bims-bicyki Biomed News
on Bicaudal-C1 and interactors in cystic kidney disease
Issue of 2022–01–16
seventeen papers selected by
Céline Gagnieux, École Polytechnique Fédérale de Lausanne
  1. Large deletion of Wdr19 in developing renal tubules disrupts primary ciliogenesis leading to polycystic kidney disease in mice.
  2. Repeated aortic dissection in a patient with autosomal dominant polycystic kidney disease.
  3. Autosomal Dominant Polycystic Kidney Disease (ADPKD) in Tunisia: from molecular genetics to the development of prognostic tools.
  4. Polycystic horseshoe kidney case report: Genetically reviewed.
  5. Prevalence and predicting factors of increased arterial stiffness in autosomal dominant polycystic kidney disease.
  6. Quantitative Determination of Primary Cilia Protein Distribution Using Immunofluorescence Staining and MATLAB Analysis.
  7. Impact of Motile Ciliopathies on Human Development and Clinical Consequences in the Newborn.
  8. RHOA inhibits chondrogenic differentiation of mesenchymal stem cells in adolescent idiopathic scoliosis.
  9. Comparative culture of human corneal endothelial cells following treatment with human platelet lysate/fibrin hydrogel versus Y-27632 ROCK inhibitor: in vitro and ex vivo study.
  10. Targeted next-generation sequencing in a large series of fetuses with severe renal diseases.
  11. Swyer-James syndrome: A cause of adult-onset dyspnea in a patient with adult polycystic kidney disease.
  12. Role of the polycystic kidney disease domain in matriptase chaperone activity and localization of hepatocyte growth factor activator inhibitor-1.
  13. The complex web of canonical and non-canonical Hedgehog signaling.
  14. CRISPR/Cas9 genome editing system confirms centriolin's role in cytokinesis.
  15. KIAA0319 influences cilia length, cell migration and mechanical cell-substrate interaction.
  16. PKA and Epac1 Reduce Nek7 to Block the NLRP3 Inflammasome Proteins in the Retinal Vasculature.
  17. FOXO3/TGF-β signal-dependent ciliogenesis and cell functions during differentiation of temperature-sensitive mouse cochlear precursor hair cells.
  1. J Pathol. 2022 Jan 10.
    Large deletion of Wdr19 in developing renal tubules disrupts primary ciliogenesis leading to polycystic kidney disease in mice.
    Shang-Shiuan Yu, Ellian Wang, Chih-Ying Chiang, Po-Hao Cheng, Yu-Shan Yeh, Ying-Ying Wu, Yuan-Yow Chiou, Si-Tse Jiang.
      WD repeat domain 19 (Wdr19) is a major component of the intraflagellar transport (IFT) machinery, which is involved in the function of primary cilia. However, the effects of Wdr19 on primary cilia formation, cystogenesis, and polycystic kidney disease (PKD) progression remain unclear. To study these effects, we generated three lines of kidney-specific conditional knockout mice: Wdr19-knockout (Wdr19-KO, Wdr19f/- ::Cdh16-CreTg/0 ), Pkd1-knockout (Pkd1-KO, Pkd1f/- ::Cdh16-CreTg/0 ), and Wdr19/Pkd1-double knockout (Wdr19&Pkd1-dKO, Wdr19f/- ;Pkd1f/- ::Cdh16-CreTg/0 ) mice. Ultrastructural analysis using transmission electron microscopy (TEM) indicated that the primary cilia were almost absent at postnatal day 10 in Wdr19-KO mice compared with Pkd1-KO and wild-type (WT) mice. However, the primary cilia appeared structurally normal even if malfunctional in Pkd1-deficient cysts. The Pkd1-KO mice had the most severe PKD progression, including the shortest lifespan (14 days) and the largest renal cysts, among the three knockout lines. Thus, the molecular mechanism of renal cystogenesis in Wdr19-KO mice (primary cilia abrogation) was different from that in Pkd1-KO mice (primary cilia malfunction). In summary, Wdr19 deficiency leads to primary cilia abrogation and renal cyst formation. Wdr19 is primarily proposed to participate in retrograde IFT and to be crucial for the construction of primary cilia, which are critical organelles for tubulogenesis in the developing kidneys. This article is protected by copyright. All rights reserved.
