Molecular Mechanisms of Fibroblast Growth Factor Signaling in Physiology and Pathology

Fibroblast growth factors (FGFs) signal in a paracrine or endocrine fashion to mediate a myriad of biological activities, ranging from issuing developmental cues, maintaining tissue homeostasis, and regulating metabolic processes. FGFs carry out their diverse functions by binding and dimerizing FGF receptors (FGFRs) in a heparan sulfate (HS) cofactor- or Klotho coreceptor-assisted manner. The accumulated wealth of structural and biophysical data in the past decade has transformed our understanding of the mechanism of FGF signaling in human health and development, and has provided novel concepts in receptor tyrosine kinase (RTK) signaling. Among these contributions are the elucidation of HS-assisted receptor dimerization, delineation of the molecular determinants of ligand–receptor specificity, tyrosine kinase regulation, receptor cis-autoinhibition, and tyrosine trans-autophosphorylation. These structural studies have also revealed how disease-associated mutations highjack the physiological mechanisms of FGFR regulation to contribute to human diseases. In this paper, we will discuss the structurally and biophysically derived mechanisms of FGF signaling, and how the insights gained may guide the development of therapies for treatment of a diverse array of human diseases.Fibroblast growth factor (FGF) signaling fulfills essential roles in metazoan development and metabolism. A wealth of literature has documented the requirement for FGF signaling in multiple processes during embryogenesis, including implantation (Feldman et al. 1995), gastrulation (Sun et al. 1999), somitogenesis (Dubrulle and Pourquie 2004; Wahl et al. 2007; Lee et al. 2009; Naiche et al. 2011; Niwa et al. 2011), body plan formation (Martin 1998; Rodriguez Esteban et al. 1999; Tanaka et al. 2005; Mariani et al. 2008), morphogenesis (Metzger et al. 2008; Makarenkova et al. 2009), and organogenesis (Goldfarb 1996; Kato and Sekine 1999; Sekine et al. 1999; Sun et al. 1999; Colvin et al. 2001; Serls et al. 2005; Vega-Hernandez et al. 2011). Recent clinical and biochemical data have uncovered unexpected roles for FGF signaling in metabolic processes, including phosphate/vitamin D homeostasis (Consortium 2000; Razzaque and Lanske 2007; Nakatani et al. 2009; Gattineni et al. 2011; Kir et al. 2011), cholesterol/bile acid homeostasis (Yu et al. 2000a; Holt et al. 2003), and glucose/lipid metabolism (Fu et al. 2004; Moyers et al. 2007). Highlighting its diverse biology, deranged FGF signaling contributes to many human diseases, such as congenital craniosynostosis and dwarfism syndromes (Naski et al. 1996; Wilkie et al. 2002, 2005), Kallmann syndrome (Dode et al. 2003; Pitteloud et al. 2006a), hearing loss (Tekin et al. 2007, 2008), and renal phosphate wasting disorders (Shimada et al. 2001; White et al. 2001), as well as many acquired forms of cancers (Rand et al. 2005; Pollock et al. 2007; Gartside et al. 2009; di Martino et al. 2012). Endocrine FGFs have also been implicated in the progression of acquired metabolic disorders, including chronic kidney disease (Fliser et al. 2007), obesity (Inagaki et al. 2007; Moyers et al. 2007; Reinehr et al. 2012), and insulin resistance (Fu et al. 2004; Chen et al. 2008b; Chateau et al. 2010; Huang et al. 2011), giving rise to many opportunities for drug discovery in the field of FGF biology (Beenken and Mohammadi 2012).Based on sequence homology and phylogeny, the 18 mammalian FGFs are grouped into six subfamilies (Ornitz and Itoh 2001; Popovici et al. 2005; Itoh and Ornitz 2011). Five of these subfamilies act in a paracrine fashion, namely, the FGF1 subfamily (FGF1 and FGF2), the FGF4 subfamily (FGF4, FGF5, and FGF6), the FGF7 subfamily (FGF3, FGF7, FGF10, and FGF22), the FGF8 subfamily (FGF8, FGF17, and FGF18), and the FGF9 subfamily (FGF9, FGF16, and FGF20). In contrast, the FGF19 subfamily (FGF19, FGF21, and FGF23) signals in an endocrine manner (Beenken and Mohammadi 2012). FGFs exert their pleiotropic effects by binding and activating the FGF receptor (FGFR) subfamily of receptor tyrosine kinases that are coded by four genes (FGFR1, FGFR2, FGFR3, and FGFR4) in mammals (Johnson and Williams 1993; Mohammadi et al. 2005b). The extracellular domain of FGFRs consists of three immunoglobulin (Ig)-like domains (D1, D2, and D3), and the intracellular domain harbors the conserved tyrosine kinase domain flanked by the flexible amino-terminal juxtamembrane linker and carboxy-terminal tail (Lee et al. 1989; Dionne et al. 1991; Givol and Yayon 1992). A unique feature of FGFRs is the presence of a contiguous segment of glutamic and aspartic acids in the D1–D2 linker, termed the acid box (AB). The two-membrane proximal D2 and D3 and the intervening D2–D3 linker are necessary and sufficient for ligand binding/specificity (Dionne et al. 1990; Johnson et al. 1990), whereas D1 and the D1–D2 linker are implicated in receptor autoinhibition (Wang et al. 1995; Roghani and Moscatelli 2007; Kalinina et al. 2012). Alternative splicing and translational initiation further diversify both ligands and receptors. The amino-terminal regions of FGF8 and FGF17 can be differentially spliced to yield FGF8a, FGF8b, FGF8e, FGF8f (Gemel et al. 1996; Blunt et al. 1997), and FGF17a and FGF17b isoforms (Xu et al. 1999), whereas cytosine-thymine-guanine (CTG)-mediated translational initiation gives rise to multiple high molecular weight isoforms of FGF2 and FGF3 (Florkiewicz and Sommer 1989; Prats et al. 1989; Acland et al. 1990). The tissue-specific alternative splicing in D3 of FGFR1, FGFR2, and FGFR3 yields “b” and “c” receptor isoforms which, along with their temporal and spatial expression patterns, is the major regulator of FGF–FGFR specificity/promiscuity (Orr-Urtreger et al. 1993; Ornitz et al. 1996; Zhang et al. 2006). A large body of structural data on FGF–FGFR complexes has begun to reveal the intricate mechanisms by which different FGFs and FGFRs combine selectively to generate quantitatively and qualitatively different intracellular signals, culminating in distinct biological responses. In addition, these structural data have unveiled how pathogenic mutations hijack the normal physiological mechanisms of FGFR regulation to lead to pathogenesis. We will discuss the current state of the structural biology of the FGF–FGFR system, lessons learned from studying the mechanism of action of pathogenic mutations, and how the structural data are beginning to shape and advance the translational research.     

