Age Structure,Changing Demography and Effective Population Size in Atlantic Salmon (Salmo salar)

Effective population size (N_e) is a central evolutionary concept, but its genetic estimation can be significantly complicated by age structure. Here we investigate N_e in Atlantic salmon (Salmo salar) populations that have undergone changes in demography and population dynamics, applying four different genetic estimators. For this purpose we use genetic data (14 microsatellite markers) from archived scale samples collected between 1951 and 2004. Through life table simulations we assess the genetic consequences of life history variation on N_e. Although variation in reproductive contribution by mature parr affects age structure, we find that its effect on N_e estimation may be relatively minor. A comparison of estimator models suggests that even low iteroparity may upwardly bias N_e estimates when ignored (semelparity assumed) and should thus empirically be accounted for. Our results indicate that N_e may have changed over time in relatively small populations, but otherwise remained stable. Our ability to detect changes in N_e in larger populations was, however, likely hindered by sampling limitations. An evaluation of N_e estimates in a demographic context suggests that life history diversity, density-dependent factors, and metapopulation dynamics may all affect the genetic stability of these populations.THE effective size of a population (N_e) is an evolutionary parameter that can be informative on the strength of stochastic evolutionary processes, the relevance of which relative to deterministic forces has been debated for decades (e.g., Lande 1988). Stochastic forces include environmental, demographic, and genetic components, the latter two of which are thought to be more prominent at reduced population size, with potentially detrimental consequences for average individual fitness and population persistence (Newman and Pilson 1997; Saccheri et al. 1998; Frankham 2005). The quantification of N_e in conservation programs is thus frequently advocated (e.g., Luikart and Cornuet 1998; Schwartz et al. 2007), although gene flow deserves equal consideration given its countering effects on genetic stochasticity (Frankham et al. 2003; Palstra and Ruzzante 2008).Effective population size is determined mainly by the lifetime reproductive success of individuals in a population (Wright 1938; Felsenstein 1971). Variance in reproductive success, sex ratio, and population size fluctuations can reduce N_e below census population size (Frankham 1995). Given the difficulty in directly estimating N_e through quantification of these demographic factors (reviewed by Caballero 1994), efforts have been directed at inferring N_e indirectly through measurement of its genetic consequences (see Leberg 2005, Wang 2005, and Palstra and Ruzzante 2008 for reviews). Studies employing this approach have quantified historical levels of genetic diversity and genetic threats to population persistence (e.g., Nielsen et al. 1999b; Miller and Waits 2003; Johnson et al. 2004). N_e has been extensively studied in (commercially important) fish species, due to the common availability of collections of archived samples that facilitate genetic estimation using the temporal method (e.g., Hauser et al. 2002; Shrimpton and Heath 2003; Gomez-Uchida and Banks 2006; Saillant and Gold 2006).Most models relating N_e to a population''s genetic behavior make simplifying assumptions regarding population dynamics. Chiefly among these is the assumption of discrete generations, frequently violated in practice given that most natural populations are age structured with overlapping generations. Here, theoretical predictions still apply, provided that population size and age structure are constant (Felsenstein 1971; Hill 1972). Ignored age structure can introduce bias into temporal genetic methods for the estimation of N_e, especially for samples separated by time spans that are short relative to generation interval (Jorde and Ryman 1995; Waples and Yokota 2007; Palstra and Ruzzante 2008). Moreover, estimation methods that do account for age structure (e.g., Jorde and Ryman 1995) still assume this structure to be constant. Population dynamics will, however, likely be altered as population size changes, thus making precise quantifications of the genetic consequences of acute population declines difficult (Nunney 1993; Engen et al. 2005; Waples and Yokota 2007). This problem may be particularly relevant when declines are driven by anthropogenic impacts, such as selective harvesting regimes, that can affect age structure and N_e simultaneously (Ryman et al. 1981; Allendorf et al. 2008). Demographic changes thus have broad conservation implications, as they can affect a population''s sensitivity to environmental stochasticity and years of poor recruitment (Warner and Chesson 1985; Ellner and Hairston 1994; Gaggiotti and Vetter 1999). Consequently, although there is an urgent need to elucidate the genetic consequences of population declines, relatively little is understood about the behavior of N_e when population dynamics change (but see Engen et al. 2005, 2007).Here we focus on age structure and N_e in Atlantic salmon (Salmo salar) river populations in Newfoundland and Labrador. The freshwater habitat in this part of the species'' distribution range is relatively pristine (Parrish et al. 1998), yet Atlantic salmon in this area have experienced demographic declines, associated with a commercial marine fishery, characterized by high exploitation rates (40–80% of anadromous runs; Dempson et al. 2001). A fishery moratorium was declared in 1992, with rivers displaying differential recovery patterns since then (Dempson et al. 2004b), suggesting a geographically variable impact of deterministic and stochastic factors, possibly including genetics. An evaluation of those genetic consequences thus requires accounting for potential changes in population dynamics as well as in life history. Life history in Atlantic salmon can be highly versatile (Fleming 1996; Hutchings and Jones 1998; Fleming and Reynolds 2004), as exemplified by the high variation in age-at-maturity displayed among and within populations (Hutchings and Jones 1998), partly reflecting high phenotypic plasticity (Hutchings 2004). This diversity is particularly evident in the reproductive biology of males, which can mature as parr during juvenile freshwater stages (Jones and King 1952; Fleming and Reynolds 2004) and/or at various ages as anadromous individuals, when returning to spawn in freshwater from ocean migration. Variability in life history strategies is further augmented by iteroparity, which can be viewed as a bet-hedging strategy to deal with environmental uncertainty (e.g., Orzack and Tuljapurkar 1989; Fleming and Reynolds 2004). Life history diversity and plasticity may allow salmonid fish populations to alter and optimize their life history under changing demography and population dynamics, potentially acting to stabilize N_e. Reduced variance in individual reproductive success at low breeder abundance (genetic compensation) will achieve similar effects and might be a realistic aspect of salmonid breeding systems (Ardren and Kapuscinski 2003; Fraser et al. 2007b). Little is currently known about the relationships between life history plasticity, demographic change and N_e, partly due to scarcity of the multivariate data required for these analyses.Our objective in this article is twofold. First, we use demographic data for rivers in Newfoundland to quantify how life history variation influences age structure in Atlantic salmon and hence N_e and its empirical estimation from genetic data. We find that variation in reproductive contribution by mature parr has a much smaller effect on the estimation of N_e than is often assumed. Second, we use temporal genetic data to estimate N_e and quantify the genetic consequences of demographic changes. We attempt to account for potential sources of bias, associated with (changes in) age structure and life history, by using four different analytical models to estimate N_e: a single-sample estimator using the linkage disequilibrium method (Hill 1981), the temporal model assuming discrete generations (Nei and Tajima 1981; Waples 1989), and two temporal models for species with overlapping generations (Waples 1990a,b; Jorde and Ryman 1995) that differ principally in assumptions regarding iteroparity. A comparison of results from these different estimators suggests that iteroparity may often warrant analytical consideration, even when it is presumably low. Although sometimes limited by statistical power, a quantification and comparison of temporal changes in N_e among river populations suggests a more prominent impact of demographic changes on N_e in relatively small river populations.     

