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

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

Alzheimer's disease-related amyloid-β induces synaptotoxicity in human iPS cell-derived neurons

Human induced pluripotent stem cell (iPSC)-derived neurons have been proposed to be a highly valuable cellular model for studying the pathomechanisms of Alzheimer''s disease (AD). Studies employing patient-specific human iPSCs as models of familial and sporadic forms of AD described elevated levels of AD-related amyloid-β (Aβ). However, none of the present AD iPSC studies could recapitulate the synaptotoxic actions of Aβ, which are crucial early events in a cascade that eventually leads to vast brain degeneration. Here we established highly reproducible, human iPSC-derived cortical cultures as a cellular model to study the synaptotoxic effects of Aβ. We developed a highly efficient immunopurification procedure yielding immature neurons that express markers of deep layer cortical pyramidal neurons and GABAergic interneurons. Upon long-term cultivation, purified cells differentiated into mature neurons exhibiting the generation of action potentials and excitatory glutamatergic and inhibitory GABAergic synapses. Most interestingly, these iPSC-derived human neurons were strongly susceptible to the synaptotoxic actions of Aβ. Application of Aβ for 8 days led to a reduction in the overall FM4–64 and vGlut1 staining of vesicles in neurites, indicating a loss of vesicle clusters. A selective analysis of presynaptic vesicle clusters on dendrites did not reveal a significant change, thus suggesting that Aβ impaired axonal vesicle clusters. In addition, electrophysiological patch-clamp recordings of AMPA receptor-mediated miniature EPSCs revealed an Aβ-induced reduction in amplitudes, indicating an impairment of postsynaptic AMPA receptors. A loss of postsynaptic AMPA receptor clusters was confirmed by immunocytochemical stainings for GluA1. Incubation with Aβ for 8 days did not result in a significant loss of neurites or cell death. In summary, we describe a highly reproducible cellular AD model based on human iPSC-derived cortical neurons that enables the mechanistic analysis of Aβ-induced synaptic pathomechanisms and the development of novel therapeutic approaches.In Alzheimer''s disease (AD), synapse damage and synapse loss are thought to underlie cognitive deficits.¹ Oligomers of the amyloid-β (Aβ) peptide appear to induce synaptic failure as an early event in the etiology of AD.^{2, 3, 4} However, despite its well-established synapse-impairing effects in rodent models,^{5, 6, 7} the synaptotoxic actions of Aβ most relevant for the human disease have not been identified in a human model system. Several studies have investigated the synaptotoxic effects of Aβ in cultured rodent neurons and in transgenic mouse models revealing a multitude of potential mechanisms affecting synapses. Postsynaptic Aβ actions result in the loss of functional (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type) glutamate receptors,^{8, 9, 10} involve long-term depression-like mechanisms,^{9, 11, 12} and lead to the degradation of the entire postsynapse (dendritic spines).^{9, 11, 13} In addition, several distinct presynaptic Aβ actions on the synaptic vesicle cycle have been described.^{10, 14} Furthermore, Aβ-induced impairments of axonal transport regulation and Aβ-induced axon degeneration have been found in rodent neurons.^{15, 16, 17} This puzzling diversity of Aβ-induced synapse-related defects raises the question whether all of them are involved in the early pathomechanisms of human AD.In addition to well-established animal systems, the modelling of human neurological disease pathologies by human induced pluripotent stem cell (hiPSC) technology¹⁸ has been proposed as an innovative approach.^{19, 20, 21} The in vitro differentiation of hiPSCs to excitable neurons has been reported using a variety of protocols.^{22, 23, 24} However, quantitative analysis of both functional glutamatergic and GABAergic synapses has been difficult to achieve.^{19, 25, 26} In addition to studying the functional properties of iPSC-derived human neurons from healthy individuals, the in vitro differentiation of patient-derived iPSCs has been used to model complex neurodevelopmental and neurodegenerative diseases.^{19, 27, 28} Recently, iPSCs derived from AD patients have been reported to exhibit increased secretion of Aβ upon in vitro neuronal differentiation; however, neither a loss of synapses nor an impairment of synapse function was detected.^{21, 29, 30, 31, 32, 33} Here we describe a hiPSC-based, carefully optimized in vitro differentiation protocol, including a novel immunopanning step, which enabled us to study the deleterious effects of application of Aβ on human cortical neurons and on human synapses.     

Alpha-Synuclein affects neurite morphology,autophagy, vesicle transport and axonal degeneration in CNS neurons

