bims-bicyki 2023-04-02 papers

bims-bicyki

Biomed News

on Bicaudal-C1 and interactors in cystic kidney disease

Issue of 2023–04–02
25 papers selected by
Céline Gagnieux, École Polytechnique Fédérale de Lausanne

Proteomic Approaches and Potential Applications in Autosomal Dominant Polycystic Kidney Disease and Fabry Disease.
Dehydration Accelerates Cytogenesis and Cyst Growth in Pkd1-/- Mice by Regulating Macrophage M2 Polarization.
HOW TO ESTIMATE KIDNEY GROWTH IN PATIENTS WITH AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE.
Time-lapse imaging of primary cilium behavior with physiological expression of fluorescent ciliary proteins.
Using mammary organoids to study cilia.
A flow cytometry-based approach for the study of primary cilia.
Protocols to induce and study ciliogenesis.
The C-terminal tail of polycystin-1 suppresses cystic disease in a mitochondrial enzyme-dependent fashion.
Complete small bowel obstruction due to ileal volvulus with autosomal dominant polycystic kidney disease: A rare case report.
Nutritional status is associated with preserved kidney function in patients with autosomal dominant polycystic kidney disease.
High-efficient CRISPR/Cas9-mediated gene targeting to establish cell models of ciliopathies.
Cholesterol and Phosphoinositides in Cilia Biology.
Evaluation of ciliary-GPCR dynamics using a validated organotypic brain slice culture method.
The Importance of Genetic Testing in the Differential Diagnosis of Atypical TSC2-PKD1 Contiguous Gene Syndrome-Case Series.
Identification of a heterogeneous and dynamic ciliome during embryonic development and cell differentiation.
Methods for Paramecium tetraurelia ciliary membrane protein identification and function.
Microscopic observation of human airway ciliary movement using wheat germ agglutinin.
Studying the morphology, composition and function of the photoreceptor primary cilium in zebrafish.
AN 84-YEAR-OLD PATIENT WITH CAROLI SYNDROME: WHAT IS THE PROGNOSIS OF THIS CONDITION?
Mucus Plugging in Primary Ciliary Dyskinesia.
Isolation of ciliary ectosomes and analysis of amidated peptide-mediated chemotaxis in Chlamydomonas.
Complex PKD1 Genetics in Early-Onset Cystic Kidney Disease.
Olfaction and Gustation in Children With Primary Ciliary Dyskinesia.
Reserpine maintains photoreceptor survival in retinal ciliopathy by resolving proteostasis imbalance and ciliogenesis defects.
Role of mucociliary clearance system in respiratory diseases.

Diagnostics (Basel). 2023 Mar 17. pii: 1152. [Epub ahead of print]13(6):

Proteomic Approaches and Potential Applications in Autosomal Dominant Polycystic Kidney Disease and Fabry Disease.

Merita Rroji, Andreja Figurek, Goce Spasovski.

  Although rare, hereditary diseases, such as autosomal dominant polycystic kidney disease (ADPKD) and Fabry disease (FD) may significantly progress towards severe nephropathy. It is crucial to characterize it accurately, predict the course of the illness and estimate treatment effectiveness. A huge effort has been undertaken to find reliable biomarkers that might be useful for an early prevention of the disease progression and/or any invasive diagnostic procedures. The study of proteomics, or the small peptide composition of a sample, is a field of study under continuous development. Over the past years, several strategies have been created to study and define the proteome of samples from widely varying origins. However, urinary proteomics has become essential for discovering novel biomarkers in kidney disease. Here, the extracellular vesicles in human urine that contain cell-specific marker proteins from every segment of the nephron, offer a source of potentially valuable urinary biomarkers, and may play an essential role in kidney development and kidney disease. This review summarizes the relevant literature investigating the proteomic approaches and potential applications in the regular studies of ADPKD and FD.

Keywords:  chronic kidney disease biomarker; extracellular vesicles; proteomic

DOI:  https://doi.org/10.3390/diagnostics13061152
Inflammation. 2023 Mar 31.

Dehydration Accelerates Cytogenesis and Cyst Growth in Pkd1-/- Mice by Regulating Macrophage M2 Polarization.

Yang Yang, Jie Zhou, Dongjuan Zhang, Jiayi Lv, Meihan Chen, Chao Wang, Minghui Song, Fagui He, Shuwei Song, Changlin Mei.

  Adult autosomal dominant polycystic kidney disease (ADPKD) has been shown to be related as a "third hit" to the occurrence of acute or chronic kidney injury. Here, we examined whether dehydration, as a common kidney risk factor, could cause cystogenesis in chronic-onset Pkd1-/- mice by regulating macrophage activation. First, we confirmed that dehydration accelerated cytogenesis in Pkd1-/- mice and that macrophages infiltrated the kidney tissues even earlier than macroscopic cyst formation. Then, microarray analysis suggested that glycolysis pathway may be involved in macrophage activation in Pkd1-/- kidneys under conditions of dehydration. Further, we confirmed glycolysis pathway was activated and lactic acid (L-LA) was overproduced in the Pkd1-/- kidney under conditions of dehydration. We have already proved that L-LA strongly stimulated M2 macrophage polarization and overproduction of polyamine in macrophage in vitro, and in the present study, we further discovered that M2 polarization-induced polyamine production shortened the primary cilia length by disrupting the PC1/PC2 complex. Finally, the activation of L-LA-arginase 1-polyamine pathway contributed to cystogenesis and progressive cyst growth in Pkd1-/- mice recurrently exposed to dehydration.

