The inner junction protein CFAP20 functions in motile and non-motile cilia and is critical for vision.

Motile and non-motile cilia are associated with mutually-exclusive genetic disorders. Motile cilia propel sperm or extracellular fluids, and their dysfunction causes primary ciliary dyskinesia. Non-motile cilia serve as sensory/signalling antennae on most cell types, and their disruption causes single-organ ciliopathies such as retinopathies or multi-system syndromes. CFAP20 is a ciliopathy candidate known to modulate motile cilia in unicellular eukaryotes. We demonstrate that in zebrafish, cfap20 is required for motile cilia function, and in C. elegans, CFAP-20 maintains the structural integrity of non-motile cilia inner junctions, influencing sensory-dependent signalling and development. Human patients and zebrafish with CFAP20 mutations both exhibit retinal dystrophy. Hence, CFAP20 functions within a structural/functional hub centered on the inner junction that is shared between motile and non-motile cilia, and is distinct from other ciliopathy-associated domains or macromolecular complexes. Our findings suggest an uncharacterised pathomechanism for retinal dystrophy, and potentially for motile and non-motile ciliopathies in general.

Interpreting ciliopathy-associated missense variants of uncertain significance (VUS) in Caenorhabditis elegans.

Better methods are required to interpret the pathogenicity of disease-associated variants of uncertain significance (VUS), which cannot be actioned clinically. In this study, we explore the use of an animal model (Caenorhabditis elegans) for in vivo interpretation of missense VUS alleles of TMEM67, a cilia gene associated with ciliopathies. CRISPR/Cas9 gene editing was used to generate homozygous knock-in C. elegans worm strains carrying TMEM67 patient variants engineered into the orthologous gene (mks-3). Quantitative phenotypic assays of sensory cilia structure and function (neuronal dye filling, roaming and chemotaxis assays) measured how the variants impacted mks-3 gene function. Effects of the variants on mks-3 function were further investigated by looking at MKS-3::GFP localization and cilia ultrastructure. The quantitative assays in C. elegans accurately distinguished between known benign (Asp359Glu, Thr360Ala) and known pathogenic (Glu361Ter, Gln376Pro) variants. Analysis of eight missense VUS generated evidence that three are benign (Cys173Arg, Thr176Ile and Gly979Arg) and five are pathogenic (Cys170Tyr, His782Arg, Gly786Glu, His790Arg and Ser961Tyr). Results from worms were validated by a genetic complementation assay in a human TMEM67 knock-out hTERT-RPE1 cell line that tests a TMEM67 signalling function. We conclude that efficient genome editing and quantitative functional assays in C. elegans make it a tractable in vivo animal model for rapid, cost-effective interpretation of ciliopathy-associated missense VUS alleles.

A complement factor H homolog, heparan sulfation, and syndecan maintain inversin compartment boundaries in C. elegans cilia.

Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly. Canonical disease models suggest that defective interactions between complement factor H (CFH) and cell surface heparan sulfate (HS) result in increased alternative complement pathway activity, cytolytic damage, and tissue inflammation in the retina. Although these factors are thought to contribute to increased disease risk, multiple studies indicate that noncanonical mechanisms that result from defective CFH and HS interaction may contribute to the progression of AMD as well. A total of 60 ciliated sensory neurons in the nematode Caenorhabditis elegans detect chemical, olfactory, mechanical, and thermal cues in the environment. Here, we find that a C. elegans CFH homolog localizes on CEP mechanosensory neuron cilia where it has noncanonical roles in maintaining inversin/NPHP-2 within its namesake proximal compartment and preventing inversin/NPHP-2 accumulation in distal cilia compartments in aging adults. CFH localization and maintenance of inversin/NPHP-2 compartment integrity depend on the HS 3-O sulfotransferase HST-3.1 and the transmembrane proteoglycan syndecan/SDN-1. Defective inversin/NPHP-2 localization in mouse and human photoreceptors with CFH mutations indicates that these functions and interactions may be conserved in vertebrate sensory neurons, suggesting that previously unappreciated defects in cilia structure may contribute to the progressive photoreceptor dysfunction associated with CFH loss-of-function mutations in some AMD patients.

