Calcium Oscillations

Calcium signaling results from a complex interplay between activation and inactivation of intracellular and extracellular calcium permeable channels. This complexity is obvious from the pattern of calcium signals observed with modest, physiological concentrations of calcium-mobilizing agonists, which typically present as sequential regenerative discharges of stored calcium, a process referred to as calcium oscillations. In this review, we discuss recent advances in understanding the underlying mechanism of calcium oscillations through the power of mathematical modeling. We also summarize recent findings on the role of calcium entry through store-operated channels in sustaining calcium oscillations and in the mechanism by which calcium oscillations couple to downstream effectors.Calcium ions participate in a multiplicity of physiological and pathological functions. Among the most intensely studied, and the major focus of this article, is the role of Ca²⁺ as a cellular signal. Elevations in cytoplasmic Ca²⁺ mediate a plethora of cellular responses, ranging from extremely rapid events (muscle contraction, neurosecretion), to slower more subtle responses (cell division, differentiation, apoptosis). In contrast to most cellular signals, it is a relatively simple matter to observe changes in cytoplasmic Ca²⁺ in real time in living cells. As a result, the truly complex nature of Ca²⁺ signaling pathways has been revealed. The challenge is to understand what regulates these signals and what the biological significance of their complexity is.In the majority of laboratory experiments examining effects of various stimulants on Ca²⁺ signaling, supramaximal concentrations of activating agonists are employed resulting in rapid, robust, and often sustained increases in cytoplasmic Ca²⁺. It has long been appreciated that these signals result from a coordinated release of intracellular stores and increased Ca²⁺ influx across the plasma membrane (Bohr, 1973; Putney et al. 1981). The intracellular release of Ca²⁺ most commonly results from the Ca²⁺ releasing action of the phospholipase C-derived second messenger, inositol 1,4,5-trisphosphate (InsP₃) (Streb et al. 1983), whereas the entry of Ca²⁺ is because of the activation of store-operated channels in the plasma membrane (Putney 1986). However, it is becoming increasingly clear that these large sustained elevations seldom occur with physiological levels of stimulants. Rather the more common pattern of Ca²⁺ signaling, in both excitable and nonexcitable cells is a pattern of periodic discharges and/or entry of Ca²⁺. In excitable cells, such as the heart for example, these may be comprised of, or initiated by regenerative all-or-none plasma membrane channel activation, the Ca²⁺ action potential (Tsien et al. 1986) with amplification by intracellular Ca²⁺ release (Fabiato 1983). In nonexcitable cells, these spikes of cytoplasmic Ca²⁺ arise from regenerative discharge of stored Ca²⁺, a process generally termed Ca²⁺ oscillations (Prince and Berridge 1973; Woods et al. 1986). Like Ca²⁺ action potentials, these all-or-none discharges of Ca²⁺ represent a form of excitable behavior of the intracellular Ca²⁺ release signaling mechanism. However, because it is not possible to easily monitor and control the transmembrane chemical and biophysical parameters, as is the case for excitable plasma membrane behavior, it has been more difficult to fully understand the basic mechanisms by which these Ca²⁺ oscillations arise. Thus, although the question has been exhaustively studied for well over twenty years, there is still uncertainty and controversy over the underlying processes that give rise to Ca²⁺ oscillations. A number of reviews have discussed these issues at some length (Berridge and Galione 1988; Rink and Jacob 1989; Berridge 1990; Petersen and Wakui 1990; Berridge 1991; Cuthbertson and Cobbold 1991; Meyer and Stryer 1991; Hellman et al. 1992; Tepikin and Petersen 1992; Thomas et al. 1992; Dupont and Goldbeter 1993; Keizer 1993; Sneyd et al. 1994; Li et al. 1995; Thomas et al. 1996; Shuttleworth 1999; Lewis 2003; Dupont et al. 2007). In the current treatment, we have chosen to focus on two important aspects of Ca²⁺ oscillations. First, we review the available evidence for various computational models of Ca²⁺ oscillations that employ a quantitative approach to validate or repudiate specific mechanisms. Second, we consider the interrelationship between Ca²⁺ oscillations and plasma membrane Ca²⁺ influx mechanisms, with the view that we may learn more of the physiological function that these intracellular discharges of Ca²⁺ provide.     

Cadherin-mediated Intercellular Adhesion and Signaling Cascades Involving Small GTPases

