bims-bicyki Biomed News
on Bicaudal-C1 and interactors in cystic kidney disease
Issue of 2021–10–10
twenty-one papers selected by
Céline Gagnieux, École Polytechnique Fédérale de Lausanne
  1. Tolvaptan in Pediatric Autosomal Dominant Polycystic Kidney Disease: From Here to Where?
  2. A radiomics method based on MR FS-T2WI sequence for diagnosing of autosomal dominant polycystic kidney disease progression.
  3. Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models.
  4. Can the Enhanced Liver Fibrosis score be used to Diagnose Children with Liver Fibrosis?
  5. Vascular Stiffening Mediated by Rho-Associated Coiled-Coil Containing Kinase Isoforms.
  6. A comprehensive PGT-M strategy for ADPKD patients with de novo PKD1 mutations using affected embryo or gametes as proband.
  7. Rho-Kinase as a Therapeutic Target for Nonalcoholic Fatty Liver Diseases.
  8. An updated SYSCILIA gold standard (SCGSv2) of known ciliary genes, revealing the vast progress that has been made in the cilia research field.
  9. Chronic activation of AMP-activated protein kinase leads to early onset polycystic kidney phenotype.
  10. Loss of the ciliary gene Bbs4 results in defective thermogenesis due to metabolic inefficiency and impaired lipid metabolism.
  11. Regulation of myosin light-chain phosphorylation and its roles in cardiovascular physiology and pathophysiology.
  12. TRIP6 functions in brain ciliogenesis.
  13. Pharmacological Modulation of Neurite Outgrowth in Human Neural Progenitor Cells by Inhibiting Non-muscle Myosin II.
  14. CCRK/CDK20 regulates ciliary retrograde protein trafficking via interacting with BROMI/TBC1D32.
  15. Phenotypic characterization of Cdkl5-knockdown neurons establishes elongated cilia as a functional assay for CDKL5 Deficiency Disorder.
  16. Hydrochlorothiazide Reduces Cardiac Hypertrophy, Fibrosis and Rho-Kinase Activation in DOCA-Salt Induced Hypertension.
  17. Diversity and function of motile ciliated cell types within ependymal lineages of the zebrafish brain.
  18. Wdr47, Camsaps, and Katanin cooperate to generate ciliary central microtubules.
  19. NEK7-Mediated Activation of NLRP3 Inflammasome Is Coordinated by Potassium Efflux/Syk/JNK Signaling During Staphylococcus aureus Infection.
  20. CARMIL3 is important for cell migration and morphogenesis during early development in zebrafish.
  21. Mice lacking PECAM-1 and Ceacam1 have an aberrant platelet and thrombus phenotype.
  1. Kidney Dis (Basel). 2021 Sep;7(5): 343-349
    Tolvaptan in Pediatric Autosomal Dominant Polycystic Kidney Disease: From Here to Where?
    Fei Liu, Chunyue Feng, Huijun Shen, Huaidong Fu, Jianhua Mao.
       Background: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disorder, accounting for approximately 5% of all ESRD cases worldwide. As a vasopressin receptor 2 antagonist, tolvaptan is the FDA-approved therapeutic agent for ADPKD, which is only made available to a limited number of adult patients; however, its efficacy in pediatric patients has not been reported widely.
    Summary: Tolvaptan was shown to delay ADPKD progression in the Tolvaptan Efficacy and Safety in Management of Autosomal Dominant Polycystic Kidney Disease and Its Outcomes (TEMPO) 3:4 study, Replicating Evidence of Preserved Renal Function: an Investigation of Tolvaptan Safety and Efficacy in ADPKD (REPRISE) trial, and other clinical studies. In addition to its effects on aquaretic adverse events and alanine aminotransferase elevation, the effect of tolvaptan on ADPKD is clear, sustained, and cumulative. While ADPKD is a progressive disease, the early intervention has been shown to be important and beneficial in hypotheses as well as in trials. The use of tolvaptan in pediatric ADPKD involves the following challenges: patient assessment, quality of life assessment, cost-effectiveness, safety, and tolerability. The ongoing, phase 3b, 2-part study (ClinicalTrials.gov identifier: NCT02964273) on the evaluation of tolvaptan in pediatric ADPKD (patients aged 12-17 years) may help obtain some insights.
    Key Messages: This review focuses on the rationality of tolvaptan use in pediatric patients with ADPKD, the associated challenges, and the suggested therapeutic approaches.
    Keywords:  Autosomal dominant polycystic kidney disease; Children; Therapy; Tolvaptan
    DOI:  https://doi.org/10.1159/000517186
  2. Eur Rev Med Pharmacol Sci. 2021 Sep;pii: 26795. [Epub ahead of print]25(18): 5769-5780
    A radiomics method based on MR FS-T2WI sequence for diagnosing of autosomal dominant polycystic kidney disease progression.
    L Cong, Q-Q Hua, Z-Q Huang, Q-L Ma, X-M Wang, C-C Huang, J-X Xu, T Ma.
       OBJECTIVE: We aimed to construct/validate a radiomics method based on MR FS-T2WI sequence for the evaluation of kidney function in patients with autosomal dominant polycystic kidney disease (ADPKD).