Genetic draft and quasi-neutrality in large facultatively sexual populations

Large populations may contain numerous simultaneously segregating polymorphisms subject to natural selection. Since selection acts on individuals whose fitness depends on many loci, different loci affect each other’s dynamics. This leads to stochastic fluctuations of allele frequencies above and beyond genetic drift—an effect known as genetic draft. Since recombination disrupts associations between alleles, draft is strong when recombination is rare. Here, we study a facultatively outcrossing population in a regime where the frequency of outcrossing and recombination, r, is small compared to the characteristic scale of fitness differences σ. In this regime, fit genotypes expand clonally, leading to large fluctuations in the number of recombinant offspring genotypes. The power law tail in the distribution of the latter makes it impossible to capture the dynamics of draft by an effective neutral model. Instead, we find that the fixation time of a neutral allele increases only slowly with the population size but depends sensitively on the ratio r/σ. The efficacy of selection is reduced dramatically and alleles behave “quasi-neutrally” even for Ns≫1, provided that |s| < s_c, where s_c depends strongly on r/σ, but only weakly on population size N. In addition, the anomalous fluctuations due to draft change the spectrum of (quasi)-neutral alleles from f(ν) ∼ ν⁻¹, corresponding to drift, to ∼ ν⁻². Finally, draft accelerates the rate of two-step adaptations through deleterious intermediates.THE genetic diversity of a population is determined by mutation, selection, recombination, and genetic drift, i.e., the stochasticity inherent in reproduction. Understanding how genetic diversity depends on these elements of evolutionary dynamics is central to population genetics, since it allows us to make inferences about the past history and to predict how rapidly populations can adapt.Population genetic inference focuses on the diversity at putatively neutral sites and assumes that the history of these sites is described by the neutral “coalescent” (Kingman 1982). Coalescent theory models the genealogy of asexual organisms or nonrecombining segments of a genome by positing that lineages merge at random, backward in time, due to common ancestry. Under this assumption, the mean time to the most recent common ancestor, T_C, of the extant N individuals, is 2N generations. The coalescence timescale is very important, since the genetic diversity of the population is given by the number of mutations that occur in all lineages descending from the most recent common ancestor of the population. Genetic diversity is therefore controlled by T_C and hence, under the assumption of neutral evolution, proportional to N. [Coalescent theory has been extended to weak selection (Krone and Neuhauser 1997) and recombination (Hudson 1983; Griffiths and Marjoram 1996).]However, the prediction that neutral genetic diversity is proportional to N is at odds with observations: Population sizes of different organisms differ by many orders of magnitude, while genetic variation among organisms is fairly constant (Lewontin 1974). To resolve this “paradox of variation”, Maynard Smith and Haigh (1974) suggested that selection acting on linked loci might reduce diversity at a neutral locus. Rapid fixation of a novel mutation at a linked locus will perturb the allele frequencies. These perturbations can bring alleles to fixation and, more generally, reduce the coalescence time and hence the average genetic diversity (Kaplan et al. 1989; Barton 1998; Gillespie 2001). Such “hitchhiking” of neutral alleles on linked selected loci will dominate over genetic drift in large populations. Since hitchhiking leads to larger perturbations for more closely linked loci, one expects genetic variation to correlate with the recombination rate, as is indeed observed in Drosophila (Begun and Aquadro 1992; Stephan and Mitchell 1992; Andolfatto and Przeworski 2001; Sella et al. 2009).A related effect was described earlier by Hill and Robertson (1966), who studied the reduction in the fixation probability of a novel beneficial mutation because of selection acting at a linked locus. This effect is commonly known as Hill–Robertson interference (Felsenstein 1974). Hitchhiking and Hill–Robertson interference are different aspects of the same phenomenon, one focusing on the effects of linked selection on genetic diversity and the other on the efficacy of selection. While hitchhiking and Hill–Robertson effects are primarily associated with positive selection for novel alleles, purifying selection against deleterious mutations also affects genetic diversity. The elimination of (neutral) alleles linked deleterious mutations is known as background selection. The lower the recombination rates are, the larger is the target for linked deleterious mutations, resulting in stronger background selection (Charlesworth et al. 1993; Hudson and Kaplan 1995; Nordborg et al. 1996).Most models used to study Hill–Robertson and hitchhiking effects between positively selected mutations consider only two loci. Deleterious mutations, however, are expected to be much more frequent, and background selection models typically consider many mutations with small deleterious effects. A systematic study of the effect of interference between many weakly selected sites in a mutation/selection/drift equilibrium was presented by McVean and Charlesworth (2000), who used computer simulations of a model of codon bias evolution. They showed that linkage-dependent interference between a large number of weakly selected sites has substantial effects on genetic diversity, fixation probability of mutations, and the degree of adaptation measured as the frequency of preferred codons. This and subsequent computational studies reinforced the understanding that the Hill–Roberson effect reduces the effectiveness of selection and made clear that a quantitative understanding of Hill–Robertson effects in multilocus systems requires an analysis that goes beyond two-locus models (Comeron and Kreitman 2002; Seger et al. 2010); see Comeron et al. (2008) for a recent review.It is common to interpret the effect of linked selection in terms of increased variance in offspring number. In this interpretation, linked selection can be thought of as a stochastic force analogous to genetic drift and is often referred to as genetic draft—a term coined by Gillespie (2000). Following Hill and Robertson (1966) and Felsenstein (1974), the increased variance in offspring number is often captured by a reduction in the “effective population” size, N_e, in a neutral model (which means enhanced drift and accelerated coalescence). It has, however, been noted that a rescaled neutral model does not consistently explain all observables (Charlesworth et al. 1993; Braverman et al. 1995; Fay and Wu 2000; McVean and Charlesworth 2000; Barton and Etheridge 2004; Seger et al. 2010) and that different effective population sizes need to be defined depending on the question and timescale of interest (Ewens 2004; Karasov et al. 2010). Furthermore, the dependence of N_e on the actual population size and other relevant parameters is not understood (Wiehe and Stephan 1993; Gillespie 2000; Lynch 2007).Here, we provide analytic results on the effect of draft in a stochastic multilocus evolution model. Instead of a mutation/selection equilibrium considered in McVean and Charlesworth (2000), our focus here is a continuously adapting and facultatively sexual population, like human immunodeficiency virus (HIV) in coevolution with the host’s immune system. Our model and its relation to the biology of HIV are described in more detail below. The scope of the model, however, extends beyond HIV and is equally applicable to scenarios where background selection is dominant. Many important and well-studied organisms such as influenza, yeast, and plants are facultatively sexual. Rice, for example, is a partly selfing species and strong selection has acted during its domestication (Caicedo et al. 2007). While dominance effects can render the selfing of diploid organisms more complicated than facultatively sexual propagation of haploid organisms (Charlesworth et al. 1991; Kelly and Williamson 2000), our analysis still provides a null model on top of which dominance effects can be investigated.Using computer simulations of an adapting population, we first demonstrate how quantities such as the coalescence time, the fixation probability of beneficial or deleterious mutations, and the allele frequency spectra depend on the rate of outcrossing relative to selection. We also show that our in silico observations cannot be described by a neutral model with a reduced effective population size. This is because single genotypes can, through clonal expansion, give rise to a wildly fluctuating number of recombinant genotypes. The distribution is so broad that its variance diverges, which in turn makes an effectively neutral diffusion limit impossible. To provide an analytic understanding of the simulation results, we use a branching process model that allows us to study the stochastic dynamics of novel mutations (neutral, beneficial, and deleterious) as they spread through the population. We calculate fixation probabilities and the typical time to fixation, T_fix (and more generally, the probability of attaining n copies after time T), for a new mutant allele, making explicit the dependence on the rate of recombination, fitness variance, and the population size. An important consequence of genetic draft is a qualitatively different frequency spectrum of rare alleles, which we also calculate analytically. Finally, we show that empirical HIV allele frequency spectra are in agreement with our theoretical prediction, confirming the relevance of our model to the dynamics of HIV adaptation.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.^{1, 2, 3, 4} Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29} As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.^{16, 17, 22, 25} Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations^{30, 31} and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.^{17, 22} Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.^{22, 32} Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.²⁵Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.^{27, 33} Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.^{27, 34} Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36} Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.^{37, 38} The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.³⁹ The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury.     

Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts

Transforming growth factor-β₁ (TGF-β₁) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β₁ may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β₁-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β₁ to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β₁ promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β₁ in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β₁-induced autophagy is required for the fibrogenic response in hATMyofbs.Interstitial fibrosis is common to many cardiovascular disease etiologies including myocardial infarction (MI),¹ diabetic cardiomyopathy² and hypertension.³ Fibrosis may arise due to maladaptive cardiac remodeling following injury and is a complex process resulting from activation of signaling pathways, such as TGF-β₁.⁴ TGF-β₁ signaling has broad-ranging effects that may affect cell growth, differentiation and the production of extracellular matrix (ECM) proteins.^{5, 6} Elevated TGF-β₁ is observed in post-MI rat heart⁷ and is associated with fibroblast-to-myofibroblast phenoconversion and concomitant activation of canonical Smad signaling.⁸ The result is a proliferation of myofibroblasts, which then leads to inappropriate deposition of fibrillar collagens, impaired cardiac function and, ultimately, heart failure.^{9, 10}Autophagy is necessary for cellular homeostasis and is involved in organelle and protein turnover.^{11, 12, 13, 14} Autophagy aids in cell survival by providing primary materials, for example, amino acids and fatty acids for anabolic pathways during starvation conditions.^{15, 16} Alternatively, autophagy may be associated with apoptosis through autodigestive cellular processes, cellular infection with pathogens or extracellular stimuli.^{17, 18, 19, 20} The overall control of cardiac fibrosis is likely due to the complex functioning of an array of regulatory factors, but to date, there is little evidence linking autophagy with fibrogenesis in cardiac tissue.^{11, 12, 13, 14, 15, 16, 17, 18, 21, 22}Recent studies have demonstrated that TGF-β₁ may not only promote autophagy in mouse fibroblasts and human tubular epithelial kidney cells^{15, 23, 24} but can also inhibit this process in fibroblasts extracted from human patients with idiopathic pulmonary fibrosis.²⁵ Moreover, it has recently been reported that autophagy can negatively¹⁵ and positively^{25, 26, 27} regulate the fibrotic process in different model cell systems. In this study, we have explored the putative link between autophagy and TGF-β₁-induced fibrogenesis in human atrial myofibroblasts (hATMyofbs) and in a model of MI rat heart.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

Characterization of the Possible Roles for B Class MADS Box Genes in Regulation of Perianth Formation in Orchid

