Neurotoxicity of Human Amylin in Rat Primary Hippocampal Cultures: Similarity to Alzheimer's Disease Amyloid-β Neurotoxicity

Amylin, a 37-amino-acid amyloidogenic peptide, bears biophysical similarities to the amyloid-β peptide (Aβ) deposited in Alzheimer's disease. Using embryonic rat hippocampal cultures we tested whether amylin induces neurotoxicity similar to that previously observed with Aβ(1–40). Treatment with human amylin (1–37) resulted in prominent toxicity as assessed by phasecontrast microscopy and quantification of lactate dehydrogenase in the medium. Amylin-induced neurotoxicity was morphologically similar to that induced by Aβ(1–40). In contrast, the nonamyloidogenic rat amylin showed negligible neurotoxicity despite having 95% sequence similarity to human amylin. Only full-length human amylin was toxic; various amylin peptide fragments including amino acid residues 20–29 were nontoxic at similar concentrations. These studies suggest that unrelated amyloidogenic peptides like human amylin and Aβ can adopt a similar neurotoxic conformation in vitro. Similar conformation-dependent neurotoxicity may drive the prominent neurite degeneration around compacted but not diffuse deposits of Aβ in Alzheimer's disease.     

Inactivation of an Iron Transporter in Lactococcus lactis Results in Resistance to Tellurite and Oxidative Stress

In Lactococcus lactis, the interactions between oxidative defense, metal metabolism, and respiratory metabolism are not fully understood. To provide an insight into these processes, we isolated and characterized mutants of L. lactis resistant to the oxidizing agent tellurite (TeO₃²⁻), which generates superoxide radicals intracellularly. A collection of tellurite-resistant mutants was obtained using random transposon mutagenesis of L. lactis. These contained insertions in genes encoding a proton-coupled Mn²⁺/Fe²⁺ transport homolog (mntH), the high-affinity phosphate transport system (pstABCDEF), a putative osmoprotectant uptake system (choQ), and a homolog of the oxidative defense regulator spx (trmA). The tellurite-resistant mutants all had better survival than the wild type following aerated growth. The mntH mutant was found to be impaired in Fe²⁺ uptake, suggesting that MntH is a Fe²⁺ transporter in L. lactis. This mutant is capable of carrying out respiration but does not generate as high a final pH and does not exhibit the long lag phase in the presence of hemin and oxygen that is characteristic of wild-type L. lactis. This study suggests that tellurite-resistant mutants also have increased resistance to oxidative stress and that intracellular Fe²⁺ can heighten tellurite and oxygen toxicity.     

Oxidative Stress Induces Dephosphorylation of τ in Rat Brain Primary Neuronal Cultures

Abstract: Oxidative stress and free radical damage have been implicated in the neurodegenerative changes characteristic of several neurodegenerative diseases, including Alzheimer's disease. There is experimental evidence that the neurotoxicity of β-amyloid is mediated via free radicals, and as the deposition of β-amyloid apparently precedes the formation of paired helical filaments (PHF) in Alzheimer's disease, we have investigated whether subjecting primary neuronal cultures to oxidative stress induces changes in the phosphorylation state of the principal PHF protein τ that resemble those found in PHF-τ. Contrary to causing an increase in τ phosphorylation, treatment of neurones with hydrogen peroxide caused a dephosphorylation of τ and so we conclude that oxidative stress is not the direct cause of τ hyperphosphorylation and hence of PHF formation.     

Oxygen Glucose Deprivation in Rat Hippocampal Slice Cultures Results in Alterations in Carnitine Homeostasis and Mitochondrial Dysfunction

