Importance Of Cilia Research Articles

Disruption of Dopamine Receptor 1 Localization to Primary Cilia Impairs Signaling in Striatal Neurons.

A rod-shaped appendage called a primary cilium projects from the soma of most central neurons in the mammalian brain. The importance of cilia within the nervous system is highlighted by the fact that human syndromes linked to primary cilia dysfunction, collectively termed ciliopathies, are associated with numerous neuropathologies, including hyperphagia-induced obesity, neuropsychiatric disorders, and learning and memory deficits. Neuronal cilia are enriched with signaling molecules, including specific G-protein-coupled receptors (GPCRs) and their downstream effectors, suggesting that they act as sensory organelles that respond to neuromodulators in the extracellular space. We previously showed that GPCR ciliary localization is disrupted in neurons from mouse models of the ciliopathy Bardet-Biedl syndrome (BBS). Based on this finding, we hypothesized that mislocalization of ciliary GPCRs may impact receptor signaling and contribute to the BBS phenotypes. Here, we show that disrupting localization of the ciliary GPCR dopamine receptor 1 (D1) in male and female mice, either by loss of a BBS protein or loss of the cilium itself, specifically in D1-expressing neurons, results in obesity. Interestingly, the weight gain is associated with reduced locomotor activity, rather than increased food intake. Moreover, the loss of a BBS protein or cilia on D1-expressing neurons leads to a reduction in D1-mediated signaling. Together, these results indicate that cilia impact D1 activity in the nervous system and underscore the importance of neuronal cilia for proper GPCR signaling.SIGNIFICANCE STATEMENT Most mammalian neurons possess solitary appendages called primary cilia. These rod-shaped structures are enriched with signaling proteins, such as G-protein-coupled receptors (GPCRs), suggesting that they respond to neuromodulators. This study examines the consequences of disrupting ciliary localization of the GPCR dopamine receptor 1 (D1) in D1-expressing neurons. Remarkably, mice that have either an abnormal accumulation of D1 in cilia or a loss of D1 ciliary localization become obese. In both cases, the obesity is associated with lower locomotor activity rather than overeating. As D1 activation increases locomotor activity, these results are consistent with a reduction in D1 signaling. Indeed, we found that D1-mediated signaling is reduced in brain slices from both mouse models. Thus, cilia impact D1 signaling in the brain.

Unraveling Molecular and Metabolic Gut‐Brain Signalling Disruptions and Potential Therapy in New Bardet‐Biedl Syndrome Mouse

Obesity is a cardinal feature of Bardet‐Biedl syndrome (BBS), a syndromic ciliopathy caused by mutations in genes for BBS 1‐21. BBS proteins control the function of primary cilia that act as a sensory antenna on virtually all mammalian cells. The mechanisms of BBS‐induced obesity remain poorly understood, resulting in a lack of therapeutic options for these patients. The overarching goal of the study was to define underlying mechanisms involved in the pathogenesis of BBS‐induced obesity and use this knowledge to identify novel therapeutic targets. Given the importance of cilia in interoceptive and homeostatic sensing, we hypothesized that disruption of cilia function by BBS mutation would impair negative feedback resulting in a proinflammatory microenvironment in adipose tissue and disruption of neuroendocrine control of feeding in hypothalamus.In this study, we first characterized the metabolic phenotype of a novel BBS5 knockout mouse (BBS5‐/‐; n=6‐12/strain/gender). Next, we performed immunophenotyping of adipocytes and unbiased RNA sequencing of the hypothalamus of BBS5‐/‐ and WT mice. To validate the predictions from transcriptomic data, we performed pharmacological‐based feeding experiments and tested a putative therapeutic for BBS‐induced obesity.We found that BBS5‐/‐ mice exhibits all the classic symptoms observed in human patients with BBS, including hyperphagia, cognitive impairments, glucose, and insulin intolerance, and 3 to 4‐fold increased fat mass on chow diet compared to wildtype mice (p<0.001). BBS5‐/‐ mice had increased proinflammatory M1 macrophages and decreased functional regulatory T‐cells in adipocytes suggesting that extrinsic factors promote fat mass gain. Pathway analysis of the transcriptomics data revealed altered endocrine receptor signaling in the hypothalamus of BBS5‐/‐ mice. Feeding experiments confirmed the development of both leptin and cholecystokinin resistance independent of body weight (p>0.05). The receptor for glucagon‐like‐peptide‐1 (GLP1) was found to be upregulated in the hypothalamus of BBS5‐/‐ mice (p<0.001). Chronic daily pharmacological administration of GLP1 receptor agonist (Semaglutide) for two weeks significantly reduced food intake, body weight, elevated glucose levels, and fat mass (p<0.001).In conclusion, BBS5‐/‐ mice are a valuable new translational tool to elucidate interoceptive and homeostatic gut‐brain axis alterations in BBS‐induced obesity. We identify adipose inflammation, and impaired hypothalamic leptin, insulin, and cholecystokinin signalling as underlying mechanisms for the metabolic pathogenesis of ciliopathy. Our results support using GLP1 receptor agonist as a novel therapeutic strategy for treating obesity in patients with BBS.

