Omentin protects against LPS-induced ARDS through suppressing pulmonary inflammation and promoting endothelial barrier via an Akt/eNOS-dependent mechanism

Acute respiratory distress syndrome (ARDS) is characterized by increased pulmonary inflammation and endothelial barrier permeability. Omentin has been shown to benefit obesity-related systemic vascular diseases; however, its effects on ARDS are unknown. In the present study, the level of circulating omentin in patients with ARDS was assessed to appraise its clinical significance in ARDS. Mice were subjected to systemic administration of adenoviral vector expressing omentin (Ad-omentin) and one-shot treatment of recombinant human omentin (rh-omentin) to examine omentin''s effects on lipopolysaccharide (LPS)-induced ARDS. Pulmonary endothelial cells (ECs) were treated with rh-omentin to further investigate its underlying mechanism. We found that a decreased level of circulating omentin negatively correlated with white blood cells and procalcitonin in patients with ARDS. Ad-omentin protected against LPS-induced ARDS by alleviating the pulmonary inflammatory response and endothelial barrier injury in mice, accompanied by Akt/eNOS pathway activation. Treatment of pulmonary ECs with rh-omentin attenuated inflammatory response and restored adherens junctions (AJs), and cytoskeleton organization promoted endothelial barrier after LPS insult. Moreover, the omentin-mediated enhancement of EC survival and differentiation was blocked by the Akt/eNOS pathway inactivation. Therapeutic rh-omentin treatment also effectively protected against LPS-induced ARDS via the Akt/eNOS pathway. Collectively, these data indicated that omentin protects against LPS-induced ARDS by suppressing inflammation and promoting the pulmonary endothelial barrier, at least partially, through an Akt/eNOS-dependent mechanism. Therapeutic strategies aiming to restore omentin levels may be valuable for the prevention or treatment of ARDS.Acute respiratory distress syndrome (ARDS) is a devastating condition with a 30–60% mortality rate.^{1, 2} Although the pathogenesis of ARDS is complex, the inflammatory response and endothelial barrier disruption play important roles in the development of ARDS.^{3, 4, 5} Therefore, in addition to conventional anti-inflammatory treatments, therapeutic strategies aim to restore pulmonary endothelial barrier integrity and function through regulating inter-endothelial AJs and the endothelial cytoskeleton to minimize protein leakage and leukocyte infiltration under ARDS conditions.^{6, 7}Obesity, especially visceral obesity, has clearly been shown to impair systemic vasculature and to lead to the initiation and progression of vascular disorders.^{8, 9, 10} Although different from the well-documented impacts of obesity on cardiovascular disease, the relationships between obesity and ARDS have not been well elucidated. Clinical and experimental data focused on pertinent physiological changes in obesity indicate that the obesity may alter ARDS pathogenesis by ‘priming'' the pulmonary endothelial barrier for insult and amplifying the early inflammatory response, thus lowering the threshold to initiate ARDS.^{11, 12} Contrary to conventional dogma, adipose tissue is now appreciated as an important endocrine tissue that secretes various bioactive molecules called adipokines, which contribute to the progression of diverse vascular diseases, including hypertension, cardiovascular disease and atherosclerosis.^{13, 14, 15, 16} Although ARDS is not a classified pulmonary vascular disease, it is a severe inflammatory lung condition with widespread pulmonary endothelial breakdown. Clinical evidence has indicated that the obesity might be an emerging risk factor for ARDS and that circulating adipokines levels are associated with the initiation and progression of ARDS.^{11, 12, 17, 18} Moreover, experimental studies have suggested that some anti-inflammatory adipokines, such as adiponectin and apelin, exert beneficial actions on ARDS.^{19, 20, 21}Omentin is an anti-inflammatory adipokine that is abundant in human visceral fat tissue.^{22, 23} Paradoxically, higher circulating omentin-1 levels are present in lean and healthy individuals compared with the obese and diabetic patients. Moreover, as a novel biomarker of endothelial dysfunction, reduced circulating omentin levels are related to the pathological mechanism of obesity-linked vascular disorders, including type 2 diabetes, atherosclerosis, hypertension and cardiovascular disease.^{24, 25, 26, 27, 28} Furthermore, experimental studies have found that omentin stimulates vasodilation in isolated blood vessels and suppresses cytokine-stimulated inflammation in endothelial cells (ECs).^{29, 30, 31} Thus, these data suggest that omentin may protect against obesity-related vascular complications through its anti-inflammatory and vascular-protective properties; however, little is known regarding its role in lung tissue. It was reported that decreased circulating omentin-1 levels could be regarded as an independent predictive marker for the obstructive sleep apnea syndrome and that omentin protects against pulmonary arterial hypertension through inhibiting vascular structure remodeling and abnormal contractile reactivity.^{32, 33, 34} However, to our knowledge, no study has assessed the impact of omentin on ARDS.Akt-related signaling pathways function as an endogenous negative feedback mechanism in response to the injurious stimulus. Our prior studies have demonstrated that Akt-related signaling contributes to protection against ARDS.^{35, 36} Moreover, omentin has been reported to exert anti-inflammatory, pro-survival and pro-angiogenic functions in various cells via an Akt-dependent mechanism.^{30, 31, 37, 38, 39, 40, 41, 42}Collectively, given that ARDS is ultimately an obesity-related disorder of vascular function and that omentin is a favorable pleiotropic adipokine capable of anti-inflammatory, pro-angiogenic and anti-apoptotic abilities; omentin may exert beneficial effects on ARDS. In the present study, we first aimed to appraise the clinical significance of omentin in ARDS and then specifically evaluated its impact on inflammation and the endothelial barrier. Furthermore, we mechanistically investigated the role of Akt-related signaling pathways in these effects induced by omentin in vivo and in vitro.     