CDKL kinase regulates the length of the ciliary proximal segment.

Cilia are organelles found throughout most unicellular eukaryotes and different metazoan cell types. To accomplish their essential roles in cell motility, fluid flow, and signaling, cilia are divided into subcompartments with variable structures, compositions, and functions. How these specific subcompartments are built remains almost completely unexplored. Here, we show that C. elegans CDKL-1, related to the human CDKL kinase family (CDKL1/CDKL2/CDKL3/CDKL4/CDKL5), specifically controls the length of the proximal segment, a ciliary subdomain conserved in evolution from Tetrahymena motile cilia to C. elegans chemosensory, mammalian olfactory, and photoreceptor non-motile cilia. CDKL-1 associates with intraflagellar transport (IFT), influences the distribution of the IFT anterograde motors heterotrimeric kinesin-II and homodimeric OSM-3-kinesin/KIF17 in the proximal segment, and shifts the boundary between the proximal and distal segments (PS/DS boundary). CDKL-1 appears to function independently from several factors that influence cilium length, namely the kinases DYF-5 (mammalian CILK1/MAK) and NEKL-1 (NEK9), as well as the depolymerizing kinesins KLP-13 (KIF19) and KLP-7 (KIF2). However, a different kinase, DYF-18 (CCRK), is needed for the correct localization and function of CDKL-1 and similarly influences the length of the proximal segment. Loss of CDKL-1, which affects proximal segment length without impairing overall ciliary microtubule structural integrity, also impairs cilium-dependent processes, namely cGMP-signaling-dependent body length control and CO2 avoidance. Collectively, our findings suggest that cilium length is regulated by various pathways and that the IFT-associated kinase CDKL-1 is essential for the construction of a specific ciliary compartment and contributes to development and sensory physiology.

Interpreting the pathogenicity of Joubert syndrome missense variants in Caenorhabditis elegans.

Ciliopathies are inherited disorders caused by defects in motile and non-motile (primary) cilia. Ciliopathy syndromes and associated gene variants are often highly pleiotropic and represent exemplars for interrogating genotype-phenotype correlations. Towards understanding disease mechanisms in the context of ciliopathy mutations, we have used a leading model organism for cilia and ciliopathy research, Caenorhabditis elegans, together with gene editing, to characterise two missense variants (P74S and G155S) in mksr-2/B9D2 associated with Joubert syndrome (JBTS). B9D2 functions within the Meckel syndrome (MKS) module at the ciliary base transition zone (TZ) compartment and regulates the molecular composition and sensory/signalling functions of the cilium. Quantitative assays of cilium/TZ structure and function, together with knock-in reporters, confirm that both variant alleles are pathogenic in worms. G155S causes a more severe overall phenotype and disrupts endogenous MKSR-2 organisation at the TZ. Recapitulation of the patient biallelic genotype shows that compound heterozygous worms phenocopy worms homozygous for P74S. The P74S and G155S alleles also reveal evidence of a very close functional association between the B9D2-associated B9 complex and MKS-2/TMEM216. Together, these data establish C. elegans as a model for interpreting JBTS mutations and provide further insight into MKS module organisation. This article has an associated First Person interview with the first author of the paper.

Ciliary Rab28 and the BBSome negatively regulate extracellular vesicle shedding.

Cilia both receive and send information, the latter in the form of extracellular vesicles (EVs). EVs are nano-communication devices that influence cell, tissue, and organism behavior. Mechanisms driving ciliary EV biogenesis are almost entirely unknown. Here, we show that the ciliary G-protein Rab28, associated with human autosomal recessive cone-rod dystrophy, negatively regulates EV levels in the sensory organs of Caenorhabditis elegans in a cilia specific manner. Sequential targeting of lipidated Rab28 to periciliary and ciliary membranes is highly dependent on the BBSome and the prenyl-binding protein phosphodiesterase 6 subunit delta (PDE6D), respectively, and BBSome loss causes excessive and ectopic EV production. We also find that EV defective mutants display abnormalities in sensory compartment morphogenesis. Together, these findings reveal that Rab28 and the BBSome are key in vivo regulators of EV production at the periciliary membrane and suggest that EVs may mediate signaling between cilia and glia to shape sensory organ compartments. Our data also suggest that defects in the biogenesis of cilia-related EVs may contribute to human ciliopathies.

