Cilioplasm is a cellular compartment for calcium signaling in response to mechanical and chemical stimuli

Primary cilia with a diameter of ~200 nm have been implicated in development and disease. Calcium signaling within a primary cilium has never been directly visualized and has therefore remained a speculation. Fluid-shear stress and dopamine receptor type-5 (DR5) agonist are among the few stimuli that require cilia for intracellular calcium signal transduction. However, it is not known if these stimuli initiate calcium signaling within the cilium or if the calcium signal originates in the cytoplasm. Using an integrated single-cell imaging technique, we demonstrate for the first time that calcium signaling triggered by fluid-shear stress initiates in the primary cilium and can be distinguished from the subsequent cytosolic calcium response through the ryanodine receptor. Importantly, this flow-induced calcium signaling depends on the ciliary polycystin-2 calcium channel. While DR5-specific agonist induces calcium signaling mainly in the cilioplasm via ciliary CaV1.2, thrombin specifically induces cytosolic calcium signaling through the IP₃ receptor. Furthermore, a non-specific calcium ionophore triggers both ciliary and cytosolic calcium responses. We suggest that cilia not only act as sensory organelles but also function as calcium signaling compartments. Cilium-dependent signaling can spread to the cytoplasm or be contained within the cilioplasm. Our study thus provides the first model to understand signaling within the cilioplasm of a living cell.     

MicroRNAs in myocardial ischemia: identifying new targets and tools for treating heart disease. New frontiers for miR-medicine

MicroRNAs (miRNAs) are natural, single-stranded, small RNA molecules which subtly control gene expression. Several studies indicate that specific miRNAs can regulate heart function both in development and disease. Despite prevention programs and new therapeutic agents, cardiovascular disease remains the main cause of death in developed countries. The elevated number of heart failure episodes is mostly due to myocardial infarction (MI). An increasing number of studies have been carried out reporting changes in miRNAs gene expression and exploring their role in MI and heart failure. In this review, we furnish a critical analysis of where the frontier of knowledge has arrived in the fields of basic and translational research on miRNAs in cardiac ischemia. We first summarize the basal information on miRNA biology and regulation, especially concentrating on the feedback loops which control cardiac-enriched miRNAs. A focus on the role of miRNAs in the pathogenesis of myocardial ischemia and in the attenuation of injury is presented. Particular attention is given to cardiomyocyte death (apoptosis and necrosis), fibrosis, neovascularization, and heart failure. Then, we address the potential of miR-diagnosis (miRNAs as disease biomarkers) and miR-drugs (miRNAs as therapeutic targets) for cardiac ischemia and heart failure. Finally, we evaluate the use of miRNAs in the emerging field of regenerative medicine.     

Role of the ubiquitin–proteasome system in the regulation of P2Y13 receptor expression: impact on hepatic HDL uptake

The protective effect of high density lipoproteins (HDL) against atherosclerosis is mainly attributed to their capacity to transport excess cholesterol from peripheral tissues back to the liver for further elimination into the bile, a process called reverse cholesterol transport (RCT). Recently, the importance of the P2Y₁₃ receptor (P2Y₁₃-R) was highlighted in HDL metabolism since HDL uptake by the liver was decreased in P2Y₁₃-R deficient mice, which translated into impaired RCT. Here, we investigated for the first time the molecular mechanisms regulating cell surface expression of P2Y₁₃-R. When transiently expressed, P2Y₁₃-R was mainly detected in the endoplasmic reticulum (ER) and strongly subjected to proteasome degradation while its homologous P2Y₁₂ receptor (P2Y₁₂-R) was efficiently targeted to the plasma membrane. We observed an inverse correlation between cell surface expression and ubiquitination level of P2Y₁₃-R in the ER, suggesting a close link between ubiquitination of P2Y₁₃-R and its efficient targeting to the plasma membrane. The C-terminus tail exchange between P2Y₁₃-R and P2Y₁₂-R strongly restored plasma membrane expression of P2Y₁₃-R, suggesting the involvement of the intra-cytoplasmic tail of P2Y₁₃-R in expression defect. Accordingly, proteasomal inhibition increased plasma membrane expression of functionally active P2Y₁₃-R in hepatocytes, and consequently stimulated P2Y₁₃-R-mediated HDL endocytosis. Importantly, proteasomal inhibition strongly potentiated HDL hepatic uptake (>200 %) in wild-type but not in P2Y₁₃-R-deficient mice, thus reinforcing the role of P2Y₁₃-R expression in regulating HDL metabolism. Therefore, specific inhibition of the ubiquitin–proteasome system might be a novel powerful HDL therapy to enhance P2Y₁₃-R expression and consequently promote the overall RCT.     

Dominant missense mutations in ABCC9 cause Cantú syndrome

Cantú syndrome is characterized by congenital hypertrichosis, distinctive facial features, osteochondrodysplasia and cardiac defects. By using family-based exome sequencing, we identified a de novo mutation in ABCC9. Subsequently, we discovered novel dominant missense mutations in ABCC9 in 14 of the 16 individuals with Cantú syndrome examined. The ABCC9 protein is part of an ATP-dependent potassium (K(ATP)) channel that couples the metabolic state of a cell with its electrical activity. All mutations altered amino acids in or close to the transmembrane domains of ABCC9. Using electrophysiological measurements, we show that mutations in ABCC9 reduce the ATP-mediated potassium channel inhibition, resulting in channel opening. Moreover, similarities between the phenotype of individuals with Cantú syndrome and side effects from the K(ATP) channel agonist minoxidil indicate that the mutations in ABCC9 result in channel opening. Given the availability of ABCC9 antagonists, our findings may have direct implications for the treatment of individuals with Cantú syndrome.     

