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.     

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.     

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.     

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.     

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.     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.     

Adiponectin receptor-mediated signaling ameliorates cerebral cell damage and regulates the neurogenesis of neural stem cells at high glucose concentrations: an in vivo and in vitro study

In the central nervous system (CNS), hyperglycemia leads to neuronal damage and cognitive decline. Recent research has focused on revealing alterations in the brain in hyperglycemia and finding therapeutic solutions for alleviating the hyperglycemia-induced cognitive dysfunction. Adiponectin is a protein hormone with a major regulatory role in diabetes and obesity; however, its role in the CNS has not been studied yet. Although the presence of adiponectin receptors has been reported in the CNS, adiponectin receptor-mediated signaling in the CNS has not been investigated. In the present study, we investigated adiponectin receptor (AdipoR)-mediated signaling in vivo using a high-fat diet and in vitro using neural stem cells (NSCs). We showed that AdipoR1 protects cell damage and synaptic dysfunction in the mouse brain in hyperglycemia. At high glucose concentrations in vitro, AdipoR1 regulated the survival of NSCs through the p53/p21 pathway and the proliferation- and differentiation-related factors of NSCs via tailless (TLX). Hence, we suggest that further investigations are necessary to understand the cerebral AdipoR1-mediated signaling in hyperglycemic conditions, because the modulation of AdipoR1 might alleviate hyperglycemia-induced neuropathogenesis.Adiponectin secreted by the adipose tissue^{1, 2} exists in either a full-length or globular form.^{3, 4, 5, 6} Adiponectin can cross the blood–brain barrier, and various forms of adiponectin are found in the cerebrospinal fluid.^{7, 8, 9, 10, 11} Adiponectin exerts its effect by binding to the adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2)^{12, 13} that have different affinities for the various circulating adiponectins.^{12, 14, 15, 16, 17} Several studies reported that both receptor subtypes are expressed in the central nervous system (CNS).^{7, 12, 18} As adiponectin modulates insulin sensitivity and inflammation,¹⁹ its deficiency induces insulin resistance and glucose intolerance in animals fed a high-fat diet (HFD).^{19, 20, 21} In addition, adiponectin can ameliorate the glucose homeostasis and increase insulin sensitivity.^{22, 23, 24} Adiponectin, which is the most well-known adipokine, acts mainly as an anti-inflammatory regulator,^{25, 26} and is associated with the onset of neurological disorders.²⁷ In addition, a recent study reported that adiponectin promotes the proliferation of hippocampal neural stem cells (NSCs).²⁸ Considering that adiponectin acts by binding to the adiponectin receptors, investigation of the adiponectin receptor-mediated signaling in the brain is crucial to understand the cerebral effects of adiponectin and the underlying cellular mechanisms.The prevalence of type II diabetes mellitus (DM2) and Alzheimer''s disease increases with aging.²⁹ According to a cross-sectional study, in people with DM2, the risk of dementia is 2.5 times higher than that in the normal population.^{30, 31} A study performed between 1980 and 2002 suggested that an elevated blood glucose level is associated with a greater risk for dementia in elderly patients with DM2.³² In addition, according to a 9-year-long longitudinal cohort study, the risk of developing Alzheimer''s disease was 65% higher in people with diabetes than in control subjects.³³ A community-based cohort study also reported that higher plasma glucose concentrations are associated with an increased risk for dementia, because the higher glucose level has detrimental effects on the brain.³¹ High blood glucose level causes mitochondria-dependent apoptosis,^{34, 35, 36} and aggravates diverse neurological functions.^{37, 38} Inflammation and oxidative stress, which are commonly observed in people with diabetes, inhibit neurogenesis.^{39, 40, 41} Similarly, neurogenesis is decreased in mice and rats with genetically induced type I diabetes.^{42, 43} In addition, diabetic rodents have a decreased proliferation rate of neural progenitors.^{43, 44} Furthermore, several studies suggested that an HFD leads to neuroinflammation, the impairment of synaptic plasticity, and cognitive decline.^{45, 46}Here, we investigated whether AdipoR1-mediated signaling is associated with cell death in the brain of mice on a HFD, and whether high glucose level modifies the proliferation and differentiation capacity of NSCs in vitro. Our study provides novel findings about the role of AdipoR1-mediated signaling in hyperglycemia-induced neuropathogenesis.     