Vascular Nox (NADPH Oxidase) Compartmentalization, Protein Hyperoxidation, and Endoplasmic Reticulum Stress Response in Hypertension.

Vascular Nox (NADPH oxidase)-derived reactive oxygen species and endoplasmic reticulum (ER) stress have been implicated in hypertension. However, relationships between these processes are unclear. We hypothesized that Nox isoforms localize in a subcellular compartment-specific manner, contributing to oxidative and ER stress, which influence the oxidative proteome and vascular function in hypertension. Nox compartmentalization (cell fractionation), O2- (lucigenin), H2O2 (amplex red), reversible protein oxidation (sulfenylation), irreversible protein oxidation (protein tyrosine phosphatase, peroxiredoxin oxidation), and ER stress (PERK [protein kinase RNA-like endoplasmic reticulum kinase], IRE1α [inositol-requiring enzyme 1], and phosphorylation/oxidation) were studied in spontaneously hypertensive rat (SHR) vascular smooth muscle cells (VSMCs). VSMC proliferation was measured by fluorescence-activated cell sorting, and vascular reactivity assessed in stroke-prone SHR arteries by myography. Noxs were downregulated by short interfering RNA and pharmacologically. In SHR, Noxs were localized in specific subcellular regions: Nox1 in plasma membrane and Nox4 in ER. In SHR, oxidative stress was associated with increased protein sulfenylation and hyperoxidation of protein tyrosine phosphatases and peroxiredoxins. Inhibition of Nox1 (NoxA1ds), Nox1/4 (GKT137831), and ER stress (4-phenylbutyric acid/tauroursodeoxycholic acid) normalized SHR vascular reactive oxygen species generation. GKT137831 reduced IRE1α sulfenylation and XBP1 (X-box binding protein 1) splicing in SHR. Increased VSMC proliferation in SHR was normalized by GKT137831, 4-phenylbutyric acid, and STF083010 (IRE1-XBP1 disruptor). Hypercontractility in the stroke-prone SHR was attenuated by 4-phenylbutyric acid. We demonstrate that protein hyperoxidation in hypertension is associated with oxidative and ER stress through upregulation of plasmalemmal-Nox1 and ER-Nox4. The IRE1-XBP1 pathway of the ER stress response is regulated by Nox4/reactive oxygen species and plays a role in the hyperproliferative VSMC phenotype in SHR. Our study highlights the importance of Nox subcellular compartmentalization and interplay between cytoplasmic reactive oxygen species and ER stress response, which contribute to the VSMC oxidative proteome and vascular dysfunction in hypertension.

Vascular dysfunction in obese diabetic db/db mice involves the interplay between aldosterone/mineralocorticoid receptor and Rho kinase signaling.

Activation of aldosterone/mineralocorticoid receptors (MR) has been implicated in vascular dysfunction of diabetes. Underlying mechanisms are elusive. Therefore, we investigated the role of Rho kinase (ROCK) in aldosterone/MR signaling and vascular dysfunction in a model of diabetes. Diabetic obese mice (db/db) and control counterparts (db/+) were treated with MR antagonist (MRA, potassium canrenoate, 30 mg/kg/day, 4 weeks) or ROCK inhibitor, fasudil (30 mg/kg/day, 3 weeks). Plasma aldosterone was increased in db/db versus db/+. This was associated with enhanced vascular MR signaling. Norepinephrine (NE)-induced contraction was increased in arteries from db/db mice. These responses were attenuated in mice treated with canrenoate or fasudil. Db/db mice displayed hypertrophic remodeling and increased arterial stiffness, improved by MR blockade. Vascular calcium sensitivity was similar between depolarized arteries from db/+ and db/db. Vascular hypercontractility in db/db mice was associated with increased myosin light chain phosphorylation and reduced expression of PKG-1α. Vascular RhoA/ROCK signaling and expression of pro-inflammatory and pro-fibrotic markers were exaggerated in db/db mice, effects that were attenuated by MRA. Fasudil, but not MRA, improved vascular insulin sensitivity in db/db mice, evidenced by normalization of Irs1 phosphorylation. Our data identify novel pathways involving MR-RhoA/ROCK-PKG-1 that underlie vascular dysfunction and injury in diabetic mice.

