the-rodent-model-nonarteritic-anterior-ischemic-optic-neuropathy

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

Nonarteritic anterior ischemic optic neuropathy (NAION) is a focal ischemic lesion of the optic nerve that affects 1/700 individuals throughout their ...

University of Maryland-Baltimore School of Medicine

In this study, the effect of bilobalide, a purified terpene lactone component of the Ginkgo biloba extract (EGb 761), and EGb 761 against ischemic injury and against glutamate-induced excitotoxic neuronal death was compared. In the case of ischemic injury, neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in the hippocampal regions of gerbils was measured. A significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons at 7-days of reperfusion after 5 min of transient global forebrain ischemia. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected hippocampal CA1 neurons against ischemia-induced neuronal death and reductions in COX III mRNA. In rat cerebellar neuronal cultures, addition of bilobalide or EGb 761 protected in a dose-dependent manner against glutamate-induced excitotoxic neuronal death [effective concentration (EC50) = 5 microg/ml (12 microM) forbilobalide and 100 microg/ml for EGb 761]. These results suggest thatboth EGb 761 and bilobalide protect against ischemia-induced neuronal death in vivo and glutamate-induced neuronal death in vitro by synergistic mechanisms involving anti-excitotoxicity, inhibition of free radical generation, scavenging of reactive oxygen species, and regulation of mitochondrial gene expression.

Bilobalide, a component of the Ginkgo biloba extract (EGb 761), protects against neuronal death in global brain ischemia and in glutamate-induced excitotoxicity.

Accelerated decrease in the levels of mitochondrial DNA-encoded mRNA (mt-mRNA) occurs in neuronal cells exposed either to the excitatory amino acid, glutamate or to the sodium ionophore, monensin, suggesting a role of mitochondrial RNase(s) on the stability of mt-mRNAs. Here we report that in mouse embryo fibroblasts that are devoid of the interferon-regulated RNase, RNase-L, the monensin-induced decrease in the half-life of mt-mRNA was reduced. In monensin (250 nM)-treated RNase-L(+/+) cells the average half-life of mt-mRNA, determined after termination of transcription with actinomycin D, was found to be 3h, whereas in monensin-treated RNase-L(-/-) cells the half-life of mt-mRNA was >6h. In contrast, the stability of nuclear DNA-encoded beta-actin mRNA was unaffected. Induction of RNase-L expression in mouse 3T3 fibroblasts further decreased the monensin-induced reduction in mt-mRNA half-life to 1.5h. The results indicate that the RNase-L-dependent decrease in mtDNA-encoded mRNA transcript levels occurs through a decrease in the half-life of mt-mRNA, and that RNase-L may play a role in the stability of mt-mRNA.

RNase-L regulates the stability of mitochondrial DNA-encoded mRNAs in mouse embryo fibroblasts.

Mitochondrial DNA (mtDNA) encodes critical subunit proteins of the oxidative phosphorylation (OXPHOS) complex that generates ATP. This study tested the hypothesis that mitochondrial gene expression in neural cells is regulated by energy demand, as modified via stimulation of cellular sodium transport. Exposure of PC12S cells to the sodium ionophore monensin (250 nm) for 1-6 h caused a 13-60% decrease in cellular ATP (from 15 to 5 nmol per mg protein at 6 h). Levels of mitochondrial DNA-encoded mRNAs (mt-mRNAs) increased significantly (150%) within the first hour of exposure to monensin, and then decreased significantly (50%) at 3-4 h. Levels of mtDNA-encoded 12S rRNA and nuclear DNA-encoded OXPHOS subunit mRNAs were not significantly affected. Exposure of primary cerebellar neuronal cultures to the excitatory amino acid glutamate caused a similar rapid and significant increase followed by a significant decrease in cell mt-mRNA levels. The monensin-induced initial increase in mt-mRNA levels was abolished by pretreatment with actinomycin D or by reducing extracellular sodium ion concentration. The monensin-induced delayed reduction in mt-mRNA levels was accelerated in the presence of actinomycin D, and was accompanied by a 67% reduction in the half-life (from 3.6 to 1.2 h). Exposure of PC12S cells to 2-deoxy-d-glucose significantly decreased cellular ATP levels (from 14.2 to 7.1 nmol per mg protein at 8 h), and increased mt-mRNA levels. These results suggest a physiological transcriptional mechanism of regulation of mitochondrial gene expression by energy demand and a post-transcriptional regulation that is independent of energy status of the cell.

Regulation of mitochondrial gene expression by energy demand in neural cells.