    PATIENTS AND METHODS: The clinical data and MRI images of 114 patients with ADPKD were retrospectively analyzed. With a glomerular filtration rate of 60 mL/min per 1.73 m2 as the cutoff value, patients were divided into two groups, where there were 59 patients with GFR ≥60 mL/min per 1.73 m2 (including CKD1 and CKD2 phase) and 55 patients with GFR <60 mL/min per 1.73 m2 (including CKD3 phase and higher). All patients underwent the 3.0T MR scan of the kidney. Then, the kidney were delineated layer by layer based on the FS-T2WI sequence to obtain the volume of interest (VOI) for radiomics features extraction. The optimal radiomics features were selected by least absolute shrinkage and selection operator (LASSO). Three kinds of data modality including the pure clinical data, the pure image data and the clinical-image fused data were utilized to establish three types of models (clinical, image and with their combination) separately by five machine learning classifiers: k-nearest-neighbors (KNN), support vector machine (SVM), logistic regression (LR), random forests (RF) and multi-layer perception (MLP). Receiver operating characteristic (ROC) curve, areas under the curve (AUC), sensitivity, specificity and precision were employed to evaluate the model's effectiveness to diagnosis the glomerular filtration rate of patients with ADPKD based on different models. Besides, Delong test was applied to compare ROCs between models.
    RESULTS: 960 radiomics features were extracted from each VOIs, and clinical information included the gender and age of each patient. After feature selection, 23 and 21 features based on pure image data and clinical-image fused data were independently used to construct models for the kidney function evaluation. The clinical-image fused model (AUC=0.89) has better performance than the pure image model (p=0.046) and pure clinical model (p<0.001). Clinical-image fused model based on LR classifier showed the best diagnostic efficiency, with AUC=0.89, sensitivity=0.8867 and specificity=0.7959.
    CONCLUSIONS: The MR FS-T2WI radiomics analysis based on clinical-image fused model is instrumental in evaluating and predicting the kidney function of patients with polycystic kidney disease.
    DOI:  https://doi.org/10.26355/eurrev_202109_26795
  3. Sci Rep. 2021 Oct 05. 11(1): 19798
    Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models.
    Juan J Muñoz, Ana C Anauate, Andressa G Amaral, Frederico M Ferreira, Elieser H Watanabe, Renata Meca, Milene S Ormanji, Mirian A Boim, Luiz F Onuchic, Ita P Heilberg.
      Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited renal disorder, characterized by renal cyst development leading to end-stage renal disease. Although the appropriate choice of suitable reference is critical for quantitative RNA analysis, no comparison of frequently used "housekeeping" genes is available. Here, we determined the validity of 7 candidate housekeeping genes (Actb, Actg1, B2m, Gapdh, Hprt, Pgam1 and Ppia) in kidney tissues from mouse models orthologous to ADPKD, including a cystic mice (CY) 10-12 weeks old (Pkd1flox/flox:Nestincre/Pkd1flox/-:Nestincre, n = 10) and non-cystic (NC) controls (Pkd1flox/flox/Pkd1flox/-, n = 10), Pkd1-haploinsufficient (HT) mice (Pkd1+/-, n = 6) and wild-type (WT) controls (Pkd1+/+, n = 6) and a severely cystic (SC) mice 15 days old (Pkd1V/V, n = 7) and their controls (CO, n = 5). Gene expression data were analyzed using six distinct statistical softwares. The estimation of the ideal number of genes suggested the use of Ppia alone as sufficient, although not ideal, to analyze groups altogether. Actb, Hprt and Ppia expression profiles were correlated in all samples. Ppia was identified as the most stable housekeeping gene, while Gapdh was the least stable for all kidney samples. Stat3 expression level was consistent with upregulation in SC compared to CO when normalized by Ppia expression. In conclusion, present findings identified Ppia as the best housekeeping gene for CY + NC and SC + CO groups, while Hprt was the best for the HT + WT group.
    DOI:  https://doi.org/10.1038/s41598-021-99366-x
  4. J Pediatr Gastroenterol Nutr. 2021 Oct 05.
    Can the Enhanced Liver Fibrosis score be used to Diagnose Children with Liver Fibrosis?
    Iben Thusing, Steen Antonsen, Benjamin Søgaard Hoffmann-Petersen, Helene Marlies Rasmussen, Per Wittenhagen.
       OBJECTIVES: The non-invasive Enhanced Liver Fibrosis (ELF) score is used in adults with liver fibrosis as a diagnostic aid. The ELF score combines three serum markers of extracellular matrix remodeling and fibrogenesis: Hyaluronic acid (HA), the N-terminal pro-peptide of collagen type III (PIIINP) and tissue inhibitor of metalloproteinase-1 (TIMP-1). We aimed to evaluate the clinical use of the ELF score in children.
    METHODS AND RESULTS: A reference interval for the ELF score was established using 343 liver-healthy children aged 6-17 years. The median ELF score of 8.9 in healthy children was significantly increased compared to healthy adults. ELF scores increased significantly in both female and male healthy controls with peak levels at puberty, driven by elevated levels of HA and PIIINP likely explained by increased growth. If adult normal values were applied to the group of liver-healthy children, only 6.4% were in the normal range. Prospectively we analysed ELF scores in patients with possible or confirmed liver fibrosis due to autosomal recessive polycystic kidney disease (ARPKD). All ELF scores in children with ARPKD were within the reference intervals generated from the group of healthy children.
