Inflammatory neurodegeneration mediated by nitric oxide,glutamate, and mitochondria

In inflammatory, infectious, ischemic, and neurodegenerative pathologies of th central nervous system (CNS) glia become “activated” by inflammatory mediators, and express new proteins such as the inducible isoform of nitric oxide synthase (iNOS). Although these activated glia have beneficial roles, in vitro they potently kill cocultured neurons, and there is increasing evidence that they contribute to pathology in vivo. Nitric oxide (NO) from iNOS appears to be a key mediator of such glial-induced neuronal death. The high sensitivity of neurons to NO is partly due to NO causing inhibition of respiration, rapid glutamate release from both astrocytes and neurons, and subsequent excitotoxic death of the neurons. NO is a potent inhibitor of mitochondrial respiration, due to reversible binding of NO to cytochrome oxidase in competition with oxygen, resulting in inhibition of energy production and sensitization to hypoxia. Activated astrocytes or microglia cause a potent inhibition of respiration in cocultured neurons due to glial NO inhibiting cytochrome oxidase within the neurons, resulting in ATP depletion and glutamate release. In some conditions, glutamate-induced neuronal death can itself be mediated by N-methyl-d-aspartate (NMDA)-receptor activation of the neuronal isoform of NO synthase (nNOS) causing mitochondrial damage. In addition NO can be converted to a number of reactive derivatives such as peroxynitrite, NO₂, N₂O₃, and S-nitrosothiols that can kill cells in part by inhibiting mitochondrial respiration or activation of mitochondrial permeability transition, triggering neuronal apoptosis or necrosis.     

Regulation of neuronal excitability by release of proteins from glial cells

Effects of glial cells on electrical isolation and shaping of synaptic transmission between neurons have been extensively studied. Here we present evidence that the release of proteins from astrocytes as well as microglia may regulate voltage-activated Na⁺ currents in neurons, thereby increasing excitability and speed of transmission in neurons kept at distance from each other by specialized glial cells. As a first example, we show that basic fibroblast growth factor and neurotrophin-3, which are released from astrocytes by exposure to thyroid hormone, influence each other to enhance Na⁺ current density in cultured hippocampal neurons. As a second example, we show that the presence of microglia in hippocampal cultures can upregulate Na⁺ current density. The effect can be boosted by lipopolysaccharides, bacterial membrane-derived stimulators of microglial activation. Comparable effects are induced by the exposure of neuron-enriched hippocampal cultures to tumour necrosis factor-α, which is released from stimulated microglia. Taken together, our findings suggest that release of proteins from various types of glial cells can alter neuronal excitability over a time course of several days. This explains changes in neuronal excitability occurring in states of thyroid hormone imbalance and possibly also in seizures triggered by infectious diseases.     

鱼藤酮帕金森大鼠多脑区铁积聚与胶质细胞激活

目的观察鱼藤酮帕金森病（Parkinson＇s disease,PD）大鼠铁积聚脑区胶质细胞是否激活。方法雄性Wistar大鼠予颈背部皮下注射鱼藤酮（2 mg/kg）葵花油乳化液,每日一次,连续注射4~6周制备PD模型,8周时做冰冻切片,行免疫组化染色观察PD大鼠铁积聚脑区小胶质细胞（OX42）和星形胶质细胞（GFAP）。结果 PD大鼠黑质致密部、海马齿状回颗粒细胞层、纹状体苍白球、小脑齿状-间位核及小脑面神经核中铁染色显著积聚区域内小胶质细胞和星形胶质细胞染色积分光密度值较正常组明显增加（P〈0.05）。结论鱼藤酮PD大鼠铁积聚脑区小胶质细胞、星型胶质细胞均呈激活状态。     

Neurons reduce glial responses to lipopolysaccharide (LPS) and prevent injury of microglial cells from over-activation by LPS

