ABSTRACT
Excess release of glutamate at the inner hair cell-type I auditory nerve synapse results in excitotoxicity characterized by rapid swelling and disintegration of the afferent synapses, but in some cases, the damage expands to the spiral ganglion soma. Cochlear excitotoxic damage is largely mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) and kainate receptor (KAR) and potentially N-methyl-D-aspartate receptors (NMDAR). Because these receptors are developmentally regulated, the pattern of excitotoxic damage could change during development. To test this hypothesis, we compared AMPAR, NMDAR and KAR immunolabeling and excitotoxic damage patterns in rat postnatal day 3 (P3) and adult cochlear cultures. At P3, AMPAR and KAR immunolabeling, but not NMDAR, was abundantly expressed on peripheral nerve terminals adjacent to IHCs. In contrast, AMPAR, KAR and NMDAR immunolabeling was minimal or undetectable on the SGN soma. In adult rats, however, AMPAR, KAR and NMDAR immunolabeling occurred on both peripheral nerve terminals near IHCs as well as the soma of SGNs. High doses of Glu and KA only damaged peripheral nerve terminals near IHCs, but not SGNs, at P3, consistent with selective expression of AMPAR and KAR expression on the terminals. However, in adults, Glu and KA damaged both peripheral nerve terminals near IHCs and SGNs both of which expressed AMPAR and KAR. These results indicate that cochlear excitotoxic damage is closely correlated with structures that express AMPAR and KAR.
Subject(s)
Spiral Ganglion , Animals , Glutamic Acid , Hair Cells, Auditory, Inner , Neurons , Rats , Receptors, N-Methyl-D-Aspartate , Up-Regulation , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicityABSTRACT
Glutamate excitotoxicity and endoplasmic reticulum (ER) recently have been found to be instrumental in the pathogenesis of various neurodegenerative diseases. However, the paucity of literature deciphering the inter-linkage among glutamate receptors, behavioral alterations, and ER demands thorough exploration. Reckoning the aforesaid concerns, a prospective study was outlined to delineate the influence of ER stress inhibition via 4-phenylbutyric acid (PBA) on α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) excitotoxicity-induced behavioral aspects and possible ER stress-glutamate linkage. Male SD rats were randomly divided into four groups namely sham (surgical control+vehicle, group 1), AMPA-induced excitotoxic group 2 receive a single intra-hippocampal injection of 10 mM AMPA, group 3 received AMPA along with PBA (i.p, 100 mg/kg body weight) for 15 days, and group 4 received PBA alone. Behavioral analyses were performed prior to the sacrifice of animals and hippocampus was extracted thereafter for further analysis. AMPA-induced excitotoxicity exhibited significant impairment of locomotion as well as cognitive functions. The levels of neurotransmitters such as dopamine, homo vanillic acid (HVA), norepinephrine, and serotonin were reduced accompanied by reduced expression of GLUR1 and GLUR4 (glutamate receptor) as well as loss of neurons in different layers of hippocampus. ER stress markers were upregulated upon AMPA excitotoxicity. However, chemical chaperone PBA supplementation remarkably mitigated the behavioral alterations along with expression of glutamate and ER stress intermediates/markers in AMPA excitotoxic animals. Therefore, the present exploration convincingly emphasizes the significance of ER stress and its inhibition via PBA in combating cognitive impairment as well as improving locomotion in excitotoxic animals.
Subject(s)
Butylamines/pharmacology , Cognitive Dysfunction/chemically induced , Cognitive Dysfunction/prevention & control , Endoplasmic Reticulum Stress/physiology , Excitatory Amino Acid Agonists/toxicity , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicity , Animals , Butylamines/therapeutic use , Cognitive Dysfunction/metabolism , Endoplasmic Reticulum Stress/drug effects , Glutamic Acid/metabolism , Locomotion/drug effects , Locomotion/physiology , Male , Rats , Rats, Sprague-DawleyABSTRACT
The endocannabinoid system has been shown to be a putative therapeutic target for retinal disease. Here, we aimed to investigate the ability of the endocannabinoid 2-arachidonoylglycerol (2-AG) and novel inhibitors of its metabolic enzymes, α/ß-hydrolase domain-containing 6 (ABHD6) and monoacylglycerol lipase (MAGL), a) to protect the retina against excitotoxicity and b) the mechanisms involved in the neuroprotection. Sprague-Dawley rats, wild type and Akt2-/- C57BL/6 mice were intravitreally administered with phosphate-buffered saline or (RS)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA). 2-AG was intravitreally co-administered with AMPA in the absence and presence of AM251 or AM630 (cannabinoid 1 and 2 receptor antagonists, respectively) or Wortmannin [Phosphoinositide 3-Kinase (PI3K)/Akt inhibitor]. Inhibitors of ABHD6 and dual ABHD6/MAGL (AM12100 and AM11920, respectively) were co-administered with AMPA intravitreally in rats. Immunohistochemistry was performed using antibodies raised against retinal neuronal markers (bNOS), microglia (Iba1) and macroglia (GFAP). TUNEL assay and real-time PCR were also employed. The CB2 receptor was expressed in rat retina (approx. 62% of CB1 expression). 2-AG attenuated the AMPA-induced increase in TUNEL+ cells. 2-AG activation of both CB1 and CB2 receptors and the PI3K/Akt downstream signaling pathway, as substantiated by the use of Akt2-/- mice, afforded neuroprotection against AMPA excitotoxicity. AM12100 and AM11920 attenuated the AMPA-induced glia activation and produced a dose-dependent partial neuroprotection, with the dual inhibitor AM11920 being more efficacious. These results show that 2-AG has the pharmacological profile of a putative therapeutic for retinal diseases characterized by neurodegeneration and neuroinflammation, when administered exogenously or by the inhibition of its metabolic enzymes.
