Motile Cilia Research Articles

#5983 PULMONARY INVOLVEMENT IN AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE

Abstract Background and Aims Autosomal dominant policystic kidney disease (ADPKD) is the most common genetic kidney disease. It is caused by mutations in PKD1 and PKD2 genes, which encode for polycystin-1 (PC-1) and polycystin-2 (PC-2), two proteins localized in the primary cilia which regulate calcium permeability and cellular signaling pathways. The major phenotype of the disease is the formation of multiple cysts in the kidneys but in many cases it also involves other organs like heart, brain and lungs. The latters are not yet well studied; some papers report a higher prevalence of bronchiectasis in ADPKD patients compared to the general population; morover, one work describes the absence of PC-1 in motile cilia of ADPKD pts compared to controls, who actually do express the protein. Based on available literature, we hypothesized that the PC-1 and PC-2 could have a role also in motile cilia. Exaled nasal nitric oxide (nNO) is an indirect marker of cilia dysfunction, standardized for the screening of primary ciliary dyskinesia, a motile ciliopathy, in which very low levels of nNO are found because of the entrapment of NO in the abundant stagnant mucus. Method We recruited 27 patients with ADPKD and 25 randomly selected controls. We compared nNO measurements between the two groups, stratifying for truncated mutations and eGFR. After stratification according to ADPKD genetic mutation, we analyzed separately each of the two subgroups with the control group. Results nNO levels were no different in ADPKD patients compared with healthy controls (mean ± standard deviation: 850±240 vs 923±290; P = .3). However in sub-group analysis, lower nNO measurements were found in patients with truncated mutation (769±164) compared to the control group (924±290, P = .05). No association was observed between renal functions expressed as eGFR and nNO levels. Discussion Overall nNO levels did not differ in ADPKD individuals compared to controls, although lower nNO measurements were observed in ADPKD patients exhibiting a truncated mutation. Conclusion Even at the edge of the statistical significance (P = .05), given it's confirmed in a wider population, this finding could signify that pts who carry a more aggressive genotype (PKD-1 truncated mutation) may not express polycystins in motile cilia; this aspect is in line with the verified knowledge that this kind of mutation ends up in the total absence of the protein. On the other hand, pts with a missense mutation may still express the protein in motile cilia, therefore without any functional impairment.

Dynein axonemal heavy chain 10 deficiency causes primary ciliary dyskinesia in humans and mice.

Primary ciliary dyskinesia (PCD) is a congenital, motile ciliopathy with pleiotropic symptoms. Although nearly 50 causative genes have been identified, they only account for approximately 70% of definitive PCD cases. Dynein axonemal heavy chain 10 (DNAH10) encodes a subunit of the inner arm dynein heavy chain in motile cilia and sperm flagella. Based on the common axoneme structure of motile cilia and sperm flagella, DNAH10 variants are likely to cause PCD. Using exome sequencing, we identified a novel DNAH10 homozygous variant (c.589C > T, p.R197W) in a patient with PCD from a consanguineous family. The patient manifested sinusitis, bronchiectasis, situs inversus, and asthenoteratozoospermia. Immunostaining analysis showed the absence of DNAH10 and DNALI1 in the respiratory cilia, and transmission electron microscopy revealed strikingly disordered axoneme 9+2 architecture and inner dynein arm defects in the respiratory cilia and sperm flagella. Subsequently, animal models of Dnah10-knockin mice harboring missense variants and Dnah10-knockout mice recapitulated the phenotypes of PCD, including chronic respiratory infection, male infertility, and hydrocephalus. To the best of our knowledge, this study is the first to report DNAH10 deficiency related to PCD in human and mouse models, which suggests that DNAH10 recessive mutation is causative of PCD.

Robust acoustic trapping and perturbation of single-cell microswimmers illuminate three-dimensional swimming and ciliary coordination

