Restless legs syndrome in patients with obstructive sleep apnea: Association between apnea severity and symptoms of depression, insomnia, and daytime sleepiness.

OBJECTIVE: To determine if the prevalence and severity of restless legs syndrome (RLS) varies with apnea severity and analyze differences between the sexes in terms of comorbid RLS with symptoms of depression, insomnia, and daytime sleepiness in patients with obstructive sleep apnea (OSA). METHODS: Symptoms of depression, insomnia, and daytime sleepiness were defined as Patient Health Questionnaire-9 score ≥10, Insomnia Severity Index score ≥15, and Epworth Sleepiness Scale score ≥11. Multivariate logistic and linear regression analyses were conducted. RESULTS: In 707 adults with OSA (85.1% males), 16.1% (n = 114) had comorbid RLS. The prevalence of RLS was markedly lower in those with moderate and severe OSA than in those with mild OSA. Similarly, the odds of RLS significantly decreased with increasing apnea-hypopnea index. After controlling for age and sex, in patients with comorbid RLS, the International RLS Study Group Rating Scale scores were negatively correlated with apnea-hypopnea index and a nadir peripheral oxygen saturation during sleep. The presence of RLS was more likely to be associated with symptoms of depression, insomnia, and daytime sleepiness after controlling for confounding variables, but only in men. CONCLUSIONS: RLS is frequently noted in combination with OSA, with a female preponderance. The severities of OSA and RLS may be negatively associated. In patients with OSA, sex-related differences in terms of comorbid RLS with symptoms of depression, insomnia, and daytime sleepiness warrant further investigations.

An integrin binding motif in TLN-1/talin plays a minor role in motility and ovulation.

TLN-1/talin is a conserved focal adhesion protein that forms part of the linkage between the cytoplasmic tail of integrin and the actin cytoskeleton. In C. elegans , TLN-1 is expressed strongly in striated muscle and the gonadal sheath cells. Here, we report that a CRISPR-generated TLN-1 allele TLN-1(W387A), predicted to affect binding of talin to integrins, results in mild phenotypes, including motility defects and ovulation defects. The arrangement of the actin cytoskeleton in the body wall muscles, spermatheca, and sheath appears identical in wild type and TLN-1(W387A) animals. This analysis suggests that W387 in TLN-1 does not have a major effect on the binding of talin to integrin in vivo .

The RGD (Arg-Gly-Asp) is a potential cell-binding motif of UNC-52/PERLECAN.

UNC-52/perlecan is a basement membrane (BM) proteoglycan playing an essential role in the muscle cell attachment of C. elegans. The UNC-52 protein contains two RGD (Arg-Gly-Asp) motifs in domains III and IV, a well-characterized tripeptide known for binding to mammalian ß integrin. To investigate the role of the RGD motif in UNC-52/perlecan, we created two mutations in the 2021RGD2023 motif: one mutation changed the RGD to an RGE, and the other deleted the RGD motif. The RGE2023 caused defective actin filaments and aberrant localization of PAT-3 ß integrin and TLN-1/talin. Additionally, the in-frame deletion of RGD2023 resulted in a paralyzed and arrested at two-fold embryonic stages (Pat) phenotype, which is the identical phenotype of the pat-3 ß integrin null allele. These results indicate that RGD2023 is a potential ligand for cell binding and is essential for development and survival. Furthermore, our analysis reveals that the RGD of an invertebrate BM molecule is a potential cell-binding motif, suggesting that the function of the RGD motif is conserved among species.

Mutations in the cell-binding motif of lam-3/laminin α reveal hypercontraction behavior and defective sensitivity to levamisole in Caenorhabditis elegans.

The amino acid sequence Arg-Gly-Asp (RGD) is a cell-binding motif for extracellular matrix proteins. Initially found in fibronectin, the RGD motif is also found in LAM-3/laminin α chain in C. elegans. Laminin, a heterotrimeric glycoprotein, is a significant component of the basement membrane. Mutations in laminin subunits disrupt the extracellular matrix hence inhibit cell adhesion. This study aims to characterize the function of the RGD motif in lam-3/laminin α. Two mutations, lam-3 RGE and lam-3 ΔRGD, were generated. Our analysis of the mutants revealed that the RGD motif is involved in the motility of animals, suggesting that the cell-laminin interaction plays a role in regulating body contraction.