Mitochondrial dysfunction characterized by depolarization of mitochondrial membranes and the initiation of mitochondrial-mediated apoptosis are pathological responses to hypoxia-ischemia (HI) in the neonatal brain. Carnitine metabolism directly supports mitochondrial metabolism by shuttling long chain fatty acids across the inner mitochondrial membrane for beta-oxidation. Our previous studies have shown that HI disrupts carnitine homeostasis in neonatal rats and that L-carnitine can be neuroprotective. Thus, this study was undertaken to elucidate the molecular mechanisms by which HI alters carnitine metabolism and to begin to elucidate the mechanism underlying the neuroprotective effect of L-carnitine (LCAR) supplementation. Utilizing neonatal rat hippocampal slice cultures we found that oxygen glucose deprivation (OGD) decreased the levels of free carnitines (FC) and increased the acylcarnitine (AC): FC ratio. These changes in carnitine homeostasis correlated with decreases in the protein levels of carnitine palmitoyl transferase (CPT) 1 and 2. LCAR supplementation prevented the decrease in CPT1 and CPT2, enhanced both FC and the AC∶FC ratio and increased slice culture metabolic viability, the mitochondrial membrane potential prior to OGD and prevented the subsequent loss of neurons during later stages of reperfusion through a reduction in apoptotic cell death. Finally, we found that LCAR supplementation preserved the structural integrity and synaptic transmission within the hippocampus after OGD. Thus, we conclude that LCAR supplementation preserves the key enzymes responsible for maintaining carnitine homeostasis and preserves both cell viability and synaptic transmission after OGD.     

SOD3 Ameliorates H2O2-Induced Oxidative Damage in SH-SY5Y Cells by Inhibiting the Mitochondrial Pathway

Neurochemical Research - This study was designed to investigate the protective effects of extracellular superoxide dismutase (SOD3) against hydrogen peroxide (H2O2) induced damage in human...     

H2O2-Mediated Damage to Lysosomal Membranes of J-774 Cells

The effects of hydrogen peroxide on cell viability and, in particular, on lysosomal integrity were investigated in a model system of cultured, established, macrophage-like J-774 cells. The cells were found to rapidly degrade added hydrogen peroxide, withstanding concentrations 250μM without cell death; however, all tested concentrations (100-500/μM) substantially decreased cellular ATP to approximately the same degree. Concentrations of hydrogen peroxide 500/μM resulted in a pronounced and rapid decrease in cell viability preceded by the loss of lysosomal integrity, as judged by the relocalization of acridine orange, a lysosomotropic weak base, in pre-labelled cells. Hydrogen peroxide-induced relocalization of acridine orange and cell death were either enhanced or much prevented, according to if the cells were initially allowed to endocytose ferric iron or the specific iron-chelator deferoxamine, respectively. Depletion of ATP, however, was not associated with the loss of lysosomal integrity and viability regardless of iron or deferoxamine pretreatment. Pre-exposure to E-64, an inhibitor of lysosomal thiol proteases, resulted in the reduction of both lysosomal membrane damage and cell death. The results are interpreted as indicating (i) generation of hydroxyl radicals within the secondary lysosomal compartment due to the occurrence of reactive ferrous iron, leading to (ii) peroxidative alterations of the lysosomal membrane resulting in (iii) loss of lysosomal membrane integrity with dissipation of the proton gradient and leakage of lysosomal contents, including hydrolytic enzymes, into the cell sap. The partial protection by E-64 may result from hydroxyl radical scavening by accumulated non-degraded autophagocytosed lysosomal material, and/or decreased availability of reactive redox-cycling iron due to decreased enzymatic digestion of autophagocytosed iron-containing metalloproteins. Moreover, our results show that the normal lysosomal content of iron, capable of redox cycling, of the cell line under study is enough to induce oxidative damage leading to loss of lysosomal integrity. It is suggested that lysosomal damage may be an important cause of cell degeneration under conditions of increased intra- or extracellular hydrogen peroxide-formation.     

Mitochondrial Aconitase is a Transglutaminase 2 Substrate: Transglutamination is a Probable Mechanism Contributing to High-Molecular-Weight Aggregates of Aconitase and Loss of Aconitase Activity in Huntington Disease Brain

Transglutaminase activity was found to be present in highly purified non-synaptosomal rat brain mitochondria. A 78-kDa protein in these organelles was shown to be a transglutaminase 2 substrate, and incubation of a non-synaptosomal mitochondrial lysate with transglutaminase 2 yielded high-M_r proteins. The 78-kDa protein was identified as mitochondrial aconitase by MALDI-TOF analysis. Aconitase activity was decreased in a dose-dependent manner when non-synaptosomal rat brain mitochondria were incubated with transglutaminase 2. Transglutaminase activity is increased about 2-fold in the mitochondrial fraction of HD caudate. Moreover, Western blotting of the mitochondrial fraction revealed that most of the mitochondrial aconitase in HD caudate is present as high-M_r aggregates. Aconitase activity was previously shown to be decreased in Huntington disease (HD) caudate (a region severely damaged by the disease). The present findings suggest that an increase of transglutaminase activity in HD caudate may contribute to mitochondrial dysfunction by incorporating aconitase into inactive polymers.     