Isolated brain mitochondria are a heterogeneous mixture from different cell types and these subsets may have differing sensitivities to Ca2+-induced membrane permeability transition (MPT) and to inhibition of the MPT by cyclosporin A (CsA). This study tested the hypothesis that mitochondria within primary cultures of astrocytes and neurons exhibit different energy-dependent Ca2+ uptake capacities and different degrees to which CsA increases their uptake capacity. Astrocytes and neurons were suspended in a cytosol-like medium containing respiratory substrates, ATP, and Mg2+ in the presence of digitonin to selectively permeabilize the plasma membrane. Uptake of added Ca2+ by mitochondria within the cells was measured by Calcium Green 5N fluorescent monitoring of the medium [Ca2+]. Permeabilized astrocytes had a fourfold higher Ca2+ uptake capacity, relative to neurons and a twofold higher content based on relative contents of mitochondria assessed by measurements of mitochondrial DNA and cytochrome oxidase subunit 1 protein. In astrocytes the Ca2+ uptake capacity was increased twofold by preincubation with 2-5 microM CsA, while in neurons CsA had no effect. Similar results were obtained using measurements of the effects of added Ca2+ on mitochondrial membrane potential. FK506, a drug similar to CsA but without MPT inhibitory activity, had no effect on either cell type. These results are consistent with the presence of a calcium-induced MPT in astrocytes, even in the presence of ATP, and indicate that the MPT in cerebellar granule neurons is resistant to CsA inhibition. Some of the protective effects of CsA in vivo may therefore be mediated by preservation of mitochondrial functional integrity within astrocytes.

Cyclosporin A increases mitochondrial calcium uptake capacity in cortical astrocytes but not cerebellar granule neurons.

The induction of GRIM-19 has been shown to be essential for interferon-beta (IFN-beta)-induced and retinoic acid (RA)-induced tumor cell death. We have studied the localization and levels of GRIM-19 in IFN/RA-induced cell death in neural cells and in focal cerebral ischemia. Exposure to IFN/RA caused a approximately 15-fold increase in GRIM-19 protein levels and induced >50% cell death in human neuroblastoma SH-SY5Y cells. In rats subjected to permanent focal cerebral ischemia, increased oxidative stress, as well as increased GRIM mRNA levels (32-fold) and increased GRIM-19 (>50%) protein levels were noted in the ipsilateral (affected) hemisphere compared with the contralateral (unaffected) hemisphere. These results suggest that GRIM-19 may play a role in ischemia-induced neuronal cell death.

The IFN-beta and retinoic acid-induced cell death regulator GRIM-19 is upregulated during focal cerebral ischemia.

Nonarteritic anterior ischemic optic neuropathy (NAION) results in optic nerve damage with retinal ganglion cell (RGC) loss. An NAION model, rodent anterior ischemic optic neuropathy (rAION), was used to determine AION-associated mechanisms of RGC death and associated regional retinal changes.

Rodent anterior ischemic optic neuropathy (rAION) induces regional retinal ganglion cell apoptosis with a unique temporal pattern.

Oxidative stress and mitochondrial dysfunction have been closely associated in many subcellular, cellular, animal, and human studies of both acute brain injury and neurodegenerative diseases. Our animal models of brain injury caused by cardiac arrest illustrate this relationship and demonstrate that both oxidative molecular modifications and mitochondrial metabolic impairment are exacerbated by reoxygenation of the brain using 100% ventilatory O(2) compared to lower levels that maintain normoxemia. Numerous molecular mechanisms may be responsible for mitochondrial dysfunction caused by oxidative stress, including oxidation and inactivation of mitochondrial proteins, promotion of the mitochondrial membrane permeability transition, and consumption of metabolic cofactors and intermediates, for example, NAD(H). Moreover, the relative contribution of these mechanisms to cell injury and death is likely different among different types of brain cells, for example, neurons and astrocytes. In order to better understand these oxidative stress mechanisms and their relevance to neurologic disorders, we have undertaken studies with primary cultures of astrocytes and neurons exposed to O(2) and glucose deprivation and reoxygenation and compared the results of these studies to those using a rat model of neonatal asphyxic brain injury. These results support the hypothesis that release and or consumption of mitochondrial NAD(H) is at least partially responsible for respiratory inhibition, particularly in neurons.

Postischemic oxidative stress promotes mitochondrial metabolic failure in neurons and astrocytes.