    Keywords:  Wdr19; intraflagellar transport; polycystic kidney disease; primary cilia; renal cyst
    DOI:  https://doi.org/10.1002/path.5863
  2. Gen Thorac Cardiovasc Surg. 2022 Jan 11.
    Repeated aortic dissection in a patient with autosomal dominant polycystic kidney disease.
    Kimihiro Kobayashi, Yoshinori Kuroda, Shingo Nakai, Tetsuro Uchida.
      Autosomal dominant polycystic kidney disease is a systemic disorder associated with cardiovascular complications. However, there are few reports on autosomal dominant polycystic kidney disease-associated aortic dissection. Herein, we present a rare case of a 46-year-old man with autosomal dominant polycystic kidney disease who underwent endovascular repair for acute type B aortic dissection three years after his initial open surgery for acute type A aortic dissection. The postoperative course was uneventful, and he is doing well two years after the endovascular repair. Clinicians should be aware of the potential for occurrence of aortic dissection in patients with autosomal dominant polycystic kidney disease, including the possibility of recurrence.
    Keywords:  Aortic dissection; Autosomal dominant polycystic kidney disease
    DOI:  https://doi.org/10.1007/s11748-021-01756-5
  3. Gene. 2022 Jan 11. pii: S0378-1119(21)00769-1. [Epub ahead of print] 146174
    Autosomal Dominant Polycystic Kidney Disease (ADPKD) in Tunisia: from molecular genetics to the development of prognostic tools.
    Mayssa Abdelwahed, Pascale Hilbert, Asma Ahmed, Mouna Dey, Salem Bouomrani, Hassen Kamoun, Leila Ammar-Keskes, Neila Belguith.
      
    DOI:  https://doi.org/10.1016/j.gene.2021.146174
  4. Saudi J Kidney Dis Transpl. 2021 Mar-Apr;32(2):32(2): 574-578
    Polycystic horseshoe kidney case report: Genetically reviewed.
    Saad S Alobaili, Sarah M Aljasser, Amal S Asseri, Daad A Alotaibi.
      Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic renal disease, with a reported prevalence ranging from one in 400 to one in 1000. ADPKD accounts for as high as 10% of end-stage renal disease cases. It is characterized by cystic formation replacing kidney parenchyma leading to renal enlargement and renal functional impairment. Consequently, it is associated with renal and extrarenal complications contributing to high mortality. On the other hand, horseshoe kidney (HSK) is a common congenital renal anomaly, with an incidence ranging between one in 400 and 600. Surprisingly, the coexistence of both distinct common clinical conditions is extremely rare, and it is thought that the incidence of polycystic HSK varies from one in 134,000 to one in 8,000,000 cases. Although the particular genetic association is not established, familial cases raise the question of whether they are related. We report this case to cultivate the current medical literature regarding this rare entity.
    DOI:  https://doi.org/10.4103/1319-2442.335474
  5. Saudi J Kidney Dis Transpl. 2021 Mar-Apr;32(2):32(2): 481-487
    Prevalence and predicting factors of increased arterial stiffness in autosomal dominant polycystic kidney disease.
    Nada Sellami, Rania Kheder-El Fekih, Hela Jebali, Ikram Mami, Wided Smaoui, Fethi Ben Hmida, Mohamed Karim Zouaghi, Lilia Ben Fatma, Lamia Rais.