Molecular Bases of Cell–Cell Junctions Stability and Dynamics

Epithelial cell–cell junctions are formed by apical adherens junctions (AJs), which are composed of cadherin adhesion molecules interacting in a dynamic way with the cortical actin cytoskeleton. Regulation of cell–cell junction stability and dynamics is crucial to maintain tissue integrity and allow tissue remodeling throughout development. Actin filament turnover and organization are tightly controlled together with myosin-II activity to produce mechanical forces that drive the assembly, maintenance, and remodeling of AJs. In this review, we will discuss these three distinct stages in the lifespan of cell–cell junctions, using several developmental contexts, which illustrate how mechanical forces are generated and transmitted at junctions, and how they impact on the integrity and the remodeling of cell–cell junctions.Cell–cell junction formation and remodeling occur repeatedly throughout development. Epithelial cells are linked by apical adherens junctions (AJs) that rely on the cadherin-catenin-actin module. Cadherins, of which epithelial E-cadherin (E-cad) is the most studied, are Ca²⁺-dependent transmembrane adhesion proteins forming homophilic and heterophilic bonds in trans between adjacent cells. Cadherins and the actin cytoskeleton are mutually interdependent (Jaffe et al. 1990; Matsuzaki et al. 1990; Hirano et al. 1992; Oyama et al. 1994; Angres et al. 1996; Orsulic and Peifer 1996; Adams et al. 1998; Zhang et al. 2005; Pilot et al. 2006). This has long been attributed to direct physical interaction of E-cad with β-catenin (β-cat) and of α-catenin (α-cat) with actin filaments (for reviews, see Gumbiner 2005; Leckband and Prakasam 2006; Pokutta and Weis 2007). Recently, biochemical and protein dynamics analyses have shown that such a link may not exist and that instead, a constant shuttling of α-cat between cadherin/β-cat complexes and actin may be key to explain the dynamic aspect of cell–cell adhesion (Drees et al. 2005; Yamada et al. 2005). Regardless of the exact nature of this link, several studies show that AJs are indeed physically attached to actin and that cadherins transmit cortical forces exerted by junctional acto-myosin networks (Costa et al. 1998; Sako et al. 1998; Pettitt et al. 2003; Dawes-Hoang et al. 2005; Cavey et al. 2008; Martin et al. 2008; Rauzi et al. 2008). In addition, physical association depends in part on α-cat (Cavey et al. 2008) and additional intermediates have been proposed to represent alternative missing links (Abe and Takeichi 2008) (reviewed in Gates and Peifer 2005; Weis and Nelson 2006). Although further work is needed to address the molecular nature of cadherin/actin dynamic interactions, association with actin is crucial all throughout the lifespan of AJs. In this article, we will review our current understanding of the molecular mechanisms at work during three different developmental stages of AJs biology: assembly, stabilization, and remodeling, with special emphasis on the mechanical forces controlling AJs integrity and development.     

Receptor Tyrosine Kinases: Legacy of the First Two Decades

Receptor tyrosine kinases (RTKs) and their cellular signaling pathways play important roles in normal development and homeostasis. Aberrations in their activation or signaling leads to many pathologies, especially cancers, motivating the development of a variety of drugs that block RTK signaling that have been successfully applied for the treatment of many cancers. As the current field of RTKs and their signaling pathways are covered by a very large amount of literature, spread over half a century, I am focusing the scope of this review on seminal discoveries made before tyrosine phosphorylation was discovered, and on the early days of research into RTKs and their cellular signaling pathways. I review the history of the early days of research in the field of RTKs. I emphasize key early findings, which provided conceptual frameworks for addressing the questions of how RTKs are activated and how they regulate intracellular signaling pathways.The family of cell-surface receptors designated receptor tyrosine kinases (RTK) received their name more that a decade after the same molecules were already known as the cell-surface receptors for insulin (insulin receptor), epidermal growth factor (EGFR), and many other growth factor receptors. Following the pioneering discoveries of nerve growth factor and epidermal growth factor (EGF; Levi-Montalcini and Booker 1960; Cohen 1962) and the establishment of the important roles of these two growth factors in the control of neuronal differentiation and cell proliferation in vivo and in vitro, it became clear that these cytokines bind specifically to cell-surface receptors. Insulin had already been discovered by this time, and had been applied successfully to treat diabetes patients since the early twentieth century. The resulting homogenous preparations of pure insulin enabled the quantitative characterization of insulin binding to its receptor on intact cells or to solubilized insulin receptor preparations using radiolabeled insulin (De Meyts et al. 1973). These studies greatly advanced understanding of the ligand binding characteristics of insulin receptor and, later on EGFR (Carpenter et al. 1975), including the establishment of negative cooperativity in insulin binding to its receptor expressed on the surface of living cells (De Meyts et al. 1973). Moreover, these studies shed important light on the dynamic nature of the cellular behavior of these receptors. The capacities of insulin receptor and EGFR to undergo ligand-dependent down-regulation and desensitization through receptor-mediated internalization and degradation (Carpenter and Cohen 1976; Gordon et al. 1978; Schlessinger et al. 1978a,b; Carpentier et al. 1979; Haigler et al. 1979) were also established well before the realization that growth factors receptors are endowed with intrinsic protein tyrosine kinase activities (Fig. 1).Open in a separate windowFigure 1.A time line of key findings during the history of RTKs, with emphasis on findings and discoveries that produced the conceptual framework in the development of the RTK field and its application for cancer therapy. References for the key findings are also presented in the text (Lee et al. 1985; Libermann et al. 1985; Margolis et al. 1990; Bottaro et al. 1991; Bae et al. 2009).Progress was also made in elucidating the role of growth factors in normal embryonic development, wound healing, and pathological conditions such as cancer. Early studies in the 1960s and 1970s showed that growth factors play an important role in oncogenesis induced by retroviruses and in the proliferation of tumor-derived cancer cells. Pioneering studies performed by Howard Temin (1966, 1967) showed that cancer cells need less insulin and serum growth factors for cell proliferation compared with normal cells, suggesting that cancer cells produce and use their own growth factors and/or use cellular processes that in normal cells are regulated by exogenously supplied growth factors; both predictions were subsequently confirmed. A variety of new polypeptide growth factors that stimulate cell proliferation by binding to receptors at the cell surface were subsequently discovered. Those include a growth factor isolated from human platelets designated platelet-derived growth factor (PDGF; Antoniades et al. 1979; Heldin et al. 1979), a growth factor isolated from bovine brain designated fibroblast growth factor (FGF; Gospodarowicz et al. 1978), a growth factor isolated from rat platelets that stimulates the proliferation of mature hepatocytes, designated hepatocyte growth factor (HGF; Nakamura et al. 1986). In addition to EGF, another growth factor that binds selectively to cells expressing EGFR was isolated from virally and chemically transformed cells, suggesting that this growth factor—designated transforming growth factor α—may play a role in oncogenesis by an autocrine mechanism (Roberts et al. 1980, 1982). This discovery provided further support to the earlier finding that transformation by murine and feline sarcoma viruses selectively interferes with EGF binding to EGFR in transformed cells (Todaro et al. 1976). Together with many other studies published since the 1980s, this work showed that growth factors and their receptors play numerous important roles during development and in many normal cellular processes as well as in pathologies such as cancer, diabetes, atherosclerosis, severe bone disorders, and tumor angiogenesis.Visualization of dynamic cellular redistribution of ligand/receptor complexes, and rapid receptor-mediated internalization of growth factors such as insulin or EGF, led to the proposal that cell-surface receptors for these ligands may play a passive role in delivering them to intracellular compartments in which internalized EGF or insulin molecules exert their actions (Vigneri et al. 1978; Podlecki et al. 1986; Jiang and Schindler 1990). In other words, according to this hypothesis, the biological signals induced by insulin or EGF were thought to be mediated by binding of the ligands themselves to intracellular target(s) in the cytoplasm or nucleus, with the role of the cell-surface receptor being to act as a “carrier” that delivers them directly to these targets. An alternative hypothesis was that insulin or EGF activates their cognate receptors at the cell surface, which in turn stimulate the production of an intracellular second messenger molecule analogous to cAMP in signaling by the G-protein-activating β-adrenergic receptor. Indeed, several potential second messengers that are generated in cells on stimulation with insulin or other growth factors were proposed before (and even after) it became clear that insulin receptor, EGFR, and other RTKs are endowed with intrinsic tyrosine kinase activity (Larner et al. 1979; Das 1980; Saltiel and Cuatrecasas 1986).A demonstration that anti-insulin receptor antibodies from the serum of certain diabetic patients could mimic cellular responses of insulin (Flier et al. 1977; Van Obberghen et al. 1979) provided the first conclusive answer to the question of whether the biological activity of growth factors is mediated directly or indirectly through their membrane receptors. This experiment ruled out the possibility that insulin receptor functions as a passive carrier that delivers insulin to an intracellular target to induce cellular responses. Studies showing that intact, bivalent antibodies against the insulin receptor can activate its signaling, whereas monovalent Fab fragments of the same antibodies cannot further argued that ligand-induced receptor dimerization or stimulation of a particular arrangement between two receptor molecules in a dimer can activate the insulin receptor (Kahn et al. 1978).A similar conclusion was reached using certain monoclonal antibodies that bind to the extracellular region of EGFR and block ligand binding (Schreiber et al. 1981). Whereas intact antibodies were able to mimic EGF in stimulating a variety of EGF-like responses including cell proliferation, monovalent Fab fragments of the same monoclonal EGFR antibodies failed to do so—and acted instead as EGFR antagonists (Schreiber et al. 1981, 1983). These experiments provided strong evidence both that EGFR plays a crucial role in mediating EGF-induced cellular responses and that EGFR is activated by ligand-induced receptor dimerization (Schreiber 1981, 1983).     