Epithelia form physical barriers that separate the internal milieu of the body from its external environment. The biogenesis of functional epithelia requires the precise coordination of many cellular processes. One of the key events in epithelial biogenesis is the establishment of cadherin-dependent cell–cell contacts, which initiate morphological changes and the formation of other adhesive structures. Cadherin-mediated adhesions generate intracellular signals that control cytoskeletal reorganization, polarity, and vesicle trafficking. Among such signaling pathways, those involving small GTPases play critical roles in epithelial biogenesis. Assembly of E-cadherin activates several small GTPases and, in turn, the activated small GTPases control the effects of E-cadherin-mediated adhesions on epithelial biogenesis. Here, we focus on small GTPase signaling at E-cadherin-mediated epithelial junctions.Cell–cell adhesions are involved in a diverse range of physiological processes, including morphological changes during tissue development, cell scattering, wound healing, and synaptogenesis (Adams and Nelson 1998; Gumbiner 2000; Halbleib and Nelson 2006; Takeichi 1995; Tepass et al. 2000). In epithelial cells, cell–cell adhesions are classified into three kinds of adhesions: adherens junction, tight junction, and desmosome (for more details, see Meng and Takeichi 2009, Furuse 2009, and Delva et al. 2009, respectively). A key event in epithelial polarization and biogenesis is the establishment of cadherin-dependent cell–cell contacts. Cadherins belong to a large family of adhesion molecules that require Ca²⁺ for their homophilic interactions (Adams and Nelson 1998; Blanpain and Fuchs 2009; Gumbiner 2000; Hartsock and Nelson 2008; Takeichi 1995; Tepass et al. 2000). Cadherins form transinteraction on the surface of neighboring cells (for details, see Shapiro and Weis 2009). For the development of strong and rigid adhesions, cadherins are clustered concomitantly with changes in the organization of the actin cytoskeleton (Tsukita et al. 1992). Classical cadherins are required, but not sufficient, to initiate cell–cell contacts, and other adhesion protein complexes subsequently assemble (for details, see Green et al. 2009). These complexes include the tight junction, which controls paracellular permeability, and desmosomes, which support the structural continuum of epithelial cells. A fundamental problem is to understand how these diverse cellular processes are regulated and coordinated. Intracellular signals, generated when cells attach with one another, mediate these complicated processes.Several signaling pathways upstream or downstream of cadherin-mediated cell–cell adhesions have been identified (Perez-Moreno et al. 2003) (see also McCrea et al. 2009). Among these pathways, small GTPases including the Rho and Ras family GTPases play critical roles in epithelial biogenesis and have been studied extensively. Many key morphological and functional changes are induced when these small GTPases act at epithelial junctions, where they mediate an interplay between cell–cell adhesion molecules and fundamental cellular processes including cytoskeletal activity, polarity, and vesicle trafficking. In addition to these small GTPases, Ca²⁺ signaling and phosphorylation of cadherin complexes also play pivotal roles in the formation and maintenance of cadherin-mediated adhesions. Here, we focus on signaling pathways involving the small GTPases in E-cadherin-mediated cell–cell adhesions. Other signaling pathways are described in recent reviews (Braga 2002; Fukata and Kaibuchi 2001; Goldstein and Macara 2007; McLachlan et al. 2007; Tsukita et al. 2008; Yap and Kovacs 2003; see also McCrea et al. 2009).     