We report a label-free acoustic microfluidic method to confine single, cilia-driven swimming cells in space without limiting their rotational degrees of freedom. Our platform integrates a surface acoustic wave (SAW) actuator and bulk acoustic wave (BAW) trapping array to enable multiplexed analysis with high spatial resolution and trapping forces that are strong enough to hold individual microswimmers. The hybrid BAW/SAW acoustic tweezers employ high-efficiency mode conversion to achieve submicron image resolution while compensating for parasitic system losses to immersion oil in contact with the microfluidic chip. We use the platform to quantify cilia and cell body motion for wildtype biciliate cells, investigating effects of environmental variables like temperature and viscosity on ciliary beating, synchronization, and three-dimensional helical swimming. We confirm and expand upon the existing understanding of these phenomena, for example determining that increasing viscosity promotes asynchronous beating. Motile cilia are subcellular organelles that propel microorganisms or direct fluid and particulate flow. Thus, cilia are critical to cell survival and human health. The unicellular alga Chlamydomonas reinhardtii is widely used to investigate the mechanisms underlying ciliary beating and coordination. However, freely swimming cells are difficult to image with sufficient resolution to capture cilia motion, necessitating that the cell body be held during experiments. Acoustic confinement is a compelling alternative to use of a micropipette, or to magnetic, electrical, and optical trapping that may modify the cells and affect their behavior. Beyond establishing our approach to studying microswimmers, we demonstrate a unique ability to mechanically perturb cells via rapid acoustic positioning.

Early postnatal microglial ablation in the Ccdc39 mouse model reveals adverse effects on brain development and in neonatal hydrocephalus

BackgroundNeonatal hydrocephalus is a congenital abnormality resulting in an inflammatory response and microglial cell activation both clinically and in animal models. Previously, we reported a mutation in a motile cilia gene, Ccdc39 that develops neonatal progressive hydrocephalus (prh) with inflammatory microglia. We discovered significantly increased amoeboid-shaped activated microglia in periventricular white matter edema, reduced mature homeostatic microglia in grey matter, and reduced myelination in the prh model. Recently, the role of microglia in animal models of adult brain disorders was examined using cell type-specific ablation by colony-stimulating factor-1 receptor (CSF1R) inhibitor, however, little information exists regarding the role of microglia in neonatal brain disorders such as hydrocephalus. Therefore, we aim to see if ablating pro-inflammatory microglia, and thus suppressing the inflammatory response, in a neonatal hydrocephalic mouse line could have beneficial effects.MethodsIn this study, Plexxikon 5622 (PLX5622), a CSF1R inhibitor, was subcutaneously administered to wild-type (WT) and prh mutant mice daily from postnatal day (P) 3 to P7. MRI-estimated brain volume was compared with untreated WT and prh mutants P7-9 and immunohistochemistry of the brain sections was performed at P8 and P18-21.ResultsPLX5622 injections successfully ablated IBA1-positive microglia in both the WT and prh mutants at P8. Of the microglia that are resistant to PLX5622 treatment, there was a higher percentage of amoeboid-shaped microglia, identified by morphology with retracted processes. In PLX-treated prh mutants, there was increased ventriculomegaly and no change in the total brain volume was observed. Also, the PLX5622 treatment significantly reduced myelination in WT mice at P8, although this was recovered after full microglia repopulation by P20. Microglia repopulation in the mutants worsened hypomyelination at P20.ConclusionsMicroglia ablation in the neonatal hydrocephalic brain does not improve white matter edema, and actually worsens ventricular enlargement and hypomyelination, suggesting critical functions of homeostatic ramified microglia to better improve brain development with neonatal hydrocephalus. Future studies with detailed examination of microglial development and status may provide a clarification of the need for microglia in neonatal brain development.

The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes.

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift-null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partially preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. Transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. These findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies.

Airway Epithelial Cultures of Children with Esophageal Atresia as a Model to Study Respiratory Tract Disorders.

Esophageal atresia (EA) is a rare birth defect in which respiratory tract disorders are a major cause of morbidity. It remains unclear whether respiratory tract disorders are in part caused by alterations in airway epithelial cell functions such as the activity of motile cilia. This can be studied using airway epithelial cell culture models of patients with EA. Therefore, the aim of this study was to evaluate the feasibility to culture and functionally characterize motile cilia function in the differentiated air-liquid interface cultured airway epithelial cells and 3D organoids derived from nasal brushings and bronchoalveolar lavage (BAL) fluid from children with EA. We demonstrate the feasibility of culturing differentiated airway epithelia and organoids of nasal brushings and BAL fluid of children with EA, which display normal motile cilia function. EA patient-derived airway epithelial cultures can be further used to examine whether alterations in epithelial functions contribute to respiratory disorders in EA.

Artificial cilia for soft and stable surface covalent immobilization of bone morphogenetic protein-2.

