doi: 10.1161/01.STR.0000033074.40202.8E
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(Stroke. 2002;33:2348.)
© 2002 American Heart Association, Inc.
Letters to the Editor |
Nitric Oxide May Contribute to the Long-Term Impairment of Synaptic Transmission After Transient Ischemia
Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York
To the Editor:
In their appealing study published in a recent issue of Stroke, Bolay et al1 extend their previous observations2 that relatively mild ischemia causes long-term dysfunction of synaptic transmission and now suggest that a likely mechanism for this phenomenon is persistent inhibition of presynaptic signaling and neurotransmitter release. In their convincing experiments, the authors demonstrate that 1-hour middle cerebral artery occlusion causes selective inhibition of synapsin-I phosphorylation in the ischemic penumbra of the rat cortex and propose this as a key reason for the suppression of vesicular neurotransmitter release. Interestingly, the postsynaptic elements of neuronal networks seem to remain intact and continue to respond to exogenously applied glutamate. These findings, together with previous data on ischemia-induced inactivation of Ca2+/calmodulin-dependent protein kinase II3 and protein kinase A,4 create a new picture of how relatively mild ischemia or transient ischemic attack may cause long-term impairment of brain function despite survival of neuronal cells.
I agree with the authors that phosphorylation defects likely contribute to the long-lasting suppression of synaptic transmission. However, I would like to attract the attention of the authors and readers to an additional possible mechanism of synaptic transmission impairment. It is a well-known fact that ischemia upregulates production of the free radical nitric oxide (NO).5 In the healthy brain, NO serves as a vasorelaxant and neuromodulator. In contrast, under pathological conditions, neuronally derived NO and several related nitrogen reactive species play major roles in mediating brain damage.5,6 One such pathological NO product, peroxynitrite (ONOO-), is formed in the reaction of NO with the superoxide radical.7 ONOO- oxidizes or modifies many cellular components, including proteins and DNA,7 and has been quantitatively linked to the degree of ischemic brain damage.8,9 In proteins, ONOO- nitrates tyrosine residues and nitrosates cysteine amino acid moieties, potently perturbing enzymatic functions.7 Synaptic vesicle membrane fusion and exocytotic neurotransmitter release involve an N-ethylmaleimide–sensitive ATPase that is especially prone to SH-group oxidation.10,11 Recently, my colleagues and I have found that nitric oxide donors potently and selectively suppress vesicular GABA release in brain synaptosomes, while marginally affecting other types of GABA transport.12 The effects of the NO donors coincided with the oxidation of intrasynaptosomal SH groups and were mimicked by N-ethylmaleimide. Therefore, under pathological conditions, NO-dependent disruption of synaptic fusion may work in parallel or in sequence with defects in synapsin-I phosphorylation, as described by Bolay et al. This alternative mechanism deserves further experimental exploration.
References
1. Bolay H, Gursoy-Ozdemir Y, Sara Y, Onur R, Can A, Dalkara T. Persistent defect in transmitter release and synapsin phosphorylation in cerebral cortex after transient moderate ischemic injury. Stroke. 2002; 33: 1369–1375.
2. Bolay H, Dalkara T. Mechanisms of motor dysfunction after transient MCA occlusion: persistent transmission failure in cortical synapses is a major determinant. Stroke. 1998; 29: 1988–1993.
3. Hanson SK, Grotta JC, Waxham MN, Aronowski J, Ostrow P. Calcium/calmodulin-dependent protein kinase II activity in focal ischemia with reperfusion in rats. Stroke. 1994; 25: 466–473.[Abstract]
4. Tanaka K, Nogawa S, Nagata E, Suzuki S, Dembo T, Kosakai A, Fukuuchi Y. Inhibition of cyclic AMP-dependent protein kinase in the acute phase of focal cerebral ischemia in the rat. Neuroscience. 1999; 94: 361–371.[CrossRef][Medline] [Order article via Infotrieve]
5. Samdani AF, Dawson TM, Dawson VL. Nitric oxide synthase in models of focal ischemia. Stroke. 1997; 28: 1283–1288.
6. Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci. 1999; 22: 391–397.[CrossRef][Medline] [Order article via Infotrieve]
7. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol. 1996; 271: C1424–C1437.[Medline] [Order article via Infotrieve]
8. Eliasson MJ, Huang Z, Ferrante RJ, Sasamata M, Molliver ME, Snyder SH, Moskowitz MA. Neuronal nitric oxide synthase activation and peroxynitrite formation in ischemic stroke linked to neural damage. J Neurosci. 1999; 19: 5910–5918.
