Amyloid-β peptide activates cultured astrocytes: morphological alterations, cytokine induction and nitric oxide release

A common feature of many neurodegenerative disorders is an abundance of activated glial cells (astrocytes and microglia). In Alzheimer's disease (AD), activated astrocytes are in close apposition to and surrounding the amyloid plaques. The mechanisms by which the astrocytes become activated in AD and the consequences of reactive astrocytosis to disease progression are not known. We examined the possibility that the amyloid-β (Aβ) peptide, a major constituent of the amyloid plaque, could act as a stimulus leading to activation. We found that treatment of rat cortical astrocyte cultures with aggregated Aβ 1–42 peptide induces activation, as assessed by reactive morphological changes and upregulation of selective glial mRNA and proteins, such as the inflammatory cytokine interleukin-1β. Aβ also stimulates inducible nitric oxide synthase (iNOS) mRNA levels and nitric oxide (NO) release. Aβ 1–42, a major form of amyloid associated with neurotoxicity, activated astrocytes in a time- and dose-dependent manner, whereas a scrambled Aβ 1–42 sequence or Aβ 17–42 had little or no effect. We also determined that the Aβ activity can be found in a supernatant fraction containing soluble Aβ oligomers. Our data suggest that Aβ plays a role in the reactive astrocytosis of AD and that the inflammatory response induced upon glial activation is a critical component of the neurodegenerative process.     

Superoxide-induced nitric oxide release from cultured glial cells

Nitric oxide (NO) has been implicated as a potential contributor to neural cell death in a variety of neurological conditions. Cultured glial cells were exposed to extracellular superoxide generated by the action of xanthine oxidase on xanthine. In this experimental paradigm, both C6 glioma cells and primary astrocytes from rat cerebral cortex produced a rapid release of nitric oxide, measured using an NO specific electrode, in response to the applied superoxide stimulus. Application of a superoxide scavenger, or over-expression of Cu/Zn superoxide dismutase decreased the observed NO release. Authenticity of the NO signal was confirmed by the addition of the NO scavenger 2-(carboxyphenyl)-4,4,5,5-tetramethyllimidazoline-1-oxyl 3-oxide (carboxy-PTIO), which abolished the observed NO release without affecting simultaneously measured superoxide. Therefore, we suggest that glial cells may produce NO under free radical stimulation, which may be relevant to several neurological disorders where superoxide radicals are generated in the vicinity of glia. This would be predicted to result in the release of NO, which may exert toxic effects on neighbouring cells.     

Modulation of glial activation by astrocyte-derived protein S100B: differential responses of astrocyte and microglial cultures

The astrocyte-derived protein S100B stimulates production of inducible nitric oxide synthase and nitric oxide (NO) in astrocytes [Hu et al., 1996, J. Biol. Chem. 271:2543], but its effect on microglia is not known. In addition, S100B's ability to modulate the activity of other glial activating agents has not been defined. In this study, we compared the ability of S100B to stimulate NO in cultures of rat primary astrocytes and the BV-2 murine microglial cell line, and investigated the effect of the combined action of S100B and other stimuli known to activate glial cells. S100B itself stimulated the production of NO in astrocytes, and did not modify or potentiated only weakly the NO production induced by interleukin-1 beta, tumor necrosis factor alpha, dibutyryl cyclic AMP, zymosan A or lipid A. In contrast, S100B alone did not induce NO in BV-2 cells but strongly potentiated NO production in the presence of lipid A but not zymosan A. The deletion of eight C-terminal amino acid residues in S100B leads to a loss of the effect of S100B on microglia but not on astrocytes. These results demonstrate that responses of glial cells to extracellular S100B can vary depending on the cell type, and suggest that different structural features of S100B are important for the protein's effects on microglia and astrocytes.     

