Number Of Cilia Research Articles

Molecular phenotyping of single pancreatic islet leader beta cells by “Flash-Seq”

AimsSpatially-organized increases in cytosolic Ca2+ within pancreatic beta cells in the pancreatic islet underlie the stimulation of insulin secretion by high glucose. Recent data have revealed the existence of subpopulations of beta cells including “leaders” which initiate Ca2+ waves. Whether leader cells possess unique molecular features, or localisation, is unknown. Main methodsHigh speed confocal Ca2+ imaging was used to identify leader cells and connectivity analysis, running under MATLAB and Python, to identify highly connected “hub” cells. To explore transcriptomic differences between beta cell sub-groups, individual leaders or followers were labelled by photo-activation of the cryptic fluorescent protein PA-mCherry and subjected to single cell RNA sequencing (“Flash-Seq”). Key findingsDistinct Ca2+ wave types were identified in individual islets, with leader cells present in 73 % (28 of 38 islets imaged). Scale-free, power law-adherent behaviour was also observed in 29 % of islets, though “hub” cells in these islets did not overlap with leaders. Transcripts differentially expressed (295; padj < 0.05) between leader and follower cells included genes involved in cilium biogenesis and transcriptional regulation. Providing some support for these findings, ADCY6 immunoreactivity tended to be higher in leader than follower cells, whereas cilia number and length tended to be lower in the former. Finally, leader cells were located significantly closer to delta, but not alpha, cells in Euclidian space than were follower cells. SignificanceThe existence of both a discrete transcriptome and unique localisation implies a role for these features in defining the specialized function of leaders. These data also raise the possibility that localised signalling between delta and leader cells contributes to the initiation and propagation of islet Ca2+ waves.

Atp7b-dependent choroid plexus dysfunction causes transient copper deficit and metabolic changes in the developing mouse brain.

Copper (Cu) has a multifaceted role in brain development, function, and metabolism. Two homologous Cu transporters, Atp7a (Menkes disease protein) and Atp7b (Wilson disease protein), maintain Cu homeostasis in the tissue. Atp7a mediates Cu entry into the brain and activates Cu-dependent enzymes, whereas the role of Atp7b is less clear. We show that during postnatal development Atp7b is necessary for normal morphology and function of choroid plexus (ChPl). Inactivation of Atp7b causes reorganization of ChPl' cytoskeleton and cell-cell contacts, loss of Slc31a1 from the apical membrane, and a decrease in the length and number of microvilli and cilia. In ChPl lacking Atp7b, Atp7a is upregulated but remains intracellular, which limits Cu transport into the brain and results in significant Cu deficit, which is reversed only in older animals. Cu deficiency is associated with down-regulation of Atp7a in locus coeruleus and catecholamine imbalance, despite normal expression of dopamine-β-hydroxylase. In addition, there are notable changes in the brain lipidome, which can be attributed to inhibition of diacylglyceride-to-phosphatidylethanolamine conversion. These results identify the new role for Atp7b in developing brain and identify metabolic changes that could be exacerbated by Cu chelation therapy.

Regulator of G protein signaling 12 drives inflammatory arthritis by activating synovial fibroblasts through MYCBP2/KIF2A signaling

Synovial fibroblasts are the active and aggressive drivers in the progression of arthritis, but the cellular and molecular mechanisms remain unknown. Here, our results showed that regulator of G protein signaling 12 (RGS12) maintained ciliogenesis in synovial fibroblasts, which is critical for the development of inflammatory arthritis. Deletion of RGS12 led to a significant decrease in ciliogenesis, adhesion, migration, and secretion of synovial fibroblasts. Mechanistically, RGS12 overexpression in synovial fibroblasts increased the length and number of cilia but decreased the protein level of kinesin family member 2A (KIF2A). The results of LC-MS analyses showed that RGS12 interacted with MYC binding protein 2 to enhance its ubiquitination activity, through which the KIF2A protein was degraded in synovial fibroblasts. Moreover, overexpression of KIF2A blocked the increases in cilia length and number. Mice with RGS12 deficiency or treatment of RGS12 shRNA nanoparticles significantly decreased the clinical score, paw swelling, synovitis, and cartilage destruction during inflammatory arthritis by inhibiting the activation of synovial fibroblasts. Therefore, this study provides evidence that RGS12 activates synovial fibroblasts' pathological function via promoting MCYBP2-mediated degradation of KIF2A and ciliogenesis. Our data suggest that RGS12 may be a potential drug target for the treatment of inflammatory arthritis.