Retrograde Traffic from the Golgi to the Endoplasmic Reticulum

Proteins to be secreted are transported from the endoplasmic reticulum (ER) to the Golgi apparatus. The transport of these proteins requires the localization and activity of proteins that create ER exit sites, coat proteins to collect cargo and to reshape the membrane into a transport container, and address labels—SNARE proteins—to target the vesicles specifically to the Golgi apparatus. In addition some proteins may need export chaperones or export receptors to enable their exit into transport vesicles. ER export factors, SNAREs, and misfolded Golgi-resident proteins must all be retrieved from the Golgi to the ER again. This retrieval is also part of the organellar homeostasis pathway essential to maintaining the identity of the ER and of the Golgi apparatus. In this review, I will discuss the different processes in retrograde transport from the Golgi to the ER and highlight the mechanistic insights we have obtained in the last couple of years.Proteins that are exposed at the plasma membrane or populate a membrane-bounded organelle are synthesized into the endoplasmic reticulum (ER). In the ER, the folding of these proteins takes place and posttranslational modifications such as N-glycosylation and disulfide bridge formation occur. Upon adopting a suitable, often correct, conformation, proteins destined to locations beyond the ER are concentrated at so-called ER exit sites (ERES) and incorporated into nascent COPII-coated vesicles. These COPII vesicles eventually bud off the ER membrane and are transported to the Golgi (in yeast, Drosophila, and C. elegans) or the ER-Golgi intermediate compartment (in mammalian cells) (Schweizer et al. 1990; Kondylis and Rabouille 2003; Spang 2009; Witte et al. 2011).It is assumed that the vesicle coat is at least partially destabilized through the hydrolysis of GTP by the small GTPase Sar1 (Oka and Nakano 1994; Springer et al. 1999). However, some of the destabilized coat components have to stay on the vesicle until it has reached the Golgi apparatus because coat components participate in the recognition and the tethering process (Barlowe 1997; Cai et al. 2007; Lord et al. 2011; Zong et al. 2012). Subsequently, SNARE proteins on the vesicles (v-SNAREs) zipper up with cognate SNAREs on the Golgi (target SNAREs, t-SNAREs) to drive membrane fusion (Hay et al. 1998; Cao and Barlowe 2000; Parlati et al. 2002). The content of the ER-derived COPII vesicles is thereby released into the lumen of the cis-cisterna of the Golgi apparatus. Most proteins will continue their journey through the Golgi apparatus and encounter further modifications such as extension of the glycosylation tree or lipidation. However, some proteins, especially those involved in the fusion process, i.e., the v-SNAREs or proteins that act as export factors of the ER, such as Vma21, which is essential for export of the correctly folded and assembled V0 sector of the V-ATPase, need to be recycled back to the ER for another round of transport (Ballensiefen et al. 1998; Malkus et al. 2004). Moreover, cis-Golgi proteins are returned to the ER for quality/functional control (Todorow et al. 2000; Sato et al. 2004; Valkova et al. 2011). Finally, some ER-resident proteins, such as the ER Hsp70 chaperone BiP/Kar2, can escape the ER, but are captured at the cis-Golgi by the H/KDEL receptor Erd2 and returned to the ER (Lewis et al. 1990; Semenza et al. 1990; Aoe et al. 1997).Unfortunately, the retrograde transport route is also hijacked by toxins. For example, endocytosed cholera toxin subunit A contains a KDEL sequence and can thereby exploit the system to access the ER (Majoul et al. 1996, 1998). From there, it is retro-translocated into the cytoplasm where it can exert its detrimental function.     

Wnt Signaling in Vertebrate Axis Specification

The Wnt pathway is a major embryonic signaling pathway that controls cell proliferation, cell fate, and body-axis determination in vertebrate embryos. Soon after egg fertilization, Wnt pathway components play a role in microtubule-dependent dorsoventral axis specification. Later in embryogenesis, another conserved function of the pathway is to specify the anteroposterior axis. The dual role of Wnt signaling in Xenopus and zebrafish embryos is regulated at different developmental stages by distinct sets of Wnt target genes. This review highlights recent progress in the discrimination of different signaling branches and the identification of specific pathway targets during vertebrate axial development.Wnt pathways play major roles in cell-fate specification, proliferation and differentiation, cell polarity, and morphogenesis (Clevers 2006; van Amerongen and Nusse 2009). Signaling is initiated in the responding cell by the interaction of Wnt ligands with different receptors and coreceptors, including Frizzled, LRP5/6, ROR1/2, RYK, PTK7, and proteoglycans (Angers and Moon 2009; Kikuchi et al. 2009; MacDonald et al. 2009). Receptor activation is accompanied by the phosphorylation of Dishev-elled (Yanagawa et al. 1995), which appears to transduce the signal to both the cell membrane and the nucleus (Cliffe et al. 2003; Itoh et al. 2005; Bilic et al. 2007). Another common pathway component is β-catenin, an abundant component of adherens junctions (Nelson and Nusse 2004; Grigoryan et al. 2008). In response to signaling, β-catenin associates with T-cell factors (TCFs) and translocates to the nucleus to stimulate Wnt target gene expression (Behrens et al. 1996; Huber et al. 1996; Molenaar et al. 1996).This β-catenin-dependent activation of specific genes is often referred to as the “canonical” pathway. In the absence of Wnt signaling, β-catenin is destroyed by the protein complex that includes Axin, GSK3, and the tumor suppressor APC (Clevers 2006; MacDonald et al. 2009). Wnt proteins, such as Wnt1, Wnt3, and Wnt8, stimulate Frizzled and LRP5/6 receptors to inactivate this β-catenin destruction complex, and, at the same time, trigger the phosphorylation of TCF proteins by homeodomain-interacting protein kinase 2 (HIPK2) (Hikasa et al. 2010; Hikasa and Sokol 2011). Both β-catenin stabilization and the regulation of TCF protein function by phosphorylation appear to represent general strategies that are conserved in multiple systems (Sokol 2011). Thus, the signaling pathway consists of two branches that together regulate target gene expression (Fig. 1).Open in a separate windowFigure 1.Conserved Wnt pathway branches and components. In the absence of Wnt signals, glycogen synthase kinase 3 (GSK3) binds Axin and APC to form the β-catenin destruction complex. Some Wnt proteins, such as Wnt8 and Wnt3a, stimulate Frizzled and LRP5/6 receptors to inhibit GSK3 activity and stabilize β-catenin (β-cat). Stabilized β-cat forms a complex with T-cell factors (e.g., TCF1/LEF1) to activate target genes. Moreover, GSK3 inhibition leads to target gene derepression by promoting TCF3 phosphorylation by homeodomain-interacting protein kinase 2 (HIPK2) through an unknown mechanism, for which β-catenin is required as a scaffold. This phosphorylation results in TCF3 removal from target promoters and gene activation. Other Wnt proteins, such as Wnt5a and Wnt11, use distinct receptors such as ROR2 and RYK, in addition to Frizzled, to control the the cytoskeletal organization through core planar cell polarity (PCP) proteins, small GTPases (Rho/Rac/Cdc42), and c-Jun amino-terminal kinase (JNK).Other Wnt proteins, such as Wnt5a or Wnt11, strongly affect the cytoskeletal organization and morphogenesis without stabilizing β-catenin (Torres et al. 1996; Angers and Moon 2009; Wu and Mlodzik 2009). These “noncanonical” ligands do not influence TCF3 phosphorylation (Hikasa and Sokol 2011), but may use distinct receptors such as ROR1/2 and RYK instead of or in addition to Frizzled (Hikasa et al. 2002; Lu et al. 2004; Mikels and Nusse 2006; Nishita et al. 2006, 2010; Schambony and Wedlich 2007; Grumolato et al. 2010; Lin et al. 2010; Gao et al. 2011). In such cases, signaling mechanisms are likely to include planar cell polarity (PCP) components, such as Vangl2, Flamingo, Prickle, Diversin, Rho GTPases, and c-Jun amino-terminal kinases (JNKs), which do not directly affect β-catenin stability (Fig. 1) (Sokol 2000; Schwarz-Romond et al. 2002; Schambony and Wedlich 2007; Komiya and Habas 2008; Axelrod 2009; Itoh et al. 2009; Tada and Kai 2009; Sato et al. 2010; Gao et al. 2011). This simplistic dichotomy of the Wnt pathway does not preclude some Wnt ligands from using both β-catenin-dependent and -independent routes in a context-specific manner.Despite the existence of many pathway branches, only the β-catenin-dependent branch has been implicated in body-axis specification. Recent experiments in lower vertebrates have identified additional pathway components and targets and provided new insights into the underlying mechanisms.     