Many neuropathological and experimental studies suggest that the degeneration of dopaminergic terminals and axons precedes the demise of dopaminergic neurons in the substantia nigra, which finally results in the clinical symptoms of Parkinson disease (PD). The mechanisms underlying this early axonal degeneration are, however, still poorly understood. Here, we examined the effects of overexpression of human wildtype alpha-synuclein (αSyn-WT), a protein associated with PD, and its mutant variants αSyn-A30P and -A53T on neurite morphology and functional parameters in rat primary midbrain neurons (PMN). Moreover, axonal degeneration after overexpression of αSyn-WT and -A30P was analyzed by live imaging in the rat optic nerve in vivo. We found that overexpression of αSyn-WT and of its mutants A30P and A53T impaired neurite outgrowth of PMN and affected neurite branching assessed by Sholl analysis in a variant-dependent manner. Surprisingly, the number of primary neurites per neuron was increased in neurons transfected with αSyn. Axonal vesicle transport was examined by live imaging of PMN co-transfected with EGFP-labeled synaptophysin. Overexpression of all αSyn variants significantly decreased the number of motile vesicles and decelerated vesicle transport compared with control. Macroautophagic flux in PMN was enhanced by αSyn-WT and -A53T but not by αSyn-A30P. Correspondingly, colocalization of αSyn and the autophagy marker LC3 was reduced for αSyn-A30P compared with the other αSyn variants. The number of mitochondria colocalizing with LC3 as a marker for mitophagy did not differ among the groups. In the rat optic nerve, both αSyn-WT and -A30P accelerated kinetics of acute axonal degeneration following crush lesion as analyzed by in vivo live imaging. We conclude that αSyn overexpression impairs neurite outgrowth and augments axonal degeneration, whereas axonal vesicle transport and autophagy are severely altered.Growing evidence suggests that Parkinson''s disease (PD) pathology starts at the presynaptic terminals and the distal axons and is then propagated back to the soma in a ''dying back'' pattern.^{1, 2} Accordingly, at the time of clinical onset, there is only a 30% loss of total substantia nigra pars compacta neurons but a far more severe loss of striatal dopaminergic markers (70–80%), suggesting that axonal terminals of the nigrostriatal pathway are affected earlier.¹ It is thus essential to understand the pathomechanisms specifically affecting the axon in PD in order to interfere with early disease progression.Neurodegeneration in PD is accompanied by the appearance of intraneuronal protein aggregates, denoted Lewy bodies (LBs).³ Interestingly, also LB pathology is initially found in the distal axons before becoming evident in the neuronal somata, and dystrophic neurites, so called ''Lewy neurites'', outnumber LBs in the early stages of PD.^{2, 4, 5} A main component of LBs is the protein alpha-synuclein (αSyn) that is not only widely used as a histopathological marker for PD but is also believed to have a major role in PD pathogenesis.^{6, 7} The importance of αSyn is further underlined by the discovery of αSyn point mutations (e.g. Ala53Thr (A53T), Ala30Pro (A30P)) and multiplications of the αSyn gene, all of which cause autosomal dominant forms of PD.^{8, 9, 10} However, neither the physiological functions nor the pathogenetic mechanisms of αSyn are well understood.⁷The biological effects of αSyn expression strongly depend on the model system. Wild-type (WT) human αSyn does not lead to major clinical or histological abnormalities when expressed in transgenic mice,^{11, 12} but its overexpression mediated by adeno-associated viral vectors (AAV) results in severe neurodegeneration, suggesting a dose-dependent toxic effect.^{13, 14} Different human αSyn-A30P and -A53T transgenic mouse lines develop severe motor impairments, partly resembling symptoms of human PD, accompanied by a degeneration of the nigrostriatal neuronal system and LB-like pathology.^{11, 12, 15} In line with the pathological findings in human PD, the axonal compartment is affected early and most prominently in these animal models.Different putative pathomechanisms of αSyn toxicity have been explored. For example, the cytoskeleton is an important molecular target of αSyn. Multimeric forms of αSyn were shown to impair the polymerization of tubulin and microtubule formation.^{16, 17} Overexpression of αSyn increased actin instability and induced actin bundling in cultured hippocampal neurons.¹⁸ There are, however, divergent data on the resulting effects of αSyn overexpression on neurite outgrowth and integrity in different model systems.^{19, 20, 21, 22}Moreover, a dysregulation of autophagy has been implicated in PD pathology. Aberrant αSyn is normally degraded by autophagy and only to a negligible degree by the proteasome.²³ Several studies have shown that the inhibition of autophagy results in an accumulation and increased toxicity of αSyn, whereas the activation of autophagy has therapeutic effects in PD models.^{23, 24, 25, 26} However, the direct effects of αSyn and its mutants on autophagy seem to rely strongly on the model system and the published data are highly controversial.^{24, 26, 27, 28, 29, 30, 31, 32}Given the central role of axonal degeneration in PD, it is likely that disturbances of axonal transport are involved.³³ In support of this proposition, the motor protein kinesin was shown to be decreased early and stage-dependently in PD patients, preceding the loss of substantia nigra neurons.³⁴ αSyn itself is actively transported along the axons, mainly by the slow component of axonal transport, but the role of αSyn in axonal vesicle transport is unclear.³⁵Here, we present a comprehensive analysis of the effects of αSyn on neurite morphology and examine important pathomechanisms.     

Inhibition of the MAP3 kinase Tpl2 protects rodent and human β-cells from apoptosis and dysfunction induced by cytokines and enhances anti-inflammatory actions of exendin-4

Proinflammatory cytokines exert cytotoxic effects on β-cells, and are involved in the pathogenesis of type I and type II diabetes and in the drastic loss of β-cells following islet transplantation. Cytokines induce apoptosis and alter the function of differentiated β-cells. Although the MAP3 kinase tumor progression locus 2 (Tpl2) is known to integrate signals from inflammatory stimuli in macrophages, fibroblasts and adipocytes, its role in β-cells is unknown. We demonstrate that Tpl2 is expressed in INS-1E β-cells, mouse and human islets, is activated and upregulated by cytokines and mediates ERK1/2, JNK and p38 activation. Tpl2 inhibition protects β-cells, mouse and human islets from cytokine-induced apoptosis and preserves glucose-induced insulin secretion in mouse and human islets exposed to cytokines. Moreover, Tpl2 inhibition does not affect survival or positive effects of glucose (i.e., ERK1/2 phosphorylation and basal insulin secretion). The protection against cytokine-induced β-cell apoptosis is strengthened when Tpl2 inhibition is combined with the glucagon-like peptide-1 (GLP-1) analog exendin-4 in INS-1E cells. Furthermore, when combined with exendin-4, Tpl2 inhibition prevents cytokine-induced death and dysfunction of human islets. This study proposes that Tpl2 inhibitors, used either alone or combined with a GLP-1 analog, represent potential novel and effective therapeutic strategies to protect diabetic β-cells.It is now clear that chronic inflammation is a hallmark of type I and type II diabetes, affecting both β-cell mass and insulin secretion.¹ Type I diabetes is characterized by drastic decreases in β-cell mass and insulin secretion, in part mediated by proinflammatory cytokines produced following autoimmune activation.¹ Proinflammatory cytokines, particularly interleukin-1β (IL-1β), in combination with interferon-γ (IFN-γ) and/or tumor necrosis factor-α (TNF-α), promote death by apoptosis and decrease function of differentiated β-cells, leading to β-cell destruction.¹ Pancreatic islet transplantation is a promising alternative therapy for some type I diabetic patients.² However, clinical outcome is not always achieved because of significant loss of islet mass during and after transplantation.³ Up to 80% of transplanted islets can die during the post-transplantation period as a result of apoptosis because of several mechanisms, notably the instant blood-mediated inflammatory response (IBMIR) and the release of a mix of cytokines including IL-1β, TNF-α and IFN-γ.⁴Immune-modulatory strategies for type I diabetes therapy and improvement of islet transplantation outcomes have emerged, targeting a single specific cytokine, such as IL-1β or TNF-α.^{2, 5} However, these strategies may only target inflammation partially.² Indeed, multiple cytokines, originating from surrounding immune cells and/or β-cells themselves, are more likely to be present simultaneously^{4, 6} and act synergistically to induce β-cell death and dysfunction.^{7, 8, 9} Preclinical and clinical studies demonstrated that glucagon-like peptide-1 (GLP-1) analogs, in addition to regulating glucose homeostasis in vivo, contribute to the restoration of normoglycemia after islet transplantation.^{10, 11, 12, 13} GLP-1 receptor (GLP-1R) analogs protect β-cell survival and function from proinflammatory cytokine attack.^{12, 14, 15} However, some studies have shown only modest and short-term anti-inflammatory effects of GLP-1 analogs when used alone.^{11, 13, 16}Mitogen-activated protein kinases (MAPKs) (i.e., extracellular-regulated kinase-1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAPK) play important roles in cytokine-induced β-cell dysfunction and death.¹ Conversely, ERK1/2 are involved in the beneficial effects of glucose and GLP-1 analogs.^{17, 18, 19} In this context, upstream protein kinases that specifically control the activation of MAPK in response to a combination of inflammatory cytokines (IL-1β, TNF-α and IFN-γ), rather than a single cytokine, may be useful targets for therapeutic interventions against pancreatic β-cell failure.The serine/threonine kinase tumor progression locus 2 (Tpl2) (also known as COT (Cancer Osaka Thyroid) in humans) is a member of the MAP3K family (the MAP3K8) whose activation stimulates primarily the ERK1/2 pathway, but also JNK and/or p38 MAPK in some cell types, specifically in response to various inflammatory stimuli.^{20, 21, 22} Dysregulation of Tpl2 expression and signaling is associated with acute and chronic inflammatory diseases,^{20, 21, 22} and several studies highlight a critical function of Tpl2 in the control of inflammatory responses and survival in adipocytes, fibroblasts and immune and epithelial cells.^{21, 22, 23, 24}However, there is currently nothing known about the effects of Tpl2 in β-cells. The aim of this study was to determine whether Tpl2 may be a new key inflammatory regulator in β-cells or islets. We demonstrate that Tpl2 contributes to cytokine-induced β-cell apoptosis and dysfunction, and suggest that Tpl2 inhibition, either alone or combined with a GLP-1 receptor agonist, represents a potential new therapeutic strategy for the treatment of diabetes.     