      Carotid-femoral pulse wave velocity (cf-PWV) is the noninvasive gold standard technique for measuring aortic stiffness. Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic renal disease in adults. It is associated with a high risk of cardiovascular complications. We aimed to assess the prevalence of increased arterial stiffness and its predicting factors in a population of ADPKD patients. Sixty-two patients with ADPKD underwent noninvasive measurement of cf-PWV using a COMPLIOR Analyse device. Recruitment period was 17 months and we used the cut-off of 10 m/s to define a high cf-PWV. Mean age was 51 ± 12.7 years. Gender ratio male/female was 0.63. Smoking, hypertension (HTN), and dyslipidemia were reported in 14%, 66%, and 27% of the cases, respectively. Mean glomerular filtration rate (GFR) was 47.7 ± 44 mL/min/1.73 m2. Among our patients, 39% had chronic kidney disease stages 1 or 2 and 45% stage 5 (40% stage 5D). Mean cf-PWV was 9 ± 2.4 m/s, and 31% of the patients had a high cf-PWV. In univariate analysis of all our patients, cf- PWV correlated with age (r = 0.565; P <10-3), GFR (r = -0.268;P = 0.035), C-reactive protein (r = 0.447; P = 0.007), peripheral systolic arterial pressure (r = 0.309; P = 0.015), and peripheral pulse pressure (r = 0.335; P = 0.008). Patients with high cf-PWV were on average nine years older than the others. Patients with HTN were 3.84 times more likely to have high cf-PWV (P = 0.046). cf-PWV did not seem to be lower with any antihypertensive treatment. A level of C-reactive protein higher than 10 mg/L was the only independent predicting factor of a high cf-PWV in multivariate analysis (P = 0.043). Our study confirmed the relationship between cf-PWV and age, renal failure, and HTN in patients with ADPKD. It also emphasized the close relationship between systemic inflammation and arterial stiffness in this nephropathy.
    DOI:  https://doi.org/10.4103/1319-2442.335460
  6. Bio Protoc. 2021 Dec 05. 11(23): e4248
    Quantitative Determination of Primary Cilia Protein Distribution Using Immunofluorescence Staining and MATLAB Analysis.
    Andrew D Jenks, Marc Fivaz, Barbara E Tanos.
      Primary cilia are microtubule-based sensory organelles surrounded by membrane. They can detect mechanical and chemical stimuli. The last few years have uncovered cilia as unique signaling hubs that host a number of receptors and effector molecules. Thus, defining how specific proteins localize and are distributed along the cilium is critical to understanding its function. Quantitative immunofluorescence can be used to accurately assess the localization of receptors and signaling molecules within the primary cilia. However, image analysis can be time consuming, and there are limited programs that can accurately determine staining intensity along the cilia. To overcome these issues, we developed a series of MATLAB scripts to accurately measure staining intensity along the length of the cilia, in both a semi-automated and automated fashion. Here, we describe the scripts and include a protocol for image analysis for each. With these scripts, the protocols can be used to analyze the distribution of any ciliary protein using immunofluorescence images.
    Keywords:  Acetylated tubulin; Arl13B; Cilia; Imaging; Immunofluorescence; Quantification
    DOI:  https://doi.org/10.21769/BioProtoc.4248
  7. Cells. 2021 Dec 31. pii: 125. [Epub ahead of print]11(1):
    Impact of Motile Ciliopathies on Human Development and Clinical Consequences in the Newborn.
    Rachael M Hyland, Steven L Brody.
      Motile cilia are hairlike organelles that project outward from a tissue-restricted subset of cells to direct fluid flow. During human development motile cilia guide determination of the left-right axis in the embryo, and in the fetal and neonatal periods they have essential roles in airway clearance in the respiratory tract and regulating cerebral spinal fluid flow in the brain. Dysregulation of motile cilia is best understood through the lens of the genetic disorder primary ciliary dyskinesia (PCD). PCD encompasses all genetic motile ciliopathies resulting from over 60 known genetic mutations and has a unique but often underrecognized neonatal presentation. Neonatal respiratory distress is now known to occur in the majority of patients with PCD, laterality defects are common, and very rarely brain ventricle enlargement occurs. The developmental function of motile cilia and the effect and pathophysiology of motile ciliopathies are incompletely understood in humans. In this review, we will examine the current understanding of the role of motile cilia in human development and clinical considerations when assessing the newborn for suspected motile ciliopathies.