Effects of Short-Term Salinity Stress on Ions, Free Amino Acids, Na+/K+-ATPase Activity, and Gill Histology in the Threatened Freshwater Shellfish Solenaia oleivora

Salinity is an important ecological factor affecting the osmolality of aquatic animals. Solenaia oleivora is an endemic and economically important freshwater shellfish in China. However, its osmotic response and osmoregulatory mechanisms under high salinity stress are still unclear. In this study, S. oleivora was exposed to saline water (salinity: 2.2‰) for 3 h, 6 h, 12 h, 24 h, and 48 h, and then the changes in osmolality, ion concentrations, free amino acid (FAA) content, Na+/K+-ATPase (NKA) activity, and gill histology were analyzed. The hemolymph osmolality increased from 3 h after salinity stress and stabilized between 24–48 h. Na+ in the hemolymph increased from 24 h after salinity stress, and Cl− increased from 3 h. The content of total FAAs in the hemolymph increased after salinity stress. The content of alanine, glycine, glutamine, proline, and other FAAs increased after salinity stress. NKA activity in the gill, hepatopancreases, adductor muscle, and axe foot decreased during 3–48 h of salinity stress. The gill filament space increased and the number of gill cilia decreased after salinity stress. Principal component analysis (PCA) showed that the first two principal components (PC1 and PC2) cumulatively explained 77.6% of the total variation. The NKA activity was positively associated with PC1, while the ion concentration and most FAAs were negatively associated with PC1. Thus, these results indicated that S. oleivora is an osmoconformer, and inorganic ions, FAA, NKA, and gill structure changes play an important role in its osmoregulation.

Renal primary cilia lengthen in the progression of diabetic kidney disease.

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease, and its early pathogenesis is critical. Shear stress caused by glomerular hyperfiltration contributes to the initiation of kidney injury in diabetes. The primary cilium of renal tubular epithelial cells (RTECs) is an important mechanical force sensor of shear stress and regulates energy metabolism homeostasis in RTECs to ensure energy supply for reabsorption functions, but little is known about the alterations in the renal cilium number and length during the progression of DKD. Here, we demonstrate that aberrant ciliogenesis and dramatic increase in the cilium length, the number of ciliated cells, and the length of cilia are positively correlated with the DKD class in the kidney biopsies of DKD patients by super-resolution imaging and appropriate statical analysis methods. This finding was further confirmed in STZ-induced or db/db diabetic mice. These results suggest that the number and length of renal cilia may be clinically relevant indicators and that cilia will be attractive therapeutic targets for DKD.

Endothelial RGS12 governs angiogenesis in inflammatory arthritis by controlling cilia formation and elongation via MYCBP2 signaling.

Angiogenesis is the formation of new capillaries that plays an essential role in the pathogenesis of inflammatory arthritis. However, the cellular and molecular mechanisms remain unclear. Here, we provide the first evidence that regulator of G-protein signaling 12 (RGS12) promotes angiogenesis in inflammatory arthritis through governing ciliogenesis and cilia elongation in endothelial cells. The knockout of RGS12 inhibits the development of inflammatory arthritis with the reduction in clinical score, paw swelling, and angiogenesis. Mechanistically, RGS12 overexpression (OE) in endothelial cells increases cilia number and length, and thereby promotes cell migration and tube-like structure formation. The knockout of cilia marker protein Intraflagellar transport (IFT) 80 blocked the increase in cilia number and length caused by RGS12 OE. Moreover, the results from LC/MS and IP analysis showed that RGS12 is associated with cilia-related protein MYC binding protein 2 (MYCBP2), which enhances the phosphorylation of MYCBP2 to promote ciliogenesis in endothelial cells. These findings demonstrate that upregulation of RGS12 by inflammation enhances angiogenesis by promoting cilia formation and elongation via activation of MYCBP2 signaling during inflammatory arthritis pathogenesis.