Subject(s)
Anti-Inflammatory Agents/administration & dosage , Arachidonic Acids/administration & dosage , Endocannabinoids/administration & dosage , Enzyme Inhibitors/administration & dosage , Glycerides/administration & dosage , Monoacylglycerol Lipases/antagonists & inhibitors , Retina/drug effects , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicity , Animals , Dose-Response Relationship, Drug , Female , Intravitreal Injections , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Monoacylglycerol Lipases/metabolism , Neuroprotection/drug effects , Neuroprotection/physiology , Rats , Rats, Sprague-Dawley , Retina/metabolism , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/administration & dosageABSTRACT
On the basis of the evidence that extracellular Zn2+ influx induced with AMPA causes Parkinson's syndrome in rats that apomorphine-induced movement disorder emerges, here we used a low dose of AMPA, which does not increase intracellular Zn2+ level in the substantia nigra pars compacta (SNpc) of young adult rats, and tested whether intracellular Zn2+ dysregulation induced with AMPA is accelerated in the SNpc of aged rats, resulting in age-related vulnerability to Parkinson's syndrome. When AMPA (1 mM) was injected at the rate of 0.05 µl/min for 20 min into the SNpc, intracellular Zn2+ level was increased in the SNpc of aged rats followed by increase in turning behavior in response to apomorphine and nigral dopaminergic degeneration. In contrast, young adult rats do not show movement disorder and nigral dopaminergic degeneration, in addition to no increase in intracellular Zn2+. In aged rats, movement disorder and nigral dopaminergic degeneration were rescued by co-injection of either extracellular (CaEDTA) or intracellular (ZnAF-2DA) Zn2+ chelators. 1-Naphthyl acetyl spermine (NASPM), a selective blocker of Ca2+- and Zn2+-permeable GluR2-lacking AMPA receptors blocked increase in intracellular Zn2+ in the SNpc of aged rats followed by rescuing nigral dopaminergic degeneration. The present study indicates that intracellular Zn2+ dysregulation is accelerated by Ca2+- and Zn2+-permeable GluR2-lacking AMPA receptor activation in the SNpc of aged rats, resulting in age-related vulnerability to Parkinson's syndrome.
Subject(s)
Dopaminergic Neurons/drug effects , Excitatory Amino Acid Agonists/toxicity , Nerve Degeneration , Parkinson Disease, Secondary/chemically induced , Pars Compacta/drug effects , Receptors, AMPA/agonists , Zinc/metabolism , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicity , Age Factors , Animals , Behavior, Animal/drug effects , Calcium/metabolism , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/pathology , Male , Motor Activity/drug effects , Parkinson Disease, Secondary/metabolism , Parkinson Disease, Secondary/pathology , Parkinson Disease, Secondary/physiopathology , Pars Compacta/metabolism , Pars Compacta/pathology , Pars Compacta/physiopathology , Rats, Wistar , Receptors, AMPA/metabolismABSTRACT
Cannabinoids have been shown to protect the retina from ischemic/excitotoxic insults. The aim of the present study was to investigate the neuroprotective and anti-inflammatory properties of the synthetic cannabinoid (R)-WIN55,212-2 (CB1/CB2 receptor agonist) when administered acutely or subchronically in control and AMPA treated retinas. Sprague-Dawley rats were intravitreally administered (acutely) with vehicle or AMPA, in the absence or presence of (R)-WIN55,212-2 (10-7-10-4M) alone or in combination with AM251 [CB1 receptor antagonist/inverse agonist,10-4M] and AM630 (CB2 receptor antagonist,10-4M). In addition, AMPA was co-administered with the racemic (R,S)-WIN55,212 (10-4Μ). (R)-WIN55,212-2 was also administered subchronically (25,100 µg/kg,i.p.,4d) in control and AMPA treated rats. Immunohistochemical studies were performed using antibodies against the CB1R, and retinal markers for retinal neurons (brain nitric oxide synthetase, bNOS) and microglia (ionized calcium binding adaptor molecule 1, Iba1). ELISA assay was employed to assess TNFα levels in AMPA treated retinas. Intravitreal administration of (R)-WIN55,212-2 reversed the AMPA induced loss of bNOS expressing amacrine cells, an effect that was blocked by both AM251 and AM630. (R,S)WIN55,212 had no effect. (R)-WIN55,212-2 also reduced a) the AMPA induced activation of microglia, by activating CB2 receptors that were shown to be colocalized with Iba1+ reactive microglial cells, and b) TNFα levels in retina. (R)-WIN55,212-2 administered subchronically led to the downregulation of CB1 receptors at the high dose of 100 µg/kg(i.p.), and to the attenuation of the WIN55,212-2 induced neuroprotection of amacrine cells. At the same dose, (R)-WIN55,212-2 did not attenuate the AMPA induced increase in the number of reactive microglia cells, suggesting CB2 receptor downregulation under subchronic conditions. This study provides new findings regarding the role of CB1 and CB2 receptor activation by the synthetic cannabinoid (R)-WIN55,212-2, administered acutely or sub-chronically, on neuron viability and microglia activation in healthy and diseased retina.
