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1.
Q Xia Q Hu H Wang H Yang F Gao H Ren D Chen C Fu L Zheng X Zhen Z Ying G Wang 《Cell death & disease》2015,6(3):e1702
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, 14Neuroinflammation, 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. 相似文献
2.
S L Petersen T T Chen D A Lawrence S A Marsters F Gonzalvez A Ashkenazi 《Cell death and differentiation》2015,22(11):1846-1857
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, 22The 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. 相似文献
3.
O Maximyuk V Khmyz C-J Lindskog V Vukojevi? T Ivanova I Bazov K F Hauser G Bakalkin O Krishtal 《Cell death & disease》2015,6(3):e1683
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 concentrations30, 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. 相似文献
4.
A Fauster M Rebsamen K V M Huber J W Bigenzahn A Stukalov C-H Lardeau S Scorzoni M Bruckner M Gridling K Parapatics J Colinge K L Bennett S Kubicek S Krautwald A Linkermann G Superti-Furga 《Cell death & disease》2015,6(5):e1767
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-mediated13, 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. 相似文献
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T Yabu H Shiba Y Shibasaki T Nakanishi S Imamura K Touhata M Yamashita 《Cell death and differentiation》2015,22(2):258-273
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 cytokines1, 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 相似文献
7.
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. 相似文献
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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 tissue1, 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, 46Here, 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. 相似文献
10.
Jamus G MacGuire Kari L Christe JoAnn L Yee Alexis L Kalman-Bowlus Nicholas W Lerche 《Comparative medicine》2009,59(2):168-173
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. 相似文献
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12.
Girdhar K. Pandey Poonam Kanwar Amarjeet Singh Leonie Steinhorst Amita Pandey Akhlilesh K. Yadav Indu Tokas Sibaji K. Sanyal Beom-Gi Kim Sung-Chul Lee Yong-Hwa Cheong J?rg Kudla Sheng Luan 《Plant physiology》2015,169(1):780-792
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. 相似文献
13.
Multivesicular bodies (MVBs) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these ILV contain Fas ligand (FasL) and are secreted as ''lethal exosomes'' following activation-induced fusion of the MVB with the plasma membrane. Diacylglycerol (DAG) and diacylglycerol kinase α (DGKα) regulate MVB maturation and polarized traffic, as well as subsequent secretion of pro-apoptotic exosomes, but the molecular basis underlying these phenomena remains unclear. Here we identify protein kinase D (PKD) family members as DAG effectors involved in MVB genesis and secretion. We show that the inducible secretion of exosomes is enhanced when a constitutively active PKD1 mutant is expressed in T lymphocytes, whereas exosome secretion is impaired in PKD2-deficient mouse T lymphoblasts and in PKD1/3-null B cells. Analysis of PKD2-deficient T lymphoblasts showed the presence of large, immature MVB-like vesicles and demonstrated defects in cytotoxic activity and in activation-induced cell death. Using pharmacological and genetic tools, we show that DGKα regulates PKD1/2 subcellular localization and activation. Our studies demonstrate that PKD1/2 is a key regulator of MVB maturation and exosome secretion, and constitutes a mediator of the DGKα effect on MVB secretory traffic.Exosomes are nanovesicles that form as intraluminal vesicles (ILVs) inside multivesicular bodies (MVBs) and are then secreted by numerous cell types.1 ILVs are generated by inward budding of late endosome limiting membrane in a precisely regulated maturation process.2, 3 Two main pathways are involved in MVB maturation.4, 5 In addition to the ESCRT (endosomal complex required for traffic) proteins,6 there is increasing evidence that lipids such as lyso-bisphosphatidic acid (LBPA),7 ceramides8 and diacylglycerol (DAG)9 contribute to this membrane invagination process.Exosomes participate in many biological processes related to T-cell receptor (TCR)-triggered immune responses, including T lymphocyte-mediated cytotoxicity and activation-induced cell death (AICD), antigen presentation and intercellular miRNA exchange.10, 11, 12, 13, 14, 15 The discovery of exosome involvement in these responses