amyloid-beta-peptides and 6-hydroxy-2-5-7-8-tetramethylchroman-2-carboxylic-acid

Previous studies suggested that amyloid β (Aβ)-induced disruption of astrocytic Ca(2+) signalling and oxidative stress play a major role in the progression towards neuronal and glial death in Alzheimer's disease. We have recently demonstrated that Ca(2+)-permeable TRPV4 channels are highly expressed in rat hippocampal astrocytes and are involved in oxidative stress-induced cell damage. The aim of this study was to test the hypothesis that TRPV4 channels also contribute to hippocampal damage evoked by Aβ. Synthetic Aβ40 evoked cell death in hippocampal slice cultures in a concentration (0-20μM) and time (12-48h) dependent manner, after cultures were preconditioned with sublethal concentration of buthionine sulfoximine (1.5μM) which enhanced endogenous ROS production. As demonstrated by propidium iodide fluorescence, damage was observed in the granule cell layer of the dentate gyrus and to a smaller degree in pyramidal neurons of the CA1-CA3 region, as well as in glia cells mainly at the edge of the slice. Immunocytochemistry revealed an altered pattern of TRPV4 and GFAP protein expression, and reactive astrogliosis surrounding pyramidal CA1-CA3 neurons. Neuronal and astrocytic damage was attenuated by the antioxidant Trolox, TRPV4 channel blockers Gd(3+) and ruthenium red (RR), and a specific inhibitor of the redox and Ca(2+)-sensitive phospholipase A2 enzyme (MAFP). In disassociated co-cultures of hippocampal neurons and astrocytes without BSO preconditioning, Aβ40 evoked pronounced neuronal damage, enhanced the expression of TRPV4 and GFAP proteins (indicative of reactive astrogliosis), and increased intracellular free Ca(2+) concentration in astrocytes. The latter effect was attenuated by RR and in Ca(2+)-free media. These data show that Aβ40 can activate astrocytic TRPV4 channels in the hippocampus, leading to neuronal and astrocytic damage in a Ca(2+) and oxidative stress-dependent manner.

Topics: Amyloid beta-Peptides; Animals; Animals, Newborn; Antioxidants; Arachidonic Acids; Astrocytes; Cadmium Chloride; Calcium Signaling; Cell Death; Cells, Cultured; Chromans; Coculture Techniques; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hippocampus; Neurons; Organ Culture Techniques; Organophosphonates; Peptide Fragments; Rats; Rats, Wistar; Time Factors; TRPV Cation Channels

Cannabinoids have been widely reported to have neuroprotective properties in vitro and in vivo. In this study we compared the effects of CB1 and CB2 receptor-selective ligands, the endocannabinoid anandamide and the phytocannabinoid cannabidiol, against oxidative stress and the toxic hallmark Alzheimer's protein, β-amyloid (Aβ) in neuronal cell lines. PC12 or SH-SY5Y cells were selectively exposed to either hydrogen peroxide, tert-butyl hydroperoxide or Aβ, alone or in the presence of the CB1 specific agonist arachidonyl-2'-chloroethylamide (ACEA), CB2 specific agonist JWH-015, anandamide or cannabidiol. Cannabidiol improved cell viability in response to tert-butyl hydroperoxide in PC12 and SH-SY5Y cells, while hydrogen peroxide-mediated toxicity was unaffected by cannabidiol pretreatment. Aβ exposure evoked a loss of cell viability in PC12 cells. Of the cannabinoids tested, only anandamide was able to inhibit Aβ-evoked neurotoxicity. ACEA had no effect on Aβ-evoked neurotoxicity, suggesting a CB1 receptor-independent effect of anandamide. JWH-015 pretreatment was also without protective influence on PC12 cells from either pro-oxidant or Aβ exposure. None of the cannabinoids directly inhibited or disrupted preformed Aβ fibrils and aggregates. In conclusion, the endocannabinoid anandamide protects neuronal cells from Aβ exposure via a pathway unrelated to CB1 or CB2 receptor activation. The protective effect of cannabidiol against oxidative stress does not confer protection against Aβ exposure, suggesting divergent pathways for neuroprotection of these two cannabinoids.