Subject(s)
Anti-Inflammatory Agents/administration & dosage , Benzoxazines/administration & dosage , Morpholines/administration & dosage , Naphthalenes/administration & dosage , Neuroprotective Agents/administration & dosage , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/metabolism , Retina/metabolism , Animals , Dose-Response Relationship, Drug , Drug Administration Schedule , Female , Male , Rats , Rats, Sprague-Dawley , Receptor, Cannabinoid, CB1/agonists , Receptor, Cannabinoid, CB2/agonists , Retina/drug effects , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicityABSTRACT
NADPH oxidases (NOX) are activated in ischemic conditions leading to increases in reactive oxygen species (ROS) and neurotoxicity. The aim of the present study was to investigate the role of NOX in the development of retinal pathologies, associated with excitotoxicity and the evaluation of NOX inhibitors as putative therapeutic agents. Sprague-Dawley rats were used for the induction of the in vivo retinal model of (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA) excitotoxicity. Rats were intravitreally administered with PBS, AMPA (42 nmoles) or AMPA + NOX inhibitors, VAS2870 (pan-NOX inhibitor, 10-6-10-4 M), ML171 (NOX1 inhibitor, 10-5, 10-4 M), and GLX7013114 (NOX4 inhibitor, 10-4 M). Immunohistochemical studies were performed using antibodies raised against nitrotyrosine, a ROS/oxidative stress marker, bNOS, a neuronal marker for nitric oxide synthase and the macro and microglia markers, glial fibrillary acidic protein and ionized calcium-binding adaptor molecule-1, respectively. VAS2870 and ML171 showed neuroprotective and anti-inflammatory actions reversing the AMPA induced reduction of bNOS expressing amacrine cells and attenuating macro/microglial activation. GLX7013114 (10-4 M) did not protect bNOS expressing amacrine cells, but it did attenuate the AMPA induced increase in nitrotyrosine positive cells and activation of glial cells. These results suggest that NOX1, NOX4 and possibly NOX2 (due to the actions of VAS2870) play an important role in the pathophysiology of the retina and that NOX inhibitors are putative neuroprotective and anti-inflammatory agents against retinal abnormalities caused by excitotoxicity.
Subject(s)
Benzoxazoles/pharmacology , Ischemia/drug therapy , NADPH Oxidase 4/antagonists & inhibitors , Retina/metabolism , Retinal Diseases/drug therapy , Triazoles/pharmacology , Animals , Disease Models, Animal , Female , Immunohistochemistry , Ischemia/chemically induced , Ischemia/metabolism , Male , Microglia/metabolism , NADPH Oxidase 4/metabolism , Oxidation-Reduction , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Retinal Diseases/chemically induced , Retinal Diseases/metabolism , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicityABSTRACT
BACKGROUND: Small conductance, calcium-activated (SK3) potassium channels control the intrinsic excitability of dopaminergic neurons (DN) in the midbrain and modulate their susceptibility to toxic insults during development. METHODS: We evaluated the age-dependency of the neuroprotective effect of an SK3 agonist, 1-Ethyl-1,3-dihydro-2H-benzimidazol-2-one (1-EBIO), on Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) excitotoxicity to DN in ventral mesencephalon (VM) organotypic cultures. RESULTS: Most tyrosine hydroxylase (TH)+ neurons were also SK3+; SK3+/TH- cells (DN+) were common at each developmental stage but more prominently at day in vitro (DIV) 8. Young DN+ neurons were small bipolar and fusiform, whereas mature ones were large and multipolar. Exposure of organotypic cultures to AMPA (100 µm, 16 h) had no effect on the survival of DN+ at DIV 8, but caused significant toxicity at DIV 15 (n = 15, p = 0.005) and DIV 22 (n = 15, p<0.001). These results indicate that susceptibility of DN to AMPA excitotoxicity is developmental stage-dependent in embryonic VM organotypic cultures. Immature DN+ (small, bipolar) were increased after AMPA (100 µm, 16 h) at DIV 8, at the expense of the number of differentiated (large, multipolar) DN+ (p = 0.039). This effect was larger at DIV 15 (p<<<0.0001) and at DIV 22 (p<<<0.0001). At DIV 8, 30 µM 1-EBIO resulted in a large increase in DN+. At DIV 15, AMPA toxicity was prevented by exposure to 30 µM, but not 100 µM 1-EBIO. At DIV 22, excitotoxicity was unaffected by 30 µM 1-EBIO, and partially reduced by 100 µM 1-EBIO. CONCLUSION: The effects of the SK3 channel agonist 1-EBIO on the survival of SK3-expressing dopaminergic neurons were concentration-dependent and influenced by neuronal developmental stage.
Subject(s)
Dopaminergic Neurons/drug effects , Neuroprotective Agents/pharmacology , Small-Conductance Calcium-Activated Potassium Channels/agonists , Animals , Benzimidazoles/pharmacology , Cell Survival/drug effects , Cells, Cultured , Dopaminergic Neurons/metabolism , Dopaminergic Neurons/pathology , Embryo, Mammalian/cytology , Embryonic Development/drug effects , Female , Mesencephalon/cytology , Mesencephalon/metabolism , Rats , Rats, Sprague-Dawley , Receptors, AMPA/metabolism , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicityABSTRACT
Assessment of the effects of chemical mixtures is a very important objective of the ecotoxicological risk assessment. This study was aimed at evaluating for the first time the effects of a mixture of glyphosate and its main breakdown product aminomethylphosphonic acid (AMPA) on various biomarkers in the mussel Mytilus galloprovincialis. Mussels were exposed for 7, 14 and 21 days to either 100 µg/L of glyphosate, 100 µg/L of AMPA or a mixture of both (100 + 100 µg/L). Various haemocyte parameters, such as total haemocyte counts, haemocyte diameter and volume, haemocyte proliferation, haemolymph lactate dehydrogenase activity and haemocyte lysate acid phosphatase activities were measured. In addition, the effects of exposure on the activity of antioxidant enzymes, acetylcholinesterase and glutathione-S-transferase were evaluated in gills and digestive gland from mussels. On the whole, this study demonstrated that the variables considered in the experimental plan, namely treatment, exposure time and their interaction, affect significantly biomarker responses in M. galloprovincialis. The effects of the mixture were comparable to those of the individual compounds, whereas their synergistic effects were occasionally observed, under the experimental conditions tested at least.
