The vertebrate primary cilium in development, homeostasis, and disease.

Cilia are complex structures that have garnered interest because of their roles in vertebrate development and their involvement in human genetic disorders. In contrast to multicellular invertebrates in which cilia are restricted to specific cell types, these organelles are found almost ubiquitously in vertebrate cells, where they serve a diverse set of signaling functions. Here, we highlight properties of vertebrate cilia, with particular emphasis on their relationship with other subcellular structures, and explore the physiological consequences of ciliary dysfunction.

Identification of proliferative and mature ß-cells in the islets of Langerhans.

Insulin-dependent diabetes is a complex multifactorial disorder characterized by loss or dysfunction of ß-cells. Pancreatic ß-cells differ in size, glucose responsiveness, insulin secretion and precursor cell potential; understanding the mechanisms that underlie this functional heterogeneity might make it possible to develop new regenerative approaches. Here we show that Fltp (also known as Flattop and Cfap126), a Wnt/planar cell polarity (PCP) effector and reporter gene acts as a marker gene that subdivides endocrine cells into two subpopulations and distinguishes proliferation-competent from mature ß-cells with distinct molecular, physiological and ultrastructural features. Genetic lineage tracing revealed that endocrine subpopulations from Fltp-negative and -positive lineages react differently to physiological and pathological changes. The expression of Fltp increases when endocrine cells cluster together to form polarized and mature 3D islet mini-organs. We show that 3D architecture and Wnt/PCP ligands are sufficient to trigger ß-cell maturation. By contrast, the Wnt/PCP effector Fltp is not necessary for ß-cell development, proliferation or maturation. We conclude that 3D architecture and Wnt/PCP signalling underlie functional ß-cell heterogeneity and induce ß-cell maturation. The identification of Fltp as a marker for endocrine subpopulations sheds light on the molecular underpinnings of islet cell heterogeneity and plasticity and might enable targeting of endocrine subpopulations for the regeneration of functional ß-cell mass in diabetic patients.

Metabolic regulation through the endosomal system.

The endosomal system plays an essential role in cell homeostasis by controlling cellular signaling, nutrient sensing, cell polarity and cell migration. However, its place in the regulation of tissue, organ and whole body physiology is less well understood. Recent studies have revealed an important role for the endosomal system in regulating glucose and lipid homeostasis, with implications for metabolic disorders such as type 2 diabetes, hypercholesterolemia and non-alcoholic fatty liver disease. By taking insights from in vitro studies of endocytosis and exploring their effects on metabolism, we can begin to connect the fields of endosomal transport and metabolic homeostasis. In this review, we explore current understanding of how the endosomal system influences the systemic regulation of glucose and lipid metabolism in mice and humans. We highlight exciting new insights that help translate findings from single cells to a wider physiological level and open up new directions for endosomal research.

Role of mitochondrial complex I and protective effect of CoQ10 supplementation in propofol induced cytotoxicity.

Propofol (2,6-diisopropylphenol) is an anaesthetic widely used for human sedation. Due to its intrinsic antioxidant properties, rapid induction of anaesthesia and fast recovery, it is employed in paediatric anaesthesia and in the intensive care of premature infants. Recent studies have pointed out that exposure to anaesthesia in the early stage of life might be responsible of long-lasting cognitive impairment. The apoptotic neurodegeneration induced by general anaesthetics (GA) involves mitochondrial impairment due to the inhibition of the OXPHOS machinery. In the present work, we aim to identify the main mitochondrial respiratory chain target of propofol toxicity and to evaluate the possible protective effect of CoQ10 supplementation. The propofol effect on the mitochondrial functionality was assayed in isolated mitochondria and in two cell lines (HeLa and T67) by measuring oxygen consumption rate. The protective effect of CoQ10 was assessed by measuring cells viability, NADH-oxidase activity and ATP/ADP ratio in cells treated with propofol. Our results show that propofol reduces cellular oxygen consumption rate acting mainly on mitochondrial Complex I. The kinetic analysis of Complex I inhibition indicates that propofol interferes with the Q module acting as a non-competitive inhibitor with higher affinity for the free form of the enzyme. Cells supplemented with CoQ10 are more resistant to propofol toxicity. Propofol exposure induces cellular damages due to mitochondrial impairment. The site of propofol inhibition on Complex I is the Q module. CoQ10 supplementation protects cells against the loss of energy suggesting its possible therapeutic role to minimizing the detrimental effects of general anaesthesia.

Functional analyses of variants reveal a significant role for dominant negative and common alleles in oligogenic Bardet-Biedl syndrome.