Origin of Microglia: Current Concepts and Past Controversies

Microglia are the resident macrophages of the central nervous system (CNS), which sit in close proximity to neural structures and are intimately involved in brain homeostasis. The microglial population also plays fundamental roles during neuronal expansion and differentiation, as well as in the perinatal establishment of synaptic circuits. Any change in the normal brain environment results in microglial activation, which can be detrimental if not appropriately regulated. Aberrant microglial function has been linked to the development of several neurological and psychiatric diseases. However, microglia also possess potent immunoregulatory and regenerative capacities, making them attractive targets for therapeutic manipulation. Such rationale manipulations will, however, require in-depth knowledge of their origins and the molecular mechanisms underlying their homeostasis. Here, we discuss the latest advances in our understanding of the origin, differentiation, and homeostasis of microglial cells and their myelomonocytic relatives in the CNS.Microglia are the resident macrophages of the central nervous system (CNS), which are uniformly distributed throughout the brain and spinal cord with increased densities in neuronal nuclei, including the Substantia nigra in the midbrain (Lawson et al. 1990; Perry 1998). They belong to the nonneuronal glial cell compartment and their function is crucial to maintenance of the CNS in both health and disease (Ransohoff and Perry 2009; Perry et al. 2010; Ransohoff and Cardona 2010; Prinz and Priller 2014).Two key functional features define microglia: immune defense and maintenance of CNS homeostasis. As part of the innate immune system, microglia constantly sample their environment, scanning and surveying for signals of external danger (Davalos et al. 2005; Nimmerjahn et al. 2005; Lehnardt 2010), such as those from invading pathogens, or internal danger signals generated locally by damaged or dying cells (Bessis et al. 2007; Hanisch and Kettenmann 2007). Detection of such signals initiates a program of microglial responses that aim to resolve the injury, protect the CNS from the effects of the inflammation, and support tissue repair and remodeling (Minghetti and Levi 1998; Goldmann and Prinz 2013).Microglia are also emerging as crucial contributors to brain homeostasis through control of neuronal proliferation and differentiation, as well as influencing formation of synaptic connections (Lawson et al. 1990; Perry 1998; Hughes 2012; Blank and Prinz 2013). Recent imaging studies revealed dynamic interactions between microglia and synaptic connections in the healthy brain, which contributed to the modification and elimination of synaptic structures (Perry et al. 2010; Tremblay et al. 2010; Bialas and Stevens 2013). In the prenatal brain, microglia regulate the wiring of forebrain circuits, controlling the growth of dopaminergic axons in the forebrain and the laminar positioning of subsets of neocortical interneurons (Squarzoni et al. 2014). In the postnatal brain, microglia-mediated synaptic pruning is similarly required for the remodeling of neural circuits (Paolicelli et al. 2011; Schafer et al. 2012). In summary, microglia occupy a central position in defense and maintenance of the CNS and, as a consequence, are a key target for the treatment of neurological and psychiatric disorders.Although microglia have been studied for decades, a long history of experimental misinterpretation meant that their true origins remained debated until recently. Although we knew that microglial progenitors invaded the brain rudiment at very early stages of embryonic development (Alliot et al. 1999; Ransohoff and Perry 2009), it has now been established that microglia arise from yolk sac (YS)-primitive macrophages, which persist in the CNS into adulthood (Davalos et al. 2005; Nimmerjahn et al. 2005; Ginhoux et al. 2010, 2013; Kierdorf and Prinz 2013; Kierdorf et al. 2013a). Moreover, early embryonic brain colonization by microglia is conserved across vertebrate species, implying that it is essential for early brain development (Herbomel et al. 2001; Bessis et al. 2007; Hanisch and Kettenmann 2007; Verney et al. 2010; Schlegelmilch et al. 2011; Swinnen et al. 2013). In this review, we will present the latest findings in the field of microglial ontogeny, which provide new insights into their roles in health and disease.     

What Was the Real Contribution of Endosymbionts to the Eukaryotic Nucleus? Insights from Photosynthetic Eukaryotes

