Multiciliated cell development and ciliary resorption at the mammalian choroid plexus.

Ciliopathies are a group of diseases caused by defects in cilia, hair-like organelles that can have many functions ranging from regulating extracellular fluid flow to sensing mechanical or chemical stimuli. Multiciliated cells (MCCs) with motile cilia are found in locations that include the central nervous system, where they are critical for homeostasis. Specifically, ependymal MCCs line the brain ventricles and central canal of the spinal cord, while other specialized MCCs occupy highly vascularized structures known as the choroid plexuses (ChPs) and produce cerebrospinal fluid (CSF). Now, a recent study has shown that murine ChP MCCs develop nodal-like cilia. Interestingly, ChP cilia were found to undergo resorption during early postnatal stages in part through axoneme regression, and this phenomenon was mirrored in human postmortem ChP samples. Taken together, these findings reveal important new insights about the ultrastructure of MCCs that comprise the mammalian ChP, and may have ramifications for other MCC populations in health and disease states.

The role of cilia in the development, survival, and regeneration of hair cells.

Mutations impacting cilia genes lead to a class of human diseases known as ciliopathies. This is due to the role of cilia in the development, survival, and regeneration of many cell types. We investigated the extent to which disrupting cilia impacted these processes in lateral line hair cells of zebrafish. We found that mutations in two intraflagellar transport (IFT) genes, ift88 and dync2h1, which lead to the loss of kinocilia, caused increased hair cell apoptosis. IFT gene mutants also have a decreased mitochondrial membrane potential, and blocking the mitochondrial uniporter causes a loss of hair cells in wild-type zebrafish but not mutants, suggesting mitochondria dysfunction may contribute to the apoptosis seen in these mutants. These mutants also showed decreased proliferation during hair cell regeneration but did not show consistent changes in support cell number or proliferation during hair cell development. These results show that the loss of hair cells seen following disruption of cilia through either mutations in anterograde or retrograde IFT genes appears to be due to impacts on hair cell survival but not necessarily development in the zebrafish lateral line.

A prioritization tool for cilia-associated genes and their in vivo resources unveil new avenues for ciliopathy research.

Defects in ciliary signaling or mutations in proteins that localize to primary cilia lead to a class of human diseases known as ciliopathies. About 10% of mammalian genes encode cilia-associated proteins and a major gap in the cilia research field is prioritizing which genes to study and finding the in vivo vertebrate mutant alleles and reagents available for their study. Here we present a unified resource listing the cilia-associated human genes cross-referenced to available mouse and zebrafish mutant alleles, their associated phenotypes as well as expression data in kidney and functional data for vertebrate Hedgehog signaling. This resource empowers researchers to easily sort and filter genes based on their own expertise and priorities, cross-reference with newly-generated -omics datasets, and quickly find in vivo resources and phenotypes associated with a gene of interest.

Two Tetrahymena kinesin-9 family members exhibit slow plus-end-directed motility in vitro.

The kinesin-9 family comprises two subfamilies specific to ciliated eukaryotic cells, and has recently attracted considerable attention because of its importance in ciliary bending and formation. However, only scattered data are available on the motor properties of kinesin-9 family members; these properties have not been compared under identical experimental conditions using kinesin-9 motors from the same species. Here, we report the comprehensive motor properties of two kinesin-9 molecules of Tetrahymena thermophila, TtK9A (Kif9/Klp1 ortholog) and TtK9B1 (Kif6 ortholog), using microtubule-based in vitro assays, including single-motor and multi-motor assays and microtubule-stimulated ATPase assays. Both subfamilies exhibit microtubule plus-end-directed, extremely slow motor activity, both in single and multiple molecules. TtK9A shows lower processivity than TtK9B1. Our findings indicate that the considerable slow movement of kinesin-9 that corresponds to low ATP hydrolysis rates is a common feature of the ciliary kinesin-9 family.

Encoding spatiotemporal asymmetry in artificial cilia with a ctenophore-inspired soft-robotic platform.