ENKD1 promotes CP110 removal through competing with CEP97 to initiate ciliogenesis.

Despite the importance of cilia in cell signaling and motility, the molecular mechanisms regulating cilium formation remain incompletely understood. Herein, we characterize enkurin domain-containing protein 1 (ENKD1) as a novel centrosomal protein that mediates the removal of centriolar coiled-coil protein 110 (CP110) from the mother centriole to promote ciliogenesis. We show that Enkd1 knockout mice possess ciliogenesis defects in multiple organs. Super-resolution microscopy reveals that ENKD1 is a stable component of the centrosome throughout the ciliogenesis process. Simultaneous knockdown of ENKD1 and CP110 significantly reverses the ciliogenesis defects induced by ENKD1 depletion. Protein interaction analysis shows that ENKD1 competes with centrosomal protein 97 (CEP97) in binding to CP110. Depletion of ENKD1 enhances the CP110-CEP97 interaction and detains CP110 at the mother centriole. These findings thus identify ENKD1 as a centrosomal protein and uncover a novel mechanism controlling CP110 removal from the mother centriole for the initiation of ciliogenesis.

Zebrafish: An In Vivo Model for Motile Ciliopathy

Cilia are highly conserved, microtubule-based organelles. The growing numbers of human diseases due to cilia dysfunction, termed ciliopathies, highlights the functional importance of cilia. Primary ciliary dyskinesia (PCD) is a genetic disorder of the motile cilia, displaying diverse clinical manifestations. With many molecular and genetic tools, zebrafish have emerged as a useful organism model, which allows the investigation of human diseases. Here, we describe the utility of zebrafish in the study of cilia and PCD. With forward and reverse genetic approaches, several novel mutations encoding essential components in motile cilia had been identified in zebrafish. These discoveries will increase our understanding toward cilia biology and have the potential to improve the diagnosis and treatment for affected individuals.

Abstract 415: Metabolic And Cardiovascular Effects Of Cilia Ablation From The Leptin Receptor-containing Neurons

Genetic defects that cause ciliary dysfunction are associated with obesity and cardiovascular diseases in humans and animal models suggesting that functional cilia are important for metabolism and cardiovascular regulation. Cilia are membrane-bound, microtubular projections present on the surface of virtually all cell types including the neurons that control metabolic and cardiovascular functions such as those expressing the leptin receptor (LepRb). To examine the importance of cilia expressed in these neurons, we tested the effect of ablating cilia. For this, we generated mice lacking a key component of the intraflagellar transport (IFT) machinery, IFT88 protein, required for cilia formation, in the LepRb-containing neurons. Breeding ift88 flox mice with LepRb Cre mice created mice deficient in ift88 gene only in the LepRb positive neurons as indicated by the pattern of tdTomato reporter expression. Importantly, ift88 flox /LepRb Cre mice displayed an obesity phenotype as indicated by the increased (P<0.05) body weight (41±2 vs. 37±1. g in controls) and fat mass measured by MRI (11±2 vs. 5±1 g in controls) in 25 weeks old mice. We found that the obesity phenotype in ift88 flox /LepRb Cre mice is not due to an increase in food intake (3.03±0.25 vs. 2.96±0.29 g in controls). In contrast, energy expenditure was reduced in the ift88 flox /LepRb Cre mice as indicated by the decreased (P<0.05) O 2 consumption (1.71±0.1 vs. 2.01±0.08 mL/100g/min in controls) and heat production (5.07±0.4 vs. 5.98±0.27 kcal/kg/h in controls). Moreover, radiotelemetry measurement of core body temperature showed that ift88 flox /LepRb Cre mice had lower 24 h body temperature (36.7±0.2 vs. 37.2±0.2 o C in controls). These results indicate that low metabolic rate accounts for the development of obesity in ift88 flox /LepRb Cre mice. Next, we assessed the hemodynamic consequence of ablating the ift88 gene in LepRb positive neurons. However, mean arterial pressure and heart rate were not significantly different in the ift88 flox /LepRb Cre mice (106±7 mmHg and 635±17 bpm, respectively) compared to controls (110±4 mmHg and 679±15 bmp). These findings demonstrate that deletion of cilia in LepRb expressing neurons alter energy homeostasis, but not cardiovascular regulation.