Thrombospondin-1 signaling through CD47 inhibits cell cycle progression and induces senescence in endothelial cells

CD47 signaling in endothelial cells has been shown to suppress angiogenesis, but little is known about the link between CD47 and endothelial senescence. Herein, we demonstrate that the thrombospondin-1 (TSP1)-CD47 signaling pathway is a major mechanism for driving endothelial cell senescence. CD47 deficiency in endothelial cells significantly improved their angiogenic function and attenuated their replicative senescence. Lack of CD47 also suppresses activation of cell cycle inhibitors and upregulates the expression of cell cycle promoters, leading to increased cell cycle progression. Furthermore, TSP1 significantly accelerates replicative senescence and associated cell cycle arrest in a CD47-dependent manner. These findings demonstrate that TSP1-CD47 signaling is an important mechanism driving endothelial cell senescence. Thus, TSP1 and CD47 provide attractive molecular targets for treatment of aging-associated cardiovascular dysfunction and diseases involving endothelial dysregulation.Endothelial cell (EC) senescence is accompanied with vascular dysfunction, including arterial stiffening and remodeling,¹ impaired angiogenesis,^{2, 3} reduced endothelial repair capability and increased incidence of cardiovascular disease.^{4, 5, 6} Cellular senescence can occur in vivo or in vitro in response to various stressors,^{7, 8, 9, 10} leading to suppression of cell proliferation. EC senescence has been reported to contribute to the pathogenesis of age-associated vascular diseases, such as atherosclerosis.¹¹ Thus, further understanding the mechanisms of EC senescence may help to identify effective targets for antisenescence therapy and treatment aging-associated cardiovascular disorders.Previous studies have shown that the secreted matricellular protein thrombospondin-1 (TSP1) is as potent inhibitor of angiogenesis¹² and its antiangiogenic activity is mediated by its receptors, CD36^{13, 14} and CD47.^{15, 16} CD47 is a ubiquitously expressed transmembrane protein that serves as a ligand for signal regulatory protein-α and is a signaling receptor of TSP1. The TSP1-CD47 pathway has an important role in several fundamental cellular functions, including proliferation, apoptosis, inflammation and atherosclerotic response.¹⁷ Ligation of CD47 by TSP1 has been shown to inhibit nitric oxide (NO)/cGMP signaling in vascular cells, leading to suppression of angiogenic responses.¹⁶ Recently, it was reported that lack of CD47 expression in ECs may enable these cells to spontaneously gain characteristics of embryonic stem cells.¹⁸ However, the potential role of CD47 in regulation of EC senescence has not been well explored. The present study was initiated to determine the role and mechanisms of TSP1-CD47 signaling pathway in regulating cell cycle progression and replicative senescence of ECs.     

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.     

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.     