Effects of Helicobacter Infection on Research: The Case for Eradication of Helicobacter from Rodent Research Colonies

Infection of mouse colonies with Helicobacter spp. has become an increasing concern for the research community. Although Helicobacter infection may cause clinical disease, investigators may be unaware that their laboratory mice are infected because the pathology of Helicobacter species is host-dependent and may not be recognized clinically. The effects of Helicobacter infections are not limited to the gastrointestinal system and can affect reproduction, the development of cancers in gastrointestinal organs and remote organs such as the breast, responses to vaccines, and other areas of research. The data we present in this review show clearly that unintentional Helicobacter infection has the potential to significantly interfere with the reliability of research studies based on murine models. Therefore, frequent screening of rodent research colonies for Helicobacter spp. and the eradication of these pathogens should be key goals of the research community.The reliability of an experiment that uses an in vivo model system depends on understanding and controlling all variables that can influence the experimental outcome. Infections of mouse colonies are important to the scientific community because they can introduce such harmful variables. Therefore, the ultimate goal of laboratory animal facilities is to maintain disease-free animals, to eliminate those unwanted variables.Numerous pathogenic microbes can interfere with animal research (reviewed in reference 57), and colonization of mouse colonies with members of the family Helicobacteriaceae is an increasing concern for the research community. Naturally acquired Helicobacter infections have been reported in all commonly used laboratory rodent species.^{3,10,36,44,45,49,82,124} A study of mice derived from 34 commercial and academic institutions in Canada, Europe, Asia, Australia, and the United States showed that 88% of these institutions had mouse colonies infected with 1 or more Helicobacter spp.¹⁰⁹ Approximately 59% of these mice were infected with Helicobacter hepaticus ; however monoinfections with other species also were encountered. In another study, at least 1 of 5 Helicobacter spp. was detected in 88% of the 40 mouse strains tested.⁴Surveys such as these have established that a broad range of Helicobacter spp. may be present in mouse research colonies. Several of those Helicobacter species cause disease in laboratory mice. H. hepaticus first was identified as a pathogen when it was discovered to be the cause of chronic hepatitis and hepatocellular carcinoma in mice,^26,31,116 either alone or in combination with other Helicobacter spp.⁷⁸ In addition, H. typhlonius causes intestinal inflammation in mice with immunodeficiency or defects in immune regulation;^28,37 H. muridarum has been associated with gastritis,⁸⁶ and H. bilis has been associated with hepatitis^35,38 and colitis.^60,61 Although, H. rodentium appears to be relatively nonpathogenic in wild-type and SCID mice,⁷⁸ combined infection with H. rodentium and H. typhlonius results in a high incidence of inflammation-associated neoplasia in IL10^−/− mice.^9,46 Further, it is becoming increasingly clear that the effects of Helicobacter infections are not limited to the gastrointestinal system. Helicobacter infections have been documented to directly or indirectly affect responses as diverse as reproduction, development of breast cancer, and altered immune responses to vaccines.^65,95,99 In addition to effects on rodents, Helicobacter spp. can infect other laboratory animals^{2,5,27,29,33,36,107} and can colonize different anatomic regions of the gastrointestinal system.³⁵ This review focuses on the potential effect of these organisms on in vivo experiments and biomedical research. The results summarized here emphasize the importance of knowledge of colony infection status and prevention of unintentional infections to achieve the goal of providing a consistent and reliable environment for research studies.     

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}     

Serologic Evaluation of Clinical and Subclinical Secondary Hepatic Amyloidosis in Rhesus Macaques (Macaca mulatta)