Assessment of Protein Carbonylation and Protein Tyrosine Phosphatase (PTP) Oxidation in Vascular Smooth Muscle Cells (VSMCs) Using Immunoblotting Approaches.

Post-translational modification of proteins, such as phosphorylation and oxidation, plays a major role in cellular signaling by influencing protein structure and function. In vascular cells, in addition to influencing phosphorylation, angiotensin II (Ang II) induces oxidation of proteins, important in redox signaling in the cardiovascular and renal systems. The present chapter describes immunoblotting approaches to assess irreversible protein carbonylation and protein tyrosine phosphatase (PTPs) oxidation status in the proteome of vascular smooth muscle cells (VSMC).Protein carbonylation is generally measured using the OxyBlot™ approach, whereby derivatization of protein carbonyl groups (C = O) on oxidized amino acids by dinitrophenylhydrazine (DNPH) results in the formation of a stable dinitrophenyl (DNP) hydrazone product. The samples are analyzed by SDS-PAGE and a primary antibody raised against the DNP moiety is used to determine levels of irreversible protein carbonylation in the sample by immunoblotting.Oxidation of PTPs can be evaluated using a monoclonal antibody against the "hyperoxidized" (SO3H) catalytic site of these enzymes. The described methodology offers the ability to discriminate between irreversible (SO3H) and reversible (SOH) PTP oxidation states. Initially, the free unmodified PTP-thiols (S-) are alkylated and the sample is split into two. One part is used to assess the PTP-SO3H form. In the other part reversibly modified PTP-thiols are first reduced and then hyperoxidized by pervanadate (PV). Both untreated and PV-treated samples are analyzed by SDS-PAGE and "hyperoxidized" PTPs are detected by immunoblotting. The proportion of reversibly oxidized PTP-SOH fraction is determined by the difference between the signals in untreated and the PV-treated samples.The above immunoassays provide general approaches to detect and quantify global levels of irreversible protein oxidation and of irreversibly/reversibly oxidized PTPs in any (patho)physiological context. Characterization of the global redox status is essential to better understand the redox-sensitive mechanisms underlying chronic diseases associated with oxidative stress. This is particularly important in systems influenced by the renin angiotensin system, because Ang II is a potent inducer of oxidative stress and redox signaling.

Urine Metabolomics in Hypertension Research.

Functional genomics requires an understanding of the complete network of changes within an organism by extensive measurements of moieties from mRNA, proteins, and metabolites. Metabolomics utilizes analytic chemistry tools to profile the complete spectrum of metabolites found in a tissue, cells, or biofluids using a wide range of tools from infrared spectroscopy, fluorescence spectroscopy, NMR spectroscopy, and mass spectrometry. In this protocol, we outline a procedure for performing metabolomic analysis of urine samples using liquid chromatography-mass spectrometry (LC-MS). We outline the advantages of using this approach and summarize some of the early promising studies in cardiovascular diseases using this approach.

Adipocyte-Specific Mineralocorticoid Receptor Overexpression in Mice Is Associated With Metabolic Syndrome and Vascular Dysfunction: Role of Redox-Sensitive PKG-1 and Rho Kinase.