Technological advances hold the promise of rapidly catalyzing the discovery of pathogenic variants for genetic disease. However, this possibility is tempered by limitations in interpreting the functional consequences of genetic variation at candidate loci. Here, we present a systematic approach, grounded on physiologically relevant assays, to evaluate the mutational content (125 alleles) of the 14 genes associated with Bardet-Biedl syndrome (BBS). A combination of in vivo assays with subsequent in vitro validation suggests that a significant fraction of BBS-associated mutations have a dominant-negative mode of action. Moreover, we find that a subset of common alleles, previously considered to be benign, are, in fact, detrimental to protein function and can interact with strong rare alleles to modulate disease presentation. These data represent a comprehensive evaluation of genetic load in a multilocus disease. Importantly, superimposition of these results to human genetics data suggests a previously underappreciated complexity in disease architecture that might be shared among diverse clinical phenotypes.

Adipocyte-derived extracellular vesicles increase insulin secretion through transport of insulinotropic protein cargo.

Adipocyte-derived extracellular vesicles (AdEVs) are membranous nanoparticles that convey communication from adipose tissue to other organs. Here, to delineate their role as messengers with glucoregulatory nature, we paired fluorescence AdEV-tracing and SILAC-labeling with (phospho)proteomics, and revealed that AdEVs transfer functional insulinotropic protein cargo into pancreatic ß-cells. Upon transfer, AdEV proteins were subjects for phosphorylation, augmented insulinotropic GPCR/cAMP/PKA signaling by increasing total protein abundances and phosphosite dynamics, and ultimately enhanced 1st-phase glucose-stimulated insulin secretion (GSIS) in murine islets. Notably, insulinotropic effects were restricted to AdEVs isolated from obese and insulin resistant, but not lean mice, which was consistent with differential protein loads and AdEV luminal morphologies. Likewise, in vivo pre-treatment with AdEVs from obese but not lean mice amplified insulin secretion and glucose tolerance in mice. This data suggests that secreted AdEVs can inform pancreatic ß-cells about insulin resistance in adipose tissue in order to amplify GSIS in times of increased insulin demand.

Bardet-Biedl syndrome-associated small GTPase ARL6 (BBS3) functions at or near the ciliary gate and modulates Wnt signaling.

The expansive family of metazoan ADP-ribosylation factor and ADP-ribosylation factor-like small GTPases is known to play essential roles in modulating membrane trafficking and cytoskeletal functions. Here, we present the crystal structure of ARL6, mutations in which cause Bardet-Biedl syndrome (BBS3), and reveal its unique ring-like localization at the distal end of basal bodies, in proximity to the so-called ciliary gate where vesicles carrying ciliary cargo fuse with the membrane. Overproduction of GDP- or GTP-locked variants of ARL6/BBS3 in vivo influences primary cilium length and abundance. ARL6/BBS3 also modulates Wnt signaling, a signal transduction pathway whose association with cilia in vertebrates is just emerging. Importantly, this signaling function is lost in ARL6 variants containing BBS-associated point mutations. By determining the structure of GTP-bound ARL6/BBS3, coupled with functional assays, we provide a mechanistic explanation for such pathogenic alterations, namely altered nucleotide binding. Our findings therefore establish a previously unknown role for ARL6/BBS3 in mammalian ciliary (dis)assembly and Wnt signaling and provide the first structural information for a BBS protein.

Islet vascularization is regulated by primary endothelial cilia via VEGF-A-dependent signaling.

Islet vascularization is essential for intact islet function and glucose homeostasis. We have previously shown that primary cilia directly regulate insulin secretion. However, it remains unclear whether they are also implicated in islet vascularization. At eight weeks, murine Bbs4-/-islets show significantly lower intra-islet capillary density with enlarged diameters. Transplanted Bbs4-/- islets exhibit delayed re-vascularization and reduced vascular fenestration after engraftment, partially impairing vascular permeability and glucose delivery to ß-cells. We identified primary cilia on endothelial cells as the underlying cause of this regulation, via the vascular endothelial growth factor-A (VEGF-A)/VEGF receptor 2 (VEGFR2) pathway. In vitro silencing of ciliary genes in endothelial cells disrupts VEGF-A/VEGFR2 internalization and downstream signaling. Consequently, key features of angiogenesis including proliferation and migration are attenuated in human BBS4 silenced endothelial cells. We conclude that endothelial cell primary cilia regulate islet vascularization and vascular barrier function via the VEGF-A/VEGFR2 signaling pathway.