ZYX-1/Zyxin plays a minor role in oocyte transit through the spermatheca in C. elegans.

In C. elegans, oocytes are ovulated into the spermatheca, where they are fertilized before being pushed into the uterus. Contraction in the C. elegans spermatheca is driven by circumferential acto-myosin fibers. The C. elegans zyxin homolog, zyx-1, is expressed in the body wall muscle, pharynx and spermatheca. To our surprise, a CRISPR-generated zyx-1 deletion allele results in no overt developmental phenotypes, and the spermathecal actin cytoskeleton appears wild type, however, oocyte transit through the spermatheca is slower than in wild type animals. This suggests ZYX-1/Zyxin may regulate spermathecal contraction magnitude or timing of spermathecal bag contraction and/or spermathecal-uterine valve dilation.

Deletion of the RGD motif in LON-2/glypican is associated with morphological abnormalities.

The lon-2 gene in Caenorhabditis elegans encodes a heparan sulfate proteoglycan family glypican that negatively regulates the BMP signaling pathway responsible for controlling body length. LON-2 contains multiple functional domains, including an RGD (Arg-Gly-Asp) motif at amino acid number from 348 to 350. A novel mutant allele of lon-2 was investigated in this study. In this mutant allele, lon-2(kq348ΔRGD), the RGD motif at position 348 was deleted. Another pre-existing mutant allele, lon-2(e678), contains a ~9kb deletion and lacks most of the genomic coding sequence. The lon-2(e678) line was used as a reference allele. The novel mutant line was significantly shorter than wild-type animals, suggesting that removal of the RGD motif in LON-2 may improve its ability to inhibit BMP signaling.

A Novel Mutation in an NPXY Motif of ß Integrin Reveals Phenotypes Similar to him-4/hemicentin.

Integrin, an αß heterodimeric cell surface receptor for the extracellular matrix (ECM), carries two tyrosine phosphorylation motifs in the cytoplasmic tail of the ß subunit. NPXY (Asn-Pro-x-Tyr) is a conserved tyrosine phosphorylation motif that binds to the phospho-tyrosine binding (PTB) domain. We generated a tyrosine to glutamic acid (E) mutation to modify tyrosine (Y) into a negatively charged amino NPXY in the ßpat-3 integrin of Caenorhabditis elegans. The transgenic rescue animal displayed defects in gonad migration and tail morphology. Also, the mutant animals produced a high number of males, suggesting that the Y to E mutation in ßpat-3 integrin causes a phenotype similar to that of Him mutant. Further analyses revealed that males of pat-3(Y804E) and him-4/hemicentin share additional phenotypes such as abnormal gonad and unsuccessful mating. A pat-3 transgenic rescue mutant with a non-polar phenylalanine (F) in NPXY, pat-3(Y792/804F), suppressed the high male number, defective mating, inviable zygote, and the abnormal gonad of him-4 mutants, indicating that Y to F mutation in both NPXY motifs suppressed the him-4 phenotypes. This finding supports the idea that the ECM determines the activation state in integrin NPXY motifs; him-4/hemicentin may directly or indirectly interact with integrins and maintain the NPXY non-charged. Our findings provide new insight into a suppressive role of an ECM molecule in integrin NPXY phosphorylation.

A novel mutation in ß integrin reveals an integrin-mediated interaction between the extracellular matrix and cki-1/p27KIP1.