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.     

Naturally Occurring Disseminated Group B Streptococcus Infections in Postnatal Rats

Group B Streptococcus (Streptococcus agalactiae, GBS) is a gram-positive commensal and occasional opportunistic pathogen of the human vaginal, respiratory, and intestinal tracts that can cause sepsis, pneumonia, or meningitis in human neonates, infants, and immunosuppressed persons. We report here on a spontaneous outbreak of postnatal GBS-associated disease in rats. Ten of 26 (38.5%) 21- to 24-d-old rat pups died or were euthanized due to a moribund state in a colony of rats transgenic for the human diphtheria toxin receptor on a Munich–Wistar–Frömter genetic background. Four pups had intralesional coccoid bacteria in various organs without accompanying inflammation. GBS was isolated from the liver of 2 of these pups and from skin abscesses in 3 littermates. A connection with the transgene could not be established. A treatment protocol was evaluated in the remaining breeding female rats. GBS is a potentially clinically significant spontaneous infection in various populations of research rats, with some features that resemble late-onset postnatal GBS infection in human infants.Abbreviations: GBS, Group B Streptococcus; MWF, Munich Wistar Frömter; hDTR, human diphtheria toxin receptorStreptococci are gram-positive, coccoid bacteria that typically are classified according to their hemolytic capacity. α-hemolytic streptococci produce a zone of partial hemolysis that appears greenish on blood agar, whereas β-hemolytic streptococci produce a zone of complete hemolysis, and γ-hemolytic organisms produce no hemolysis on blood agar.²⁴ The β-hemolytic streptococci are further subdivided into Lancefield groups (A through G), according to cell-wall carbohydrate antigens.^24,29,39 The group B β-hemolytic Streptococcus (GBS) have been speciated as Streptococcus agalactiae.^28,39 It was first isolated as a causative agent of mastitis in cattle.²⁹ This organism has since been recognized as a cause of severe infection in human neonates.^28,39 In humans, GBS is harbored asymptomatically in the maternal genitourinary tract.^24,28 Infants can be infected and present with serious systemic disease in the first week of life (early-onset GBS) or from 1 wk to 3 mo of age (late-onset GBS).³⁹ In laboratory animals, rats have been used experimentally as models for neonatal^{1,6,7,20,37,38,43,44,47,50,51} or adult⁴⁵ GBS infection, but to our knowledge, GBS has not been associated with spontaneous disease in rats.     

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.     

Cerebral Amyloid Angiopathy in an Aged Sooty Mangabey (Cercocebus atys)

A 26-y-old male sooty mangabey (Cercocebus atys) was found at necropsy to have a moderate degree of cerebral amyloid β (Aβ) angiopathy in superficial and parenchymal blood vessels of the brain. Senile (Aβ) plaques were absent, as were neurofibrillary tangles and other signs of neurodegeneration. Affected blood vessels were arterial, capillary, and, less frequently, venous in nature. Histologically, the Aβ40 isoform was more prevalent than was Aβ42. As in humans but unlike in squirrel monkeys, the density of lesions in this mangabey increased along a rostral-to-caudal gradient. Therefore mangabeys appear to conform to the general tendency of nonhuman primates by developing cerebral Aβ angiopathy in the absence of other indices of Alzheimer-type neuropathology.Abbreviations: Aβ, amyloid β, CAA, cerebral amyloid angiopathy, GFAP, glial fibrillary acidic protein, Iba 1, microglia-expressed calcium-binding proteinOne of the most common microvasculopathies in the aging human brain is cerebral amyloid angiopathy (CAA), a disorder in which various aggregation-prone proteins accumulate in the walls of parenchymal and meningeal blood vessels.^4,9 Most often, the amyloidogenic protein is amyloid β (Aβ), a cleavage product of the Aβ precursor protein and the essential component of senile plaques in Alzheimer disease.^13,43 In the brain vasculature, the basal lamina is a primary site of Aβ deposition.^25,35 Severely affected arterioles show a loss of smooth muscle cells in the tunica media, a weakening of the vascular wall and a propensity to rupture.^3,34 CAA thus increases the risk of intracerebral bleeding and may be responsible for as much as 20% of nontraumatic hemorrhagic stroke in elderly humans.^15,18,35 CAA is present to various degrees in virtually all cases of Alzheimer disease,^15,16,21 but it also occurs independently.²⁴ As is the case for other proteopathies, advancing age is a significant risk factor for CAA.^8,19In humans, CAA most often affects the arteries and arterioles of the brain, particularly those in the leptomeninges and cortex.^2,25 CAA is less frequent in veins and capillaries,²⁵ but capillary CAA can be prominent in some cases.^26,33 The occipital lobe is affected most often^1,32,37 but all cortical regions are vulnerable. CAA is variable in occurrence in the cerebellum and uncommon in deep telencephalic gray structures, white matter, and the brainstem,³⁶ except in severely affected cases.³²Although its specific role in the pathogenesis of Alzheimer disease remains uncertain, there is now strong evidence that dementia is exacerbated by CAA.¹⁴ Furthermore, CAA is independently linked to cognitive decline both in rare familial cases²⁰ and in older humans with idiopathic CAA.^2,20 Despite the prevalence of cerebrovascular amyloidosis in elderly humans, surprisingly little is known about its effect on the brain, in part because of a paucity of natural animal models that closely mimic the human disorder.^17,38Nonhuman primates offer a unique opportunity to view CAA from a comparative perspective, given that they normally generate human-sequence Aβ and develop severe cerebral Aβ amyloidosis in old age, generally in the absence of other changes that characterize Alzheimer disease.¹² Nonhuman primates have the additional advantage that, compared with humans, their relatively small brains enable exhaustive regional analysis of microscopic lesions, something that, for practical reasons, is seldom undertaken in the human brain. Here we present the first investigation of age-associated brain changes in sooty mangabeys, focusing in particular on Aβ deposition and related abnormalities. One of the 2 aged mangabeys analyzed had Aβ deposition in the brain which was almost exclusively in the form of CAA. Remarkably, the vessel types affected and the regional distribution of CAA more closely resembled the pattern seen in humans than that in other nonhuman primates, particularly squirrel monkeys.⁶ Differences and similarities in CAA among primate species could provide fresh insights into the development of cerebral amyloidosis and related disorders in older humans.     