Mutations in the RNA exosome component gene EXOSC3 cause pontocerebellar hypoplasia and spinal motor neuron degeneration

RNA exosomes are multi-subunit complexes conserved throughout evolution and are emerging as the major cellular machinery for processing, surveillance and turnover of a diverse spectrum of coding and noncoding RNA substrates essential for viability. By exome sequencing, we discovered recessive mutations in EXOSC3 (encoding exosome component 3) in four siblings with infantile spinal motor neuron disease, cerebellar atrophy, progressive microcephaly and profound global developmental delay, consistent with pontocerebellar hypoplasia type 1 (PCH1; MIM 607596). We identified mutations in EXOSC3 in an additional 8 of 12 families with PCH1. Morpholino knockdown of exosc3 in zebrafish embryos caused embryonic maldevelopment, resulting in small brain size and poor motility, reminiscent of human clinical features, and these defects were largely rescued by co-injection with wild-type but not mutant exosc3 mRNA. These findings represent the first example of an RNA exosome core component gene that is responsible for a human disease and further implicate dysregulation of RNA processing in cerebellar and spinal motor neuron maldevelopment and degeneration.     

Mutations in NMNAT1 cause Leber congenital amaurosis and identify a new disease pathway for retinal degeneration

Leber congenital amaurosis (LCA) is a blinding retinal disease that presents within the first year after birth. Using exome sequencing, we identified mutations in the nicotinamide adenine dinucleotide (NAD) synthase gene NMNAT1 encoding nicotinamide mononucleotide adenylyltransferase 1 in eight families with LCA, including the family in which LCA was originally linked to the LCA9 locus. Notably, all individuals with NMNAT1 mutations also have macular colobomas, which are severe degenerative entities of the central retina (fovea) devoid of tissue and photoreceptors. Functional assays of the proteins encoded by the mutant alleles identified in our study showed that the mutations reduce the enzymatic activity of NMNAT1 in NAD biosynthesis and affect protein folding. Of note, recent characterization of the slow Wallerian degeneration (Wld(s)) mouse model, in which prolonged axonal survival after injury is observed, identified NMNAT1 as a neuroprotective protein when ectopically expressed. Our findings identify a new disease mechanism underlying LCA and provide the first link between endogenous NMNAT1 dysfunction and a human nervous system disorder.     

Common variants near TARDBP and EGR2 are associated with susceptibility to Ewing sarcoma

Ewing sarcoma, a pediatric tumor characterized by EWSR1-ETS fusions, is predominantly observed in populations of European ancestry. We performed a genome-wide association study (GWAS) of 401 French individuals with Ewing sarcoma, 684 unaffected French individuals and 3,668 unaffected individuals of European descent and living in the United States. We identified candidate risk loci at 1p36.22, 10q21 and 15q15. We replicated these loci in two independent sets of cases and controls. Joint analysis identified associations with rs9430161 (P = 1.4 × 10(-20); odds ratio (OR) = 2.2) located 25 kb upstream of TARDBP, rs224278 (P = 4.0 × 10(-17); OR = 1.7) located 5 kb upstream of EGR2 and, to a lesser extent, rs4924410 at 15q15 (P = 6.6 × 10(-9); OR = 1.5). The major risk haplotypes were less prevalent in Africans, suggesting that these loci could contribute to geographical differences in Ewing sarcoma incidence. TARDBP shares structural similarities with EWSR1 and FUS, which encode RNA binding proteins, and EGR2 is a target gene of EWSR1-ETS. Variants at these loci were associated with expression levels of TARDBP, ADO (encoding cysteamine dioxygenase) and EGR2.     

The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left-right axis formation

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous autosomal recessive disorder characterized by recurrent infections of the respiratory tract associated with the abnormal function of motile cilia. Approximately half of individuals with PCD also have alterations in the left-right organization of their internal organ positioning, including situs inversus and situs ambiguous (Kartagener's syndrome). Here, we identify an uncharacterized coiled-coil domain containing a protein, CCDC40, essential for correct left-right patterning in mouse, zebrafish and human. In mouse and zebrafish, Ccdc40 is expressed in tissues that contain motile cilia, and mutations in Ccdc40 result in cilia with reduced ranges of motility. We further show that CCDC40 mutations in humans result in a variant of PCD characterized by misplacement of the central pair of microtubules and defective assembly of inner dynein arms and dynein regulatory complexes. CCDC40 localizes to motile cilia and the apical cytoplasm and is required for axonemal recruitment of CCDC39, disruption of which underlies a similar variant of PCD.     

Genome-wide association study reveals three susceptibility loci for common migraine in the general population

Migraine is a common, heterogeneous and heritable neurological disorder. Its pathophysiology is incompletely understood, and its genetic influences at the population level are unknown. In a population-based genome-wide analysis including 5,122 migraineurs and 18,108 non-migraineurs, rs2651899 (1p36.32, PRDM16), rs10166942 (2q37.1, TRPM8) and rs11172113 (12q13.3, LRP1) were among the top seven associations (P < 5 × 10(-6)) with migraine. These SNPs were significant in a meta-analysis among three replication cohorts and met genome-wide significance in a meta-analysis combining the discovery and replication cohorts (rs2651899, odds ratio (OR) = 1.11, P = 3.8 × 10(-9); rs10166942, OR = 0.85, P = 5.5 × 10(-12); and rs11172113, OR = 0.90, P = 4.3 × 10(-9)). The associations at rs2651899 and rs10166942 were specific for migraine compared with non-migraine headache. None of the three SNP associations was preferential for migraine with aura or without aura, nor were any associations specific for migraine features. TRPM8 has been the focus of neuropathic pain models, whereas LRP1 modulates neuronal glutamate signaling, plausibly linking both genes to migraine pathophysiology.