    CONCLUSIONS: The usual diagnostic cut-off ranges for the ELF score in adults are not applicable; instead age and gender appropriate cut-off values should be used in children. The clinical value of ELF scores in children is questionable, since children during pubertal growth showed elevated ELF scores and patients with ARPKD and liver fibrosis showed normal levels.
    DOI:  https://doi.org/10.1097/MPG.0000000000003316
  5. J Am Heart Assoc. 2021 Oct 06. e022568
    Vascular Stiffening Mediated by Rho-Associated Coiled-Coil Containing Kinase Isoforms.
    Yuxin Li, Haw-Chih Tai, Nikola Sladojevic, Hyung-Hwan Kim, James K Liao.
      Background The pathogenesis of vascular stiffening and hypertension is marked by non-compliance of vessel wall because of deposition of collagen fibers, loss of elastin fibers, and increased vascular thickening. Rho/Rho-associated coiled-coil containing kinases 1 and 2 (ROCK1 and ROCK2) have been shown to regulate cellular contraction and vascular remodeling. However, the role of ROCK isoforms in mediating pathogenesis of vascular stiffening and hypertension is not known. Methods and Results Hemizygous Rock mice (Rock1+/- and Rock2+/-) were used to determine the role of ROCK1 and ROCK2 in age-related vascular dysfunction. Both ROCK activity and aortic stiffness increased to a greater extent with age in wild-type mice compared with that of Rock1+/- and Rock2+/- mice. As a model for age-related vascular stiffening, we administered angiotensin II (500 ng/kg per minute) combined with nitric oxide synthase inhibitor, L-Nω-nitroarginine methyl ester (0.5 g/L) for 4 weeks to 12-week-old male Rock1+/- and Rock2+/- mice. Similar to advancing age, angiotensin II/L-Nω-nitroarginine methyl ester caused increased blood pressure, aortic stiffening, and vascular remodeling, which were attenuated in Rock2+/-, and to a lesser extent, Rock1+/- mice. The reduction of aortic stiffening in Rock2+/- mice was accompanied by decreased collagen deposition, relatively preserved elastin content, and less aortic wall hypertrophy. Indeed, the upregulation of collagen I by transforming growth factor-β1 or angiotensin II was greatly attenuated in Rock2-/- mouse embryonic fibroblasts. Conclusions These findings indicate that ROCK1 and ROCK2 mediate both age-related and pharmacologically induced aortic stiffening, and suggest that inhibition of ROCK2, and to a lesser extent ROCK1, may have therapeutic benefits in preventing age-related vascular stiffening.
    Keywords:  Rho kinase; aging; aortic stiffness; eEF1A1; vascular remodeling
    DOI:  https://doi.org/10.1161/JAHA.121.022568
  6. J Assist Reprod Genet. 2021 Sep;38(9): 2425-2434
    A comprehensive PGT-M strategy for ADPKD patients with de novo PKD1 mutations using affected embryo or gametes as proband.
    Yuqian Wang, Fan Zhai, Shuo Guan, Zhiqiang Yan, Xiaohui Zhu, Ying Kuo, Nan Wang, Xu Zhi, Ying Lian, Jin Huang, Jialin Jia, Ping Liu, Rong Li, Jie Qiao, Liying Yan.
      Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease characterized by the development of renal cysts and progression to renal failure. Preimplantation genetic testing-monogenic disease (PGT-M) is an alternative option to obtain healthy babies. However, de novo PKD1 mutation of one of the spouses or the absence of a positive family history poses a serious challenge to PGT-M. Here, we described a comprehensive strategy which includes preimplantation genetic testing for aneuploidies (PGT-A) study and monogenic diagnosis study for ADPKD patients bearing de novo mutations. The innovation of our strategy is to use the gamete (polar body or single sperm) as proband for single-nucleotide polymorphism (SNP) linkage analysis to detect an embryo's carrier status. Nine ADPKD couples with either de novo mutation or without a positive family history were recruited and a total of 34 embryos from 13 PGT-M cycles were examined. Within these nine couples, two successfully delivered healthy babies had their genetic status confirmed by amniocentesis. This study provides a creative approach for embryo diagnosis of patients with de novo mutations or patients who lack essential family members for linkage analysis.
    Keywords:  ADPKD; De novo mutation; Haplotype analysis; PGT-M
    DOI:  https://doi.org/10.1007/s10815-021-02188-z
  7. Diabetes Metab J. 2021 Sep;45(5): 655-674
    Rho-Kinase as a Therapeutic Target for Nonalcoholic Fatty Liver Diseases.
    Inês Sousa-Lima, Hyun Jeong Kim, John Jones, Young-Bum Kim.
