oxadiazoles and Nerve-Degeneration

FTY720 is a known sphingosine 1-phosphate receptor agonist. In the present study, we investigated the neuroprotective effect of postischemic administration of FTY720 in rats with 2 hours transient middle cerebral artery occlusion (MCAO).. One hundred eleven male rats were randomly assigned to sham-operated and MCAO treated with vehicle, 0.25 mg/kg and 1 mg/kg of FTY720, another selective sphingosine 1-phosphate receptor-1 agonist SEW2871 (5 mg/kg), or 0.25 mg/kg of FTY720 plus a sphingosine 1-phosphate antagonist, VPC23019 (0.5 mg/kg). Drugs were injected intraperitoneally immediately after reperfusion. Neurological score and infarct volume were assessed at 24 and 72 hours after MCAO. Western blotting, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling were conducted at 24 hours after MCAO.. FTY720 significantly reduced infarct volume and improved neurological score at 24 and 72 hours after MCAO compared with the vehicle group. SEW2871 showed similar neuroprotective effects to FTY720, whereas VPC 20319 abolished the neuroprotective effects of FTY720. FTY720 significantly retained Akt and extracellular signal-regulated kinase phosphorylation and Bcl-2 expression and decreased cleaved caspase-3 expression and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling-positive neurons at 24 hours after MCAO. VPC23019 blocked the antiapoptotic effects of FTY720.. These data suggest that activation of sphingosine 1-phosphate-1 by FTY720 reduces neuronal death after transient MCAO.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Brain; Brain Ischemia; Disease Models, Animal; Drug Administration Schedule; Fingolimod Hydrochloride; Immunosuppressive Agents; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Male; MAP Kinase Signaling System; Nerve Degeneration; Neuroprotective Agents; Oxadiazoles; Propylene Glycols; Rats; Rats, Sprague-Dawley; Receptors, Lysosphingolipid; Sphingosine; Stroke; Thiophenes; Treatment Outcome

Applications for noninvasive real-time imaging of apoptosis and neuronal degeneration are hindered by technical limitations in imaging strategies and by existing probes. Monitoring the progression of a cell through apoptosis could provide valuable insight into the temporal events that initiate cell death as well as the potential for rescue of apoptotic cells. We engineered an annexin-based biosensor to function as a polarity-sensitive indicator for viability and apoptosis (known as pSIVA) by binding to externalized phosphatidylserine (PS) exposed on apoptotic cell membranes. Constructed from a structure-based design strategy, pSIVA fluoresces only when bound to PS and remains effectively undetectable in solution. In this paper, we describe protocols for the design, expression, purification and labeling of pSIVA as well as for its application in time-lapse imaging of degenerating neurons in culture; the entire protocol can be completed in 2 weeks. The primary advantage of this method is the flexibility to use pSIVA, in combination with other probes and without perturbing experimental conditions, to explore the cellular mechanisms involved in apoptosis and degeneration in real time.

Topics: Animals; Annexins; Apoptosis; Biosensing Techniques; Calcium; Cell Culture Techniques; Cell Membrane; Cell Survival; Culture Media; Escherichia coli; Female; Ganglia, Spinal; Microscopy, Fluorescence; Nerve Degeneration; Neurons; Oxadiazoles; Phosphatidylserines; Rats; Rats, Sprague-Dawley

Mitochondrial membrane potential (DeltaPsim)-dependent Ca2+ uptake plays a central role in neurodegeneration after NMDA receptor activation. NMDA-induced DeltaPsim dissipation increases during postnatal development, coincident with increasing vulnerability to NMDA. NMDA receptor activation also produces nitric oxide (NO), which can inhibit mitochondrial respiration, dissipating DeltaPsim. Because DeltaPsim dissipation reduces mitochondrial Ca2+ uptake, we hypothesized that NO mediates the NMDA-induced DeltaPsim dissipation in immature neurons, underlying their decreased vulnerability to excitotoxicity. Using hippocampal neurons cultured from 5- and 19-d-old rats, we measured NMDA-induced changes in [Ca2+]cytosol, DeltaPsim, NO, and [Ca2+]mito. In postnatal day 5 (P5) neurons, NMDA mildly dissipated DeltaPsim in a NO synthase (NOS)-dependent manner and increased NO. The NMDA-induced NO increase was abolished with carbonyl cyanide 4-(trifluoromethoxy)phenyl-hydrazone and regulated by [Ca2+]mito. Mitochondrial Ca2+ uptake inhibition prevented the NO increase, whereas inhibition of mitochondrial Ca2+ extrusion increased it. Consistent with this mitochondrial regulation, NOS and cytochrome oxidase immunoreactivity demonstrated mitochondrial localization of NOS. Furthermore, NOS blockade increased mitochondrial Ca2+ uptake during NMDA. Finally, at physiologic O2 tensions (3% O2), NMDA had little effect on survival of P5 neurons, but NOS blockade during NMDA markedly worsened survival, demonstrating marked neuroprotection by mitochondrial NO. In P19 neurons, NMDA dissipated DeltaPsim in an NO-insensitive manner. NMDA-induced NO production was not regulated by DeltaPsim, and NOS immunoreactivity was cytosolic, without mitochondrial localization. NOS blockade also protected P19 neurons from NMDA. These data demonstrate that mitochondrial NOS mediates much of the decreased vulnerability to NMDA in immature hippocampal neurons and that cytosolic NOS contributes to NMDA toxicity in mature neurons.