Subject(s)
Gills/drug effects , Glycine/analogs & derivatives , Hemocytes/drug effects , Mytilus/drug effects , Water Pollutants, Chemical/toxicity , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicity , Animals , Biomarkers/metabolism , Ecotoxicology , Glycine/toxicity , GlyphosateABSTRACT
Glyphosate (GLY) is the active ingredient of several herbicide formulations widely used to control weeds in agricultural and non-agricultural areas. Due to the intensive use of GLY-based herbicides and their direct application on soils, some of their components, including the active ingredient, may reach the aquatic environment through direct run-off and leaching. The present study assessed the acute toxicity and genotoxicity of the GLY-based formulation Atanor 48 (ATN) and its major constituents GLY, surfactant polyethoxylated tallow amine (POEA), as well as the main metabolite of GLY aminomethylphosphonic acid (AMPA) on non-target aquatic organisms. The toxic effects of these chemicals were evaluated in the fish embryo acute toxicity test with zebrafish (Danio rerio), while genotoxic effects were investigated in the comet assays with cells from zebrafish larvae and rainbow trout gonad-2 (RTG-2). GLY and AMPA caused no acute toxic effect, while ATN and POEA induced significant lethal effects in zebrafish (LC50-96 h 76.50 mg/L and 5.49 mg/L, respectively). All compounds were genotoxic in comet experiments with zebrafish larvae (LOEC 1.7 mg/L for GLY, ATN, AMPA and 0.4 mg/L for POEA). Unlike in vivo, only POEA induced DNA damage in RTG-2 cells (LOEC 1.6 mg/L), suggesting that it is a direct acting genotoxic agent. In summary, these data indicate that the lethal effects on zebrafish early-life stages can be ranked in the following order from most to least toxic: surfactant POEA > formulation ATN > active ingredient GLY ≈ metabolite AMPA. Genotoxic effects were observed in both RTG-2 cells (only POEA) and zebrafish (all test compounds) with the lowest tested concentrations. Therefore, it is important to evaluate different toxicological endpoints as well as use different non-target organisms to predict the hazards of GLY-based formulations and their components and breakdown product to aquatic biota.
Subject(s)
Aquatic Organisms/drug effects , Glycine/analogs & derivatives , Herbicides/toxicity , Water Pollutants, Chemical/toxicity , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicity , Amines/toxicity , Animals , Comet Assay/methods , DNA Damage/drug effects , Female , Glycine/toxicity , Larva/drug effects , Male , Mutagens/toxicity , Oncorhynchus mykiss , Organophosphorus Compounds/toxicity , Zebrafish , GlyphosateABSTRACT
Excessive stimulation of ionotropic glutamate receptor is associated with glutamate-mediated excitotoxicity, thereby causing oxidative imbalance and mitochondrial dysfunction, resulting in the excitotoxic death of neurons. Eminent role of endoplasmic reticulum under glutamate-induced excitotoxicity has been highlighted in numerous literatures which have been observed to trigger endoplasmic reticulum stress (ER stress) as well as regulating oxidative stress. However, combating ER stress in excitotoxic neurons can provide a novel approach to alleviate the mitochondrial dysfunctioning and ROS generation. Therefore, we propose to investigate the cross-communication of α-amino-3-hydroxy-5-methyl-4-isoxzole-propionate (AMPA) excitotoxicity-induced oxidative injury with ER stress by employing ER stress inhibitor-4-phenlybutyric acid (4-PBA). Male SD rats were divided into four groups viz sham group (group 1), AMPA (10 mM)-induced excitotoxic group (group 2), curative group of AMPA-induced excitotoxic animals given 4-PBA at a dose of 100 mg/kg body weight (group 3), and alone 4-PBA treatment group (100 mg/kg body weight) (group 4). Animals were sacrificed after 15 days of treatment, and hippocampi were analyzed for histopathological examination, ROS, inflammatory markers, mitochondrial dysfunction, and ER stress markers. AMPA-induced excitotoxicity exhibited a significant increase in the levels of ROS, upregulated ER stress markers, inflammation markers, and compromised mitochondrial functioning in the hippocampus. However, 4-PBA administration significantly curtailed the AMPA-induced excitotoxic insult. This study suggests that targeting ER stress with a chemical chaperone can provide a better therapeutic intervention for neurological disorders involving excitotoxicity, and thus, it opens a new avenue to screen chemical chaperones for the therapeutic modalities.
Subject(s)
Antineoplastic Agents/pharmacology , Endoplasmic Reticulum Stress/drug effects , Hippocampus/drug effects , Hippocampus/ultrastructure , Phenylbutyrates/pharmacology , Activating Transcription Factor 4/genetics , Activating Transcription Factor 4/metabolism , Animals , Cardiolipins/metabolism , Electron Transport Complex I/metabolism , Excitatory Amino Acid Agonists/toxicity , Gene Expression Regulation/drug effects , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Male , Membrane Potential, Mitochondrial/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Statistics, Nonparametric , X-Box Binding Protein 1/metabolism , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicityABSTRACT
On the basis of the findings that the rapid influx of extracellular Zn2+ into nigral dopaminergic neurons causes dopaminergic neurodegeneration, here we report that AMPA causes movement disorder in rats. AMPA markedly increased turning behavior in response to apomorphine 1 and 2 weeks after AMPA injection into the substantia nigra pars compacta (SNpc), while AMPA-induced movement disorder was suppressed by co-injection of intracellular Zn2+ chelators, i.e., ZnAF-2DA and TPEN, suggesting that AMPA-induced movement disorder is due to intracellular Zn2+ dysregulation. Furthermore, AMPA markedly induced loss of nigrostriatal dopaminergic neurons 2 weeks after AMPA injection into the SNpc, while AMPA-induced neurodegeneration was also suppressed in the SNpc and the striatum by co-injection of ZnAF-2DA and TPEN. AMPA rapidly increased nigral intracellular Zn2+ after AMPA injection into the SNpc and this increase was blocked by co-injection of TPEN. These results indicate that AMPA receptor activation rapidly increases influx of extracellular Zn2+ into nigral dopaminergic neurons and causes nigrostriatal dopaminergic neurodegeneration, resulting in movement disorder in rats. The evidence that AMPA-induced intracellular Zn2+ dysregulation causes movement disorder via nigrostriatal dopaminergic neurodegeneration suggests that AMPA receptors, probably Ca2+- and Zn2+-permeable GluR2-lacking AMPA receptors are potential targets for overcoming Parkinson's syndrome.