    Keywords:  human development; motile cilia; neonate; primary ciliary dyskinesia
    DOI:  https://doi.org/10.3390/cells11010125
  8. Connect Tissue Res. 2022 Jan 12. 1-10
    RHOA inhibits chondrogenic differentiation of mesenchymal stem cells in adolescent idiopathic scoliosis.
    Mingyuan Yang, Kai Chen, Canglong Hou, Yilin Yang, Xiao Zhai, Kai Chen, Xianzhao Wei, Yushu Bai, Ming Li.
       PURPOSE: The etiology of adolescent idiopathic scoliosis (AIS) remains unclear. The chondrogenic differentiation of mesenchymal stem cells (MSCs) is important in AIS, and the Ras homolog gene family member A (RHOA) is associated with chondrogenesis. The purpose of this study was to explore the effect of RHOA on the chondrogenic differentiation of MSCs in AIS.
    METHODS: We isolated MSCs from patients with AIS (AIS MSCs) and individuals without AIS (control MSCs). The inhibitor Y27632 was used to inhibit the function of RHOA/ROCK signaling, and plasmid-based overexpression and siRNA-mediated knockdown were used to manipulate RHOA expression. CCK-8 was used to detect cell viability. The phosphorylation levels of LIMK1, MLC2 and cofilin were detected by Western blotting. The mRNA expression of aggrecan, SOX9, and COL2A1 were confirmed using RT-PCR. Immunofluorescence was used to analyze F-actin and collagen II. Alcian blue staining was performed to assess the secretion of glycosaminoglycans (GAGs).
    RESULTS: We found that RHOA was significantly upregulated in AIS MSCs, and the phosphorylation levels of LIMK1, MLC2, and cofilin were increased. The mRNA expressions of aggrecan, SOX9, and COL2A1 were notably reduced in AIS MSCs. However, these effects were abolished by Y27632 treatment and RHOA knockdown in AIS MSCs. In addition, RHOA knockdown in AIS MSCs increased the content of collagen II and GAGs. RHOA overexpression in the control MSCs markedly activated the RHOA/ROCK signaling and decreased the expression of aggrecan, SOX9, and COL2A1, F-actin, and GAGs.
    CONCLUSION: RHOA regulates the chondrogenic differentiation ability of MSCs in AIS via the RHOA/ROCK signaling pathway and this regulation may involve SOX9.
    Keywords:  Chondrogenic differentiation; RHOA/ROCK signaling pathway; adolescent idiopathic scoliosis; mesenchymal stem cells
    DOI:  https://doi.org/10.1080/03008207.2021.2019247
  9. Int Ophthalmol. 2022 Jan 13.
    Comparative culture of human corneal endothelial cells following treatment with human platelet lysate/fibrin hydrogel versus Y-27632 ROCK inhibitor: in vitro and ex vivo study.
    Mohammad Amir Mishan, Sahar Balagholi, Tahereh Chamani, Sepehr Feizi, Zahra-Soheila Soheili, Mozhgan Rezaei Kanavi.
       PURPOSE: The advancement of tissue engineering and cell therapy research has resulted in innovative therapeutic options for patients with corneal endothelial diseases. The aim of this study was to compare the potential effect of using human platelet lysate (HPL)/Fibrin hydrogel versus using a Y-27632 ROCK inhibitor, on the culture of human corneal endothelial cells (HCECs) under in vitro and ex vivo conditions.
    METHODS: HCECs were isolated from human donors and treated separately with HPL/Fibrin hydrogel, a Y-27632 ROCK inhibitor, and fetal bovine serum (FBS). MTT viability assay and cell counting were performed on the treated cells. Subsequently, we prepared ex vivo models of human corneal endothelial dysfunction and incubated them with DiI-labeled-HCECs. Specular and fluorescence microscopy were then performed on each of the ex vivo models.