To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, three paleoAPETALA3 genes, O. Gower Ramsey MADS box gene5 (OMADS5; clade 1), OMADS3 (clade 2), and OMADS9 (clade 3), and one PISTILLATA gene, OMADS8, were characterized. The OMADS8 and OMADS3 mRNAs were expressed in all four floral organs as well as in vegetative leaves. The OMADS9 mRNA was only strongly detected in petals and lips. The mRNA for OMADS5 was only strongly detected in sepals and petals and was significantly down-regulated in lip-like petals and lip-like sepals of peloric mutant flowers. This result revealed a possible negative role for OMADS5 in regulating lip formation. Yeast two-hybrid analysis indicated that OMADS5 formed homodimers and heterodimers with OMADS3 and OMADS9. OMADS8 only formed heterodimers with OMADS3, whereas OMADS3 and OMADS9 formed homodimers and heterodimers with each other. We proposed that sepal/petal/lip formation needs the presence of OMADS3/8 and/or OMADS9. The determination of the final organ identity for the sepal/petal/lip likely depended on the presence or absence of OMADS5. The presence of OMADS5 caused short sepal/petal formation. When OMADS5 was absent, cells could proliferate, resulting in the possible formation of large lips and the conversion of the sepal/petal into lips in peloric mutants. Further analysis indicated that only ectopic expression of OMADS8 but not OMADS5/9 caused the conversion of the sepal into an expanded petal-like structure in transgenic Arabidopsis (Arabidopsis thaliana) plants.The ABCDE model predicts the formation of any flower organ by the interaction of five classes of homeotic genes in plants (Yanofsky et al., 1990; Jack et al., 1992; Mandel et al., 1992; Goto and Meyerowitz, 1994; Jofuku et al., 1994; Pelaz et al., 2000, 2001; Theißen and Saedler, 2001; Pinyopich et al., 2003; Ditta et al., 2004; Jack, 2004). The A class genes control sepal formation. The A, B, and E class genes work together to regulate petal formation. The B, C, and E class genes control stamen formation. The C and E class genes work to regulate carpel formation, whereas the D class gene is involved in ovule development. MADS box genes seem to have a central role in flower development, because most ABCDE genes encode MADS box proteins (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994; Purugganan et al., 1995; Rounsley et al., 1995; Theißen and Saedler, 1995; Theißen et al., 2000; Theißen, 2001).The function of B group genes, such as APETALA3 (AP3) and PISTILLATA (PI), has been thought to have a major role in specifying petal and stamen development (Jack et al., 1992; Goto and Meyerowitz, 1994; Krizek and Meyerowitz, 1996; Kramer et al., 1998; Hernandez-Hernandez et al., 2007; Kanno et al., 2007; Whipple et al., 2007; Irish, 2009). In Arabidopsis (Arabidopsis thaliana), mutation in AP3 or PI caused identical phenotypes of second whorl petal conversion into a sepal structure and third flower whorl stamen into a carpel structure (Bowman et al., 1989; Jack et al., 1992; Goto and Meyerowitz, 1994). Similar homeotic conversions for petal and stamen were observed in the mutants of the AP3 and PI orthologs from a number of core eudicots such as Antirrhinum majus, Petunia hybrida, Gerbera hybrida, Solanum lycopersicum, and Nicotiana benthamiana (Sommer et al., 1990; Tröbner et al., 1992; Angenent et al., 1993; van der Krol et al., 1993; Yu et al., 1999; Liu et al., 2004; Vandenbussche et al., 2004; de Martino et al., 2006), from basal eudicot species such as Papaver somniferum and Aquilegia vulgaris (Drea et al., 2007; Kramer et al., 2007), as well as from monocot species such as Zea mays and Oryza sativa (Ambrose et al., 2000; Nagasawa et al., 2003; Prasad and Vijayraghavan, 2003; Yadav et al., 2007; Yao et al., 2008). This indicated that the function of the B class genes AP3 and PI is highly conserved during evolution.It has been thought that B group genes may have arisen from an ancestral gene through multiple gene duplication events (Doyle, 1994; Theißen et al., 1996, 2000; Purugganan, 1997; Kramer et al., 1998; Kramer and Irish, 1999; Lamb and Irish, 2003; Kim et al., 2004; Stellari et al., 2004; Zahn et al., 2005; Hernandez-Hernandez et al., 2007). In the gymnosperms, there was a single putative B class lineage that duplicated to generate the paleoAP3 and PI lineages in angiosperms (Kramer et al., 1998; Theißen et al., 2000; Irish, 2009). The paleoAP3 lineage is composed of AP3 orthologs identified in lower eudicots, magnolid dicots, and monocots (Kramer et al., 1998). Genes in this lineage contain the conserved paleoAP3- and PI-derived motifs in the C-terminal end of the proteins, which have been thought to be characteristics of the B class ancestral gene (Kramer et al., 1998; Tzeng and Yang, 2001; Hsu and Yang, 2002). The PI lineage is composed of PI orthologs that contain a highly conserved PI motif identified in most plant species (Kramer et al., 1998). Subsequently, there was a second duplication at the base of the core eudicots that produced the euAP3 and TM6 lineages, which have been subject to substantial sequence changes in eudicots during evolution (Kramer et al., 1998; Kramer and Irish, 1999). The paleoAP3 motif in the C-terminal end of the proteins was retained in the TM6 lineage and replaced by a conserved euAP3 motif in the euAP3 lineage of most eudicot species (Kramer et al., 1998). In addition, many lineage-specific duplications for paleoAP3 lineage have occurred in plants such as orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009), Ranunculaceae, and Ranunculales (Kramer et al., 2003; Di Stilio et al., 2005; Shan et al., 2006; Kramer, 2009).Unlike the A or C class MADS box proteins, which form homodimers that regulate flower development, the ability of B class proteins to form homodimers has only been reported in gymnosperms and in the paleoAP3 and PI lineages of some monocots. For example, LMADS1 of the lily Lilium longiflorum (Tzeng and Yang, 2001), OMADS3 of the orchid Oncidium Gower Ramsey (Hsu and Yang, 2002), and PeMADS4 of the orchid Phalaenopsis equestris (Tsai et al., 2004) in the paleoAP3 lineage, LRGLOA and LRGLOB of the lily Lilium regale (Winter et al., 2002), TGGLO of the tulip Tulipa gesneriana (Kanno et al., 2003), and PeMADS6 of the orchid P. equestris (Tsai et al., 2005) in the PI lineage, and GGM2 of the gymnosperm Gnetum gnemon (Winter et al., 1999) were able to form homodimers that regulate flower development. Proteins in the euAP3 lineage and in most paleoAP3 lineages were not able to form homodimers and had to interact with PI to form heterodimers in order to regulate petal and stamen development in various plant species (Schwarz-Sommer et al., 1992; Tröbner et al., 1992; Riechmann et al., 1996; Moon et al., 1999; Winter et al., 2002; Kanno et al., 2003; Vandenbussche et al., 2004; Yao et al., 2008). In addition to forming dimers, AP3 and PI were able to interact with other MADS box proteins, such as SEPALLATA1 (SEP1), SEP2, and SEP3, to regulate petal and stamen development (Pelaz et al., 2000; Honma and Goto, 2001; Theißen and Saedler, 2001; Castillejo et al., 2005).Orchids are among the most important plants in the flower market around the world, and research on MADS box genes has been reported for several species of orchids during the past few years (Lu et al., 1993, 2007; Yu and Goh, 2000; Hsu and Yang, 2002; Yu et al., 2002; Hsu et al., 2003; Tsai et al., 2004, 2008; Xu et al., 2006; Guo et al., 2007; Kim et al., 2007; Chang et al., 2009). Unlike the flowers in eudicots, the nearly identical shape of the sepals and petals as well as the production of a unique lip in orchid flowers make them a very special plant species for the study of flower development. Four clades (1–4) of genes in the paleoAP3 lineage have been identified in several orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009). Several works have described the possible interactions among these four clades of paleoAP3 genes and one PI gene that are involved in regulating the differentiation and formation of the sepal/petal/lip of orchids (Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009). However, the exact mechanism that involves the orchid B class genes remains unclear and needs to be clarified by more experimental investigations.O. Gower Ramsey is a popular orchid with important economic value in cut flower markets. Only a few studies have been reported on the role of MADS box genes in regulating flower formation in this plant species (Hsu and Yang, 2002; Hsu et al., 2003; Chang et al., 2009). An AP3-like MADS gene that regulates both floral formation and initiation in transgenic Arabidopsis has been reported (Hsu and Yang, 2002). In addition, four AP1/AGAMOUS-LIKE9 (AGL9)-like MADS box genes have been characterized that show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis (Hsu et al., 2003; Chang et al., 2009). Compared with other orchids, the production of a large and well-expanded lip and five small identical sepals/petals makes O. Gower Ramsey a special case for the study of the diverse functions of B class MADS box genes during evolution. Therefore, the isolation of more B class MADS box genes and further study of their roles in the regulation of perianth (sepal/petal/lip) formation during O. Gower Ramsey flower development are necessary. In addition to the clade 2 paleoAP3 gene OMADS3, which was previously characterized in our laboratory (Hsu and Yang, 2002), three more B class MADS box genes, OMADS5, OMADS8, and OMADS9, were characterized from O. Gower Ramsey in this study. Based on the different expression patterns and the protein interactions among these four orchid B class genes, we propose that the presence of OMADS3/8 and/or OMADS9 is required for sepal/petal/lip formation. Further sepal and petal formation at least requires the additional presence of OMADS5, whereas large lip formation was seen when OMADS5 expression was absent. Our results provide a new finding and information pertaining to the roles for orchid B class MADS box genes in the regulation of sepal/petal/lip formation.     