Keywords:  adult autosomal dominant polycystic kidney disease; dehydration; macrophage; metabolism.

DOI:  https://doi.org/10.1007/s10753-023-01806-5
J Am Soc Nephrol. 2023 Mar 30.

HOW TO ESTIMATE KIDNEY GROWTH IN PATIENTS WITH AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE.

Francisco José Borrego Utiel, Y Mario Espinosa Hernández.

ABSTRACT: Autosomal dominant polycystic kidney disease (ADPKD) is a disease characterized by a progressive kidney growth due to the development of cysts that lead to gradual destruction of the surrounding parenchyma. In the first stage, the estimated glomerular filtration rate will remain stable despite the reduction of the renal parenchyma, because of an increase in glomerular hyperfiltration. The total kidney volume (TKV) measured with computed tomography or magnetic-resonance imaging is related to the future glomerular filtration rate decline. Thus, TKV has become an early marker to be analyzed in all patients with ADPKD. In addition, in recent years it has been pointed out that kidney growth rate estimated with a single TKV measurement can be a clear prognostic marker for future glomerular filtration decline. However, there is no consensus on how to measure kidney volume growth in ADPKD, so each author has used different models that, not having the same meaning, have been handled as if they produced similar values. This may lead to erroneous estimates of kidney growth rate with the consequent prognostic error. The Mayo Clinic classification is now the most widely accepted prognostic model in clinical practice to predict patients that will deteriorate faster and to decide what patients should be treated with tolvaptan. However, some aspects of this model have not been discussed in depth. Our aim in this review is to present the models that can be used to estimate kidney volume growth rate in ADPKD, to facilitate their applicability in daily clinical practice.

DOI: https://doi.org/10.1681/ASN.0000000000000130
Methods Cell Biol. 2023 ;pii: S0091-679X(22)00167-4. [Epub ahead of print]175 45-68

Time-lapse imaging of primary cilium behavior with physiological expression of fluorescent ciliary proteins.

Ryota Nakazato, Hiroshi Otani, Faryal Ijaz, Koji Ikegami.

  Almost all cell types of mammals have a small protrusion named a primary cilium on their surface. Primary cilia are enriched by cilia-specific ion channels and G-protein-coupled receptors. They are known to regulate various cellular functions that contribute to the development and homeostasis of living organisms by receiving extracellular signals and transfusing them to the cell body. All functions are performed when the structure of the primary cilia is maintained properly. Abnormalities in primary cilia or their signaling can lead to a collection of diseases in various organs called ciliopathies. The primary cilium is dynamic, static, or fixed. The length of primary cilia varies as the cell cycle progresses and is also altered by extracellular stimuli. Ligand binding to cilia-specific receptors is also known to alter the length. Thus, there is a need for a method to study the morphological changes of the primary cilium in a time-dependent manner, especially under stimuli or mechanical shocks. Time-lapse imaging of primary cilia is one of the most powerful methods to capture the time-dependent behavior of primary cilia. Overexpression of ciliary proteins fused to fluorescent proteins is commonly used for the time-lapse imaging of primary cilia. However, overexpression has drawbacks in terms of artifacts. In addition, the time-lapse imaging of the tiny primary cilia requires some technical tricks. Here, we present a detailed description of the methods for time-lapse imaging of primary cilium, from the generation of cell lines that stably express fluorescent protein-labeled cilia-localized proteins at the physiological level to image analysis, including quantification through image acquisition.

Keywords:  CRISPR; Fluorescent tags; Knock-In; NHEJ; Primary cilium; Time-lapse imaging

DOI:  https://doi.org/10.1016/bs.mcb.2022.10.003
Methods Cell Biol. 2023 ;pii: S0091-679X(22)00151-0. [Epub ahead of print]175 221-233

Using mammary organoids to study cilia.

Aurore M M Dupuy, Philippe P Juin, Vincent J Guen.

  Cilia are hair-like projections that assemble at the surface of cells in various tissues of multicellular organisms through a complex cell biological process called ciliogenesis. Cilia can assemble as single structures per cell (i.e. non-motile primary cilia), which act as cell signaling centers that dictate cell fate, or can be assembled in distinct cell types as many copies per cell (i.e. motile cilia) that beat to move fluids at the cell surface. The mechanisms that orchestrate formation and function of cilia, which are dysregulated in pathological settings such as ciliopathies, remain incompletely understood. Stem cell-derived organoids represent valuable models to study the mechanisms of ciliogenesis, ciliary signaling, and ciliary beating that collectively promote tissue development and homeostasis. Here, we present a comprehensive protocol for the growth of mammary organoids derived from mouse mammary stem cells and for immunofluorescence staining of primary cilia in these three-dimensional structures.

Keywords:  Cilia; Ciliogenesis; Immunofluorescence; Organoids; Signaling

DOI:  https://doi.org/10.1016/bs.mcb.2022.09.010
Methods Cell Biol. 2023 ;pii: S0091-679X(22)00109-1. [Epub ahead of print]175 17-31

A flow cytometry-based approach for the study of primary cilia.

Katja Baur, Gabriele Hölzl-Wenig, Francesca Ciccolini.