Relationship between S100beta and GFAP expression in astrocytes during infarction and glial scar formation after mild transient ischemia

The expression of astrocyte marker proteins (S100beta and GFAP) during infarction and glial scar formation after transient middle cerebral artery (MCA) occlusion was examined using double immunostaining. S100beta immunoreactivity markedly decreased in the core of the injured area when observed immediately after reperfusion and did not increase again. In the periphery, however, S100beta expression increased, showing that S100beta synthesis was up-regulated. S100beta+/iNOS+ astrocytes in the periphery were observed from day 1, when small infarct areas were detectable, up to day 5, when infarct expansion had almost ended. TUNEL+ cells in the periphery were present from days 1 to 5. S100beta+/TUNEL+ cells were observed centrally and around the periphery of the injured area, predicting that cell death contributes to the increase of S100beta concentration in the injured area. Our results suggest that (1) higher concentration of S100beta in the extracellular space due to S100beta leakage from damaged astrocytes leads to up-regulation of S100beta synthesis and induction of inducible nitric oxide synthase (iNOS) synthesis in astrocytes, contributing to infarct expansion that results in DNA damage or cell death via NO and ROS production, and (2) GFAP, but not S100beta, is a main contributor to glial scar formation. On day 1 postreperfusion, the microdiascopic images of the injured areas from the unstained thick sections or the areas detected by S100beta immunoreactivity were larger than those of the infarct areas detected by hematoxylin--eosin (HE)-staining. The difference between these sizes might be useful to predict infarct expansion.     

Antiepileptogenic and anticonvulsant activity of interleukin-1 beta in amygdala-kindled rats

Ischaemic, excitotoxic and traumatic brain injuries have been associated with the occurrence of epileptic seizures. Microglia, the principal immune cells in the brain, produce a variety of proinflammatory and cytotoxic factors especially interleukin-1 (IL-1) early after an acute insult. We studied the effect of intracerebroventricularly administered IL-1beta on seizure acquisition and on fully kindled seizures in amygdala kindling model of epilepsy. IL-1beta (0.01 ng/rat) retarded acquisition of kindled behavioral seizures and growth of afterdischarges (AD). IL-1beta (0.01-10 ng/rat) also exhibited significant anticonvulsant effect on established kindled seizures and AD duration. This effect began 0.5 h after administration and was continued up to 72 h. Pretreatment of the kindled animals with nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester, or cyclooxygenase inhibitor, piroxicam, reversed the anticonvulsant effect of IL-1beta at early time points. Although most of the previous studies indicate a proconvulsant or convulsant property of IL-1, our results support a protective and antiepileptogenic role of IL-1beta.     

冈田酸对拟AD模型大鼠海马CA1区Aβ1-40和nNOS表达的影响

目的观察冈田酸(OA)对大鼠海马CA1区Aβ1-40和nNOS表达的影响,建立更接近临床表现的拟AD大鼠模型。方法在大鼠海马CA1区多次微量注射OA,水迷宫实验观测大鼠行为学改变;Bielschowsky染色观察海马CA1区神经原纤维缠结(NFT)和老年斑(SP)等特征性病理变化;免疫组化方法观察海马CA1区Aβ1-40和nNOS的表达。结果模型组学习记忆减退;海马CA1区出现NFT(P<0.05)和SP特征性病理变化,及Aβ1-40的高表达(P<0.05)和nNOS的低表达(P<0.05)。结论冈田酸海马CA1区微量多次注射,可建立更接近AD临床表现和病理特征的大鼠模型,大鼠海马CA1区SP和NFT的形成,以及Aβ1-40表达增加、nNOS表达减少,是该模型大鼠学习记忆能力减退的可能机制。     

Reduced expression of NO-sensitive guanylyl cyclase in reactive astrocytes of Alzheimer disease, Creutzfeldt-Jakob disease, and multiple sclerosis brains