Characteristics of Sorbitol Uptake in Rat Glial Primary Cultures

Uptake of [U-14C]sorbitol was studied in astrogliarich rat primary cultures. Initial rate of sorbitol uptake is proportional to sorbitol concentration between 20 microM and 400 mM. Sorbitol transport is not inhibited by glucose, fructose, and a variety of structurally related polyols, or by cytochalasin B, an inhibitor of glucose transport. Phloretin, phlorizin, filipin, and n-hexanol, all compounds that alter the properties of biological membranes, and the sulfhydryl reagent p-chloromercuribenzoate inhibit sorbitol uptake to various degrees. Variation in the concentrations of extracellular Na+ and K+ does not affect transfer of sorbitol across the cell membrane. It is concluded that sorbitol is taken up into glial cells by a diffusion process, not involving a carrier and probably not through the lipid bilayer, but through a proteinaceous channel-like structure.     

植物细胞的氧化猝发和H2O2的信号转导

概述了植物细胞氧化猝发的特性、产生机理、生理作用以及Ｈ２Ｏ２信号转导途径及其对基因表达的调控等的研究进展。     

Metabolism of Guanine and Guanine Nucleotides in Primary Rat Neuronal Cultures

The metabolic fate of guanine and of guanine ribonucleotides (GuRNs) in cultured rat neurons was studied using labeled guanine. 8-Aminoguanosine (8-AGuo), an inhibitor of purine nucleoside phosphorylase, was used to clarify the pathways of GMP degradation, and mycophenolic acid, an inhibitor of IMP dehydrogenase, was used to assess the flux from IMP to GMP and, indirectly, the activity of the guanine nucleotide cycle (GMP----IMP----XMP----GMP). The main metabolic fate of guanine in the neurons was deamination to xanthine, but significant incorporation of guanine into GuRNs, at a rate of approximately 8.5-13.1% of that of the deamination, was also demonstrated. The turnover rate of GuRNs was fast (loss of 80% of the radioactivity of the prelabeled pool in 22 h), reflecting synthesis of nucleic acids (32.8% of the loss in radioactivity) and degradation to xanthine, guanine, hypoxanthine, guanosine, and inosine (49.3, 4.3, 4.1, 1.1, and 0.5% of the loss, respectively). Of the radioactivity in GuRNs, 7.9% was shifted to adenine nucleotides. The accumulation of label in xanthine indicates (in the absence of xanthine oxidase) that the main degradative pathway from GMP is that to xanthine through guanosine and guanine. The use of 8-AGuo confirmed this pathway but indicated the operation of an additional, relatively slower degradative pathway, that from GMP through IMP to inosine and hypoxanthine. Hypoxanthine was incorporated mainly into adenine nucleotide (91.5%), but a significant proportion (6%) was found in GuRNs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ethanol Differentially Inhibits Homoquinolinic Acid- and NMDA-Induced Neurotoxicity in Primary Cultures of Cerebellar Granule Cells

The potency of ethanol to inhibit N-methyl-D-aspartate (NMDA) receptor functions may depend on the subunit composition of the NMDA receptors. We used a NR2A-B subunit-selective NMDA receptor agonist, homoquinolinic acid (HQ), and a subunit-unselective agonist, NMDA, to induce neurotoxicity in cerebellar granule cells and examined the neuroprotective actions of ethanol, as well as NR2A- and NR2B-subunit selective antagonists, respectively. HQ was a more potent neurotoxic agent than NMDA, as measured by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. NR2A- and NR2B-selective NMDA receptor antagonists displayed quite similar neuroprotective potencies against the NMDA- and HQ-produced cell death, indicating that the higher potency of HQ to induce neurotoxicity cannot be simply explained by NR2A- or NR2B-subunit selectivity. As expected, ethanol (25 and 50 mM) attenuated the NMDA-induced neurotoxicity in a non-competitive manner by significantly reducing the maximum neurotoxicity produced by NMDA. By contrast, ethanol inhibited the HQ-induced neurotoxicity in a manner resembling a competitive-like interaction significantly increasing the EC₅₀ value for HQ, without reducing the maximum neurotoxicity produced by HQ. These results suggest that HQ reveals either a novel site or a not previously observed mechanism of interaction between ethanol and NMDA receptors in rat cerebellar granule cell cultures.     