      Nonalcoholic fatty liver disease (NAFLD) is a major public health problem and the most common form of chronic liver disease, affecting 25% of the global population. Although NAFLD is closely linked with obesity, insulin resistance, and type 2 diabetes mellitus, knowledge on its pathogenesis remains incomplete. Emerging data have underscored the importance of Rho-kinase (Rho-associated coiled-coil-containing kinase [ROCK]) action in the maintenance of normal hepatic lipid homeostasis. In particular, pharmacological blockade of ROCK in hepatocytes or hepatic stellate cells prevents the progression of liver diseases such as NAFLD and fibrosis. Moreover, mice lacking hepatic ROCK1 are protected against obesity-induced fatty liver diseases by suppressing hepatic de novo lipogenesis. Here we review the roles of ROCK as an indispensable regulator of obesity-induced fatty liver disease and highlight the key cellular pathway governing hepatic lipid accumulation, with focus on de novo lipogenesis and its impact on therapeutic potential. Consequently, a comprehensive understanding of the metabolic milieu linking to liver dysfunction triggered by ROCK activation may help identify new targets for treating fatty liver diseases such as NAFLD.
    Keywords:  AMP-activated protein kinase; Diet, high-fat; Lipogenesis; Nonalcoholic fatty liver disease; Rho-kinase
    DOI:  https://doi.org/10.4093/dmj.2021.0197
  8. Mol Biol Cell. 2021 Oct 06. mbcE21020089
    An updated SYSCILIA gold standard (SCGSv2) of known ciliary genes, revealing the vast progress that has been made in the cilia research field.
    Suly Saray Villa Vasquez, John van Dam, Gabrielle Wheway.
      Cilia are microtubule-based organelles with important functions in motility and sensation. They contribute to a broad spectrum of developmental disorders called ciliopathies, and have recently been linked to common conditions such as cancers and congenital heart disease. There has been increasing interest in the biology of cilia and their contribution to disease over the past two decades. As a result, in 2013 we published a 'Gold Standard' list of genes confirmed to be associated with cilia. This was published as part of the SYSCILIA consortium systems biology study dissecting the contribution of cilia to human health and disease, and was named the Syscilia Gold Standard (SCGS). Since this publication, interest in cilia and understanding of their functions has continued to grow, and we now present an updated SCGS version 2. This includes an additional 383 genes, more than doubling the size of SCGSv1. We use this dataset to conduct a review of advances in understanding of cilia biology 2013-2021, and perspectives on the future of cilia research. We hope that this continues to be a useful resource for the cilia community.
    DOI:  https://doi.org/10.1091/mbc.E21-05-0226
  9. Clin Sci (Lond). 2021 Oct 08. pii: CS20210821. [Epub ahead of print]
    Chronic activation of AMP-activated protein kinase leads to early onset polycystic kidney phenotype.
    Laura Wilson, Alice Pollard, Lucy Penfold, Phillip Muckett, Chad Whilding, Mohammad Bohlooly-Y, Patricia D Wilson, David Carling.
      AMP-activated protein kinase (AMPK) plays a key role in the cellular response to low energy stress and has emerged as an attractive therapeutic target for tackling metabolic diseases. Whilst significant progress has been made regarding the physiological role of AMPK, its function in the kidney remains only partially understood. We use a mouse model expressing a constitutively active mutant of AMPK to investigate the effect of AMPK activation on kidney function in vivo. Kidney morphology and changes in gene and protein expression were monitored and serum and urine markers were measured to assess kidney function in vivo. Global AMPK activation resulted in an early onset polycystic kidney phenotype, featuring collecting duct cysts and compromised renal function in adult mice. Mechanistically, the cystic kidneys had increased cAMP levels and ERK activation, increased hexokinase I expression, glycogen accumulation and altered expression of proteins associated with autophagy. Kidney tubule-specific activation of AMPK also resulted in a polycystic phenotype, demonstrating that renal tubular AMPK activation caused the cystogenesis. Importantly, human ADPKD kidney sections revealed similar protein localisation patterns to that observed in the murine cystic kidneys. Our findings show that early onset chronic AMPK activation leads to a polycystic kidney phenotype, suggesting dysregulated AMPK signalling is a contributing factor in cystogenesis.
    Keywords:  AMPK; chronic kidney disease; metabolic regulation
    DOI:  https://doi.org/10.1042/CS20210821
  10. FASEB J. 2021 Nov;35(11): e21966
    Loss of the ciliary gene Bbs4 results in defective thermogenesis due to metabolic inefficiency and impaired lipid metabolism.
    Sabrina Gohlke, Carola Mancini, Francisco Garcia-Carrizo, Tim J Schulz.
      Adipose tissue is central to the regulation of energy balance. While white adipose tissue (WAT) is responsible for triglyceride storage, brown adipose tissue specializes in energy expenditure. Deterioration of brown adipocyte function contributes to the development of metabolic complications like obesity and diabetes. These disorders are also leading symptoms of the Bardet-Biedl syndrome (BBS), a hereditary disorder in humans which is caused by dysfunctions of the primary cilium and which therefore belongs to the group of ciliopathies. The cilium is a hair-like organelle involved in cellular signal transduction. The BBSome, a supercomplex of several Bbs gene products, localizes to the basal body of cilia and is thought to be involved in protein sorting to and from the ciliary membrane. The effects of a functional BBSome on energy metabolism and lipid mobilization in brown and white adipocytes were tested in whole-body Bbs4 knockout mice that were subjected to metabolic challenges. Chronic cold exposure reveals cold-intolerance of knockout mice but also ameliorates the markers of metabolic pathology detected in knockouts prior to cold. Hepatic triglyceride content is markedly reduced in knockout mice while circulating lipids are elevated, altogether suggesting that defective lipid metabolism in adipose tissue creates increased demand for systemic lipid mobilization to meet energetic demands of reduced body temperatures. These findings taken together suggest that Bbs4 is essential for the regulation of adipose tissue lipid metabolism, representing a potential target to treat metabolic disorders.
