DLG1 functions upstream of SDCCAG3 and IFT20 to control ciliary targeting of polycystin-2.

Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney caused ciliary elongation and cystogenesis, and cell-based proximity labelling proteomics and fluorescence microscopy showed alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20 and polycystin-2 (PC2) were reduced in cilia of DLG1 deficient cells compared to control cells. This phenotype was recapitulated in vivo and rescuable by re-expression of wildtype DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggested that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.

Plastic debris in lakes and reservoirs.

Plastic debris is thought to be widespread in freshwater ecosystems globally1. However, a lack of comprehensive and comparable data makes rigorous assessment of its distribution challenging2,3. Here we present a standardized cross-national survey that assesses the abundance and type of plastic debris (>250 µm) in freshwater ecosystems. We sample surface waters of 38 lakes and reservoirs, distributed across gradients of geographical position and limnological attributes, with the aim to identify factors associated with an increased observation of plastics. We find plastic debris in all studied lakes and reservoirs, suggesting that these ecosystems play a key role in the plastic-pollution cycle. Our results indicate that two types of lakes are particularly vulnerable to plastic contamination: lakes and reservoirs in densely populated and urbanized areas and large lakes and reservoirs with elevated deposition areas, long water-retention times and high levels of anthropogenic influence. Plastic concentrations vary widely among lakes; in the most polluted, concentrations reach or even exceed those reported in the subtropical oceanic gyres, marine areas collecting large amounts of debris4. Our findings highlight the importance of including lakes and reservoirs when addressing plastic pollution, in the context of pollution management and for the continued provision of lake ecosystem services.

Primary cilia as dynamic and diverse signalling hubs in development and disease.

Primary cilia, antenna-like sensory organelles protruding from the surface of most vertebrate cell types, are essential for regulating signalling pathways during development and adult homeostasis. Mutations in genes affecting cilia cause an overlapping spectrum of >30 human diseases and syndromes, the ciliopathies. Given the immense structural and functional diversity of the mammalian cilia repertoire, there is a growing disconnect between patient genotype and associated phenotypes, with variable severity and expressivity characteristic of the ciliopathies as a group. Recent technological developments are rapidly advancing our understanding of the complex mechanisms that control biogenesis and function of primary cilia across a range of cell types and are starting to tackle this diversity. Here, we examine the structural and functional diversity of primary cilia, their dynamic regulation in different cellular and developmental contexts and their disruption in disease.

The genetic background of hydrocephalus in a population-based cohort: implication of ciliary involvement.

Hydrocephalus is one of the most common congenital disorders of the central nervous system and often displays psychiatric co-morbidities, in particular autism spectrum disorder. The disease mechanisms behind hydrocephalus are complex and not well understood, but some association with dysfunctional cilia in the brain ventricles and subarachnoid space has been indicated. A better understanding of the genetic aetiology of hydrocephalus, including the role of ciliopathies, may bring insights into a potentially shared genetic aetiology. In this population-based case-cohort study, we, for the first time, investigated variants of postulated hydrocephalus candidate genes. Using these data, we aimed to investigate potential involvement of the ciliome in hydrocephalus and describe genotype-phenotype associations with an autism spectrum disorder. One-hundred and twenty-one hydrocephalus candidate genes were screened in a whole-exome-sequenced sub-cohort of the Lundbeck Foundation Initiative for Integrative Psychiatric Research study, comprising 72 hydrocephalus patients and 4181 background population controls. Candidate genes containing high-impact variants of interest were systematically evaluated for their involvement in ciliary function and an autism spectrum disorder. The median age at diagnosis for the hydrocephalus patients was 0 years (range 0-27 years), the median age at analysis was 22 years (11-35 years), and 70.5% were males. The median age for controls was 18 years (range 11-26 years) and 53.3% were males. Fifty-two putative hydrocephalus-associated variants in 34 genes were identified in 42 patients (58.3%). In hydrocephalus cases, we found increased, but not significant, enrichment of high-impact protein altering variants (odds ratio 1.51, 95% confidence interval 0.92-2.51, P = 0.096), which was driven by a significant enrichment of rare protein truncating variants (odds ratio 2.71, 95% confidence interval 1.17-5.58, P = 0.011). Fourteen of the genes with high-impact variants are part of the ciliome, whereas another six genes affect cilia-dependent processes during neurogenesis. Furthermore, 15 of the 34 genes with high-impact variants and three of eight genes with protein truncating variants were associated with an autism spectrum disorder. Because symptoms of other diseases may be neglected or masked by the hydrocephalus-associated symptoms, we suggest that patients with congenital hydrocephalus undergo clinical genetic assessment with respect to ciliopathies and an autism spectrum disorder. Our results point to the significance of hydrocephalus as a ciliary disease in some cases. Future studies in brain ciliopathies may not only reveal new insights into hydrocephalus but also, brain disease in the broadest sense, given the essential role of cilia in neurodevelopment.