Eukaryotic genomes are composed of genes of different evolutionary origins. This is especially true in the case of photosynthetic eukaryotes, which, in addition to typical eukaryotic genes and genes of mitochondrial origin, also contain genes coming from the primary plastids and, in the case of secondary photosynthetic eukaryotes, many genes provided by the nuclei of red or green algal endosymbionts. Phylogenomic analyses have been applied to detect those genes and, in some cases, have led to proposing the existence of cryptic, no longer visible endosymbionts. However, detecting them is a very difficult task because, most often, those genes were acquired a long time ago and their phylogenetic signal has been heavily erased. We revisit here two examples, the putative cryptic endosymbiosis of green algae in diatoms and chromerids and of Chlamydiae in the first photosynthetic eukaryotes. We show that the evidence sustaining them has been largely overestimated, and we insist on the necessity of careful, accurate phylogenetic analyses to obtain reliable results.Today it is widely accepted that photosynthesis originated in eukaryotes by the endosymbiosis of a cyanobacterium within a heterotrophic eukaryotic host. This occurred in a lineage that subsequently diversified to give rise to the three contemporary groups of primary photosynthetic eukaryotes: Viridiplantae (including green algae and land plants), Rhodophyta and Glaucophyta, grouped collectively within a unique eukaryotic superphylum called Archaeplastida (Adl et al. 2005) or Plantae (Cavalier-Smith 1982). Recently, a second case of primary endosymbioses has been unveiled thanks to the characterization of Paulinella chromatophora, a filose amoeba that hosts a cyanobacterium with a reduced genome that has been described as “a plastid in the making” (Marin et al. 2005; Keeling and Archibald 2008; Nowack et al. 2008). Primary endosymbioses resulted in the establishment of plastids with two membranes. However, a vast variety of eukaryotes possess plastids with three or more membranes. They derive from the endosymbioses of primary photosynthetic eukaryotes within other eukaryotic cells (Delwiche 1999; Keeling 2013). Such secondary endosymbioses have spread photosynthesis across the eukaryotic tree, either by the endosymbiosis of red or of green algae. Whereas it is almost certain that secondary endosymbioses of green algae occurred twice (in euglenids and chlorarachniophytes), secondary red algal plastids are found in a variety of alveolates, stramenopiles, cryptophytes, and haptophytes, and the number of red algal endosymbioses at the origin of these groups has been matter of intense debate (Baurain et al. 2010; Keeling 2010, 2013; Burki et al. 2012b). Moreover, the existence of tertiary endosymbioses (namely, the symbiosis of a secondary photosynthetic eukaryote within another eukaryotic cell) and of plastid replacements makes the picture of plastid evolution in eukaryotes even more complex. Dinoflagellates, some of which have replaced their ancestral red algal plastids by green algae, diatoms, haptophytes, or cryptophytes, are paradigmatic examples of such complex situations (Keeling 2013).The evolution of plastids has been studied using genes from the plastid genome as well as typical eukaryotic nuclear genes, which allow inferring the phylogenies of both the plastids and their hosts. The use of those markers has led to interesting discoveries, such as the monophyly of the Archaeplastida (Moreira et al. 2000; Rodríguez-Ezpeleta et al. 2005) or the difficulties in reconciling the plastid and host histories in eukaryotes with red algal plastids (Baurain et al. 2010; Burki et al. 2012b). However, a third class of genes can also provide useful complementary information: the genes of plastid origin retrieved within the nuclear genome of the host. In fact, contemporary plastids have small genomes, which is due to the fact that most of the original cyanobacterial symbiont genes were lost or transferred to the host nucleus (by a process called endosymbiotic gene transfer, EGT) during the evolution of plastids (Weeden 1981; Martin et al. 1998). These transfer events are not restricted to plastid endosymbioses—the same phenomenon occurred during the endosymbiosis that gave rise to the mitochondria (Gray et al. 1999; Burger et al. 2003).EGT genes may serve to study the evolutionary history of plastids and, in particular, the presence of cryptic endosymbioses. In fact, species that had a plastid in the past but lost photosynthesis may have conserved genes of plastid origin in their nuclear genomes. This has been shown for a variety of nonphotosynthetic eukaryotes, such as, for example, apicomplexan parasites (Fast et al. 2001; Roos et al. 2002; Williams and Keeling 2003; Huang et al. 2004), perkinsids (Stelter et al. 2007; Matsuzaki et al. 2008; Fernández Robledo et al. 2011) or nonphotosynthetic dinoflagellates (Sanchez-Puerta et al. 2007; Slamovits and Keeling 2008), and green algae (de Koning and Keeling 2004). Although much more controversial, potential EGTs have also been used to propose a photosynthetic ancestry for ciliates (Reyes-Prieto et al. 2008) or that algae with secondary plastids of red algal origin, such as diatoms and chromerids, may have contained green algal endosymbionts in their past (Moustafa et al. 2009; Woehle et al. 2011). Likewise, several dozens of potential EGTs have been detected in algae and plants that appear to have been acquired from Chlamydiae, a group of parasitic bacteria (Huang and Gogarten 2007; Becker et al. 2008; Moustafa et al. 2008), which led to proposing that cryptic chlamydial endosymbionts may have helped to establish the first plastids, in particular, by providing essential functions for plastid activity (Greub and Raoult 2003; Ball et al. 2013; Baum 2013).We revise here some of these cases of cryptic endosymbiosis, with special attention on the difficulties in accurately detecting EGT and the importance of proper phylogenetic analysis and of an adequate taxonomic sampling to achieve that task.     

Origin and Evolution of the Self-Organizing Cytoskeleton in the Network of Eukaryotic Organelles

The eukaryotic cytoskeleton evolved from prokaryotic cytomotive filaments. Prokaryotic filament systems show bewildering structural and dynamic complexity and, in many aspects, prefigure the self-organizing properties of the eukaryotic cytoskeleton. Here, the dynamic properties of the prokaryotic and eukaryotic cytoskeleton are compared, and how these relate to function and evolution of organellar networks is discussed. The evolution of new aspects of filament dynamics in eukaryotes, including severing and branching, and the advent of molecular motors converted the eukaryotic cytoskeleton into a self-organizing “active gel,” the dynamics of which can only be described with computational models. Advances in modeling and comparative genomics hold promise of a better understanding of the evolution of the self-organizing cytoskeleton in early eukaryotes, and its role in the evolution of novel eukaryotic functions, such as amoeboid motility, mitosis, and ciliary swimming.The eukaryotic cytoskeleton organizes space on the cellular scale and this organization influences almost every process in the cell. Organization depends on the mechanochemical properties of the cytoskeleton that dynamically maintain cell shape, position organelles, and macromolecules by trafficking, and drive locomotion via actin-rich cellular protrusions, ciliary beating, or ciliary gliding. The eukaryotic cytoskeleton is best described as an “active gel,” a cross-linked network of polymers (gel) in which many of the links are active motors that can move the polymers relative to each other (Karsenti et al. 2006). Because prokaryotes have only cytoskeletal polymers but lack motor proteins, this “active gel” property clearly sets the eukaryotic cytoskeleton apart from prokaryotic filament systems.Prokaryotes contain elaborate systems of several cytomotive filaments (Löwe and Amos 2009) that share many structural and dynamic features with eukaryotic actin filaments and microtubules (Löwe and Amos 1998; van den Ent et al. 2001). Prokaryotic cytoskeletal filaments may trace back to the first cells and may have originated as higher-order assemblies of enzymes (Noree et al. 2010; Barry and Gitai 2011). These cytomotive filaments are required for the segregation of low copy number plasmids, cell rigidity and cell-wall synthesis, cell division, and occasionally the organization of membranous organelles (Komeili et al. 2006; Thanbichler and Shapiro 2008; Löwe and Amos 2009). These functions are performed by dynamic filament-forming systems that harness the energy from nucleotide hydrolysis to generate forces either via bending or polymerization (Löwe and Amos 2009; Pilhofer and Jensen 2013). Although the identification of actin and tubulin homologs in prokaryotes is a major breakthrough, we are far from understanding the origin of the structural and dynamic complexity of the eukaryotic cytoskeleton.Advances in genome sequencing and comparative genomics now allow a detailed reconstruction of the cytoskeletal components present in the last common ancestor of eukaryotes. These studies all point to an ancestrally complex cytoskeleton, with several families of motors (Wickstead and Gull 2007; Wickstead et al. 2010) and filament-associated proteins and other regulators in place (Jékely 2003; Richards and Cavalier-Smith 2005; Rivero and Cvrcková 2007; Chalkia et al. 2008; Eme et al. 2009; Fritz-Laylin et al. 2010; Eckert et al. 2011; Hammesfahr and Kollmar 2012). Genomic reconstructions and comparative cell biology of single-celled eukaryotes (Raikov 1994; Cavalier-Smith 2013) allow us to infer the cellular features of the ancestral eukaryote. These analyses indicate that amoeboid motility (Fritz-Laylin et al. 2010; although, see Cavalier-Smith 2013), cilia (Cavalier-Smith 2002; Mitchell 2004; Jékely and Arendt 2006; Satir et al. 2008), centrioles (Carvalho-Santos et al. 2010), phagocytosis (Cavalier-Smith 2002; Jékely 2007; Yutin et al. 2009), a midbody during cell division (Eme et al. 2009), mitosis (Raikov 1994), and meiosis (Ramesh et al. 2005) were all ancestral eukaryotic cellular features. The availability of functional information from organisms other than animals and yeasts (e.g., Chlamydomonas, Tetrahymena, Trypanosoma) also allow more reliable inferences about the ancestral functions of cytoskeletal components (i.e., not only their ancestral presence or absence) and their regulation (Demonchy et al. 2009; Lechtreck et al. 2009; Suryavanshi et al. 2010).The ancestral complexity of the cytoskeleton in eukaryotes leaves a huge gap between prokaryotes and the earliest eukaryote we can reconstruct (provided that our rooting of the tree is correct) (Cavalier-Smith 2013). Nevertheless, we can attempt to infer the series of events that happened along the stem lineage, leading to the last common ancestor of eukaryotes. Meaningful answers will require the use of a combination of gene family history reconstructions (Wickstead and Gull 2007; Wickstead et al. 2010), transition analyses (Cavalier-Smith 2002), and computer simulations relevant to cell evolution (Jékely 2008).     

