noc-18 and Nerve-Degeneration

We addressed the role of nitric oxide (NO) in orexin neuron degeneration that has been observed under various pathological conditions. Administration of an NO donor NOC18 (50 nmol) into the third ventricle of mice resulted in a significant decrease of orexin-immunoreactive (-IR) neurons, in contrast to a modest change in melanin-concentrating hormone-IR neurons. In addition, NOC18 promoted formation of orexin-A-IR aggregates within orexin neurons. An endoplasmic reticulum stress inducer tunicamycin replicated the effect of NOC18 with regard to decrease of orexin-IR neurons and formation of aggregates. We also found that NOC18 caused an increase in S-nitrosation of protein disulfide isomerase (PDI) and a decrease in PDI activity in hypothalamic tissues. Moreover, PDI inhibitors, such as cystamine and securinine, caused a selective decrease of orexin neurons and promoted formation of orexin-A-IR aggregates. Aggregate formation in orexin-IR neurons was also induced by local injection of small interfering RNA targeting PDI. Interestingly, sleep deprivation for 7 consecutive days induced a selective decrease of orexin-IR neurons, which was preceded by aggregate formation in orexin-IR neurons and an increase in S-nitrosated PDI in the hypothalamus. Activity of neuronal NO synthase (nNOS)-positive neurons in the lateral hypothalamus as assessed by c-Fos expression was elevated in response to sleep deprivation. Finally, sleep deprivation-induced decrease of orexin-IR neurons, formation of aggregates, and S-nitrosation of PDI were not observed in nNOS knock-out mice. These results indicate that nNOS-derived NO may mediate specific pathological events in orexin neurons, including neuropeptide misfolding via S-nitrosation and inactivation of PDI.

Topics: Animals; Cell Count; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Gene Expression Regulation; Hypothalamus; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; NADPH Dehydrogenase; Nerve Degeneration; Neuropeptides; Nitric Oxide; Nitric Oxide Donors; Nitrosation; Nitroso Compounds; Orexins; Protein Disulfide-Isomerases; Time Factors

Nitric oxide has been investigated widely both during neurodevelopment and in neurological diseases. However, whilst it has been established that nitric oxide-producing enzymes of nitric oxide synthase family are expressed in cerebellar Purkinje neurons, the effects of nitric oxide on the viability and morphology of these neurons remain unknown. Here, we have demonstrated that the activity of neuronal nitric oxide synthase, but not the inducible or endothelial forms of this enzyme, is required to support the survival of a proportion of cerebellar Purkinje neurons in vitro. We discovered that donation of high concentrations of exogenous nitric oxide reduces Purkinje neuron survival in culture and that peroxynitrite is also toxic to these cells. Finally, we demonstrated that exogenous nitric oxide and peroxynitrite reduce both the magnitude and the complexity of the neurite arbour extended by cerebellar Purkinje neurons. Taken together, these findings reveal that whilst a low level of endogenous nitric oxide, released by the activity of neuronal nitric oxide synthase, is beneficial to cerebellar Purkinje neurons in vitro, high levels of exogenous nitric oxide and peroxynitrite are detrimental to both the survival of these neurons and to their ability to extend processes and form functional neural networks.

Topics: Animals; Benzoates; Cell Count; Cells, Cultured; Cerebellar Cortex; Citrulline; Female; Imidazoles; Isothiuronium; Mice; Mice, Inbred C57BL; Nerve Degeneration; Nerve Tissue Proteins; Neurites; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroso Compounds; Peroxynitrous Acid; Pregnancy; Purkinje Cells; Reactive Nitrogen Species; Thiourea

Rat striatum was microinjected with 50 nmol sodium nitroprusside (SNP) and neural cell death as well as the binding of dopaminergic receptors were followed for 24 h after the infusion using TTC staining, cresyl violet staining, and quantitative autoradiography. Striatal cell death was observed 3 h after the infusion of SNP. A widespread area of cell death, including part of the cerebral cortex, was seen at 24 h after the infusion. A decrease of more than 80% in dopamine D1 receptor binding was seen in rat brain slices prepared 2 h after the infusion of SNP, whereas only a slight decrease in dopamine D2 receptor binding and almost no changes in dopamine transporter binding were observed. One day after the infusion, less than 10% of the binding of all three types of dopaminergic receptors remained in a widespread area in the infused side of the striatum and part of the cerebral cortex. Microinjection of either NOC-18 (50 nmol), another type of NO donor, or sodium cyanide (50 nmol) did not caused cell death. In addition, microinjection of FeCl2 (50 nmol) into the striatum caused cell death and reduction in dopamine D() receptor binding. These results suggest that iron-related radical reactions, but not NO itself, might have important roles on SNP-caused cell death. The current receptor binding study also indicated that dopamine D1 receptor binding is the most sensitive indicator for detection of cell death or cell damage induced by radical reactions in the rat striatum.

Topics: Animals; Benzazepines; Binding Sites; Binding, Competitive; Cell Death; Cocaine; Dopamine Antagonists; Down-Regulation; Ferrous Compounds; Male; Microinjections; Neostriatum; Nerve Degeneration; Neural Pathways; Neurons; Nitric Oxide; Nitroprusside; Nitroso Compounds; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Dopamine; Receptors, Dopamine D1; Sodium Cyanide; Substantia Nigra

We studied the mechanism of nitric oxide (NO) toxicity in cultured rat spinal motoneurons. Treatment with the NO donor NOC-18 (NOC) resulted in slow motoneuron death, ending in apoptosis. The observed motoneuron death was completely prevented by hemoglobin. Treatment with inhibitors of the known intracellular targets of NO, soluble guanylate cyclase, polyADP-ribose polymerase (PARP) and superoxide, did not result in any significant protection against NOC-induced motoneuron death. ATP levels were reduced as soon as 3 h after the start of NOC treatment, suggesting a direct inhibition of cellular energy production. NOC toxicity could be blocked by the general voltage-gated calcium channel blocker cobalt, but not by specific blockers of various subtypes of calcium channels.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Calcium; Cells, Cultured; Cobalt; Cyclic GMP; Electrophysiology; Motor Neurons; Nerve Degeneration; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteins; Rats