Induction of COX-2-PGE2 synthesis by activation of the MAPK/ERK pathway contributes to neuronal death triggered by TDP-43-depleted microglia

Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.¹ Most cases of ALS are sporadic, but 10% are familial. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.^{1, 2} The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.^{3, 4, 5} Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.¹ TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,^{6, 7} Alzheimer''s disease (AD)^{8, 9} and Parkinson''s disease (PD).^{10, 11} TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.^{5, 12, 13, 14}Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.^{15, 16} Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,^{17, 18, 19, 20, 21, 22} indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.^{23, 24, 25, 26, 27, 28, 29} The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.^{30, 31, 32} One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.^{33, 34, 35, 36, 37} The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,^{38, 39, 40} and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.^{25, 28, 32, 41, 42, 43, 44, 45, 46} Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.²⁸Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.^{47, 48, 49, 50, 51} Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,^{50, 51} indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.     

TRAF2 is a biologically important necroptosis suppressor

Tumor necrosis factor α (TNFα) triggers necroptotic cell death through an intracellular signaling complex containing receptor-interacting protein kinase (RIPK) 1 and RIPK3, called the necrosome. RIPK1 phosphorylates RIPK3, which phosphorylates the pseudokinase mixed lineage kinase-domain-like (MLKL)—driving its oligomerization and membrane-disrupting necroptotic activity. Here, we show that TNF receptor-associated factor 2 (TRAF2)—previously implicated in apoptosis suppression—also inhibits necroptotic signaling by TNFα. TRAF2 disruption in mouse fibroblasts augmented TNFα–driven necrosome formation and RIPK3-MLKL association, promoting necroptosis. TRAF2 constitutively associated with MLKL, whereas TNFα reversed this via cylindromatosis-dependent TRAF2 deubiquitination. Ectopic interaction of TRAF2 and MLKL required the C-terminal portion but not the N-terminal, RING, or CIM region of TRAF2. Induced TRAF2 knockout (KO) in adult mice caused rapid lethality, in conjunction with increased hepatic necrosome assembly. By contrast, TRAF2 KO on a RIPK3 KO background caused delayed mortality, in concert with elevated intestinal caspase-8 protein and activity. Combined injection of TNFR1-Fc, Fas-Fc and DR5-Fc decoys prevented death upon TRAF2 KO. However, Fas-Fc and DR5-Fc were ineffective, whereas TNFR1-Fc and interferon α receptor (IFNAR1)-Fc were partially protective against lethality upon combined TRAF2 and RIPK3 KO. These results identify TRAF2 as an important biological suppressor of necroptosis in vitro and in vivo.Apoptotic cell death is mediated by caspases and has distinct morphological features, including membrane blebbing, cell shrinkage and nuclear fragmentation.^{1, 2, 3, 4} In contrast, necroptotic cell death is caspase-independent and is characterized by loss of membrane integrity, cell swelling and implosion.^{1, 2, 5} Nevertheless, necroptosis is a highly regulated process, requiring activation of RIPK1 and RIPK3, which form the core necrosome complex.^{1, 2, 5} Necrosome assembly can be induced via specific death receptors or toll-like receptors, among other modules.^{6, 7, 8, 9} The activated necrosome engages MLKL by RIPK3-mediated phosphorylation.^{6, 10, 11} MLKL then oligomerizes and binds to membrane phospholipids, forming pores that cause necroptotic cell death.^{10, 12, 13, 14, 15} Unchecked necroptosis disrupts embryonic development in mice and contributes to several human diseases.^{7, 8, 16, 17, 18, 19, 20, 21, 22}The apoptotic mediators FADD, caspase-8 and cFLIP suppress necroptosis.^{19, 20, 21, 23, 24} Elimination of any of these genes in mice causes embryonic lethality, subverted by additional deletion of RIPK3 or MLKL.^{19, 20, 21, 25} Necroptosis is also regulated at the level of RIPK1. Whereas TNFα engagement of TNFR1 leads to K63-linked ubiquitination of RIPK1 by cellular inhibitor of apoptosis proteins (cIAPs) to promote nuclear factor (NF)-κB activation,²⁶ necroptosis requires suppression or reversal of this modification to allow RIPK1 autophosphorylation and consequent RIPK3 activation.^{2, 23, 27, 28} CYLD promotes necroptotic signaling by deubiquitinating RIPK1, augmenting its interaction with RIPK3.²⁹ Conversely, caspase-8-mediated CYLD cleavage inhibits necroptosis.²⁴TRAF2 recruits cIAPs to the TNFα-TNFR1 signaling complex, facilitating NF-κB activation.^{30, 31, 32, 33} TRAF2 also supports K48-linked ubiquitination and proteasomal degradation of death-receptor-activated caspase-8, curbing apoptosis.³⁴ TRAF2 KO mice display embryonic lethality; some survive through birth but have severe developmental and immune deficiencies and die prematurely.^{35, 36} Conditional TRAF2 KO leads to rapid intestinal inflammation and mortality.³⁷ Furthermore, hepatic TRAF2 depletion augments apoptosis activation via Fas/CD95.³⁴ TRAF2 attenuates necroptosis induction in vitro by the death ligands Apo2L/TRAIL and Fas/CD95L.³⁸ However, it remains unclear whether TRAF2 regulates TNFα-induced necroptosis—and if so—how. Our present findings reveal that TRAF2 inhibits TNFα necroptotic signaling. Furthermore, our results establish TRAF2 as a biologically important necroptosis suppressor in vitro and in vivo and provide initial insight into the mechanisms underlying this function.     

Sex-Linked Inheritance in Macaque Monkeys: Implications for Effective Population Size and Dispersal to Sulawesi