Clinical and Molecular Features of POLG-Related Mitochondrial Disease

The inability to replicate mitochondrial genomes (mtDNA) by the mitochondrial DNA polymerase (pol γ) leads to a subset of mitochondrial diseases. Many mutations in POLG, the gene that encodes pol γ, have been associated with mitochondrial diseases such as myocerebrohepatopathy spectrum (MCHS) disorders, Alpers-Huttenlocher syndrome, myoclonic epilepsy myopathy sensory ataxia (MEMSA), ataxia neuropathy spectrum (ANS), and progressive external ophthalmoplegia (PEO). This chapter explores five important topics in POLG-related disease: (1) clinical symptoms that identify and distinguish POLG-related diseases, (2) molecular characterization of defects in polymerase activity by POLG disease variants, (3) the importance of holoenzyme formation in disease presentation, (4) the role of pol γ exonuclease activity and mutagenesis in disease and aging, and (5) novel approaches to therapy and avoidance of toxicity based on primary research in pol γ replication.While many polymerases are responsible for replicating billions of nucleotides in the nucleus, DNA polymerase γ replicates thousands of copies of the 16-kilobase mitochondrial genome (mtDNA) in each human cell. The holoenzyme of DNA polymerase γ (pol γ) consists of a catalytic subunit (encoded by POLG at chromosomal locus 15q25) and a dimeric form of its accessory subunit (encoded by POLG2 at chromosomal locus 17q24.1) (Longley et al. 1998b; Lim et al. 1999). MtDNA encodes 13 proteins that are essential for the electron transport chain that provides most of the ATP in the cell. Therefore, mtDNA replication is essential for life as demonstrated by the embryonic lethality of POLG knockout mice (Hance et al. 2005). Studies over the last decade have identified over 200 mutations in POLG that are associated with certain mitochondrial diseases (http://tools.niehs.nih.gov/polg/) (Longley et al. 2005: Copeland 2008; Longley et al. 2010; Walter et al. 2010; Stumpf and Copeland 2011; Tang et al. 2011). POLG-related disorders are currently defined by at least five major phenotypes of neurodegenerative disease that include: (1) Alpers-Huttenlocher syndrome (AHS), (2) childhood myocerebrohepatopathy spectrum (MCHS), (3) myoclonic epilepsy myopathy sensory ataxia (MEMSA), (4) the ataxia neuropathy spectrum (ANS), and (5) progressive external ophthalmoplegia (PEO) with or without sensory ataxic neuropathy and dysarthria (SANDO) (Wong et al. 2008; Cohen et al. 2010; Saneto and Naviaux 2010; Saneto et al. 2010). These diseases associate with mutations that have been shown to decrease the activity of the polymerase, the fidelity of replication, and the formation of the holoenzyme. This work reviews the important conclusions regarding POLG-related diseases and discusses some strategies for disease treatment options.     

Biology of the TAM Receptors

The TAM receptors—Tyro3, Axl, and Mer—comprise a unique family of receptor tyrosine kinases, in that as a group they play no essential role in embryonic development. Instead, they function as homeostatic regulators in adult tissues and organ systems that are subject to continuous challenge and renewal throughout life. Their regulatory roles are prominent in the mature immune, reproductive, hematopoietic, vascular, and nervous systems. The TAMs and their ligands—Gas6 and Protein S—are essential for the efficient phagocytosis of apoptotic cells and membranes in these tissues; and in the immune system, they act as pleiotropic inhibitors of the innate inflammatory response to pathogens. Deficiencies in TAM signaling are thought to contribute to chronic inflammatory and autoimmune disease in humans, and aberrantly elevated TAM signaling is strongly associated with cancer progression, metastasis, and resistance to targeted therapies.The name of the TAM family is derived from the first letter of its three constituents—Tyro3, Axl, and Mer (Prasad et al. 2006). As detailed in Figure 1, members of this receptor tyrosine kinase (RTK) family were independently identified by several different groups and appear in the early literature under multiple alternative names. However, Tyro3, Axl, and Mer (officially c-Mer or MerTK for the protein, Mertk for the gene) have now been adopted as the NCBI designations. The TAMs were first grouped into a distinct RTK family (the Tyro3/7/12 cluster) in 1991, through PCR cloning of their kinase domains (Lai and Lemke 1991). The isolation of full-length cDNAs for Axl (O''Bryan et al. 1991), Mer (Graham et al. 1994), and Tyro3 (Lai et al. 1994) confirmed their segregation into a structurally distinctive family of orphan RTKs (Manning et al. 2002b). The two ligands that bind and activate the TAMs—Gas6 and Protein S (Pros1)—were identified shortly thereafter (Ohashi et al. 1995; Stitt et al. 1995; Mark et al. 1996; Nagata et al. 1996).Open in a separate windowFigure 1.TAM receptors and ligands. The TAM receptors (red) are Tyro3 (Lai and Lemke 1991; Lai et al. 1994)—also designated Brt (Fujimoto and Yamamoto 1994), Dtk (Crosier et al. 1994), Rse (Mark et al. 1994), Sky (Ohashi et al. 1994), and Tif (Dai et al. 1994); Axl (O''Bryan et al. 1991)—also designated Ark (Rescigno et al. 1991), Tyro7 (Lai and Lemke 1991), and Ufo (Janssen et al. 1991); and Mer (Graham et al. 1994)—also designated Eyk (Jia and Hanafusa 1994), Nyk (Ling and Kung 1995), and Tyro12 (Lai and Lemke 1991). The TAMs are widely expressed by cells of the mature immune, nervous, vascular, and reproductive systems. The TAM ligands (blue) are Gas6 and Protein S (Pros1). The carboxy-terminal SHBG domains of the ligands bind to the immunoglobulin (Ig) domains of the receptors, induce dimerization, and activate the TAM tyrosine kinases. When γ-carboxylated in a vitamin-K-dependent reaction, the amino-terminal Gla domains of the dimeric ligands bind to the phospholipid phosphatidylserine expressed on the surface on an apposed apoptotic cell or enveloped virus. See text for details. (From Lemke and Burstyn-Cohen 2010; adapted, with permission, from the authors.)Subsequent progress on elucidating the biological roles of the TAM receptors was considerably slower and ultimately required the derivation of mouse loss-of-function mutants (Camenisch et al. 1999; Lu et al. 1999). The fact that Tyro3^−/−, Axl^−/−, and Mer^−/− mice are all viable and fertile permitted the generation of a complete TAM mutant series that included all possible double mutants and even triple mutants that lack all three receptors (Lu et al. 1999). Remarkably, these Tyro3^−/−Axl^−/−Mer^−/− triple knockouts (TAM TKOs) are viable, and for the first 2–3 wk after birth, superficially indistinguishable from their wild-type counterparts (Lu et al. 1999). Because many RTKs play essential roles in embryonic development, even single loss-of-function mutations in RTK genes often result in an embryonic-lethal phenotype (Gassmann et al. 1995; Lee et al. 1995; Soriano 1997; Arman et al. 1998). The postnatal viability of mice in which an entire RTK family is ablated completely—the TAM TKOs can survive for more than a year (Lu et al. 1999)—is therefore highly unusual. Their viability notwithstanding, the TAM mutants go on to develop a plethora of phenotypes, some of them debilitating (Camenisch et al. 1999; Lu et al. 1999; Lu and Lemke 2001; Scott et al. 2001; Duncan et al. 2003; Prasad et al. 2006). Almost without exception, these phenotypes are degenerative in nature and reflect the loss of TAM signaling activities in adult tissues that are subject to regular challenge, renewal, and remodeling. These activities are the subject of this review.     