Mitochondrial impairment and intracellular reactive oxygen species alter primary cilia morphology.

Primary cilia have recently emerged as cellular signaling organelles. Their homeostasis and function require a high amount of energy. However, how energy depletion and mitochondria impairment affect cilia have barely been addressed. We first studied the spatial relationship between a mitochondria subset in proximity to the cilium in vitro, finding similar mitochondrial activity measured as mitochondrial membrane potential compared with the cellular network. Next, using common primary cilia cell models and inhibitors of mitochondrial energy production, we found alterations in cilia number and/or length due to energy depletion and mitochondrial reactive oxygen species (ROS) overproduction. Finally, by using a mouse model of type 2 diabetes mellitus, we provided in vivo evidence that cilia morphology is impaired in diabetic nephropathy, which is characterized by ROS overproduction and impaired mitochondrial metabolism. In conclusion, we showed that energy imbalance and mitochondrial ROS affect cilia morphology and number, indicating that conditions characterized by mitochondria and radicals imbalances might lead to ciliary impairment.

Glioblastoma disrupts the ependymal wall and extracellular matrix structures of the subventricular zone

BackgroundGlioblastoma (GBM) is the most aggressive and common type of primary brain tumor in adults. Tumor location plays a role in patient prognosis, with tumors proximal to the lateral ventricles (LVs) presenting with worse overall survival, increased expression of stem cell genes, and increased incidence of distal tumor recurrence. This may be due in part to interaction of GBM with factors of the subventricular zone (SVZ), including those contained within the cerebrospinal fluid (CSF). However, direct interaction of GBM tumors with CSF has not been proved and would be hindered in the presence of an intact ependymal cell layer.MethodsHere, we investigate the ependymal cell barrier and its derived extracellular matrix (ECM) fractones in the vicinity of a GBM tumor. Patient-derived GBM cells were orthotopically implanted into immunosuppressed athymic mice in locations distal and proximal to the LV. A PBS vehicle injection in the proximal location was included as a control. At four weeks post-xenograft, brain tissue was examined for alterations in ependymal cell health via immunohistochemistry, scanning electron microscopy, and transmission electron microscopy.ResultsWe identified local invading GBM cells within the LV wall and increased influx of CSF into the LV-proximal GBM tumor bulk compared to controls. In addition to the physical disruption of the ependymal cell barrier, we also identified increased signs of compromised ependymal cell health in LV-proximal tumor-bearing mice. These signs include increased accumulation of lipid droplets, decreased cilia length and number, and decreased expression of cell channel proteins. We additionally identified elevated numbers of small fractones in the SVZ within this group, suggesting increased indirect CSF-contained molecule signaling to tumor cells.ConclusionsOur data is the first to show that LV-proximal GBMs physically disrupt the ependymal cell barrier in animal models, resulting in disruptions in ependymal cell biology and increased CSF interaction with the tumor bulk. These findings point to ependymal cell health and CSF-contained molecules as potential axes for therapeutic targeting in the treatment of GBM.