Secondary hepatic amyloidosis in nonhuman primates carries a grave prognosis once animals become clinically ill. The purpose of this study was to establish serologic parameters that potentially could be used to identify rhesus macaques undergoing subclinical development of secondary hepatic amyloidosis. A retrospective analysis was completed by using serum biochemical profiles from 26 histologically diagnosed amyloidotic macaques evaluated at 2 stages of disease, clinical and subclinical (3 to 32 mo prior to clinical signs of disease). Standard serum biochemistry values for cases were compared with institutional age- and gender-specific references ranges by construction of 95% confidence intervals for the difference between means. In addition, 19 histologically diagnosed amyloidotic macaques and 19 age-matched controls were assayed for changes in various parameters by using routinely banked, frozen (–80 °C) sera available from clinical and subclinical time points. Clinically amyloidotic animals displayed increased levels of alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase, gamma glutamyltranspeptidase, and macrophage colony-stimulating factor and significantly decreased quantities of albumin and total cholesterol. Subclinical amyloidotic animals displayed increased levels of alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase, and serum amyloid A and decreased concentrations of albumin and total cholesterol. The serologic parameters studied indicate a temporal relationship of these factors not previously described, show a clear pattern of disease progression, and could be useful in subclinical disease detection.Abbreviations: mCSF, macrophage colony stimulating factor; SAA, serum amyloid AAmyloid is an eosinophilic substance made of insoluble fibrillar protein.³² When deposited extracellularly, amyloid causes displacement of tissue form and disruption of organ function.³² Persistent accretion of amyloid can result in organ failure and ultimately animal death.²² Clinical signs of disease depend on the tissues affected and the degree of involvement.³² Amyloidosis has been well documented in humans, other mammals, birds, and reptiles.³⁸ In humans, amyloidosis plays a key role in many diseases, including Alzheimer disease, type II diabetes, rheumatoid arthritis, and Down syndrome.^15,20,35,38Amyloidosis generally is classified into 3 categories: primary, secondary, and hereditary. Primary amyloidosis consists of the immunoglobulin- and myeloma-associated types. Secondary (reactive) amyloidosis is associated with chronic inflammation.²⁴ Common causes of secondary amyloidosis in humans include rheumatoid arthritis, idiopathic colitis, infectious diseases, such as tuberculosis and leprosy, and malignant tumors, such as mesothelioma and Hodgkins disease.²⁸ Hereditary amyloid syndromes are rare and include Mediterranean fever, Muckle–Wells syndrome, and familial amyloid cardiomyopathy.^32,38Secondary amyloidosis is the most common form of amyloidosis in animals.³⁸ Amyloidosis occurs in many species of nonhuman primates including the common marmoset (Callithrix jacchus),²³ squirrel monkey (Saimiri sciureus),³⁴ rhesus macaque (Macaca mulatta),^9,10 pigtailed macaque (Macaca nemestrina),^18,27 crab-eating macaque (Macaca fascicularis),²⁷ barbary ape (Macaca sylvanus),⁶ baboon (Papio spp.),¹⁷ mandrill (Papio sphinx), and chimpanzee (Pan troglodytes).^16,39 Although a definitive cause of secondary amyloidosis has not been identified in nonhuman primates, this condition has been associated with chronic inflammation due to rheumatoid arthritis,⁶ viral infection,¹⁸ parasitism,¹ respiratory disease,^27,30 trauma,³⁰ and bacterial enterocolitis.^27,30,31 Shigella spp. have received particular attention as a common etiology linking enterocolitis with amyloidosis.^4,7,38Previous research on amyloidosis in nonhuman primates has yielded clinical and serologic profiles in end-stage amyloidotic animals, but little is known about the serologic status in the subclinical stages of disease. Amyloid can accumulate for as long as 3 y before severe organ disruption occurs¹⁴ and clinical signs of amyloidosis become evident.¹⁶ With appropriate analysis, detection of amyloidosis could occur much earlier than typically now achieved, thus allowing for targeted preventative therapy to potentially halt the progression of this insidious disease.     

Effects of Route of Inoculation and Viral Genetic Variation on Antibody Responses to Polyomavirus SV40 in Syrian Golden Hamsters

Genetic variants of polyomavirus SV40 are powerful agents with which to define viral effects on cells and carcinogenesis pathways. We hypothesized that differences in biologic variation among viral strains affect the process of viral infection and are reflected in antibody responses to the viral nonstructural large T-antigen (TAg) protein but not in neutralizing antibody responses against the inoculated viral particles. We analyzed the production of TAg antibody and neutralizing antibody in Syrian golden hamsters that were inoculated with SV40 viral strains by intracardiac, intravenous, or intraperitoneal routes and remained tumor free. Compared with the intraperitoneal route, intravascular (that is, intravenous, intracardiac) inoculation resulted in increased frequency of responsiveness to TAg but not in higher TAg antibody titers. The intravascular route was superior both for eliciting neutralizing antibody responses and for higher titers of those responses. Viruses with complex regulatory regions induced TAg antibody more often than did viruses with simple regulatory regions after intraperitoneal but not intravascular injections, with no differences in antibody titers. This viral genetic variation had no effect on neutralizing antibody production after intraperitoneal or intravascular inoculations or on neutralizing antibody titers achieved. These findings confirm that SV40 variants differ in their biologic properties. Route of inoculation combined with viral genetic variation significantly influence the development of serum antibodies to SV40 TAg in tumor-free hamsters. Route of inoculation—but not viral genetic variation—is an important factor in production of neutralizing antibody to SV40.Abbreviations: TAg, large T antigenPolyomavirus SV40 was discovered as an unrecognized contaminant of early poliovaccines³⁸ and was shown promptly to be an oncogenic virus.^9,15,17,18 Syrian golden hamsters are the small animal model that is susceptible to SV40 tumorigenicity.^{7,9-13,18,27,35} Since its discovery, SV40 has proven to be an outstanding tool for the discovery of mechanisms underlying carcinogenesis and for viral influences on cellular processes.^{1,3,5,19,23,30}Genetic variants of SV40 exist.^{16,20,26,28,33,34,36,37,41} This variation typically occurs in the viral regulatory region, in which some strains have duplications or rearrangements (or both) in the enhancer region, and in the C-terminal region of the large T-antigen (TAg) gene, in which nucleic acid variations may result in amino acid changes in the protein. TAg is an essential viral replication protein and the major viral oncoprotein. An important question is whether SV40 viral variants differ in their biologic properties, including in host responses to infections, as this could affect the spectrum of viral disease pathogenesis. We previously have shown that the viral regulatory region influences SV40 tumor induction in hamsters^35,40 and vertical transmission of the virus in hamsters²⁹ and that the route of inoculation influences SV40-mediated carcinogenesis.²⁷ Because TAg is not a component of the virus particle but instead is synthesized in virus-infected cells, we hypothesized that differences in antibody responses to TAg reflect biologic variation among viral strains with respect to the process of viral infection. In contrast, we expected that neutralizing antibody responses arise primarily against the injected viral particles, which represent a single serotype, and therefore are less informative about viral variation. We report here that an analysis revealed that the route of inoculation—in combination with viral genetic variation—significantly influences the development of serum antibodies to SV40 large TAg but not the titer of those antibodies in virus-exposed, tumor-free hamsters. The route of inoculation—but not viral genetic variation—influenced both the frequency of development and the titers of virus-neutralizing antibodies in the same animals.     