Sex-specific differences in dispersal, survival, reproductive success, and natural selection differentially affect the effective population size (N_e) of genomic regions with different modes of inheritance such as sex chromosomes and mitochondrial DNA. In papionin monkeys (macaques, baboons, geladas, mandrills, drills, and mangabeys), for example, these factors are expected to reduce N_e of paternally inherited portions of the genome compared to maternally inherited portions. To explore this further, we quantified relative N_e of autosomal DNA, X and Y chromosomes, and mitochondrial DNA using molecular polymorphism and divergence information from pigtail macaque monkeys (Macaca nemestrina). Consistent with demographic expectations, we found that N_e of the Y is lower than expected from a Wright–Fisher idealized population with an equal proportion of males and females, whereas N_e of mitochondrial DNA is higher. However, N_e of 11 loci on the X chromosome was lower than expected, a finding that could be explained by pervasive hitchhiking effects on this chromosome. We evaluated the fit of these data to various models involving natural selection or sex-biased demography. Significant support was recovered for natural selection acting on the Y chromosome. A demographic model with a skewed sex ratio was more likely than one with sex-biased migration and explained the data about as well as an ideal model without sex-biased demography. We then incorporated these results into an evaluation of macaque divergence and migration on Borneo and Sulawesi islands. One X-linked locus was not monophyletic on Sulawesi, but multilocus data analyzed in a coalescent framework failed to reject a model without migration between these islands after both were colonized.THE effective size of a population (N_e) determines the relative impact of genetic drift and natural selection on mutations with mild effects on fitness (Charlesworth 2009). Differences in N_e are hypothesized to affect virtually every aspect of genome evolution, including rates of molecular evolution, abundance of introns and transposable elements, and persistence of duplicate genes, and this has important implications for the evolution of complexity via both adaptive and degenerative processes (Lynch 2007). Of relevance are not only the number of different individuals in a population, but also the number of copies of a gene within each individual. In diploid species with separate sexes, sex chromosomes and mitochondrial DNA (mtDNA) differ in copy number from autosomal DNA (aDNA): both sexes have two alleles at autosomal loci whereas in species with male heterogamy, males have one X and one Y chromosome, females have two Xs, and a female/male pair has effectively only one copy of mtDNA due to maternal inheritance. Sex-specific differences in demographic parameters such as migration, adult sex ratio, and variance in reproductive success also affect relative copy number and associated levels of neutral polymorphism at mtDNA, aDNA, the X chromosome (xDNA), and the Y chromosome (yDNA) (Hedrick 2007).The effective population size is the number of individuals in a Wright–Fisher idealized population (Fisher 1930; Wright 1931) that have the same magnitude of genetic drift as an observed population, where ideal individuals are diploid, and have discrete (nonoverlapping) generations, constant population size, and random mating. N_e can be quantified in terms of variance in allele frequency over generations (variance N_e) or variance in inbreeding over time (inbreeding N_e). If population size is constant with random mating, these approaches for quantifying N_e produce identical results (Kimura and Crow 1963; Whitlock and Barton 1997). At mutation–drift equilibrium with an equal number of males and females and a Poisson distributed number of offspring with a mean of two offspring per individual, N_e-aDNA and N_e-xDNA are expected to be four and three times as large, respectively, as N_e-yDNA and N_e-mtDNA; we refer to this as the “ideal expectation with an equal proportion of males and females.”Demography can alter relationships between N_e of different parts of the genome. For example, extreme skew in adult sex ratio can cause N_e of uniparentally inherited portions of the genome to exceed N_e of biparentally inherited portions (Figure 1A; Nunney 1993; Caballero 1994; Hoelzer 1997; Hedrick 2007). With a skewed sex ratio, the more common sex has a higher variance in reproductive success than the rare one, and this causes the overall variance in reproductive success to increase as the sex-ratio bias increases (Nunney 1993). Sex-biased dispersal such as female philopatry also alters relationships between N_e-aDNA, N_e-xDNA, N_e-yDNA, and N_e-mtDNA (Figure 1B), causing N_e of portions of the genome that disperse less to increase (Nei and Takahata 1993; Hoelzer 1997; Wang and Caballero 1999).Open in a separate windowFigure 1.—N_e of aDNA, xDNA, mtDNA, and yDNA as a function of (A) sex ratio skew and (B) the probability of female dispersal. In B, a finite island model of subdivided populations of constant size is assumed with a population size of 10,000 individuals, 10 subpopulations, and a male probability of migration equal to 0.1.At least five factors related to natural selection also can cause the relative N_e of aDNA, xDNA, yDNA, and mtDNA to depart from expectations: (1) very low or absent recombination in mtDNA and a portion of yDNA, (2) haploidy of mtDNA and yDNA, (3) hemizygosity of xDNA in males, (4) sexual selection and differences in gene content, and (5) differences in the rate and variance of mutation. “Selective sweeps” in which an advantageous mutation is fixed by natural selection, reduces N_e of linked sites (Maynard Smith and Haigh 1974) and this can affect the entire mitochondrial genome and nonrecombining portion of the Y chromosome. Nonrecombining portions of yDNA and mtDNA are also affected by stochastic loss of alleles containing the fewest deleterious mutations (“Muller''s ratchet”; Muller 1964; Felsenstein 1974), which results in a gradual decline of fitness of these chromosomes over time. N_e of nonrecombining DNA is further reduced by elimination of variation linked to substantially deleterious mutations (“background selection”; Charlesworth et al. 1993), by interference between linked polymorphisms that impedes fixation of advantageous alleles and extinction of deleterious ones (the “Hill–Robertson effect”; Hill and Robertson 1966; McVean and Charlesworth 2000), and by increased frequency of deleterious mutations linked to advantageous ones during a selective sweep (“genetic hitchhiking”; Rice 1987). Hemizygous X-linked and haploid Y-linked loci in males and mtDNA loci in both sexes are more vulnerable to recessive deleterious mutations because they are not masked by a second allele (Otto and Goldstein 1992). Hemizygosity on the X chromosome can also increase the rate of selective sweeps when advantageous mutations are recessive (Charlesworth et al. 1987). Similarly, these loci are also susceptible to recessive species incompatibilities—a factor that at least partially accounts for Haldane''s rule for hybrid sterility (Haldane 1922; Orr 1997). Sexual selection differentially influences the probability of fixation of mutations depending on mode of inheritance (Wade and Shuster 2004), especially mutations with antagonistic fitness effects between the sexes (Gibson et al. 2002). Additionally, the rate of evolution of animal mtDNA is much higher than aDNA, xDNA, and yDNA (Haag-Liautard et al. 2008) and this presumably contributes to variation in the frequency of nonneutral mutations in different parts of the genome.Differences among N_e of mtDNA, yDNA, xDNA, and aDNA are thought to be particularly pronounced in papionin monkeys (macaques, baboons, geladas, mandrills, drills, and mangabeys). These monkeys have a highly sex-biased adult demography; females form stable philopatric groups of close relatives, whereas males generally change social groups and disperse more widely (Dittus 1975). Often adult sex ratio of papionins is female biased (Dittus 1975; Melnick and Pearl 1987; O''Brien and Kinnard 1997; Okamoto and Matsumura 2001), and males have higher variance in reproductive success than females (Dittus 1975; de Ruiter et al. 1992; Keane et al. 1997; Van Noordwijk and Van Schaik 2002; Widdig et al. 2004). These sex differences predict strong population subdivision of mtDNA with little or no subdivision of aDNA, deep mtDNA coalescence times, and frequent mtDNA paraphyly among species, and discordant genealogical relationships between mtDNA and yDNA—and this has been observed in multiple studies (Melnick and Pearl 1987; Melnick 1988; Melnick and Hoelzer 1992; Melnick et al. 1993; Hoelzer et al. 1994; Evans et al. 1999, 2001, 2003; Tosi et al. 2000, 2002, 2003; Newman et al. 2004). Female philopatry and obligate male migration is a common social system in mammals (Greenwood 1980; Dobson 1982; Johnson 1986), though less so in humans (Seielstad et al. 1998), and molecular variation provides an effective tool for exploring the impact of natural selection and demography on aDNA, the sex chromosomes, and mtDNA (Nachman 1997; Bachtrog and Charlesworth 2002; Stone et al. 2002; Berlin and Ellegren 2004; Hellborg and Ellegren 2004; Wilder et al. 2004; Hammer et al. 2008).We explored the genetic effects of demography and linked selection in structuring sequence polymorphism of a papionin monkey—the macaques—at two levels. We first tested whether levels of polymorphism in aDNA, xDNA, yDNA, and mtDNA in a Bornean population of the pigtail macaque, Macaca nemestrina, match expectations under scenarios involving natural selection and also whether the data might be explained by simple demographic models with sex-specific dispersal or a biased sex ratio. We then explored demography on a larger, inter-island scale by estimating the time of divergence between macaques on Borneo and Sulawesi islands and by testing for evidence of ongoing migration between these islands.     

