Ciliopathies: an expanding disease spectrum

Ciliopathies comprise a group of disorders associated with genetic mutations encoding defective proteins, which result in either abnormal formation or function of cilia. As cilia are a component of almost all vertebrate cells, cilia dysfunction can manifest as a constellation of features that include characteristically, retinal degeneration, renal disease and cerebral anomalies. Additional manifestations include congenital fibrocystic diseases of the liver, diabetes, obesity and skeletal dysplasias. Ciliopathic features have been associated with mutations in over 40 genes to date. However, with over 1,000 polypeptides currently identified within the ciliary proteome, several other disorders associated with this constellation of clinical features will likely be ascribed to mutations in other ciliary genes. The mechanisms underlying many of the disease phenotypes associated with ciliary dysfunction have yet to be fully elucidated. Several elegant studies have crucially demonstrated the dynamic ciliary localisation of components of the Hedgehog and Wnt signalling pathways during signal transduction. Given the critical role of the cilium in transducing “outside-in” signals, it is not surprising therefore, that the disease phenotypes consequent to ciliary dysfunction are a manifestation of aberrant signal transduction. Further investigation is now needed to explore the developmental and physiological roles of aberrant signal transduction in the manifestation of ciliopathy phenotypes. Utilisation of conditional and inducible murine models to delete or overexpress individual ciliary genes in a spatiotemporal and organ/cell-specific manner should help clarify some of the functional roles of ciliary proteins in the manifestation of phenotypic features

Widespread GLI expression but limited canonical hedgehog signaling restricted to the ductular reaction in human chronic liver disease

Canonical Hedgehog (Hh) signaling in vertebrate cells occurs following Smoothened activation/translocation into the primary cilia (Pc), followed by a GLI transcriptional response. Nonetheless, GLI activation can occur independently of the canonical Hh pathway. Using a murine model of liver injury, we previously identified the importance of canonical Hh signaling within the Pc+ liver progenitor cell (LPC) population and noted that SMO-independent, GLI-mediated signals were important in multiple Pc-ve GLI2+ intrahepatic populations. This study extends these observations to human liver tissue, and analyses the effect of GLI inhibition on LPC viability/gene expression. Human donor and cirrhotic liver tissue specimens were evaluated for SHH, GLI2 and Pc expression using immunofluorescence and qRT-PCR. Changes to viability and gene expression in LPCs in vitro were assessed following GLI inhibition. Identification of Pc (as a marker of canonical Hh signaling) in human cirrhosis was predominantly confined to the ductular reaction and LPCs. In contrast, GLI2 was expressed in multiple cell populations including Pc-ve endothelium, hepatocytes, and leukocytes. HSCs/myofibroblasts (gt;99%) expressed GLI2, with only 1.92% displaying Pc. In vitro GLI signals maintained proliferation/viability within LPCs and GLI inhibition affected the expression of genes related to stemness, hepatocyte/biliary differentiation and Hh/Wnt signaling. At least two mechanisms of GLI signaling (Pc/SMOdependent and Pc/SMO-independent) mediate chronic liver disease pathogenesis. This may have significant ramifications for the choice of Hh inhibitor (anti-SMO or anti-GLI) suitable for clinical trials. We also postulate GLI delivers a pro-survival signal to LPCs whilst maintaining stemness

The dynamic cilium in human diseases

Cilia are specialized organelles protruding from the cell surface of almost all mammalian cells. They consist of a basal body, composed of two centrioles, and a protruding body, named the axoneme. Although the basic structure of all cilia is the same, numerous differences emerge in different cell types, suggesting diverse functions. In recent years many studies have elucidated the function of 9+0 primary cilia. The primary cilium acts as an antenna for the cell, and several important pathways such as Hedgehog, Wnt and planar cell polarity (PCP) are transduced through it. Many studies on animal models have revealed that during embryogenesis the primary cilium has an essential role in defining the correct patterning of the body. Cilia are composed of hundreds of proteins and the impairment or dysfunction of one protein alone can cause complete loss of cilia or the formation of abnormal cilia. Mutations in ciliary proteins cause ciliopathies which can affect many organs at different levels of severity and are characterized by a wide spectrum of phenotypes. Ciliary proteins can be mutated in more than one ciliopathy, suggesting an interaction between proteins. To date, little is known about the role of primary cilia in adult life and it is tempting to speculate about their role in the maintenance of adult organs. The state of the art in primary cilia studies reveals a very intricate role. Analysis of cilia-related pathways and of the different clinical phenotypes of ciliopathies helps to shed light on the function of these sophisticated organelles. The aim of this review is to evaluate the recent advances in cilia function and the molecular mechanisms at the basis of their activity

Physiology and pathophysiology of the vasopressin-regulated renal water reabsorption

To prevent dehydration, terrestrial animals and humans have developed a sensitive and versatile system to maintain their water homeostasis. In states of hypernatremia or hypovolemia, the antidiuretic hormone vasopressin (AVP) is released from the pituitary and binds its type-2 receptor in renal principal cells. This triggers an intracellular cAMP signaling cascade, which phosphorylates aquaporin-2 (AQP2) and targets the channel to the apical plasma membrane. Driven by an osmotic gradient, pro-urinary water then passes the membrane through AQP2 and leaves the cell on the basolateral side via AQP3 and AQP4 water channels. When water homeostasis is restored, AVP levels decline, and AQP2 is internalized from the plasma membrane, leaving the plasma membrane watertight again. The action of AVP is counterbalanced by several hormones like prostaglandin E2, bradykinin, dopamine, endothelin-1, acetylcholine, epidermal growth factor, and purines. Moreover, AQP2 is strongly involved in the pathophysiology of disorders characterized by renal concentrating defects, as well as conditions associated with severe water retention. This review focuses on our recent increase in understanding of the molecular mechanisms underlying AVP-regulated renal water transport in both health and disease

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

The Role of Cilia in the Regulation of Bile Flow

Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, are ciliated cells. Each cholangiocyte has a primary cilium consisting of (i) a microtubule-based axoneme and (ii) the basal body, centriole-derived, microtubule-organizing center from which the axoneme emerges. Primary cilia in cholangiocytes were described decades ago, but their physiological and pathophysiological significance remained unclear until recently. We now recognize that cholangiocyte cilia extend from the apical plasma membrane into the bile duct lumen and, as such, are ideally positioned to detect changes in bile flow, bile composition and bile osmolality. These sensory organelles act as cellular antennae that can detect and transmit signals that influence cholangiocyte function. Indeed, recent data show that cholangiocyte primary cilia can activate intracellular signaling pathways when they sense modifications in the flow, molecular constituents and osmolarity of bile. Their ability to sense and transmit signals depends on the participation of a growing number of specific ciliary-associated proteins that act as receptors, channels and transporters. Cholangiocyte cilia, in addition to being important in normal biliary physiology, likely contribute to the cholangiopathies when their normal structure or function is disturbed. Indeed, the polycystic liver diseases that occur in combination with autosomal dominant and recessive polycystic kidney disease (i.e. ADPKD and ARPKD) are two important examples of such conditions. Recent insights into the role of cholangiocyte cilia in cystic liver disease using in vitro and animal models have already resulted in clinical trials that have influenced the management of cystic liver disease