Identification and functional analysis of novel centrosomal proteins to study their implication in human disease

The physiological importance of cilia is underscored by the ever-growing list of ‘ciliopathies’ associated with ciliary dysfunction. The many overlapping phenotypes of these syndromes might reflect the complexity of the ciliary proteome and the numerous functions of cilia. Likewise, dysfunction of a number of centrosomal proteins has been linked to human diseases. As centrosomes and cilia are closely linked, centrosomal abnormalities can also cause disease by affecting cilia formation, for example by affecting the docking of the basal body to the cell membrane, transport processes relying on microtubule interactions with the centrosome, targeting of membrane vesicles or the intraflagellar transport required for cilia assembly. It might thus be that a number of diseases could be more correctly referred to as ‘centrosomeopathies’. Despite the identification of many centrosomal and ciliary proteins, it is still not known how many of these components interact and function. We have recently characterized the human centrosome proteome in depth using a quantitative mass spectrometry-based method in combination with an antibody-based screen, identifying several novel and candidate centrosomal proteins. The thereby obtained human centrosome proteome provides an excellent basis for further experiments to elucidate the multiple functions of the centrosome, including its role in human disease such as ciliopathies and cancer development. We are currently undertaking more functional experiments based on the improved centrosome proteome to investigate a possible role of these proteins in processes such as centriole duplication and ciliogenesis, both of which are processes with links to human disease.

Tau tubulin kinase 2 is required to initiate mammalian ciliogenesis

Despite the critical importance of cilia in development and disease, the biochemical pathways that initiate ciliogenesis at the mother centriole are not well understood. Here we describe the identification of a serine-threonine protein kinase, Ttbk2, as an essential component of the ciliary initiation pathway. The mouse mutant bartleby (bby) was isolated in a genetic screen in our lab based on phenotypes consistent with a severe disruption in Hh signaling, and immunostaining and scanning electron microscopy revealed that bby mutants lack cilia. The bby phenotype is caused by a nonsense mutation in the gene encoding Tau Tubulin Kinase 2 (Ttbk2), truncating the protein within the kinase domain. Independent studies have shown that human mutations in Ttbk2 are associated with the human neurodegenerative disorder spinocerebellar ataxia type 11. We show that Ttbk2::GFP localizes to the distal mother centriole in cells prior to ciliary axoneme extension, and to the transition zone between the basal body and axoneme in ciliated cells. In Ttbk2bby mutant cells, components of the IFT complexes, such as IFT88 and IFT140, are not recruited to the transition zone. Thus Ttbk2 acts at a step upstream to IFT in the process of cilia formation and is a critical component of a pathway regulating the initiation of ciliogenesis. Although several kinases have roles in ciliogenesis, Ttbk2 is thus far the only kinase found to have an essential role in cilia formation. Moreover, identification of Ttbk2 substrates and upstream regulators has the potential to reveal other regulators of this important step in ciliogenesis.

Ciliopathies: The Central Role of Cilia in a Spectrum of Pediatric Disorders

Cilia are evolutionarily conserved hair-like structures with key roles in cell locomotion, fluid movement, and sexual reproduction. Recent studies have shown that cilia also sense the extracellular environment and are important signaling organelles, which direct embryonic development and organ function. Abnormal ciliary axonemal structure and function have been linked to the growing class of genetic disorders collectively known as ciliopathies. The prototypical ciliopathy, primary ciliary dyskinesia (PCD), was the first human disorder linked to ciliary dysfunction, described over a century ago, and since then, our understanding of the genetic and molecular abnormalities of this disease have greatly advanced [1-3]. The importance of cilia in other human diseases are just beginning to be elucidated, and defects have been associated with a growing number of pediatric conditions, including obesity, renal disease, hepatic fibrosis, skeletal dysplasias, endocrinopathies, neurodevelopmental defects, central nervous system (CNS) anomalies, laterality defects, and congenital heart disease (CHD) [2-5]. Research into ciliopathies has rapidly expanded, involving multidisciplinary efforts to define the complex genetics and functional phenotypes of cilia. In this review, we will describe ciliary ultrastructure and function, genetics, clinical characteristics, and overlapping features of these diverse diseases.