Preservation of growth factor sensitivity and bioactivity (e.g., bone morphogenetic protein-2 (BMP-2)) post-immobilization to tissue engineering scaffolds remains a great challenge. Here, we develop a stable and soft surface modification strategy to address this issue. BMP-2 (a model growth factor) is covalently immobilized onto homogeneous poly (glycidyl methacrylate) (PGMA) polymer brushes which are grafted onto substrate surfaces (Au, quartz glass, silica wafer, or common biomaterials) via surface-initiated atom transfer radical polymerization. This surface modification method multiplies the functionalized interfacial area; it is simple, fast, gentle, and has little effect on the loaded protein owing to the cilia motility. The immobilized BMP-2 (i-BMP-2) on the surface of homogeneous PGMA polymer brushes exhibits excellent bioactivity (⁓87% bioactivity of free BMP-2 in vitro and 20%-50% higher than scaffolds with free BMP-2 in vivo), with conformation and secondary structure well-preserved after covalent immobilization and ethanol sterilization. Moreover, the osteogenic activity of i-BMP-2 on the nanoline pattern (PGMA-poly (N-isopropylacrylamide)) shows ⁓110% bioactivity of free BMP-2. This is superior compared to conventional protein covalent immobilization strategies in terms of both bioactivity preservation and therapeutic efficacy. PGMA polymer brushes can be used to modify surfaces of different tissue-engineered scaffolds, which facilitates in situ immobilization of growth factors, and accelerates repair of a wide range of tissue types.

Axonemal structures reveal mechanoregulatory and disease mechanisms

Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections1. Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes2. The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures.

Primary Ciliary Dyskinesia Patient-Specific hiPSC-Derived Airway Epithelium in Air-Liquid Interface Culture Recapitulates Disease Specific Phenotypes In Vitro.

Primary ciliary dyskinesia (PCD) is a rare heterogenic genetic disorder associated with perturbed biogenesis or function of motile cilia. Motile cilia dysfunction results in diminished mucociliary clearance (MCC) of pathogens in the respiratory tract and chronic airway inflammation and infections successively causing progressive lung damage. Current approaches to treat PCD are symptomatic, only, indicating an urgent need for curative therapeutic options. Here, we developed an in vitro model for PCD based on human induced pluripotent stem cell (hiPSC)-derived airway epithelium in Air-Liquid-Interface cultures. Applying transmission electron microscopy, immunofluorescence staining, ciliary beat frequency, and mucociliary transport measurements, we could demonstrate that ciliated respiratory epithelia cells derived from two PCD patient-specific hiPSC lines carrying mutations in DNAH5 and NME5, respectively, recapitulate the respective diseased phenotype on a molecular, structural and functional level.

BBOF1 is required for sperm motility and male fertility by stabilizing the flagellar axoneme in mice.

The sperm flagellum is a specialized type of motile cilium composed of a typical "9 + 2" axonemal structure with peri-axonemal structures, such as outer dense fibers (ODFs). This flagellar arrangement is crucial for sperm movement and fertilization. However, the association of axonemal integrity with ODFs remains poorly understood. Here, we demonstrate that mouse BBOF1 could interact with both MNS1, an axonemal component, and ODF2, an ODF protein, and is required for sperm flagellar axoneme maintenance and male fertility. BBOF1 is expressed exclusively in male germ cells from the pachytene stage onwards and is detected in sperm axoneme fraction. Spermatozoa derived from Bbof1-knockout mice exhibit a normal morphology, however, reduced motility due to the absence of certain microtubule doublets, resulting in the failure to fertilize mature oocytes. Furthermore, BBOF1 is found to interact with ODF2 and MNS1 and is also required for their stability. Our findings in mice suggest that Bbof1 could also be essential for human sperm motility and male fertility, thus is a novel potential candidate gene for asthenozoospermia diagnosis.

Rab8, Rab11, and Rab35 coordinate lumen and cilia formation during zebrafish left-right organizer development.

An essential process during Danio rerio's left-right organizer (Kupffer's Vesicle, KV) formation is the formation of a motile cilium by developing KV cells which extends into the KV lumen. Beating of motile cilia within the KV lumen directs fluid flow to establish the embryo's left-right axis. However, the timepoint at which KV cells start to form cilia and how cilia formation is coordinated with KV lumen formation have not been examined. We identified that nascent KV cells form cilia at their centrosomes at random intracellular positions that then move towards a forming apical membrane containing cystic fibrosis transmembrane conductance regulator (CFTR). Using optogenetic clustering approaches, we found that Rab35 positive membranes recruit Rab11 to modulate CFTR delivery to the apical membrane, which is required for lumen opening, and subsequent cilia extension into the lumen. Once the intracellular cilia reach the CFTR positive apical membrane, Arl13b-positive cilia extend and elongate in a Rab8 dependent manner into the forming lumen once the lumen reaches an area of 300 μm2. These studies demonstrate the need to acutely coordinate Rab8, Rab11, and Rab35-mediated membrane trafficking events to ensure appropriate timing in lumen and cilia formation during KV development.