The microenvironment of the CNS has been considered to tonically inhibit glial activities. It has been shown that glia become activated where neuronal death occurs in the aging brain. We have previously demonstrated that neurons tonically inhibit glial activities including their responses to the bacterial endotoxin lipopolysaccharide (LPS). It is not clear whether activation of glia, especially microglia in the aging brain, is the consequence of disinhibition due to neuronal death. This study was designed to determine if glia regain their responsiveness to LPS once the neurons have died in aged cultures. When cultured alone, glia from postnatal day one rat mesencephalons stimulated with LPS (0.1-1000 ng/mL) produced both nitric oxide (NO) and tumor necrosis factor alpha (TNFalpha), yielding a sigmoid and a bell-shaped curve, respectively. When neuron-containing cultures were prepared from embryonic day 14/15 mesencephalons, the shape of the dose-response curve for NO was monotonic and the bell-shaped curve for TNFalpha production was shifted to the right. After 1 month of culture under conditions where neurons die, the production curves for NO and TNFalpha in LPS-stimulated glia shifted back to the left compared to mixed neuron-glia cultures. Immunostaining of rat microglia for the marker CR3 (the receptor for complement component C3) demonstrated that high concentrations of LPS (1 microg/mL) reduced the number of microglia in mixed-glial cultures. In contrast, reduction of CR3 immunostaining was not observed in LPS-stimulated mixed neuron-glia cultures. Taken together, the results demonstrate that disinhibition of the glial response to LPS occurs after neurons die in aged cultures. Once neurons have died, the responsiveness of glia to LPS is restored. Neurons prevented injury to microglia by reducing their responsiveness to LPS. This study broadens our understanding of the ways in which the CNS microenvironment affects cerebral inflammation.     

Salvianolic Acid B Attenuates Toxin-Induced Neuronal Damage via Nrf2-Dependent Glial Cells-Mediated Protective Activity in Parkinson’s Disease Models

Salvianolic acid B (SalB), a bioactive compound isolated from the plant-derived medicinal herb Danshen, has been shown to exert various anti-oxidative and anti-inflammatory activities in several neurological disorders. In this study, we sought to investigate the potential protective effects and associated molecular mechanisms of SalB in Parkinson’s disease (PD) models. To determine the neuroprotective effects of SalB in vitro, MPP⁺- or lipopolysaccharide (LPS)-induced neuronal injury was achieved using primary cultures with different compositions of neurons, microglia and astrocytes. Our results showed that SalB reduced both LPS- and MPP⁺-induced toxicity of dopamine neurons in a dose-dependent manner. Additionally, SalB treatment inhibited the release of microglial pro-inflammatory cytokines and resulted in an increase in the expression and release of glial cell line-derived neurotrophic factor (GDNF) from astrocytes. Western blot analysis illustrated that SalB increased the expression and nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). The knockdown of Nrf2 using specific small interfering RNA (siRNA) partially reversed the SalB-induced GDNF expression and anti-inflammatory activity. Moreover, SalB treatment significantly attenuated dopaminergic (DA) neuronal loss, inhibited neuroinflammation, increased GDNF expression and improved the neurological function in MPTP-treated mice. Collectively, these findings demonstrated that SalB protects DA neurons by an Nrf-2 -mediated dual action: reducing microglia activation-mediated neuroinflammation and inducing astrocyte activation-dependent GDNF expression. Importantly the present study also highlights critical roles of glial cells as targets for developing new strategies to alter the progression of neurodegenerative disorders.     