Quality Control of Mitochondrial Proteostasis

A decline in mitochondrial activity has been associated with aging and is a hallmark of many neurological diseases. Surveillance mechanisms acting at the molecular, organellar, and cellular level monitor mitochondrial integrity and ensure the maintenance of mitochondrial proteostasis. Here we will review the central role of mitochondrial chaperones and proteases, the cytosolic ubiquitin-proteasome system, and the mitochondrial unfolded response in this interconnected quality control network, highlighting the dual function of some proteases in protein quality control within the organelle and for the regulation of mitochondrial fusion and mitophagy.In all cellular compartments, correct protein folding is critical to maintain cellular homeostasis. In cases where proteins become misfolded or damaged, it is imperative that they are turned over and removed to prevent the formation of toxic folding intermediates or the accumulation of aggregates to levels that can be deleterious for the cell. Several neurodegenerative diseases share a common pathogenic mechanism, which involves the formation of fibrillar aggregates of a particular protein that can accumulate in the cytosol, the nucleus, or the mitochondria. Examples of this include accumulation of the amyloid-β peptide in Alzheimer’s disease (Kayed et al. 2003; Tanzi and Bertram 2005), accumulation of α-synuclein in Parkinson’s disease (Spillantini et al. 1997; Zarranz et al. 2004), and aggregation of a mutant form of the huntingtin protein caused by extended polyglutamine stretches in Huntington’s disease (DiFiglia et al. 1997). Although the exact mechanism of pathogenesis for these diseases remains unresolved, mitochondrial dysfunction is implicated in their progression, which may in turn be responsible for the loss of neurological cell populations because of their sensitivity and requirement for functional mitochondria (Rodolfo et al. 2010).The evolution of mitochondria began approximately 1.5 billion years ago after an α-proteobacterium was engulfed by a preeukaryotic cell (Gray et al. 1999). Since that time, mitochondria have retained two phospholipid bilayers that segregate two aqueous compartments, the mitochondrial intermembrane space (IMS) and the mitochondrial matrix (Palade 1953). Mitochondria are found in essentially all eukaryotic cells and play integral roles in a number of the cell''s metabolic pathways. For example, mitochondria are the key players in cellular ATP production through an elaborate respiratory chain network found in the organelles inner membrane (IM) (Mitchell 1961; Leonard and Schapira 2000). Mitochondria are also required for the β-oxidation of fatty acids, Fe-S biosynthesis, and Ca²⁺ homeostasis (Pinton et al. 1998; Rizzuto et al. 2000; Lill 2009; Modre-Osprian et al. 2009). Moreover, mitochondria are key regulators of programmed cell death and they participate in developmental processes as well as aging (Singh 2004; Green 2005).In contrast to early depictions of mitochondria as singular kidney bean shaped entities, it is now well established that mitochondria form elaborate, reticular networks in many tissues (Bereiter-Hahn 1990). The ability of mitochondria to form such networks arises from two major factors: (1) Specialized machineries in the mitochondrial outer membrane (OM) and the IM allow mitochondria to fuse and divide and (2) mitochondria are able to be shuttled along cytoskeletal elements (Anesti and Scorrano 2006; Hoppins et al. 2007). This plasticity of mitochondria ensures that they are able to respond to different cellular cues, which is potentially important for their numerous functions. In different cell types, mitochondria adopt varying morphologies (Kuznetsov et al. 2009). For example, in cultured fibroblasts mitochondria form extensive reticular networks, whereas in neuronal cells, mitochondria can be found enriched at areas of high-energy demand, including presynaptic termini, axon initial segments, and growth cones. Furthermore, in muscle cells, mitochondria adopt a very uniform intermyofibrillar conformation (Vendelin et al. 2005). The dynamic nature of mitochondria provides an explanation as to how they adopt varying organizations in different cell populations. The importance of mitochondrial networks is highlighted by the fact that mutations in components involved in maintaining mitochondrial dynamics results in neurodegenerative diseases (Chan 2006; Olichon et al. 2006; Knott et al. 2008; Martinelli and Rugarli 2010; Winklhofer and Haass 2010).     

NF-��B in the Immune Response of Drosophila

The nuclear factor κB (NF-κB) pathways play a major role in Drosophila host defense. Two recognition and signaling cascades control this immune response. The Toll pathway is activated by Gram-positive bacteria and by fungi, whereas the immune deficiency (Imd) pathway responds to Gram-negative bacterial infection. The basic mechanisms of recognition of these various types of microbial infections by the adult fly are now globally understood. Even though some elements are missing in the intracellular pathways, numerous proteins and interactions have been identified. In this article, we present a general picture of the immune functions of NF-κB in Drosophila with all the partners involved in recognition and in the signaling cascades.The paramount roles of NF-κB family members in Drosophila development and host defense are now relatively well established and have been the subject of several in-depth reviews in recent years, including some from this laboratory (e.g., Hoffmann 2003; Minakhina and Steward 2006; Ferrandon et al. 2007; Lemaitre and Hoffmann 2007; Aggarwal and Silverman 2008). To avoid excessive duplication, we limit this text to the general picture that has evolved over nearly two decades—since the initial demonstration that the dorsal gene plays a role in dorsoventral patterning in embryogenesis of Drosophila and that it encodes a member of the NF-κB family of inducible transactivators (Nüsslein-Volhard et al. 1980; Steward 1987; Roth et al. 1989). In the early nineties, it became apparent that NF-κB also plays a role in the antimicrobial host defense of Drosophila (Engström et al. 1993; Ip et al. 1993; Kappler et al. 1993; Reichhart et al. 1993). We focus in this article on the immune functions of NF-κB and refer the reader to recent reviews for the roles of NF-κB in development (Roth 2003; Brennan and Anderson 2004; Moussian and Roth 2005; Minakhina and Steward 2006).The Drosophila genome codes for three NF-κB family members (Fig. 1). Dorsal and DIF (for dorsal-related immunity factor) are 70 kDa proteins, with a typical Rel homology domain, which is 45% identical to that of the mammalian counterparts c-Rel, Rel A, and Rel B. Dorsal and DIF lie some 10 kbp apart on the second chromosome and probably arose from a recent duplication (Meng et al. 1999). Both proteins are retained in the cytoplasm by binding to the same 54-kDa inhibitor protein Cactus, which is homologous to mammalian IκBs (Schüpbach and Wieshaus 1989; Geisler et al. 1992). The single Drosophila Cactus gene is closest to mammalian IκBα (Huguet et al. 1997). The third member of the family in Drosophila, Relish, is a 100-kDa protein with an amino-terminal Rel domain and a carboxy-terminal extension with typical ankyrin repeats, as found in Cactus and mammalian IκBs. Relish is similar to mammalian p100 and p105 and its activation requires proteolytic cleavage as in the case for these mammalian counterparts (reviewed in Hultmark 2003).Open in a separate windowFigure 1.The NF-κB and IκB proteins in Drosophila. The length in amino acids is indicated by numbers. REL, Rel-homology domain; NLS, nuclear localization sequence; PEST, proline, glutamic acid, serine, and threonine-rich segment; Ac, acidic domain.Put in simple terms, NF-κB family members function in the host defense of Drosophila to control the expression of genes encoding immune-responsive peptides and proteins. Prominent among the induced genes are those encoding peptides with direct antimicrobial activity. To exert this function, Dorsal and DIF are translocated to the nucleus following stimulus-induced degradation of the inhibitor Cactus, whereas Relish requires stimulus-induced proteolytic cleavage for nuclear translocation of its amino-terminal Rel domain. This paradigm is similar to that observed in mammalian immunity. Again, for the sake of simplicity, we may say that the stimulus-induced degradation of Cactus, and the concomitant release of Dorsal or DIF, is primarily observed during Gram-positive bacterial and fungal infections and mediated by the Toll signaling pathway. In contrast, stimulus-induced proteolytic cleavage of Relish, and concomitant nuclear translocation of its amino-terminal Rel domain, is the hallmark of the response to Gram-negative bacterial infection and mediated by the Imd signaling pathway. Whether these pathways are also involved in the multifaceted defense against viruses remains an open question (Zambon et al. 2005). The Toll pathway was further shown to be involved in hematopoiesis of flies (Qiu et al. 1998). Of note, the Cactus-NF-κB module also plays a central role in the elimination of Plasmodium parasites in infected mosquitoes (Frolet et al. 2006). In the following, we review our information of the two established signaling pathways, Toll and Imd, which lead to gene reprogramming through NF-κB in response to bacterial and fungal infections. We first consider the upstream mechanisms that mediate the recognition of infection and allow for a certain level of discrimination between invading microorganisms. Gene reprogramming in this context is best illustrated by the induction of the antimicrobial peptide genes, which serve as the most convenient readouts of the antimicrobial defense of Drosophila (see Samakovlis et al. 1990; Reichhart et al. 1992; Ferrandon et al. 1998). Flies produce at least seven families of mostly cationic, small-sized, membrane-active peptides, with spectra variously directed against Gram-positive (defensins) and Gram-negative (diptericins, attacins, and drosocin) bacteria, and against fungi (drosomycins and metchnikowins), or with overlapping spectra (cecropins) (reviewed in Bulet et al. 1999; Hetru et al. 2003). The primary site of biosynthesis of these peptides is the fat body, a functional equivalent of the mammalian liver. Blood cells also participate in the production of antimicrobial peptides. As a rule, these molecules are secreted into the hemolymph where they reach remarkably high concentrations to oppose invading microorganisms (Hetru et al. 2003). This facet of the antimicrobial host defense is generally referred to as systemic immune response. Of note, the gut and the tracheae also produce antimicrobial peptides in response to microbes (see Tzou et al. 2000; Onfelt Tingvall et al. 2001; Liehl et al. 2006; Nehme et al. 2007).During infection, the Toll and Imd pathways control the expression of hundreds of genes. In addition to the antimicrobial peptides, these genes encode proteases, putative cytokines, cytoskeletal proteins, and many peptides and proteins whose function in the host defense are still not understood (De Gregorio et al. 2001; Irving et al. 2001).     