Store-Operated Calcium Channels: New Perspectives on Mechanism and Function

Store-operated calcium channels (SOCs) are a nearly ubiquitous Ca²⁺ entry pathway stimulated by numerous cell surface receptors via the reduction of Ca²⁺ concentration in the ER. The discovery of STIM proteins as ER Ca²⁺ sensors and Orai proteins as structural components of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, a prototypic SOC, opened the floodgates for exploring the molecular mechanism of this pathway and its functions. This review focuses on recent advances made possible by the use of STIM and Orai as molecular tools. I will describe our current understanding of the store-operated Ca²⁺ entry mechanism and its emerging roles in physiology and disease, areas of uncertainty in which further progress is needed, and recent findings that are opening new directions for research in this rapidly growing field.Calcium is a remarkably multifunctional signaling ion, at the heart of diverse biological processes that direct the birth, development, function, and eventual death of cells, tissues, and organisms. Cells use a diverse array of transporters and channels to regulate intracellular Ca²⁺ concentration ([Ca²⁺]_i). A major pathway present in nearly all metazoan cells is the store-operated Ca²⁺ channel (SOC). The defining feature of SOCs, the one that distinguishes them from all other classes of Ca²⁺ channels discussed in this volume, is their activation by the reduction of Ca²⁺ concentration in the lumen of the ER ([Ca²⁺]_ER). Though they were originally described in nonexcitable cells (cells lacking the ability to fire action potentials), they are now known to be present in virtually all cells, including excitable cells like skeletal muscle and neurons (Parekh and Putney 2005).Physiologically, SOCs are most commonly activated by stimuli that release Ca²⁺ from the ER. This generally involves receptors that activate phospholipase C to produce inositol 1,4,5-trisphosphate (IP₃) and activate IP₃ receptors in the ER, but can also result from Ca²⁺-induced Ca²⁺ release through ER/SR ryanodine receptors. The notion that ER Ca²⁺ depletion can control Ca²⁺ entry was first formulated by Jim Putney 25 years ago as the “capacitative calcium entry” hypothesis based on observations that Ca²⁺ entry triggered by muscarinic agonists was more closely linked to the emptiness of the ER store than to IP₃ elevation or occupation of the muscarinic receptor (Putney 1986). The introduction of thapsigargin (TG) (Thastrup et al. 1989), a sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor that depletes ER Ca²⁺ independently of receptors and IP₃, and methods for measuring [Ca²⁺]_i in single cells (fura-2, indo-1, etc.) (Grynkiewicz et al. 1985) provided powerful tools that greatly accelerated progress in establishing store-operated Ca²⁺ entry (SOCE, as it was later renamed) as a ubiquitous Ca²⁺ entry pathway. TG-induced Ca²⁺ entry was soon shown to occur in dozens of cell types (Putney and Bird 1993; Parekh and Penner 1997), though nothing was known about the diversity of pathways that might be involved, let alone their molecular basis.A major step forward was the identification of store-operated Ca²⁺ currents in mast cells and T cells. This achievement arose initially from attempts to identify Ca²⁺ conductances triggered by secretory agonists in mast cells and antigen receptors in T cells (Penner et al. 1988; Lewis and Cahalan 1989). In both cases, extremely small currents were detected and linked to large [Ca²⁺]_i rises, suggesting a high Ca²⁺ selectivity. In T cells, the current was shown to activate spontaneously during whole-cell recordings and in response to the T-cell mitogen phytohemagglutinin in perforated-patch recordings (Lewis and Cahalan 1989). Soon after, fluorescence-based studies showed that Ca²⁺ influx triggered through T cell mitogens shared several features with TG-induced influx, suggesting that T cell receptor agonists activated the store-dependent pathway (Mason et al. 1991; Sarkadi et al. 1991). These two paths of research converged when Hoth and Penner described a highly Ca²⁺-selective current in mast cells that was activated in whole-cell recordings spontaneously (by Ca²⁺ chelators), by IP₃, and by ionomycin, and called it the Ca²⁺ release-activated Ca²⁺ (CRAC) current (Hoth and Penner 1992). In Jurkat T cells, Zweifach and Lewis showed that TG activated a similar current, which appeared to be identical to the mitogen-stimulated current described earlier, and made the first estimate of its characteristically tiny conductance (∼20 femtosiemens, far too small to resolve single-channel currents) (Zweifach and Lewis 1993). These initial studies defined a membrane conductance that over the next decade would be described biophysically and pharmacologically in detail, providing a characteristic “fingerprint” culled from its ion selectivity, unitary conductance, and regulation by intra- and extracellular Ca²⁺ and pharmacological inhibitors (Parekh and Penner 1997; Prakriya et al. 2004).Among several currents that were described as store-operated in different cells, the CRAC current emerged as the prototype because of its extensive characterization and the weight of evidence showing that it could be activated by ER Ca²⁺ depletion independently of surface receptors or changes in cytosolic [Ca²⁺]. This included activation by intracellular Ca²⁺ chelators, SERCA inhibitors or ionomycin at constant intracellular [Ca²⁺]_i, and by the Ca²⁺ chelator TPEN loaded into the ER (Prakriya et al. 2004; Parekh and Putney 2005). In fact, the CRAC channel is the only store-operated channel whose input–output relation is known. This relation, first examined by Hofer et al. (1998) and later quantified by Luik et al. (2008) using an ER-targeted cameleon protein, shows that I_CRAC is a highly nonlinear function of [Ca²⁺]_ER, with a Hill coefficient of ∼4 and a K_1/2 of 170 µM. Given a resting [Ca²⁺]_ER of ∼400 µM, these results suggest that the ER must be depleted by ∼25% before I_CRAC begins to activate significantly.Over the two decades after Putney formalized the capacitative Ca²⁺ entry hypothesis, many mechanisms were proposed as the link between Ca²⁺ store depletion and SOCE. Among these, diffusible messengers released from the ER, insertion of CRAC channels into the plasma membrane, and conformational coupling of CRAC channels with IP₃ receptors in the ER were the most extensively studied, but in the absence of molecular substrates were difficult to establish (Prakriya et al. 2004; Parekh and Putney 2005). The CRAC channel fingerprint proved useful in ruling out a number of candidate genes for the CRAC channel itself, particularly members of the transient receptor potential (TRP) protein family, but its identity remained a mystery (Prakriya et al. 2004; Parekh and Putney 2005). The discoveries of the ER Ca²⁺ sensor STIM1 in 2005 and the CRAC channel protein Orai1 a year later marked an unmistakable turning point in the field, as they provided the first and most essential molecular tools with which to dissect the SOCE mechanism. The history of these discoveries and the early revelations they afforded have been reviewed extensively (Cahalan et al. 2007; Wu et al. 2007; Fahrner et al. 2009; Putney 2009; Várnai et al. 2009; Hogan et al. 2010). In this review, I will summarize our current understanding of how Ca²⁺ store depletion leads to Ca²⁺ entry at a molecular level, and the role of STIM oligomerization and additional proteins in this process. I will also describe how these discoveries and the ensuing studies have increased awareness of SOCE roles in physiology and disease, and have created entirely new directions for research. Throughout I will emphasize work on STIM1 and Orai1 mainly because they have been the most extensively studied isoforms, but will discuss other STIM and Orai isoforms to highlight important functional differences. For more information on these other isoforms, the reader is referred to the reviews cited above.     

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.     