  Primary cilia provide a specialized subcellular environment favoring ordered and timely interaction and modification of signaling molecules, necessary for the sensing and transduction of extracellular signals and environmental conditions. Crucial to the understanding of ciliary function is the knowledge of the signaling molecules composing the ciliary compartment. While proteomes of primary cilia have been published recently, the selective isolation of primary cilia from specific cell types and whole tissue still proves difficult, and many laboratories instead resort to the analysis of cultured cells, which may introduce experimental artifacts. Here we present a flow cytometry-based method to isolate and characterize primary cilia from the murine ventricular-subventricular zone. After deciliation, primary cilia are immunolabeled with antibodies against ciliary markers. As an example, we here use a double-staining with acetylated tubulin, which stains the ciliary axoneme, and ciliary membrane protein ADP-ribosylation-like factor 13b (Arl13b); additionally, we triple-labeled primary cilia using the ciliary marker adenylate cyclase 3 (AC3). Besides analysis at the single particle level, fluorescence activated cell sorting (FACS) allows collection of pure preparations of primary cilia suited for subsequent proteomic analyses like mass spectrometry or western blot. As an example of analytical application, we performed triple immunostaining and FACS analysis to reveal cilia heterogeneity. Thus, our cilia isolation method, which can readily be applied to other tissues or cell culture, will facilitate the study of this key cellular organelle and shed light on its role in normal conditions and disease.

Keywords:  AC3; Acetylated tubulin; Arl13b; Flow cytometry; Mammalian brain; Neural stem cells; Primary cilia; Ventricular-subventricular zone

DOI:  https://doi.org/10.1016/bs.mcb.2022.07.018
Methods Cell Biol. 2023 ;pii: S0091-679X(22)00166-2. [Epub ahead of print]175 1-15

Protocols to induce and study ciliogenesis.

Paula Moreno-Cruz, Yaiza Corral Nieto, Laura Manrique Garcia, Amanda Gabrielly Pereira, José Manuel Bravo-San Pedro.

  Primary cilia (PC) are sensory organelles that function as cellular antennas, transmitting signals between the extracellular and intracellular spaces in many vertebrate tissues. The cell generates and assembles PC through a highly regulated process called ciliogenesis. This complex process is involved in several physiological functions, including embryonic development, locomotion, cell cycle regulation or energetic homeostasis control. In general, when a cell finishes its cell division, the oldest centriole usually migrates to the plasma membrane and becomes a basal body that gives rise to the formation of a cilium. For this reason, the presence of cilia is incompatible with cell division, so when a cell is going to divide, the cilium and the basal body disappear. Ciliogenesis is triggered by various stimuli, all of them related to cell cycle blockade. This cell cycle, and ciliogenesis induction, can be observed by: (1) the influence of growth factors (lack of serum and consequent inability to promote cell cycle exit and increase the proportion of cells in G0); (2) pharmacological cell cycle inhibitors (staurosporine or etoposide); or (3) physiological cell cycle inhibition (excessive contact between neighboring cells). Evaluation of ciliogenesis induction is vitally important for the study of diseases related to ciliary dysfunction, called ciliopathies. That is why the use of correct protocols for inducing cilia formation and an accurate posterior visualization of the cilia after performing said protocols are essential parts in the study of these diseases. To facilitate this task, here we described detailed protocols to induce ciliogenesis in vitro and visualize PC by immunofluorescence microscopy in cultured cells.

Keywords:  Axoneme; Basal body; Cell cycle; Cilia; Ciliogenesis; Immunofluorescence

DOI:  https://doi.org/10.1016/bs.mcb.2022.10.002
Nat Commun. 2023 Mar 30. 14(1): 1790

The C-terminal tail of polycystin-1 suppresses cystic disease in a mitochondrial enzyme-dependent fashion.

Laura Onuchic, Valeria Padovano, Giorgia Schena, Vanathy Rajendran, Ke Dong, Xiaojian Shi, Raj Pandya, Victoria Rai, Nikolay P Gresko, Omair Ahmed, TuKiet T Lam, Weiwei Wang, Hongying Shen, Stefan Somlo, Michael J Caplan.

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent potentially lethal monogenic disorder. Mutations in the PKD1 gene, which encodes polycystin-1 (PC1), account for approximately 78% of cases. PC1 is a large 462-kDa protein that undergoes cleavage in its N and C-terminal domains. C-terminal cleavage produces fragments that translocate to mitochondria. We show that transgenic expression of a protein corresponding to the final 200 amino acid (aa) residues of PC1 in two Pkd1-KO orthologous murine models of ADPKD suppresses cystic phenotype and preserves renal function. This suppression depends upon an interaction between the C-terminal tail of PC1 and the mitochondrial enzyme Nicotinamide Nucleotide Transhydrogenase (NNT). This interaction modulates tubular/cyst cell proliferation, the metabolic profile, mitochondrial function, and the redox state. Together, these results suggest that a short fragment of PC1 is sufficient to suppress cystic phenotype and open the door to the exploration of gene therapy strategies for ADPKD.

DOI: https://doi.org/10.1038/s41467-023-37449-1
Int J Surg Case Rep. 2023 Mar 29. pii: S2210-2612(23)00180-3. [Epub ahead of print]105 108052

Complete small bowel obstruction due to ileal volvulus with autosomal dominant polycystic kidney disease: A rare case report.

Pashupati Pokharel, Milan K C, Sagun Ghimire, Arjun Yadav, Kabi Raj Bhusal, Prasan Bir Singh Kansakar.