In Alzheimer's disease (AD) brains increased NO synthase (NOS) expression is found in reactive astrocytes surrounding amyloid plaques. We have recently shown that treatment with beta-amyloid peptides or IL-1beta down-regulates NO-sensitive soluble guanylyl cyclase (sGC) in cultured astrocytes and in adult rat brain. In this work, we have examined sGC activity and expression in postmortem brain tissue of AD patients and matched controls. No significant alteration was observed in basal or NO-stimulated sGC activity, nor in sGC beta1 and alpha1 subunit levels in cortical extracts of AD brains. Immunohistochemistry showed intense and widespread labeling of sGC beta1 in cortical and hippocampal neurons and white matter fibrillar astrocytes, while grey matter astrocytes were faintly stained. In AD, expression of sGC in neurons and fibrillar astrocytes is not altered but is markedly reduced in reactive astrocytes surrounding amyloid plaques. Immunostaining for sGC beta1 was also lacking in reactive astrocytes in cortex and subcortical white matter in Creutzfeldt-Jakob disease brains and in subacute and chronic plaques in multiple sclerosis (MS) brains. Thus, induction of astrocyte reactivity is associated with decreased capacity to generate cGMP in response to NO both in vitro and in vivo. This effect may be related to the development of the astroglial inflammatory response.     

The acute and the long-term effects of nigral lipopolysaccharide administration on dopaminergic dysfunction and glial cell activation

Sustained reactive microgliosis may contribute to the progressive degeneration of nigral dopaminergic neurons in Parkinson's disease (PD), in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) exposed human and in non-human primates. However, the temporal relationship between glial cell activation and nigral cell death is relatively unexplored. Consequently, the effects of acute (24 h) and chronic (30 days) glial cell activation induced by unilateral supranigral lipopolysaccharide (LPS) administration were studied in rats. At 24 h, LPS administration caused a marked reduction in the number of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the substantia nigra (SN) but striatal TH-ir was unaffected. By 30 days, the loss of TH-positive neurons in the LPS-treated nigra was no greater than at 24 h although a heterogeneous loss of striatal TH-ir was present. The loss of nigrostriatal neurons was of functional significance, as at 30 days, LPS-treated rats exhibited ipsiversive circling in response to (+)-amphetamine administration. At 24 h, there was a moderate increase in glial fibrillary acidic protein (GFAP)-ir astrocytes in the SN but a marked elevation of p47phox positive OX-42-ir microglia, and intense inducible nitric oxide synthase (iNOS)-ir and 3-nitrotyrosine (3-NT)-ir was present. However, by 30 days the morphology of OX-42-ir microglia returned to a resting state, the numbers were greatly reduced and no 3-NT-ir was present. At 30 days, GFAP-ir astrocytes were markedly increased in number and iNOS-ir was present in fibrillar astrocyte-like cells. This study shows that acute glial activation leading to dopaminergic neuron degeneration is an acute short-lasting response that does not itself perpetuate cell death or lead to prolonged microglial activation.     

Mitochondrial toxins in models of neurodegenerative diseases. I: in vivo brain hydroxyl radical production during sytemic MPTP treatment or following microdialysis infusion of methylpyridinium or azide ions

Mitochondrial electron transport chain (ETC) function is selectively reduced in multiple tissues, including brain, from patients with Parkinson's disease (PD) and Alzheimer's disease (AD). The ETC defects are specific to each illness, involve complex I in PD and complex IV in AD, are transferable with mitochondrial DNA (mtDNA) and lead to increased production of reactive oxygen species (ROS) in mtDNA-deficient clonal neuronal cells hybridized with mtDNA (`cybrids') from PD or AD patients. C57BL/6 mice treated with MPTP developed elevated tissue hydroxyl radical (^⋅OH) levels in striatum and ventral midbrain but not cerebellum. In brain microdialysis in awake rats, striatal ^⋅OH output increased 3–5-fold after infusion of methylpyridinium ion (MPP+), a complex I inhibitor, or sodium azide, a complex IV inhibitor. Elevated ^⋅OH after MPP+ was blocked stereospecifically by infusion of the nitric oxide synthase (NOS) inhibitor nitro-l-arginine or by the NMDA channel blocker MK801. Neither NOS inhibition nor NMDA blockade altered azide-induced ^⋅OH production. ETC inhibition in vivo increases production of toxic ^⋅OH, but the underlying mechanisms vary as a function of which ETC complex is inhibited. These results support the concept of developing oxygen free radical scavengers for both AD and PD and further suggest that inhibition of NOS and blockade of NMDA receptor function may alter progression of idiopathic PD.     