    Keywords:  BBsome; Bbs4; adipose tissue; browning; cilium; lipid metabolism
    DOI:  https://doi.org/10.1096/fj.202100772RR
  11. Hypertens Res. 2021 Oct 06.
    Regulation of myosin light-chain phosphorylation and its roles in cardiovascular physiology and pathophysiology.
    Masaaki Ito, Ryuji Okamoto, Hiromasa Ito, Ye Zhe, Kaoru Dohi.
      The regulation of muscle contraction is a critical function in the cardiovascular system, and abnormalities may be life-threatening or cause illness. The common basic mechanism in muscle contraction is the interaction between the protein filaments myosin and actin. Although this interaction is primarily regulated by intracellular Ca2+, the primary targets and intracellular signaling pathways differ in vascular smooth muscle and cardiac muscle. Phosphorylation of the myosin regulatory light chain (RLC) is a primary molecular switch for smooth muscle contraction. The equilibrium between phosphorylated and unphosphorylated RLC is dynamically achieved through two enzymes, myosin light chain kinase, a Ca2+-dependent enzyme, and myosin phosphatase, which modifies the Ca2+ sensitivity of contractions. In cardiac muscle, the primary target protein for Ca2+ is troponin C on thin filaments; however, RLC phosphorylation also plays a modulatory role in contraction. This review summarizes recent advances in our understanding of the regulation, physiological function, and pathophysiological involvement of RLC phosphorylation in smooth and cardiac muscles.
    Keywords:  Myosin light chain kinase; Myosin light chain phosphorylation; Myosin phosphatase; Rho-kinase; RhoA
    DOI:  https://doi.org/10.1038/s41440-021-00733-y
  12. Nat Commun. 2021 Oct 07. 12(1): 5887
    TRIP6 functions in brain ciliogenesis.
    Shalmali Shukla, Ronny Haenold, Pavel Urbánek, Lucien Frappart, Shamci Monajembashi, Paulius Grigaravicius, Sigrun Nagel, Woo Kee Min, Alicia Tapias, Olivier Kassel, Heike Heuer, Zhao-Qi Wang, Aspasia Ploubidou, Peter Herrlich.
      TRIP6, a member of the ZYXIN-family of LIM domain proteins, is a focal adhesion component. Trip6 deletion in the mouse, reported here, reveals a function in the brain: ependymal and choroid plexus epithelial cells are carrying, unexpectedly, fewer and shorter cilia, are poorly differentiated, and the mice develop hydrocephalus. TRIP6 carries numerous protein interaction domains and its functions require homodimerization. Indeed, TRIP6 disruption in vitro (in a choroid plexus epithelial cell line), via RNAi or inhibition of its homodimerization, confirms its function in ciliogenesis. Using super-resolution microscopy, we demonstrate TRIP6 localization at the pericentriolar material and along the ciliary axoneme. The requirement for homodimerization which doubles its interaction sites, its punctate localization along the axoneme, and its co-localization with other cilia components suggest a scaffold/co-transporter function for TRIP6 in cilia. Thus, this work uncovers an essential role of a LIM-domain protein assembly factor in mammalian ciliogenesis.
    DOI:  https://doi.org/10.1038/s41467-021-26057-6
  13. Front Cell Dev Biol. 2021 ;9 719636
    Pharmacological Modulation of Neurite Outgrowth in Human Neural Progenitor Cells by Inhibiting Non-muscle Myosin II.
    Julianna Lilienberg, Zoltán Hegyi, Eszter Szabó, Edit Hathy, András Málnási-Csizmadia, János M Réthelyi, Ágota Apáti, László Homolya.
      Studies on neural development and neuronal regeneration after injury are mainly based on animal models. The establishment of pluripotent stem cell (PSC) technology, however, opened new perspectives for better understanding these processes in human models by providing unlimited cell source for hard-to-obtain human tissues. Here, we aimed at identifying the molecular factors that confine and modulate an early step of neural regeneration, the formation of neurites in human neural progenitor cells (NPCs). Enhanced green fluorescent protein (eGFP) was stably expressed in NPCs differentiated from human embryonic and induced PSC lines, and the neurite outgrowth was investigated under normal and injury-related conditions using a high-content screening system. We found that inhibitors of the non-muscle myosin II (NMII), blebbistatin and its novel, non-toxic derivatives, initiated extensive neurite outgrowth in human NPCs. The extracellular matrix components strongly influenced the rate of neurite formation but NMII inhibitors were able to override the inhibitory effect of a restrictive environment. Non-additive stimulatory effect on neurite generation was also detected by the inhibition of Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), the upstream regulator of NMII. In contrast, inhibition of c-Jun N-terminal kinases (JNKs) had only a negligible effect, suggesting that the ROCK1 signal is dominantly manifested by actomyosin activity. In addition to providing a reliable cell-based in vitro model for identifying intrinsic mechanisms and environmental factors responsible for impeded axonal regeneration in humans, our results demonstrate that NMII and ROCK1 are important pharmacological targets for the augmentation of neural regeneration at the progenitor level. These studies may open novel perspectives for development of more effective pharmacological treatments and cell therapies for various neurodegenerative disorders.