Haploinsufficiency of cathepsin D leads to lysosomal dysfunction and promotes cell-to-cell transmission of α-synuclein aggregates

Lysosomal dysfunction has been implicated both pathologically and genetically in neurodegenerative disorders, such as Alzheimer''s disease and Parkinson''s disease (PD). Lysosomal gene deficiencies cause lysosomal storage disorders, many of which involve neurodegeneration. Heterozygous mutations of some of these genes, such as GBA1, are associated with PD. CTSD is the gene encoding Cathepsin D (CTSD), a lysosomal protein hydrolase, and homozygous CTSD deficiency results in neuronal ceroid-lipofuscinosis, which is characterized by the early onset, progressive neurodegeneration. CTSD deficiency was also associated with deposition of α-synuclein aggregates, the hallmark of PD. However, whether partial deficiency of CTSD has a role in the late onset progressive neurodegenerative disorders, including PD, remains unknown. Here, we generated cell lines harboring heterozygous nonsense mutations in CTSD with genomic editing using the zinc finger nucleases. Heterozygous mutation in CTSD resulted in partial loss of CTSD activity, leading to reduced lysosomal activity. The CTSD mutation also resulted in increased accumulation of intracellular α-synuclein aggregates and the secretion of the aggregates. When α-synuclein was introduced in the media, internalized α-synuclein aggregates accumulated at higher levels in CTSD+/− cells than in the wild-type cells. Consistent with these results, transcellular transmission of α-synuclein aggregates was increased in CTSD+/− cells. The increased transmission of α-synuclein aggregates sustained during the successive passages of CTSD+/− cells. These results suggest that partial loss of CTSD activity is sufficient to cause a reduction in lysosomal function, which in turn leads to α-synuclein aggregation and propagation of the aggregates.Maintaining protein homeostasis (proteostasis) is crucial in not only maintenance of physiological functions of cells, but survival of cells. Proteostasis is a particularly important issue for the survival of post-mitotic cells, such as neurons, while dividing cells can dilute aged and misfolded proteins during the mitosis process.^{1, 2} For the clearance of protein burden, cells utilize two major protein degradation systems, ubiquitin proteasome system and lysosomal degradation, the latter degrades endosomal and autophagosomal cargos.^{3, 4, 5, 6} Dysregulation of ubiquitin proteasome system and lysosome has been shown to cause protein conformational diseases, including neurodegenerative disorders and metabolic disorders.^{7, 8} Genetic studies have suggested that impairment of lysosomal functions has important roles in the pathogenesis of neurodegenerative diseases. Mutations in ATP13A2, GBA1 and VPS35 have been associated with PD.^{9, 10, 11, 12} Mutations in progranulin and charged multivesicular body protein 2B (CHMP2B) have been identified as genetic causes of amyotrophic lateral sclerosis and frontotemporal dementia.^{13, 14, 15} Postmortem brain tissues of neurodegenerative diseases have exhibited deposition of endosomal and autophagic vesicles.¹⁶ Therefore, neurodegenerative proteinopathies might be attributed to lysosomal dysfunction.Pathological examinations of patient tissues have exhibited that protein aggregates, such as amyloid beta (Aβ), tau and α-synuclein aggregates, spread to larger brain regions as disease progresses.¹⁷ In animal models, intracerebrally injected α-synuclein aggregates could spread into larger brain regions both in α-synuclein transgenic and non-transgenic mice.^{18, 19, 20, 21} Inoculation of Aβ or tau aggregates into either non-transgenic or transgenic models of AD also exhibited propagation of those aggregates.^{22, 23, 24, 25, 26, 27, 28} Studies have suggested that cell-to-cell transmission of protein aggregates is the underlying mechanism of the pathological propagation.^{29, 30}Mounting evidence have suggested that lysosomal function is important for the clearance of the transferred aggregates in recipient neurons during cell-to-cell aggregate transmission.³¹ This has been extensively studied in cell culture models for α-synuclein transmission. Previous studies showed α-synculein aggregates can be internalized and transported through the endolysosomal pathway.³² Lyososomal dysfunction led to increased accumulation of the internalized α-synuclein aggregates, suggesting that the lysosomal activity in recipient cells is critical in the clearance of the transmitted α-synuclein aggregates.^{32, 33}Lysosomal storage diseases (LSDs) are caused by defects in the lysosomal degradation process. Mutations in genes encoding lysosomal catabolic enzymes and transporters manifest excessive deposition of the enzyme substrates in various organs.³⁴ Though different LSDs show different symptoms, most of LSD patients exhibit neurological symptoms such as mental retardation, motor dysfunction and progressive neurodegeneration, as well as specific pathological changes in the nervous system.^{35, 36} In addition, some of progressive neurodegenerative disorders such as AD, PD and Huntington''s disease also show similar pathological features with LSD: accumulations of endosomal and autophagosomal vesicles and undegraded macromolecules, and inflammatory responses in brain.¹⁶Gaucher''s disease (GD) is the most common LSD, which is inherited in an autosomal recessive manner. Homozygous mutations of GBA1 gene, encoding β-glucocerebrosidase 1 (GCase 1), a lysosomal hydrolase, is responsible for GD.³⁷ Evidence has suggested that GD is closely related to PD. Patients with type-1 GD, the most common form of GD, frequently develop parkinsonism.³⁸ Heterozygous carriers of GBA1 mutations are at a higher risk for PD.^{39, 40} It has been shown that about 75% of Lewy bodies, a pathological hallmark of PD, colocalized with GCase 1 in brains of PD and DLB patients with heterozygous GBA1 mutations.⁴¹ These results suggest that lysosomal enzyme deficiency is associated with the development of PD.Cathepsin D (CTSD) is a major lysosomal endopeptidase, which is critical in the degradation of long-lived proteins.⁴² Genetic and clinical studies have shown that the homozygous deficiency of CTSD results in the early onset, progressive neurodegeneration, such as congenital neuronal ceroid-lipofuscinosis.⁴³ The heterozygous missense mutations in CTSD have been known to cause the early onset motor and visual problems, brain atrophy, and progressive psychomotor symptoms.⁴⁴ However, the effects of CTSD deficiency on the late onset progressive neurodegenerative disorders, including AD and PD, remain unclear. Nevertheless, it has become clear that CTSD activity is crucial in the degradation of pathogenic protein aggregates.^{45, 46}Herein, we generated a cell line with a heterozygous nonsense mutation in CTSD and investigated the roles of the CTSD activity in lysosomal function, α-synuclein aggregation and transcellular transmission of α-synuclein aggregates.     