&#x867D;&#x7136;&#x9499; (EC 3.4.22) &#x86CB;&#x767D;&#x9176;&#x86CB;&#x767D;&#x9176;&#x6FC0;&#x6D3B;&#x6709;&#x4EBA;&#x5EFA;&#x8BAE;&#x6709;&#x52A9;&#x4E8E;&#x5174;&#x594B;&#x6BD2;&#x6027;&#x5EF6;&#x8FDF;&#x9499;&#x653E;&#x677E; (DCD) &#x901A;&#x8FC7;&#x86CB;&#x767D;&#x8D28;&#x964D;&#x89E3;&#x7684; Na + Ca2 + &#x6362;&#x70ED;&#x5668; 3 &#xFF08;NCX3&#xFF09;&#xFF0C;&#x4E0E; DCD &#x7EC6;&#x80DE;&#x8D28;&#x9499;&#x86CB;&#x767D;&#x9176;&#x6FC0;&#x6D3B;&#x5DF2;&#x6C38;&#x8FDC;&#x4E0D;&#x4F1A;&#x88AB;&#x5B9E;&#x65F6;&#x53EF;&#x89C6;&#x5316;&#x5904;&#x7406;&#x3002;&#x6211;&#x4EEC;&#x96C7;&#x7528;&#x5230;&#x540C;&#x65F6;&#x56FE;&#x50CF;&#x9499;&#x86CB;&#x767D;&#x9176;&#x6FC0;&#x6D3B;&#x548C;&#x9499;&#x653E;&#x677E;&#x6D3B;&#x8111;&#x76AE;&#x8D28;&#x795E;&#x7ECF;&#x5143;&#x9499;&#x86CB;&#x767D;&#x9176;&#x8367;&#x5149;&#x5171;&#x632F;&#x80FD;&#x91CF;&#x8F6C;&#x79FB;&#x57FA;&#x8D28;&#x3002;&#x8367;&#x5149;&#x5171;&#x632F;&#x80FD;&#x91CF;&#x8F6C;&#x79FB;&#x9499;&#x86CB;&#x767D;&#x9176;&#x6291;&#x5236;&#x5242;&#x654F;&#x611F;&#x4E0B;&#x964D; DCD &#x906D;&#x53D7;&#x8FDE;&#x7EED;&#x8C37;&#x6C28;&#x9178; &#xFF08;100 microM&#xFF09; &#x7684;&#x795E;&#x7ECF;&#x5143;&#x53D1;&#x751F;&#x540E;&#x5728; + /-5 &#x5206;&#x949F; 39 &#x7EA2;&#x6F6E;&#x3002;Calpeptin &#x7531;&#x9499;&#x86CB;&#x767D;&#x9176;&#x7684;&#x6291;&#x5236;&#x4F5C;&#x7528;&#x4E0D;&#x62D6;&#x5EF6; DCD&#xFF0C;&#x6062;&#x590D;&#x4ECE;&#x6D77;&#x62D4; DCD &#x6837;&#x53EF;&#x9006;&#x9499;&#x6216;&#x5C3D;&#x7BA1;&#x6291;&#x5236; &alpha;-&#x8840;&#x5F71;&#x5904;&#x7406; &gt; 90%&#x7684;&#x7EC6;&#x80DE;&#x6B7B;&#x4EA1;&#x7684;&#x6765;&#x4E34;&#x3002;NCXs &#x9006;&#x8F6C;&#x671F;&#x95F4;&#x8C37;&#x6C28;&#x9178;&#x66B4;&#x9732;&#xFF0C;NCX KB R7943 &#x5EF6;&#x957F; DCD&#xFF0C;&#x548C;&#x91CD;&#x5927; NCX3 &#x5288;&#x88C2;&#x672A;&#x770B;&#x5230;&#x4EE5;&#x4E0B; 90 &#x5206;&#x949F;&#x7684;&#x8C37;&#x6C28;&#x9178;&#x66B4;&#x9732;&#x65F6;&#x95F4;&#x7684;&#x62EE;&#x6297;&#x5242;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x8868;&#x660E;&#x4E0E;&#x6025;&#x6027;&#x8C37;&#x6C28;&#x9178;&#x6BD2;&#x6027;&#x76F8;&#x5173;&#x7684;&#x9C81;&#x68D2;&#x9499;&#x86CB;&#x767D;&#x9176;&#x6FC0;&#x6D3B;&#x53D1;&#x751F;&#x5728;&#x9499;&#x7A33;&#x6001;&#x7684;&#x6301;&#x7EED;&#x635F;&#x5931;&#x540E;&#x624D;&#x3002;&#x886C;&#x5E95;&#x662F; NCX3 &#x6216;&#x5176;&#x4ED6;&#x9499;&#x86CB;&#x767D;&#x9176;&#x7684;&#x5904;&#x7406;&#x53EF;&#x80FD;&#x4E0D;&#x9700;&#x8981;&#x5728;&#x76AE;&#x5C42;&#x795E;&#x7ECF;&#x5143;&#x7684;&#x6025;&#x6027;&#x6BD2;&#x6027;&#x7684;&#x4E3B;&#x8981;&#x539F;&#x56E0;&#x3002;&#x4F46;&#x662F;&#xFF0C;&#x4E0D;&#x6392;&#x9664;&#x9499;&#x86CB;&#x767D;&#x9176;&#x7684;&#x4F5C;&#x7528;&#x4F5C;&#x4E3A;&#x4E00;&#x4E2A;&#x56E0;&#x7D20;&#x6216;&#x6E29;&#x548C;&#x7684;&#x8C37;&#x6C28;&#x9178;&#x4FAE;&#x8FB1;&#x7684;&#x54CD;&#x5E94;&#x3002;

&#x6025;&#x6027;&#x8C37;&#x6C28;&#x9178;&#x5174;&#x594B;&#x6BD2;&#x6027;&#x7684;&#x7EC6;&#x80DE;&#x8D28;&#x9499;&#x86CB;&#x767D;&#x9176;&#x6FC0;&#x6D3B;&#x548C;&#x9499;&#x9010;&#x6B65;&#x653E;&#x5F00;&#x7684;&#x5B9E;&#x65F6;&#x53EF;&#x89C6;&#x5316;&#x3002;