    RESULTS: In comparison, similar viability results were achieved in the cells treated with HPL/Fibrin hydrogel versus those treated with the Y-27632 ROCK inhibitor, but both treatments showed higher viability than the control group (FBS). More importantly, based on the specular and fluorescence microscopic results, the HPL/Fibrin hydrogel and the Y-27632 ROCK inhibitor treatments showed similar inducible effects on the attachment of the cells to the Descemet membranes of the ex vivo models.
    CONCLUSION: HPL/Fibrin hydrogel and Y-27632 ROCK inhibitor have similar inducible effects on the viability and attachment of the HCECs. A definite advantage of treating cells with HPL/Fibrin hydrogel is that it serves as a xeno-free and biocompatible material which can have autologous applications in future usage by clinics.
    Keywords:  Corneal endothelial cells; Fibrin; Human platelet lysate; Y-27632 ROCK inhibitor
    DOI:  https://doi.org/10.1007/s10792-021-02136-x
  10. Hum Mutat. 2022 Jan 10.
    Targeted next-generation sequencing in a large series of fetuses with severe renal diseases.
    Penelope Jordan, Guillaume Dorval, Christelle Arrondel, Vincent Morinière, Carole Tournant, Marie-Pierre Audrezet, Laurence Michel-Calemard, Audrey Putoux, Gaethan Lesca, Audrey Labalme, Sandra Whalen, Laurence Loeuillet, Jelena Martinovic, Tania Attie-Bitach, Bettina Bessières, Elise Schaefer, Sophie Scheidecker, Laetitia Lambert, Claire Beneteau, Olivier Patat, Odile Boute-Benejean, Arnaud Molin, Fabien Guimiot, Nicolas Fontanarosa, Mathilde Nizon, Mathilde Lefebvre, Cécile Jeanpierre, Sophie Saunier, Laurence Heidet.
      We report the screening of a large panel of genes in a series of 100 fetuses (98 families) affected with severe renal defects. Causative variants were identified in 22% of cases, greatly improving genetic counseling. The percentage of variants explaining the phenotype was different according to the type of phenotype. The highest diagnostic yield was found in cases affected with the ciliopathy-like phenotype (11/15 families and, in addition, a single heterozygous or a homozygous Class 3 variant in PKHD1 in three unrelated cases with autosomal recessive polycystic kidney disease). The lowest diagnostic yield was observed in cases with congenital anomalies of the kidney and urinary tract (9/78 families and, in addition, Class 3 variants in GREB1L in three unrelated cases with bilateral renal agenesis). Inheritance was autosomal recessive in nine genes (PKHD1, NPHP3, CEP290, TMEM67, DNAJB11, FRAS1, ACE, AGT, and AGTR1), and autosomal dominant in six genes (PKD1, PKD2, PAX2, EYA1, BICC1, and MYOCD). Finally, we developed an original approach of next-generation sequencing targeted RNA sequencing using the custom capture panel used for the sequencing of DNA, to validate one MYOCD heterozygous splicing variant identified in two male siblings with megabladder and inherited from their healthy mother.
    Keywords:  NGS targeted RNA sequencing; congenital abnormalities of the kidney and urinary tract; fetal renal diseases; renal ciliopathies; renal tubular dysgenesis
    DOI:  https://doi.org/10.1002/humu.24324
  11. Respir Med Case Rep. 2022 ;36 101569
    Swyer-James syndrome: A cause of adult-onset dyspnea in a patient with adult polycystic kidney disease.
    Ahel El Haj Chehade, Himanshu Bhardwaj.