Dopamine Selectively Sensitizes Dopaminergic Neurons to Rotenone-Induced Apoptosis

Among various types of neurons affected in Parkinson’s disease, dopamine (DA) neurons of the substantia nigra undergo the most pronounced degeneration. Products of DA oxidation and consequent cellular damage have been hypothesized to contribute to neuronal death. To examine whether elevated intracellular DA will selectively predispose the dopaminergic subpopulation of nigral neurons to damage by an oxidative insult, we first cultured rat primary mesencephalic cells in the presence of rotenone to elevate reactive oxygen species. Although MAP2⁺ neurons were more sensitive to rotenone-induced toxicity than type 1 astrocytes, rotenone affected equally both DA (TH⁺) neurons and MAP2⁺ neurons. In contrast, when intracellular DA concentration was elevated, DA neurons became selectively sensitized to rotenone. Raising intracellular DA levels in primary DA neurons resulted in dopaminergic neuron death in the presence of subtoxic concentrations of rotenone. Furthermore, mitochondrial superoxide dismutase mimetic, manganese (III) meso-tetrakis (4-benzoic acid) porphyrin, blocked activation of caspase-3, and consequent cell death. Our results demonstrate that an inhibitor of mitochondrial complex I and increased cytosolic DA may cooperatively lead to conditions of elevated oxidative stress and thereby promote selective demise of dopaminergic neurons.     

Activated Scavenger Receptor A Promotes Glial Internalization of Aβ

Beta-amyloid (Aβ) aggregates have a pivotal role in pathological processing of Alzheimer’s disease (AD). The clearance of Aβ monomer or aggregates is a causal strategy for AD treatment. Microglia and astrocytes are the main macrophages that exert critical neuroprotective roles in the brain. They may effectively clear the toxic accumulation of Aβ at the initial stage of AD, however, their functions are attenuated because of glial overactivation. In this study, we first showed that heptapeptide XD4 activates the class A scavenger receptor (SR-A) on the glia by increasing the binding of Aβ to SR-A, thereby promoting glial phagocytosis of Aβ oligomer in microglia and astrocytes and triggering intracellular mitogen-activated protein kinase (MAPK) signaling cascades. Moreover, XD4 enhances the internalization of Aβ monomers to microglia and astrocytes through macropinocytosis or SR-A-mediated phagocytosis. Furthermore, XD4 significantly inhibits Aβ oligomer-induced cytotoxicity to glial cells and decreases the production of proinflammatory cytokines, such as TNF-α and IL-1β, in vitro and in vivo. Our findings may provide a novel strategy for AD treatment by activating SR-A.     

Effect of NDP-α-MSH on PPAR-γ and –β Expression and Anti-Inflammatory Cytokine Release in Rat Astrocytes and Microglia

Brain inflammation plays a central role in numerous brain pathologies. Microglia and astrocytes are the main effector cells that become activated when an inflammatory process takes place within the central nervous system. α-melanocyte-stimulating hormone (α-MSH) is a neuropeptide with proven anti-inflammatory properties. It binds with highest affinity to the melanocortin receptor 4 (MC4R), which is present in astrocytes and upon activation triggers anti-inflammatory pathways. The aim of this research was to identify anti-inflammatory mediators that may participate in the immunomodulatory effects of melanocortins in glial cells. Since peroxisome proliferator-activated receptors (PPARs) have recently been implicated in the modulation of inflammation, we investigated the effect of an α-MSH analog, [Nle⁴, D-Phe⁷]-α-MSH (NDP-α-MSH), on PPAR-β and PPAR-γ gene and protein expression in rat primary astrocytes and microglia. We initially demonstrated that rat primary microglia express MC4R and showed that treatment with NDP-α-MSH increases PPAR-γ protein levels and strongly decreases PPAR-β levels in both astrocytes and microglia. We also showed that extracellular signal-regulated kinase 1/2 (ERK1/2)–mediated signaling is partially involved in these effects in a cell-specific fashion. Finally, we showed that NDP-α-MSH stimulates the release of the anti-inflammatory cytokines IL-10 and TGF-β from microglia and astrocytes, respectively. The presented data suggest a role for IL-10 and TGF-β in the protective action of melanocortins and a connection between MC4R pathway and that of the nuclear receptor PPAR-γ. This is the first report providing evidence that MC4R is expressed in rat primary microglia and that melanocortins modulate PPAR levels in glial cells. Our findings provide new insights into the mechanisms underlying the activation of glial MC4R and open perspectives for new therapeutic strategies for the treatment of inflammation-mediated brain diseases.     