Although calpain (EC 3.4.22) protease activation was suggested to contribute to excitotoxic delayed calcium deregulation (DCD) via proteolysis of Na+/Ca2+ exchanger 3 (NCX3), cytoplasmic calpain activation in relation to DCD has never been visualized in real-time. We employed a calpain fluorescence resonance energy transfer substrate to simultaneously image calpain activation and calcium deregulation in live cortical neurons. A calpain inhibitor-sensitive decline in fluorescence resonance energy transfer was observed at 39 +/- 5 min after the occurrence of DCD in neurons exposed to continuous glutamate (100 microM). Inhibition of calpain by calpeptin did not delay the onset of DCD, recovery from DCD-like reversible calcium elevations, or cell death despite inhibiting alpha-spectrin processing by > 90%. NCXs reversed during glutamate exposure, the NCX antagonist KB-R7943 prolonged the time to DCD, and significant NCX3 cleavage following 90 min of glutamate exposure was not observed. Our findings suggest that robust calpain activation associated with acute glutamate toxicity occurs only after a sustained loss in calcium homeostasis. Processing of NCX3 or other calpain substrates is unlikely to be the primary cause of acute excitotoxicity in cortical neurons. However, a role for calpain as a contributing factor or in response to milder glutamate insults is not excluded.

Real-time visualization of cytoplasmic calpain activation and calcium deregulation in acute glutamate excitotoxicity.

Bien que l&#39;activation de prot&eacute;ases calpa&iuml;ne (EC 3.4.22) a &eacute;t&eacute; propos&eacute;e de contribuer &agrave; excitotoxique retard&eacute; la d&eacute;r&eacute;glementation (DCD) de calcium par prot&eacute;olyse de Na + / Ca2 + &eacute;changeur 3 (NCX3), activation de la calpa&iuml;ne cytoplasmique en ce qui concerne la DCD n&#39;a jamais &eacute;t&eacute; visualis&eacute;e en temps r&eacute;el. Nous avons utilis&eacute; un substrat calpa&iuml;ne fluorescence par r&eacute;sonance &eacute;nerg&eacute;tique transfert &agrave; simultan&eacute;ment image calpa&iuml;ne activation et calcium d&eacute;r&eacute;gulation dans des neurones corticaux direct. Une baisse sensible inhibiteur calpa&iuml;ne transfert d&#39;&eacute;nergie de fluorescence resonance a &eacute;t&eacute; observ&eacute;e &agrave; 39 +/-5 min apr&egrave;s la survenance du DCD dans les neurones expos&eacute;s au glutamate continu (100 microM). L&#39;inhibition de la calpa&iuml;ne par calpeptin ne retarde pas le d&eacute;but de la DCD, r&eacute;cup&eacute;ration d&#39;&eacute;l&eacute;vations de calcium r&eacute;versible de DCD-type, ou la mort des cellules malgr&eacute; inhibant la transformation de l&#39;alpha-spectrine &gt; 90 %. NCXs reprises au cours de l&#39;exposition de glutamate, l&#39;antagoniste du NCX KB-R7943 prolong&eacute; le temps de DCD et le clivage NCX3 significatif apr&egrave;s 90 min d&#39;exposition glutamate n&#39;a pas &eacute;t&eacute; observ&eacute;e. Nos r&eacute;sultats sugg&egrave;rent que la calpa&iuml;ne robuste activation associ&eacute;e &agrave; la toxicit&eacute; aigu&euml; de glutamate se produit qu&#39;apr&egrave;s une perte soutenue dans l&#39;hom&eacute;ostasie calcique. Traitement des NCX3 ou autre calpa&iuml;ne substrats est peu susceptible d&#39;&ecirc;tre la principale cause de l&#39;excitotoxicit&eacute; aigu&euml; dans les neurones corticaux. Toutefois, un r&ocirc;le pour la calpa&iuml;ne comme un facteur contributif ou en r&eacute;ponse &agrave; des insultes de glutamate n&#39;est pas exclu.

Visualisation en temps r&eacute;el de la calpa&iuml;ne cytoplasmique d&#39;activation et de calcium d&eacute;r&eacute;glementation en glutamate aigu&euml; excitotoxicit&eacute;.