Alpha 2-macroglobulin-mediated degradation of amyloid beta 1--42: a mechanism to enhance amyloid beta catabolism

Peptides derived from proteolytic degradation of the amyloid precursor protein, e.g., amyloid beta (A beta), are considered to be central to the pathology of Alzheimer's disease (AD). Soluble A beta is present in measurable concentrations in cerebrospinal fluid and blood. There are indications that soluble A beta present in circulation can cross the blood-brain barrier via transcytosis mediated by brain capillary endothelial cells. It implies that A beta originating from circulation may contribute to vascular and parenchymal A beta deposition in AD. Enhancing of A beta catabolism mediated by proteolytic degradation or receptor-mediated endocytosis could be a key mechanism to maintain low concentrations of soluble A beta. To launch A beta clearance we have exploited the A beta-degrading activity of diverse alpha 2-macroglobulin (alpha 2-M)-proteinase complexes. Complexes with trypsin, alpha-chymotrypsin, and bromelain strongly degrade (125)I-A beta 1--42 whereas complexes with endogenous proteinases, e.g., plasmin and prostate-specific antigen, were not effective. A beta degradation by the complexes was not inhibited by alpha 1-antichymotrypsin and soybean trypsin inhibitor which normally would inactivate the free serine proteinases. A prerequisite for A beta degradation is its binding to specific binding sites in alpha 2-M that may direct A beta to the active site of the caged proteinase. Ex vivo, enhanced degradation of (125)I-A beta 1--42 in blood could be achieved upon oral administration of high doses of proteinases to volunteers. These results suggest that up-regulation of A beta catabolism could probably reduce the risk of developing AD by preventing A beta accumulation in brain and vasculature.     

Induction of nitric oxide synthase activity in human astrocytes by interleukin-1β and interferon-γ

Nitric oxide (NO) is a short-lived, diffusible molecule that has a variety of biological activities including vasorelaxation, neurotransmission, and cytotoxicity. In the central nervous system, a constitutive form of nitric oxide synthase (NOS) has been localized in a subset of neurons and in endothelial cells. In addition, both constitutive and LPS-inducible NOS has been demonstrated in rat astrocytes and microglia in vitro. In this report, we present evidence for the production of NO, as measured by the production of nitrite, in highly enriched human fetal astrocyte cultures stimulated with IL-1β. The production of nitrite paralleled the induction of NADPH diaphorase enzyme activity in the perikarya of the majority of stimulated astrocytes. The IL-1β-induced nitrite production by astrocytes was markedly enhanced when cells were co-stimulated with IFN-γ or TNF-α (IFN-γ > TNF-α); LPS had no effect used as a single agent or in combination with other cytokines. N^GMMA and N^G-nitro-arginine, competitive inhibitors of NOS, diminished the accumulation of nitrite, but calmodulin antagonists (trifluoperazine, W-5 and W-7) had little or no inhibitory effect. Human fetal microglia, in contrast to astrocytes, failed to secrete significant amounts of nitrite in response to various stimuli. The results demonstrate the presence of an inducible form of NOS in human fetal astrocytes; human microglia, in turn, may control astrocyte NO production by providing IL-1β as an activating signal.     

Time-related changes in constitutive and inducible nitric oxide synthases in the rat striatum in a model of Huntington's disease