increased interest in the regulation of exosome biogenesis and secretory traffic, with special attention to the contribution of lipids such as ceramide and DAG, as well as DAG-binding proteins.14, 16, 17, 18, 19, 20, 21 These studies suggest that positive and negative DAG regulators may control secretory traffic. By transforming DAG into phosphatidic acid (PA), diacylglycerol kinase α (DGKα) is essential for the negative control of DAG function in T lymphocytes.22 DGKα translocates transiently to the T-cell membrane after human muscarinic type 1 receptor (HM1R) triggering or to the immune synapse (IS) after TCR stimulation; at these subcellular locations, DGKα acts as a negative modulator of phospholipase C (PLC)-generated DAG.23, 24The secretory vesicle pathway involves several DAG-controlled checkpoints at which DGKα may act; these include vesicle formation and fission at the trans-Golgi network (TGN), MVB maturation, as well as their transport, docking and fusion to the plasma membrane.9, 16, 17, 18, 19, 20 The molecular components that regulate some of these trafficking processes include protein kinase D (PKD) family members.21 PKD1 activity, for instance, regulates fission of transport vesicles from TGN via direct interaction with the pre-existing DAG pool at this site.19 The cytosolic serine/threonine kinases PKD1, PKD2 and PKD3(ref. 21) are expressed in a wide range of cells, with PKD2 the most abundant isotype in T lymphocytes.25, 26 PKD have two DAG-binding domains (C1a and C1b) at the N terminus,21 which mediate PKD recruitment to cell membranes. Protein kinase C (PKC) phosphorylation at the PKD activation loop further promotes PKD autophosphorylation and activation.27Based on our previous studies showing DGKα regulation of DAG in MVB formation and exosome secretion,9, 14, 28 and the identification of PKD1/2 association to MVB,14 we hypothesized that DGKα control of DAG mediates these events, at least in part, through PKD. Here we explored whether, in addition to its role in vesicle fission from TGN,19 PKD regulates other steps in the DAG-controlled secretory traffic pathway. Using PKD-deficient cell models, we analyzed the role of PKD1/2 in MVB formation and function, and demonstrate their implication in exosome secretory traffic. 相似文献
14.
B Oettinghaus J M Schulz L M Restelli M Licci C Savoia A Schmidt K Schmitt A Grimm L Morè J Hench M Tolnay A Eckert P D'Adamo P Franken N Ishihara K Mihara J Bischofberger L Scorrano S Frank 《Cell death and differentiation》2016,23(1):18-28
Well-balanced mitochondrial fission and fusion processes are essential for nervous system development. Loss of function of the main mitochondrial fission mediator, dynamin-related protein 1 (Drp1), is lethal early during embryonic development or around birth, but the role of mitochondrial fission in adult neurons remains unclear. Here we show that inducible Drp1 ablation in neurons of the adult mouse forebrain results in progressive, neuronal subtype-specific alterations of mitochondrial morphology in the hippocampus that are marginally responsive to antioxidant treatment. Furthermore, DRP1 loss affects synaptic transmission and memory function. Although these changes culminate in hippocampal atrophy, they are not sufficient to cause neuronal cell death within 10 weeks of genetic Drp1 ablation. Collectively, our in vivo observations clarify the role of mitochondrial fission in neurons, demonstrating that Drp1 ablation in adult forebrain neurons compromises critical neuronal functions without causing overt neurodegeneration.In addition to their crucial importance in energy conversion, mitochondria serve many other housekeeping functions, including calcium buffering, amino-acid and steroid biosynthesis as well as fatty acids beta-oxidation and regulation of cell death. During the past decade, it has become increasingly clear that processes regulating mitochondrial morphology and ultrastructure are influenced by specific cellular requirements upon which mitochondria, in a precisely regulated manner, undergo fusion and division events.1 Maintaining this balance is especially important for highly energy-consuming, polarized cells such as neurons, where single organellar units sprouting from the mitochondrial network are transported along the cytoskeleton into dendrites and spines to meet local energy requirements.2 In addition, elaborate quality-control mechanisms also rely on mitochondrial dynamics: whereas defective organelles are sequestered