Subject(s)
Dopaminergic Neurons/metabolism , Dyskinesia, Drug-Induced/metabolism , Extracellular Fluid/metabolism , Substantia Nigra/metabolism , Zinc/metabolism , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicity , Animals , Chelating Agents/administration & dosage , Dopaminergic Neurons/drug effects , Dyskinesia, Drug-Induced/prevention & control , Extracellular Fluid/drug effects , Injections, Intraventricular , Male , Rats , Rats, Wistar , Substantia Nigra/drug effects , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/administration & dosageABSTRACT
In the CNS, including the optic nerve, oligodendrocytes play a critical role in the myelination of axons. Oligodendrocytes are exceptionally sensitive to insults to the CNS, such as injury, ischemia, or inflammation, which result in the loss of oligodendrocytes and myelin and eventually secondary axon degeneration. Oligodendrocytes are sensitive to excitotoxic insults mediated by overactivation of their AMPA ionotropic glutamate receptors. Phenolic compounds, which are widely distributed in fruits and vegetables, received the great attention of scientists due to their antioxidant activities and free radical scavenging abilities. Chlorogenic acid (CGA) has been demonstrated to possess potent neuroprotective activities against oxidative stress in various cellular models and pathological conditions. Hence, CGA protect against oxidative stress and excitotoxic insults mediated by AMPA receptors and that the protective mechanisms involve free radical scavenging, Ca2+ handling in the cytosol, and modulating antioxidant enzyme system. CGA was associated with the protein kinase A (PKC) signaling pathways transduction. Caspases and calpains have been studied as apoptotic mediators and cell death in this model of AMPA toxicity. Inhibitors of caspases initiators, caspases 1, 8, and 9, the upstream of caspase 3 effectors, have totally abrogated the protective activity of CGA. Inhibitors of calpains also totally abrogated the protective activity of CGA. In addition, a potential role for the CGA in inhibiting Bax in oligodendrocyte cell model undergoing AMPA is inducing excitotoxic death. Our results indicate that CGA exhibits a protective potential via antioxidant and apoptosis caspases and calpains dependent against AMPA-mediated excitotoxicity, and these finding indicate that CGA is able to be a good candidate for preventive approach for neurodegenerative disorders associated with loss and damage in oligodendrocytes and AMPA-mediated excitotoxicity.
Subject(s)
Caspases/metabolism , Chlorogenic Acid/pharmacology , Oligodendroglia/drug effects , Optic Nerve/cytology , Protein Kinase C/metabolism , Signal Transduction/drug effects , Animals , Animals, Newborn , Cell Survival/drug effects , Cells, Cultured , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Agonists/toxicity , Hydrogen Peroxide/metabolism , Iron/metabolism , Mitochondria/drug effects , Rats , Rats, Sprague-Dawley , Rats, Wistar , Reactive Oxygen Species/metabolism , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicityABSTRACT
Inhibitory GABAergic and glycinergic neurotransmission in the spinal cord play a central role in the regulation of neuronal excitability, by maintaining a balance with the glutamate-mediated excitatory transmission. Glutamatergic agonists infusion in the spinal cord induce motor neuron death by excitotoxicity, leading to motor deficits and paralysis, but little is known on the effect of the blockade of inhibitory transmission. In this work we studied the effects of GABAergic and glycinergic blockade, by means of microdialysis perfusion (acute administration) and osmotic minipumps infusion (chronic administration) of GABA and glycine receptors antagonists directly in the lumbar spinal cord. We show that acute glycinergic blockade with strychnine or GABAergic blockade with bicuculline had no significant effects on motor activity and on motor neuron survival. However, chronic bicuculline infusion, but not strychnine, induced ipsilateral gait alterations, phalange flaccidity and significant motor neuron loss, and these effects were prevented by AMPA receptor blockade with CNQX but not by NMDA receptor blockade with MK801. In addition, we demonstrate that the chronic infusion of bicuculline enhanced the excitotoxic effect of AMPA, causing faster bilateral paralysis and increasing motor neuron loss. These findings indicate a relevant role of GABAergic inhibitory circuits in the regulation of motor neuron excitability and suggest that their alterations may be involved in the neurodegeneration processes characteristic of motor neuron diseases such as amyotrophic lateral sclerosis.
Subject(s)
Bicuculline/toxicity , GABA Antagonists/toxicity , Motor Activity/drug effects , Motor Neurons/drug effects , Nerve Degeneration/chemically induced , Spinal Cord/drug effects , Strychnine/toxicity , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Animals , Atrophy/chemically induced , Bicuculline/antagonists & inhibitors , Dizocilpine Maleate/pharmacology , Drug Interactions , Gait/drug effects , Male , Muscle Hypotonia/chemically induced , Rats , Receptors, Glycine/antagonists & inhibitors , Strychnine/antagonists & inhibitors , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicityABSTRACT
Glutamate excitotoxicity and mitochondrial dysfunction are involved in motor neuron degeneration process during amyotrophic lateral sclerosis (ALS). We have previously shown that microdialysis perfusion of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) in the lumbar region of the rat spinal cord produces permanent paralysis of the ipsilateral hindlimb and death of motor neurons by a Ca(2+)-dependent mechanism, in a process that starts 2-3 h after AMPA perfusion. Co-perfusion with different energy metabolic substrates, mainly pyruvate, prevented the paralysis and motor neuron degeneration induced by AMPA, suggesting that mitochondrial energetic deficiencies are involved in this excitotoxic motor neuron death. To test this, in the present work, we studied the functional and ultrastructural characteristics of mitochondria isolated from the ventral horns of lumbar spinal cords of rats, at the beginning of the AMPA-induced degeneration process, when motor neurons are still alive. Animals were divided in four groups: perfused with AMPA, AMPA + pyruvate, and pyruvate alone and Krebs-Ringer medium as controls. Mitochondria from the AMPA-treated group showed decreased oxygen consumption rates, respiratory controls, and transmembrane potentials. Additionally, activities of the respiratory chain complexes I and IV were significantly decreased. Electron microscopy showed that mitochondria from AMPA-treated rats presented swelling, disorganized cristae and disrupted membranes. Remarkably, in the animals co-perfused with AMPA and pyruvate all these abnormalities were prevented. We conclude that mitochondrial dysfunction plays a crucial role in spinal motor neuron degeneration induced by overactivation of AMPA receptors in vivo. These mechanisms could be involved in ALS motor neuron degeneration.