Ca2+ Signaling During Mammalian Fertilization: Requirements,Players, and Adaptations

Changes in the intracellular concentration of calcium ([Ca²⁺]_i) represent a vital signaling mechanism enabling communication among cells and between cells and the environment. The initiation of embryo development depends on a [Ca²⁺]_i increase(s) in the egg, which is generally induced during fertilization. The [Ca²⁺]_i increase signals egg activation, which is the first stage in embryo development, and that consist of biochemical and structural changes that transform eggs into zygotes. The spatiotemporal patterns of [Ca²⁺]_i at fertilization show variability, most likely reflecting adaptations to fertilizing conditions and to the duration of embryonic cell cycles. In mammals, the focus of this review, the fertilization [Ca²⁺]_i signal displays unique properties in that it is initiated after gamete fusion by release of a sperm-derived factor and by periodic and extended [Ca²⁺]_i responses. Here, we will discuss the events of egg activation regulated by increases in [Ca²⁺]_i, the possible downstream targets that effect these egg activation events, and the property and identity of molecules both in sperm and eggs that underpin the initiation and persistence of the [Ca²⁺]_i responses in these species.An increase in the intracellular concentration of calcium ([Ca²⁺]_i) underlies the initiation, progression and/or completion of a wide variety of cellular processes, including fertilization, muscle contraction, secretion, cell division, and apoptosis (Berridge et al. 2000). To survive and proliferate, cells and organisms must communicate, and changes in [Ca²⁺]_i allow them to quickly respond to environmental, nutritional, or ligand challenges with responses that regulate cell fate and function. Cells devote significant amounts of their energy reserves to create and maintain ionic gradients between extracellular and intracellular milieus and also within the latter, thereby allowing brief alterations in these gradients to have profound signaling effects. In the case of Ca²⁺, myriad proteins have acquired the ability to bind Ca²⁺, which allows them to interpret and transform these elevations into cellular functions. This review will examine the cellular modifications induced by [Ca²⁺]_i changes during fertilization in mature mammalian oocytes, henceforth referred to as eggs.Oocytes during maturation ready themselves for fertilization and the initiation of embryogenesis. During this transition, oocytes undergo changes that include the resumption and progression of meiosis, the development of polyspermy-preventing mechanisms, the reorganization of the cytoskeleton with spindle formation and displacement to the cortex, and the translation, accumulation, and degradation of specific mRNAs and proteins involved in development (Horner and Wolfner 2008b). In most species, and in all mammals, a [Ca²⁺]_i signal is responsible for breaking the meiosis-imposed developmental pause, causing egg activation, which is the first stage of embryo development (Whitaker 2006; Horner and Wolfner 2008b). The egg activating [Ca²⁺]_i signal is generally associated with sperm-egg fusion, which occurs at different stages of meiosis depending on the species (Stricker 1999), although in insects, where fertilization is dissociated from activation and where embryos can develop parthenogenetically, the presumed [Ca²⁺]_i increase is thought to be induced by mechanical stimulation during ovulation/oviductal transport (Page and Orr-Weaver 1997; Horner and Wolfner 2008a).The [Ca²⁺]_i responses that underlie egg activation offer a great deal of diversity regarding their spatiotemporal configuration, reflecting both the plasticity of the Ca²⁺ signaling machinery as well as the dissimilar Ca²⁺ requirements for egg activation among species. Generally speaking, species can be categorized either as displaying a single [Ca²⁺]_i increase, which is the case of sea urchins, starfish, frogs, and fish, or showing multiple [Ca²⁺]_i changes, also known as oscillations, which is the case of nemertian worms, ascidians, and mammals (Stricker 1999; Miyazaki and Ito 2006). Elucidation of the signaling cascades and identification of the molecules/receptor(s) that initiate the Ca²⁺ signal at fertilization has proven elusive, and this review will not dwell on that literature; readers are referred to excellent recent reviews on the subject (Whitaker 2006; Parrington et al. 2007). Nonetheless, research has found that Src-family kinases (SFKs) and phospholipase Cγ (PLCγ) are involved in the activation of the phosphoinositide pathway and production of inositol 1,4,5-trisphosphate (IP₃) during fertilization in sea urchins, starfish, and frogs, which reflects the contribution of a plasma membrane receptor/signaling complex (Giusti et al. 1999; Sato et al. 2000). Remarkably, a receptor responsible for recruiting and activating SFKs during fertilization remains undiscovered (Mahbub Hasan et al. 2005). Similarly, it has proved difficult to uncover how the sperm initiates oscillations. Research now suggests that this may be accomplished by a novel mechanism whereby the signaling molecule/cargo, known as the sperm factor (SF), is released by the sperm into the ooplasm after fusion of the gametes. Importantly, the SF is not IP₃ or Ca²⁺ but rather it contains a protein moiety (Swann 1990; Wu et al. 1997; Kyozuka et al. 1998; Harada et al. 2007). To date, only the mammalian SF’s molecular identity has been resolved, and found to be another member of the PLC family, a novel sperm-specific isoform named PLCζ (Saunders et al. 2002). This review will examine the literature on mammalian PLCζs and will focus as well on the egg molecules that are required to initiate and sustain [Ca²⁺]_i oscillations in these species.     

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.     

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).     

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.     

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).     