A remarkable variety of organisms use metachronal coordination (i.e., numerous neighboring appendages beating sequentially with a fixed phase lag) to swim or pump fluid. This coordination strategy is used by microorganisms to break symmetry at small scales where viscous effects dominate and flow is time-reversible. Some larger organisms use this swimming strategy at intermediate scales, where viscosity and inertia both play important roles. However, the role of individual propulsor kinematics-especially across hydrodynamic scales-is not well-understood, though the details of propulsor motion can be crucial for the efficient generation of flow. To investigate this behavior, we developed a new soft robotic platform using magnetoactive silicone elastomers to mimic the metachronally coordinated propulsors found in swimming organisms. Furthermore, we present a method to passively encode spatially asymmetric beating patterns in our artificial propulsors. We investigated the kinematics and hydrodynamics of three propulsor types, with varying degrees of asymmetry, using Particle Image Velocimetry and high-speed videography. We find that asymmetric beating patterns can move considerably more fluid relative to symmetric beating at the same frequency and phase lag, and that asymmetry can be passively encoded into propulsors via the interplay between elastic and magnetic torques. Our results demonstrate that nuanced differences in propulsor kinematics can substantially impact fluid pumping performance. Our soft robotic platform also provides an avenue to explore metachronal coordination at the meso-scale, which in turn can inform the design of future bioinspired pumping devices and swimming robots.

Fly Fam161 is an essential centriole and cilium transition zone protein with unique and diverse cell type-specific localizations.

Family with sequence similarity 161 (Fam161) is an ancient family of microtubule-binding proteins located at the centriole and cilium transition zone (TZ) lumen that exhibit rapid evolution in mice. However, their adaptive role is unclear. Here, we used flies to gain insight into their cell type-specific adaptations. Fam161 is the sole orthologue of FAM161A and FAM161B found in flies. Mutating Fam161 results in reduced male reproduction and abnormal geotaxis behaviour. Fam161 localizes to sensory neuron centrioles and their specialized TZ (the connecting cilium) in a cell type-specific manner, sometimes labelling only the centrioles, sometimes labelling the centrioles and cilium TZ and sometimes labelling the TZ with varying lengths that are longer than other TZ proteins, defining a new ciliary compartment, the extra distal TZ. These findings suggest that Fam161 is an essential centriole and TZ protein with a unique cell type-specific localization in fruit flies that can produce cell type-specific adaptations.

mTOR potentiates senescent phenotypes and primary cilia formation after cisplatin-induced G2 arrest in retinal pigment epithelial cells.

Cisplatin, a platinum-based anticancer drug, is used to treat several types of cancer. Despite its effectiveness, cisplatin-induced side effects have often been reported. Although cisplatin-induced toxicities, such as apoptosis and/or necrosis, have been well studied, the fate of cells after exposure to sublethal doses of cisplatin needs further elucidation. Treatment with a sublethal dose of cisplatin-induced cell cycle arrest at the G2 phase in retinal pigment epithelial cells. Following cisplatin withdrawal, the cells irreversibly exit the cell cycle and become senescent. Notably, the progression from the G2 to the G1 phase occurred without mitotic entry, a phenomenon referred to as mitotic bypass, resulting in the accumulation of cells containing 4 N DNA content. Cisplatin-exposed cells exhibited morphological changes associated with senescence, including an enlarged size of cell and nucleus and increased granularity. In addition, the senescent cells possessed primary cilia and persistent DNA lesions. Senescence induced by transient exposure to cisplatin involves mTOR activation. Although transient co-exposure with the mTORC1 inhibitor rapamycin did not prevent mitotic bypass and entry into senescence, it delayed the progression of senescence and attenuated senescent phenotypes, resulting in shorter primary cilia formation. Conclusively, cisplatin induces senescence in retinal pigment epithelial cells by promoting mTOR activation.

Cdon is essential for organ left-right patterning by regulating dorsal forerunner cells clustering and Kupffer's vesicle morphogenesis.