9. Osuka K, Feustel PJ, Mongin AA, Tranmer BI, Kimelberg HK. Tamoxifen inhibits nitrotyrosine formation after reversible middle cerebral artery occlusion in the rat. J Neurochem. 2001; 76: 1842–1850.[CrossRef][Medline] [Order article via Infotrieve]
10. Whiteheart SW, Rossnagel K, Buhrow SA, Brunner M, Jaenicke R, Rothman JE. N-ethylmaleimide-sensitive fusion protein: a trimeric ATPase whose hydrolysis of ATP is required for membrane fusion. J Cell Biol. 1994; 126: 945–954.
11. Schiavo G, Gmachl MJ, Stenbeck G, Sollner TH, Rothman JE. A possible docking and fusion particle for synaptic transmission. Nature. 1995; 378: 733–736.[CrossRef][Medline] [Order article via Infotrieve]
12. Nedvetsky PI., Konev SV, Rakovich AA, Petrenko SV, Mongin AA. Effects of nitric oxide donors on Ca2+-dependent [14C]GABA release from brain synaptosomes: the role of SH-groups. Biochemistry (Mosc). 2000; 65: 1027–1035.[Medline] [Order article via Infotrieve]
Department of Neurology, Faculty of Medicine, Gazi University, Ankara, Turkey
Department of Neurology, Faculty of Medicine, Hacettepe University, Ankara, Turkey
Response
We thank Dr Mongin for his interest in our study and his comments. Dr Mongin proposes an intriguing mechanism that may contribute to persistent synaptic transmission defect seen after transient cerebral ischemia.1 He and his colleagues demonstrated that NO donors selectively suppressed GABA release in synaptosomes and that this effect was correlated with their ability to oxidize intrasynaptosomal SH groups.2 Based on these in vitro data, the author suggests that N-ethylmaleimide–sensitive ATPase, a key enzyme for the fusion step of vesicular exocytosis, might be involved in ischemia-induced impairment of neurotransmitter release because this enzyme is particularly prone to SH-group oxidation by peroxynitrite. The idea is appealing and may provide an additional mechanism that may disrupt neurotransmitter release after an ischemic insult. During ischemia/reperfusion, a considerable amount of peroxynitrite is generated in the penumbral cortex,3 where we detected the synaptic transmission defect.1,4 However, it should be noted that neurons in the peri-infarct area are reversibly injured and peroxynitrite toxicity may be reversed by protein denitrosylation after reperfusion. Therefore, peroxynitrite-induced protein modifications may not be sustained. This interesting point may be clarified in future experiments.
Supporting Dr Mongin’s view, several studies suggest that peroxynitrite may biochemically modify presynaptic proteins involved in transmitter release including SNARE proteins.2,5–7 Even small changes in the formation of SNARE complex and other vesicular proteins could have significant consequences for vesicle docking and fusion.8 Di Stasi and colleagues7 demonstrated that peroxynitrite stimulated vesicle exocytosis and induced glutamate release from synaptosomes through nitration of SNAP25 and Munc-18. Peroxynitrite seems to exert opposing effects on release of various neurotransmitters in that it inhibits ACh synthesis6 and GABA release,2 whereas it stimulates excitatory amino acid release.5,7 It would be interesting to know whether NO/peroxynitrite impairs N-ethylmaleimide–sensitive ATPase differentially between inhibitory and excitatory synapses, because selective suppression of GABA release may account for the ischemia-induced cortical hyperexcitability.9–12
As we emphasized before,1 mechanisms of failure of transmitter release are likely to be complex after an ischemic insult; swelling and depolarization of the presynaptic membrane may adversely affect evoked transmitter release. Synaptic terminals may suffer from significant free radical damage due to a high rate of oxidative phosphorylation and Ca2+ shuttling across the mitochondrial membrane during ischemia.13 Oxidative and nitrosative stress may damage several macromolecules in synaptic boutons. Mitochondria of synaptic origin have been reported to be much more sensitive to inhibition of complex 1 and to depletion of glutathione than are nonsynaptosomal mitochondria.14 Abrupt decrease in ATP synthesis and respiration rate occurred when complex I activity was inhibited by 25% in synaptic mitochondria, whereas this threshold was found to be 72% in nonsynaptic mitochondria.14 Therefore, mitochondria in presynaptic terminals may be selectively damaged despite recovery of oxidative phosphorylation in other cellular compartments after recirculation. In that case, a generalized deficiency in phosphorylation of presynaptic proteins is expected in addition to phosphorylation defects induced by selective inactivation of some kinases. However, after a mild injury, most of these perturbations may quickly recover, whereas some of them remain dysfunctional and cause long-lasting disturbances in transmitter release. In our study, we detected the deficiency in synapsin-1 phosphorylation as one of the possible mechanisms; certainly, several other mechanisms may be identified in future studies, as suggested by Dr Mongin.