   INTRODUCTION AND IMPORTANCE: Volvulus is the twisting of the mesentery of the bowel along its axis. Ileal volvulus is a rare cause of small bowel obstruction. Ileal volvulus coexisting with autosomal dominant polycystic kidney disease has not been reported in the literature previously.
CASE PRESENTATION: 65-year male with known history of autosomal dominant polycystic kidney disease (ADPKD) presented with pain abdomen for 5 days, obstipation for 3 days, and multiple episodes of bilious vomiting in the emergency department. Being a suspect of bowel obstruction, X-ray abdomen was done which showed features of small intestine obstruction. Further, to find the etiology of obstruction, contrast enhanced computed tomography (CECT) abdomen was done which showed swirling of the ileal loop and the ileal mesenteric vessels along with transition point in the ileal loop suggestive of ileal volvulus. Exploratory laparotomy with detorsion of the volvulus was done for management.
CLINICAL DISCUSSION: Small bowel volvulus, more specifically ileal volvulus, is a rare cause of intestinal obstruction. Patients present with the cardinal features of bowel obstruction, i.e., abdominal pain, distension, vomiting, and constipation/obstipation. Our patient had coexisting ADPKD which further aggravated the clinical presentation. Definitive management of the volvulus includes exploratory laparotomy and detorsion along with resection of the bowel if found ischemic. In our case the bowel was healthy so only detorsion was done.
CONCLUSION: Early diagnosis and meticulous exploratory laparotomy is utmost for the management of ileal volvulus. Besides, the secondary etiology (ADPKD in our case) should be managed to prevent future recurrences.

Keywords:  ADPKD; Ileal volvulus; Jejunal diverticula; Small bowel obstruction; Volvulus

DOI:  https://doi.org/10.1016/j.ijscr.2023.108052
J Ren Nutr. 2023 Mar 23. pii: S1051-2276(23)00042-0. [Epub ahead of print]

Nutritional status is associated with preserved kidney function in patients with autosomal dominant polycystic kidney disease.

Jinwoo Lee, Hyunjin Ryu, Yong-Chul Kim, Hayne Cho Park, Curie Ahn, Kyu-Beck Lee, Yeong Hoon Kim, Yaerim Kim, Seungyeop Han, Eun Hui Bae, Kook-Hwan Oh, Yun Kyu Oh.

   OBJECTIVE: Malnutrition is a common complication in autosomal dominant polycystic kidney disease (ADPKD). We examined whether nutritional status is associated with the preservation of kidney function, using a cohort of typical ADPKD.
METHODS: We enrolled ambulatory ADPKD patients in nine tertiary medical centers in Korea from May 2019 to December 2021. We excluded patients who were aged less than 18 years, who had known end-stage kidney disease at the time of enrollment, who had a diagnosis of atypical ADPKD, and who were Tolvaptan users. The primary outcome was an estimated glomerular filtration rate (eGFR) decline >3 mL/min/1.73m2, based on nutritional status assessed by subjective global assessment (SGA). We also evaluated an eGFR decline >1 mL/min/1.73m2, an increase in urine protein-creatinine ratio (UPCR) >0, and an increase in UPCR >0.3 as secondary outcomes, based on SGA after the one-year follow-up. A logistic regression (LR) model was used to calculate the odds ratio (OR) for the primary outcome. Because there were differences in several baseline variables, such as Mayo classification, serum hemoglobin, serum creatinine, and UPCR between SGA groups, we matched propensity scores.
RESULTS: In total, 805 patients were prospectively enrolled in nine tertiary medical centers in Korea from May 2019 to December 2021. Among them, 236 patients who had one-year follow-up data and typical imaging findings were analyzed to evaluate the effect of nutritional status on kidney function. SGA was used to assess the nutritional status. The mean age was 45.0 ± 13.3 years, and 49.6% of the patients were female. The mean eGFR was 81.9 ml/min/1.73m2. Among the 236 patients, 91 (38.6%) experienced a one-year eGFR decline >3 mL/min/1.73m2. When a multivariable LR was applied, SGA 3-6 was identified as a significant factor related to a one-year eGFR decline >3 mL/min/1.73m2 (adjusted OR = 1.22 [1.04-1.43]; P = 0.017). Despite matching propensity scores, the one-year eGFR decline >3 mL/min/1.73m2 was still higher in the SGA 3-6 group regardless of proteinuria.
CONCLUSION: Good nutritional status is associated with better-preserved kidney function in non-obese typical ADPKD patients who do not take Tolvaptan.

Keywords:  Autosomal dominant polycystic kidney disease; Kidney function; Nutrition; Proteinuria; Sarcopenia

DOI:  https://doi.org/10.1053/j.jrn.2023.02.006
Methods Cell Biol. 2023 ;pii: S0091-679X(22)00111-X. [Epub ahead of print]175 85-95

High-efficient CRISPR/Cas9-mediated gene targeting to establish cell models of ciliopathies.

Kosuke Hosoba, Tomoka Morita, Ying Zhang, Hiroko Kishi, Takashi Yamamoto, Tatsuo Miyamoto.