Topics: Amyloid beta-Peptides; Analysis of Variance; Animals; Antioxidants; Arachidonic Acids; Benzothiazoles; Cannabinoids; Cell Line, Tumor; Cell Survival; Chromans; Drug Interactions; Gene Expression Regulation; Humans; Hydrogen Peroxide; Indoles; Lipid Peroxidation; Microscopy, Electron, Transmission; Neuroblastoma; Oxidative Stress; PC12 Cells; Peptide Fragments; Rats; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Thiazoles

Alzheimer's disease (AD) in the early stages is characterized by memory impairment, which may be attributable to synaptic dysfunction. Oxidative stress, mitochondrial dysfunction, and Ca²⁺ dysregulation are key factors in the pathogenesis of AD, but the causal relationship between these factors and synaptic dysfunction is not clearly understood. We found that in the hippocampus of an AD mouse model (Tg2576), mitochondrial Ca²⁺ handling in dentate granule cells was impaired as early as the second postnatal month, and this Ca²⁺ dysregulation caused an impairment of post-tetanic potentiation in mossy fiber-CA3 synapses. The alteration of cellular Ca²⁺ clearance in Tg2576 mice is region-specific within hippocampus because in another region, CA1 pyramidal neuron, no significant difference in Ca²⁺ clearance was detected between wild-type and Tg2576 mice at this early stage. Impairment of mitochondrial Ca²⁺ uptake was associated with increased mitochondrial reactive oxygen species and depolarization of mitochondrial membrane potential. Mitochondrial dysfunctions in dentate granule cells and impairment of post-tetanic potentiation in mossy fiber-CA3 synapses were fully restored when brain slices obtained from Tg2576 were pretreated with antioxidant, suggesting that mitochondrial oxidative stress initiates other dysfunctions. Reversibility of early dysfunctions by antioxidants at the preclinical stage of AD highlights the importance of early diagnosis and antioxidant therapy to delay or prevent the disease processes.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Animals, Genetically Modified; Antioxidants; Biophysics; Calcium; Chromans; Dentate Gyrus; Disease Models, Animal; Drug Interactions; Electric Stimulation; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Humans; In Vitro Techniques; Male; Membrane Potential, Mitochondrial; Mice; Mitochondria; Mossy Fibers, Hippocampal; Mutation; Neuronal Plasticity; Neurons; Patch-Clamp Techniques; Peptide Fragments; Plasma Membrane Calcium-Transporting ATPases; Reactive Oxygen Species; Ruthenium Compounds; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sodium-Calcium Exchanger; Synaptic Transmission

Thiamine pyrophosphate (TPP) and the activities of thiamine-dependent enzymes are reduced in Alzheimer's disease (AD) patients. In this study, we analyzed the relationship between thiamine deficiency (TD) and amyloid precursor protein (APP) processing in both cellular and animal models of TD. In SH-SY5Y neuroblastoma cells overexpressing APP, TD promoted maturation of β-site APP cleaving enzyme 1 (BACE1) and increased β-secretase activity which resulted in elevated levels of β-amyloid (Aβ) as well as β-secretase cleaved C-terminal fragment (β-CTF). An inhibitor of β-secretase efficiently reduced TD-induced up-regulation of Aβ and β-CTF. Importantly, thiamine supplementation reversed the TD-induced alterations. Furthermore, TD treatment caused a significant accumulation of reactive oxygen species (ROS); antioxidants suppressed ROS production and maturation of BACE1, as well as TD-induced Aβ accumulation. On the other hand, exogenous Aβ(1-40) enhanced TD-induced production of ROS. A study on mice indicated that TD also caused Aβ accumulation in the brain, which was reversed by thiamine supplementation. Taken together, our study suggests that TD could enhance Aβ generation by promoting β-secretase activity, and the accumulation of Aβ subsequently exacerbated TD-induced oxidative stress.