Role of Oxidative Stress,Apoptosis, and Intracellular Homeostasis in Primary Cultures of Rat Proximal Tubular Cells Exposed to Cadmium

Cadmium (Cd) is a known nephrotoxic element. In this study, the primary cultures of rat proximal tubular (rPT) cells were treated with low doses of cadmium acetate (2.5 and 5 μM) to investigate its cytotoxic mechanism. A progressive loss in cell viability, together with a significant increase in the number of apoptotic and necrotic cells, were seen in the experiment. Simultaneously, elevation of intracellular [Ca²⁺]i and reactive oxygen species (ROS) levels, significant depletion of mitochondrial membrane potential(Δ Ψ) and cellular glutathione (GSH), intracellular acidification, and inhibition of Na⁺, K⁺-ATPase and Ca²⁺-ATPase activities were revealed in a dose-dependent manner during the exposure, while the cellular death and the apoptosis could be markedly reversed by N-acetyl-l-cysteine (NAC). Also, the calcium overload and GSH depletion were significantly affected by NAC. In conclusion, exposure of rPT cells to low-dose cadmium led to cellular death, mediated by an apoptotic and a necrotic mechanism. The apoptotic death might be the chief mechanism, which may be mediated by oxidative stress. Also, a disorder of intracellular homeostasis induced by oxidative stress and mitochondrial dysfunction is a trigger of apoptosis in rPT cells.     

Curcumin Ameliorates Copper-Induced Neurotoxicity Through Inhibiting Oxidative Stress and Mitochondrial Apoptosis in SH-SY5Y Cells

Neurochemical Research - Impaired homeostasis of copper has been linked to different pathophysiological mechanisms in neurodegenerative diseases and oxidative injury has been proposed as the main...     

Mitochondrial Oxidative Stress and Dysfunction in Rat Brain Induced by Carbofuran Exposure

Repeated low-dose exposure to carbofuran exerts its neurotoxic effects by non-cholinergic mechanisms. Emerging evidence indicates that oxidative stress plays an important role in carbofuran neurotoxicity after sub-chronic exposure. The purpose of the present study is to evaluate the role of mitochondrial oxidative stress and dysfunction as a primary event responsible for neurotoxic effects observed after sub-chronic carbofuran exposure. Carbofuran was administered to rats at a dose of 1 mg/kg orally for a period of 28 days. There was a significant inhibition in the activity of acetylcholinesterase (66.6%) in brain samples after 28 days of carbofuran exposure. Mitochondrial respiratory chain functions were assessed in terms of MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) reduction and activity of succinate dehydrogenase in isolated mitochondria. It was observed that carbofuran exposure significantly inhibited MTT reduction (31%) and succinate dehydrogenase activity (57%). This was accompanied by decrease in low-molecular weight thiols (66.6%) and total thiols (37.4%) and an increase in lipid peroxidation (43.7%) in the mitochondria isolated from carbofuran-exposed rat brain. The changes in mitochondrial oxidative stress and functions were associated with impaired cognitive and motor functions in the animals exposed to carbofuran as compared to the control animals. Based on these results, it is clear that carbofuran exerts its neurotoxicity by impairing mitochondrial functions leading to oxidative stress and neurobehavioral deficits.     

Mitochondrial O2*- and H2O2 mediate glucose deprivation-induced stress in human cancer cells