Mineralocorticoid receptor (MR) expression is increased in adipose tissue from obese individuals and animals. We previously demonstrated that adipocyte-MR overexpression (Adipo-MROE) in mice is associated with metabolic changes. Whether adipocyte MR directly influences vascular function in these mice is unknown. We tested this hypothesis in resistant mesenteric arteries from Adipo-MROE mice using myography and in cultured adipocytes. Molecular mechanisms were probed in vessels/vascular smooth muscle cells and adipose tissue/adipocytes and focused on redox-sensitive pathways, Rho kinase activity, and protein kinase G type-1 (PKG-1) signaling. Adipo-MROE versus control-MR mice exhibited reduced vascular contractility, associated with increased generation of adipocyte-derived hydrogen peroxide, activation of vascular redox-sensitive PKG-1, and downregulation of Rho kinase activity. Associated with these vascular changes was increased elastin content in Adipo-MROE. Inhibition of PKG-1 with Rp-8-Br-PET-cGMPS normalized vascular contractility in Adipo-MROE. In the presence of adipocyte-conditioned culture medium, anticontractile effects of the adipose tissue were lost in Adipo-MROE mice but not in control-MR mice. In conclusion, adipocyte-MR upregulation leads to impaired contractility with preserved endothelial function and normal blood pressure. Increased elasticity may contribute to hypocontractility. We also identify functional cross talk between adipocyte MR and arteries and describe novel mechanisms involving redox-sensitive PKG-1 and Rho kinase. Our results suggest that adipose tissue from Adipo-MROE secrete vasoactive factors that preferentially influence vascular smooth muscle cells rather than endothelial cells. Our findings may be important in obesity/adiposity where adipocyte-MR expression/signaling is amplified and vascular risk increased.

Redox signaling, Nox5 and vascular remodeling in hypertension.

PURPOSE OF REVIEW: Extensive data indicate a role for reactive oxygen species (ROS) and redox signaling in vascular damage in hypertension. However, molecular mechanisms underlying these processes remain unclear, but oxidative post-translational modification of vascular proteins is critical. This review discusses how proteins are oxidatively modified and how redox signaling influences vascular smooth muscle cell growth and vascular remodeling in hypertension. We also highlight Nox5 as a novel vascular ROS-generating oxidase. RECENT FINDINGS: Oxidative stress in hypertension leads to oxidative imbalance that affects vascular cell function through redox signaling. Many Nox isoforms produce ROS in the vascular wall, and recent findings show that Nox5 may be important in humans. ROS regulate signaling by numerous processes including cysteine oxidative post-translational modification such as S-nitrosylation, S-glutathionylation and sulfydration. In vascular smooth muscle cells, this influences cellular responses to oxidative stimuli promoting changes from a contractile to a proliferative phenotype. SUMMARY: In hypertension, Nox-induced ROS production is increased, leading to perturbed redox signaling through oxidative modifications of vascular proteins. This influences mitogenic signaling and cell cycle regulation, leading to altered cell growth and vascular remodeling in hypertension.

Oxidative stress and human hypertension: vascular mechanisms, biomarkers, and novel therapies.

Hypertension is a major cardiovascular risk factor. Of the many processes involved in the pathophysiology of hypertension, vascular damage due to oxidative stress (excess bioavailability of reactive oxygen species [ROS]) is particularly important. Physiologically, ROS regulate vascular function through redox-sensitive signalling pathways. In hypertension, oxidative stress promotes endothelial dysfunction, vascular remodelling, and inflammation, leading to vascular damage. Vascular ROS are derived primarily by nicotinamide adenine dinucleotide phosphate oxidases, which are prime targets for therapeutic development. Although experimental evidence indicates a causative role for oxidative stress in hypertension, human data are less convincing. This might relate, in part, to suboptimal methods to accurately assess the redox state. Herein we review current knowledge on oxidative stress in vascular pathobiology and implications in human hypertension. We also discuss biomarkers to assess the redox state in the clinic, highlight novel strategies to inhibit ROS production, and summarize how lifestyle modifications promote vascular health by reducing oxidative stress.