  Primary cilia are antenna-like structures developed on the cell surface of mammalian cells during the quiescent G0 phase. Primary cilia in mammalian cells receive extracellular signals for early development and cell tissue homeostasis. Ciliopathies characterized with congenital anomalies such as cerebellar hypoplasia, polycystic kidney and polydactyly are caused by germline mutations of ciliary structure- and function-related genes. Gene knock-out techniques in ciliated cultured cells with the uniformed genetic background are useful to evaluate the pathophysiological roles of ciliopathy-related gene products. Genome editing technology has been applied into the gene knock-out in many types of cultured cell lines. However, the frequency of genome editing varies according to cell species and cycle because of dependency on error-free homology-directed repair (HDR) activity. The human telomerase reverse transcriptase-immortalized retinal pigmented epithelial cell line (hTERT-RPE1) is well known for its suitability in cilia research. However, the efficacy of the HDR-mediated knock-out clone isolation was low. Here, we introduce the clustered regularly interspaced short palindromic repeats-obligate ligation-gated recombination (CRISPR-ObLiGaRe) system, which is a nonhomologous end-joining (NHEJ)-mediated gene targeting method, to generate the knock-out clones effectively even in the lower-HDR activity cell lines including hTERT-RPE1 cell. This CRISPR-ObLiGaRe system is a powerful tool for establishing ciliopathy model cell libraries and identifying each gene function in cilia-related phenotypes.

Keywords:  CRISPR/Cas9 system; Ciliopathy; Genome editing technology; INPP5E; Primary cilia

DOI:  https://doi.org/10.1016/bs.mcb.2022.07.020
Adv Exp Med Biol. 2023 ;1422 121-142

Cholesterol and Phosphoinositides in Cilia Biology.

Steffen-Alexander Sailer, Martin D Burkhalter, Melanie Philipp.

  Cilia are evolutionarily conserved organelles that can be found on virtually every cell. They appear as hair-like structures emanating from the cellular surface either as single or as bundles of cilia. There, they sense external stimuli and translate them into intracellular signals. Motile cilia beat for the generation of locomotion of unicellular organisms or fluid flow in certain body cavities of vertebrate organisms. Defects in cilia are detrimental and account for the development of ciliopathies, one of the fastest-growing family of afflictions. In the past decade, membrane lipids, such as cholesterol and phosphoinositides, have emerged as essential elements in both the signal transduction via cilia and the building of cilia itself. Here, we summarize the current knowledge on the impact of cholesterol and phosphoinositides on cilium biology.

Keywords:  Cholesterol; Cilia; Ciliopathy; Phosphoinositide

DOI:  https://doi.org/10.1007/978-3-031-21547-6_4
Methods Cell Biol. 2023 ;pii: S0091-679X(22)00148-0. [Epub ahead of print]175 69-83

Evaluation of ciliary-GPCR dynamics using a validated organotypic brain slice culture method.

Yuki Kobayashi, Yumiko Saito.

  The primary cilium is a structural organelle present in most mammalian cells. Primary cilia are enriched with a unique protein repertoire distinct from that of the cytosol and the plasma membrane. Such a highly organized microenvironment creates effective machinery for translating extracellular cues into intracellular signals. G protein-coupled receptors (GPCRs) are key receptors in sensing environmental stimuli transmitted via a second messenger into a cellular response. Recent data has demonstrated that a limited number of non-olfactory GPCRs, including melanin-concentrating hormone receptor 1 (MCHR1), are preferentially localized to ciliary membranes of several mammalian cell types, including neuronal cells. Evidence was accumulated to support the functional importance of ciliary-GPCR signaling accompanying ciliary structural changes using cilia-specific cell and molecular biology techniques. Thus, cilia are now considered to function as a unique sensory platform for the integration of GPCR signaling and various cytoplasmic domains. Dissociated neurons expressing ciliary-GPCRs can be a useful tool for examining ciliary dynamics. However, losing preexisting neuronal connectivity may alter neuronal ciliary morphology, such as abnormal elongation. Brain slices prepared under ex vitro conditions are a powerful approach that maintains the cytoarchitecture, enabling researchers to have accurate control over experimental conditions and to study individual cells from subregions of the brain. Here, we present a detailed description of our novel modified method for organotypic culture of rat brain slice and a validated immunostaining protocol to characterize ciliary-GPCR dynamics in coupling with neuropeptides or aminergic activation.

Keywords:  Brain slice; G protein-coupled receptor; Hippocampus; Melanin-concentrating hormone; Primary Cilium

DOI:  https://doi.org/10.1016/bs.mcb.2022.09.007
Children (Basel). 2023 Feb 22. pii: 420. [Epub ahead of print]10(3):

The Importance of Genetic Testing in the Differential Diagnosis of Atypical TSC2-PKD1 Contiguous Gene Syndrome-Case Series.

Petronella Orosz, Zita Kollák, Ákos Pethő, András Fogarasi, György Reusz, Kinga Hadzsiev, Tamás Szabó.

   BACKGROUND: In clinical practice, the possible diagnosis of tuberous sclerosis or polycystic kidney disease is primarily based on clinical criteria, which can later be verified by genetic testing. But in the case of TSC2/PKD1 contiguous gene syndrome (TSC2/PKD1-CGS), the renal appearance of the disease is more serious. Therefore, early genetic analysis is recommended.
METHODS: Herein we present the report of four children with TSC2/PKD1-CGS, one involving the NTHL1 gene. We aim to emphasize the importance of genetic testing in this rare syndrome.
RESULTS: During the follow-up of tuberous sclerosis and polycystic kidney disease patients, it is essential to reappraise the diagnosis if the clinical symptoms' appearance or onset time is unusual. Targeted genetic testing is recommended. However, early tumor formation necessitates the extension of genetic analysis.
CONCLUSIONS: An appropriate evaluation of the phenotype is the cornerstone of diagnosing the rare TSC2/PKD1-CGS with the help of genetic results. In addition, malignant tumors could draw attention to an infrequent large deletion.