by fission, enabling their removal from the mitochondrial network,3, 4 fusion supports qualitative homogeneity of the syncytium through complementation.5Mitochondrial fusion and fission are mediated by large GTPases of the dynamin superfamily.6 The outer mitochondrial membrane mitofusins 1 (MFN1) and 2 (MFN2) tether mitochondrial membranes by homodimer or heterodimer formation,7 thereby initiating fusion of the organelles, a process that also involves the inner mitochondrial membrane-associated GTPase Optic Atrophy 1.8 In addition, MFN2 also mediates contacts between mitochondria and endoplasmic reticulum.9 The only known mammalian mitochondrial fission protein, Dynamin-Related Protein 1 (Drp1), translocates upon dephosphorylation by calcineurin10 to fission sites where it binds to mitochondrial fission factor.11 Drp1 translocation is preceded by ER membranes wrapping around mitochondria to constrict the organelles,12 thereby facilitating the formation of multimeric Drp1 complexes that, upon GTP hydrolysis, further tighten to complete the process of mitochondrial fission.13Genetic evidence in mice and humans indicates that mitochondrial dynamics are crucially important in neurons: in humans, a sporadic dominant-negative DRP1 mutation caused a lethal syndromic defect with abnormal brain development;14 similarly, constitutive Drp1 knockout in the mouse brain leads to lethal neurodevelopmental defects.15, 16 Although the crucial role of Drp1 during brain development is undisputed, studies on Drp1 function in postmitotic (adult) neurons are scarce; likewise, Drp1 ablation studies in primary cultures have so far failed to yield a conclusive picture. In vitro, Drp1 ablation is reported to lead to a super-elongated neuroprotective17, 18, 19, 20, 21, 22, 23, 24 or an aggregated mitochondrial phenotype associated with neurodegeneration.15, 16, 25, 26, 27 These discrepancies are probably due to different experimental conditions: neuronal health is indeed influenced by the onset and duration of Drp1 inhibition, which varies considerably among the cited reports,28 and different types of neuronal cultures studied display different sensitivity to Drp1 inhibition. In vivo, Drp1 ablation in Purkinje cells results in oxidative stress and neurodegeneration,29 demonstrating that Drp1 is essential for postmitotic neurons'' health. In contrast, transient pharmacological Drp1 inhibition is neuroprotective in several mouse ischemia models, indicating that temporarily blocking mitochondrial fission holds therapeutic potential.30, 31, 32To elucidate the consequences of blocked mitochondrial fission in the central nervous system in vivo, we bypassed the critical role of Drp1 during brain development by generating Drp1flx/flx mice15 expressing tamoxifen-inducible Cre recombinase under the control of the CaMKIIα promoter.33 Upon induced Drp1 deletion in postmitotic adult mouse forebrain neurons, mice develop progressive, neuronal subtype-specific alterations in mitochondrial shape and distribution in the absence of overt neurodegeneration. In addition, respiratory capacity, ATP content, synaptic reserve pool vesicle recruitment as well as spatial working memory are impaired, demonstrating that severely dysregulated mitochondrial dynamics can compromise critical neuronal functions in vivo without causing neuronal cell death. 相似文献
15.
A C Daquinag C Tseng A Salameh Y Zhang F Amaya-Manzanares A Dadbin F Florez Y Xu Q Tong M G Kolonin 《Cell death and differentiation》2015,22(2):351-363
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 factors5,19 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. 相似文献
16.
Amelia C Jones James G Herndon Cynthia L Courtney Lynn Collura Joyce K Cohen 《Comparative medicine》2014,64(3):200-210
In 2008, clinical observations in our colony of sooty mangabeys (Cercocebus atys) suggested a high frequency of type 2 diabetes. Postmortem studies of diabetic animals revealed dense amyloid deposits in pancreatic islets. To investigate these findings, we screened our colony (97 male mangabeys; 99 female mangabeys) for the disease from 2008 to 2012. The overall prevalence of diabetes was 11% and of prediabetes was 7%, which is nearly double that reported for other primate species (less than 6%). Fructosamine and triglyceride levels were the best indicators of diabetes; total cholesterol and glycated hemoglobin were not associated with disease. Increasing age was a significant risk factor: prevalence increased from 0% in