      Swyer-James syndrome (SJS) is a rare lung condition characterized by abnormal lung growth secondary to childhood post-infectious bronchiolitis obliterans. Usually, one lung is affected more than the other leading to asymmetrical lungs with one lung being significantly smaller. The disease can lead to pulmonary obstructive airflow physiology, bronchiectasis, and fibrosis. Dyspnea usually presents early on in infancy and symptoms can mimic asthma, however, they can go unnoticed until adulthood. We present a case of SJS in a patient with adult polycystic kidney disease (ADPKD) and color vision deficiency. The patient presented to our clinic for evaluation for progressively worsening dyspnea and cough. His imaging revealed a hypoplastic left lung with fibrosis, cystic airway disease, and a small left pulmonary artery. His spirometry revealed an obstructive defect. A Ventilation-Perfusion scan (V/Q) showed a significant reduction of ventilation and perfusion to his left lung confirming the diagnosis of SJS. Both conditions - SJS and ADPKD-are not pathologically or genetically related and are very rare. Having both conditions is even rarer yielding interesting radiological imaging.
    Keywords:  Adult polycystic kidney disease; Bronchiectasis; Dyspnea; Lung hypoplasia; Swyer-james syndrome
    DOI:  https://doi.org/10.1016/j.rmcr.2021.101569
  12. FEBS J. 2022 Jan 12.
    Role of the polycystic kidney disease domain in matriptase chaperone activity and localization of hepatocyte growth factor activator inhibitor-1.
    Fumiki Yamashita, Takashi Kaieda, Takeshi Shimomura, Makiko Kawaguchi, Chen-Yong Lin, Michael D Johnson, Hiroyuki Tanaka, Takumi Kiwaki, Tsuyoshi Fukushima, Hiroaki Kataoka.
      Hepatocyte growth factor activator inhibitor-1 (HAI-1, also known as SPINT1) is an inhibitor of matriptase, a type-2 transmembrane protease widely expressed in epithelial cells. HAI-1 also functions as a chaperone to maintain the processing and localization of matriptase required for epithelial integrity. However, mechanisms underpinning the chaperone function remain to be elucidated. Here, we show that the first Kunitz domain (KD1) and the adjacent polycystic kidney disease (PKD) domain-like internal domain of HAI-1 are essential for the chaperone function. In HEK293T cells, which do not express endogenous HAI-1 or matriptase, forced matriptase overexpression was unsuccessful unless sufficient HAI-1 was co-expressed. Among mutant HAI-1 constructs, HAI-1 with inactivation mutation in KD1 (HAI-1mKD1) or HAI-1 lacking the PKD domain (HAI-1dPKD) was unable to support matriptase expression, and neither mutant formed a complex with activated matriptase. Matriptase did not localize to the cell surface when co-expressed with HAI-1dPKD. Moreover, HAI-1dPKD accumulated in the cytoplasm of HEK293T and HaCaT cells rather than localizing to the cell surface, presumably due to misfolding as judged by altered antibody recognition. On the other hand, activation-locked and activity-incompetent matriptase were stable and readily overexpressed and localized to the cell surface without HAI-1. Therefore, the observed matriptase instability was caused by its own catalytic activity in the absence of inhibitory HAI-1. The matriptase chaperone function of HAI-1 is thus mediated primarily by the inhibition of undesired intracellular matriptase activity, and the PKD domain is essential for the proper folding and trafficking of inhibitory HAI-1 and its chaperone function.
    Keywords:   SPINT1 ; HAI-1; Kunitz domain; PKD domain; matriptase
    DOI:  https://doi.org/10.1111/febs.16348
  13. Bioessays. 2022 Jan 09. e2100183
    The complex web of canonical and non-canonical Hedgehog signaling.
    Tara Akhshi, Rachel Shannon, William S Trimble.
      Hedgehog (Hh) signaling is a widely studied signaling pathway because of its critical roles during development and in cell homeostasis. Vertebrate canonical and non-canonical Hh signaling are typically assumed to be distinct and occur in different cellular compartments. While research has primarily focused on the canonical form of Hh signaling and its dependency on primary cilia - microtubule-based signaling hubs - an extensive list of crucial functions mediated by non-canonical Hh signaling has emerged. Moreover, amounting evidence indicates that canonical and non-canonical modes of Hh signaling are interlinked, and that they can overlap spatially, and in many cases interact functionally. Here, we discuss some of the many cellular effects of non-canonical signaling and discuss new evidence indicating inter-relationships with canonical signaling. We discuss how Smoothened (Smo), a key component of the Hh pathway, might coordinate such diverse downstream effects. Collectively, pursuit of questions such as those proposed here will aid in elucidating the full extent of Smo function in development and advance its use as a target for cancer therapeutics.