Smooth muscle ATP-sensitive potassium channels mediate migraine-relevant hypersensitivity in mouse models.

BACKGROUND: Opening of KATP channels by systemic levcromakalim treatment triggers attacks in migraine patients and hypersensitivity to von Frey stimulation in a mouse model. Blocking of these channels is effective in several preclinical migraine models. It is unknown in what tissue and cell type KATP-induced migraine attacks are initiated and which KATP channel subtype is targeted. METHODS: In mouse models, we administered levcromakalim intracerebroventricularly, intraperitoneally and intraplantarily and compared the nociceptive responses by von Frey and hotplate tests. Mice with a conditional loss-of-function mutation in the smooth muscle KATP channel subunit Kir6.1 were given levcromakalim and GTN and examined with von Frey filaments. Arteries were tested for their ability to dilate ex vivo. mRNA expression, western blotting and immunohistochemical stainings were made to identify relevant target tissue for migraine induced by KATP channel opening. RESULTS: Systemic administration of levcromakalim induced hypersensitivity but central and local administration provided antinociception respectively no effect. The Kir6.1 smooth muscle knockout mouse was protected from both GTN and levcromakalim induced hypersensitivity, and their arteries had impaired dilatory response to the latter. mRNA and protein expression studies showed that trigeminal ganglia did not have significant KATP channel expression of any subtype, whereas brain arteries and dura mater primarily expressed the Kir6.1 + SUR2B subtype. CONCLUSION: Hypersensitivity provoked by GTN and levcromakalim in mice is dependent on functional smooth muscle KATP channels of extracerebral origin. These results suggest a vascular contribution to hypersensitivity induced by migraine triggers.

CGRP-dependent signalling pathways involved in mouse models of GTN- cilostazol- and levcromakalim-induced migraine.

BACKGROUND: Knowledge of exact signalling events during migraine attacks is lacking. Various substances are known to trigger migraine attacks in patients and calcitonin gene-related peptide antagonising drugs are effective against migraine pain. Here, we investigated the signalling pathways involved in three different mouse models of provoked migraine and relate them to calcitonin gene-related peptide and other migraine-relevant targets. METHODS: In vivo mouse models of glyceryl trinitrate-, cilostazol- and levcromakalim-induced migraine were applied utilising tactile sensitivity to von Frey filaments as measuring readout. Signalling pathways involved in the three models were dissected by use of specific knockout mice and chemical inhibitors. In vivo results were supported by ex vivo wire myograph experiments measuring arterial dilatory responses and ex vivo calcitonin gene-related peptide release from trigeminal ganglion and trigeminal nucleus caudalis from mice. RESULTS: Glyceryl trinitrate-induced hypersensitivity was dependent on both prostaglandins and transient receptor potential cation channel, subfamily A, member 1, whereas cilostazol- and levcromakalim-induced hypersensitivity were independent of both. All three migraine triggers activated calcitonin gene-related peptide signalling, as both receptor antagonism and antibody neutralisation of calcitonin gene-related peptide were effective inhibitors of hypersensitivity in all three models. Stimulation of trigeminal ganglia and brain stem tissue samples with cilostazol and levcromakalim did not result in release of calcitonin gene-related peptide, and vasodilation following levcromakalim stimulation was independent of CGRP receptor antagonism. CONCLUSION: The mouse models of glyceryl trinitrate-, cilostazol- and levcromakalim- induced migraine all involve calcitonin gene-related peptide signalling in a complex interplay between different cell/tissue types. These models are useful in the study of migraine mechanisms.

ALMS1 Regulates TGF-ß Signaling and Morphology of Primary Cilia.

In this study, we aimed to evaluate the role of ALMS1 in the morphology of primary cilia and regulation of cellular signaling using a knockdown model of the hTERT-RPE1 cell line. ALMS1 depletion resulted in the formation of longer cilia, which often displayed altered morphology as evidenced by extensive twisting and bending of the axoneme. Transforming growth factor beta/bone morphogenetic protein (TGF-ß/BMP) signaling, which is regulated by primary cilia, was similarly affected by ALMS1 depletion as judged by reduced levels of TGFß-1-mediated activation of SMAD2/3. These results provide novel information on the role of ALMS1 in the function of primary cilia and processing of cellular signaling, which when aberrantly regulated may underlie Alström syndrome.