Keywords:  NTHL1; angiomyolipoma; contiguous gene syndrome; polycystic kidney; tuberous sclerosis

DOI:  https://doi.org/10.3390/children10030420
Development. 2023 Mar 27. pii: dev.201237. [Epub ahead of print]

Identification of a heterogeneous and dynamic ciliome during embryonic development and cell differentiation.

Kelsey H Elliott, Sai K Balchand, Christian Louis Bonatto Paese, Ching-Fang Chang, Yanfen Yang, Kari M Brown, Daniel T Rasicci, Hao He, Konrad Thorner, Praneet Chaturvedi, Stephen A Murray, Jing Chen, Aleksey Porollo, Kevin A Peterson, Samantha A Brugmann.

  Primary cilia are nearly ubiquitous organelles that transduce molecular and mechanical signals. While the basic structure of the cilium and the cadre of genes that contribute to ciliary formation and function (the ciliome) are believed to be evolutionarily conserved, the presentation of ciliopathies with narrow, tissue-specific phenotypes and distinct molecular readouts suggests an unappreciated heterogeneity exists within this organelle. Here, we provide a searchable transcriptomic resource for a curated primary ciliome detailing various subgroups of differentially expressed genes within the ciliome that display tissue and temporal specificity (https://research.cchmc.org/Ciliome_Gene_Expression/). Genes within the differentially expressed ciliome exhibited a lower level of functional constraint across species suggesting organism and cell-specific function adaptation. The biological relevance of ciliary heterogeneity was functionally validated by utilizing Cas9 gene-editing to disrupt ciliary genes that displayed dynamic gene expression profiles during osteogenic differentiation of multipotent neural crest cells. Collectively, this novel primary cilia-focused resource will allow researchers to explore long-standing questions related to how tissue and cell-type specific functions and ciliary heterogeneity may contribute to the range of phenotypes associated with ciliopathies.

Keywords:  Ciliopathy; Craniofacial; Primary cilia; Skeletogenesis; Tissue heterogeneity

DOI:  https://doi.org/10.1242/dev.201237
Methods Cell Biol. 2023 ;pii: S0091-679X(22)00191-1. [Epub ahead of print]175 177-219

Methods for Paramecium tetraurelia ciliary membrane protein identification and function.

Megan Valentine, Junji Yano, Sukanya Lodh, Ashikun Nabi, Bin Deng, Judith Van Houten.

  In this chapter we provide some tools to study the ciliary proteins that make it possible for Paramecium cells to swim by beating their cilia. These proteins include many ion channels, accessory proteins, peripheral proteins, structural proteins, rootlets of cilia, and enzymes. Some of these proteins are also found in the soma membrane, but their distinct and critical functions are in the cilia. Paramecium has 4000 or more cilia per cell, giving it an advantage for biochemical studies over cells that have one primarily cilium per cell. Nonetheless, a challenge for studies of many ciliary proteins in Paramecium is their low abundance. We discuss here several strategies to overcome this challenge and other challenges such as working with very large channel proteins. We also include for completeness other techniques that are critical to the study of swimming behavior, such as genetic crosses, recording of swimming patterns, electrical recordings, expression of very large channel proteins, RNA Interference, among others.

Keywords:  Calcium; Channels; Cilia; Mutants; Paramecium

DOI:  https://doi.org/10.1016/bs.mcb.2022.12.003
Methods Cell Biol. 2023 ;pii: S0091-679X(22)00110-8. [Epub ahead of print]175 33-43

Microscopic observation of human airway ciliary movement using wheat germ agglutinin.

Ryosuke Nakamura, Seiji Oyagi, Tatsuya Katsuno, Yo Kishimoto, Koichi Omori.

  Ciliated cells in the airway epithelium generate mucus streams to remove extraneous particles and microorganisms by beating the motile cilia. This defense mechanism is crucial for maintaining homeostasis and preventing infection in the airway. Conventional methods to assess ciliary beating have revealed that rapid (>10 times per second) and metachronal beating of cilia enables efficient mucus transport. Cilia are oriented to excrete mucus toward the outside of the body. However, conventional methods to directly observe ciliary movements uses transmitted light, which requires translucent samples. Sliced or fragmented tissues are used to observe ciliary movements in thick human airway tissues. Therefore, conventional methods are unsuitable for assessing in situ orientation of ciliary movements. The orientation of ciliary beating can be indirectly analyzed by tracking particles spread onto the epithelium; however, the particles are not efficiently transported by immature cilia. To address this issue, we developed a method for labeling airway motile cilia with fluorescently labeled wheat germ agglutinin (FL-WGA). The new method enables microscopic observation of ciliary movements without slicing or fragmenting the airway tissues. Since the airway epithelium is observed from the apical side, in situ orientation of ciliary beating can be analyzed using this method. Additionally, epithelial damage, and the number and maturity of cilia can be assessed during the observation of ciliary beating. The new method, in combination with other methods, can provide more comprehensive data regarding ciliary movements.

Keywords:  Airway; Cilium; Mucociliary; Mucus; Multiciliated; Trachea

DOI:  https://doi.org/10.1016/bs.mcb.2022.07.019
Methods Cell Biol. 2023 ;pii: S0091-679X(22)00168-6. [Epub ahead of print]175 97-128

Studying the morphology, composition and function of the photoreceptor primary cilium in zebrafish.

Markus Masek, Jingjing Zang, José M Mateos, Marco Garbelli, Urs Ziegler, Stephan C F Neuhauss, Ruxandra Bachmann-Gagescu.