Mer receptor tyrosine kinase mediates both tethering and phagocytosis of apoptotic cells

Billions of inflammatory leukocytes die and are phagocytically cleared each day. This regular renewal facilitates the normal termination of inflammatory responses, suppressing pro-inflammatory mediators and inducing their anti-inflammatory counterparts. Here we investigate the role of the receptor tyrosine kinase (RTK) Mer and its ligands Protein S and Gas6 in the initial recognition and capture of apoptotic cells (ACs) by macrophages. We demonstrate extremely rapid binding kinetics of both ligands to phosphatidylserine (PtdSer)-displaying ACs, and show that ACs can be co-opsonized with multiple PtdSer opsonins. We further show that macrophage phagocytosis of ACs opsonized with Mer ligands can occur independently of a requirement for αV integrins. Finally, we demonstrate a novel role for Mer in the tethering of ACs to the macrophage surface, and show that Mer-mediated tethering and subsequent AC engulfment can be distinguished by their requirement for Mer kinase activity. Our results identify Mer as a receptor uniquely capable of both tethering ACs to the macrophage surface and driving their subsequent internalization.Many diseases, including rheumatoid arthritis, pulmonary fibrosis, adult respiratory distress syndrome, and inflammatory bowel disease,^{1, 2, 3, 4} are commonly marked by impaired resolution of inflammation that is linked to defects in the phagocytic clearance of apoptotic cells.^{5, 6, 7} Apoptotic cell (AC) clearance normally eliminates a plethora of pro-inflammatory stimuli,^{8, 9} and the recognition of ACs by phagocytes¹⁰ limits progression to necrosis,¹¹ suppresses pro-inflammatory mediator production, and induces IL-10 and TGF-β release.^{12, 13} As defective clearance of ACs is associated with the development of inflammatory disease and autoimmunity,^{14, 15} new therapeutic approaches designed to increase the capacity of phagocytes to remove ACs could effectively promote the resolution of inflammation.Phagocytosis of ACs can be regulated by soluble mediators, including cytokines,^{16, 17} prostaglandins and lipoxins,^{17, 18, 19} serum proteins,²⁰ agonists of Liver X receptors (LXRs),^{17, 21} and glucocorticoids (GC).^{17, 22} In particular, LXR agonists and GCs promote phagocytosis of ACs predominantly via a Tyro3/Axl/Mer (TAM) receptor tyrosine kinase (RTK)-dependent pathway.^{17, 21, 23} There are two established ligands for the TAM RTKs, Protein S (gene name Pros1), which activates Tyro3 and Mer, and Gas6, which activates all three TAMs,^{24, 25} although other ligands have been suggested.^{26, 27} The amino terminal Gla domains of Protein S and Gas6 bind to phosphatidylserine (PtdSer) on the plasma membrane of ACs,²⁸ a potent ‘eat-me'' signal by which ACs are recognized by phagocytes.²⁹ TAM receptors bind to the carboxy terminal domains of Protein S and Gas6, which effectively act as molecular ‘bridges'' between PtdSer on the AC and TAM receptors on the phagocyte.^{17, 30, 31} TAM receptor- and ligand-deficient mice exhibit defective phagocytic pruning of photoreceptor outer segments by retinal pigment epithelial (RPE) cells of the eye,^{32, 33, 34} defective clearance of apoptotic germ cells by Sertoli cells of the testis,³⁵ and defective clearance of ACs by macrophages/dendritic cells in lymphoid organs.³⁶ These phenotypes are also detectable in Mer (gene name Mertk) single knockouts.³⁷ In addition to phagocytic clearance, TAM signaling also has a pivotal role in controlling the innate immune response to pathogenic stimuli.^{13, 17, 38}Although the importance of Mer in the internalization of ACs by macrophages is now well-established, this receptor has been thought not to have a significant role in the initial ‘tethering'' of ACs to the macrophage surface.^{36, 39} In their studies, Scott et al.³⁶ used peritoneal macrophages for which tethering of ACs has now been shown to be mediated by T-cell immunoglobulin and mucin domain-containing molecule 4 (TIM4).³⁹ Subsequent internalization of tethered ACs is then mediated by either integrin αvβ3- or Mer-mediated signaling.^{39, 40} Similarly, for RPE cells, the initial capture of photoreceptor outer segments by RPE cells required the integrin αvβ5,⁴¹ with Mer-dependent signaling necessary for subsequent internalization. To further probe the mechanistic role of Mer in AC recognition and engulfment, we have now examined macrophages that predominantly use a Mer-dependent AC phagocytosis mechanism.^{17, 23} We show that in these cells, which do not express TIM4, Mer has the capacity to serve a unique dual role in mediating both tethering of ACs to the macrophage surface as well as subsequent AC engulfment.     