Alteration of primary cilia morphology and associated signalling in ameloblastoma

ObjectivesPrimary cilium is a cellular organelle with growing significance confirmed in tumour biology. Primary cilia have been associated with fine tuning of numerous cell signalling pathways and the role of this structure in cancer initiation and progression is recently at the forefront of attention. Here, we investigated possible alterations in the occurrence of primary cilia and changes of associated signalling in ameloblastoma, which represents the most common odontogenic tumour. MethodsWe performed immunohistochemistry to assess the number and morphology of primary cilia in ameloblastoma tissues. The gene expression of key SHH pathway members was analysed by qPCR. As a functional experiment, we treated a primary ameloblastoma cell line by a SHH pathway inhibitor Sonidegib (LDE225). ResultsWe uncovered differences in primary cilia distribution and appearance in histological subtypes of ameloblastoma with the highest number of ciliated cells in plexiform and follicular subtypes. SHH protein was located close to primary cilia in ameloblastoma epithelial cells and the expression of molecules downstream of SHH signalling was upregulated. Moreover, the inhibition of SHH pathway by Sonidegib caused downregulation of SHH effector gene GLI1 and cell cycle regulator CCND1 in ameloblastoma primary cell line. The inhibition of SHH signalling also altered the expression of molecules involved in intraflagellar transport. ConclusionsIn conclusion, our study uncovered alterations in number of ciliated cells and associated signalling in ameloblastoma, which indicate SHH inhibitors as potential therapeutic target to treat this disease.

Ciliogenesis mechanisms mediated by PAK2-ARL13B signaling in brain endothelial cells is responsible for vascular stability

In the developing vasculature, cilia, microtubule-based organelles that project from the apical surface of endothelial cells (ECs), have been identified to function cell autonomously to promote vascular integrity and prevent hemorrhage. To date, the underlying mechanisms of endothelial cilia formation (ciliogenesis) are not fully understood. Understanding these mechanisms is likely to open new avenues for targeting EC-cilia to promote vascular stability. Here, we hypothesized that brain ECs ciliogenesis and the underlying mechanisms that control this process are critical for brain vascular stability. To investigate this hypothesis, we utilized multiple approaches including developmental zebrafish model system and primary cell culture systems. In the p21 activated kinase 2 (pak2a) zebrafish vascular stability mutant [redhead (rhd)] that shows cerebral hemorrhage, we observed significant decrease in cilia-inducing protein ADP Ribosylation Factor Like GTPase 13B (Arl13b), and a 4-fold decrease in cilia numbers. Overexpressing ARL13B-GFP fusion mRNA rescues the cilia numbers (1–2-fold) in brain vessels, and the cerebral hemorrhage phenotype. Further, this phenotypic rescue occurs at a critical time in development (24 h post fertilization), prior to initiation of blood flow to the brain vessels. Extensive biochemical mechanistic studies in primary human brain microvascular ECs implicate ligands platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor-A (VEGF-A) trigger PAK2-ARL13B ciliogenesis and signal through cell surface VEGFR-2 receptor. Thus, collectively, we have implicated a critical brain ECs ciliogenesis signal that converges on PAK2-ARL13B proteins to promote vascular stability.

Abnormal accumulation of OFD1 in endometrial cancer with poor prognosis inhibits ciliogenesis.

The aim of the present study was to examine primary cilia in endometrial tissue during the menstrual cycle and to clarify their morphological changes with different grades of endometrial cancer. Images of fluorescence immunostaining taken by confocal microscopy were used to count the number of primary cilia in normal endometrium and endometrioid carcinoma Grade 1 and Grade 3 specimens. To examine the association between autophagy and ciliogenesis in endometrioid carcinoma, the expression of p62/Sequestosome-1, a selective substrate for autophagy, and oral-facial-digital syndrome 1 protein (OFD1), a protein associated with ciliogenesis, were examined using images of fluorescence immunostaining taken by confocal microscopy. The level of p62 expression was confirmed by western blotting. In proliferative and secretory endometrial stromal cells, the percentage of cells that were ciliated was 7.2 and 32.7% (95% confidence interval=21.61-39.79; P<0.01), and the length of the primary cilia was 1.24 µm and 2.34 µm (0.92-1.26; P<0.01), respectively. In stromal cells of endometrioid carcinoma Grade 1 and Grade 3, the percentage of ciliated cells was 13.5 and 2.9% (7.89-15.05; P<0.001), and the length of the primary cilia was 2.02 and 1.14 µm (0.76-0.99; P<0.001), respectively. In both normal menstrual cycle tissue and endometrial carcinomas, the percentage of primary cilia was lower and their length was shorter in tissues with higher proliferative potential. The expression of OFD1 was significantly higher in Grade 3 compared with Grade 1 as indicated by quantifying the intensity of the fluorescence images (133–12248; P=0.046). To the best of our knowledge, this is the first study concerning the distribution of primary cilia in normal endometrium and endometrial cancer tissues. Overall, fewer ciliated cells in the highly malignant endometrial cancer tissues may be associated not only to the proliferation of cancer cells, but also to the excessive accumulation of OFD1 due to dysfunctional autophagy.