    Keywords:  blebbistatin; extracellular matrix (ECM); human neural progenitor cells (hNPCs); neurite; non-muscle myosin II
    DOI:  https://doi.org/10.3389/fcell.2021.719636
  14. PLoS One. 2021 ;16(10): e0258497
    CCRK/CDK20 regulates ciliary retrograde protein trafficking via interacting with BROMI/TBC1D32.
    Tatsuro Noguchi, Kentaro Nakamura, Yuuki Satoda, Yohei Katoh, Kazuhisa Nakayama.
      CCRK/CDK20 was reported to interact with BROMI/TBC1D32 and regulate ciliary Hedgehog signaling. In various organisms, mutations in the orthologs of CCRK and those of the kinase ICK/CILK1, which is phosphorylated by CCRK, are known to result in cilia elongation. Furthermore, we recently showed that ICK regulates retrograde ciliary protein trafficking and/or the turnaround event at the ciliary tips, and that its mutations result in the elimination of intraflagellar transport (IFT) proteins that have overaccumulated at the bulged ciliary tips as extracellular vesicles, in addition to cilia elongation. However, how these proteins cooperate to regulate ciliary protein trafficking has remained unclear. We here show that the phenotypes of CCRK-knockout (KO) cells closely resemble those of ICK-KO cells; namely, the overaccumulation of IFT proteins at the bulged ciliary tips, which appear to be eliminated as extracellular vesicles, and the enrichment of GPR161 and Smoothened on the ciliary membrane. The abnormal phenotypes of CCRK-KO cells were rescued by the exogenous expression of wild-type CCRK but not its kinase-dead mutant or a mutant defective in BROMI binding. These results together indicate that CCRK regulates the turnaround process at the ciliary tips in concert with BROMI and probably via activating ICK.
    DOI:  https://doi.org/10.1371/journal.pone.0258497
  15. Neurosci Res. 2021 Oct 05. pii: S0168-0102(21)00209-1. [Epub ahead of print]
    Phenotypic characterization of Cdkl5-knockdown neurons establishes elongated cilia as a functional assay for CDKL5 Deficiency Disorder.
    Alessia Di Nardo, Alina Rühmkorf, Patricia Award, Ashton Brennecke, Michela Fagiolini, Mustafa Sahin.
      CDKL5 Deficiency Disorder (CDD) is a severe encephalopathy characterized by intractable epilepsy, infantile spasms, and cognitive disabilities. The detrimental CNS manifestations and lack of therapeutic interventions represent unmet needs, necessitating identification of CDD-dependent phenotypes for in vitro disease modeling and therapeutic testing. Here, we optimized a high-content assay to quantify cilia in CDKL5-deficient neurons. Our work shows that Cdkl5-knockdown neurons have elongated cilia and uncovers cilium lengthening in hippocampi of Cdkl5 knockout mice. Collectively, our findings identify cilia length alterations under CDKL5 activity loss in vitro and in vivo and reveal elongated cilia as a robust functional phenotype for CDD.
    Keywords:  CDKL5; autism; cilia; neurodevelopmental disorder; seizure
    DOI:  https://doi.org/10.1016/j.neures.2021.10.001
  16. J Cardiovasc Pharmacol Ther. 2021 Oct 08. 10742484211053109
    Hydrochlorothiazide Reduces Cardiac Hypertrophy, Fibrosis and Rho-Kinase Activation in DOCA-Salt Induced Hypertension.
    David Mondaca-Ruff, Patricio Araos, Cristián E Yañez, Ulises F Novoa, Italo G Mora, María Paz Ocaranza, Jorge E Jalil.
       BACKGROUND: Thiazides are one of the most common antihypertensive drugs used for hypertension treatment and hydrochlorothiazide (HCTZ) is the most frequently used diuretic for hypertension treatment. The Rho/Rho-kinase (ROCK) path plays a key function in cardiovascular remodeling. We hypothesized that in preclinical hypertension HCTZ reduces myocardial ROCK activation and consequent myocardial remodeling.
    METHODS: The preclinical model of deoxycorticosterone (DOCA)-salt hypertension was used (Sprague-Dawley male rats). After 3 weeks, in 3 different groups: HCTZ, the ROCK inhibitor fasudil or spironolactone was added (3 weeks). After 6 weeks myocardial hypertrophy and fibrosis, cardiac levels of profibrotic proteins, mRNA levels (RT PCR) of pro remodeling and pro oxidative molecules and ROCK activity were determined.
    RESULTS: Blood pressure, myocardial hypertrophy and fibrosis were reduced significantly by HCTZ, fasudil and spironolactone. In the heart, increased levels of the pro-fibrotic proteins Col-I, Col-III and TGF-β1 and gene expression of pro-remodeling molecules TGF-β1, CTGF, MCP-1 and PAI-1 and the pro-oxidative molecules gp91phox and p22phox were significantly reduced by HCTZ, fasudil and spironolactone. ROCK activity in the myocardium was increased by 54% (P < 0.05) as related to the sham group and HCTZ, spironolactone and fasudil, reduced ROCK activation to control levels.