Topics: Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Analysis of Variance; Animals; Antioxidants; Aspartic Acid Endopeptidases; Brain; Cell Death; Cell Line, Tumor; Chromans; Disease Models, Animal; Gene Expression Regulation; Humans; Male; Mice; Mice, Inbred C57BL; Neuroblastoma; Peptide Fragments; Pyrithiamine; Reactive Oxygen Species; Thiamine; Thiamine Deficiency; Time Factors

Different mechanisms including oxidative stress are proposed for amyloid-β peptide (Aβ) neurotoxicity, and here we contribute to demonstrate that nitro-oxidative stress is playing a key role. Yeasts are a well-known model for H2O2 toxicity. Interestingly, yeast cell wall prevents interaction of Aβ fibrils with membrane receptors or calcium channels and we found a significant viability reduction in yeasts when challenged with Aβ fibrils. Furthermore, iron and copper chelators, as well as the antioxidants glutathione and trolox, were neuroprotective on neuroblastoma cells and mouse hippocampal neurons challenged with Aβ fibrils. Glutathione prevents the oxidation, glycation and nitrotyrosination of cell proteins induced by Aβ. Trolox protected neurons in cell viability studies, maintaining the vesicular transport integrity and preventing the trigger of apoptotic mechanisms. Interestingly, we have also found that brain derived neuronal factor (BDNF) and neurotrophin-3 (NT-3) were able to protect mouse hippocampal and cortical neurons against H2O2 and Aβ fibrils. Considering that superoxide anion, produced by Aβ cell damage, and nitric oxide, whose production is altered in AD, react to form the highly reactive peroxynitrite anion, we studied the role of trolox to ameliorate the peroxynitrite cell damage. Finally, one of the major proteins to be nitrotyrosinated in AD, the triose phosphate isomerase (TPI) was assayed searching for a denitrase activity that could reverse intracellular nitrotyrosination. We have found that human neuroblastoma SH-SY5Y cells express a constitutive denitrase activity that partially denitrated nitro-TPI. Altogether, our results support a key role of nitro-oxidative stress in the neuronal damage induced by Aβ fibrils.

Topics: Aged; Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Animals; Antioxidants; Apoptosis; Caspase 3; Cell Survival; Cells, Cultured; Cerebral Cortex; Chromans; Deferoxamine; Dose-Response Relationship, Drug; Embryo, Mammalian; Glutathione; Hippocampus; Humans; Hydrogen Peroxide; Immunoprecipitation; In Situ Nick-End Labeling; Male; Mice; Models, Biological; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Oxidative Stress; Peptide Fragments; Rats; Siderophores; Thymus Extracts

Amyloid-beta protein (A beta) deposition in the cerebral vascular walls is one of the key features of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). A beta(1-40) carrying the 'Dutch' mutation (HCHWA-D A beta(1-40)) induces pronounced degeneration of cultured human brain pericytes. In this study, we aimed to identify inhibitors of A beta-induced toxicity in human brain pericytes. The toxic effect of HCHWA-D A beta(1-40) on human brain pericytes was inhibited by co-incubation with catalase, but not with superoxide dismutase, glutathione or vitamin E analogue Trolox. Catalase interacts with A beta, both in cell cultures and in cell-free assays, and has a prominent effect on the amount and conformational state of A beta binding to the cell surface of human brain pericytes. This activity of catalase is likely based on its ability to bind and slowly degrade A beta and not by its usual capacity to convert hydrogen peroxide. Our data confirm that assembly of A beta at the cell surface of human brain pericytes is a crucial step in A beta-induced cellular degeneration of human brain pericytes. Inhibition of fibril formation at the cell surface could be an important factor in therapy aimed at reducing cerebral amyloid angiopathy.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloidosis; Antioxidants; Blotting, Western; Brain; Catalase; Cell Death; Cells, Cultured; Chromans; Glutathione; Humans; In Vitro Techniques; Microscopy, Immunoelectron; Oxidative Stress; Peptide Fragments; Pericytes; Protein Binding; Superoxide Dismutase