Cerium oxide nanoparticles protect against Aβ-induced mitochondrial fragmentation and neuronal cell death

Evidence indicates that nitrosative stress and mitochondrial dysfunction participate in the pathogenesis of Alzheimer''s disease (AD). Amyloid beta (Aβ) and peroxynitrite induce mitochondrial fragmentation and neuronal cell death by abnormal activation of dynamin-related protein 1 (DRP1), a large GTPase that regulates mitochondrial fission. The exact mechanisms of mitochondrial fragmentation and DRP1 overactivation in AD remain unknown; however, DRP1 serine 616 (S616) phosphorylation is likely involved. Although it is clear that nitrosative stress caused by peroxynitrite has a role in AD, effective antioxidant therapies are lacking. Cerium oxide nanoparticles, or nanoceria, switch between their Ce³⁺ and Ce⁴⁺ states and are able to scavenge superoxide anions, hydrogen peroxide and peroxynitrite. Therefore, nanoceria might protect against neurodegeneration. Here we report that nanoceria are internalized by neurons and accumulate at the mitochondrial outer membrane and plasma membrane. Furthermore, nanoceria reduce levels of reactive nitrogen species and protein tyrosine nitration in neurons exposed to peroxynitrite. Importantly, nanoceria reduce endogenous peroxynitrite and Aβ-induced mitochondrial fragmentation, DRP1 S616 hyperphosphorylation and neuronal cell death.Nitric oxide (NO) is a neurotransmitter and neuromodulator required for learning and memory.¹ NO is generated by NO synthases, a group of enzymes that produce NO from L-arginine. In addition to its normal role in physiology, NO is implicated in pathophysiology. When overproduced, NO combines with superoxide anions (O₂·⁻), byproducts of aerobic metabolism and mitochondrial oxidative phosphorylation, to form peroxynitrite anions (ONOO⁻) that are highly reactive and neurotoxic. Accumulation of these reactive oxygen species (ROS) and reactive nitrogen species (RNS), known as oxidative and nitrosative stress, respectively, is a common feature of aging, neurodegeneration and Alzheimer''s disease (AD).¹Nitrosative stress caused by peroxynitrite has a critical role in the etiology and pathogenesis of AD.^{2, 3, 4, 5, 6, 7} Peroxynitrite is implicated in the formation of the two hallmarks of AD, Aβ aggregates and neurofibrillary tangles containing hyperphosphorylated Tau protein.^{1, 4, 7} In addition, peroxynitrite promotes the nitrotyrosination of presenilin 1, the catalytic subunit of the γ-secretase complex, which shifts production of Aβ to amyloid beta (Aβ)42 and increases the Aβ42/Aβ40 ratio, ultimately resulting in an increased propensity for aggregation and neurotoxicity.⁵ Furthermore, nitration of Aβ tyrosine 10 enhances its aggregation.⁶ Peroxynitrite can also modify enzymes, such as triosephosphate isomerase,⁴ and activate kinases, including Jun amino-terminal kinase and p38 mitogen-activated protein kinase, which enhance neuronal cell death.^{8, 9} Moreover, peroxynitrite can trigger the release of free metals such as Zn²⁺ from intracellular stores with consequent inhibition of mitochondrial function and enhancement of neuronal cell death.^{10, 11, 12} Finally, peroxynitrite can irreversibly inhibit complexes I and IV of the mitochondrial respiratory chain.^{11, 13}Because mitochondria have a critical role in neurons as energy producers to fuel vital processes such as synaptic transmission and axonal transport,¹⁴ and mitochondrial dysfunction is a well-documented and early event in AD,¹⁵ it is important to consider how peroxynitrite and nitrosative stress affect mitochondria. Although the ultimate cause of mitochondrial dysfunction in AD remains unclear, an imbalance in mitochondrial fission and fusion is one possibility.^{1, 14, 16, 17, 18} Notably, peroxynitrite, N-methyl D-aspartate (NMDA) receptor activation and Aβ can induce mitochondrial fragmentation by activating mitochondrial fission and/or inhibiting fusion.¹⁶ Mitochondrial fission and fusion is regulated by large GTPases of the dynamin family, including dynamin-related protein 1 (DRP1) that is required for mitochondrial division,¹⁹ and inhibition of mitochondrial division by overexpression of the GTPase-defective DRP1^K38A mutant provides protection against peroxynitrite-, NMDA- and Aβ-induced mitochondrial fragmentation and neuronal cell death.¹⁶The exact mechanism of peroxynitrite-induced mitochondrial fragmentation remains unclear. A recent report suggested that S-nitrosylation of DRP1 at cysteine 644 increases DRP1 activity and is the cause of peroxynitrite-induced mitochondrial fragmentation in AD;²⁰ however, the work remains controversial, suggesting that alternative pathways might be involved.²¹ For example, peroxynitrite also causes rapid DRP1 S616 phosphorylation that promotes its translocation to mitochondria and organelle division.^{21, 22} In mitotic cells, DRP1 S616 phosphorylation is mediated by Cdk1/cyclinB1 and synchronizes mitochondrial division with cell division.²³ Interestingly, DRP1 is S616 hyperphosphorylated in AD brains, suggesting that this event might contribute to mitochondrial fragmentation in the disease.^{21, 22} A recent report indicates that Cdk5/p35 is responsible for DRP1 S616 phosphorylation,²⁴ and notably aberrant Cdk5/p35/p25 signaling is associated with AD pathogenesis.²⁵ Thus, we explored here the possible role of DRP1 S616 hyperphosphorylation in Aβ- and peroxynitrite-mediated mitochondrial fragmentation.Under normal conditions, accumulated mitochondrial superoxide anions and hydrogen peroxide (H₂O₂) can be neutralized by superoxide dismutase (SOD) and catalase. Nitrosative stress in aging and AD might be explained by a loss of antioxidant enzymes. Previous studies suggest that expression of SOD subtypes is decreased in the human AD brain.^{26, 27} Furthermore, SOD1 deletion in a mouse model of AD increased the burden of amyloid plaques.²⁶ By contrast, overexpression of SOD2 in a mouse model of AD decreased the Aβ42/Aβ40 ratio and alleviated memory deficits.^{28, 29} There is currently a lack of antioxidants that can effectively quench superoxide anions, H₂O₂ or peroxynitrite and provide lasting effects. Cerium is a rare earth element and cerium oxide (CeO₂) nanoparticles, or nanoceria, shuttle between their 3+ or 4+ states. Oxidation of Ce⁴⁺ to Ce³⁺ causes oxygen vacancies and defects on the surface of the crystalline lattice structure of the nanoparticles, generating a cage for redox reactions to occur.³⁰ Accordingly, nanoceria mimic the catalytic activities of antioxidant enzymes, such as SOD^{31, 32} and catalase,³³ and are able to neutralize peroxynitrite.³⁴ Because of these antioxidant properties, we hypothesized that nanoceria could detoxify peroxynitrite and protect against Aβ-induced DRP1 S616 hyperphosphorylation, mitochondrial fragmentation and neuronal cell death.     