Functional Differentiation of Adult-Born Neurons along the Septotemporal Axis of the Dentate Gyrus

Over the past several decades, the proliferation and integration of adult-born neurons into existing hippocampal circuitry has been implicated in a wide range of behaviors, including novelty recognition, pattern separation, spatial learning, anxiety behaviors, and antidepressant response. In this review, we suggest that the diversity in behavioral requirements for new neurons may be partly caused by separate functional roles of individual neurogenic niches. Growing evidence shows that the hippocampal formation can be compartmentalized not only along the classic trisynaptic circuit, but also along a longitudinal septotemporal axis. We suggest that subpopulations of hippocampal adult-born neurons may be specialized for distinct mnemonic- or mood-related behavioral tasks. We will examine the literature supporting a functional and anatomical dissociation of the hippocampus along the longitudinal axis and discuss techniques to functionally dissect the roles of adult-born hippocampal neurons in these distinct subregions.Since the presence of dividing cells in the mostly postmitotic adult brain was first described (Altman and Das 1965), the generation of new neurons in adulthood has been proposed to be involved in a variety of behaviors (Doetsch and Hen 2005; Becker and Wojtowicz 2007; Sahay and Hen 2007; Deng et al. 2010; Ming and Song 2011; Miller and Hen 2014). Adult neurogenesis in the healthy mammalian brain is consistently seen in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). Recent studies have implicated hippocampal neurogenesis in learning- and memory-related tasks, such as contextual discrimination and spatial navigation and, specifically, in behavioral pattern separation (Clelland et al. 2009; Sahay et al. 2011; Nakashiba et al. 2012; Niibori et al. 2012; see also reviews in Deng et al. 2010; Ming and Song 2011; Marin-Burgin and Schinder 2012), but also in some behavioral effects of antidepressants (Santarelli et al. 2003; see also reviews in Sahay and Hen 2007; Kheirbek et al. 2012; Tanti and Belzung 2013). However, the exact role of adult hippocampal neurogenesis in some of these behaviors has been debated as some studies have shown no effects of altering adult neurogenesis on spatial navigation or antidepressant response. Proposed explanations have included differences in the behavioral tasks used to measure cognition or emotion, motivational state of subjects, species differences, or in how neurogenesis is defined, either as proliferation, survival, or differentiation (see reviews in Zhao et al. 2008; Aimone et al. 2011; Petrik et al. 2012b; Miller and Hen 2014).It must also be noted, however, that these hippocampal neurons are not born into a singular structure. Work in the past several decades has shown that the hippocampus can be divided, not only along the classic trisynaptic loop, but also longitudinally along a septotemporal axis. The septal (dorsal in rodents; posterior in primates) and temporal (ventral in rodents; anterior in primates) poles, as well as potential intermediate zones of the hippocampus, have different anatomic connections and electrophysiological properties, express a gradient of molecular markers, and play different functional roles, such as performance in spatial learning tasks and stress responses (see reviews in Moser and Moser 1998; Fanselow and Dong 2010). Consequently, adult-born neurons in the hippocampal DG may also be segregated along this longitudinal axis, and conflicting functional roles for neurogenesis may be a result of attempting to examine hippocampal neurogenesis as a unitary phenomenon. It is possible that there are intrinsic, cell-autonomous differences in adult-born neurons generated at opposite poles of the DG. An alternative, although not mutually exclusive, hypothesis is that progenitor cells are initially identical, but differentiate in a dissimilar manner as a result of integration into distinct network circuitry. We will, therefore, first discuss heterogeneity of the hippocampus along its longitudinal axis before reviewing differences in neurogenesis between the septal and temporal poles of the DG. As these topics have been reviewed extensively elsewhere (Moser and Moser 1998; Deng et al. 2010; Fanselow and Dong 2010; Koehl and Abrous 2011; Samuels and Hen 2011; Kheirbek et al. 2012; Petrik et al. 2012b), we will not try to exhaustively cover all the current literature. Rather, we attempt to gather key studies examining a septotemporal gradient of the hippocampus and hippocampal neurogenesis. We will then suggest possible approaches to examine neurogenesis in specific subregions of the hippocampal DG. Finally, a short section will examine segregation of the DG along its transverse axis.     

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.     

CD38/cADPR/Ca2+ Pathway Promotes Cell Proliferation and Delays Nerve Growth Factor-induced Differentiation in PC12 Cells