    CONCLUSIONS: HCTZ reduced pathologic LVH by controlling blood pressure, hypertrophy and myocardial fibrosis and by decreasing myocardial ROCK activation, expression of pro remodeling, pro fibrotic and pro oxidative genes. In hypertension, the observed effects of HCTZ on the myocardium might explain preventive outcomes of thiazides in hypertension, specifically on LVH regression and incident heart failure.
    Keywords:  Rho-kinase; cardiac remodeling; hydrochlorothiazide; hypertension; hypertrophy
    DOI:  https://doi.org/10.1177/10742484211053109
  17. Cell Rep. 2021 Oct 05. pii: S2211-1247(21)01229-8. [Epub ahead of print]37(1): 109775
    Diversity and function of motile ciliated cell types within ependymal lineages of the zebrafish brain.
    Percival P D'Gama, Tao Qiu, Mehmet Ilyas Cosacak, Dheeraj Rayamajhi, Ahsen Konac, Jan Niklas Hansen, Christa Ringers, Francisca Acuña-Hinrichsen, Subhra P Hui, Emilie W Olstad, Yan Ling Chong, Charlton Kang An Lim, Astha Gupta, Chee Peng Ng, Benedikt S Nilges, Nachiket D Kashikar, Dagmar Wachten, David Liebl, Kazu Kikuchi, Caghan Kizil, Emre Yaksi, Sudipto Roy, Nathalie Jurisch-Yaksi.
      Motile cilia defects impair cerebrospinal fluid (CSF) flow and can cause brain and spine disorders. The development of ciliated cells, their impact on CSF flow, and their function in brain and axial morphogenesis are not fully understood. We have characterized motile ciliated cells within the zebrafish brain ventricles. We show that the ventricles undergo restructuring through development, involving a transition from mono- to multiciliated cells (MCCs) driven by gmnc. MCCs co-exist with monociliated cells and generate directional flow patterns. These ciliated cells have different developmental origins and are genetically heterogenous with respect to expression of the Foxj1 family of ciliary master regulators. Finally, we show that cilia loss from the tela choroida and choroid plexus or global perturbation of multiciliation does not affect overall brain or spine morphogenesis but results in enlarged ventricles. Our findings establish that motile ciliated cells are generated by complementary and sequential transcriptional programs to support ventricular development.
    Keywords:  brain; cerebrospinal fluid; choroid plexus; cilia; ependymal cell; foxj1; gmnc; multiciliated cells; scoliosis; zebrafish
    DOI:  https://doi.org/10.1016/j.celrep.2021.109775
  18. Nat Commun. 2021 Oct 04. 12(1): 5796
    Wdr47, Camsaps, and Katanin cooperate to generate ciliary central microtubules.
    Hao Liu, Jianqun Zheng, Lei Zhu, Lele Xie, Yawen Chen, Yirong Zhang, Wei Zhang, Yue Yin, Chao Peng, Jun Zhou, Xueliang Zhu, Xiumin Yan.
      The axonemal central pair (CP) are non-centrosomal microtubules critical for planar ciliary beat. How they form, however, is poorly understood. Here, we show that mammalian CP formation requires Wdr47, Camsaps, and microtubule-severing activity of Katanin. Katanin severs peripheral microtubules to produce central microtubule seeds in nascent cilia. Camsaps stabilize minus ends of the seeds to facilitate microtubule outgrowth, whereas Wdr47 concentrates Camsaps into the axonemal central lumen to properly position central microtubules. Wdr47 deficiency in mouse multicilia results in complete loss of CP, rotatory beat, and primary ciliary dyskinesia. Overexpression of Camsaps or their microtubule-binding regions induces central microtubules in Wdr47-/- ependymal cells but at the expense of low efficiency, abnormal numbers, and wrong location. Katanin levels and activity also impact the central microtubule number. We propose that Wdr47, Camsaps, and Katanin function together for the generation of non-centrosomal microtubule arrays in polarized subcellular compartments.
    DOI:  https://doi.org/10.1038/s41467-021-26058-5
  19. Front Immunol. 2021 ;12 747370
    NEK7-Mediated Activation of NLRP3 Inflammasome Is Coordinated by Potassium Efflux/Syk/JNK Signaling During Staphylococcus aureus Infection.
    Ruiqing Liu, Yashan Liu, Chang Liu, Aijiao Gao, Lin Wang, Huixin Tang, Qiang Wu, Xia Wang, Derun Tian, Zhi Qi, Yanna Shen.
      Staphylococcus aureus (S. aureus) is a foodborne pathogen that causes severe diseases, such as endocarditis, sepsis, and bacteremia. As an important component of innate immune system, the NLR family pyrin domain-containing 3 (NLRP3) inflammasome plays a critical role in defense against pathogen infection. However, the cellular mechanism of NLRP3 inflammasome activation during S. aureus infection remains unknown. In the present study, we found that spleen tyrosine kinase (Syk) and c-Jun N-terminal kinase (JNK) were rapidly phosphorylated during S. aureus infection. Moreover, a Syk/JNK inhibitor and Syk/JNK siRNA not only reduced NLRP3 inflammasome-associated molecule expression at the protein and mRNA levels, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) speck formation, and interleukin-1β (IL-1β), and IL-18 release but also rescued the decreased NIMA-related kinase 7 (NEK7) expression level following suppression of the NEK7-NLRP3 interaction in macrophages. Interestingly, Syk/JNK phosphorylation levels and NLRP3 inflammasome-associated molecule expression were decreased by blockade of K+ efflux. Furthermore, activation of the NLRP3 inflammasome and a lower NEK7 protein level were found in vivo upon S. aureus infection. Taken together, our data indicated that S. aureus infection induces a K+ efflux/Syk/JNK/NEK7-NLRP3 signaling pathway and the subsequent activation of the NLRP3 inflammasome for the release of proinflammatory cytokines. This study expands our understanding of the basic molecular mechanism regulating inflammation and provides potential value for anti-infective drug development against S. aureus infection.