In Vivo Fate Mapping and Expression Analysis Reveals Molecular Hallmarks of Prospectively Isolated Adult Neural Stem Cells

Until now, limitations in the ability to enrich adult NSCs (aNSCs) have hampered meaningful analysis of these cells at the transcriptome level. Here we show via a split-Cre technology that coincident activity of the hGFAP and prominin1 promoters is a hallmark of aNSCs in vivo. Sorting of cells from the adult mouse subependymal zone (SEZ) based on their expression of GFAP and prominin1 isolates all self-renewing, multipotent stem cells at high purity. Comparison of the transcriptome of these purified aNSCs to parenchymal nonneurogenic astrocytes and other SEZ cells reveals aNSC hallmarks, including neuronal lineage priming and the importance of cilia- and Ca-dependent signaling pathways. Inducible deletion of the ciliary protein IFT88 in aNSCs validates the role of ciliary function in aNSCs. Our work reveals candidate molecular regulators for unique features of aNSCs and facilitates future selective analysis of aNSCs in other functional contexts, such as aging and injury.

Notch signalling regulates left-right asymmetry through ciliary length control

The importance of cilia in embryonic development and adult physiology is emphasized by human ciliopathies. Despite its relevance, molecular signalling pathways behind cilia formation are poorly understood. We show that Notch signalling is a key pathway for cilia length control. In deltaD zebrafish mutants, cilia length is reduced in Kupffer's vesicle and can be rescued by the ciliogenic factor foxj1a. Conversely, cilia length increases when Notch signalling is hyperactivated. Short cilia found in deltaD mutants reduce the fluid flow velocity inside Kupffer's vesicle, thus compromising the asymmetric expression of the flow sensor charon. Notch signalling brings together ciliary length control and fluid flow hydrodynamics with transcriptional activation of laterality genes. In addition, our deltaD mutant analysis discloses an uncoupling between gut and heart laterality.

Gli2 and Gli3 Localize to Cilia and Require the Intraflagellar Transport Protein Polaris for Processing and Function

Intraflagellar transport (IFT) proteins are essential for cilia assembly and have recently been associated with a number of developmental processes, such as left–right axis specification and limb and neural tube patterning. Genetic studies indicate that IFT proteins are required for Sonic hedgehog (Shh) signaling downstream of the Smoothened and Patched membrane proteins but upstream of the Glioma (Gli) transcription factors. However, the role that IFT proteins play in transduction of Shh signaling and the importance of cilia in this process remain unknown. Here we provide insights into the mechanism by which defects in an IFT protein, Tg737/Polaris, affect Shh signaling in the murine limb bud. Our data show that loss of Tg737 results in altered Gli3 processing that abrogates Gli3-mediated repression of Gli1 transcriptional activity. In contrast to the conclusions drawn from genetic analysis, the activity of Gli1 and truncated forms of Gli3 (Gli3R) are unaffected in Tg737 mutants at the molecular level, indicating that Tg737/Polaris is differentially involved in specific activities of the Gli proteins. Most important, a negative regulator of Shh signaling, Suppressor of fused, and the three full-length Gli transcription factors localize to the distal tip of cilia in addition to the nucleus. Thus, our data support a model where cilia have a direct role in Gli processing and Shh signal transduction.

Some aspects of the fine structure of the statocysts of the Molluscs Pecten and Pterotrachea.

The statocyst ofPecten is composed of hair cells and supporting cells. The hair cells bear kinocilia and microvilli at their distal ends and the supporting cells bear microvilli. The cilia have a 9+2 internal filament content, and arise from basal bodies that have roots, basal feet and microtubular connections. Two different ciliary arrangements are described, one with a small number of cilia arranged in a ring, and another with many more cilia arranged in rows. Below the hair cells are probable synapses. A ciliated duct connects to the lumen of the static sac and passes through the centre of the static nerve. The hair cells in the statocyst ofPterotrachea bear kinocilia and microvilli. The possible importance of cilia and microvilli in the transduction process is discussed.