Intracellular Ca²⁺ mobilization plays an important role in a wide variety of cellular processes, and multiple second messengers are responsible for mediating intracellular Ca²⁺ changes. Here we explored the role of one endogenous Ca²⁺-mobilizing nucleotide, cyclic adenosine diphosphoribose (cADPR), in the proliferation and differentiation of neurosecretory PC12 cells. We found that cADPR induced Ca²⁺ release in PC12 cells and that CD38 is the main ADP-ribosyl cyclase responsible for the acetylcholine (ACh)-induced cADPR production in PC12 cells. In addition, the CD38/cADPR signaling pathway is shown to be required for the ACh-induced Ca²⁺ increase and cell proliferation. Inhibition of the pathway, on the other hand, accelerated nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells. Conversely, overexpression of CD38 increased cell proliferation but delayed NGF-induced differentiation. Our data indicate that cADPR plays a dichotomic role in regulating proliferation and neuronal differentiation of PC12 cells.Mobilization of intracellular Ca²⁺ stores is involved in diverse cell functions, including fertilization, cell proliferation, and differentiation (1–4). At least three endogenous Ca²⁺-mobilizing messengers have been identified, including inositol trisphosphate (IP₃),³ nicotinic adenine acid dinucleotide phosphate (NAADP), and cyclic adenosine diphosphoribose (cADPR). Similar to IP₃, cADPR can mobilize calcium release in a wide variety of cell types and species, from protozoa to animals. The cADPR-mediated Ca²⁺ signaling has been indicated in a variety of cellular processes (5–7), from abscisic acid signaling and regulation of the circadian clock in plants, to mediating long-term synaptic depression in hippocampus.Ample evidence shows that the ryanodine receptors are the main intracellular targets for cADPR (1, 2, 8). Ryanodine receptors (RyRs) are intracellular Ca²⁺ channels widely expressed in various cells and tissues, including muscles and neurons. It is the major cellular mediator of Ca²⁺-induced Ca²⁺ release (CICR) in cells. There are three isoforms of ryanodine receptors: RyR1, RyR2, and RyR3, all of which have been implicated in the cADPR signaling (1, 2, 8). However, evidence regarding cADPR acting directly on the receptors is lacking (9). It has been suggested that accessory proteins, such as calmodulin and FK506-binding protein (FKBP), may be involved instead (10–15).cADPR is formed from nicotinamide adenine dinucleotide (NAD) by ADP-ribosyl cyclases. Six ADP-ribosyl cyclases have been identified so far: Aplysia ADP-ribosyl cyclase, three sea urchin homologues (16, 17), and two mammalian homologues, CD38 and CD157 (18). CD38 is a membrane-bound protein and the main mammalian ADP-ribosyl cyclase. As a novel multifunctional enzyme, CD38 catalyzes the synthesis and hydrolysis of both cADPR and NAADP, two structurally and functionally distinct Ca²⁺ messengers. Virtually all mammalian tissues ever examined have been shown to express CD38. CD38 knock-out mice exhibit multiple physiological defects, ranging from impaired immune responses, metabolic disturbances, to behavioral modifications (1, 6, 18).CD38 was originally identified as a lymphocyte differentiation antigen (18). Indeed, CD38/cADPR has been linked to cell differentiation (5). For example, in human HL-60 cells, CD38 expression and the consequential accumulation of cADPR play a causal role in mediating granulocytic differentiation (19). In addition, expression of CD38 in HeLa and 3T3 cells not only increased intracellular Ca²⁺ concentration but also induced cell proliferation by significantly reducing the S phase duration, leading to shortened cell doubling time (20). The ability of cADPR to increase cell proliferation has also been observed in human T cells (21), human hemopoietic progenitors (22), human peripheral blood mononuclear cells (23), human mesenchymal stem cells (24), and murine mesangial cells (25).The PC12 cell line was derived from rat adrenal medulla and has been used extensively as a neuronal model, since it exhibits many of the functions observed in primary neuronal cultures (26). Most importantly, PC12 cells can be induced by nerve growth factor (NGF) to differentiate into cells with extensive neurite outgrowths, resembling neuronal dendritic trees (26, 27). In contrast to NGF, numerous growth factors and neurotransmitters can induce the proliferation of PC12 cells instead (26). Both IP₃ receptor- and ryanodine receptor-mediated Ca²⁺ stores have been shown to be present in PC12 cells (28–31). The type 2 ryanodine receptor is expressed in PC12 cells and activation of the NO/cGMP pathway in PC12 cells results in calcium mobilization, which is mediated by cADPR and similar to that seen in sea urchin eggs (32). It has been demonstrated that NAADP, another Ca²⁺-mobilizing messenger, is also a potent neuronal differentiation inducer in PC12 cells, while IP₃ exhibits no such role (33, 34). Whether cADPR is involved in the proliferation and differentiation of PC12 cells is unknown.Here we show that activation of the CD38/cADPR/Ca²⁺ signaling is required for the ACh-induced proliferation in PC12 cells, while inhibition of the pathway accelerates NGF-induced neuronal differentiation. Our data indicate that cADPR is important in regulating cell proliferation and neuronal differentiation in PC12 cells.     

A Ca2+ Release-activated Ca2+ (CRAC) Modulatory Domain (CMD) within STIM1 Mediates Fast Ca2+-dependent Inactivation of ORAI1 Channels