The hypothesis that glucose deprivation-induced cytotoxicity in transformed human cells is mediated by mitochondrial O2*- and H2O2 was first tested by exposing glucose-deprived SV40-transformed human fibroblasts (GM00637G) to electron transport chain blockers (ETCBs) known to increase mitochondrial O2*- and H2O2 production (antimycin A (AntA), myxothiazol (Myx), or rotenone (Rot)). Glucose deprivation (2-8 h) in the presence of ETCBs enhanced parameters indicative of oxidative stress (i.e. GSSG and steady-state levels of oxygen-centered radicals) as well as cytotoxicity. Glucose deprivation in the presence of AntA also significantly enhanced cytotoxicity and parameters indicative of oxidative stress in several different human cancer cell lines (PC-3, DU145, MDA-MB231, and HT-29). In addition, human osteosarcoma cells lacking functional mitochondrial electron transport chains (rho0) were resistant to glucose deprivation-induced cytotoxicity and oxidative stress in the presence of AntA. In the absence of ETCBs, aminotriazole-mediated inactivation of catalase in PC-3 cells demonstrated increases in intracellular steady-state levels of H2O2 during glucose deprivation. Finally, in the absence of ETCBs, overexpression of manganese containing superoxide dismutase and/or mitochondrial targeted catalase using adenoviral vectors significantly protected PC-3 cells from toxicity and oxidative stress induced by glucose deprivation with expression of both enzymes providing greater protection than was seen with either alone. Overall, these findings strongly support the hypothesis that mitochondrial O2*- and H2O2 significantly contribute to glucose deprivation-induced cytotoxicity and metabolic oxidative stress in human cancer cells.     

Comparison of Primary and Secondary Rat Astrocyte Cultures Regarding Glucose and Glutathione Metabolism and the Accumulation of Iron Oxide Nanoparticles

Astrocyte-rich primary cultures (APCs) are frequently used as a model system for the investigation of properties of brain astrocytes. However, as APCs contain a substantial number of microglial and oligodendroglial cells, biochemical parameters determined for such cultures may at least in part reflect also the presence of the contaminating cell types. To lower the potential contributions of microglial and oligodendroglial cells on properties of the astrocytes in APCs we prepared rat astrocyte-rich secondary cultures (ASCs) by subculturing of APCs and compared these ASCs with APCs regarding basal metabolic parameters, specific enzyme activities and the accumulation of iron oxide nanoparticles. Immunocytochemical characterization revealed that ASCs contained only minute amounts of microglial and oligodendroglial cells. ASCs and APCs did not significantly differ in their specific glucose consumption and lactate production rates, in their specific iron and glutathione contents, in their specific activities of various enzymes involved in glucose and glutathione metabolism nor in their accumulation of iron oxide nanoparticles. Thus, the absence or presence of some contaminating microglial and oligodendroglial cells appears not to substantially modulate the investigated metabolic parameters of astrocyte cultures.     

Oxidative modification of lens crystallins by H2O2 and chelated iron

Crystallins are the soluble structural proteins that constitute approximately 90% of the dry mass of the eye lens. The present study attempts to elucidate possible mechanisms whereby the H2O2 present in the eye could contribute to the oxidative modification of lens crystallins. The data indicate that exposure of solutions of crystallins to H2O2 and EDTA-chelated iron leads to covalent crosslinking of polypeptides, loss of intrinsic protein fluorescence, and the generation of a novel fluorophor emitting in the 420 nm range. These changes closely mimic oxidative modifications that occur in lens proteins in vivo. Exposure of the proteins to H2O2 in the absence of chelated iron failed to generate detectable levels of these modifications. These findings are contrasted with earlier studies of lenses in organ culture where H2O2 alone produced marked damage while the further addition of chelated iron protected the lenses from oxidation.     

Glutamate Neurotoxicity Is Independent of Calpain I Inhibition in Primary Cultures of Cerebellar Granule Cells

Glutamate-induced neurotoxicity and calpain activity were studied in primary cultures of rat cerebellar granule neurons and glial cells. Calpain activation, as monitored by quantitative immunoblotting of spectrin, required micromolar concentrations of Ca2+ in neuronal homogenates (calpain I) and millimolar Ca2+ concentrations in glial homogenates (calpain II). Glutamate-induced toxicity and calpain activation were observed in neuronal, but not in glial, cultures. In neurons, calpain I activation by glutamate was dose-dependent and persisted after withdrawal of neurotoxic doses of glutamate. Natural (GM1) and semisynthetic (LIGA4) gangliosides or the glutamate receptor blocker MK-801 prevented calpain I activation and delayed neuronal death elicited by glutamate. GM1 and LIGA4 had no effect on calpain I activity in neuronal homogenates, however. Furthermore, two calpain I inhibitors (leupeptin and N-acetyl-Leu-Leu-norleucinal) prevented glutamate-induced spectrin degradation, but failed to affect glutamate neurotoxicity. These results thus suggest that glutamate-induced neurotoxicity is independent of calpain I activation.