infants, juveniles, and young adults to 11% in adults and 19% in geriatric mangabeys. Sex, medroxyprogesterone acetate exposure, and SIV status were unrelated to disease. Weight was marginally higher in prediabetics, but body condition did not indicate obesity. Of the 49 mangabeys that were necropsied after clinical euthanasia or death from natural causes, 22 were diabetic; all 22 animals demonstrated pancreatic amyloid, and most had more than 75% of islets replaced with amyloid. We conclude that type 2 diabetes is more common in mangabeys than in other primate species. Diabetes in mangabeys has some unusual pathologic characteristics, including the absence of altered cholesterol levels and glycated hemoglobin but a robust association of pancreatic insular amyloidosis with clinical diabetes. Future research will examine the genetic basis of mangabey diabetes and evaluate additional diagnostic tools using imaging and serum markers.Abbreviations: HbA1c, glycated hemoglobin; MPA, medroxyprogesterone acetate; YNPRC, Yerkes National Primate Research CenterSooty mangabeys (Cercocebus atys) are Old World NHP that are native to West Africa. Historically their use in research has been limited to infectious disease studies, leprosy studies, and behavioral research.14,25 Over the past 20 to 30 y, they have been used in HIV–AIDS research. Mangabeys are natural hosts of SIVsmm, which is recognized as the origin of HIV2 infection in humans.7,8,30,36,42 SIV typically is nonpathogenic in mangabeys despite high levels of virus replication, which makes this species a unique and invaluable model in AIDS research.7,30,36,42 Our facility maintains a colony of approximately 200 sooty mangabeys. In 2008 clinical observations of relative hyperglycemia, glucosuria, and weight loss in our colony suggested that type 2 diabetes mellitus occurred at a relatively high frequency in this population. Spontaneous diabetes was found in 10% of the colony, and 5% of animals were prediabetic; this incidence is higher than that typically reported for other NHP species, such as cynomolgus macaques (less than 1% to 2%)22 and chimpanzees (less than 1%).37 The prevalence of spontaneous diabetes in humans is typically 8.3%.2,6,22,37 In addition, necropsies revealed that many affected animals had dense amyloid deposits in pancreatic islet cells. Insular amyloidosis was seen on histology, with a total replacement of islets by amyloid deposition in advanced diabetes. Advanced diabetes was determined by increased weight loss and severity of relative hyperglycemia. The increased clinical prevalence of diabetes in our mangabey colony prompted additional characterization of the clinicopathologic profile, risk factors, and prevalence of diabetes in our mangabey colony.The form of diabetes in this mangabey colony is characterized as type 2 diabetes mellitus, as they have hyperglycemia, hypertriglyceridemia, and islet amyloidosis. Type 2 diabetes mellitus is the most common of the 3 forms of diabetes, and has been documented in humans and NHP,22,31,37,55 including rhesus macaques (Macaca mulatta), cynomolgus macaques (Macaca fascicularis), Celebes crested macaques (Macaca nigra), bonnet macaques (Macaca radiate), pigtailed macaques (Macaca nemestrina), vervet monkeys (Chlorocebus pygerythrus), squirrel monkeys (Saimiri sciureus), chimpanzees (Pan troglodytes), and woolly monkeys (Lagothrix spp.).1,24,31,52,55 Type 2 diabetes is a chronic metabolic disorder in which insulin resistance occurs in liver, muscle, and adipose tissue. As type 2 diabetes progresses, it also can be characterized as a relative insulin deficiency.1,6,15,22,29,31,37,55 The initial clinical presentation of diabetes in humans and NHP includes polydipsia, polyuria, polyphagia, weight loss, and lethargy.1,6,22,27,31,37,55 Similar presentation was observed in our colony of diabetic mangabeys.Diagnostic criteria of diabetes in NHP species is similar to that for humans and is based on clinical symptoms and routine lab tests, including serum chemistry panel to evaluate persistent fasting hyperglycemia, hypertriglyceridemia, and hypercholesterolemia.2,6,11,16-18,21,22,29,31,37,48-50,52,55 Hypertriglyceridemia and hypercholesterolemia frequently are elevated due to diabetes and therefore are used as supportive diagnostic markers. In addition, the disease is characterized by transient hyperinsulinemia followed by insulin deficiency subsequent to glucose challenge. Urinalysis is used to evaluate glucosuria and