Obwohl Calpain (EG 3.4.22) Protease Aktivierung angeregt wurde, Beitrag zur excitotoxischem verz&ouml;gert Kalzium Deregulierung (DCD) &uuml;ber Proteolyse der Na + / Ca2 +-Austauscher 3 (NCX3), zytoplasmatische Calpain-Aktivierung im Zusammenhang mit DCD hat nie in Echtzeit visualisiert wurde. Wir besch&auml;ftigten Calpain Fluoreszenz Resonanz Energie Transfer Substrat gleichzeitig Bild Calpain Aktivierung und Kalzium Deregulierung im Leben kortikale Neuronen. Ein Calpain-Inhibitor-empfindlichen R&uuml;ckgang der Fluoreszenz-Resonanz-Energie-Transfer wurde um 39 +/-5 min nach Eintritt des DCD in Neuronen ausgesetzt kontinuierliche Glutamat (100 MicroM) beobachtet. Hemmung der Calpain von Calpeptin f&uuml;hrte zu keiner Verz&ouml;gerung der Beginn der DCD, Wiederherstellung nach DCD-wie reversibel Kalzium Erhebungen oder Zelltod trotz Hemmung der Alpha-Spektrin Verarbeitung durch &gt; 90 %. NCXs umgekehrt w&auml;hrend der Glutamat-Exposition, der NCX-Antagonist KB-R7943 verl&auml;ngert die Zeit, DCD und erhebliche NCX3-Spaltung, die folgenden 90 min der Glutamat-Exposition nicht beobachtet wurde. Unsere Ergebnisse weisen, dass erst nach einem anhaltenden Verlust an Kalzium-Hom&ouml;ostase robuste Calpain-Aktivierung verbunden mit akuten Glutamat Toxizit&auml;t auftritt. Verarbeitung von NCX3 oder anderen Calpain Substrate ist unwahrscheinlich, dass die prim&auml;re Ursache des akuten Exzitotoxit&auml;t in kortikalen Neuronen. Eine Rolle f&uuml;r Calpain als entscheidender Faktor oder als Reaktion auf milder Glutamat Beleidigungen ist jedoch nicht ausgeschlossen.

Echtzeit-Visualisierung der zytoplasmatischen Calpain-Aktivierung und Kalzium Deregulierung in akuten Glutamat Exzitotoxit&auml;t.

Sebbene attivazione proteasi calpaina (EC 3.4.22) &egrave; stato suggerito di contribuire alla excitotoxic ritardato deregolamentazione calcio (DCD) tramite proteolisi del Na + / Ca2 + scambiatore 3 (NCX3), attivazione citoplasmatica calpain in relazione alla DCD non ha mai stato visualizzato in tempo reale. Abbiamo impiegato un substrato di trasferimento calpain energia di risonanza fluorescenza a contemporaneamente immagine calpain attivazione e calcio deregolamentazione in diretta dei neuroni corticali. Un calo sensibile inibitore calpain il trasferimento di energia di risonanza di fluorescenza &egrave; stato osservato a 39 + 5 min dopo l&#39;occorrenza di DCD nei neuroni esposti al continuo glutammato (100 microM). Inibizione di calpaina da calpeptin non ha fatto ritardare l&#39;insorgenza di DCD, recupero da elevazioni DCD-come calcio reversibile, o morte cellulare nonostante inibizione alfa-spectrina elaborazione &amp;gt; 90%. NCXs invertito durante l&#39;esposizione di glutammato, l&#39;antagonista NCX KB-R7943 prolungato il tempo di DCD e significativo NCX3 clivaggio seguenti 90 min di glutammato esposizione non &egrave; stata osservata. I nostri risultati suggeriscono che robusto calpain attivazione associata con tossicit&agrave; acuta glutammato si verifica solo dopo una perdita sostenuta nell&#39;omeostasi del calcio. Elaborazione di NCX3 o altri calpain substrati &egrave; improbabile da essere la causa primaria di eccitotossicit&agrave; acuta nei neuroni corticali. Tuttavia, non &egrave; escluso un ruolo per calpain come concausa o in risposta agli insulti pi&ugrave; miti di glutammato.

Visualizzazione in tempo reale di calpaina citoplasmatica attivazione e calcio deregolamentazione in eccitotossicit&agrave; del glutammato acuta.

