismo , Pneumonia Suína Micoplasmática/microbiologia , Animais , Proteínas de Bactérias/genética , Biomarcadores/análise , Células Cultivadas , Cílios/metabolismo , Células Epiteliais/microbiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , MicroRNAs/análise , Mycoplasma hyopneumoniae/crescimento & desenvolvimento , Pneumonia Suína Micoplasmática/genética , Pneumonia Suína Micoplasmática/metabolismo , RNA Mensageiro/análise , SuínosRESUMO
Melanin-concentrating hormone (MCH) is a ubiquitous vertebrate neuropeptide predominantly synthesized by neurons of the diencephalon that can act through two G protein-coupled receptors, called MCHR1 and MCHR2. The expression of Mchr1 has been investigated in both rats and mice, but its synthesis remains poorly described. After identifying an antibody that detects MCHR1 with high specificity, we employed immunohistochemistry to map the distribution of MCHR1 in the CNS of rats and mice. Multiple neurochemical markers were also employed to characterize some of the neuronal populations that synthesize MCHR1. Our results show that MCHR1 is abundantly found in a subcellular structure called the primary cilium, which has been associated, among other functions, with the detection of free neurochemical messengers present in the extracellular space. Ciliary MCHR1 was found in a wide range of areas, including the olfactory bulb, cortical mantle, striatum, hippocampal formation, amygdala, midline thalamic nuclei, periventricular hypothalamic nuclei, midbrain areas, and in the spinal cord. No differences were observed between male and female mice, and interspecies differences were found in the caudate-putamen nucleus and the subgranular zone. Ciliary MCHR1 was found in close association with several neurochemical markers, including tyrosine hydroxylase, calretinin, kisspeptin, estrogen receptor, oxytocin, vasopressin, and corticotropin-releasing factor. Given the role of neuronal primary cilia in sensing free neurochemical messengers in the extracellular fluid, the widespread distribution of ciliary MCHR1, and the diverse neurochemical populations who synthesize MCHR1, our data indicate that nonsynaptic communication plays a prominent role in the normal function of the MCH system.
Assuntos
Encéfalo/metabolismo , Cílios/metabolismo , Receptores de Somatostatina/biossíntese , Caracteres Sexuais , Animais , Cílios/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Receptores de Somatostatina/genéticaRESUMO
The melanin-concentrating hormone (MCH) is a peptidergic neuromodulator synthesized by neurons of the posterior hypothalamus and incerto-hypothalamic area. These neurons project throughout the central nervous system, including the dorsal raphe nucleus (DRN). In rodents, MCH exerts its biological functions through the MCHR-1 receptor. We previously demonstrated that intra-DRN MCH administration increases REM sleep time and induces a pro-depressive behavior. We also determined that MCH modulates the neuronal firing rate and serotonin release within this nucleus. Previous studies in mice identified the presence of MCHR-1 in neurons located in the olfactory tubercle, hypothalamus, and nucleus accumbens, in a specialized neuronal appendage: the neuronal primary cilia. However, the subcellular location of MCHR-1 protein in the DRN is still unknown. Hence, the aim of the present study was to explore, by means of single and double immunohistochemical procedures, whether MCHR-1 is present in neuronal primary cilia in serotonergic and GABAergic neurons located in the DRN of the rat. We demonstrated colocalization of MCHR-1 with type III adenylyl cyclase (AC-III), a neuronal ciliary marker, in the DRN and confirmed their colocalization in the hippocampus and cerebral cortex of the rat. We quantified the proportion of serotoninergic and GABAergic neurons that coexpress MCHR-1 at the mid-caudal and mid-rostral levels of the DRN: 4% and 12%, respectively. Furthermore, approximately 10% of the total number of MCHR-1 immunoreactive primary cilia belonged to serotonergic neurons, whilst 12% were appendages of GABAergic neurons. These morphological data allow us to conclude that the mechanism by which MCH modulates the activity of DRN neurons is through MCHR-1 receptors present in the primary cilia of different neurochemical phenotypes. New experiments are needed to understand the functional rationale of the unexpected localization of these receptors and to explore their presence in other neuronal phenotypes.
Assuntos
Núcleo Dorsal da Rafe/metabolismo , Neurônios/metabolismo , Receptores de Somatostatina/metabolismo , Animais , Cílios/metabolismo , Masculino , Ratos , Ratos WistarRESUMO
The cilia and flagella of eukaryotic cells serve many functions, exhibiting remarkable conservation of both structure and molecular composition in widely divergent eukaryotic organisms. SPAG6 and SPAG16 are the homologous in the mice to Chlamydomonas reinhardtii PF16 and PF20. Both proteins are associated with the axonemal central apparatus and are essential for ciliary and flagellar motility in mammals. Recent data derived from high-throughput studies revealed expression of these genes in tissues that do not contain motile cilia. However, the distribution of SPAG6 and SPAG16 in ciliated and non-ciliated tissues is not completely understood. In this work, we performed a quantitative analysis of the expression of Spag6 and Spag16 genes in parallel with the immune-localization of the proteins in several tissues of adult mice. Expression of mRNA was higher in the testis and tissues bearing motile cilia than in the other analyzed tissues. Both proteins were present in ciliated and non-ciliated tissues. In the testis, SPAG6 was detected in spermatogonia, spermatocytes, and in the sperm flagella whereas SPAG16 was found in spermatocytes and in the sperm flagella. In addition, both proteins were detected in the cytoplasm of cells from the brain, spinal cord, and ovary. A small isoform of SPAG16 was localized in the nucleus of germ cells and some neurons. In a parallel set of experiments, we overexpressed EGFP-SPAG6 in cultured cells and observed that the protein co-localized with a subset of acetylated cytoplasmic microtubules. A role of these proteins stabilizing the cytoplasmic microtubules of eukaryotic cells is discussed.