The role of primary cilia in pulmonary hypertension

IntroductionVascular remodeling in pulmonary hypertension (PH) is characterized by endothelial dysfunction and smooth muscle cell proliferation and hypertrophy ultimately causing right heart failure and death. Up to now, the molecular mechanisms of PH pathogenesis have not been fully resolved, preventing the development of novel therapies against PH.Based on increased expression and activity levels in both patients and animal models with PH, respectively, PDGF‐BB, TGF‐β and mTOR signaling have been proposed to play critical roles as drivers of lung vascular remodeling. Among other effects, PDGF‐BB, TGF‐β and mTOR signaling cause shortening of the primary cilium – an antenna‐like organelle that functions as a flow‐sensor and signaling hub. Loss or shortening of cilia is characteristically associated with cell proliferation and promotes mTOR signaling, thus potentially establishing a positive feedback loop. Here, we hypothesized that PH is associated with a loss of primary cilia in pulmonary artery endothelial (PAECs) and smooth muscle cells (PASMCs) which in turn drives cell proliferation and thus, remodeling in the pulmonary arterial wall.MethodsPulmonary artery tissue from PH‐patients and non‐PH donors was fixed and stained for acetylated α‐tubulin to determine the number of primary cilia. PAECs and PASMCs from PH patients and non‐PH donors were exposed in vitro to three different characteristic stimuli or mediators, respectively, of PH, namely PDGF‐BB, TGF‐β1, or hypoxia (1%). Cells were fixed and stained for measurement of primary cilia length. IFT88 ‐ an essential structural protein of the primary cilium ‐ was knocked down by siRNA in PAECs and PASMCs to assess the effect of cilium loss on migration and proliferation. Mice with an endothelial‐specific deletion of IFT88 (Cdh5‐CreERT2+, IFT88fl/fl) were housed for 5 weeks in normoxia or hypoxia (10% O2) to test for the role of endothelial primary cilia in the development of PH.ResultsIn pulmonary arteries of PH patients, the number of primary cilia per area was reduced by 80% as compared to healthy donor lungs. Analogously, PASMCs from PH patients had shorter cilia. Following IFT88 knock‐down, PAECs and PASMCs showed increased migration and proliferation as compared to control cells. In both PAECs and PASMCs, stimulation with PDGF‐BB, TGF‐β1, or hypoxia shortened primary cilia as compared to controls. Mice lacking primary cilia in endothelial cells showed a significant increase in right ventricular systolic pressure after 5 weeks of hypoxia as compared to control mice.ConclusionsHere, we demonstrate that PH is associated with a loss of cilia in vivo and reduced cilium length in vitro. Primary cilium loss promotes proliferation and migration of PAECs and PASMCs in vitro, as well as the development of PH in vivo. Cilium loss may as such be an important propagator of vascular remodeling in PH and may present a target for novel therapeutic interventions.