Brain ischemia downregulates the neuroprotective GDNF-Ret signaling by a calpain-dependent mechanism in cultured hippocampal neurons

The glial cell line-derived neurotrophic factor (GDNF) has an important role in neuronal survival through binding to the GFRα1 (GDNF family receptor alpha-1) receptor and activation of the receptor tyrosine kinase Ret. Transient brain ischemia alters the expression of the GDNF signaling machinery but whether the GDNF receptor proteins are also affected, and the functional consequences, have not been investigated. We found that excitotoxic stimulation of cultured hippocampal neurons leads to a calpain-dependent downregulation of the long isoform of Ret (Ret51), but no changes were observed for Ret9 or GFRα1 under the same conditions. Cleavage of Ret51 by calpains was selectively mediated by activation of the extrasynaptic pool of N-methyl-d-aspartate receptors and leads to the formation of a stable cleavage product. Calpain-mediated cleavage of Ret51 was also observed in hippocampal neurons subjected to transient oxygen and glucose deprivation (OGD), a model of global brain ischemia, as well as in the ischemic region in the cerebral cortex of mice exposed to transient middle cerebral artery occlusion. Although the reduction of Ret51 protein levels decreased the total GDNF-induced receptor activity (as determined by assessing total phospho-Ret51 protein levels) and their downstream signaling activity, the remaining receptors still showed an increase in phosphorylation after incubation of hippocampal neurons with GDNF. Furthermore, GDNF protected hippocampal neurons when present before, during or after OGD, and the effects under the latter conditions were more significant in neurons transfected with human Ret51. These results indicate that the loss of Ret51 in brain ischemia partially impairs the neuroprotective effects of GDNF.Neuronal injury induced by brain ischemia is partly owing to an excessive release of excitatory amino acids followed by toxic overactivation of glutamate receptors (excitotoxicity).^{1, 2, 3} The subsequent increase in [Ca²⁺]_i activates Ca²⁺-dependent proteolytic enzymes (e.g., calpains), which contribute to ischemic neurodegeneration.^{4, 5, 6} The extrasynaptic N-methyl-d-aspartate receptors (NMDAR) for glutamate are preferentially coupled to the activation of excitotoxic signaling mechanisms, including the activation of calpains, and were therefore suggested to have a key role in neuronal death.^{7, 8, 9}The glial cell line-derived neurotrophic factor (GDNF) protects neurons from excitotoxicity-induced cell death^{10, 11, 12, 13} and from ischemic damage.^{14, 15, 16} GDNF dimers bind to GFRα1 (GDNF family receptor alpha-1) with a high affinity, and this complex signals through the transmembrane Ret receptor tyrosine kinase.^{17, 18} The Ret receptor pre-mRNA is alternatively spliced in three isoforms that differ in the composition and length of the C-terminus tail. Once activated by the complex GDNF–GFRα1, Ret isoforms undergo transphosphorylation on several tyrosine residues, activating the Ras/mitogen-activated protein kinase (MAPK) and the phosphatidylinositol-3-kinase/AKT, responsible for cell survival or differentiation, and the phospholipase C-γ (PLCγ) pathway.^{19, 20, 21, 22, 23}Cerebral ischemia is known to alter the expression of the GDNF signaling machinery.¹⁶ Thus, the GDNF mRNA and protein were both found to be upregulated in experimental models of transient focal and global ischemia.^{16, 24, 25, 26} An increased expression of the mRNA for GFRα1^{24, 27} and Ret^{27, 28} was observed in damaged areas after transient middle cerebral artery occlusion (MCAO), a model of focal ischemia, and an upregulation of GFRα1 mRNA was also observed in the hippocampus following transient global ischemia in rats.²⁶ However, whether GFRα1 and Ret protein levels are altered in transient brain ischemia, the impact on their signaling activity and the functional consequences, have not been investigated. In this work, we show that the Ret51 protein is selectively downregulated following excitotoxic injury and in brain ischemia, by a calpain-dependent mechanism, with a consequent decrease in GDNF signaling activity. This loss of Ret51 receptors impairs the neuroprotective effects of GDNF after transient in vitro ischemia.     