The toxicity of the beta-amyloid (Abeta) peptide of Alzheimer's disease may relate to its polymerisation state (i.e. fibril content). We have shown previously that plasma lipoproteins, particularly when oxidised, greatly enhance Abeta polymerisation. In the present study the nature of the interactions between both native and oxidised lipoproteins and Abeta1-40 was investigated employing various chemical treatments. The addition of ascorbic acid or the vitamin E analogue, trolox, to lipoprotein/Abeta coincubations failed to inhibit Abeta fibrillogenesis, as did the treatment of lipoproteins with the aldehyde reductant, sodium borohydride. The putative lipid peroxide-derived aldehyde scavenger, aminoguanidine, however, inhibited Abeta-oxidised lipoprotein-potentiated polymerisation, but in a manner consistent with an antioxidant action for the drug. Lipoprotein treatment with the reactive aldehyde 4-hydroxy-2-trans-nonenal enhanced Abeta polymerisation in a concentration-dependent fashion. Incubation of Abeta with lipoprotein fractions from which the apoprotein components had been removed resulted in extents of polymerisation comparable to those observed with Abeta alone. These data indicate that the apoprotein components of plasma lipoproteins play a key role in promoting Abeta polymerisation, possibly via interactions with aldehydes.

Topics: Aldehydes; Alzheimer Disease; Amyloid beta-Peptides; Antioxidants; Apolipoproteins; Ascorbic Acid; Biopolymers; Borohydrides; Chromans; Guanidines; Humans; Kinetics; Lipoproteins; Oxidation-Reduction; Peptide Fragments

Beta amyloid (Abeta) is implicated in Alzheimer's disease (AD). Abeta(1 - 42) (5, 10, or 20 microM) was able to increase NO release and decrease cellular viability in primary rat cortical mixed cultures. L-NOARG and SMTC (both at 10 or 100 microM) - type I NOS inhibitors - reduced cellular NO release in the absence of Abeta(1 - 42). At 100 microM, both drugs decreased cell viability. L-NIL (10 or 100 microM), and 1400W (1 or 5 microM) - type II NOS inhibitors - reduced NO release and improved viability when either drug was administered up to 4 h post Abeta(1 - 42) (10 microM) treatment. L-NOARG and SMTC (both at 10 or 100 microM) were only able to decrease NO release. Carboxy-PTIO or Trolox (both at 10 or 100 microM) - a NO scavenger and an antioxidant, respectively - increased viability when administered up to 1 h post Abeta(1 - 42) treatment. Either L-NIL (50 microM) or 1400W (3 microM) and Trolox (50 microM) showed synergistic actions. Peroxynitrite (100 or 200 microM) reduced cell viability. Viabilities were improved by L-NIL (100 microM), 1400W (5 microM), carboxy-PTIO (10 or 100 microM), and Trolox (10 or 100 microM). Hence, the data show that Abeta(1 - 42) induced NO release in neurons and glial cells, and that Abeta neurotoxicity is, at least in part, mediated by NO. NO concentration modulating compounds and antioxidant may have therapeutic importance in neurological disorders where oxidative stress is likely involved such as in AD.

Topics: Amyloid beta-Peptides; Animals; Antioxidants; Benzoates; Cell Survival; Cells, Cultured; Cerebral Cortex; Chromans; Citrulline; Dose-Response Relationship, Drug; Enzyme Inhibitors; Imidazoles; Isoenzymes; Lysine; Neuroprotective Agents; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Oxidants; Peptide Fragments; Rats; Rats, Sprague-Dawley; Thiourea; Time Factors