Localization of Cdc14 Isoforms to Ciliary Structures in Tetrahymena thermophila

Cdc14 protein phosphatase is highly conserved across the eukaryotic kingdom, from single‐celled yeast and protozoa to multicellular organisms including mammals. In budding yeast, where it was first studied, Cdc14 is required for mitotic exit; however, this function is not widely conserved. In humans and mice, Cdc14A mutations cause deficiencies in hearing and male fertility due to defects in cilia formation and maintenance. There is evidence for Cdc14 localization to the basal bodies at the proximal end of cilia. The large number of cilia in the free‐living protozoan Tetrahymena thermophila make it an attractive model for molecular studies of cilia structure and function. Our goal is to determine the localization of Cdc14 isoforms to gain some insight into their cellular function and to test if Cdc14 cilia localization is broadly conserved. Interestingly, T. thermophilahas a larger number of Cdc14 isoforms than most organisms. Thus far, genes encoding three of the seven T. thermophilaCdc14 isoforms have been used to create C‐terminal gene fusions with YFP that are expressed in vivo. The constructs were inserted into the T. thermophilamacronuclear genome adjacent to the RPL29 gene and cell lines were selected due to the resulting conversion to cycloheximide resistance. Expression of the transgenes was regulated by a metallothionine promoter. Cells were examined via fluorescence microscopy in the presence and absence of cadmium. All three isoforms localize along ciliary rows and in the oral apparatus after induction during vegetative growth, consistent with basal body localization. No differences in localization have been noted between the three isoforms thus far. The presence of multiple isoforms with the same localization raises questions regarding the redundancy and/or functional specialization of Cdc14 in this highly ciliated organism.

The Primary Ciliary Deficits in Cerebellar Bergmann Glia of the Mouse Model of Fragile X Syndrome.

Primary cilia are non-motile cilia that function as antennae for cells to sense signals. Deficits of primary cilia cause ciliopathies, leading to the pathogenesis of various developmental disorders; however, the contribution of primary cilia to neurodevelopmental disorders is largely unknown. Fragile X syndrome (FXS) is a genetically inherited disorder and isthe most common known cause of autism spectrum disorders. FXS is caused by the silencing of the fragile X mental retardation 1 (FMR1) gene, which encodes for the fragile X mental retardation protein (FMRP). Here, we discovered a reduction in the number of primary cilia and the Sonic hedgehog (Shh) signaling in cerebellar Bergmann glia of Fmr1 KO mice. We further found reduced granule neuron precursor (GNP) proliferation and thickness of the external germinal layer (EGL) in Fmr1 KO mice, implicating that primary ciliary deficits in Bergmann glia may contribute to cerebellar developmental phenotypes in FXS, as Shh signaling through primary cilia in Bergmann glia is known to mediate proper GNP proliferation in the EGL. Taken together, our study demonstrates that FMRP loss leads to primary ciliary deficits in cerebellar Bergmann glia which may contribute to cerebellar deficits in FXS.

From Water to Land: The Structural Construction and Molecular Switches in Lungs during Metamorphosis of Microhyla fissipes.

Simple SummaryThe functionalization of lungs is a necessity for most anurans to breathe on land. Previous studies have focused on the morphological and physiological functions of amphibian lungs, while the microstructural changes and molecular mechanisms that underpin the functional maturation of lungs remain under-researched. We used integrated histology and transcriptomics to study the critical cytological and molecular events associated with lung maturation in Microhyla fissipes. The results illuminated the molecular processes and their coordination in lung development, providing an insight into the transition of amphibians from aquatic to terrestrial life stages.Most anurans must undergo metamorphosis to adapt to terrestrial life. This process enhances the air-breathing ability of the lungs to cope with the change in oxygen medium from water to air. Revealing the structural construction and molecular switches of lung organogenesis is essential to understanding the realization of the air-breathing function. In this study, histology and transcriptomics were conducted in combination to explore these issues in Microhyla fissipes’ lungs during metamorphosis. During the pro-metamorphic phase, histological structural improvement of the alveolar wall is accompanied by robust substrate metabolism and protein turnover. The lungs, at the metamorphic climax phase, are characterized by an increased number of cilia in the alveolar epithelial cells and collagenous fibers in the connective tissues, corresponding to the transcriptional upregulation of cilia and extracellular matrix-related genes. Post-metamorphic lungs strengthen their contracting function, as suggested by the thickened muscle layer and the upregulated expression of genes involved in muscle contraction. The blood–gas barrier is fully developed in adult lungs, the transcriptional features of which are tissue growth and regulation of differentiation and immunity. Importantly, significant transcriptional switches of pulmonary surfactant protein and hemoglobin facilitate air breathing. Our results illuminated four key steps of lung development for amphibians to transition from water to land.