&#x30AB;&#x30EB;&#x30D1;&#x30A4;&#x30F3; &#xFF08;EC 3.4.22&#xFF09; &#x30D7;&#x30ED;&#x30C6;&#x30A2;&#x30FC;&#x30BC;&#x6D3B;&#x6027;&#x5316;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x86CB;&#x767D;&#x3092;&#x4ECB;&#x3057;&#x3066; &#xFF08;DCD&#xFF09; &#x898F;&#x5236;&#x7DE9;&#x548C; Na &#x306E; excitotoxic &#x306B;&#x8CA2;&#x732E;&#x3059;&#x308B;&#x9045;&#x5EF6;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x305F;&#x304C; +/2 + &#x4EA4;&#x63DB; 3 (NCX3) &#x7D30;&#x80DE;&#x8CEA;&#x30AB;&#x30EB;&#x30D1;&#x30A4;&#x30F3;&#x6D3B;&#x6027;&#x5316; DCD &#x306B;&#x95A2;&#x9023;&#x3057;&#x3066;&#x3044;&#x308B;&#x6C7A;&#x3057;&#x3066;&#x3055;&#x308C;&#x3066;&#x8996;&#x899A;&#x5316;&#x30EA;&#x30A2;&#x30EB;&#x30BF;&#x30A4;&#x30E0;&#x3067;&#x3002;&#x30AB;&#x30EB;&#x30D1;&#x30A4;&#x30F3;&#x86CD;&#x5149;&#x5171;&#x9CF4;&#x30A8;&#x30CD;&#x30EB;&#x30AE;&#x30FC;&#x4F1D;&#x9054;&#x57FA;&#x677F;&#x306B;&#x540C;&#x6642;&#x306B;&#x30A4;&#x30E1;&#x30FC;&#x30B8; &#x30AB;&#x30EB;&#x30D1;&#x30A4;&#x30F3;&#x6D3B;&#x6027;&#x5316;&#x3068;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x3067;&#x306E;&#x898F;&#x5236;&#x7DE9;&#x548C;&#x30E9;&#x30A4;&#x30D6;&#x76AE;&#x8CEA;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x3092;&#x63A1;&#x7528;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x86CD;&#x5149;&#x5171;&#x9CF4;&#x30A8;&#x30CD;&#x30EB;&#x30AE;&#x30FC;&#x79FB;&#x52D5;&#x306E;&#x30AB;&#x30EB;&#x30D1;&#x30A4;&#x30F3;&#x963B;&#x5BB3;&#x5264;&#x611F;&#x53D7;&#x6027;&#x4F4E;&#x4E0B;&#x306F; DCD &#x306E;&#x9023;&#x7D9A;&#x30B0;&#x30EB;&#x30BF;&#x30DF;&#x30F3;&#x9178; (100 microM) &#x516C;&#x958B;&#x3055;&#x308C;&#x308B;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x767A;&#x751F;&#x5F8C; 5 &#x5206; +/-39 &#x3067;&#x89B3;&#x5BDF;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x30AB;&#x30EB;&#x30D1;&#x30A4;&#x30F3;&#x306B;&#x3088;&#x308B; calpeptin &#x306E;&#x6291;&#x5236;&#x306F; DCD&#x3001;DCD &#x306E;&#x3088;&#x3046;&#x306A;&#x53EF;&#x9006;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x7ACB;&#x9762;&#x56F3;&#x307E;&#x305F;&#x306F;&#x30A2;&#x30EB;&#x30D5;&#x30A1; &#x30B9;&#x30DA;&#x30AF;&#x30C8;&#x30EA;&#x30F3;&#x51E6;&#x7406;&#x76EE; 90% &#x6291;&#x5236;&#x306B;&#x3082;&#x304B;&#x304B;&#x308F;&#x3089;&#x305A;&#x7D30;&#x80DE;&#x6B7B;&#x304B;&#x3089;&#x306E;&#x56DE;&#x5FA9;&#x306E;&#x767A;&#x75C7;&#x3092;&#x9045;&#x3089;&#x305B;&#x308B;&#x304B;&#x306A;&#x3044;&#x3067;&#x3059;&#x3002;NCXs KB R7943 DCD &#x3068;&#x6B21;&#x306E; 90 &#x5206;&#x306E;&#x30B0;&#x30EB;&#x30BF;&#x30DF;&#x30F3;&#x9178;&#x306E;&#x9732;&#x51FA;&#x306F;&#x307F;&#x3089;&#x308C;&#x306A;&#x304B;&#x3063;&#x305F;&#x91CD;&#x8981;&#x306A; NCX3 &#x80F8;&#x306E;&#x8C37;&#x9593;&#x306B;&#x6642;&#x9593;&#x5EF6;&#x9577; NCX &#x62EE;&#x6297;&#x30B0;&#x30EB;&#x30BF;&#x30DF;&#x30F3;&#x9178;&#x9732;&#x5149;&#x4E2D;&#x306B;&#x9006;&#x8EE2;&#x3002;&#x5805;&#x7262;&#x306A;&#x30AB;&#x30EB;&#x30D1;&#x30A4;&#x30F3;&#x6D3B;&#x6027;&#x5316;&#x6025;&#x6027;&#x30B0;&#x30EB;&#x30BF;&#x30DF;&#x30F3;&#x9178;&#x6BD2;&#x6027;&#x306B;&#x3088;&#x308B;&#x6295;&#x4E0E;&#x304C;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x6052;&#x5E38;&#x6027;&#x306E;&#x6301;&#x7D9A;&#x7684;&#x306A;&#x640D;&#x5931;&#x306E;&#x5F8C;&#x306B;&#x306E;&#x307F;&#x767A;&#x751F;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;NCX3 &#x307E;&#x305F;&#x306F;&#x4ED6;&#x306E;&#x30AB;&#x30EB;&#x30D1;&#x30A4;&#x30F3;&#x306E;&#x51E6;&#x7406;&#x57FA;&#x677F;&#x3067;&#x3059;&#x76AE;&#x8CEA;&#x30CB;&#x30E5;&#x30FC;&#x30ED;&#x30F3;&#x306E;&#x6025;&#x6027;&#x6BD2;&#x6027;&#x306E;&#x4E3B;&#x306A;&#x539F;&#x56E0;&#x3068;&#x306A;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x306A;&#x3044;&#x3067;&#x3059;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;&#x8981;&#x56E0;&#x3068;&#x3057;&#x3066;&#x307E;&#x305F;&#x306F;&#x7A4F;&#x3084;&#x304B;&#x306A;&#x30B0;&#x30EB;&#x30BF;&#x30DF;&#x30F3;&#x9178;&#x4FAE;&#x8FB1;&#x306B;&#x5FDC;&#x3048;&#x3066;&#x30AB;&#x30EB;&#x30D1;&#x30A4;&#x30F3;&#x306E;&#x5F79;&#x5272;&#x306F;&#x9664;&#x5916;&#x3055;&#x308C;&#x307E;&#x305B;&#x3093;&#x3002;