Excitotoxicity and oxidative stress are mechanisms involved in the neuronal cell death induced by the intrastriatal injection of quinolinic acid (QUIN) as a model of Huntington's disease. Production of nitric oxide by nitric oxide synthase (NOS) has been proposed to participate in QUIN-induced neurotoxicity; however, the precise role of NOS in QUIN-induced toxicity still remains controversial. In order to provide further information on the role of NOS isoforms in QUIN toxicity, we performed real time RT-PCR and immunohistochemistry of inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS) and determined Ca(2+)-dependent and Ca(2+)-independent NOS activity in a temporal course (3-48h), after an intrastriatal injection of QUIN to rats. NOS isoforms exhibited a transitory expression of mRNA and protein after QUIN infusion: eNOS increased between 3 and 24h, iNOS between 12 and 24h, while nNOS at 35 and 48h. Ca(2+)-independent activity (iNOS) did not show any change, while Ca(2+)-dependent activity (constitutive NOS: eNOS/nNOS) exhibited increased levels at 3h. Our results support the participation of Ca(2+)-dependent NOS isoforms during the toxic events produced at early times after QUIN injection.     

河南汉族人群eNOS基因VNTR多态性与缺血性脑血管病的关系

目的 探讨河南汉族人群内皮细胞性一氧化氮合酶(eNOS)基因内含子4可变性重复序列(VN-TR)的多态性与缺血性脑血管病(ICVD)的关系.方法 应用聚合酶链反应(PCR)技术,检测488例缺血性脑血管病患者的基因型,并与对照组比较.结果 缺血性脑血管病组eNOS基因ab基因型的频率(18.4%)明显高于对照组(13.57%),a等位基因的频率(11.5%)也明显高于对照组(7.7%),差异均有显著性(P＜0.05).结论 eNOS基因ab基因型与缺血性脑血管病有相关性,等位基因a可能是缺血性脑血管病的危险因素.     

Blockade of quinolinic acid-induced neurotoxicity by pyruvate is associated with inhibition of glial activation in a model of Huntington's disease

In this study, we have examined the mechanisms involved in pyruvate-mediated neuroprotection against quinolinic acid (QA)-induced striatal damage. QA injection into the striatum caused widespread neuronal damage and extensive areas of lesions in core and penumbra. The involvement of oxidative-mediated striatal damage was suggested by increased expressions of peroxynitrite, marked lipid peroxidation, and formation of DNA oxidative damage products. Administration of pyruvate, a glycolysis end product with antioxidant activity, significantly reduced QA-mediated striatal lesions, neuronal degeneration, and oxidative damage, whereas another energy substrate, lactate, was ineffective against oxidative damage and only partially effective in reducing lesions and neuronal degeneration. Treatment with the iNOS inhibitor aminoguanidine attenuated QA-mediated striatal lesions and reduced oxidative damage, indicating that iNOS activation may be involved in the striatal oxidative damage induced by QA. A role for glial cells in mediating oxidative damage was suggested because pyruvate blocked the expression of iNOS and nitrotyrosine in activated microglia and astrocytes in QA-injected striatum. These data suggest that pyruvate reduces oxidative free radical damage in QA-injected striatum and could have clinical utility in the treatment of Huntington's disease (HD).     

Heterogeneity of astroglia cultured from adult human temporal lobe

This study characterized the morphological and electrophysiological diversity of astroglia cultured from adult human cerebral temporal lobe, and explored the influence of the cytokine interleukin-1beta on these cells. The cultures contained astroglia positive for glial fibrillary acidic protein which were flat, bipolar or multipolar in shape and variable in size. A subpopulation of the bipolar and multipolar cells was positive for S100 protein. The most striking feature of these cultures was the presence of glia with long (600 micrometer) processes with few branches or only terminal branches. Patch clamp recordings of the non-stellate process bearing cells revealed prominent inward Na(+) and transient and sustained outward K(+) conductances. Distinct differences in the relative proportion of these conductances were evident among cells but did not appear to be correlated with cell morphology. Treatment of cultures with interleukin-1beta for 96 h did not change total protein content, but increased the content of S100beta protein and decreased the content of glial fibrillary acidic protein. The findings indicate that cultures of adult human cerebrum contain subpopulations of morphologically and electrophysiologically pleomorphic glial fibrillary acidic protein positive astroglia, exhibit increased levels of the neurotrophic factor S100beta when exposed to interleukin-1beta, and may serve as a useful model for investigation of glial involvement in neuropathology.     