Phosphoproteomics Reveals the GSK3-PDX1 Axis as a Key Pathogenic Signaling Node in Diabetic Islets.

Progressive decline of pancreatic beta cell function is central to the pathogenesis of type 2 diabetes. Protein phosphorylation regulates glucose-stimulated insulin secretion from beta cells, but how signaling networks are remodeled in diabetic islets in vivo remains unknown. Using high-sensitivity mass spectrometry-based proteomics, we quantified 6,500 proteins and 13,000 phosphopeptides in islets of obese diabetic mice and matched controls, revealing drastic remodeling of key kinase hubs and signaling pathways. Integration with a literature-derived signaling network implicated GSK3 kinase in the control of the beta cell-specific transcription factor PDX1. Deep phosphoproteomic analysis of human islets chronically treated with high glucose demonstrated a conserved glucotoxicity-dependent role of GSK3 kinase in regulating insulin secretion. Remarkably, the ability of beta cells to secrete insulin in response to glucose was rescued almost completely by pharmacological inhibition of GSK3. Thus, our resource enables investigation of mechanisms and drug targets in type 2 diabetes.

Glucose homeostasis is regulated by pancreatic ß-cell cilia via endosomal EphA-processing.

Diabetes mellitus affects one in eleven adults worldwide. Most suffer from Type 2 Diabetes which features elevated blood glucose levels and an inability to adequately secrete or respond to insulin. Insulin producing ß-cells have primary cilia which are implicated in the regulation of glucose metabolism, insulin signaling and secretion. To better understand how ß-cell cilia affect glucose handling, we ablate cilia from mature ß-cells by deleting key cilia component Ift88. Here we report that glucose homeostasis and insulin secretion deteriorate over 12 weeks post-induction. Cilia/basal body components are required to suppress spontaneous auto-activation of EphA3 and hyper-phosphorylation of EphA receptors inhibits insulin secretion. In ß-cells, loss of cilia/basal body function leads to polarity defects and epithelial-to-mesenchymal transition. Defective insulin secretion from IFT88-depleted human islets and elevated pEPHA3 in islets from diabetic donors both point to a role for cilia/basal body proteins in human glucose homeostasis.

Decreased Expression of Cilia Genes in Pancreatic Islets as a Risk Factor for Type 2 Diabetes in Mice and Humans.

An insufficient adaptive beta-cell compensation is a hallmark of type 2 diabetes (T2D). Primary cilia function as versatile sensory antennae regulating various cellular processes, but their role on compensatory beta-cell replication has not been examined. Here, we identify a significant enrichment of downregulated, cilia-annotated genes in pancreatic islets of diabetes-prone NZO mice as compared with diabetes-resistant B6-ob/ob mice. Among 327 differentially expressed mouse cilia genes, 81 human orthologs are also affected in islets of diabetic donors. Islets of nondiabetic mice and humans show a substantial overlap of upregulated cilia genes that are linked to cell-cycle progression. The shRNA-mediated suppression of KIF3A, essential for ciliogenesis, impairs division of MIN6 beta cells as well as in dispersed primary mouse and human islet cells, as shown by decreased BrdU incorporation. These findings demonstrate the substantial role of cilia-gene regulation on islet function and T2D risk.

The role of primary cilia in obesity and diabetes.

One in 12 people worldwide suffers from diabetes and more than 90% of affected adult individuals are diagnosed with type 2 diabetes mellitus (T2DM). Obesity adds to the personal risk to develop T2DM, and both metabolic diseases are rampantly increasing worldwide. Over recent years, primary cilia have moved into the focus of basic and clinical research, after several human diseases have been identified as ciliopathies (i.e., they are linked to ciliary dysfunction). A subset of ciliopathies presents with obesity and diabetes, either as core symptoms or major complications. Several studies have shown a role for ciliary signaling in the satiety signaling centers of the hypothalamus and in other metabolically active tissues, such as pancreatic islets. Here, we discuss recent advances and perspectives in ciliary metabolic research.

Short and prolonged exposure to hyperglycaemia in human fibroblasts and endothelial cells: metabolic and osmotic effects.