Stress-induced ceramide generation and apoptosis via the phosphorylation and activation of nSMase1 by JNK signaling

Neutral sphingomyelinase (nSMase) activation in response to environmental stress or inflammatory cytokine stimuli generates the second messenger ceramide, which mediates the stress-induced apoptosis. However, the signaling pathways and activation mechanism underlying this process have yet to be elucidated. Here we show that the phosphorylation of nSMase1 (sphingomyelin phosphodiesterase 2, SMPD2) by c-Jun N-terminal kinase (JNK) signaling stimulates ceramide generation and apoptosis and provide evidence for a signaling mechanism that integrates stress- and cytokine-activated apoptosis in vertebrate cells. An nSMase1 was identified as a JNK substrate, and the phosphorylation site responsible for its effects on stress and cytokine induction was Ser-270. In zebrafish cells, the substitution of Ser-270 for alanine blocked the phosphorylation and activation of nSMase1, whereas the substitution of Ser-270 for negatively charged glutamic acid mimicked the effect of phosphorylation. The JNK inhibitor SP600125 blocked the phosphorylation and activation of nSMase1, which in turn blocked ceramide signaling and apoptosis. A variety of stress conditions, including heat shock, UV exposure, hydrogen peroxide treatment, and anti-Fas antibody stimulation, led to the phosphorylation of nSMase1, activated nSMase1, and induced ceramide generation and apoptosis in zebrafish embryonic ZE and human Jurkat T cells. In addition, the depletion of MAPK8/9 or SMPD2 by RNAi knockdown decreased ceramide generation and stress- and cytokine-induced apoptosis in Jurkat cells. Therefore the phosphorylation of nSMase1 is a pivotal step in JNK signaling, which leads to ceramide generation and apoptosis under stress conditions and in response to cytokine stimulation. nSMase1 has a common central role in ceramide signaling during the stress and cytokine responses and apoptosis.The sphingomyelin pathway is initiated by the hydrolysis of sphingomyelin to generate the second messenger ceramide.¹ Sphingomyelin hydrolysis is a major pathway for stress-induced ceramide generation. Neutral sphingomyelinase (nSMase) is activated by a variety of environmental stress conditions, such as heat shock,^{1, 2, 3} oxidative stress (hydrogen peroxide (H₂O₂), oxidized lipoproteins),¹ ultraviolet (UV) radiation,¹ chemotherapeutic agents,⁴ and β-amyloid peptides.^{5, 6} Cytokines, including tumor necrosis factor (TNF)-α,^{7, 8, 9} interleukin (IL)-1β,¹⁰ Fas ligand,¹¹ and their associated proteins, also trigger the activation of nSMase.¹² Membrane-bound Mg²⁺-dependent nSMase is considered to be a strong candidate for mediating the effects of stress and inflammatory cytokines on ceramide.³Among the four vertebrate nSMases, nSMase1 (SMPD2) was the first to be cloned and is localized in the endoplasmic reticulum (ER) and Golgi apparatus.¹³ Several studies have focused on the potential signaling roles of nSMase1, and some reports have suggested that nSMase1 is important for ceramide generation in response to stress.^{5, 6, 14, 15} In addition, nSMase1 is responsible for heat-induced apoptosis in zebrafish embryonic cultured (ZE) cells, and a loss-of-function study showed a reduction in ceramide generation, caspase-3 activation, and apoptosis in zebrafish embryos.¹⁶ However, nSMase1-knockout mice showed no lipid storage diseases or abnormalities in sphingomyelin metabolism.¹⁷ Therefore, the molecular mechanisms by which nSMase1 is activated have yet to be elucidated.Environmental stress and inflammatory cytokines^{1, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27} stimulate stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) signaling, which involves the sequential activation of members of the mitogen-activated protein kinase (MAPK) family, including MAPK/ERK kinase kinase (MEKK)1/MAPK kinase (MKK)4, and/or SAPK/ERK kinase (SEK)1/MKK7, JNK, and c-jun. Both the JNK and sphingomyelin signaling pathways coordinately mediate the induction of apoptosis.¹ However, possible crosstalk between the JNK and sphingomyelin signaling pathways has not yet been characterized. Previously, we used SDS-PAGE to determine that nSMase1 polypeptides migrated at higher molecular masses,¹⁶ suggesting that the sphingomyelin signaling pathway might cause the production of a chemically modified phosphorylated nSMase1, which is stimulated under stressed conditions in ZE cells.¹⁶ Here, we demonstrate that JNK signaling results in the phosphorylation of Ser-270 of nSMase1, which initiates ceramide generation and apoptosis. We also provide evidence for a signaling mechanism that integrates cytokine- and stress-activated apoptosis in vertebrate cells. We studied stress-induced ceramide generation in two cell types: ZE cells and human leukemia Jurkat T-lymphoid cells. Stress-induced apoptosis has been investigated in these systems previously.^{16, 28}     

Housing Conditions Modulate the Severity of Mycoplasma pulmonis Infection in Mice Deficient in Class A Scavenger Receptor