&#x6025;&#x6027;&#x30B0;&#x30EB;&#x30BF;&#x30DF;&#x30F3;&#x9178;&#x5FDC;&#x9700;&#x578B;&#x306E;&#x7D30;&#x80DE;&#x8CEA;&#x306E;&#x30AB;&#x30EB;&#x30D1;&#x30A4;&#x30F3;&#x6D3B;&#x6027;&#x5316;&#x3068;&#x30AB;&#x30EB;&#x30B7;&#x30A6;&#x30E0;&#x898F;&#x5236;&#x7DE9;&#x548C;&#x306E;&#x30EA;&#x30A2;&#x30EB;&#x30BF;&#x30A4;&#x30E0;&#x53EF;&#x8996;&#x5316;&#x3002;

&#xBE44;&#xB85D; calpain (EC 3.4.22) &#xB2E8;&#xBC31;&#xC9C8; &#xD65C;&#xC131;&#xD654; &#xC9C0;&#xC5F0; &#xB41C; Na&#xC758; &#xBCA0;&#xC774;&#xC2A4;&#xB97C; &#xD1B5;&#xD574; &#xCE7C;&#xC298; &#xADDC;&#xC81C; (DCD) excitotoxic&#xC5D0; &#xAE30;&#xC5EC;&#xD560; &#xC81C;&#xC548; &#xD588;&#xB2E4; + Ca2 + &#xAD50;&#xD658;&#xAE30; 3 (NCX3), DCD&#xC5D0; &#xAD00;&#xD558;&#xC5EC; &#xC138;&#xD3EC;&#xC9C8; calpain &#xD65C;&#xC131;&#xD654; &#xC2E4;&#xC2DC;&#xAC04; &#xC2DC;&#xAC01; &#xC801;&#xC774; &#xC788;&#xB2E4;. &#xC6B0;&#xB9AC; calpain &#xD615;&#xAD11; &#xACF5;&#xBA85; &#xC5D0;&#xB108;&#xC9C0; &#xC804;&#xC1A1; &#xAE30;&#xD310;&#xC5D0; &#xB3D9;&#xC2DC;&#xC5D0; &#xC774;&#xBBF8;&#xC9C0; calpain &#xD65C;&#xC131;&#xD654; &#xBC0F; &#xCE7C;&#xC298; &#xADDC;&#xC81C; &#xB77C;&#xC774;&#xBE0C; &#xB300;&#xB1CC; &#xD53C; &#xC9C8;&#xC758; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xC0AC;&#xC6A9; &#xB429;&#xB2C8;&#xB2E4;. &#xD615;&#xAD11; &#xACF5;&#xBA85; &#xC5D0;&#xB108;&#xC9C0; &#xC804;&#xB2EC; calpain &#xC5B5;&#xC81C;&#xC81C; &#xBBFC;&#xAC10;&#xD55C; &#xAC10;&#xC18C; DCD &#xC5F0;&#xC18D; &#xC870;&#xBBF8;&#xB8CC; (100 microM)&#xC5D0; &#xB178;&#xCD9C; &#xB41C; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xBC1C;&#xC0DD; &#xD55C; &#xD6C4; 5 &#xBD84; + 39&#xC5D0;&#xC11C; &#xAD00;&#xCC30; &#xB418;&#xC5C8;&#xB2E4;. Calpeptin&#xC5D0; &#xC758;&#xD574; calpain &#xC800;&#xD574; DCD, &#xBCF5;&#xAD6C; DCD &#xAC19;&#xC740; &#xAC00;&#xC5ED; &#xCE7C;&#xC298; &#xACE0;&#xB3C4; &#xB610;&#xB294; &#xC54C;&#xD30C; spectrin &#xCC98;&#xB9AC; &amp;gt; 90% &#xC5B5;&#xC81C;&#xC5D0; &#xBD88;&#xAD6C; &#xD558; &#xACE0; &#xC138;&#xD3EC; &#xC8FD;&#xC74C;&#xC758; &#xBC1C;&#xBCD1;&#xC744; &#xC9C0;&#xC5F0; &#xD558;&#xC9C0; &#xC54A;&#xC558;&#xB2E4;. NCXs &#xC870;&#xBBF8;&#xB8CC; &#xB178;&#xCD9C;, KB R7943 &#xC5F0;&#xC7A5; &#xC2DC;&#xAC04; DCD, &#xC870;&#xBBF8;&#xB8CC; &#xB178;&#xCD9C; &#xB2E4;&#xC74C; 90 &#xBD84; &#xAD00;&#xCC30; &#xD558;&#xC9C0; &#xC911;&#xC694; &#xD55C; NCX3 &#xBD84;&#xC5F4;&#xC744; NCX &#xAE38; &#xD56D; &#xC81C; &#xC911; &#xBC18;&#xC804;. &#xC6B0;&#xB9AC;&#xC758; &#xBC1C;&#xACAC; &#xAC74;&#xC758; &#xAC15;&#xB825;&#xD55C; calpain &#xD65C;&#xC131;&#xD654; &#xC870;&#xBBF8;&#xB8CC; &#xAE09;&#xC131; &#xB3C5;&#xC131;&#xACFC; &#xAD00;&#xB828; &#xB41C; &#xCE7C;&#xC298; &#xD56D;&#xC0C1;&#xC131;&#xC5D0;&#xC11C; &#xC9C0;&#xC18D;&#xC801;&#xC778;&#xB41C; &#xC190;&#xC2E4; &#xD6C4;&#xC5D0; &#xBC1C;&#xC0DD; &#xD569;&#xB2C8;&#xB2E4;. NCX3 &#xB610;&#xB294; &#xB2E4;&#xB978; calpain &#xCC98;&#xB9AC; &#xAE30;&#xD310; &#xAE09;&#xC131; excitotoxicity &#xB300;&#xB1CC; &#xD53C; &#xC9C8;&#xC758; &#xC2E0;&#xACBD; &#xC138;&#xD3EC;&#xC5D0;&#xC11C;&#xC758; &#xC8FC;&#xC694; &#xC6D0;&#xC778;&#xC774; &#xB420; &#xAC83;&#xC785;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xAE30;&#xC5EC; &#xD558;&#xB294; &#xC694;&#xC18C; &#xB610;&#xB294; &#xC628;&#xD654;&#xD55C; &#xC870;&#xBBF8;&#xB8CC; &#xBAA8;&#xC695;&#xC5D0; &#xB300; &#xD55C; &#xC751;&#xB2F5;&#xC5D0;&#xC11C; calpain &#xC5ED;&#xD560;&#xC744; &#xBC30;&#xC81C; &#xD558;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xAE09;&#xC131; &#xC870;&#xBBF8;&#xB8CC; excitotoxicity&#xC758; &#xC138;&#xD3EC;&#xC9C8; calpain &#xD65C;&#xC131;&#xD654; &#xBC0F; &#xCE7C;&#xC298; &#xADDC;&#xC81C; &#xC644;&#xD654;&#xC758; &#xC2E4;&#xC2DC;&#xAC04; &#xC2DC;&#xAC01;&#xD654;.