A Novel Subtype of Astrocytes Expressing TRPV4 (Transient Receptor Potential Vanilloid 4) Regulates Neuronal Excitability via Release of Gliotransmitters

Astrocytes play active roles in the regulation of synaptic transmission. Neuronal excitation can evoke Ca²⁺ transients in astrocytes, and these Ca²⁺ transients can modulate neuronal excitability. Although only a subset of astrocytes appears to communicate with neurons, the types of astrocytes that can regulate neuronal excitability are poorly characterized. We found that ∼30% of astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be classified on the basis of their expression patterns. When TRPV4⁺ astrocytes are activated by ligands such as arachidonic acid, the activation propagates to neighboring astrocytes through gap junctions and by ATP release from the TRPV4⁺ astrocytes. After activation, both TRPV4⁺ and TRPV4⁻ astrocytes release glutamate, which acts as an excitatory gliotransmitter to increase synaptic transmission through type 1 metabotropic glutamate receptor (mGluR). Our results indicate that TRPV4⁺ astrocytes constitute a novel subtype of the population and are solely responsible for initiating excitatory gliotransmitter release to enhance synaptic transmission. We propose that TRPV4⁺ astrocytes form a core of excitatory glial assembly in the brain and function to efficiently increase neuronal excitation in response to endogenous TRPV4 ligands.     

Role of Astrocytes in Pain

Over the last decade, a series of studies has demonstrated that glia in the central nervous system play roles in many aspects of neuronal functioning including pain processing. Peripheral tissue damage or inflammation initiates signals that alter the function of the glial cells (microglia and astrocytes in particular), which in turn release factors that regulate nociceptive neuronal excitability. Like immune cells, these glial cells not only react at sites of central and/or peripheral nervous system damage but also exert their action at remote sites from the focus of injury or disease. As well as extensive evidence of microglial involvement in various pain states, there is also documentation that astrocytes are involved, sometimes seemingly playing a more dominant role than microglia. The interactions between astrocytes, microglia and neurons are now recognized as fundamental mechanisms underlying acute and chronic pain states. This review focuses on recent advances in understanding of the role of astrocytes in pain states.     

The effect of methionine-sulphoximine on nerve and glia cells in vitro

Summary The effect of methionine-sulphoximine (MSI) on the behaviour of the nerve and glia elements from brain and cerebellum of rat and chick in tissue culture was studied. It was observed that a concentration of MSI^–3 M inhibits the growth of early brain expiants. Concentrations of 10^–4 and 10^–5 M did not show this effect. Time lapse cinematographic recordings evaluating continually the behaviour of living glial and neuronal elements in tissue culture showed that MSI in a concentration of 10^–3 caused, very shortly after administration, a conspicuous oedema of cellular elements. The highest degree of swelling occurred in the perikarya and processes of astrocytes. Cell bodies of the oligodendroglia swelled only in the beginning and later shrank again considerably. This shrinkage was accompanied by a retraction of the glial processes, their fragmentation, and destruction. The perikaryon of the oligodendroglia contracted discontinually. Similarly the volumetric increase of astrocytes was accompanied by rhythmic contractions. In neuronal as well as oligodendroglial perikarya movements of mitochondria and other cytoplasmic particles first accelerated, later slowed down. Synchronously with cytoplasmic swelling of neurons the formation of many vacuoles is observed. From a comparison of these changes, their time course, and the degree of damage in various cell types it may be concluded that MSI attacks in the first place glial elements, among them most intensely the oligodendroglia.     