Subject(s)
Excitatory Amino Acid Agonists/toxicity , Mitochondria/pathology , Motor Neuron Disease , Nerve Degeneration/etiology , Spinal Cord/pathology , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicity , Animals , Disease Models, Animal , Male , Membrane Potential, Mitochondrial/drug effects , Microscopy, Electron, Transmission , Mitochondria/metabolism , Mitochondria/physiology , Mitochondria/ultrastructure , Motor Neuron Disease/chemically induced , Motor Neuron Disease/complications , Motor Neuron Disease/pathology , Motor Neurons/pathology , Motor Neurons/ultrastructure , Multienzyme Complexes/metabolism , Oxygen Consumption/drug effects , Pyruvic Acid/pharmacology , Rats , Rats, Wistar , Spinal Cord/ultrastructureABSTRACT
Cannabinoids have been suggested to protect retinal ganglion cells in different models of toxicity, but their effects on other retinal neurons are poorly known. We investigated the neuroprotective actions of the endocannabinoid N-arachidonoyl ethanolamine (Anandamide/AEA) and the synthetic cannabinoids R1-Methanandamide (MethAEA) and HU-210, in an in vivo retinal model of AMPA excitotoxicity, and the mechanisms involved in the neuroprotection. Sprague-Dawley rats were intravitreally injected with PBS or AMPA in the absence or presence of the cannabinoid agonists. Brain nitric oxide synthase (bNOS) and choline acetyltransferase (ChAT) immunoreactivity (IR), as well as TUNEL staining, assessed the AMPA-induced retinal amacrine cell loss and the dose-dependent neuroprotection afforded by cannabinoids. The CB1 receptor selective antagonist AM251 and the PI3K/Akt inhibitor wortmannin reversed the cannabinoid-induced neuroprotection, suggesting the involvement of CB1 receptors and the PI3K/Akt pathway in cannabinoids' actions. Experiments with the CB2 agonist JWH015 and [(3)H]CP55940 radioligand binding suggested that the CB2 receptor is not involved in the neuroprotection. AEA and HU-210 induced phosphorylation of Akt but only AEA induced phosphorylation of ERK1/2 kinases, as revealed by western blot analysis. To investigate the role of caspase-3 in the AMPA-induced cell death, the caspase-3 inhibitor Z-DEVD-FMK was co-injected with AMPA. Z-DEVD-FMK had no effect on AMPA excitotoxicity. Moreover, no difference was observed in the phosphorylation of SAPK/JNK kinases between PBS- and AMPA-treated retinas. These results suggest that endogenous and synthetic cannabinoids protect retinal amacrine neurons from AMPA excitotoxicity in vivo via a mechanism involving the CB1 receptors, and the PI3K/Akt and/or MEK/ERK1/2 signaling pathways.
Subject(s)
Amacrine Cells/drug effects , MAP Kinase Signaling System/physiology , Neuroprotective Agents/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Receptor, Cannabinoid, CB1/metabolism , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicity , Amacrine Cells/metabolism , Amacrine Cells/pathology , Animals , Apoptosis , Arachidonic Acids/pharmacology , Cannabinoid Receptor Agonists/pharmacology , Choline O-Acetyltransferase/metabolism , Dose-Response Relationship, Drug , Dronabinol/analogs & derivatives , Dronabinol/pharmacology , Endocannabinoids/pharmacology , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Agonists/toxicity , Fluorescent Antibody Technique, Indirect , In Situ Nick-End Labeling , Intravitreal Injections , Male , Nitric Oxide Synthase Type I/metabolism , Phosphoinositide-3 Kinase Inhibitors , Polyunsaturated Alkamides/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Rats , Rats, Sprague-Dawley , Receptor, Cannabinoid, CB1/antagonists & inhibitorsABSTRACT
INTRODUCTION: Several data suggest that excitotoxicity due to excessive glutamatergic neurotransmission may be an important factor in the mechanisms of motor neuron (MN) death occurring in amyotrophic lateral sclerosis (ALS). We have previously shown that the overactivation of the Ca(2+)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) glutamate receptor type, through the continuous infusion of AMPA in the lumbar spinal cord of adult rats during several days, results in progressive rear limb paralysis and bilateral MN degeneration. Because it has been shown that energy failure and oxidative stress are involved in MN degeneration, in both ALS and experimental models of spinal MN degeneration, including excitotoxicity, in this work we tested the protective effect of the energy substrates pyruvate and ß-hydroxybutyrate (ßHB) and the antioxidants glutathione ethyl ester (GEE) and ascorbate in this chronic AMPA-induced neurodegeneration. RESULTS: AMPA infusion induced remarkable progressive motor deficits, assessed by two motor tasks, that by day seven reach bilateral rear limb paralysis. These effects correlate with the death of >80% of lumbar spinal MNs in the infused and the neighbor spinal cord segments, as well as with notable astrogliosis in the ventral horns, detected by glial fibrillary acidic protein immunohistochemistry. Co-infusion with pyruvate or ßHB notably prevented the motor deficits and paralysis, decreased MN loss to <25% and completely prevented the induction of astrogliosis. In contrast, the antioxidants tested were ineffective regarding all parameters analyzed. CONCLUSIONS: Chronic progressive excitotoxicity due to AMPA receptors overactivation results in MN death and astrogliosis, with consequent motor deficits and paralysis. Because of the notable protection against these effects exerted by pyruvate and ßHB, which are well established mitochondrial energy substrates, we conclude that deficits in mitochondrial energy metabolism are an important factor in the mechanisms of this slowly developed excitotoxic MN death, while the lack of protective effect of the antioxidants indicates that oxidative stress seems to be less significant factor. Because excitotoxicity may be involved in MN degeneration in ALS, these findings suggest possible preventive or therapeutic strategies for the disease.