    Keywords:  JNK; K+ efflux; NEK7; NLRP3 inflammasome; S. aureus; Syk
    DOI:  https://doi.org/10.3389/fimmu.2021.747370
  20. Dev Biol. 2021 Sep 29. pii: S0012-1606(21)00215-3. [Epub ahead of print]
    CARMIL3 is important for cell migration and morphogenesis during early development in zebrafish.
    Benjamin C Stark, Yuanyuan Gao, Diane S Sepich, Lakyn Belk, Matthew A Culver, Bo Hu, Marlene Mekel, Wyndham Ferris, Jimann Shin, Lilianna Solnica-Krezel, Fang Lin, John A Cooper.
      Cell migration is important during early animal embryogenesis. Cell migration and cell shape are controlled by actin assembly and dynamics, which depend on capping proteins, including the barbed-end heterodimeric actin capping protein (CP). CP activity can be regulated by capping-protein-interacting (CPI) motif proteins, including CARMIL (capping protein Arp2/3 myosin-I linker) family proteins. Previous studies of CARMIL3, one of the three highly conserved CARMIL genes in vertebrates, have largely been limited to cells in culture. Towards understanding CARMIL function during embryogenesis in vivo, we analyzed zebrafish lines carrying mutations of carmil3. Maternal-zygotic mutants showed impaired endodermal migration during gastrulation, along with defects in dorsal forerunner cell (DFC) cluster formation, which affected the morphogenesis of Kupffer's vesicle (KV). Mutant KVs were smaller, contained fewer cells and displayed decreased numbers of cilia, leading to defects in left/right (L/R) patterning with variable penetrance and expressivity. The penetrance and expressivity of the KV phenotype in carmil3 mutants correlated well with the L/R heart positioning defect at the end of embryogenesis. This in vivo animal study of CARMIL3 reveals its new role during morphogenesis of the vertebrate embryo. This role involves migration of endodermal cells and DFCs, along with subsequent morphogenesis of the KV and L/R asymmetry.
    Keywords:  Actin; Capping protein; Cell migration; Endoderm; Gastrulation; Kupffer's vesicle; Morphogenesis; Zebrafish
    DOI:  https://doi.org/10.1016/j.ydbio.2021.09.008
  21. Thromb Haemost. 2021 Oct 07.
    Mice lacking PECAM-1 and Ceacam1 have an aberrant platelet and thrombus phenotype.
    Fahd Kuriri, Genia Burchall, Fehaid Alanazi, Juliana Antonipillai, Gasim Dobie, Nicole Beauchemin, Denise Edna Jackson.
      The immunoglobulin (Ig)-immunoreceptor tyrosine-based inhibitory motif (ITIM) bearing receptors, PECAM-1 and CEACAM1 have been shown net negative regulators of platelet-collagen interactions and hemi-ITAM signalling pathways. In this study, a double knockout (DKO) mouse was developed with deleted PECAM-1 and CEACAM1 to study their combined contribution in platelet activation by glycoprotein VI, C-type lectin-like receptor 2 (CLEC-2), protease activated receptor PAR-4, ADP purinergic receptors and thromboxane receptor TP A2 pathways. Additionally, their collective contribution was examined in thrombus formation under high shear and microvascular thrombosis using in vivo models. DKO platelets responded normally to ADP purinergic receptors and TP A2 pathway. However, DKO platelets released significantly higher amounts of P-selectin compared to hyper-responsive Pecam-1-/- or Ceacam1-/- versus wild-type (WT) upon stimulation with collagen related peptide or rhodocytin. Contrastingly, DKO platelets released increased amounts of P-selectin upon stimulation with PAR-4 agonist peptide or thrombin but not Pecam-1-/-, Ceacam1-/- or WT platelets. Blockade of phospholipase C (PLC) or Rho A kinase revealed that DKO platelets enhanced alpha granule release via PAR-4/Gαq/PLC signalling without crosstalk with Src/Syk or G12/13 signalling pathways. This DKO model showed a significant increase in thrombus formation compared to the hyper-responsive Ceacam1-/- or Pecam-1-/- versus WT phenotype. DKO platelets have similar glycoprotein surface expression compared to Pecam-1-/-, Ceacam1-/- and WT platelets. PECAM-1 and CEACAM1 work in concert to negatively regulate hemiITAM signalling, platelet-collagen interactions and PAR-4 Gαq protein coupled signalling pathways. Both PECAM-1 and CEACAM1 are required for negative regulation of platelet activation and microvascular thrombosis in vivo.
    DOI:  https://doi.org/10.1055/a-1663-8108
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