Glial Contributions to Neural Function and Disease

The nervous system consists of neurons and glial cells. Neurons generate and propagate electrical and chemical signals, whereas glia function mainly to modulate neuron function and signaling. Just as there are many different kinds of neurons with different roles, there are also many types of glia that perform diverse functions. For example, glia make myelin; modulate synapse formation, function, and elimination; regulate blood flow and metabolism; and maintain ionic and water homeostasis to name only a few. Although proteomic approaches have been used extensively to understand neurons, the same cannot be said for glia. Importantly, like neurons, glial cells have unique protein compositions that reflect their diverse functions, and these compositions can change depending on activity or disease. Here, I discuss the major classes and functions of glial cells in the central and peripheral nervous systems. I describe proteomic approaches that have been used to investigate glial cell function and composition and the experimental limitations faced by investigators working with glia.The nervous system is composed of neurons and glial cells that function together to create complex behaviors. Traditionally, glia have been considered to be merely passive contributors to brain function, resulting in a pronounced neurocentric bias among neuroscientists. Some of this bias reflects a paucity of knowledge and tools available to study glia. However, this view is rapidly changing as new tools, model systems (culture and genetic), and technologies have permitted investigators to show that glia actively sculpt and modulate neuronal properties and functions in many ways. Glia have been thought to outnumber neurons by 10:1, although more recent studies suggest the ratio in the human brain is closer to 1:1 with region-specific differences (1). There are many different types of glia, some of which are specific to the central nervous system (CNS),¹ whereas others are found only in the peripheral nervous system (PNS). The main types of CNS glia include astrocytes, oligodendrocytes, ependymal cells, radial glia, and microglia. In the PNS, the main glial cells are Schwann cells, satellite cells, and enteric glia. These cells differ and are classified according to their morphologies, distinct anatomical locations in the nervous system, functions, developmental origins, and unique molecular compositions. Among the different classes of glia there are additional subclasses that reflect further degrees of specialization. In this review, I will discuss the characteristics and functions of the major glial cell types including astrocytes, microglia, and the myelin-forming oligodendrocytes (CNS) and Schwann cells (PNS). Because of space limitations, it is impossible to give a complete accounting of all glia and what is known about each of these cell types. Therefore, I encourage the interested reader to refer to some of the many excellent reviews referenced below that focus on individual glial cell types. Finally, I will discuss proteomic studies of glial cell function and some of the unique challenges investigators face when working with these cells.     

Berberine Protects against Neuronal Damage via Suppression of Glia-Mediated Inflammation in Traumatic Brain Injury

Traumatic brain injury (TBI) triggers a series of neuroinflammatory processes that contribute to evolution of neuronal injury. The present study investigated the neuroprotective effects and anti-inflammatory actions of berberine, an isoquinoline alkaloid, in both in vitro and in vivo TBI models. Mice subjected to controlled cortical impact injury were injected with berberine (10 mg·kg⁻¹) or vehicle 10 min after injury. In addition to behavioral studies and histology analysis, blood-brain barrier (BBB) permeability and brain water content were determined. Expression of PI3K/Akt and Erk signaling and inflammatory mediators were also analyzed. The protective effect of berberine was also investigated in cultured neurons either subjected to stretch injury or exposed to conditioned media with activated microglia. Berberine significantly attenuated functional deficits and brain damage associated with TBI up to day 28 post-injury. Berberine also reduced neuronal death, apoptosis, BBB permeability, and brain edema at day 1 post-injury. These changes coincided with a marked reduction in leukocyte infiltration, microglial activation, matrix metalloproteinase-9 activity, and expression of inflammatory mediators. Berberine had no effect on Akt or Erk 1/2 phosphorylation. In mixed glial cultures, berberine reduced TLR4/MyD88/NF-κB signaling. Berberine also attenuated neuronal death induced by microglial conditioned media; however, it did not directly protect cultured neurons subjected to stretch injury. Moreover, administration of berberine at 3 h post-injury also reduced TBI-induced neuronal damage, apoptosis and inflammation in vivo. Berberine reduces TBI-induced brain damage by limiting the production of inflammatory mediators by glial cells, rather than by a direct neuroprotective effect.     