Novel Aspects of Cilia-Driven Flow of Viscoelastic Fluid through a Non-Darcy Medium under the Influence of an Induced Magnetic Field and Heat Transfer

The spontaneous movement of natural motile cilia in the form of metachronal waves is responsible for fluid transport. These cilia, in particular, play important roles in locomotion, feeding, liquid pumping, and cell delivery. On the other hand, artificial cilia can be useful in lab-on-a-chip devices for manipulation processes. In this study, a novel model for the ciliated tapered channel in Sutterby fluid flow under the impact of an induced magnetic field and heat transport is proposed. The Darcy–Brinkman–Forchheimer law for porous media with a viscous dissipation function is considered. With the help of lubrication theory, the simplified non-linear form of the leading equation with cilia-oriented boundary conditions is achieved. The analytical results of differential equations are based on the topological perturbation approach. The numerical simulation is performed to elaborate on the physical interpretations of emerging parameters through computer software.

Congenital heart defects caused by FOXJ1.

FOXJ1 is expressed in ciliated cells of the airways, testis, oviduct, central nervous system and the embryonic left-right organizer. Ablation or targeted mutation of Foxj1 in mice, zebrafish and frogs results in loss of ciliary motility and/or reduced length and number of motile cilia, affecting the establishment of the left-right axis. In humans, heterozygous pathogenic variants in FOXJ1 cause ciliopathy leading to situs inversus, obstructive hydrocephalus and chronic airway disease. Here, we report a novel truncating FOXJ1 variant (c.784_799dup; p.Glu267Glyfs*12) identified by clinical exome sequencing from a patient with isolated congenital heart defects (CHD) which included atrial and ventricular septal defects, double outlet right ventricle (DORV) and transposition of the great arteries. Functional experiments show that FOXJ1 c.784_799dup; p.Glu267Glyfs*12, unlike FOXJ1, fails to induce ectopic cilia in frog epidermis in vivo or to activate the ADGB promoter, a downstream target of FOXJ1 in cilia, in transactivation assays in vitro. Variant analysis of patients with heterotaxy or heterotaxy-related CHD indicates that pathogenic variants in FOXJ1 are an infrequent cause of heterotaxy. Finally, we characterize embryonic-stage CHD in Foxj1 loss-of-function mice, demonstrating randomized heart looping. Abnormal heart looping includes reversed looping (dextrocardia), ventral looping and no looping/single ventricle hearts. Complex CHDs revealed by histological analysis include atrioventricular septal defects, DORV, single ventricle defects as well as abnormal position of the great arteries. These results indicate that pathogenic variants in FOXJ1 can cause isolated CHD.

The Function of Spag6 to Repair Brain Edema Damage After Cerebral Ischemic Stroke-reperfusion

Sperm associated antigen 6 (Spag6) is the PF16 homolog of Chlamydomonas and participates in the regulation of cilia movement. Studies have shown that Spag6 is expressed in the brain, and its loss will lead to cerebral edema caused by a defect in motor cilium function in ependymal cells. However, it has not been reported whether the limited or extensive cerebral edema resulting from ischemic strokes is related to the expression regulation of Spag6. Therefore, this study aimed to investigate the effect and related mechanism of Spag6 in alleviating Cerebral Ischemic stroke-reperfusion (CIS/R) damage. Our experimental results showed that Spag6 overexpression alleviated CIS/R-mediated motor cilia structural disorder, improved cerebral edema, inhibited nerve injuries in rats with cerebral ischemia, and alleviated synaptic and dendritic spinal injuries by regulating the expressions of synaptic-related proteins such as CaMKII, PSD95, and CREB. Based on significant changes in PI3K/AKT-mTOR signaling pathway activity after CIS/R determination, we determined that Spag6 regulates the abnormal expression of CIS/R-induced inflammatory factors NF-κB, NLRP3, IL-10, and the autophagy-related proteins Beclin-1, LC3, and P62 by activating the PI3K/AKT-mTOR signaling pathway. This inhibits inflammation and autophagy in the brain tissue. In summary, this study revealed that Spag6 alleviates brain edema damage after CIS/R by maintaining the structural function of the motor cilium, regulating the PI3K/AKT-mTOR signaling pathway, and inhibiting inflammation and autophagy reaction.