Cdon and boc are members of the cell adhesion molecule subfamily III Ig/fibronectin. Although they have been reported to be involved in muscle and neural development at late developmental stage, their early roles in embryonic development remain unknown. Here, we discovered that in zebrafish, cdon, but not boc, is expressed in dorsal forerunner cells (DFCs) and the epithelium of Kupffer's vesicle (KV), suggesting a potential role for cdon in organ left-right (LR) patterning. Further data showed that liver and heart LR patterning were disrupted in cdon morphants and cdon mutants. Mechanistically, we found that loss of cdon function led to defect in DFCs clustering, reduced KV lumen, and defective cilia, resulting in randomized Nodal/spaw signaling and subsequent organ LR patterning defects. Additionally, predominant distribution of a cdon morpholino (MO) in DFCs caused defects in DFC clustering, KV morphogenesis, cilia number/length, Nodal/spaw signaling, and organ LR asymmetry, similar to those observed in cdon morphants and cdon -/- embryos, indicating a cell-autonomous role for cdon in regulating KV formation during LR patterning. In conclusion, our data demonstrate that during gastrulation and early somitogenesis, cdon is essential for proper DFC clustering, KV formation, and normal cilia, thereby playing a critical role in establishing organ LR asymmetry.

Multiciliated ependymal cells: an update on biology and pathology in the adult brain.

Mature multiciliated ependymal cells line the cerebral ventricles where they form a partial barrier between the cerebrospinal fluid (CSF) and brain parenchyma and regulate local CSF microcirculation through coordinated ciliary beating. Although the ependyma is a highly specialized brain interface with barrier, trophic, and perhaps even regenerative capacity, it remains a misfit in the canon of glial neurobiology. We provide an update to seminal reviews in the field by conducting a scoping review of the post-2010 mature multiciliated ependymal cell literature. We delineate how recent findings have either called into question or substantiated classical views of the ependymal cell. Beyond this synthesis, we document the basic methodologies and study characteristics used to describe multiciliated ependymal cells since 1980. Our review serves as a comprehensive resource for future investigations of mature multiciliated ependymal cells.

Haploinsufficiency of intraflagellar transport protein 172 causes autism-like behavioral phenotypes in mice through BDNF.

INTRODUCTION: Primary cilia are hair-like solitary organelles growing on most mammalian cells that play fundamental roles in embryonic patterning and organogenesis. Defective cilia often cause a suite of inherited diseases called ciliopathies with multifaceted manifestations. Intraflagellar transport (IFT), a bidirectional protein trafficking along the cilium, actively facilitates the formation and absorption of primary cilia. IFT172 is the largest component of the IFT-B complex, and its roles in Bardet-Biedl Syndrome (BBS) have been appreciated with unclear mechanisms. OBJECTIVES: We performed a battery of behavioral tests with Ift172 haploinsufficiency (Ift172+/-) and WT littermates. We use RNA sequencing to identify the genes and signaling pathways that are differentially expressed and enriched in the hippocampus of Ift172+/- mice. Using AAV-mediated sparse labeling, electron microscopic examination, patch clamp and local field potential recording, western blot, luciferase reporter assay, chromatin immunoprecipitation, and neuropharmacological approach, we investigated the underlying mechanisms for the aberrant phenotypes presented by Ift172+/- mice. RESULTS: Ift172+/- mice displayed excessive self-grooming, elevated anxiety, and impaired cognition. RNA sequencing revealed enrichment of differentially expressed genes in pathways relevant to axonogenesis and synaptic plasticity, which were further confirmed by less spine density and synaptic number. Ift172+/- mice demonstrated fewer parvalbumin-expressing neurons, decreased inhibitory synaptic transmission, augmented theta oscillation, and sharp-wave ripples in the CA1 region. Moreover, Ift172 haploinsufficiency caused less BDNF production and less activated BDNF-TrkB signaling pathway through transcription factor Gli3. Application of 7,8-Dihydroxyflavone, a potent small molecular TrkB agonist, fully restored BDNF-TrkB signaling activity and abnormal behavioral phenotypes presented by Ift172+/- mice. With luciferase and chip assays, we provided further evidence that Gli3 may physically interact with BDNF promoter I and regulate BDNF expression. CONCLUSIONS: Our data suggest that Ift172 per se drives neurotrophic effects and, when defective, could cause neurodevelopmental disorders reminiscent of autism-like disorders.