Functional Characterization of Cfap206 for Bivalve Ciliogenesis by RNAi and CRISPR/Cas9 Technologies

Cilia are organelles located on the surface of eukaryotic cells and play important roles in numerous physiological and developmental processes. Cilia- and flagella-associated proteins (CFAP family) are well known due to their close relation to the assembly and functionalization of both cilia and flagella. In vertebrates, Cfap206 is reported to function during motile cilia assembly, and its abolished expression could lead to abnormal ciliary beating and decreased sperm motility. However, there is scarcely any information on the function of Cfap206 in marine invertebrates. Here, through using an ideal shellfish research model, the dwarf surf calm Mulinia lateralis, we explored the role of Cfap206 during embryonic ciliogenesis as well as during male gametogenesis. In trochophore larvae, effective knocking down or knocking out of Cfap206 was detected respectively through RNAi and CRISPR/Cas9 technology, showing an obviously decreased number of cilia. During the period of gonadal maturation, we managed to deliver the dsRNA of Cfap206 to male individuals for 14 days through carrier vector feeding. Significant suppression of Cfap206 was observed, together with impaired sperm motility and aberrant sperm tail assembly. The present study provided valuable information on gene function exploration methods in M. lateralis and further enriched the understanding of Cfap206 on ciliogenesis in marine invertebrates. Furthermore, by revealing the relationship between Cfap206 and spermatozoa flagellum, it is possible to generate male sterile populations in hermaphroditic marine organisms to improve crossbreeding efficiency.

Microgravity and space radiation inhibit autophagy in human capillary endothelial cells, through either opposite or synergistic effects on specific molecular pathways.

Microgravity and space radiation (SR) are two highly influential factors affecting humans in space flight (SF). Many health problems reported by astronauts derive from endothelial dysfunction and impaired homeostasis. Here, we describe the adaptive response of human, capillary endothelial cells to SF. Reference samples on the ground and at 1g onboard permitted discrimination between the contribution of microgravity and SR within the combined responses to SF. Cell softening and reduced motility occurred in SF cells, with a loss of actin stress fibers and a broader distribution of microtubules and intermediate filaments within the cytoplasm than in control cells. Furthermore, in spacethe number of primary cilia per cell increased and DNA repair mechanisms were found to be activated. Transcriptomics revealed the opposing effects of microgravity from SR for specific molecular pathways: SR, unlike microgravity, stimulated pathways for endothelial activation, such as hypoxia and inflammation, DNA repair and apoptosis, inhibiting autophagic flux and promoting an aged-like phenotype. Conversely, microgravity, unlike SR, activated pathways for metabolism and a pro-proliferative phenotype. Therefore, we suggest microgravity and SR should be considered separately to tailor effective countermeasures to protect astronauts' health.

Primary Cilia Structure Is Prolonged in Enteric Neurons of 5xFAD Alzheimer's Disease Model Mice.