The MIR34B/C genomic region contains multiple potential regulators of multiciliogenesis.

The MIR449 genomic locus encompasses several regulators of multiciliated cell (MCC) formation (multiciliogenesis). The miR-449 homologs miR-34b/c represent additional regulators of multiciliogenesis that are transcribed from another locus. Here, we characterized the expression of BTG4, LAYN, and HOATZ, located in the MIR34B/C locus using single-cell RNA-seq and super-resolution microscopy from human, mouse, or pig multiciliogenesis models. BTG4, LAYN, and HOATZ transcripts were expressed in both precursors and mature MCCs. The Layilin/LAYN protein was absent from primary cilia, but it was expressed in apical membrane regions or throughout motile cilia. LAYN silencing altered apical actin cap formation and multiciliogenesis. HOATZ protein was detected in primary cilia or throughout motile cilia. Altogether, our data suggest that the MIR34B/C locus may gather potential actors of multiciliogenesis.

Altered differentiation in proximal airway epithelium in trisomy 21

Rationale: Trisomy 21 (T21), resulting in Down Syndrome (DS), is the most prevalent chromosomal abnormality worldwide. Recent studies have indicated that individuals with DS present with ciliopathies, as well as impaired mucociliary clearance. Whereas these defects have been determined in children, we recently reported that structural changes initiate prenatally in T21 lungs. Thus, our goal is to determine whether proximal airway differentiation and specifically ciliary structure and function are altered during T21 lung development. Methods: Immunofluorescent (IF) staining was performed on prenatal T21 and non-T21 age and sex matched control lungs to assess proximal airway cellular composition and differentiation. Gene expression was assessed by RT-qPCR. In vitro proximal airway differentiation was performed using primary basal cells isolated from prenatal T21 and non-T21 tracheas. Cells were plated into transwells and differentiated on air liquid interface (ALI). IF staining, RT-qPCR, and trans-epithelial electrical resistance (TEER) analyses were performed on ALI cultures at Day 0, 7, 14, and 21. Results: Prenatal T21 lungs were assessed for all major proximal airway cell types (basal, club, ciliated, goblet) and compared to non-T21 controls. IF staining of club cell markers (SCGB1A1, SCGB3A2) were increased in T21 lungs, with a significantly greater number of SCGB1A1 cells (p=0.0114) (n=20). A comparable trend was observed with goblet cells, which showed a larger quantifiable area of MUC5B staining in T21 lungs as compared to non-T21. These results were confirmed by RT-qPCR, which demonstrated a trend of increased expression for both SCGB3A2 and MUC5B, and a significant increase of SCGB1A1 (p=0.0169) (n=14). IF staining of ciliated cell markers (FOXJ1, RSPH1, ARL13B) were also increased in T21 lungs as compared to non-T21. These results were validated by RT-qPCR, with a significant increase in RSPH1, a gene responsible for motile cilia and encoded on chromosome 21 (p=0.0168) (n=11). We then cultured primary T21 and non-T21 basal cells in ALI and achieved successful airway epithelium differentiation. Confluent transwell IF staining shows a steady rate of P63+ cells, and an increase of ciliated cells (ARL13B, FOXJ1) over 21 days in culture. TEER measurements were lower in T21 cultures as compared to age matched non-T21. Conclusion: Our data demonstrate that prenatal T21 lungs undergo precocious proximal airway differentiation with possibly compromised tight junctions. These aberrancies may possibly contribute to the high infection rate, ciliopathies, and faulty mucociliary clearance observed in DS. California Institute for Regenerative Medicine (CIRM) EDUC4 - 12837 (CC) and NIH NHLBI/Office of the Director R01HL155104 (SD). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Concurrent Actuation and Sensing in Fluid by Cilia‐Like Transducers

Motile cilia are miniature, hair‐like organelles whose beating moves fluid in various organisms and the human body. Motile cilia are also able to sense many mechanical or fluidic cues. Inspired by the motile cilia in nature, a type of artificial cilia that can both actuate and sense is investigated. These artificial cilia can operate their sensing and actuation individually to sense the flow speed and collaboratively transport objects. More importantly, they can also sense the viscosity of the fluid and the distance from the wall by beating and feeling their own motion. In the latter function, two different sensing modes (modes 1 and 2) are proposed for low and high Reynolds numbers. They are investigated through both computational fluid dynamics and experiments. It is found that mode 1 can sense a wall distance up to 3 times the body length of the cilium, while mode 2 can sense up to 2 times. Such sensing of distance can be further converted into the ability to compute the morphology of the boundary inside the fluid. This work can be useful for small‐scale soft robots that work inside fluid and other flow control applications.