STIM1 and ORAI1, the two limiting components in the Ca²⁺ release-activated Ca²⁺ (CRAC) signaling cascade, have been reported to interact upon store depletion, culminating in CRAC current activation. We have recently identified a modulatory domain between amino acids 474 and 485 in the cytosolic part of STIM1 that comprises 7 negatively charged residues. A STIM1 C-terminal fragment lacking this domain exhibits enhanced interaction with ORAI1 and 2–3-fold higher ORAI1/CRAC current densities. Here we focused on the role of this CRAC modulatory domain (CMD) in the fast inactivation of ORAI1/CRAC channels, utilizing the whole-cell patch clamp technique. STIM1 mutants either with C-terminal deletions including CMD or with 7 alanines replacing the negative amino acids within CMD gave rise to ORAI1 currents that displayed significantly reduced or even abolished inactivation when compared with STIM1 mutants with preserved CMD. Consistent results were obtained with cytosolic C-terminal fragments of STIM1, both in ORAI1-expressing HEK 293 cells and in RBL-2H3 mast cells containing endogenous CRAC channels. Inactivation of the latter, however, was much more pronounced than that of ORAI1. The extent of inactivation of ORAI3 channels, which is also considerably more prominent than that of ORAI1, was also substantially reduced by co-expression of STIM1 constructs missing CMD. Regarding the dependence of inactivation on Ca²⁺, a decrease in intracellular Ca²⁺ chelator concentrations promoted ORAI1 current fast inactivation, whereas Ba²⁺ substitution for extracellular Ca²⁺ completely abrogated it. In summary, CMD within the STIM1 cytosolic part provides a negative feedback signal to Ca²⁺ entry by triggering fast Ca²⁺-dependent inactivation of ORAI/CRAC channels.The Ca²⁺ release-activated Ca²⁺ (CRAC)⁵ channel is one of the best characterized store-operated entry pathways (1–7). Substantial efforts have led to identification of two key components of the CRAC channel machinery: the stromal interaction molecule 1 (STIM1), which is located in the endoplasmic reticulum and acts as a Ca²⁺ sensor (8–10), and ORAI1/CRACM1, the pore-forming subunit of the CRAC channel (11–13). Besides ORAI1, two further homologues named ORAI2 and ORAI3 belong to the ORAI channel family (12, 14).STIM1 senses endoplasmic reticulum store depletion primarily by its luminal EF-hand in its N terminus (8, 15), redistributes close to the plasma membrane, where it forms puncta-like structures, and co-clusters with ORAI1, leading to inward Ca²⁺ currents (12, 16–19). The STIM1 C terminus, located in the cytosol, contains two coiled-coil regions overlapping with an ezrin-radixin-moesin (ERM)-like domain followed by a serine/proline- and a lysine-rich region (2, 8, 20–22). Three recent studies have described the essential ORAI-activating region within the ERM domain, termed SOAR (Stim ORAI-activating region) (23), OASF (ORAI-activating small fragment) (24), and CAD (CRAC-activating domain) (25), including the second coiled coil domain and the following ∼55 amino acids. We and others have provided evidence that store depletion leads to a dynamic coupling of STIM1 to ORAI1 (26–28) that is mediated by a direct interaction of the STIM1 C terminus with ORAI1 C terminus probably involving the putative coiled-coil domain in the latter (27).Furthermore, different groups have proven that the C terminus of STIM1 is sufficient to activate CRAC as well as ORAI1 channels independent of store depletion (22–25, 27, 29). We have identified that OASF-(233–474) or shorter fragments exhibit further enhanced coupling to ORAI1 resulting in 3-fold increased constitutive Ca²⁺ currents. A STIM1 fragment containing an additional cluster of anionic amino acids C-terminal to position 474 displays weaker interaction with ORAI1 as well as reduced Ca²⁺ current comparable with that mediated by wild-type STIM1 C terminus. Hence, we have suggested that these 11 amino acids (474–485) act in a modulatory manner onto ORAI1; however, their detailed mechanistic impact within the STIM1/ORAI1 signaling machinery has remained so far unclear.In this study, we focused on the impact of this negative cluster on fast inactivation of STIM1-mediated ORAI Ca²⁺ currents. Lis et al. (30) have shown that all three ORAI homologues display distinct inactivation profiles, where ORAI2 and ORAI3 show a much more pronounced fast inactivation than ORAI1. Moreover, it has been reported (31) that different expression levels of STIM1 to ORAI1 affect the properties of CRAC current inactivation. Yamashita et al. (32) have demonstrated a linkage between the selectivity filter of ORAI1 and its Ca²⁺-dependent fast inactivation. Here we provide evidence that a cluster of acidic residues within the C terminus of STIM1 is involved in the fast inactivation of ORAI1 and further promotes that of ORAI3 and native CRAC currents.     