Cilia-Inspired Magnetic Flexible Shear Force Sensors for Tactile and Fluid Monitoring.

Recently, there has been a burgeoning interest in flexible shear force sensors capable of precisely detecting both magnitude and direction. Despite considerable efforts, the challenge of achieving accurate direction recognition persists, primarily due to the inherent structural characteristics and sensing mechanisms. Here, we present a shear force sensor constructed by a magnetically induced assembled Ni/PDMS composite membrane, which is magnetized and integrated with a three-axis Hall sensor, facilitating its ability to simultaneously monitor both shear force magnitude (0.7-87 mN) and direction (0-360°). The cilia-inspired shear force magnetic sensor (CISFMS) exhibits admirable attributes, including exceptional flexibility, high sensitivity (0.76 mN-1), an exceedingly low detection limit (1° and 0.7 mN), and remarkable durability (over 10,000 bending cycles). Further, our results demonstrate the capacity of the CISFMS in detecting tactile properties, fluid velocity, and direction, offering substantial potential for future developments in wearable electronics.

Activation of angiotensin II type 2 receptor leads to preservation of primary cilia in tubular cells during renal ischaemia-reperfusion injury.

Ischaemia-reperfusion (IR)-associated acute kidney injury (AKI) is a severe clinical condition that lacks effective pharmacological treatments. Our recent research revealed that pretreatment with the angiotensin II type 2 receptor (AT2R) agonist C21 alleviates kidney damage during IR. Primary cilia are organelles crucial for regulation of epithelial cell homeostasis, which are significantly affected by IR injury. This study aimed to evaluate the impact of AT2R activation on cilia integrity during IR and to identify pathways involved in the nephroprotective effect of C21. Rats were subjected to 40 min of unilateral ischaemia followed by 24 h of reperfusion. Immunofluorescence analysis of the kidneys showed that the nephroprotective effect of C21 was associated with preservation of cilia integrity in tubular cells. AT2R agonists increased α-tubulin acetylation in primary cilia in tubular cells in vivo and in a cell model. Analysis of ERK phosphorylation indicated that AT2R activation led to diminished activation of ERK1/2 in tubular cells. Similar to AT2R agonists, inhibitors of α-tubulin deacetylase HDAC6 or inhibitors of ERK activation ameliorated IR-induced cell death and preserved cilia integrity. Immunofluorescence analysis of tubular cells revealed significant ERK localization at primary cilia and demonstrated that ERK inhibition increased cilia levels of acetylated α-tubulin. Overall, our findings demonstrate that C21 elicits a preconditioning effect that enhances cilia stability in renal tubular cells, thereby preserving their integrity when exposed to IR injury. Furthermore, our results indicate that this effect might be mediated by AT2R-induced inhibition of ERK activation. These findings offer potential insights for the development of pharmacological interventions to mitigate IR-associated AKI. KEY POINTS: The AT2R agonist C21 prevents primary cilia shortening and tubular cell deciliation during renal ischaemia-reperfusion. AT2R activation inhibits ERK1/2 in renal tubular cells. Both AT2R agonists and ERK1/2 inhibitors increase alpha-tubulin acetylation at the primary cilium in tubular cells. AT2R activation, ERK1/2 inhibition or inhibition of alpha-tubulin deacetylation elicit protective effects in tubular cells subjected to ischaemia-reperfusion injury.

Rare host variants in ciliary expressed genes contribute to COVID-19 severity in Bulgarian patients.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), a pneumonia with extremely heterogeneous clinical presentation, ranging from asymptomatic to severely ill patients. Previous studies have reported links between the presence of host genetic variants and the outcome of the COVID-19 infection. In our study, we used whole exome sequencing in a cohort of 444 SARS-CoV-2 patients, admitted to hospital in the period October-2020-April-2022, to search for associations between rare pathogenic/potentially pathogenic variants and COVID-19 progression. We used gene prioritization-based analysis in genes that have been reported by host genetic studies. Although we did not identify correlation between the presence of rare pathogenic variants and COVID-19 outcome, in critically ill patients we detected known mutations in a number of genes associated with severe disease related to cardiovascular disease, primary ciliary dyskinesia, cystic fibrosis, DNA damage repair response, coagulation, primary immune disorder, hemoglobin subunit ß, and others. Additionally, we report 93 novel pathogenic variants found in severely infected patients who required intubation or died. A network analysis showed main component, consisting of 13 highly interconnected genes related to epithelial cilium. In conclusion, we have detected rare pathogenic host variants that may have influenced the COVID-19 outcome in Bulgarian patients.