References
1. Bolay H, Gursoy-Ozdemir Y, Sara Y, Onur R, Can A, Dalkara T. Persistent defect in transmitter release and synapsin phosphorylation in cerebral cortex after transient moderate ischemic injury. Stroke. 2002; 33: 1369–1375.
2. Nedvetsky PI, Konev SV, Rakovich AA, Petrenko SV, Mongin AA. Effects of nitric oxide donors on Ca2+-dependent [14C]GABA release from brain synaptosomes: the role of SH-groups. Biochemistry (Mosc). 2000; 65: 1027–1035.[Medline] [Order article via Infotrieve]
3. Fukuyama N, Takizawa S, Ishida H, Hoshiai K, Shinohara Y, Nakazawa H. Peroxynitrite formation in focal cerebral ischemia-reperfusion in rats occurs predominantly in the peri-infarct region. J Cereb Blood Flow Metab. 1998; 18: 123–129.[CrossRef][Medline] [Order article via Infotrieve]
4. Bolay H, Dalkara T. Mechanisms of motor dysfunction after transient MCA occlusion: a persistent transmission failure in cortical synapses is a major determinant. Stroke. 1998; 29: 1988–1993.
5. Moro MA, Leza JC, Lorenzo P, Lizasoain I. Peroxynitrite causes aspartate release from dissociated rat cerebellar granule neurones. Free Radic Res. 1998; 28: 193–204.[Medline] [Order article via Infotrieve]
6. Guermonprez L, Ducrocq C, Gaudry-Talarmain YM. Inhibition of acetylcholine synthesis and tyrosine nitration induced by peroxynitrite are differentially prevented by antioxidants. Mol Pharmacol. 2001; 60: 838–846.
7. Di Stasi AM, Mallozzi C, Macchia G, Maura G, Petrucci TM, Minetti M. Peroxynitrite affects exocytosis and SNARE complex formation and induces tyrosine nitration of synaptic proteins. J Neurochem. 2002; 82: 420–429.[CrossRef][Medline] [Order article via Infotrieve]
8. Mochida S. Protein-protein interactions in neurotransmitter release. Neurosci Res. 2000; 36: 175–182.[CrossRef][Medline] [Order article via Infotrieve]
9. Luhmann HJ. Ischemia and lesion induced imbalances in cortical function. Prog Neurobiol. 1996; 48: 131–166.[CrossRef][Medline] [Order article via Infotrieve]
10. Schiene K, Bruehl C, Zilles K, Qu M, Hagemann G, Kraemer M, Witte OW. Neuronal hyperexcitability and reduction of GABAA-receptor expression in the surround of cerebral photothrombosis. J Cereb Blood Flow Metab. 996; 16: 906–914.
11. Xu ZC, Pulsinelli WA. Electrophysiological changes of CA1 pyramidal neurons following transient forebrain ischemia: an in vivo intracellular recording and staining study. J Neurophysiol. 1996; 76: 1689–1697.
12. Bolay H, Gürsoy-Özdemir Y, Ünal I, Dalkara T. Altered mechanisms of motor evoked potential generation after transient focal cerebral ischemia in the rat: implications for transcranial magnetic stimulation. Brain Res,. 2000; 873: 26–33.[CrossRef][Medline] [Order article via Infotrieve]
13. Nicholls DG, Budd SL. Mitochondria and neuronal survival. Physiol Rev. 2000; 80: 315–360.
14. Davey GP, Peuchen S, Clark JB. Energy thresholds in brain mitochondria: potential involvement in neurodegeneration. J Biol Chem. 1998; 273: 12753–12757.
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