The Ca2+ Pumps of the Endoplasmic Reticulum and Golgi Apparatus

The various splice variants of the three SERCA- and the two SPCA-pump genes in higher vertebrates encode P-type ATPases of the P_2A group found respectively in the membranes of the endoplasmic reticulum and the secretory pathway. Of these, SERCA2b and SPCA1a represent the housekeeping isoforms. The SERCA2b form is characterized by a luminal carboxy terminus imposing a higher affinity for cytosolic Ca²⁺ compared to the other SERCAs. This is mediated by intramembrane and luminal interactions of this extension with the pump. Other known affinity modulators like phospholamban and sarcolipin decrease the affinity for Ca²⁺. The number of proteins reported to interact with SERCA is rapidly growing. Here, we limit the discussion to those for which the interaction site with the ATPase is specified: HAX-1, calumenin, histidine-rich Ca²⁺-binding protein, and indirectly calreticulin, calnexin, and ERp57. The role of the phylogenetically older and structurally simpler SPCAs as transporters of Ca²⁺, but also of Mn²⁺, is also addressed.All cells invest a considerable part of their total energy budget in active transport to keep up transmembrane (TM) ion gradients (Rolfe and Brown 1997). Prokaryotes already evolved P-type ion-transport ATPases/ion pumps to that aim (Axelsen and Palmgren 1998). The name P-type refers to the transient transfer of the γ-phosphate group of ATP to a highly conserved aspartate group in the enzyme forming a phospho-intermediate. This autophosphorylation is an important step in the pump’s catalytic cycle (Kuhlbrandt 2004). Based on amino-acid sequence comparisons and on the exon/intron layout of the corresponding genes, three types of P-type Ca²⁺ pumps can be discerned in Eumetazoa: the SERCA-, the SPCA-, and the PMCA-type. Whereas ancestral representatives of each type are recognized in some Eubacteria and Archaea, it is also remarkable that some Eukaryotes have apparently lost either SERCA or SPCA pumps. Yeast for instance lacks SERCA pumps whereas plants thrive well without SPCAs (Mills et al. 2008). The SERCA pumps, which are found in the endoplasmic reticulum (ER) or in the sarcoplasmic reticulum (SR) of eukaryotic cells and the evolutionary older secretory pathway ATPases (SPCA) found in the Golgi apparatus, are closely related to each other and together belong to the P_2A subfamily. They form the topic of this review. The plasma-membrane Ca²⁺-pumps (PMCA), on the other hand, appear to be phylogenetically the oldest of the three and form the P_2B-subfamily branch. PMCAs are addressed in an article by Brini and Carafoli (2009). Some further information on the evolution of the three types of ATPases was recently reviewed by Palmgren and Axelsen (1998) and Vangheluwe et al. (2009). Of the three families, only SERCA pumps translocate two Ca²⁺ ions and hydrolyze one ATP for each catalytic turnover. They possess two Ca²⁺-transport sites: site I and site II; the numbers specify the sequence of filling of the respective sites. The single Ca²⁺-binding site of the SPCA and PMCA pumps structurally corresponds to site II of SERCA (Toyoshima 2009).     

Self-avoidance and Tiling: Mechanisms of Dendrite and Axon Spacing

The spatial pattern of branches within axonal or dendritic arbors and the relative arrangement of neighboring arbors with respect to one another impact a neuron''s potential connectivity. Although arbors can adopt diverse branching patterns to suit their functions, evenly spread branches that avoid clumping or overlap are a common feature of many axonal and dendritic arbors. The degree of overlap between neighboring arbors innervating a surface is also characteristic within particular neuron types. The arbors of some populations of neurons innervate a target with a comprehensive and nonoverlapping “tiled” arrangement, whereas those of others show substantial territory overlap. This review focuses on cellular and molecular studies that have provided insight into the regulation of spatial arrangements of neurite branches within and between arbors. These studies have revealed principles that govern arbor arrangements in dendrites and axons in both vertebrates and invertebrates. Diverse molecular mechanisms controlling the spatial patterning of sister branches and neighboring arbors have begun to be elucidated.Axonal and dendritic arbors adopt complex and morphologically diverse shapes that influence neural connectivity and information processing. In this article we review anatomical and molecular studies that elucidate how the arrangements of branches within neuronal arbors are established during development (isoneuronal spacing) and how the relative spacing of arbors is determined when multiple neurons together innervate a defined territory (heteroneuronal spacing). Together these mechanisms ensure that arbors achieve functionally appropriate coverage of input or output territories.Isoneuronal and heteroneuronal processes display a variety of spacing arrangements, suggesting a diversity of underlying molecular mechanisms. Self-avoidance can occur between branches that arise from a single soma (Yau 1976; Kramer and Kuwada 1983; Kramer and Stent 1985), implying that neurons are able to discriminate “self,” which they avoid, from “nonself” arbors, with which they coexist (Kramer and Kuwada 1983). Similarly, arbors from different cells that share the same function and together innervate a defined territory can create a pattern of minimally overlapping neighboring dendritic or axonal fields, known as tiling. Such spacing mechanisms ensure that arbors maximize their spread across a territory while minimizing the redundancy with which the territory is innervated. In contrast, adhesive interactions between arbors can operate to maintain coherence of dendrites at specific targets (Zhu and Luo 2004), or to bundle functionally similar processes and possibly coordinate their activity (Campbell et al. 2009). Understanding how processes are patterned relative to one another can help to uncover the functional logic of neural circuit organization.Here we focus primarily on mechanisms of isoneuronal and heteroneuronal avoidance that result in complete and nonredundant innervation of sensory or synaptic space. Such mechanisms have been studied extensively in systems where neuronal arbors innervate a two-dimensional plane, such as the retina or body wall (Wassle et al. 1981; Perry and Linden 1982; Hitchcock 1989; Lin and Masland 2004; Fuerst et al. 2009; Kramer and Stent 1985; Grueber et al. 2003; Sugimura et al. 2003; Sagasti et al. 2005). However, the principles regulating process spacing in these regions likely also apply in three dimensions, most prominently where processes are segregated into nonoverlapping domains or columns (Huckfeldt et al. 2009). It is also notable that nonneuronal cell types might similarly engage in self-avoidance and form tiling arrangements, including leech comb cells (Jellies and Kristan 1991) and mammalian astrocytes (Bushong et al. 2002; Ogata and Kosaka 2002; Livet et al. 2007). Elucidating the mechanisms of process spacing during development is therefore relevant for understanding principles of tissue organization inside and outside of the nervous system.