Subject(s)
Hydroxybutyrates/pharmacology , Mitochondria/drug effects , Motor Neurons/pathology , Nerve Degeneration/drug therapy , Neuroprotection/drug effects , Pyruvic Acid/pharmacology , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicity , Animals , Antioxidants/administration & dosage , Antioxidants/pharmacology , Ascorbic Acid/administration & dosage , Ascorbic Acid/pharmacology , Cell Death/drug effects , Disease Models, Animal , Extremities/physiopathology , Gliosis/chemically induced , Gliosis/drug therapy , Gliosis/pathology , Glutathione/administration & dosage , Glutathione/analogs & derivatives , Glutathione/pharmacology , Hydroxybutyrates/administration & dosage , Male , Mitochondria/metabolism , Motor Activity/drug effects , Motor Neurons/cytology , Motor Neurons/drug effects , Nerve Degeneration/chemically induced , Nerve Degeneration/metabolism , Nerve Degeneration/pathology , Nerve Degeneration/physiopathology , Oxidative Stress/drug effects , Paralysis/chemically induced , Paralysis/drug therapy , Pyruvic Acid/administration & dosage , Rats , Rats, Wistar , Receptors, AMPA/metabolism , Spinal Cord/cytology , Spinal Cord/physiopathology , Treatment OutcomeABSTRACT
PURPOSE: The in vivo binding parameters of the novel imidazopyridine TSPO ligand [(18)F]PBR102 were assessed and compared with those of [(18)F]PBR111 in a rodent model of neuroinflammation. The validity of the key assumptions of the simplified reference tissue model (SRTM) for estimation of binding potential (BP) was determined, with validation against a two-tissue compartment model (2TC). METHODS: Acute neuroinflammation was assessed 7 days after unilateral stereotaxic administration of (R,S)-α-amino-3-hydroxy-5-methyl-4-isoxazolopropionique (AMPA) in anaesthetized adult Wistar rats. Anaesthetized rats were implanted with a femoral arterial cannula then injected with a low mass of [(18)F]PBR102 or [(18)F]PBR111 and dynamic images were acquired over 60 min using an INVEON PET/CT camera. Another population of rats underwent the same PET protocol after pretreatment with a presaturating mass of the same unlabelled tracer (1 mg/kg) to assess the validity of the reference region for SRTM analysis. Arterial blood was sampled during imaging, allowing pharmacokinetic determination of radiotracer concentrations. Plasma activity concentration-time curves were corrected for unchanged tracer based on metabolic characterization experiments in a separate cohort of Wistar rats. The stability of neuroinflammation in both imaging cohorts was assessed by [(125)I] CLINDE TSPO quantitative autoradiography, OX42/GFAP immunohistochemistry, Fluoro-Jade C histology, and elemental mapping using microparticle-induced x-ray emission spectroscopy. The BP of each ligand were assessed in the two cohorts of lesioned animals using both SRTM and a 2TC with arterial parent compound concentration, coupled with the results from the presaturation cohort for comparison and validation of the SRTM. RESULTS: The BPs of [(18)F]PBR102 [(18)F]PBR111 were equivalent, with improved signal-to-noise ratio and sensitivity compared with [(11)C]PK11195. The presaturation study showed differences in the volume of distribution between the ipsilateral striatum and the striatum contralateral to the injury (0.7) indicating that an assumption of the SRTM was not met. The modelling indicated that the BPs were consistent for both ligands. Between the SRTM and 2TC model, the BPs were highly correlated, but there was a bias in BP. CONCLUSION: [(18)F]PBR102 and [(18)F]PBR111 have equivalent binding properties in vivo, displaying significantly greater BPs with lower signal-to-noise ratio than [(11)C]PK11195. While an assumption of the SRTM was not met, this modelling approach was validated against 2TC modelling for both ligands, facilitating future use in longitudinal PET imaging of neuroinflammation.