Neurodegenerative diseases such as Alzheimer’s disease (AD) have long been acknowledged as mere disorders of the central nervous system (CNS). However, in recent years the gut with its autonomous nervous system and the multitude of microbial commensals has come into focus. Changes in gut properties have been described in patients and animal disease models such as altered enzyme secretion or architecture of the enteric nervous system. The underlying cellular mechanisms have so far only been poorly investigated. An important organelle for integrating potentially toxic signals such as the AD characteristic A-beta peptide is the primary cilium. This microtubule-based signaling organelle regulates numerous cellular processes. Even though the role of primary cilia in a variety of developmental and disease processes has recently been recognized, the contribution of defective ciliary signaling to neurodegenerative diseases such as AD, however, has not been investigated in detail so far. The AD mouse model 5xFAD was used to analyze possible changes in gut functionality by organ bath measurement of peristalsis movement. Subsequently, we cultured primary enteric neurons from mutant mice and wild type littermate controls and assessed for cellular pathomechanisms. Neurite mass was quantified within transwell culturing experiments. Using a combination of different markers for the primary cilium, cilia number and length were determined using fluorescence microscopy. 5xFAD mice showed altered gut anatomy, motility, and neurite mass of enteric neurons. Moreover, primary cilia could be demonstrated on the surface of enteric neurons and exhibited an elongated phenotype in 5xFAD mice. In parallel, we observed reduced β-Catenin expression, a key signaling molecule that regulates Wnt signaling, which is regulated in part via ciliary associated mechanisms. Both results could be recapitulated via in vitro treatments of enteric neurons from wild type mice with A-beta. So far, only a few reports on the probable role of primary cilia in AD can be found. Here, we reveal for the first time an architectural altered phenotype of primary cilia in the enteric nervous system of AD model mice, elicited potentially by neurotoxic A-beta. Potential changes on the sub-organelle level—also in CNS-derived neurons—require further investigations.

Diabetes impairs fracture healing through disruption of cilia formation in osteoblasts.

Diabetes-associated fracture risk and impaired fracture healing represents a serious health threat. It is well known that type 1 diabetes mellitus (T1DM) impairs fracture healing due to its effect on osteoblasts and their progenitor cells. Previous studies have showed that primary cilia and intraflagellar transport protein 80 (IFT80) are critical for bone formation. However, whether TIDM impairs fracture healing due to influencing ciliary gene expression and cilia formation is unknown. Here, we investigated the effect of T1DM on primary cilia in a streptozotocin induced diabetes mouse model and examined the impact of cilia on fracture healing in osteoblasts by deletion of IFT80 in osteoblast linage using osterix (OSX)-cre (OSXcretTAIFT80f/f). The results showed that diabetes inhibited ciliary gene expression and primary cilia formation to an extent that was similar to normoglycemic mice with IFT80 deletion. Moreover, diabetic mice and normoglycemic mice with cilia loss in osteoblasts (OSXcretTAIFT80f/f) both exhibited delayed fracture healing with significantly reduced bone density and mechanical strength as well as with reduced expression of osteoblast markers, decreased angiogenesis and proliferation of bone lining cells at the fracture sites. In vitro studies showed that advanced glycation end products (AGEs) downregulated IFT80 expression in osteoblast progenitors. Moreover, AGEs and IFT80 deletion significantly reduced cilia number and length which inhibited differentiation of primary osteoblast precursors. Thus, this study for the first time report that primary cilia are essential for bone regeneration during fracture healing and loss of cilia caused by diabetes in osteoblasts resulted in defective diabetic fracture healing.

Ciliary Type III Adenylyl Cyclase in the VMH Is Crucial for High-Fat Diet-Induced Obesity Mediated by Autophagy.

Neuronal primary cilia are crucial for body weight maintenance. Type III adenylyl cyclase (AC3) is abundantly enriched in neuronal cilia, and mice with global AC3 ablation are obese. However, whether AC3 regulates body weight through its ciliary expression and the mechanism underlying this potential regulation are not clear. In this study, humanized AC3 knock‐in mice that are resistant to high‐fat diet (HFD)‐induced obesity are generated, and increases in the number and length of cilia in the ventromedial hypothalamus (VMH) are shown. It is demonstrated that mice with specifically knocked down ciliary AC3 expression in the VMH show pronounced HFD‐induced obesity. In addition, in vitro and in vivo analyses of the VMH show that ciliary AC3 regulates autophagy by binding an autophagy‐related gene, gamma‐aminobutyric acid A receptor‐associated protein (GABARAP). Mice with GABARAP knockdown in the VMH exhibit exacerbated HFD‐induced obesity. Overall, the findings may reveal a potential mechanism by which ciliary AC3 expression regulates body weight in the mouse VMH.