Genetic inactivation of the pancreatitis-inducible gene Nupr1 impairs PanIN formation by modulating KrasG12D-induced senescence

Nuclear protein 1 (Nupr1), a small chromatin protein, has a critical role in cancer development, progression and resistance to therapy. Previously, we had demonstrated that Nupr1 cooperates with Kras^G12D to induce pancreas intraepithelial neoplasias (PanIN) formation and pancreatic ductal adenocarcinoma development in mice. However, the molecular mechanisms by which Nupr1 influences Kras-mediated preneoplastic growth remain to be fully characterized. In the current study, we report evidence supporting a role for Nupr1 as a gene modifier of Kras^G12D-induced senescence, which must be overcome to promote PanIN formation. We found that genetic inactivation of Nupr1 in mice impairs Kras-induced PanIN, leading to an increase in β-galactosidase-positive cells and an upregulation of surrogate marker genes for senescence. More importantly, both of these cellular and molecular changes are recapitulated by the results of mechanistic experiments using RNAi-based inactivation of Nupr1 in human pancreatic cancer cell models. In addition, the senescent phenotype, which results from Nupr1 inactivation, is accompanied by activation of the FoxO3a-Skp2-p27^Kip1-pRb-E2F pathway in vivo and in vitro. Thus, combined, these results show, for the first time, that Nupr1 aids oncogenic Kras to bypass senescence in a manner that cooperatively promotes PanIN formation. Besides its mechanistic importance, this new knowledge bears medical relevance as it delineates early pathobiological events that may be targeted in the future as a means to interfere with the formation of preneoplastic lesions early during pancreatic carcinogenesis.Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with <5% survival after 5 years and a median survival of <6 months after diagnosis.¹ PDAC progresses from precursor lesions named pancreas intraepithelial neoplasias (PanINs). In this regard, it has been firmly established that oncogenic mutations in KRAS behave as one of the earliest stimuli for the formation of PanINs.^{2, 3} These data are strongly supported by animal models, such as the Pdx1-Cre; LSL-Kras^G12D transgenic mice, in which the pancreas-specific expression of oncogenic Kras promotes PanIN occurrence⁴ and, at a lower frequency, pancreatic cancer. Thus, the role of Kras as an initiating cancer mutation is one of the best-established pathobiological mechanisms required for the development of pancreatic cancer. Noteworthy, however, during the initiation stage, pancreatic cells not only trigger protumoral processes but also cellular events that aim at counteracting transformation. One of these tumor-suppressive processes elicited by Kras activation is cellular senescence (oncogene-induced senescence). In the pancreas, the induction of senescence underlies the resistance of exocrine cells to oncogenic Kras-mediated transformation⁵ so as to prevent tumor promotion, which is often supported by common diseases such as chronic pancreatitis.⁶ Indeed, tissue injury, as it occurs in pancreatitis, weakens the defense mechanism posed by senescence leading to its bypass by exocrine cells, which can then readily form PanINs.⁵ Therefore, the molecular mechanism that supports the development of oncogene-induced senescence (OIS) needs to be fully elucidated, if we want to advance our understanding of pancreatic cancer development.The nuclear protein 1 (Nupr1) is a basic helix–loop–helix molecule that is strongly induced by acute pancreatitis and several other cell stresses.^{7, 8} Nupr1 is also overexpressed in several types of human cancers, including PDAC. In this regard, the expression of genes that are targets for regulation by Nupr1 has been implicated in key protumorigenic pathways, including cell cycle regulation, matrix remodeling, autophagy, cell cannibalism and apoptosis inhibition.^{9, 10, 11, 12, 13, 14, 15, 16} Moreover, the fundamental role that Nupr1 has in pancreatic tumorigenesis is underscorded by recent results, which showed that, in mice, the oncogenic form of Kras^G12D is unable to promote PanINs in the absence of this chromatin protein,¹⁷ although the mechanisms responsible for this effect remain an area of active investigation. Consequently, we designed the current study with the aim of testing the hypothesis that Nupr1 cooperates with oncogenic Kras to induce PanIN formation by modulating the expression of gene networks that are necessary for bypassing senescence. To address this question, we characterized the effects that Nupr1 inactivation has on Kras-induced senescence using genome-wide expression profiling, as well as both cellular and molecular assays for this process. As a result of these experiments, we found that, indeed, the genetic inactivation of Nupr1 induces cellular senescence in exocrine pancreatic cells and reduces Kras-induced PanIN formation. At the molecular level, we demonstrated that this phenomenon is characterized by the upregulation of gene networks, which are known mediators of this phenomenon, by regulating at the G1/S transition. Taken together, these results provide mechanistic insights into how Nupr1 cooperates with Kras to promote the development of pancreatic preneoplastic lesions by discovering and characterizing a role for this pancreatitis-inducible protein in modulating cellular senescence. Thus, the new information emerging from this study has both mechanistic and biomedical implications for a better understanding of the pathobiology of pancreatic cancer.     

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.     

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.     

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.     

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.     