Depletion of white adipocyte progenitors induces beige adipocyte differentiation and suppresses obesity development

Overgrowth of white adipose tissue (WAT) in obesity occurs as a result of adipocyte hypertrophy and hyperplasia. Expansion and renewal of adipocytes relies on proliferation and differentiation of white adipocyte progenitors (WAP); however, the requirement of WAP for obesity development has not been proven. Here, we investigate whether depletion of WAP can be used to prevent WAT expansion. We test this approach by using a hunter-killer peptide designed to induce apoptosis selectively in WAP. We show that targeted WAP cytoablation results in a long-term WAT growth suppression despite increased caloric intake in a mouse diet-induced obesity model. Our data indicate that WAP depletion results in a compensatory population of adipose tissue with beige adipocytes. Consistent with reported thermogenic capacity of beige adipose tissue, WAP-depleted mice display increased energy expenditure. We conclude that targeting of white adipocyte progenitors could be developed as a strategy to sustained modulation of WAT metabolic activity.Obesity, a medical condition predisposing to diabetes, cardiovascular diseases, cancer, and complicating other life-threatening diseases, is becoming an increasingly important social problem.^{1, 2, 3} Development of pharmacological approaches to reduction of body fat has remained a daunting task.⁴ Approved obesity treatments typically produce only moderate and temporary effects.^2,5 White adipocytes are the differentiated cells of white adipose tissue (WAT) that store triglycerides in lipid droplets.^6,7 In contrast, adipocytes of brown adipose tissue (BAT) dissipate excess energy through adaptive thermogenesis. Under certain conditions, white adipocytes can become partially replaced with brown-like ‘beige'' (‘brite'') adipocytes that simulate the thermogenic function of BAT adipocytes.^7,8 Obesity develops in the context of positive energy balance as a result of hypertrophy and hyperplasia of white adipocytes.⁹Expansion and renewal of the white adipocyte pool in WAT continues in adulthood.^10,11 This process is believed to rely on proliferation and self-renewal of mesenchymal precursor cells¹² that we term white adipocyte progenitors (WAPs). WAPs reside within the population of adipose stromal cells (ASCs)¹³ and are functionally similar to bone marrow mesenchymal stem cells (MSCs).^{14, 15, 16} ASCs can be isolated from the stromal/vascular fraction (SVF) of WAT based on negativity for hematopoietic (CD45) and endothelial (CD31) markers.^17,18 ASCs support vascularization as mural/adventitial cells secreting angiogenic factors^5,19 and, unlike bone marrow MSCs, express CD34.^19,20 WAPs have been identified within the ASC population based on expression of mesenchymal markers, such as platelet-derived growth factor receptor-β (PDGFRβ, aka CD140b) and pericyte markers.^17,18 Recently, a distinct ASC progenitor population capable of differentiating into both white and brown adipocytes has been identified in WAT based on PDGFRα (CD140a) expression and lack of PDGFRβ expression.^21,22 The physiological relevance of the two precursor populations residing in WAT has not been explored.We have previously established an approach to isolate peptide ligands binding to receptors selectively expressed on the surface of cell populations of interest.^{23, 24, 25, 26, 27} Such cell-targeted peptides can be used for targeted delivery of experimental therapeutic agents in vivo. A number of ‘hunter-killer'' peptides²⁸ composed of a cell-homing domain binding to a surface marker and of KLAKLAK₂ (sequence KLAKLAKKLAKLAK), a moiety inducing apoptosis upon receptor-mediated internalization, has been described by our group.^26,29 Such bimodal peptides have been used for depletion of malignant cells and organ-specific endothelial cells in preclinical animal models.^26,30,31 Recently, we isolated a cyclic peptide WAT7 (amino acid sequence CSWKYWFGEC) based on its specific binding to ASCs.²⁰ We identified Δ-decorin (ΔDCN), a proteolytic cleavage fragment of decorin, as the WAT7 receptor specifically expressed on the surface of CD34+PDGFRβ+CD31-CD45- WAPs and absent on MSCs in other organs.²⁰Here, we investigated whether WAPs are required for obesity development in adulthood. By designing a new hunter-killer peptide that directs KLAKLAK₂ to WAPs through WAT7/ΔDCN interaction, we depleted WAP in the mouse diet-induced obesity model. We demonstrate that WAP depletion suppresses WAT growth. We show that, in response to WAP deficiency, WAT becomes populated with beige adipocytes. Consistent with the reported thermogenic function of beige adipocytes,^32,33 the observed WAT remodeling is associated with increased energy expenditure. We identify a population of PDGFRα-positive, PDGFRβ-negative ASCs reported recently²² as a population surviving WAP depletion and responsible for WAT browning.     