Extra-Large G Proteins Expand the Repertoire of Subunits in Arabidopsis Heterotrimeric G Protein Signaling

Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, are a conserved signal transduction mechanism in eukaryotes. However, G protein subunit numbers in diploid plant genomes are greatly reduced as compared with animals and do not correlate with the diversity of functions and phenotypes in which heterotrimeric G proteins have been implicated. In addition to GPA1, the sole canonical Arabidopsis (Arabidopsis thaliana) Gα subunit, Arabidopsis has three related proteins: the extra-large GTP-binding proteins XLG1, XLG2, and XLG3. We demonstrate that the XLGs can bind Gβγ dimers (AGB1 plus a Gγ subunit: AGG1, AGG2, or AGG3) with differing specificity in yeast (Saccharomyces cerevisiae) three-hybrid assays. Our in silico structural analysis shows that XLG3 aligns closely to the crystal structure of GPA1, and XLG3 also competes with GPA1 for Gβγ binding in yeast. We observed interaction of the XLGs with all three Gβγ dimers at the plasma membrane in planta by bimolecular fluorescence complementation. Bioinformatic and localization studies identified and confirmed nuclear localization signals in XLG2 and XLG3 and a nuclear export signal in XLG3, which may facilitate intracellular shuttling. We found that tunicamycin, salt, and glucose hypersensitivity and increased stomatal density are agb1-specific phenotypes that are not observed in gpa1 mutants but are recapitulated in xlg mutants. Thus, XLG-Gβγ heterotrimers provide additional signaling modalities for tuning plant G protein responses and increase the repertoire of G protein heterotrimer combinations from three to 12. The potential for signal partitioning and competition between the XLGs and GPA1 is a new paradigm for plant-specific cell signaling.The classical heterotrimeric G protein consists of a GDP/GTP-binding Gα subunit with GTPase activity bound to an obligate dimer formed by Gβ and Gγ subunits. In the signaling paradigm largely elucidated from mammalian systems, the plasma membrane-associated heterotrimer contains Gα in its GDP-bound form. Upon receiving a molecular signal, typically transduced by a transmembrane protein (e.g. a G protein-coupled receptor), Gα exchanges GDP for GTP and dissociates from the Gβγ dimer. Both Gα and Gβγ interact with intracellular effectors to initiate downstream signaling cascades. The intrinsic GTPase activity of Gα restores Gα to the GDP-bound form, which binds Gβγ, thereby reconstituting the heterotrimer (McCudden et al., 2005; Oldham and Hamm, 2008).Signal transduction through a heterotrimeric G protein complex is an evolutionarily conserved eukaryotic mechanism common to metazoa and plants, although there are distinct differences in the functional intricacies between the evolutionary branches (Jones et al., 2011a, 2011b; Bradford et al., 2013). The numbers of each subunit encoded within genomes, and therefore the potential for combinatorial complexity within the heterotrimer, is one of the most striking differences between plants and animals. For example, the human genome encodes 23 Gα (encoded by 16 genes), five Gβ, and 12 Gγ subunits (Hurowitz et al., 2000; McCudden et al., 2005; Birnbaumer, 2007). The Arabidopsis (Arabidopsis thaliana) genome, however, only encodes one canonical Gα (GPA1; Ma et al., 1990), one Gβ (AGB1; Weiss et al., 1994), and three Gγ (AGG1, AGG2, and AGG3) subunits (Mason and Botella, 2000, 2001; Chakravorty et al., 2011), while the rice (Oryza sativa) genome encodes one Gα (Ishikawa et al., 1995), one Gβ (Ishikawa et al., 1996), and either four or five Gγ subunits (Kato et al., 2004; Chakravorty et al., 2011; Botella, 2012). As expected, genomes of polyploid plants have more copies due to genome duplication, with the soybean (Glycine max) genome encoding four Gα, four Gβ (Bisht et al., 2011), and 10 Gγ subunits (Choudhury et al., 2011). However, Arabidopsis heterotrimeric G proteins have been implicated in a surprisingly large number of phenotypes, which is seemingly contradictory given the relative scarcity of subunits. Arabidopsis G proteins have been implicated in cell division (Ullah et al., 2001; Chen et al., 2006) and morphological development in various tissues, including hypocotyls (Ullah et al., 2001, 2003), roots (Ullah et al., 2003; Chen et al., 2006; Li et al., 2012), leaves (Lease et al., 2001; Ullah et al., 2001), inflorescences (Ullah et al., 2003), and flowers and siliques (Lease et al., 2001), as well as in pathogen responses (Llorente et al., 2005; Trusov et al., 2006; Cheng et al., 2015), regulation of stomatal movement (Wang et al., 2001; Coursol et al., 2003; Fan et al., 2008) and development (Zhang et al., 2008; Nilson and Assmann, 2010), cell wall composition (Delgado-Cerezo et al., 2012), responses to various light stimuli (Warpeha et al., 2007; Botto et al., 2009), responses to multiple abiotic stimuli (Huang et al., 2006; Pandey et al., 2006; Trusov et al., 2007; Zhang et al., 2008; Colaneri et al., 2014), responses to various hormones during germination (Ullah et al., 2002), and postgermination development (Ullah et al., 2002; Pandey et al., 2006; Trusov et al., 2007). Since the Gγ subunit appeared to be the only subunit that provides diversity in heterotrimer composition in Arabidopsis, it was proposed that all functional specificity in heterotrimeric G protein signaling was provided by the Gγ subunit (Trusov et al., 2007; Chakravorty et al., 2011; Thung et al., 2012, 2013). This allowed for only three heterotrimer combinations to account for the wide range of G protein-associated phenotypes.In addition to the above typical G protein subunits, the plant kingdom contains a conserved protein family of extra-large GTP-binding proteins (XLGs). XLGs differ from typical Gα subunits in that they possess a long N-terminal extension of unknown function, but they are similar in that they all have a typical C-terminal Gα-like region, with five semiconserved G-box (G1–G5) motifs. The XLGs also possess the two sequence features that differentiate heterotrimeric G protein Gα subunits from monomeric G proteins: a helical region between the G1 and G2 motifs and an Asp/Glu-rich loop between the G3 and G4 motifs (Lee and Assmann, 1999; Ding et al., 2008; Heo et al., 2012). The Arabidopsis XLG family comprises XLG1, XLG2, and XLG3, and all three have demonstrated GTP-binding and GTPase activities, although they differ from GPA1 in exhibiting a much slower rate of GTP hydrolysis, with a Ca²⁺ cofactor requirement instead of an Mg²⁺ requirement, as for canonical Gα proteins (Heo et al., 2012). All three Arabidopsis XLGs were observed to be nuclear localized (Ding et al., 2008). Although much less is known about XLGs than canonical Gα subunits, XLG2 positively regulates resistance to the bacterial pathogen Pseudomonas syringae and was immunoprecipitated with AGB1 from tissue infected with P. syringae (Zhu et al., 2009). xlg3 mutants, like agb1 mutants, are impaired in root-waving and root-skewing responses (Pandey et al., 2008). During the preparation of this report, Maruta et al. (2015) further investigated XLG2, particularly focusing on the link between XLG2 and Gβγ in pathogen responses. Based on symptom progression in xlg mutants, they found that XLG2 is a positive regulator of resistance to both bacterial and fungal pathogens, with a minor contribution from XLG3 in resistance to Fusarium oxysporum. XLG2 and XLG3 are also positive regulators of reactive oxygen species (ROS) production in response to pathogen-associated molecular pattern elicitors. The resistance and pathogen-associated molecular pattern-induced ROS phenotypes of the agg1 agg2 and xlg2 xlg3 double mutants were not additive in an agg1 agg2 xlg2 xlg3 quadruple mutant, indicating that these two XLGs and the two Gγ subunits function in the same, rather than parallel, pathways. Unfortunately, the close proximity of XLG2 and AGB1 on chromosome 4 precluded the generation of an agb1 xlg2 double mutant; therefore, direct genetic evidence of XLG2 and AGB1 interaction is still lacking, but physical interactions between XLG2 and the Gβγ dimers were shown by yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescence complementation (BiFC) assays (Maruta et al., 2015). Localization of all three XLGs was also reexamined, indicating that XLGs are capable of localizing to the plasma membrane in addition to the nucleus (Maruta et al., 2015).Interestingly, several other plant G protein-related phenotypes, in addition to pathogen resistance, have been observed only in Gβ and Gγ mutants, with opposite phenotypes observed in Gα (gpa1) mutants. Traditionally, the observation of opposite phenotypes in Gα versus Gβγ mutants in plants and other organisms has mechanistically been attributed to signaling mediated by free Gβγ, which increases in abundance in the absence of Gα. However, an intriguing alternative is that XLG proteins fulfill a Gα-like role in forming heterotrimeric complexes with Gβγ and function in non-GPA1-based G protein signaling processes. If XLGs function like Gα subunits, the corresponding increase in subunit diversity could potentially account for the diversity of G protein phenotypes. In light of this possibility, we assessed the heterotrimerization potential of all possible XLG and Gβγ dimer combinations, XLG localization and its regulation by Gβγ, and the effect of xlg mutation on selected known phenotypes associated with heterotrimeric G proteins. Our results provide compelling evidence for the formation of XLG-Gβγ heterotrimers and reveal that plant G protein signaling is substantially more complex than previously thought.     