Transcriptional regulation of CCNO during the formation of multiple motile cilia.

Primary ciliary dyskinesia (PCD) is a group of genetically heterogeneous disorders characterized by clinical manifestations resulting from abnormal ciliary motility. Mutations in critical genes, such as Cyclin O (CCNO), have been associated with severe respiratory disease, though limited data are currently available. Here we show that CCNO deficient ciliated cells can only form a reduced number of fully functional centrioles that can mature into ciliated basal bodies, and their transport and anchoring to the top of the plasma membrane are abnormal. Furthermore, we observed that CCNO localizes not only in the cytoplasm but also in the nucleus during the early stages of ciliogenesis, and this dual localization persists into adulthood. Transcriptome analysis revealed downregulation of genes involved in cilia assembly and movement, along with altered transcription factors associated with ciliation upon CCNO depletion. These findings indicate that CCNO may serve as a key regulator in the transcriptional regulation of multiciliogenesis.

Loss of primary cilia and dopaminergic neuroprotection in pathogenic LRRK2-driven and idiopathic Parkinson's disease.

Activating leucine-rich repeat kinase 2 (LRRK2) mutations cause Parkinson's and phosphorylation of Rab10 by pathogenic LRRK2 blocks primary ciliogenesis in cultured cells. In the mouse brain, LRRK2 blockade of primary cilia is highly cell type specific: For example, cholinergic interneurons and astrocytes but not medium spiny neurons of the dorsal striatum lose primary cilia in LRRK2-pathway mutant mice. We show here that the cell type specificity of LRRK2-mediated cilia loss is also seen in human postmortem striatum from patients with LRRK2 pathway mutations and idiopathic Parkinson's. Single nucleus RNA sequencing shows that cilia loss in mouse cholinergic interneurons is accompanied by decreased glial-derived neurotrophic factor transcription, decreasing neuroprotection for dopamine neurons. Nevertheless, LRRK2 expression differences cannot explain the unique vulnerability of cholinergic neurons to LRRK2 kinase as much higher LRRK2 expression is seen in medium spiny neurons that have normal cilia. In parallel with decreased striatal dopaminergic neurite density, LRRK2 G2019S neurons show increased autism-linked CNTN5 adhesion protein expression; glial cells show significant loss of ferritin heavy chain. These data strongly suggest that loss of cilia in specific striatal cell types decreases neuroprotection for dopamine neurons in mice and human Parkinson's.

Congenital hydrocephalus: a review of recent advances in genetic etiology and molecular mechanisms.

The global prevalence rate for congenital hydrocephalus (CH) is approximately one out of every five hundred births with multifaceted predisposing factors at play. Genetic influences stand as a major contributor to CH pathogenesis, and epidemiological evidence suggests their involvement in up to 40% of all cases observed globally. Knowledge about an individual's genetic susceptibility can significantly improve prognostic precision while aiding clinical decision-making processes. However, the precise genetic etiology has only been pinpointed in fewer than 5% of human instances. More occurrences of CH cases are required for comprehensive gene sequencing aimed at uncovering additional potential genetic loci. A deeper comprehension of its underlying genetics may offer invaluable insights into the molecular and cellular basis of this brain disorder. This review provides a summary of pertinent genes identified through gene sequencing technologies in humans, in addition to the 4 genes currently associated with CH (two X-linked genes L1CAM and AP1S2, two autosomal recessive MPDZ and CCDC88C). Others predominantly participate in aqueduct abnormalities, ciliary movement, and nervous system development. The prospective CH-related genes revealed through animal model gene-editing techniques are further outlined, focusing mainly on 4 pathways, namely cilia synthesis and movement, ion channels and transportation, Reissner's fiber (RF) synthesis, cell apoptosis, and neurogenesis. Notably, the proper functioning of motile cilia provides significant impulsion for cerebrospinal fluid (CSF) circulation within the brain ventricles while mutations in cilia-related genes constitute a primary cause underlying this condition. So far, only a limited number of CH-associated genes have been identified in humans. The integration of genotype and phenotype for disease diagnosis represents a new trend in the medical field. Animal models provide insights into the pathogenesis of CH and contribute to our understanding of its association with related complications, such as renal cysts, scoliosis, and cardiomyopathy, as these genes may also play a role in the development of these diseases. Genes discovered in animals present potential targets for new treatments but require further validation through future human studies.