Attenuation of Reactive Gliosis Does Not Affect Infarct Volume in Neonatal Hypoxic-Ischemic Brain Injury in Mice

Background

Astroglial cells are activated following injury and up-regulate the expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Adult mice lacking the intermediate filament proteins GFAP and vimentin (GFAP^−/−Vim^−/−) show attenuated reactive gliosis, reduced glial scar formation and improved regeneration of neuronal synapses after neurotrauma. GFAP^−/−Vim^−/− mice exhibit larger brain infarcts after middle cerebral artery occlusion suggesting protective role of reactive gliosis after adult focal brain ischemia. However, the role of astrocyte activation and reactive gliosis in the injured developing brain is unknown.

Methodology/Principal Findings

We subjected GFAP^−/−Vim^−/− and wild-type mice to unilateral hypoxia-ischemia (HI) at postnatal day 9 (P9). Bromodeoxyuridine (BrdU; 25 mg/kg) was injected intraperitoneally twice daily from P9 to P12. On P12 and P31, the animals were perfused intracardially. Immunohistochemistry with MAP-2, BrdU, NeuN, and S100 antibodies was performed on coronal sections. We found no difference in the hemisphere or infarct volume between GFAP^−/−Vim^−/− and wild-type mice at P12 and P31, i.e. 3 and 22 days after HI. At P31, the number of NeuN⁺ neurons in the ischemic and contralateral hemisphere was comparable between GFAP^−/−Vim^−/− and wild-type mice. In wild-type mice, the number of S100⁺ astrocytes was lower in the ipsilateral compared to contralateral hemisphere (65.0±50.1 vs. 85.6±34.0, p<0.05). In the GFAP^−/−Vim^−/− mice, the number of S100⁺ astrocytes did not differ between the ischemic and contralateral hemisphere at P31. At P31, GFAP^−/−Vim^−/− mice showed an increase in NeuN⁺BrdU⁺ (surviving newly born) neurons in the ischemic cortex compared to wild-type mice (6.7±7.7; n = 29 versus 2.9±3.6; n = 28, respectively, p<0.05), but a comparable number of S100⁺BrdU⁺ (surviving newly born) astrocytes.

Conclusions/Significance

Our results suggest that attenuation of reactive gliosis in the developing brain does not affect the hemisphere or infarct volume after HI, but increases the number of surviving newborn neurons.     

Disruption in Connexin-Based Communication Is Associated with Intracellular Ca2+ Signal Alterations in Astrocytes from Niemann-Pick Type C Mice

Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC) disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the NPC1 gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1^−/−) showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1^−/− hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1^−/− astrocytes also showed more intracellular Ca²⁺ signal oscillations mediated by functional connexin 43 hemichannels and P2Y₁ receptors. Therefore, Npc1^−/− astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.     

Differential Pro-Inflammatory Responses of Astrocytes and Microglia Involve STAT3 Activation in Response to 1800 MHz Radiofrequency Fields

Microglia and astrocytes play important role in maintaining the homeostasis of central nervous system (CNS). Several CNS impacts have been postulated to be associated with radiofrequency (RF) electromagnetic fields exposure. Given the important role of inflammation in neural physiopathologic processes, we investigated the pro-inflammatory responses of microglia and astrocytes and the involved mechanism in response to RF fields. Microglial N9 and astroglial C8-D1A cells were exposed to 1800 MHz RF for different time with or without pretreatment with STAT3 inhibitor. Microglia and astrocytes were activated by RF exposure indicated by up-regulated CD11b and glial fibrillary acidic protein (GFAP). However, RF exposure induced differential pro-inflammatory responses in astrocytes and microglia, characterized by different expression and release profiles of IL-1β, TNF-α, IL-6, PGE2, nitric oxide (NO), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). Moreover, the RF exposure activated STAT3 in microglia but not in astrocytes. Furthermore, the STAT3 inhibitor Stattic ameliorated the RF-induced release of pro-inflammatory cytokines in microglia but not in astrocytes. Our results demonstrated that RF exposure differentially induced pro-inflammatory responses in microglia and astrocytes, which involved differential activation of STAT3 in microglia and astrocytes. Our data provide novel insights into the potential mechanisms of the reported CNS impacts associated with mobile phone use and present STAT3 as a promising target to protect humans against increasing RF exposure.     