    Keywords:  cancer therapy; development; non-canonical Hedgehog signaling; primary cilia; smoothened
    DOI:  https://doi.org/10.1002/bies.202100183
  14. BMC Res Notes. 2022 Jan 10. 15(1): 8
    CRISPR/Cas9 genome editing system confirms centriolin's role in cytokinesis.
    Eric Seronick, Jae Son, Cameron Michael, Hannah Fogg, Zeynep Gromley, Adam Gromley.
       OBJECTIVE: In addition to its function as the microtubule organizing center of the cell, the centrosome has functions in many other cellular processes including primary cilia formation, DNA damage checkpoints, and cell cycle progression. But the role of individual components of the centrosome in these processes remains unclear. Previous studies used siRNA (small interfering RNA) to "knock down" protein levels of the centrosome component centriolin, resulting in failed cytokinesis. Since this approach was transient, only targeting centriolin at the mRNA level, we sought to confirm these findings by permanently disrupting the gene encoding centriolin using the CRISPR/Cas9 system of genome editing.
    RESULTS: This study provides evidence that the CRISPR/Cas9 system is capable of effectively reducing centriolin protein levels in the cell. Furthermore, this disruption leads to a failure of cytokinesis that is reminiscent of the phenotype previously reported for the siRNA-mediated disruption of centriolin. Furthermore, no additional defects in cell division were observed, consistent with results seen with previous siRNA studies. We conclude that the CRISPR/Cas9 system is an effective means of permanently removing the cellular pools of centriolin and that the disruption of centriolin at both the mRNA level and genomic level lead to similar cell division defects.
    Keywords:  CRISPR; Centriolin; Centrosome; Cytokinesis
    DOI:  https://doi.org/10.1186/s13104-021-05898-w
  15. Sci Rep. 2022 Jan 14. 12(1): 722
    KIAA0319 influences cilia length, cell migration and mechanical cell-substrate interaction.
    Rebeca Diaz, Nils M Kronenberg, Angela Martinelli, Philipp Liehm, Andrew C Riches, Malte C Gather, Silvia Paracchini.
      Following its association with dyslexia in multiple genetic studies, the KIAA0319 gene has been extensively investigated in different animal models but its function in neurodevelopment remains poorly understood. We developed the first human cellular knockout model for KIAA0319 in RPE1 retinal pigment epithelia cells via CRISPR-Cas9n to investigate its role in processes suggested but not confirmed in previous studies, including cilia formation and cell migration. We observed in the KIAA0319 knockout increased cilia length and accelerated cell migration. Using Elastic Resonator Interference Stress Microscopy (ERISM), we detected an increase in cellular force for the knockout cells that was restored by a rescue experiment. Combining ERISM and immunostaining we show that RPE1 cells exert highly dynamic, piconewton vertical pushing forces through actin-rich protrusions that are surrounded by vinculin-rich pulling sites. This protein arrangement and force pattern has previously been associated to podosomes in other cells. KIAA0319 depletion reduces the fraction of cells forming these actin-rich protrusions. Our results suggest an involvement of KIAA0319 in cilia biology and cell-substrate force regulation.
    DOI:  https://doi.org/10.1038/s41598-021-04539-3
  16. Invest Ophthalmol Vis Sci. 2022 Jan 03. 63(1): 14
    PKA and Epac1 Reduce Nek7 to Block the NLRP3 Inflammasome Proteins in the Retinal Vasculature.
    Li Liu, Youde Jiang, Jena J Steinle.