  Vision is one of our dominant senses and its loss has a profound impact on the life quality of affected individuals. Highly specialized neurons in the retina called photoreceptors convert photons into neuronal responses. This conversion of photons is mediated by light sensitive opsin proteins, which are found in the outer segments of the photoreceptors. These outer segments are highly specialized primary cilia, explaining why retinal dystrophy is a key feature of ciliopathies, a group of diseases resulting from abnormal and dysfunctional cilia. Therefore, research on ciliopathies often includes the analysis of the retina with special focus on the photoreceptor and its outer segment. In the last decade, the zebrafish has emerged as an excellent model organism to study human diseases, in particular with respect to the retina. The cone-rich retina of zebrafish resembles the fovea of the human macula and thus represents an excellent model to study human retinal diseases. Here we give detailed guidance on how to analyze the morphological and ultra-structural integrity of photoreceptors in the zebrafish using various histological and imaging techniques. We further describe how to conduct functional analysis of the retina by electroretinography and how to prepare isolated outer segment fractions for different -omic approaches. These different methods allow a comprehensive analysis of photoreceptors, helping to enhance our understanding of the molecular and structural basis of ciliary function in health and of the consequences of its dysfunction in disease.

Keywords:  Cilia; Correlative light and electron microscopy (CLEM); Immunohistochemistry; Outer segment isolation; Plastic sections; Transmission electron microscopy (TEM); Zebrafish

DOI:  https://doi.org/10.1016/bs.mcb.2022.10.004
Eur J Case Rep Intern Med. 2023 ;10(3): 003794

AN 84-YEAR-OLD PATIENT WITH CAROLI SYNDROME: WHAT IS THE PROGNOSIS OF THIS CONDITION?

Fabio Caleça Emidio, Rafaela Costa Pereira, Rosário Blanco, Pedro Santos, Teresa Abegão, Claudia Fitas.

  Caroli disease is a rare congenital pathology caused by mutation of the PKHD1 gene (polycystic kidney and hepatic disease 1), also responsible for autosomal recessive polycystic kidney disease. Characterized by segmental and multifocal dilatation of the large intrahepatic bile ducts, classic disease involves only malformation of the biliary tract. The association with congenital hepatic fibrosis is called Caroli syndrome. We describe the case of an 84-year-old man with Caroli syndrome diagnosed in 1997 by liver biopsy. The CT scan revealed massive hepatomegaly, extending to the pelvic region, and almost total replacement of the parenchyma by numerous cystic formations, no evidence of bile duct dilatation, and no ascites or splenomegaly suggestive of portal hypertension. The atypical clinical presentation, with no reported complications, resembles that of a space-occupying lesion with an indolent course, previously misdiagnosed as metastatic neoplasm.
LEARNING POINTS: We describe a case of advanced and rare Caroli syndrome with an atypical clinical presentation of a space-occupying lesion with slow progression.The atypical presentation could be misdiagnosed as metastatic neoplasm.

Keywords:  Caroli disease; hepatomegaly; intrahepatic bile ducts; polycystic kidney disease

DOI:  https://doi.org/10.12890/2023_003794
Ann Am Thorac Soc. 2023 Apr;20(4): 514-515

Mucus Plugging in Primary Ciliary Dyskinesia.

Stephanie Adaikalam, Benjamin Gaston.

DOI: https://doi.org/10.1513/AnnalsATS.202301-021ED
Methods Cell Biol. 2023 ;pii: S0091-679X(22)00150-9. [Epub ahead of print]175 163-175

Isolation of ciliary ectosomes and analysis of amidated peptide-mediated chemotaxis in Chlamydomonas.

Raj Luxmi, Stephen M King.

  Ciliary ectosomes are vesicles that bud from the ciliary membrane. Isolation and analysis of these structures can shed light on their bioactive cargoes and identify proteins and biomolecules involved in intercellular communication and various physiological processes. Most published methods to isolate ciliary ectosomes are based on their size (100nm to 1μm) to separate cilia-derived vesicles from isolated cilia and/or intact cells. However, it is often difficult to determine the origin of extracellular vesicles and to distinguish ciliary ectosomes from ectosomes budded from the plasma membrane or from exosomes that derive from multivesicular bodies. Here, we describe procedures to isolate and purify ciliary ectosomes from the unicellular green alga, Chlamydomonas reinhardtii, through differential and iodixanol density gradient ultracentrifugation; in this organism, the ciliary membrane is the only membrane directly exposed to the environment and thus ectosomes are of known origin. Ciliary ectosomes contain enzymes and α-amidated peptide products required to mediate peptidergic-signaling cascades; one identified amidated peptide acts as a chemotactic modulator for C. reinhardtii gametes. Classical methods used to assess chemotaxis do not provide quantitative measurements of the chemotactic gradient or the real-time effects on the migration of fast moving cells. Consequently, we developed a chemotaxis assay protocol using microfluidic channel slides that provides quantitative and qualitative measurements of the chemotactic gradient and cell migration. Here, we describe how to establish a stable gradient of a bioactive substance in microfluidic channel slides and perform quantitative assays to assess chemotaxis of both individual cells and populations of C. reinhardtii.

Keywords:  Chemotaxis; Chlamydomonas; Cilia; Ectosome; Extracellular vesicle; Peptide amidation; Peptidergic signaling

DOI:  https://doi.org/10.1016/bs.mcb.2022.09.009
Kidney360. 2023 Mar 01. 4(3): 297-298

Complex PKD1 Genetics in Early-Onset Cystic Kidney Disease.