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.     

p73 is required for endothelial cell differentiation,migration and the formation of vascular networks regulating VEGF and TGFβ signaling

Vasculogenesis, the establishment of the vascular plexus and angiogenesis, branching of new vessels from the preexisting vasculature, involves coordinated endothelial differentiation, proliferation and migration. Disturbances in these coordinated processes may accompany diseases such as cancer. We hypothesized that the p53 family member p73, which regulates cell differentiation in several contexts, may be important in vascular development. We demonstrate that p73 deficiency perturbed vascular development in the mouse retina, decreasing vascular branching, density and stability. Furthermore, p73 deficiency could affect non endothelial cells (ECs) resulting in reduced in vivo proangiogenic milieu. Moreover, p73 functional inhibition, as well as p73 deficiency, hindered vessel sprouting, tubulogenesis and the assembly of vascular structures in mouse embryonic stem cell and induced pluripotent stem cell cultures. Therefore, p73 is necessary for EC biology and vasculogenesis and, in particular, that DNp73 regulates EC migration and tube formation capacity by regulation of expression of pro-angiogenic factors such as transforming growth factor-β and vascular endothelial growth factors. DNp73 expression is upregulated in the tumor environment, resulting in enhanced angiogenic potential of B16-F10 melanoma cells. Our results demonstrate, by the first time, that differential p73-isoform regulation is necessary for physiological vasculogenesis and angiogenesis and DNp73 overexpression becomes a positive advantage for tumor progression due to its pro-angiogenic capacity.Vascular system formation is one of the earliest events during organogenesis.¹ The original vascular plexus is established by vasculogenesis, through differentiation and assembly of mesodermal precursors.² The angiogenesis process allows the formation of new blood vessels from the existing vasculature and is perturbed in many diseases, including cancer.³ Although efforts have been made to identify factors that control vascular development, the understanding of the molecular networks remains incomplete.The formation of new capillaries and the remodeling of preexisting blood vessels is linked by signal transduction pathways.⁴ The members of the p53 family (p53, p73 and p63) coordinate cell proliferation, migration and differentiation, and could act as regulators of vascular development. TP73 function in angiogenesis is quite controversial,^{5, 6, 7} and it has never been addressed using developmental models.TP73 has a dual nature that resides in the existence of TA and DNp73 variants. TAp73 is capable of transactivating p53 targets^{8, 9, 10} whereas DNp73 can act as p53 and TAp73 repressor.^{11, 12, 13} TP73 final outcome will depend upon the differential expression of the TA/DNp73 isoforms in each cellular context, as they can execute synergic, as well as antagonist, functions.TP73 role during development is emphasized by the p73-knockout mice (Trp73−/−, p73KO from now on) multiple growth defects.¹⁴ These mice, which lack all p73 isoforms, exhibit gastrointestinal and cranial hemorrhages,¹⁴ suggestive of vascular fragility. Furthermore, TAp73 directly regulates GATA-1,⁸ which is essential for endothelial and hematopoietic differentiation.^{15, 16} This compounded information led us to hypothesize that p73 could be implicated in the regulation of vasculogenesis/angiogenesis.Regulation of these processes involves a broad range of signaling molecules essential for vascular growth and stability,¹⁷ such as vascular endothelial growth factor (VEGF)¹⁸ and transforming growth factor-β (TGF-β).¹⁹ TGF-β operates as a rheostat that controls endothelial cell (EC) differentiation, having an inhibitory effect on EC migration and proliferation by the TGF-β/TGFRI (ALK5)/Smad2/3 pathway, while the TβRII–ALK5/ALK1 complex activates Smad1/5/8, ID1 expression and a pro-angiogenic state.^{20, 21, 22}Regulation of the TGF-β and VEGF pathways by p53 family members has been documented.^{23, 24} However, p73''s function in these pathways during development remains largely unexplored. In this work, we have used mouse embryonic stem cells (mESC) and induced pluripotent stem cells (iPSCs) as models that recapitulate early vascular morphogenesis.^{25, 26, 27} ESC and iPSC form multi-cellular aggregates (embryoid bodies, EBs) that, under appropriate conditions, generate functional EC.²⁸ mESC and iPSC differentiation capacity into ECs has been fully addressed.^{29, 30} We have also performed retinal vascularization analysis to assess vascular processes in vivo.^{31, 32}We demonstrate that p73 deficiency perturbs density and stability of mouse retinal development by affecting VEGF and TGF-β signaling. Furthermore, p73 is necessary for the assembly of vascular structures under physiological conditions in mESC and iPSC. Moreover, DNp73 positively affects angiogenesis through regulation of the TGF-β pathway in human umbilical vein cells (HUVEC) and DNp73-overexpression results in enhanced angiogenic potential of B16-F10 melanoma cells.     

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.     

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.     