ketonuria. These tests are not exclusive for diagnosing diabetes and can be inconsistent between species, thus making conclusive diagnosis challenging. For example, hyperglycemia can be a transient finding associated with recent food intake or stress associated with restraint for blood sample collection or anesthetic access, whereas hypertriglyceridemia can be seen in obese animals and those with other metabolic diseases such as pancreatitis and hypothyroidism.1,22,37,55The typical clinical approach to the diagnosis of diabetes in NHP and other veterinary patients includes evaluation of fructosamine and glycated hemoglobin (HbA1c) levels and glucose tolerance testing. These tests are indices of glycemic control and are used in clinical settings primarily to assess prognosis and response to treatment; they are also useful for the initial diagnosis of diabetes when used in parallel with serum chemistry markers. Fructosamine and HbA1c can both provide information on long-term glycemic control, because fructosamine reflects average blood glucose levels over 2 to 3 wk whereas HbA1c reflects average blood glucose over 2 to 3 mo preceding blood collection. HbA1c is the primary test for diabetes in human medicine,6,31,35,37 whereas fructosamine is commonly used in veterinary medicine. Glucose tolerance testing provides an indirect measure of insulin sensitivity, but it is not frequently used clinically in NHP because of the requirement for prolonged physical restraint or sedation.1,21,22,26,27,34,37,55Prevention and management of diabetes in NHP and humans can be achieved by identifying potential risk factors, including age, weight, sex, genetics, hormone drug exposure, and viral status.1,6,15,22,29,31,37,42,55 Advanced age, obesity, sex, and genetics are associated with diabetes in some species of NHP and humans.1,6,15,22,29,31,37,55 In addition, exposure to drugs such as medroxyprogesterone acetate (MPA) is suspected to be linked to diabetes due to the hormonal effects of progesterone impacting glucoregulatory function.1,6,10,22,23,31,34,55 MPA exposure is of interest, because it is used regularly in our mangabey colony as both a contraceptive and as therapy for endometriosis. In addition, SIV status is being evaluated as a risk factor, because a portion of our colony is SIV positive. Although HIV is not thought to be associated with diabetes in people, SIV pathogenesis in mangabeys differs; therefore it was of interest to explore the possible association of SIV and diabetes in mangabeys.7,30,36,42 Pancreatic insular amyloidosis has been documented to be associated with type 2 diabetes in several species. Amyloidosis is a group of disorders that are caused by extracellular deposition of misfolded proteins that can result in impaired function of any organ.15,20,23,28,32,43,45,48,49 Because a high incidence of pancreatic insular amyloid was noted at necropsy, we sought to document the relationship with clinical diabetes in mangabeys.Spontaneous type 2 diabetes mellitus has been well documented in several species of NHP. Because the literature contains little information regarding the clinicopathologic features (the ‘profile’), risk factors, and prevalence of spontaneous diabetes mellitus in sooty mangabeys, the primary aims of the current study were 1) to determine whether elevated levels of fasting blood glucose, fructosamine, HbA1c, triglycerides, and total cholesterol levels are reliable diagnostic markers of type 2 diabetes mellitus in this NHP species; 2) to determine whether age, sex, MPA exposure, and SIV status influence the risk of diabetes; 3) to determine whether body weight influences diabetic status; 4) to evaluate the relationship between pancreatic amyloidosis and diabetes mellitus; and 5) to characterize the prevalence of diabetes mellitus in the mangabey population at our institution. To our knowledge, this report is the first to describe the natural occurrence of type 2 diabetes mellitus within a captive colony of sooty mangabeys. We hypothesized that blood glucose, fructosamine, HbA1c, triglyceride, and total cholesterol would be reliable diagnostic markers and that age, sex, and MPA exposure would influence the risk of diabetes in this species. 相似文献
17.