Human RTEL1 associates with Poldip3 to facilitate responses to replication stress and R-loop resolution.

RTEL1 helicase is a component of DNA repair and telomere maintenance machineries. While RTEL1's role in DNA replication is emerging, how RTEL1 preserves genomic stability during replication remains elusive. Here we used a range of proteomic, biochemical, cell, and molecular biology and gene editing approaches to provide further insights into potential role(s) of RTEL1 in DNA replication and genome integrity maintenance. Our results from complementary human cell culture models established that RTEL1 and the Polδ subunit Poldip3 form a complex and are/function mutually dependent in chromatin binding after replication stress. Loss of RTEL1 and Poldip3 leads to marked R-loop accumulation that is confined to sites of active replication, enhances endogenous replication stress, and fuels ensuing genomic instability. The impact of depleting RTEL1 and Poldip3 is epistatic, consistent with our proposed concept of these two proteins operating in a shared pathway involved in DNA replication control under stress conditions. Overall, our data highlight a previously unsuspected role of RTEL1 and Poldip3 in R-loop suppression at genomic regions where transcription and replication intersect, with implications for human diseases including cancer.

Analysis of Caveolin in Primary Cilia.

Recent evidence has indicated that caveolins are localized at the base of primary cilia, which are microtubule-based sensory organelles present on the cell surface, and that Caveolin-1 (CAV1) plays important roles in regulating ciliary membrane composition and function. Here we describe methods to analyze the localization and function of CAV1 in primary cilia of cultured mammalian cells. These include methods for culturing and transfecting mammalian cells with a CAV1-encoding plasmid or small interfering RNA (siRNA), analysis of mammalian cells by immunofluorescence microscopy (IFM) with antibodies against ciliary markers and CAV1, as well as methods for analyzing ciliary CAV1 function in siRNA-treated cells by IFM and cell-based signaling assays.

TGFß Signaling Increases Net Acid Extrusion, Proliferation and Invasion in Panc-1 Pancreatic Cancer Cells: SMAD4 Dependence and Link to Merlin/NF2 Signaling.

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer-related death, with a 5-year survival of <10% and severely limited treatment options. PDAC hallmarks include profound metabolic acid production and aggressive local proliferation and invasiveness. This phenotype is supported by upregulated net acid extrusion and epithelial-to-mesenchymal transition (EMT), the latter typically induced by aberrant transforming growth factor-ß (TGFß) signaling. It is, however, unknown whether TGFß-induced EMT and upregulation of acid extrusion are causally related. Here, we show that mRNA and protein expression of the net acid extruding transporters Na+/H+ exchanger 1 (NHE1, SLC9A1) and Na+, HCO 3 - cotransporter 1 (NBCn1, SLC4A7) are increased in a panel of human PDAC cell lines compared to immortalized human pancreatic ductal epithelial (HPDE) cells. Treatment of Panc-1 cells (which express SMAD4, required for canonical TGFß signaling) with TGFß-1 for 48 h elicited classical EMT with down- and upregulation of epithelial and mesenchymal markers, respectively, in a manner inhibited by SMAD4 knockdown. Accordingly, less pronounced EMT was induced in BxPC-3 cells, which do not express SMAD4. TGFß-1 treatment elicited a SMAD4-dependent increase in NHE1 expression, and a smaller, SMAD4-independent increase in NBCn1 in Panc-1 cells. Consistent with this, TGFß-1 treatment led to elevated intracellular pH and increased net acid extrusion capacity in Panc-1 cells, but not in BxPC-3 cells, in an NHE1-dependent manner. Proliferation was increased in Panc-1 cells and decreased in BxPC-3 cells, upon TGFß-1 treatment, and this, as well as EMT per se, was unaffected by NHE1- or NBCn1 inhibition. TGFß-1-induced EMT was associated with a 4-fold increase in Panc-1 cell invasiveness, which further increased ~10-fold upon knockdown of the tumor suppressor Merlin (Neurofibromatosis type 2). Knockdown of NHE1 or NBCn1 abolished Merlin-induced invasiveness, but not that induced by TGFß-1 alone. In conclusion, NHE1 and NBCn1 expression and NHE-dependent acid extrusion are upregulated during TGFß-1-induced EMT of Panc-1 cells. NHE1 upregulation is SMAD4-dependent, and SMAD4-deficient BxPC-3 cells show no change in pHi regulation. NHE1 and NBCn1 are not required for EMT per se or EMT-associated proliferation changes, but are essential for the potentiation of invasiveness induced by Merlin knockdown.