Pharmacologic inhibition of reactive gliosis blocks TNF-α-mediated neuronal apoptosis

Reactive gliosis is an early pathological feature common to most neurodegenerative diseases, yet its regulation and impact remain poorly understood. Normally astrocytes maintain a critical homeostatic balance. After stress or injury they undergo rapid parainflammatory activation, characterized by hypertrophy, and increased polymerization of type III intermediate filaments (IFs), particularly glial fibrillary acidic protein and vimentin. However, the consequences of IF dynamics in the adult CNS remains unclear, and no pharmacologic tools have been available to target this mechanism in vivo. The mammalian retina is an accessible model to study the regulation of astrocyte stress responses, and their influence on retinal neuronal homeostasis. In particular, our work and others have implicated p38 mitogen-activated protein kinase (MAPK) signaling as a key regulator of glutamate recycling, antioxidant activity and cytokine secretion by astrocytes and related Müller glia, with potent influences on neighboring neurons. Here we report experiments with the small molecule inhibitor, withaferin A (WFA), to specifically block type III IF dynamics in vivo. WFA was administered in a model of metabolic retinal injury induced by kainic acid, and in combination with a recent model of debridement-induced astrocyte reactivity. We show that WFA specifically targets IFs and reduces astrocyte and Müller glial reactivity in vivo. Inhibition of glial IF polymerization blocked p38 MAPK-dependent secretion of TNF-α, resulting in markedly reduced neuronal apoptosis. To our knowledge this is the first study to demonstrate that pharmacologic inhibition of IF dynamics in reactive glia protects neurons in vivo.Astrocyte reactivity (reactive gliosis) is an early pathological feature common to most neurodegenerative diseases, yet its regulation and impact remains poorly understood. In the healthy central nervous system (CNS), astrocytes coordinate homeostatic vascular perfusion, free radical detoxification and neurotransmitter recycling.^{1, 2} Injury or stress induces a phenotypic switch, whose cardinal features are cellular hypertrophy and increased expression and polymerization of type III intermediate filaments (IFs), particularly glial fibrillary acidic protein (GFAP).^{3, 4, 5} The role of intermediate filaments in reactive gliosis remains unclear.^{3, 6, 7, 8, 9} Genetic deletion of IFs GFAP and vimentin have been shown to promote axonal outgrowth and regeneration in developing neurons and models of CNS injury,^{10, 11, 12} yet result in developmental defects to inner retinal function¹³ and increased damage in models of Alzheimer''s disease.¹⁴ Genetically, GFAP gain of function mutations associated with Alexander''s disease induce a p38 mitogen-activated protein kinase (MAPK)-dependent pathology.¹⁵ However, no pharmacologic tools have been available to specifically modulate and explore this reactive switch in the context of pathological CNS injury. Consequently, strategies to therapeutically target the reactive switch have remain challenging to explore.Withaferin A (WFA) is a small molecule withanolide that is a potent and specific inhibitor of type III intermediate filament dynamics.^{16, 17, 18} Its activity has been most closely studied with respect to vimentin rearrangement and phosphorylation in the context of angiogenesis, fibrosis and cancer, through downstream effects on inflammatory signaling and cell proliferation.^{19, 20, 21, 22, 23, 24} Interestingly, WFA has been reported to regulate vimentin-mediated activation of MAPKs in a context dependent manner, as well as NFκB.^{25, 26} Recently Bargagna-Mohan et al.²⁷ reported that, in addition to vimentin, WFA also binds covalently to GFAP at cysteine 294. In these studies WFA impaired GFAP filament assembly and polymerization in cultured astrocytes, and in vivo in retinal astrocytes and related Müller glia in a model of injury-induced gliosis.²⁷ Therefore, WFA presents a novel tool to test the pharmacologic blockade of intermediate filament remodeling during gliosis. However, the consequences of WFA disruption of IFs on neuronal damage has not been studied.We have previously used the retina as a uniquely accessible model to study the regulation of astrocyte stress responses, and their influence on retinal neuronal survival.^{28, 29, 30} In the human and rodent eye retinal ganglion cells (RGCs) and amacrine cells of the inner retina maintain a delicate homeostatic balance and are particularly vulnerable to excitotoxic and metabolic damage, mediated in part through non-cell autonomous interactions with neighboring glia.^{31, 32, 33, 34} In addition, our work and others has implicated signaling through p38 MAPKs as key regulators of glutamate recycling, antioxidant activity, and cytokine secretion in neighboring stress-activated retinal astrocytes and Müller glia.^{29, 35, 36, 37} Here we take advantage of a model of induced retinal astrocyte reactivity to establish whether WFA, and the selective p38 MAPK inhibitor SB203580 (SB), affect neuronal apoptosis in a mouse model of excitotoxic injury.     