Topics: Animals; Animals, Newborn; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Clonazepam; Hippocampus; Indazoles; Ion Transport; Mitochondria; N-Methylaspartate; Nerve Degeneration; Nerve Tissue Proteins; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type I; Oxadiazoles; Oxygen; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Ruthenium Compounds; Thiazepines

Previous studies with the N18-RE-105 neuronal-like cell line and primary cortical cultures demonstrate that glutamate can produce a calcium-dependent, delayed form of neuronal degeneration that results from its competitive inhibition of cystine transport, which leads to cellular glutathione depletion and death by oxidative stress. Idebenone, a centrally active antioxidant used to treat multiinfarct dementia, protects cells from this form of glutamate-induced cytotoxicity in vitro. In the present study, we have examined the effects of systemic treatment with idebenone on the neurotoxic consequences of intrastriatal injection of kainic acid, quisqualic acid, or quinolinic acid, an NMDA receptor agonist, on neuronal degeneration. Striatal damage was assessed by quantitative neurochemistry with measurement of choline acetyltransferase activity and glutamate decarboxylase activity, by histochemical analysis for acetylcholinesterase and NADPH diaphorase staining and by behavioral assessment of circling produced by systemic apomorphine treatment 10 days after the unilateral lesion. The results indicate that treatment with idebenone provides significant protection against the neuronal degeneration induced by intrastriatal injection of kainic acid and quisqualic acid, but not the NMDA receptor agonist, quinolinic acid. The results suggest that oxidative stress may contribute to the proximate cause of neuronal degeneration induced by quisqualate and by kainate receptor agonists and that the mechanisms of neuronal degeneration caused by quisqualate/kainate receptor agonists differ from those associated with NMDA receptor agonists.

Topics: Animals; Apomorphine; Benzoquinones; Choline O-Acetyltransferase; Corpus Striatum; Glutamate Decarboxylase; Histocytochemistry; Kainic Acid; Kinetics; Male; Motor Activity; NADPH Dehydrogenase; Nerve Degeneration; Oxadiazoles; Pyridines; Quinolinic Acid; Quinolinic Acids; Quinones; Quisqualic Acid; Rats; Rats, Inbred Strains; Ubiquinone

The density of calcium channel antagonist receptors labeled by (+)-[3H]PN 200-110 was reduced by 75% in striata from patients with Huntington's disease, but unchanged in patients with Parkinson's disease, compared with control subjects. These receptors are therefore likely to be localized to neurons with cell bodies in striatum, rather than nigrostriatal nerve terminals or glia, and their loss may contribute to the pathophysiology of basal ganglia disorders.

Topics: Aged; Calcium Channel Blockers; Corpus Striatum; Humans; Huntington Disease; Isradipine; Middle Aged; Nerve Degeneration; Neural Pathways; Neurons; Oxadiazoles; Parkinson Disease; Substantia Nigra

Intravitreal (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleacetic acid (AMPA) is a powerful excitotoxin on the chicken retina. Doses of 10 nmol/eye produce half-maximum destruction of cholinergic amacrine cells, making AMPA equipotent with kainic acid. The effects of AMPA can be distinguished from those of kainic acid morphologically, and from those of N-methyl-D-aspartic acid by the failure of 2-amino-5-phosphonopentanoic acid (2-AP5) to block those of AMPA. Both morphologically, and in response to 2-AP5, the effects of AMPA and quisqualic acid are indistinguishable, but AMPA is much more potent than quisqualic acid, presumably due to the uptake and inactivation of quisqualic acid.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Aspartic Acid; Chickens; Choline O-Acetyltransferase; Dose-Response Relationship, Drug; Ibotenic Acid; Injections; Kainic Acid; N-Methylaspartate; Nerve Degeneration; Oxadiazoles; Oxazoles; Quisqualic Acid; Retina; Retinal Ganglion Cells; Vitreous Body