Electro convulsive therapy: Modification of its effect on the autonomic nervous system using anti-cholinergic drugs.

The antidepressant efficacy of electroconvulsive therapy (ECT) is correlated to the quality of the seizure as measured by EEG but has also been linked to the magnitude of changes in hemodynamic variables. Muscarinic receptor antagonists are frequently used in the treatment, and are known to affect the hemodynamic response. We hypothesized that atropine and glycopyrrolate alter the hemodynamic and autonomic hormonal response to ECT. In a randomized, cross-over study design 23 patients received either atropine, glycopyrrolate or placebo before ECT. Hemodynamic variable, EEG and EMG, and blood adrenaline, noradrenaline and pancreatic polypeptide was determined. No geriatric patients were included. Hemodynamic changes with ECT can be divided into three phases: Drop in blood pressure and pulse rate in 1st post-stimulus phase was less when using 1 mg atropine. In 2nd post-stimulus phase atropine gave a higher systolic blood pressure. No differences were seen in hormone levels after ECT in the three interventions. A significant longer tonic clonic seizure was seen in the glycopyrrolate group and a tendency of the same was seen with atropine. The study found that the changes in hemodynamic variables induced by ECT can be altered by concomitant administration of muscarinic receptor antagonist.

CEP128 Localizes to the Subdistal Appendages of the Mother Centriole and Regulates TGF-ß/BMP Signaling at the Primary Cilium.

The centrosome is the main microtubule-organizing center in animal cells and comprises a mother and daughter centriole surrounded by pericentriolar material. During formation of primary cilia, the mother centriole transforms into a basal body that templates the ciliary axoneme. Ciliogenesis depends on mother centriole-specific distal appendages, whereas the role of subdistal appendages in ciliary function is unclear. Here, we identify CEP128 as a centriole subdistal appendage protein required for regulating ciliary signaling. Loss of CEP128 did not grossly affect centrosomal or ciliary structure but caused impaired transforming growth factor-ß/bone morphogenetic protein (TGF-ß/BMP) signaling in zebrafish and at the primary cilium in cultured mammalian cells. This phenotype is likely the result of defective vesicle trafficking at the cilium as ciliary localization of RAB11 was impaired upon loss of CEP128, and quantitative phosphoproteomics revealed that CEP128 loss affects TGF-ß1-induced phosphorylation of multiple proteins that regulate cilium-associated vesicle trafficking.

KIF13B establishes a CAV1-enriched microdomain at the ciliary transition zone to promote Sonic hedgehog signalling.

Ciliary membrane composition is controlled by transition zone (TZ) proteins such as RPGRIP1, RPGRIPL and NPHP4, which are vital for balanced coordination of diverse signalling systems like the Sonic hedgehog (Shh) pathway. Activation of this pathway involves Shh-induced ciliary accumulation of Smoothened (SMO), which is disrupted by disease-causing mutations in TZ components. Here we identify kinesin-3 motor protein KIF13B as a novel member of the RPGRIP1N-C2 domain-containing protein family and show that KIF13B regulates TZ membrane composition and ciliary SMO accumulation. KIF13B is upregulated during ciliogenesis and is recruited to the ciliary base by NPHP4, which binds to two distinct sites in the KIF13B tail region, including an RPGRIP1N-C2 domain. KIF13B and NPHP4 are both essential for establishment of a CAV1 membrane microdomain at the TZ, which in turn is required for Shh-induced ciliary SMO accumulation. Thus KIF13B is a novel regulator of ciliary TZ configuration, membrane composition and Shh signalling.

Mutation of the Planar Cell Polarity Gene VANGL1 in Adolescent Idiopathic Scoliosis.

STUDY DESIGN: Mutation analysis of a candidate disease gene in a cohort of patients with moderate to severe Adolescent idiopathic scoliosis (AIS). OBJECTIVE: To investigate if damaging mutations in the planar cell polarity gene VANGL1 could be identified in AIS patients. SUMMARY OF BACKGROUND DATA: AIS is a spinal deformity which occurs in 1% to 3% of the population. The cause of AIS is often unknown, but genetic factors are important in the etiology. Rare variants in genes encoding regulators of WNT/planar cell polarity (PCP) signaling were recently identified in AIS patients. METHODS: We analyzed the coding region of the VANGL1 gene for mutations using Sanger sequencing in 157 unrelated patients with moderate to severe AIS. The frequency of mutations in the patient cohort was compared with their frequency in a large cohort of controls. Functional effect of mutations were predicted in silico and analyzed in vitro by transfection of normal and mutant recombinant VANGL1 protein in Madin-Darby Canine Kidney (MDCK) cells. Cellular localization of recombinant proteins was analyzed by immunofluorescence microscopy analysis. RESULTS: In the patient cohort, we identified two rare missense mutations in VANGL1, encoding a receptor involved in WNT/PCP signaling. The mutations, p.I136N and p.F440 V, are very rare in the normal population. Both mutations are predicted to be damaging, and to affect evolutionary conserved amino acid residues of VANGL1. Functional analysis in MDCK cells showed that the mutations abolished the normal translocation of VANGL1 to the cell membrane. CONCLUSION: Our data support that mutations in genes involved in WNT/PCP signaling may be associated with AIS, but replication in other patient cohorts and further analysis of the role of WNT/PCP signaling in AIS is needed. LEVEL OF EVIDENCE: 4.