Primary Cilia Elongation in Early-Onset Polycystic Kidney Disease with 2 Hypomorphic PKD1 Alleles: A Case Report.

Recent studies have described several children with very early-onset polycystic kidney disease (PKD) that mimicked autosomal recessive polycystic kidney disease because of 2 hypomorphic PKD1 gene variants. However, no reports have described pathological changes in the primary cilia in these cases. We analyzed the primary cilia in the kidney tubules of an early elementary school child who had very early-onset PKD and a history of large, echogenic kidneys in utero. There was no family history of autosomal dominant PKD. The patient developed kidney failure and received a living-donor kidney transplant from his father. Genetic analysis revealed compound heterozygous variants in the PKD1 gene: c.3876C>A (p. Phe1292Leu) and c.5957C>T (p. Thr1986Met). These variants were likely pathogenic based on in silico analysis. The absence of kidney cysts in the parents suggested that these variants were hypomorphic alleles. Pathological examination of the patient's excised kidney showed prominent dilatation of the proximal and distal tubules. Immunofluorescence staining for α-tubulin showed pronounced elongation of the primary cilia. These findings suggest that the hypomorphic PKD1 variants expressed in this patient with very early-onset PKD were pathogenic.

Ciliary length variations impact cilia-mediated signaling and biological responses.

Primary cilia are thin hair-like organelles that protrude from the surface of most mammalian cells. They act as specialized cell antennas that can vary widely in response to specific stimuli. However, the effect of changes in cilia length on cellular signaling and behavior remains unclear. Therefore, we aimed to characterize the elongated primary cilia induced by different chemical agents, lithium chloride (LiCl), cobalt chloride (CoCl2), and rotenone, using human retinal pigmented epithelial 1 (hRPE1) cells expressing ciliary G protein-coupled receptor (GPCR), melanin-concentrating hormone (MCH) receptor 1 (MCHR1). MCH induces cilia shortening mainly via MCHR1-mediated Akt phosphorylation. Therefore, we verified the proper functioning of the MCH-MCHR1 axis in elongated cilia. Although MCH shortened cilia that were elongated by LiCl and rotenone, it did not shorten CoCl2-induced elongated cilia, which exhibited lesser Akt phosphorylation. Furthermore, serum readdition was found to delay cilia shortening in CoCl2-induced elongated cilia. In contrast, rotenone-induced elongated cilia rapidly shortened via a chopping mechanism at the tip of the cilia. Conclusively, we found that each chemical exerted different effects on ciliary GPCR signaling and serum-mediated ciliary structure dynamics in cells with elongated cilia. These results provide a basis for understanding the functional consequences of changes in ciliary length.

Near-field hydrodynamic interactions determine travelling wave directions of collectively beating cilia.

Cilia can beat collectively in the form of a metachronal wave, and we investigate how near-field hydrodynamic interactions between cilia can influence the collective response of the beating cilia. Based on the theoretical framework developed in the work of Meng et al. (Meng et al. 2021 Proc. Natl Acad. Sci. USA 118, e2102828118), we find that the first harmonic mode in the driving force acting on each individual cilium can determine the direction of the metachronal wave after considering the finite size of the beating trajectories, which is confirmed by our agent-based numerical simulations. The stable wave patterns, e.g. the travelling direction, can be controlled by the driving forces acting on the cilia, based on which one can change the flow field generated by the cilia. This work can not only help to understand the role of the hydrodynamic interactions in the collective behaviours of cilia, but can also guide future designs of artificial cilia beating in the desired dynamic mode.