The cell-extracellular matrix (ECM) interaction plays an essential role in maintaining tissue shapes and regulates cell behaviors such as cell adhesion, differentiation and proliferation. The mechanism by which the ECM influences the cell cycle in vivo is poorly understood. Here we demonstrate that the ß integrin PAT-3 regulates the localization and expression of CKI-1, a C. elegans homologue of the cyclin dependent kinase inhibitor p27(KIP1). In nematodes expressing wild type PAT-3, CKI-1::GFP localizes primarily to nucleoli in hypodermal cells, whereas in animals expressing mutant pat-3 with a defective splice junction, CKI-1::GFP appears clumped and disorganized in nucleoplasm. RNAi analysis links cell adhesion genes to the regulation of CKI-1. RNAi of unc-52/perlecan, ina-1/α integrin, pat-4/ILK, and unc-97/PINCH resulted in abnormal CKI-1::GFP localization. Additional RNAi experiments revealed that the SCF E3 ubiquitin-ligase complex genes, skpt-1/SKP2, cul-1/CUL1 and lin-23/F-box, are required for the proper localization and expression of CKI-1, suggesting that integrin signaling and SCF E3 ligase work together to regulate the cellular distribution of CKI-1. These data also suggest that integrin plays a major role in maintaining proper CKI-1/p27(KIP1) levels in the cell. Perturbed integrin signaling may lead to the inhibition of SCF ligase activity, mislocalization and elevation of CKI-1/p27(KIP1). These results suggest that adhesion signaling is crucial for cell cycle regulation in vivo.

Differential subcellular localization of DNA topoisomerase-1 isoforms and their roles during Caenorhabditis elegans development.

DNA topoisomerase-1 (TOP-1) resolves the topological problems associated with DNA replication, transcription and recombination by introducing temporary single-strand breaks in the DNA. Caenorhabditis elegans TOP-1 has two isoforms, TOP-1α and TOP-1ß. TOP-1ß is broadly localized to the nuclei of many cells at all developmental stages and concentrated in nucleoli in embryo gut and oogenic cells. However, TOP-1α is specifically localized to centrosomes, neuronal cells, excretory cells and chromosomes of germ cells in embryonic and larval stages. Reporter gene analysis also shows that top-1 transcription is highly activated in several sensory neurons, speculating the possible role of TOP-1α in neuronal development. From RNA interference (RNAi) experiments, we demonstrated that C. elegans TOP-1 is required for chromosomal segregation, germline proliferation and gonadal migration, which are all correlated with the expression and activity of TOP-1. Therefore, our findings may provide an insight into a new role of TOP-1 in development of multicellular organisms.

CACN-1/Cactin interacts genetically with MIG-2 GTPase signaling to control distal tip cell migration in C. elegans.

The two specialized C. elegans distal tip cells (DTCs) provide an in vivo model system for the study of developmentally regulated cell migration. We identified cacn-1/cactin, a well-conserved, novel regulator of cell migration in a genome-wide RNAi screen for regulators of DTC migration. RNAi depletion experiments and analysis of the hypomorphic allele cacn-1(tm3126) indicate that CACN-1 is required during DTC migration for proper pathfinding and for cessation of DTC migration at the end of larval morphogenesis. Strong expression of CACN-1 in the DTCs, and data from cell-specific RNAi depletion experiments, suggest that CACN-1 is required cell-autonomously to control DTC migration. Importantly, genetic interaction data with Rac GTPase activators and effectors suggest that CACN-1 acts specifically to inhibit the mig-2/Rac pathway, and in parallel to ced-10/Rac, to control DTC pathfinding.

Analysis of conserved residues in the betapat-3 cytoplasmic tail reveals important functions of integrin in multiple tissues.

Integrin cytoplasmic tails contain motifs that link extracellular information to cell behavior such as cell migration and contraction. To investigate the cell functions mediated by the conserved motifs, we created mutations in the Caenorhabditis elegans betapat-3 cytoplasmic tail. The beta1D (799FK800), NPXY, tryptophan (784W), and threonine (797TT798) motifs were disrupted to identify their functions in vivo. Animals expressing integrins with disrupted NPXY motifs were viable, but displayed distal tip cell migration and ovulation defects. The conserved threonines were required for gonad migration and contraction as well as tail morphogenesis, whereas disruption of the beta1D and tryptophan motifs produced only mild defects. To abolish multiple conserved motifs, a beta1C-like variant, which results in a frameshift, was constructed. The betapat-3(beta1C) transgenic animals showed cold-sensitive larval arrests and defective muscle structure and gonad migration and contraction. Our study suggests that the conserved NPXY and TT motifs play important roles in the tissue-specific function of integrin.