Matthew B Lanktree.

DOI: https://doi.org/10.34067/KID.0000000000000110
OTO Open. 2023 Jan-Mar;7(1):7(1): e28

Olfaction and Gustation in Children With Primary Ciliary Dyskinesia.

Faisal Zawawi, Sharon Dell, Nikolaus E Wolter, Blake C Papsin, Evan J Propst.

   Objective: Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder whereby abnormal cilia cause a wide array of respiratory tract manifestations including chronic rhinosinusitis. The purpose of this study was to determine whether olfaction and gustation are impaired in children with PCD.
Study Design: Cross-sectional study.
Setting: Tertiary pediatric academic hospital.
Methods: Children with confirmed PCD based on having at least 1 of 3 approved diagnostic criteria as per The American Thoracic Society guidelines were recruited from The PCD Clinic in our tertiary care pediatric hospital. Odor identification ability was tested using the Universal Sniff (U-Sniff) test and taste threshold was measured using an electrogustometer. The main outcome of this study is to determine the incidence of olfactory dysfunction in children with PCD and investigate if there is an associated gustatory dysfunction.
Results: Twenty-five children participated (14 male, 11 female), The median age was 10.8 years (range: 4.1-17.9 years). Only 4/25 (16%) complained of olfactory dysfunction prior to testing. None of the patients complained of dysgeusia. However, 48% (12/25) scored less than 7 on the U-Sniff, signifying hyposmia or anosmia. In contrast, scores obtained by electrogustometry were in the normal range. There was no correlation between performance on the U-Sniff and electrogustometry testing.
Conclusion: Olfactory impairment in children with PCD is common but underrecognized by patients. This is not associated with abnormal gustation. Among other, this places children with PCD at an increased risk with respect to smelling a fire or detecting spoiled or poisonous food.

Keywords:  U‐Sniff; children; gustation; olfaction; primary ciliary dyskinesia

DOI:  https://doi.org/10.1002/oto2.28
Elife. 2023 Mar 28. pii: e83205. [Epub ahead of print]12

Reserpine maintains photoreceptor survival in retinal ciliopathy by resolving proteostasis imbalance and ciliogenesis defects.

Holly Y Chen, Manju Swaroop, Samantha Papal, Anupam Mondal, Hyun Beom Song, Laura Campello, Gregory Tawa, Florian Regent, Hiroko Shimada, Kunio Nagashima, Natalia de Val, Samuel G Jacobson, Wei Zheng, Anand Swaroop.

  Ciliopathies manifest from sensory abnormalities to syndromic disorders with multi-organ pathologies, with retinal degeneration a highly penetrant phenotype. Photoreceptor cell death is a major cause of incurable blindness in retinal ciliopathies. To identify drug candidates to maintain photoreceptor survival, we performed an unbiased, high-throughput screening of over 6,000 bioactive small molecules using retinal organoids differentiated from induced pluripotent stem cells (iPSC) of rd16 mouse, which is a model of Leber congenital amaurosis (LCA) type 10 caused by mutations in the cilia-centrosomal gene CEP290. We identified five non-toxic positive hits, including the lead molecule reserpine, which maintained photoreceptor development and survival in rd16 organoids. Reserpine also improved photoreceptors in retinal organoids derived from induced pluripotent stem cells of LCA10 patients and in rd16 mouse retina in vivo. Reserpine-treated patient organoids revealed modulation of signaling pathways related to cell survival/death, metabolism, and proteostasis. Further investigation uncovered dysregulation of autophagy associated with compromised primary cilium biogenesis in patient organoids and rd16 mouse retina. Reserpine partially restored the balance between autophagy and the ubiquitin-proteasome system at least in part by increasing the cargo adaptor p62, resulting in improved primary cilium assembly. Our study identifies effective drug candidates in preclinical studies of CEP290 retinal ciliopathies through cross-species drug discovery using iPSC-derived organoids, highlights the impact of proteostasis in the pathogenesis of ciliopathies, and provides new insights for treatments of retinal neurodegeneration.

Keywords:  biochemistry; chemical biology; mouse

DOI:  https://doi.org/10.7554/eLife.83205
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2023 Feb 28. pii: 1672-7347(2023)02-0275-10. [Epub ahead of print]48(2): 275-284

Role of mucociliary clearance system in respiratory diseases.

Di Wu, Yang Xiang.

  Mucociliary clearance system is the primary innate defense mechanism of the lung. It plays a vital role in protecting airways from microbes and irritants infection. Mucociliary clearance system, which is mediated by the actions of airway and submucosal gland epithelial cells, plays a critical role in a multilayered defense system via secreting fluids, electrolytes, antimicrobial and anti-inflammatory proteins, and mucus onto airway surfaces. Changes in environment, drugs or diseases can lead to mucus overproduction and cilia dysfunction, which in turn decrease the rate of mucociliary clearance and enhance mucus gathering. The dysfunction of mucociliary clearance system often occurs in several respiratory diseases, such as primary ciliary dysfunction, cystic fibrosis, asthma and chronic obstructive pulmonary disease, which are characterized by goblet cell metaplasia, submucosal gland cell hypertrophy, mucus hypersecretion, cilia adhesion, lodging and loss, and airway obstruction.

Keywords:  asthma; chronic obstructive pulmonary disease; cystic fibrosis; mucociliary clearance system; primary ciliary dysfunction

DOI:  https://doi.org/10.11817/j.issn.1672-7347.2023.220372