Safety and Colonization of Two Novel virG(icsA)-based Live Shigella sonnei Vaccine Strains in Rhesus Macaques (Macaca mulatta)

Shigella are gram-negative bacterium that cause bacillary dysentery (shigellosis). Symptoms include diarrhea and discharge of bloody mucoid stools, accompanied by severe abdominal pain, nausea, vomiting, malaise, and fever. Persons traveling to regions with poor sanitation and crowded conditions become particularly susceptible to shigellosis. Currently a vaccine for Shigella has not been licensed in the United States, and the organism quickly becomes resistant to medications. During the past 10 y, several live attenuated oral Shigella vaccines, including the strain WRSS1, have been tested in humans with considerable success. These Phase I vaccines lack the gene for the protein VirG also known as IcsA, which enables the organism to disseminate in the host target tissue. However, 5% to 20% of the vaccinated volunteers developed mild fever and brief diarrhea, and the removal of additional virulence-associated genes from the vaccine strain may reduce or eliminate these side effects. We administered 2 Shigella sonnei vaccines, WRSs2 and WRSs3, along with WRSS1 to compare their rates of colonization and clinical safety in groups of 5 rhesus macaques. The primate model provides the most physiologically relevant animal system to test the validity and efficacy of vaccine candidates. In this pilot study using a gastrointestinal model of infection, the vaccine candidates WRSs2 and WRSs3, which have additional deletions in the enterotoxin and LPS modification genes, provided better safety and comparable immunogenicity to those of WRSS1.Abbreviations: IcsA, intercellular spread protein A (VirG protein); Ipa, invasion plasmid antigen; LPS, lipopolysaccharideVarious species of Shigella cause bacillary dysentery, also known as shigellosis, in humans and other primates.^{1,18,31,37,49} Symptoms include diarrhea, with various degrees of mucus and hematochezia, accompanied by severe abdominal pain, nausea, vomiting, malaise, and fever. Shigella is a gram-negative bacterium whose genomic sequence is very similar to that of Escherichia coli and is phylogenetically considered a pathotype of E. coli.²³ Shigella infections are spread by the fecal–oral route through the consumption of contaminated food and water or by mechanical vectors such as insects. Persons traveling in areas with poor sanitation and crowded conditions become particularly susceptible to such diseases.^2,39,48,56 In colonies of research macaques, Shigella infections are spread by the fecal–oral route, originating from addition of animals that are asymptomatic carriers, either from the wild or from other colonies.^1,55 Currently no Shigella vaccine has been licensed in the United States, although several are under development.^{25,32,35,38,54} Antibiotics are used as treatment therapy, but many Shigella isolates are multidrug-resistant, increasing the need for a preventive vaccine for travelers, military personnel, and children in endemic areas.^{16,34,41,44,57} Rhesus macaques represent an excellent model for studying Shigella because primates are the only known animal model that simulate natural human infection, including dysentery after oral challenge.^{11-13,17,28,40,47,46} Other animal models, such as the mouse pulmonary model and the guineapig ocular and intranasal models, mimic specific, isolated steps in Shigella pathogenesis.^14,19,26There are 4 major serogroups of Shigella and one or more serotypes within each serogroup. This classification is based on antigenic differences in the O-antigen polysaccharide component of the outer membrane-associated lipopolysaccharide (LPS). Estimates from the Centers for Disease Control (Atlanta, GA) indicate that approximately 400,000 cases of shigellosis occur in the US annually, with an estimated 165 million cases worldwide each year.²² These occur predominantly in developing countries, where the population most affected is children younger than 5 y.²²Various in vitro cell culture models of infection, as well as studies in animal models including gastrointestinal infection in nonhuman primates, have contributed to the current understanding of Shigella pathogenesis.