       Purpose: To determine whether protein kinase a (PKA) and exchange protein for cAMP 1 (Epac1) inhibit NIMA-related kinase 7 (Nek7) to block the NOD-like receptor family pyrin domain-containing family member 3 (NLRP3) signaling pathway.
    Methods: Retinal endothelial cells (RECs) were grown in normal (5 mM) or high (25 mM) glucose. Some cells were treated with a Nek7 cDNA plasmid, Nek7 siRNA; an Epac1 agonist, forskolin; a PKA agonist; or an empty vector. Epac1 floxed and Cdh5-cre Epac1 mice and Nek7 floxed and Cdh5-cre Nek7 mice were also used. Western blot analyses were done on cell culture or whole retinal lysates for NLRP3, cleaved caspase 1, interleukin-1-beta (IL-1β). A PKA activity assay was also done.
    Results: Nek7 cDNA increased NLRP3 signaling proteins, but Nek7 siRNA inhibited high-glucose induction of these proteins in retinal endothelial cells. Epac1 and forskolin both reduced Nek7 and NLRP3 pathway proteins, even when given in combination with Nek7 cDNA. Elimination of Nek7 in endothelial cells reduced NLRP3 signaling proteins in whole retinal lysates from mice.
    Conclusions: Nek7 regulated NLRP3 inflammasome protein levels both in vitro and in vivo. Both Epac1 and PKA lie upstream of Nek7 and NLRP3 and can overcome excessive Nek7 levels. These studies establish that cAMP proteins can inhibit Nek7 and block activation of the NLRP3 inflammasome proteins.
    DOI:  https://doi.org/10.1167/iovs.63.1.14
  17. Histochem Cell Biol. 2022 Jan 13.
    FOXO3/TGF-β signal-dependent ciliogenesis and cell functions during differentiation of temperature-sensitive mouse cochlear precursor hair cells.
    Takuya Kakuki, Takayuki Kohno, Soshi Nishida, Takumi Konno, Shin Kikuchi, Kizuku Ohwada, Masaya Nakano, Mitsuki Tezuka, Kenichi Takano, Takashi Kojima.
      The transcription factor FOXO3 is necessary to preserve cochlear hair cells. Growth factors, including TGF-β, closely contribute to cochlear hair cell regeneration. In the present study, to investigate the roles of FOXO3 in the ciliogenesis and cell functions of cochlear hair cells, UB/OC-2 temperature-sensitive mouse cochlear precursor hair cells were treated with TGF-β receptor type 1 inhibitor EW-7197 or EGF receptor inhibitor AG-1478 after transfection with or without siRNA-FOXO3a. GeneChip analysis revealed that treatment with EW-7197 increased Foxo3 genes and decreased genes of Smads. During cell differentiation, treatment with EW-7197 or AG-1478 induced an increase in length of cilia-like structures that were positive for acetylated tubulin and inhibited cell migration. Treatment with EW-7197 also increased cell metabolism measured as mitochondrial basal respiration (oxygen consumption rate). The effects of EW-7197 were stronger than those of AG-1478. Knockdown of FOXO3 prevented the growth of cilia-like structures induced by EW-7197 or AG-1478 and induced cell migration under treatment with EW-7197. No change of the epithelial cell polarity molecule PAR3 was observed with any treatment. Treatment with the antimicrobial agent amikacin prevented the growth of cilia-like structures induced by EW-7197 and induced apoptosis. Pretreatment with the glucocorticoid dexamethasone inhibited the apoptosis induced by amikacin. This in vitro model of mouse cochlear hair cells suggests that FOXO3/TGF-β signaling plays a crucial role in ciliogenesis and cell functions during differentiation of cochlear hair cells. This model is useful for analysis of the mechanisms of hearing loss and to find therapeutic agents to prevent it.
    Keywords:  Cell metabolism; Cell migration; Ciliogenesis; FOXO3; Mouse cochlear hair cells; TGF-β
    DOI:  https://doi.org/10.1007/s00418-021-02068-8
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