Quantitative analysis of distal tip cell migration in C. elegans.

Correct distal tip cell (DTC) migration in the nematode C. elegans requires sensing soluble and matrix cues, remodeling extracellular matrix, and signaling through conserved integrin and netrin pathways. The DTC executes a complex path and coordinates its migration with the developmental stages of larval morphogenesis. This chapter outlines a method for investigating DTC migration in C. elegans using feeding RNA interference (RNAi) and light microscopy. To deplete a candidate gene of interest, nematode eggs are added to plates seeded with RNAi-inducing bacterial lawns. The animals hatch and begin to eat the RNAi bacteria, releasing dsRNA and causing the targeted gene to be depleted during larval development. Positions of migratory cells are monitored in larvae and young adults using differential interference contrast (DIC) and epifluorescence microscopy.

Role of phosphatidylinositol-4-phosphate 5' kinase (ppk-1) in ovulation of Caenorhabditis elegans.

During Caenorhabditis elegans ovulation, the somatic gonad integrates signals from germ cells and propels a mature oocyte into the spermatheca for fertilization. Previous work suggests that phosphoinositide signaling plays important roles in C. elegans fertility. To fully understand inositol-1,4,5-trisphosphate (IP(3)) signaling in ovulation, we have examined the function of phosphatidylinositol-4-phosphate 5' kinase (PIP5K) in C. elegans. Our results show that the C. elegans PIP5K homolog, ppk-1, is essential for ovulation in C. elegans; ppk-1 is mainly expressed in somatic gonad, and depletion of ppk-1 expression causes defective ovulation, reduced gonad sheath contractility, and sterility. Increased IP(3) signaling compensates for ppk-1 (RNAi)-induced sterility, suggesting that ppk-1 is linked to IP(3) signaling. These results demonstrate that ppk-1 plays an essential role in IP(3) signaling and cytoskeleton organization in somatic gonad.

pat-4/ILK and unc-112/Mig-2 are required for gonad function in Caenorhabditis elegans.

Tissue morphogenesis requires proper interaction between cells and the extracellular matrix (ECM), which is mediated by alphabeta heterodimeric receptor integrin. In Caenorhabditis elegans, integrin signaling is essential for formation of gonad. Here, we probe the role of several integrin-associated molecules in ovulation and cell migration. Function of pat-4/integrin-linked kinase (ILK) and unc-112/Mig-2 was examined using RNA-mediated interference (RNAi). Depletion of these messages caused oocyte accumulation in the proximal gonad and distal tip cells (DTC) migration defects. It was further determined that failed ovulation was due to defective contraction and dilation of somatic gonad structures, including spermatheca and gonad sheath. Actin cytoskeleton in the proximal gonad of RNAi animals appeared disorganized, indicating that RNAi of pat-4 or unc-112 inhibited the overall assembly of actin cytoskeleton in somatic gonad. Taken together, our analysis confirms the role of integrin and integrin-associated proteins in gonad function.

Connections between integrins and Rac GTPase pathways control gonad formation and function in C. elegans.

The integrins are a family of alphabeta heterodimeric transmembrane receptors that link extracellular matrix (ECM) proteins to the cytoskeleton and orchestrate cell behaviors. It's been suggested that integrins interact with Rho family small GTPases, such as Rho and Rac. We took advantage of a C. elegans nematode line expressing HA-betatail, a beta integrin transgene inhibiting the functions of endogenous integrins, to determine the combined effects of reducing PAT-3 beta integrin and Rac pathway activities. Double mutants of HA-betatail and unc-73, a guanine nucleotide exchange factor GEF for MIG-2/Rac, had body wall and vulval muscle abnormalities. On the other hand, HA-betatail combined with mutant CED-5, another Rac interacting protein, showed ovulation defects and sterility. RNA-mediated interference (RNAi) of pat-3 on Rac mutant backgrounds also affected gonad structure and function. These results show a functional link between integrins and Rac signaling in muscles and gonads. Furthermore, data showing distinct phenotypes of HA-betatail with unc-73 versus ced-5 suggest some tissue-specificity in the usage of Rac signaling pathways.