^4,42 Shigella organisms target the distal region of the colon and rectum, where the bacteria are captured by specialized M-cells located within the follicle-associated epithelium. The M-cells deliver bacterial antigens, which include bacterial LPS and invasion plasmid antigen (Ipa) proteins, to the underlying antigen-presenting macrophages and dendritic cells.³⁶ Virulent Shigella strains escape macrophages by a cytotoxic effect, causing release of the bacteria and inflammatory cytokines such as IL1β, TNFα, and IL18. The bacteria then invade adjacent intestinal epithelial cells at the basolateral side, through interaction between bacterial proteins and multiple signaling molecules within the host cell.^36,42Shigella are capable of orchestrating this uptake into nonphagocytic epithelial cells, a process termed invasion, through the secretion of proteins that are highly conserved among all virulent strains. These proteins are encoded by a large, 213-kb plasmid, referred to as the invasion plasmid or the virulence plasmid.^50-52 Each bacterium ultimately is engulfed within an endocytic vacuole. Subsequent lysis of the vacuole sets the bacterium free within the epithelial cell cytoplasm, where it replicates and moves to adjacent epithelial cells, with the help of the intercellular spread (Ics) A protein, also known as VirG.^3,24,45 The VirG(IcsA)-assisted intra- and intercellular spread of the bacteria within the epithelial tissue contributes significantly to the loss of epithelial cell integrity and accompanying tissue injury. Shigella strains with loss of the virG(icsA) gene are significantly attenuated in all animal models of virulence.⁵ Several Shigella vaccine candidates that have undergone Phase 1 clinical trials are principally attenuated due to lack of the VirG(IcsA) protein, these include S. flexneri 2a strain SC602, S. sonnei strain WRSS1, and S. dysenteriae 1 vaccine strain WRSd1.^{5,10,15,21,53}The S. sonnei first-generation vaccine candidate WRSS1 has been tested in several Phase I inpatient and outpatient trials.^15,21,33 WRSS1, like the previous S. flexneri 2a vaccine candidate, SC602, was shown to be safe in Phase I trials when orally administered at a dose of 10³ to 10⁴ CFU.^15,21,33 Both SC602 and WRSS1 colonized well as evidenced by fecal excretion of the vaccine strain for 5 to 7 d. The vigorous immune response seen with both these vaccine candidates was correlated with the robust colonization. However, 5% to 20% of the SC602- and WRSS1-immunized volunteers showed mild and transient symptoms of diarrhea and fever. In light of new knowledge about Shigella pathogenesis as well as data from other clinical trials, 2 new and presumably safer S. sonnei derivatives have been constructed (WRSs2 and WRSs3). In addition to loss of virG(icsA), WRSs2 has deletions in 2 genes, senA and senB, that are present on the virulence plasmid.^29,52 senA (also known as shet2-1 and ospD3) encodes the enterotoxin ShET2-1, which has been shown to cause fluid accumulation in rabbit ileal loops.^7,8 senB (also known as shet2-2 and ospD2) encodes a similarly sized protein as SenA and shows 40% identity at the amino acid level. The enterotoxic activity of ShET2-2 remains to be demonstrated.^20,29 Like WRSs2, WRSs3 lacks virG(icsA) and the 2 enterotoxin genes with the additional deletion of the virulence plasmid-based msbB2 gene. Lack of the msbB2 gene product has previously been shown to produce a less-toxic LPS molecule, which might help to reduce the symptoms of fever seen in WRSS1-immunized volunteers.^20,29 How these additional mutations, especially that of msbB2, will affect colonization of these strains is unclear, given that effective colonization is critical in the elicitation of immune response and protection. The primary goal of this pilot study was to compare the safety and colonization of 2 novel S. sonnei vaccine candidate strains (WRSs2 and WRSs3), with those of the clinically tested vaccine strain WRSS1, by using a gastrointestinal model of infection. The hypothesis tested was that the 2 novel Shigella vaccine strains would colonize the vaccinated animal and induce an immune response similar to that of WRSS1 but with less frequent and less severe clinical side effects.