Role of Retinoic Acid and Fibroblast Growth Factor 2 in Neural Differentiation from Cynomolgus Monkey (Macaca fascicularis) Embryonic Stem Cells

Retinoic acid is a widely used factor in both mouse and human embryonic stem cells. It suppresses differentiation to mesoderm and enhances differentiation to ectoderm. Fibroblast growth factor 2 (FGF2) is widely used to induce differentiation to neurons in mice, yet in primates, including humans, it maintains embryonic stem cells in the undifferentiated state. In this study, we established an FGF2 low-dose-dependent embryonic stem cell line from cynomolgus monkeys and then analyzed neural differentiation in cultures supplemented with retinoic acid and FGF2. When only retinoic acid was added to culture, neurons differentiated from FGF2 low-dose-dependent embryonic stem cells. When both retinoic acid and FGF2 were added, neurons and astrocytes differentiated from the same embryonic stem cell line. Thus, retinoic acid promotes the differentiation from embryonic stem cells to neuroectoderm. Although FGF2 seems to promote self-renewal in stem cells, its effects on the differentiation of stem cells are influenced by the presence or absence of supplemental retinoic acid.Abbreviations: EB, embryoid body; ES, embryonic stem; ESM, embryonic stem cell medium; FGF, fibroblast growth factor; GFAP, glial fibrillary acidic protein; LIF, leukemia inhibitory factor; MBP, myelin basic protein; RA, retinoic acid; SSEA, stage-specific embryonic antigen; TRA, tumor-related antigenPluripotent stem cells are potential sources of material for cell replacement therapy and are useful experimental tools for in vitro models of human disease and drug screening. Embryonic stem (ES) cells are capable of extensive proliferation and multilineage differentiation, and thus ES-derived cells are suitable for use in cell-replacement therapies.^18,23 Reported ES cell characteristics including tumorigenic potential, DNA methylation status, expression of imprinted genes, and chromatin structure were elucidated by using induced pluripotent stem cells.^2,11,17 Because the social expectations of regeneration medicine are growing, we must perform basic research with ES cells, which differ from induced pluripotent stem cells in terms of origin, differentiation ability, and epigenetic status.^2,8Several advances in research have been made by using mouse ES cells. Furthermore, primate ES cell lines have been established from rhesus monkeys (Macaca mulatta),²⁴ common marmosets (Callithrix jacchus),²⁵ cynomolgus monkeys (M. fascicularis),²⁰ and African green monkeys (Chlorocebus aethiops).¹⁹ Mouse and other mammalian ES cells differ markedly in their responses to the signaling pathways that support self-renewal.^8,28 Mouse ES cells require leukemia inhibitory factor (LIF)–STAT3 signaling.¹⁴ In contrast, primate ES cells do not respond to LIF. Fibroblast growth factor 2 (FGF2) appears to be the most upstream self-renewal factor in primate ES cells. FGF2 also exerts its effects through indirect mechanisms, such as the TGFβ–Activin–Nodal signaling pathway, in primate ES cells.²¹ In addition to the biologic similarities between monkeys and humans, ES cells derived from cynomolgus monkeys or human blastocysts have extensive similarities that are not apparent in mouse ES cells.^8,14,21,28 Numerous monkey ES cell lines are now available, and cynomolgus monkeys are an efficient model for developing strategies to investigate the efficacy of ES-cell–based medical treatments in humans.Several growth factors and chemical compounds, including retinoic acid (RA),^4,9,13,22,26 FGF2,^9,10,16,22 epidermal growth factor,^9,22 SB431542,^1,4,10 dorsomorphin,^10,27 sonic hedgehog,^{12,13,16,27,29} and noggin,^1,4,9,27 are essential for the differentiation and proliferation or maintenance of neural stem cells derived from primate ES cells. Of these factors, active RA signaling suppresses a mesodermal fate by inhibiting Wnt and Nodal signaling pathways during in vitro culture and leads to neuroectoderm differentiation in ES cells.^4,13,26 RA is an indispensable factor for the specialization to neural cells. FGF2 is important during nervous system development,¹² and FGF2 and RA both are believed to influence the differentiation to neural cells. The current study was done to clarify the mechanism of RA and FGF2 in the induction of differentiation along the neural lineage.We recently established a monkey ES cell line that does not need FGF2 supplementation for maintenance of the undifferentiated state. This ES cell line allowed us to study the role of differentiation to neural cells with RA and enabled us to compare ES cell differentiation in the context of supplementation with RA or FGF2 in culture. To this end, we established a novel cynomolgus monkey cell line derived from ES cells and maintained it in an undifferentiated state in the absence of FGF2 supplementation.     

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}