Neutrophil-Derived Myeloperoxidase Aggravates Non-Alcoholic Steatohepatitis in Low-Density Lipoprotein Receptor-Deficient Mice

Background

Chronic inflammation and oxidative stress play fundamental roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). Previously, we reported that myeloperoxidase (MPO), an aggressive oxidant-generating neutrophil enzyme, is associated with NASH severity in man. We now investigated the hypothesis that MPO contributes to the development and progression of NASH.

Methodology

Low-density lipoprotein receptor-deficient mice with an MPO-deficient hematopoietic system (LDLR^−/−/MPO^−/−tp mice) were generated and compared with LDLR^−/−/MPO^+/+tp mice after induction of NASH by high-fat feeding.

Results

High-fat feeding caused a ∼4-fold induction of liver MPO in LDLR^−/−/MPO^+/+ mice which was associated with hepatic sequestration of MPO-positive neutrophils and high levels of nitrotyrosine, a marker of MPO activity. Importantly, LDLR^−/−/MPO^−/−tp mice displayed markedly reduced hepatic neutrophil and T-lymphocyte infiltration (p<0.05), and strong down regulation of pro-inflammatory genes such as TNF-α and IL-6 (p<0.05, p<0.01) in comparison with LDLR^−/−/MPO^+/+tp mice. Next to the generalized reduction of inflammation, liver cholesterol accumulation was significantly diminished in LDLR^−/−/MPO^−/−tp mice (p = 0.01). Moreover, MPO deficiency appeared to attenuate the development of hepatic fibrosis as evident from reduced hydroxyproline levels (p<0.01). Interestingly, visceral adipose tissue inflammation was markedly reduced in LDLR^−/−/MPO^−/−tp mice, with a complete lack of macrophage crown-like structures. In conclusion, MPO deficiency attenuates the development of NASH and diminishes adipose tissue inflammation in response to a high fat diet, supporting an important role for neutrophils in the pathogenesis of metabolic disease.     

Non-cell autonomous influence of the astrocyte system xc? on hypoglycaemic neuronal cell death

Despite longstanding evidence that hypoglycaemic neuronal injury is mediated by glutamate excitotoxicity, the cellular and molecular mechanisms involved remain incompletely defined. Here, we demonstrate that the excitotoxic neuronal death that follows GD (glucose deprivation) is initiated by glutamate extruded from astrocytes via system x_c⁻ – an amino acid transporter that imports l-cystine and exports l-glutamate. Specifically, we find that depriving mixed cortical cell cultures of glucose for up to 8 h injures neurons, but not astrocytes. Neuronal death is prevented by ionotropic glutamate receptor antagonism and is partially sensitive to tetanus toxin. Removal of amino acids during the deprivation period prevents – whereas addition of l-cystine restores – GD-induced neuronal death, implicating the cystine/glutamate antiporter, system x_c⁻. Indeed, drugs known to inhibit system x_c⁻ ameliorate GD-induced neuronal death. Further, a dramatic reduction in neuronal death is observed in chimaeric cultures consisting of neurons derived from WT (wild-type) mice plated on top of astrocytes derived from sut mice, which harbour a naturally occurring null mutation in the gene (Slc7a11) that encodes the substrate-specific light chain of system x_c⁻ (xCT). Finally, enhancement of astrocytic system x_c⁻ expression and function via IL-1β (interleukin-1β) exposure potentiates hypoglycaemic neuronal death, the process of which is prevented by removal of l-cystine and/or addition of system x_c⁻ inhibitors. Thus, under the conditions of GD, our studies demonstrate that astrocytes, via system x_c⁻, have a direct, non-cell autonomous effect on cortical neuron survival.