Subject(s)
Brain/diagnostic imaging , Carrier Proteins/metabolism , Imidazoles/pharmacokinetics , Pyridines/pharmacokinetics , Radiopharmaceuticals/pharmacokinetics , Receptors, GABA-A/metabolism , Animals , Brain/drug effects , Brain/pathology , Imidazoles/chemical synthesis , Inflammation/diagnostic imaging , Inflammation/etiology , Male , Positron-Emission Tomography , Protein Binding , Pyridines/chemical synthesis , Radiopharmaceuticals/chemical synthesis , Rats , Rats, Wistar , Signal-To-Noise Ratio , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicityABSTRACT
Oligodendroglia (OL) are highly susceptible to damage and, like neurons, are terminally differentiated. It is important to protect OL precursors (OPC) because they are reservoirs of differentiating cells capable of myelination following perinatal insult and remyelination in white matter diseases, including multiple sclerosis (MS). Patients with relapsing-remitting MS are commonly treated with high-dose corticosteroids (CS) when experiencing an exacerbation. Adrenocorticotropin hormone (ACTH), a primary component of another approved MS exacerbation treatment, is a melanocortin peptide that stimulates production of CS by the adrenals. Melanocortin receptors are also found in the central nervous system (CNS) and on immune cells. ACTH is produced within the CNS and may have CS-independent effects on glia. We found that ACTH 1-39 stimulated proliferation of OPC, and to a lesser extent astroglia (AS) and microglia (MG), in rat glial cultures. ACTH accelerated differentiation of PDGFRα(+) OPC to a later stage marked by galactolipid expression and caused greater expansion of OL myelin-like sheets compared with untreated cells. Protective effects of ACTH on OPC were assessed by treating cultures with selected toxic agents, with or without ACTH. At 200 nM, ACTH protected OPC from death induced by staurosporine, glutamate, NMDA, AMPA, kainate, quinolinic acid, H2 O2 , and slow NO release, but not against kynurenic acid or rapid NO release. These agents and ACTH were not toxic to AS or MG. Our findings indicate that ACTH 1-39 provides benefits by increasing the number of OPC, accelerating their development into mature OL, and reducing OPC death from toxic insults.
Subject(s)
Adrenocorticotropic Hormone/pharmacology , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Oligodendroglia/drug effects , Stem Cells/drug effects , Animals , Animals, Newborn , Cell Death/drug effects , Cells, Cultured , Central Nervous System/cytology , Enzyme Inhibitors/toxicity , Excitatory Amino Acid Agonists/toxicity , Glial Fibrillary Acidic Protein/metabolism , Lectins/metabolism , Oligodendroglia/metabolism , Rats , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Staurosporine/toxicity , Time Factors , Transcriptome/drug effects , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicityABSTRACT
Excitatory transmission in the brain is commonly mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. In amyotrophic lateral sclerosis (ALS), AMPA receptors allow cytotoxic levels of calcium into neurons, contributing to motor neuron injury. We have previously shown that oculomotor neurons resistant to the disease process in ALS show reduced AMPA-mediated inward calcium currents compared with vulnerable spinal motor neurons. We have also shown that PTEN (phosphatase and tensin homolog deleted on chromosome 10) knockdown via siRNA promotes motor neuron survival in models of spinal muscular atrophy (SMA) and ALS. It has been reported that inhibition of PTEN attenuates the death of hippocampal neurons post injury by decreasing the effective translocation of the GluR2 subunit into the membrane. In addition, leptin can regulate AMPA receptor trafficking via PTEN inhibition. Thus, we speculate that manipulation of AMPA receptors by PTEN may represent a potential therapeutic strategy for neuroprotective intervention in ALS and other neurodegenerative disorders. To this end, the first step is to establish a fibroblast-iPS-motor neuron in vitro cell model to study AMPA receptor manipulation. Here we report that iPS-derived motor neurons from human fibroblasts express AMPA receptors. PTEN depletion decreases AMPA receptor expression and AMPA-mediated whole-cell currents, resulting in inhibition of AMPA-induced neuronal death in primary cultured and iPS-derived motor neurons. Taken together, our results imply that PTEN depletion may protect motor neurons by inhibition of excitatory transmission that represents a therapeutic strategy of potential benefit for the amelioration of excitotoxicity in ALS and other neurodegenerative disorders.
Subject(s)
Fibroblasts/enzymology , Induced Pluripotent Stem Cells/enzymology , Motor Neurons/enzymology , Neural Stem Cells/enzymology , PTEN Phosphohydrolase/metabolism , Receptors, AMPA/metabolism , Adult , Animals , Cell Survival , Cells, Cultured , Excitatory Amino Acid Agonists/toxicity , Female , Fibroblasts/drug effects , Fibroblasts/pathology , Fibroblasts/transplantation , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/pathology , Induced Pluripotent Stem Cells/transplantation , Membrane Potentials , Mice , Mice, Inbred NOD , Mice, SCID , Motor Neurons/drug effects , Motor Neurons/pathology , Motor Neurons/transplantation , Neural Stem Cells/drug effects , Neural Stem Cells/pathology , Neural Stem Cells/transplantation , PTEN Phosphohydrolase/genetics , Primary Cell Culture , RNA Interference , Signal Transduction , Synaptic Transmission , Teratoma/enzymology , Teratoma/genetics , Teratoma/pathology , Time Factors , Transfection , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicityABSTRACT
BACKGROUND: 5'-deoxy-5'-methylthioadenosine (MTA) is an endogenous compound produced through the metabolism of polyamines. The therapeutic potential of MTA has been assayed mainly in liver diseases and, more recently, in animal models of multiple sclerosis. The aim of this study was to determine the neuroprotective effect of this molecule in vitro and to assess whether MTA can cross the blood brain barrier (BBB) in order to also analyze its potential neuroprotective efficacy in vivo. METHODS: Neuroprotection was assessed in vitro using models of excitotoxicity in primary neurons, mixed astrocyte-neuron and primary oligodendrocyte cultures. The capacity of MTA to cross the BBB was measured in an artificial membrane assay and using an in vitro cell model. Finally, in vivo tests were performed in models of hypoxic brain damage, Parkinson's disease and epilepsy. RESULTS: MTA displays a wide array of neuroprotective activities against different insults in vitro. While the data from the two complementary approaches adopted indicate that MTA is likely to cross the BBB, the in vivo data showed that MTA may provide therapeutic benefits in specific circumstances. Whereas MTA reduced the neuronal cell death in pilocarpine-induced status epilepticus and the size of the lesion in global but not focal ischemic brain damage, it was ineffective in preserving dopaminergic neurons of the substantia nigra in the 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-mice model. However, in this model of Parkinson's disease the combined administration of MTA and an A2A adenosine receptor antagonist did produce significant neuroprotection in this brain region. CONCLUSION: MTA may potentially offer therapeutic neuroprotection.