The deregulation of miR-101 and DNMT3a has been implicated in the pathogenesis of multiple tumor types, but whether and how miR-101 silencing and DNMT3a overexpression contribute to lung tumorigenesis remain elusive. Here we show that miR-101 downregulation associates with DNMT3a overexpression in lung cancer cell lines and patient tissues. Ectopic miR-101 expression remarkably abrogated the DNMT3a 3′-UTR luciferase activity corresponding to the miR-101 binding site and caused an attenuated expression of endogenous DNMT3a, which led to a reduction of global DNA methylation and the re-expression of tumor suppressor CDH1 via its promoter DNA hypomethylation. Functionally, restoration of miR-101 expression suppressed lung cancer cell clonability and migration, which recapitulated the DNMT3a knockdown effects. Interestingly, miR-101 synergized with decitabine to downregulate DNMT3a and to reduce DNA methylation. Importantly, ectopic miR-101 expression was sufficient to trigger in vivo lung tumor regression and the blockage of metastasis. Consistent with these phenotypes, examination of xenograft tumors disclosed an increase of miR-101, a decrease of DNMT3a and the subsequent DNA demethylation. These findings support that the loss or suppression of miR-101 function accelerates lung tumorigenesis through DNMT3a-dependent DNA methylation, and suggest that miR-101-DNMT3a axis may have therapeutic value in treating refractory lung cancer.Owing to a high propensity for recurrence and a high rate of metastasis at the advanced stages,1, 2, 3 lung cancer remains the leading cause of cancer-related mortality. DNA methylation is a major epigenetic rule controlling chromosomal stability and gene expression.4, 5 It is under control of DNA methyltransferases (DNMTs), whose overexpression in lung cancer cells predicts worse outcomes.6, 7 It is postulated that DNMT overexpression induces DNA hypermethylation and silencing of tumor suppressor genes (TSGs), leading to an aggressive lung cancer. Indeed, enforced expression of DNMT1 or DNMT3a increases DNA methylation, while the abolition of DNMT expression by genetic depletion, microRNAs (miRs) or small molecules reduces genome-wide and gene-specific DNA methylation and restores TSG expression.8, 9, 10, 11, 12, 13 As TSGs are the master controllers for cell multiplicity and their silencing predicts poor prognosis,14, 15 TSG re-expression via promoter DNA hypomethylation inhibits cell proliferation and induces cell differentiation.13, 16 Thus, DNMT gene abundance could serve as a target for anticancer therapy, but how DNMT upregulation occurs in lung cancer is incompletely understood.MiRs are small non-coding RNAs that crucially regulate target gene expression. Up to 30% of all protein-coding genes are predicted to be targeted by miRs,17, 18 supporting the key roles of miRs in controlling cell fate.19, 20, 21, 22 Research is showing that certain miRs are frequently dysregulated in cancers, including lung cancer.7, 23, 24 As miR targets can promote or inhibit cancer cell expansion, miRs have huge potential for acting as bona fide oncogenes (i.e., miR-21) or TSGs (i.e., miR-29b).7, 25 We and others demonstrated that the levels of DNMT1 or DNMT3a or DNMT3b are regulated by miR-29b, miR-148, miR-152 or miR-30c,7, 13, 26, 27 and overexpression of these miRs results in DNA hypomethylation and TSG reactivation with the concurrent blockage of cancer cell proliferation.7, 13 These findings underscore the importance of miRs as epigenetic modulators and highlight their therapeutic applications.MiR-101 is frequently silenced in human cancers28, 29, 30, 31 and, importantly, exhibits antitumorigenic properties when overexpressed. Mechanistically, miR-101 inactivation by genomic loss causes the overexpression of EZH2, a histone methyltransferase, via 3′-UTR targeting, which is followed by histone hypermethylation and aggressive tumorigenesis.29, 30, 32 However, whether and how miR-101 silencing contributes to DNA hypermethylation patterning in lung cancer is unclear. In this study, we explore the role of miR-101 in regulating DNMT3a expression and the impacts of miR-101-DNMT3a nexus on lung cancer pathogenesis. We showed that the expression of miR-101 and DNMT3a was negatively correlated in lung cancer. We presented evidence that ectopic miR-101 expression decreased DNMT3a levels, reduced global DNA methylation and upregulated CDH1 via its promoter DNA demethylation. The biological significance of miR-101-mediated DNA hypomethylation and CDH1 re-expression was evident by its inhibition of lung tumor cell growth in vitro and in vivo. Thus, our findings mechanistically and functionally link miR-101 silencing to DNA hypermethylation in lung cancer cells. 相似文献
18.