The effects of excitotoxic lesions on the neuronal marker enzymes choline acetyltransferase and glutamate decarboxylase and on the levels of 'peripheral type' benzodiazepine binding sites (PTBBS) (a putative glial marker) have been compared to see whether PTBBS provide a suitable if indirect quantitative index of neuronal damage. Intrastriatal injection of excitotoxic compounds provoked a dose-dependent increase in the levels of PTBBS. The potency order was the following: kainate greater than AMPA greater than N-methyl-D-aspartate (NMDA) greater than quisqualate. The maximal increases in this parameter were 400, 470, 320 and 210% for kainate (12 nmol), AMPA (100 nmol), NMDA (500 nmol) and quisqualate (250 nmol), respectively. 2-Amino-5-phosphonovalerate (100 nmol)--an antagonist of the NMDA receptor subtype--completely blocked the increase in PTBBS induced by NMDA (250 nmol), but was without effect against the other excitotoxins. Increases in binding levels were in general mirrored by a decrease in choline acetyltransferase and glutamate decarboxylase activity. However, PTBBS were a more sensitive indirect index of neuronal damage than neuronal enzymes because the alterations in binding were statistically significant at doses of excitotoxins lower than those causing a loss of marker enzymes. It is concluded that PTBBS are a suitable and sensitive means of detecting discrete neurotoxic changes and that its measurement will help in the study of other pathological and experimental models.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Aspartic Acid; Binding Sites; Choline O-Acetyltransferase; Corpus Striatum; Glutamate Decarboxylase; Ibotenic Acid; Kainic Acid; Male; N-Methylaspartate; Nerve Degeneration; Neurotoxins; Oxadiazoles; Quisqualic Acid; Rats; Rats, Inbred Strains; Receptors, GABA-A

Topics: Amino Acids, Dicarboxylic; Animals; Anticonvulsants; Aspartic Acid; Epilepsy; Glutamates; Kainic Acid; N-Methylaspartate; Nerve Degeneration; Nervous System Diseases; Oxadiazoles; Quisqualic Acid; Receptors, Amino Acid; Receptors, Cell Surface; Structure-Activity Relationship

The cytotoxicity of glutamate and several analogues was investigated using a well characterized glutamatergic system; the neuromuscular system of the locust leg. In the presence of Con A (10(-6) M) (which blocks glutamate receptor desensitization) bath application on L-glutamate to isolated nerve-muscle preparations induced degeneration of the muscle cells in a dose-dependent manner. The ability of glutamate analogues to cause similar damage corresponded to their pharmacological potency, i.e. L-quisqualate greater than L-glutamate greater than L-cysteine greater than L-aspartate and L-kainate. Glutamate and the more potent agonists initially caused muscle swelling. This was followed by an increase in opacity of the muscle due to vacuolation resulting from disruption of the sarcoplasmic reticulum. Ca2+-free saline slowed the cytotoxic action of these amino acids, whilst saline containing high concentrations of Ca2+ (20 mM; substituted for Na+) accelerated muscle destruction. Denervation induces supersensitivity of locust muscle to L-glutamate; in denervated muscles the cytotoxicity of L-glutamate was enhanced. Muscles swollen by exposure to high-potassium saline (100 mM; substituted for sodium) were not damaged. We conclude that in this insect glutamatergic system, when desensitization is prevented, activated glutamate receptors gate the influx of Ca2+ and Na2+ causing an ionic imbalance which results in cellular damage. This mechanism could also account for at least some of the neurotoxic effects of amino acids in the vertebrate central nervous system. The results of our studies also indicate that other transmitters which gate non-desensitizing cationic channels should, in principle, also be cytotoxic.

Topics: Animals; Aspartic Acid; Concanavalin A; Cysteine; Dose-Response Relationship, Drug; Glutamates; Glutamic Acid; Grasshoppers; Kainic Acid; Nerve Degeneration; Neuromuscular Junction; Neurotransmitter Agents; Oxadiazoles; Quisqualic Acid; Receptors, Cell Surface; Receptors, Glutamate; Synaptic Transmission