Proton pump inhibitors protect mice from acute systemic inflammation and induce long-term cross-tolerance

Incidence of sepsis is increasing, representing a tremendous burden for health-care systems. Death in acute sepsis is attributed to hyperinflammatory responses, but the underlying mechanisms are still unclear. We report here that proton pump inhibitors (PPIs), which block gastric acid secretion, selectively inhibited tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) secretion by Toll-like receptor (TLR)-activated human monocytes in vitro, in the absence of toxic effects. Remarkably, the oversecretion of IL-1β that represents a hallmark of monocytes from patients affected by cryopyrin-associated periodic syndrome is also blocked. Based on these propaedeutic experiments, we tested the effects of high doses of PPIs in vivo in the mouse model of endotoxic shock. Our data show that a single administration of PPI protected mice from death (60% survival versus 5% of untreated mice) and decreased TNF-α and IL-1β systemic production. PPIs were efficacious even when administered after lipopolysaccharide (LPS) injection. PPI-treated mice that survived developed a long-term cross-tolerance, becoming resistant to LPS- and zymosan-induced sepsis. In vitro, their macrophages displayed impaired TNF-α and IL-1β to different TLR ligands. PPIs also prevented sodium thioglycollate-induced peritoneal inflammation, indicating their efficacy also in a non-infectious setting independent of TLR stimulation. Lack of toxicity and therapeutic effectiveness make PPIs promising new drugs against sepsis and other severe inflammatory conditions.Systemic inflammatory response is a critical clinical response to insults of either infectious or non-infectious origin.¹ Severe sepsis and septic shock are more serious clinical forms with a poor outcome.² The incidence of sepsis is continuously increasing;^{1, 2, 3, 4} the mortality rate ranges between 30 and 50% in severe sepsis and septic shock, and patients who survive have a higher risk of mortality compared with the normal population for months and even years.⁵ Although treatment of the underlying infection and circulatory support decrease mortality, sepsis remains a leading cause of death in critically ill patients, and efficacious therapy is missing.⁶Traditionally, the physiopathology of sepsis is attributed to a hyperinflammatory response, the ‘cytokine storm'', that can directly lead to death or favor the insurgence of an immunosuppressive phase during which multiple organ dysfunction occurs.¹ We have recently reproduced in vitro on primary monocytes the cytokine storm: the simultaneous activation of multiple Toll-like receptors (TLRs) results in oxidative stress responsible for a marked enhancement of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) secretion.⁷Proinflammatory cytokines are indeed increased in sepsis. TNF-α⁸ is the first cytokine detected in the serum of septic patients followed by IL-1β.⁹ High-mobility group box chromosomal protein-1 appears well after TNF-α and IL-1β, supporting its role in mortality due to late sepsis.¹⁰The ‘cytokine storm'' theory is presently debated, mainly because clinical trials with cytokine antagonists were unsuccessful.¹¹ Timing of administration and non-optimal association of cytokine-neutralizing agents may be responsible for the failure of clinical trials; however, other factors besides cytokine hypersecretion, including genetic polymorphism in genes for coagulation and fibrinolysis and the insurgence of oxidative stress,² may participate in the genesis of sepsis. A common trait in septic patients is acidosis, which is more pronounced in non-survivors.¹² Acidosis is mainly due to a shift from oxidative phosphorylation to glycolysis, with accumulation of lactate and decrease of pH,¹³ and occurs upstream of the pathologic events (release of toxic substances, vasoconstriction, endothelial damage) that lead to cell and patient death.¹² Acidosis is also a feature of inflammatory microenvironments:^{14, 15} upon activation, inflammatory cell metabolism shifts toward aerobic glycolysis,¹⁶ with consequent decrease of extracellular pH. Extracellular acidosis is proinflammatory: it induces inflammatory genes^{14, 15} and increases processing and secretion of IL-1β¹⁷ in a NOD-like receptor (NLR) family, pyrin domain containing 3 (NLRP3) inflammasome-dependent^{18, 19} or -independent²⁰ manner.Proton pump inhibitors (PPIs) are a family of prodrugs that, activated by low pH,²¹ are highly efficacious in reducing acidic secretion by gastric cells and therefore largely used in the treatment of peptic ulcers and reflux esophagitis.²² PPIs are also effective against tumors, which are similarly characterized by low pH.^{23, 24} Furthermore, PPIs have been found to exert anti-inflammatory effects unrelated to the inhibition of gastric acid production, although the underlying mechanisms remain to be elucidated.²⁵Here we show that, in vitro, PPIs inhibit the production of proinflammatory cytokines by monocytes stimulated with TLR agonists; in vivo, in a murine model of lethal endotoxic shock,^{8, 26} PPIs protect against lipopolysaccharide (LPS)-induced mortality. Interestingly, mice cured by PPIs develop cross-tolerance: not only are they resistant to a second challenge with LPS but also respond better to zymosan injection.