A Trocoli P Bensadoun E Richard G Labrunie F Merhi A M Schl?fli D Brigger S Souquere G Pierron J-M Pasquet P Soubeyran J Reiffers E Ségal-Bendirdjian M P Tschan M Djavaheri-Mergny 《Cell death and differentiation》2014,21(12):1852-1861
The p62/SQSTM1 adapter protein has an important role in the regulation of several key signaling pathways and helps transport ubiquitinated proteins to the autophagosomes and proteasome for degradation. Here, we investigate the regulation and roles of p62/SQSTM1 during acute myeloid leukemia (AML) cell maturation into granulocytes. Levels of p62/SQSTM1 mRNA and protein were both significantly increased during all-trans retinoic acid (ATRA)-induced differentiation of AML cells through a mechanism that depends on NF-κB activation. We show that this response constitutes a survival mechanism that prolongs the life span of mature AML cells and mitigates the effects of accumulation of aggregated proteins that occurs during granulocytic differentiation. Interestingly, ATRA-induced p62/SQSTM1 upregulation was impaired in maturation-resistant AML cells but was reactivated when differentiation was restored in these cells. Primary blast cells of AML patients and CD34+ progenitors exhibited significantly lower p62/SQSTM1 mRNA levels than did mature granulocytes from healthy donors. Our results demonstrate that p62/SQSTM1 expression is upregulated in mature compared with immature myeloid cells and reveal a pro-survival function of the NF-κB/SQSTM1 signaling axis during granulocytic differentiation of AML cells. These findings may help our understanding of neutrophil/granulocyte development and will guide the development of novel therapeutic strategies for refractory and relapsed AML patients with previous exposure to ATRA.p62 or sequestosome 1 (p62/SQSTM1) is a scaffold protein, implicated in a variety of biological processes including those that control cell death, inflammation, and metabolism.1, 2 Through its multi-domain structure, p62/SQSTM1 interacts specifically with key signaling proteins, including atypical PKC family members, NF-κB, and mTOR to control cellular responses.3, 4, 5, 6, 7 p62/SQSTM1 functions also as a key mediator of autophagy. Through its interaction with LC3, an essential protein involved in autophagy, p62/SQSTM1 selectively directs ubiquitinated substrates to autophagosomes leading to their subsequent degradation in lysosomes.8, 9 At the molecular level, p62/SQSTM1 acts as a pro-tumoral molecule by ensuring efficient and selective activation of cell signaling axes involved in cell survival, proliferation, and metabolism (i.e., NF-κB, mTOR, and Nrf-2 pathways).3, 5, 6, 7, 10, 11, 12, 13 p62/SQSTM1 can also signal anti-tumoral responses either by inactivating the pro-oncogenic signaling through BCR-ABL14 and Wnt pathways15, 16 or by inducing the activation of caspase 8, a pro-death protein.17, 18 Interestingly, in response to stress, autophagy promotes the degradation of p62, thus limits the activation of p62-regulatory pathways that control tumorigenesis.10 In addition, p62/SQSTM1 controls pathways that modulate differentiation of normal and cancerous cells. For example, p62/SQSTM1 has been shown to antagonize basal ERK activity and adipocyte differentiation.19 In contrast, p62/SQSTM1 favors differentiation of osteoclasts,20 osteoblasts,21 neurons,22 megakaryocytes23 and macrophages.24 The role and regulation of p62/SQSTM1 during leukemia cell differentiation has been poorly documented.Acute myeloid leukemia (AML) is a hematological disease characterized by multiple deregulated pathways resulting in a blockade of myeloid precursors at different stages of maturation.25, 26 Acute promyelocyte leukemia (APL) is the M3 type of AML characterized by an arrest of the terminal differentiation of promyelocytes into granulocytes and frequently associated with the expression of the oncogenic PML-RAR alpha fusion gene.27, 28 All-trans retinoic acid (ATRA), a potent activator of cellular growth arrest, differentiation, and death of APL cells, has been shown to effectively promote complete clinical remission of APL when combined with chemotherapy.29, 30, 31 Despite the success of this treatment, some APL patients are refractory to ATRA treatment or relapse owing to the development of resistance to ATRA in leukemia cells.32, 33, 34Our previous results revealed that autophagy flux is activated during granulocyte differentiation of myeloid leukemia cell lines induced by ATRA.35 In the present study, we observed that p62/SQSTM1, an autophagic substrate, is markedly upregulated at both mRNA and protein levels during the granulocytic differentiation process. Here, we investigated the regulation and the function of p62/SQSTM1 during AML cells differentiation into neutrophils/granulocytes. 相似文献
19.
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, 38Although 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. 相似文献
20.