Reduced activity and protein expression of NOS in R6/2 HD transgenic mice: effects of -NAME on symptom progression

Previous work found that dietary l-arginine alters symptom progression in mice transgenic for Huntington’s disease (HD), and that cerebral blood flow (CBF) is abnormal in early stage HD patients. Both of these findings potentially implicate nitric oxide (NO) and its converting enzyme, nitric oxide synthase (NOS), in HD. The current experiment found that both NOS enzymatic activity and neuronal NOS (nNOS) protein expression were reduced (P<0.05) in R6/2 HD transgenic mice compared to non-HD controls (CON). Conversely, inducible NOS (iNOS) protein expression was not significantly different between groups. The changes in nNOS were accompanied by changes in protein expression of calmodulin kinase II (CaMKII) (P<0.05) and calmodulin kinase IV (CaMKIV) (P<0.05). Protein expression of 3-nitrotyrosine (3-NT), a marker for the neurotoxin peroxynitrite, was slightly increased in non-drug treated HD and was accompanied by increased immunostaining of 3-NT in cells adhering to the vasculature and choroid plexus. Mice that received the broad-spectrum NOS inhibitor N^g-nitro- -arginine methyl ester hydrochloride ( -NAME) via their drinking water had reduced NOS enzyme activity. NOS activity varied as a function of -NAME dose, was virtually eliminated in the 500-mg/l groups, and correlated (P<0.05) with the behavioral scores as revealed by regression and correlation analyses. High dose -NAME (500 mg/l) accelerated symptom onset in HD transgenics. These results support the hypothesis that nNOS activity and NO production are abnormal in HD, this in the setting of a more global dysregulation of calcium protein expression. Taken collectively with earlier data from our laboratory demonstrating abnormal CBF findings in early-stage HD patients, these results suggest that abnormalities in NOS function may significantly contribute to the neurodegeneration found in HD.     

β-Amyloid stimulates glial cells in vitro to produce growth factors that accumulate in senile plaques in Alzheimer's disease

The effects of a synthetic homolog of β-amyloid (β1–42) on the secretion of interleukin-1 (IL-1) and basic fibroblast growth factor (bFGF) from cultures of microglia and astrocytes, cells that surround β-amyloid-containing plaques in Alzheimer's disease, were examined. Our results show that β-amyloid not only enhances glial cell secretion of these factors, it stimulates the proliferation and morphological transformation of microglia. Since IL-1 and bFGF are known to elevate the synthesis of the β-amyloid precursor protein and other plaque components, it is suggested that in this way, cascades may arise that contribute to the process of plaque development.     

β-Amyloid protein-dependent nitric oxide production from microglial cells and neurotoxicity

β-Amyloid protein (Aβ) is the major component of the senile plaques in Alzheimer's disease (AD), and microglial cells have been shown to be closely associated with these plaques. However, the roles of Aβ and microglial cells in pathogenesis of AD remain unclear. Incubation of rat microglial cells with Aβ(1–40) caused a significant increase in nitrite, a stable metabolite of nitric oxide (NO), in culture media, while there was no detectable increase in nitrite in astrocyte-rich glial cells or cortical neurons after incubation with Aβ(1–40). Nitrite production by microglial cells was also induced by Aβ(1–42), but not Aβ(25–35). An inhibitor of NO synthase, N^G-monomethyl-l-arginine (NMMA), as well as dexamethasone and actinomycin D, dose-dependently inhibited this nitrite production. Among the various cytokines investigated such as interleukin-1, interleukin-6, tumor necrosis factor- and interferon-γ (IFN-γ), only IFN-γ markedly enhanced Aβ-dependent nitrite production. Cultured cortical neurons were injured by microglial cells stimulated with Aβ in a dose-dependent manner in the presence of IFN-γ. Neurotoxicity caused by the Aβ plus IFN-γ-stimulated microglial cells was significantly attenuated by NMMA. Thus, although further investigations into the effect of Aβ on human microglial cells are needed, it is likely that Aβ-induced NO production by microglial cells is one mechanism of the neuronal death in AD.