Mycoplasmosis is a frequent causative microbial agent of community-acquired pneumonia and has been linked to exacerbation of chronic obstructive pulmonary disease. The macrophage class A scavenger receptor (SRA) facilitates the clearance of noxious particles, oxidants, and infectious organisms by alveolar macrophages. We examined wildtype and SRA^−/− mice, housed in either individually ventilated or static filter-top cages that were cycled with fresh bedding every 14 d, as a model of gene–environment interaction on the outcome of pulmonary Mycoplasma pulmonis infection. Intracage NH₃ gas measurements were recorded daily prior to infection. Mice were intranasally infected with 1 × 10⁷ cfu M. pulmonis UAB CT and evaluated at 3, 7, and 14 d after inoculation. Wildtype mice cleared 99.5% of pulmonary M. pulmonis by 3 d after infection but remained chronically infected through the study. SRA^−/− mice were chronically infected with 40-fold higher mycoplasma numbers than were wildtype mice. M. pulmonis caused a chronic mixed inflammatory response that was accompanied with high levels of IL1β, KC, MCP1, and TNFα in SRA^−/− mice, whereas pulmonary inflammation in WT mice was represented by a monocytosis with elevation of IL1β. Housing had a prominent influence on the severity and persistence of mycoplasmosis in SRA^−/− mice. SRA^-/- mice housed in static cages had an improved recovery and significant changes in surfactant proteins SPA and SPD compared with baseline levels. These results indicate that SRA is required to prevent chronic mycoplasma infection of the lung. Furthermore, environmental conditions may exacerbate chronic inflammation in M. pulmonis-infected SRA^−/− mice.Abbreviations: BAL, bronchoalveolar lavage; COPD, chronic obstructive pulmonary disease; KC, keratinocyte-derived chemokine (CXCL1); MCP1, monocyte chemotactic protein 1; SPA, surfactant protein A (SFTPA1); SPB, surfactant protein B (SFTPB); SPD, surfactant protein D (SFTPD); SRA, class A scavenger receptor (MSR1); WT, wildtypeThere are numerous options for the housing and husbandry of rodents in the laboratory setting. Various available choices in caging, bedding material, and cage-change frequency have the potential to effect physiologic values and thus experimental outcomes.^20,108 In many facilities, current practices involve performing cage changes every 1 to 2 wk, with some facilities exploring the possibility of extending these practices to every 4 wk.⁹⁷ Cage-change frequency practices are established at various institutions after consideration of several variables that affect animal health, welfare, and cost. Ideally, an appropriate sanitation program provides clean and dry bedding, adequate air quality, and clean cage surfaces and accessories.⁴⁴ When establishing performance standards for a sanitation program that are different from those which are recommended in the Guide for the Care and Use of Animals in Research,⁴⁴ microenvironmental conditions, including intracage humidity, temperature, animal behavior and appearance, microbiologic loads, and levels of pollutants such as CO₂ and NH₃, should be evaluated and verified. Although there are currently no established NH₃ exposure limits for laboratory animals, the human occupational exposure limit of 25 ppm as an 8-h time-weighted average, established by the National Institute for Occupational Safety and Health, is often referenced as a guideline for animals.⁹⁵ Multiple factors, such as animal cage density, sex, age, bedding type, reusable compared with disposable caging, static caging compared with IVC, and cage-change frequency, influence intracage and ambient NH₃ levels.^82,83,97 Only limited information is available that addresses the effect of natural intracage NH₃ levels on respiratory function in experimental rodents and whether exposure to high NH₃ levels under current standard practices affects the results of respiratory disease research.Ammonia is an alkaline, corrosive, and irritant gas that is very water soluble. It reacts with the moisture of the mucous membranes of the eyes, mouth, and respiratory tract to form ammonium hydroxide in an exothermic reaction, resulting in thermal and chemical burns.⁶⁸ Clinical symptoms in humans exposed to high levels of NH₃ include eye irritation, headaches, and multiple acute and chronic respiratory symptoms, such as irritation of the nose, pharynx, and sinuses, and in severe cases, development of bronchitis and hyper-reactive airway disease.⁷⁹ Animals are similarly susceptible to NH₃-induced pulmonary disease.^23,31,48Mice exposed to naturally increasing levels of intracage NH₃ can develop lesions in the rostral nasal cavity, with decreasing severity of the lesions moving caudally into the nasopharynx, and no lesions in the lung.⁹⁷ However, dust is another common environmental pollutant that is often present in animal settings. Dust particles readily absorb NH₃, which then serve as a source of NH₃ deposition into the lower respiratory tract. Dust particulate can range from large (300 µm), minimally respirable particles to very fine (< 50 µm) particulate matter, which can settle deep within the alveoli.^10,102 The mucociliary system of the respiratory tract is the first line of defense against inspired noxious stimuli and pathogens. Exposure of the ciliated respiratory epithelium to the damaging effects of NH₃ are known to cause decreased mucociliary beating.⁵⁶ Disruption of the respiratory mucociliary escalator initiated by NH₃ exposure can then promote establishment of chronic infections and inflammation of the airway mucosa.^11,87 Therefore, NH₃ potentially can cause pathophysiologic changes of the lung in the absence of histopathologic lesions.Our primary goal was to analyze the effect of 2 housing modalities, which result in different intracage NH₃ concentrations, on mice that were challenged with a respiratory pathogen. Mycoplasma pulmonis was chosen as a model because it is a well-established model in rodents which causes chronic mycoplasmosis and reproduces the features of M. pneumoniae in humans.^22,41 M. pneumoniae infection is a frequent and contagious etiology of community-acquired pneumonia causing tracheobronchitis, sneezing, cough, and inflammation of the respiratory tract.^8,12,47,63 Moreover, atypical and difficult-to-detect respiratory pathogens such as Chlamydophila pneumoniae and Mycoplasma pneumoniae that can establish chronic asymptomatic infections may contribute to both the development and exacerbation of COPD^{26,45,57,58,62,63,66,72,96,103} and asthma.^8,51,65 Infection with M. pulmonis in rodents causes rhinitis, otitis media, tracheitis, and pneumonia, which can be exacerbated by housing conditions and genetic background.^14,32,85 The mechanism of pathogenicity of mycoplasmas continues to be an area of interest in the research.The innate host factors protecting against pulmonary mycoplasmosis include the secreted surfactant protein opsonins SPA and SPD, surfactant phospholipids, and the molecular pattern-recognition receptor TLR2.^15,16,54,74 Therefore, compared with their wildtype (WT) counterparts, SPA-deficient mice infected with either M. pulmonis or M. pneumoniae develop more severe inflammation and have decreased capacity to clear these infections from the lungs.⁴³ In addition, TLR2-deficient mice exhibit decreased clearance and increased inflammation in response to mycoplasma infection.^60,104Second, we wanted to study the effects of SRA deficiency in mycoplasmosis. The class A scavenger receptor (SRA) modulates inflammatory responses and mediates the clearance of airborne oxidants, particulates, and respiratory pathogens.^{3,17,18,49,88,101} Inhibition of SRA expression in alveolar macrophages in an elastase–LPS model of COPD was associated with decreased clearance of Haemophilus influenzae.³³ Lack of SRA similarly impaired alveolar macrophage-mediated clearance of Streptococcus pneumoniae,⁵ environmental particles,⁶ and ozone-oxidized lipids¹⁸ by alveolar macrophages. Absence of SRA also enhanced hyperoxia-induced lung injury⁴⁹ and exacerbated inflammation in response to Staphylococcus aureus infection.⁸⁸ SRA appears to have antiinflammatory properties with the capacity to modify macrophage phenotype and suppress polarization toward the M1 alternative macrophage activation state.¹³ The SRA gene (MSR1) is polymorphic in both mice and humans.^19,29,105 Genetic association studies in humans, however, showed that subjects with truncations or point mutations in MSR1 have significantly increased risk for the development of pulmonary diseases such as COPD^33,38,71,94 and asthma.⁵ Our understanding of the immune factors that contribute to mycoplasmosis is far from complete.In the present study, by investigating the role of SRA in mycoplasmosis jointly with the effects of housing, we demonstrated that genetic and environmental factors both serve as critical players in disease progression. We show that SRA-deficient mice are susceptible to chronic colonization with M. pulmonis and development of chronic mycoplasma-induced bronchopneumonia characterized by persistent multicellular inflammation. Furthermore, we show that housing conditions influence the effect of SRA deficiency on the severity of mycoplasmosis. Taken together, these results indicate that lack of SRA function impairs host protection against both infectious and environmental insults.