Anja Drinkut Karsten Tillack Durga P Meka Jorg B Schulz Sebastian Kügler Edgar R Kramer 《Cell death & disease》2016,7(9):e2359
Glial cell line-derived neurotrophic factor (GDNF) is a potent survival and regeneration-promoting factor for dopaminergic neurons in cell and animal models of Parkinson disease (PD). GDNF is currently tested in clinical trials on PD patients with so far inconclusive results. The receptor tyrosine kinase Ret is the canonical GDNF receptor, but several alternative GDNF receptors have been proposed, raising the question of which signaling receptor mediates here the beneficial GDNF effects. To address this question we overexpressed GDNF in the striatum of mice deficient for Ret in dopaminergic neurons and subsequently challenged these mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Strikingly, in this established PD mouse model, the absence of Ret completely abolished GDNF''s neuroprotective and regenerative effect on the midbrain dopaminergic system. This establishes Ret signaling as absolutely required for GDNF''s effects to prevent and compensate dopaminergic system degeneration and suggests Ret activation as the primary target of GDNF therapy in PD.Glial cell line-derived neurotrophic factor (GDNF) is the founding member of the four ligands in the GDNF family, which belong to the transforming growth factor-β superfamily.1 GDNF was characterized as a potent survival factor for many neurons in culture such as dopaminergic, motor, sympathetic, parasympathetic, sensory and enteric neurons.1, 2 In addition, in dopaminergic neuron cultures GDNF stimulates neuronal differentiation, neurite outgrowth, synapse formation and dopamine release.1, 2As degeneration of midbrain dopaminergic neurons in the substantia nigra pars compacta (SNpc) represents a major hallmark of Parkinson disease (PD), the most common neurodegenerative movement disorder, GDNF has raised considerable interest as a therapeutic molecule for the treatment of PD.3, 4, 5 PD affects >2% of individuals over the age of 60 years, but no curative treatment is available to date, mainly due to a lack of understanding disease etiology.6, 7, 8 Preclinical studies in the established 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) rodent and primate models of PD demonstrated a substantial neuroprotection and regeneration effect by striatal provided GDNF or its close relative neurturin.3, 4, 9 However, clinical phase II trials on PD patients using GDNF or neurturin did so far not convincingly recapitulate their beneficial effects on the dopaminergic system in humans most likely due to technical problems and the selection of advanced PD patients.10, 11, 12, 13GDNF signaling is highly complex as this neurotrophic factor can bind to a variety of receptors, thus being able to induce pleiotropic effects. GDNF efficiently binds to the GPI-linked GDNF family receptor α1 (GFRα1).1, 2 It has been shown that the GDNF/GFRα1 complex can activate not only the canonical GDNF receptor Ret, a receptor tyrosine kinase which signals through the sarcoma protein (Src)/rat sarcoma (Ras)/mitogen-activated protein kinase (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt, NF-κB (nuclear factor ''kappa-light-chain-enhancer'' of activated B cells), JNK (c-Jun N-terminal kinases) and PLCγ (phospholipase γ) pathway, but also with other signaling inducing receptors.1, 2, 4, 5, 13 So far, at least four alternative GDNF receptors have been described which are all expressed in midbrain dopaminergic neurons, NCAM,14, 15 the integrins αV and βI,14, 16 syndecan 317 and N-cadherin.18 Interestingly, Ret is not essential during pre- and postnatal development of the mouse dopaminergic system,19, 20, 21, 22, 23 but specifically required for the maintenance of SNpc dopaminergic neurons and their striatal innervation in aged mice.23, 24, 25 In contrast, GDNF seems most likely under physiological conditions to be dispensable during development and maintenance of midbrain dopaminergic neurons in mice, although conflicting results exist.26, 27, 28 Thus, Ret might be activated by a GDNF-independent mechanism to stimulate SNpc dopaminergic neuron survival. In addition, the in vivo function of the alternative GDNF receptors in the dopaminergic system under physiological and pathophysiological conditions, like PD, and their dependence on GDNF has not yet been addressed in detail. This raised the important question which GDNF receptor might be required to mediate GDNF''s reported neuroprotective and regenerative effect in the dopaminergic system in PD animal models and potentially in PD patients.5, 29Previously, we showed in dopaminergic neuron-specific Ret knockout mice that Ret receptor loss does not result in a higher vulnerability of midbrain dopaminergic neurons against MPTP but to less resprouting of left over dopaminergic neuron axons in the striatum after MPTP intoxication.30 In adult mice endogenous GDNF levels are rather low.26, 31 Therefore, we could not rule out in that study the possibility, that higher levels of GDNF—as also used in the clinical GDNF trials in PD patients—might have neuroprotective and regenerating effects even in the absence of the Ret receptor. Here we addressed now this question by viral overexpression of GDNF in MPTP-treated mice lacking expression of Ret again specifically in dopaminergic neurons.23, 30 We found that in the absence of Ret in dopaminergic neurons even a substantial overexpression of GDNF in the striatum does not have a neuroprotective and regenerative effect. Thus, despite the expression of alternative GDNF receptors on midbrain dopaminergic neurons, the presence of the canonical GDNF receptor Ret seems to be mandatory for mediating GDNF''s beneficial survival and axonal resprouting effect in these neurons. 相似文献