Primary Cilia and Coordination of Receptor Tyrosine Kinase (RTK) and Transforming Growth Factor ß (TGF-ß) Signaling.

Since the beginning of the millennium, research in primary cilia has revolutionized our way of understanding how cells integrate and organize diverse signaling pathways during vertebrate development and in tissue homeostasis. Primary cilia are unique sensory organelles that detect changes in their extracellular environment and integrate and transmit signaling information to the cell to regulate various cellular, developmental, and physiological processes. Many different signaling pathways have now been shown to rely on primary cilia to function properly, and mutations that lead to ciliary dysfunction are at the root of a pleiotropic group of diseases and syndromic disorders called ciliopathies. In this review, we present an overview of primary cilia-mediated regulation of receptor tyrosine kinase (RTK) and transforming growth factor ß (TGF-ß) signaling. Further, we discuss how defects in the coordination of these pathways may be linked to ciliopathies.

TGFß1 - induced recruitment of human bone mesenchymal stem cells is mediated by the primary cilium in a SMAD3-dependent manner.

The recruitment of mesenchymal stem cells (MSCs) is a crucial process in the development, maintenance and repair of tissues throughout the body. Transforming growth factor-ß1 (TGFß1) is a potent chemokine essential for the recruitment of MSCs in bone, coupling the remodelling cycle. The primary cilium is a sensory organelle with important roles in bone and has been associated with cell migration and more recently TGFß signalling. Dysregulation of TGFß signalling or cilia has been linked to a number of skeletal pathologies. Therefore, this study aimed to determine the role of the primary cilium in TGFß1 signalling and associated migration in human MSCs. In this study we demonstrate that low levels of TGFß1 induce the recruitment of MSCs, which relies on proper formation of the cilium. Furthermore, we demonstrate that receptors and downstream signalling components in canonical TGFß signalling localize to the cilium and that TGFß1 signalling is associated with activation of SMAD3 at the ciliary base. These findings demonstrate a novel role for the primary cilium in the regulation of TGFß signalling and subsequent migration of MSCs, and highlight the cilium as a target to manipulate this key pathway and enhance MSC recruitment for the treatment of skeletal diseases.

Endocytic Control of Cellular Signaling at the Primary Cilium.

Primary cilia are dynamic signaling organelles that project from the cell surface to sense diverse chemical, physical and morphogenetic cues. Ciliary defects therefore cause diseases (ciliopathies) that affect multiple organs in developing and adult organisms. Cilia-mediated signaling involves the orchestrated movement of signaling proteins in and out of the ciliary compartment, including movement of receptors such as the Sonic Hedgehog (Shh) receptor Patched 1 (PTCH1), Smoothened (SMO), and various other G protein-coupled receptors (GPCRs), as well as transforming growth factor ß (TGF-ß) receptors I and II (TGF-ß-RI/II). We provide here a current understanding of trafficking events associated with cilia-mediated signaling, with emphasis on the involvement of clathrin-dependent receptor-mediated endocytosis in regulating ciliary Shh and TGF-ß signaling.

The intraflagellar transport machinery in ciliary signaling.

Cilia and flagella on eukaryotic cells are slender microtubule-based projections surrounded by a membrane with a unique lipid and protein composition. It is now appreciated that cilia in addition to their established roles in motility also constitute hubs for cellular signaling by sensing external environmental cues necessary for organ development and maintenance of human health. Pathways reported to rely on the cilium organelle include Hedgehog, TGF-ß, Wnt, PDGFRα, integrin and DNA damage repair signaling. An emerging theme in ciliary signaling is the requirement for active transport of signaling components into and out of the cilium proper. Here, we review the current state-of-the-art regarding the importance of intraflagellar transport and BBSome multi-subunit complexes in ciliary signaling.