Identification of TEKTIN1-expressing multiciliated cells during spontaneous differentiation of non-human primate embryonic stem cells.

Tektins are a group of microtubule-stabilizing proteins necessary for cilia and flagella assembly. TEKTIN1 (TEKT1) is used as a sperm marker for monitoring germ cell differentiation in embryonic stem (ES) and induced pluripotent stem (iPS) cells. Although upregulation of TEKT1 has been reported during spontaneous differentiation of ES and iPS cells, it is unclear which cells express TEKT1. To identify TEKT1-expressing cells, we established an ES cell line derived from cynomolgus monkeys (Macaca fascicularis), which expresses Venus controlled by the TEKT1 promoter. Venus expression was detected at 5 weeks of differentiation on the surface of the embryoid body (EB), and it gradually increased with the concomitant formation of a leash-like structure at the EB periphery. Motile cilia were observed on the surface of the Venus-positive leash-like structure after 8 weeks of differentiation. The expression of cilia markers as well as TEKT1-5 and 9 + 2 microtubule structures, which are characteristic of motile cilia, were detected in Venus-positive cells. These results demonstrated that TEKT1-expressing cells are multiciliated epithelial-like cells that form a leash-like structure during the spontaneous differentiation of ES and iPS cells. These findings will provide a new research strategy for studying cilia biology, including ciliogenesis and ciliopathies.

CEP162 deficiency causes human retinal degeneration and reveals a dual role in ciliogenesis and neurogenesis.

Defects in primary or motile cilia result in a variety of human pathologies, and retinal degeneration is frequently associated with these so-called ciliopathies. We found that homozygosity for a truncating variant in CEP162, a centrosome and microtubule-associated protein required for transition zone assembly during ciliogenesis and neuronal differentiation in the retina, caused late-onset retinitis pigmentosa in 2 unrelated families. The mutant CEP162-E646R*5 protein was expressed and properly localized to the mitotic spindle, but it was missing from the basal body in primary and photoreceptor cilia. This impaired recruitment of transition zone components to the basal body and corresponded to complete loss of CEP162 function at the ciliary compartment, reflected by delayed formation of dysmorphic cilia. In contrast, shRNA knockdown of Cep162 in the developing mouse retina increased cell death, which was rescued by expression of CEP162-E646R*5, indicating that the mutant retains its role for retinal neurogenesis. Human retinal degeneration thus resulted from specific loss of the ciliary function of CEP162.

Cerebrospinal Fluid Production and Absorption and Ventricular Enlargement Mechanisms in Hydrocephalus.

Cerebrospinal fluid (CSF) production and absorption concept significantly changed in the early 2010s from "third circulation theory" and "classical bulk flow theory" to a whole new one as follows: First, CSF is mainly produced from interstitial fluid excreted from the brain parenchyma, and CSF produced from the choroid plexus plays an important role in maintaining brain homeostasis. Second, CSF is not absorbed in the venous sinus via the arachnoid granules, but mainly in the dural lymphatic vessels. Finally, the ventricles and subarachnoid spaces have several compensatory direct CSF pathways at the borders attached to the choroid plexus, e.g., the inferior choroidal point of the choroidal fissure, other than the foramina of Luschka and Magendie. In idiopathic normal pressure hydrocephalus (iNPH), the lateral ventricles and basal cistern are enlarged simultaneously due to the compensatory direct CSF pathways. The average total intracranial CSF volume increased from about 150 mL at 20 years to about 350 mL at 70 years due to the decrease in brain volume with aging and further increased above 400 mL in patients with iNPH. CSF movements are composed of a steady microflow produced by the rhythmic wavy movement of motile cilia on the ventricular surface and dynamic pulsatile flow produced by the brain and cerebral artery pulsation, respiration, and head movement. Pulsatile CSF movements might totally decrease with aging, but it in the ventricles might increase at the foramina of Magendie and Luschka dilation. Aging CSF dynamics are strongly associated with ventricular dilatation in iNPH.