Central Role of RET in Thyroid Cancer

RET (rearranged during transfection) is a receptor tyrosine kinase involved in the development of neural crest derived cell lineages, kidney, and male germ cells. Different human cancers, including papillary and medullary thyroid carcinomas, lung adenocarcinomas, and myeloproliferative disorders display gain-of-function mutations in RET. Accordingly, RET protein has become a promising molecular target for cancer treatment.The human RET (rearranged during transfection) gene maps on 10q11.2 and is composed of 21 exons spanning a region of 55,000 bp. It encodes a single-pass trans-membrane protein, RET, that belongs to the receptor tyrosine kinase (RTK) family (Pasini et al. 1995). The RET extracellular segment contains four cadherin-like domains, followed by a domain containing cysteine residues involved in the formation of intramolecular disulfide bonds (Fig. 1A) (Anders et al. 2001; Airaksinen and Saarma 2002). RET protein is highly glycosylated and N-glycosylation is necessary for its transport to the cell surface. Only the fully mature glycosylated 170 kDa RET protein isoform is exposed to the extracellular compartment, whereas the mannose-rich 150 kDa isoform is confined to the Golgi (Takahashi et al. 1993; Carlomagno et al. 1996). The transmembrane segment is composed of 22 amino acids, among which S649 and S653 mediate self-association and dimerization of RET, possibly via formation of inter-molecular hydrogen bonding (Kjaer et al. 2006). The intracellular portion of RET contains the tyrosine kinase domain split into two subdomains by the insertion of 27 amino acids. The RET COOH-terminal tail varies in length as a result of alternative splicing of the 3′ end (carboxy terminal with respect to glycine 1063), generating three different isoforms that contain 9 (RET9), 43 (RET43), or 51 (RET51) amino acids (Myers et al. 1995). RET9 and RET51 are the most abundant isoforms, and they activate similar signaling pathways through interaction with diverse protein complexes, and may exert a differential role in development (Fig. 1A) (de Graaff et al. 2001).Open in a separate windowFigure 1.Illustration of the mechanisms of activation of wild-type (wt) RET and RET-derived oncoproteins. (A) Wild-type RET activation is mediated by ligand (GFL)-induced dimerization; ligand binding to RET is not direct and mediated by GFR-α coreceptors (not shown); major RET autophosphorylation sites and downstream signaling pathways are indicated. RET extracellular cadherin-like domains are represented in red. The split intracellular RET tyrosine kinase domain, as well as the three alternative carboxy-terminal RET tails, are also depicted. (B) RET/PTC activation is mediated by coiled-coil-induced dimerization (left); activation of RET cysteine mutants associated with MEN2A or FMTC is mediated by disulfide bonds-mediated dimerization (right).RET shows several autophosphorylation sites (Fig. 1A) (Liu et al. 1996; Kawamoto et al. 2004). RET tyrosine 1062 (Y1062) functions as a multidocking site for signaling molecules containing a phosphotyrosine-binding (PTB) domain (Asai et al. 1996). Phospho-Y1062 binding proteins include SHC, N-SHC (RAI), FRS2, IRS1/2, DOK1, and DOK4/5 that, in turn, contribute to the activation of RAS-MAPK (mitogen-activated protein kinases) and PI3K (phosphatidyl inositol 3 kinase)-AKT pathways. Y1096, specific to the RET51 splicing variant, couples to the PI3K-AKT and RAS-MAPK pathways, as well. These signaling cascades mediate RET-dependent cell survival, proliferation, and motility (Alberti et al. 1998; Murakami et al. 1999; Segouffin-Cariou and Billaud 2000; Melillo et al. 2001a,b; Schuetz et al. 2004). Y905 is located in the activation loop of the RET kinase and its phosphorylation is associated with RET kinase activation (Knowles et al. 2006). Finally, Y981 and Y1015 have been shown to be coupled to important signaling molecules such as SRC and PLC-γ, respectively (Borrello et al. 1996; Encinas et al. 2004).RET is the receptor for a group of neurotrophic growth factors that belong to the glial cell line-derived neurotrophic factor (GDNF) family (GFLs, GDNF family ligands), namely, GDNF, Neurturin (NRT), Artemin (ART), and Persephin (PSF) (Airaksinen and Saarma 2002). GFLs mediate RET protein dimerization and activation (Fig. 1A). GFLs are presented to RET by GPI (glycosylphosphatidylinositol)-anchored coreceptors, called GFR-α (GDNF family receptor α 1-4). Differential tissue expression dictates the specificity of action displayed by alternative GLF-GFR-α pairs during development and adult life (Baloh et al. 2000; Airaksinen and Saarma 2002).Together with other membrane (DCC and p75NTR) or nuclear (androgen receptor, AR) receptors, RET belongs to the family of so-called “dependence” receptors (Mehlen and Bredesen 2011). In the absence of ligand, RET exerts a proapoptotic activity, that is blocked on ligand stimulation (Bordeaux et al. 2000). Such pro-apoptotic activity is RET kinase-independent and mediated by cleavage of RET cytosolic portion by caspase-3, which, in turn, releases a carboxy-terminal RET peptide that is able to induce cell death (Bordeaux et al. 2000). It is feasible that such activity is important for RET developmental function, because it may control migration of RET-expressing cells by limiting survival of cells that move beyond ligand availability (Bordeaux et al. 2000; Cañibano et al. 2007). Whether modulation of this function is also important for RET-associated diseases is still unknown. However, it is interesting to note that a cancer-associated RET mutant (RET-C634R, see below) does not exert cleavage-dependent proapoptotic effects, whereas RET mutants associated with defective development (Hirschsprung disease, see below) exert strong proapoptotic activity that is refractory to modulation by ligand (Bordeaux et al. 2000).RET is expressed in enteric ganglia, adrenal medulla chromaffin cells, thyroid C cells, sensory and autonomic ganglia of the peripheral nervous system, a subset of central nervous system nuclei, developing kidney and testis germ cells (Manié et al. 2001; de Graaff et al. 2001). RET null mice display impaired development of superior cervical ganglia and enteric nervous system, kidney agenesia, reduction of thyroid C cells, and impaired spermatogenesis (Manié et al. 2001). Accordingly, individuals with germline loss-of-function mutations of RET are affected by intestinal aganglionosis causing congenital megacolon (Hirschsprung disease) (Brooks et al. 2005). RET loss-of-function mutations have also been identified in congenital anomalies of kidney and urinary tract (CAKUT), either isolated or in combination with Hirschsprung disease (Jain 2009).Several genetic alterations convert RET into a dominantly transforming oncogene. This review will describe RET-derived oncogenes that are associated with different types of human neoplasia (Fig. 1B).