High blood glucose levels are the main feature of diabetes. However, the underlying mechanism linking high glucose concentration to diabetic complications is still not fully elucidated, particularly with regard to human physiology. Excess of glucose is likely to trigger a metabolic response depending on the cell features, activating deleterious pathways involved in the complications of diabetes. In this study, we aim to elucidate how acute and prolonged hyperglycaemia alters the biology and metabolism in human fibroblasts and endothelial cells. We found that hyperglycaemia triggers a metabolic switch from oxidative phosphorylation to glycolysis that is maintained over prolonged time. Moreover, osmotic pressure is a major factor in the early metabolic response, decreasing both mitochondrial transmembrane potential and cellular proliferation. After prolonged exposure to hyperglycaemia we observed decreased mitochondrial steady-state and uncoupled respiration, together with a reduced ATP/ADP ratio. At the same time, we could not detect major changes in mitochondrial transmembrane potential and reactive oxygen species. We suggest that the physiological and metabolic alterations observed in healthy human primary fibroblasts and endothelial cells are an adaptive response to hyperglycaemia. The severity of metabolic and bioenergetics impairment associated with diabetic complications may occur after longer glucose exposure or due to interactions with cell types more sensitive to hyperglycaemia.

Ciliary dysfunction impairs beta-cell insulin secretion and promotes development of type 2 diabetes in rodents.

Type 2 diabetes mellitus is affecting more than 382 million people worldwide. Although much progress has been made, a comprehensive understanding of the underlying disease mechanism is still lacking. Here we report a role for the ß-cell primary cilium in type 2 diabetes susceptibility. We find impaired glucose handling in young Bbs4(-/-) mice before the onset of obesity. Basal body/ciliary perturbation in murine pancreatic islets leads to impaired first phase insulin release ex and in vivo. Insulin receptor is recruited to the cilium of stimulated ß-cells and ciliary/basal body integrity is required for activation of downstream targets of insulin signalling. We also observe a reduction in the number of ciliated ß-cells along with misregulated ciliary/basal body gene expression in pancreatic islets in a diabetic rat model. We suggest that ciliary function is implicated in insulin secretion and insulin signalling in the ß-cell and that ciliary dysfunction could contribute to type 2 diabetes susceptibility.

Ciliary function and Wnt signal modulation.

With the increase in complexity of morphogenetic signaling cascades over the course of evolution and the emergence of broadly ciliated organisms, the cilium seems to have acquired a role as regulator of paracrine signal transduction. Recently, several lines of evidence have provided a link between basal body and ciliary proteins and Wnt signaling. In this chapter, we will evaluate the evidence linking the basal body and cilium with the regulation of beta-catenin-dependent (canonical) and beta-catenin-independent (noncanonical) signaling processes as well as which role(s) Wnt signaling might play in ciliogenesis. In addition, we will discuss aberrant Wnt signaling could contribute to phenotypes common to most ciliopathies and why these phenotypes might be driven by loss of noncanonical rather than gain of noncanonical Wnt signaling.

Disruption of the basal body compromises proteasomal function and perturbs intracellular Wnt response.

Primary cilia and basal bodies are evolutionarily conserved organelles that mediate communication between the intracellular and extracellular environments. Here we show that bbs1, bbs4 and mkks (also known as bbs6), which encode basal body proteins, are required for convergence and extension in zebrafish and interact with wnt11 and wnt5b. Suppression of bbs1, bbs4 and mkks transcripts results in stabilization of beta-catenin with concomitant upregulation of T-cell factor (TCF)-dependent transcription in both zebrafish embryos and mammalian ciliated cells, a defect phenocopied by the silencing of the axonemal kinesin subunit KIF3A but not by chemical disruption of the cytoplasmic microtubule network. These observations are attributable partly to defective degradation by the proteasome; suppression of BBS4 leads to perturbed proteasomal targeting and concomitant accumulation of cytoplasmic beta-catenin. Cumulatively, our data indicate that the basal body is an important regulator of Wnt signal interpretation through selective proteolysis and suggest that defects in this system may contribute to phenotypes pathognomonic of human ciliopathies.

Small molecule intervention in microtubule-associated human disease.

Microtubules are essential for a number of cellular processes that include the transport of intracellular cargo or organelles across long distances and the assembly of the mitotic spindle. The identification of numerous microtubule-associated proteins and the progressive elucidation of the mechanisms of microtubule assembly and transport are beginning to have a profound impact on the study and treatment of human genetic disease. A number of seemingly unrelated phenotypes have now been linked to microtubular dysfunction, especially in systems dependent heavily on microtubule-based transport, such as neurons and ciliated cells. In parallel, the association of microtubule transport defects with human genetic disease has led to the realization that targeting various aspects of microtubular biology with small molecules might offer new therapeutic paradigms, including the development of new therapeutic utility for seemingly old drugs. In this review, we discuss the use of small molecules in the investigation of microtubule-associated processes and particularly the screens of chemical compound libraries for the identification of lead compounds with potential utility in microtubule-associated disease processes.