Aunque la activaci&oacute;n de proteasas de calpain (EC 3.4.22) sugiri&oacute; a contribuir a excitatoria retrasado desregulaci&oacute;n de calcio (DCD) mediante proteolisis de Na + / Ca2 + cambiador 3 (NCX3), activaci&oacute;n de calpain citopl&aacute;smicas en lo referente a DCD nunca ha sido visualizada en tiempo real. Empleamos un sustrato calpain fluorescencia resonancia energ&iacute;a transferencia a la vez imagen calpain activaci&oacute;n y calcio desregulaci&oacute;n en neuronas corticales vivos. Se observ&oacute; un descenso de inhibidor sensible de calpain en transferencia de energ&iacute;a de resonancia de fluorescencia en 39 +/-5 min despu&eacute;s de la ocurrencia de DCD en neuronas expuestas a glutamato continuo (100 microM). Inhibici&oacute;n de calpain por calpeptin no retrasar la aparici&oacute;n de DCD, recuperaci&oacute;n de calcio reversible DCD-como elevaciones, o muerte celular a pesar de inhibir el procesamiento de alfa-spectrin &gt; 90%. NCXs invertido durante la exposici&oacute;n de glutamato, el antagonista NCX KB-R7943 prolonga el tiempo de DCD y gran escote de NCX3 siguiente 90 min de exposici&oacute;n de glutamato no se observ&oacute;. Nuestros resultados sugieren que robusto calpain activaci&oacute;n asociada con toxicidad aguda glutamato ocurre solamente despu&eacute;s de una p&eacute;rdida sostenida en la homeostasis del calcio. Procesamiento de NCX3 u otro calpain sustratos es poco probable que la causa primaria de excitotoxicidad aguda en neuronas corticales. Sin embargo, no se excluye un papel de calpain como un factor contribuyente o en respuesta a los insultos m&aacute;s leves de glutamato.

Visualizaci&oacute;n en tiempo real de calpain citopl&aacute;smico desregulaci&oacute;n activaci&oacute;n y calcio en glutamato aguda excitotoxicidad.

Multiple neurodegenerative disorders are associated with altered mitochondrial bioenergetics. Although mitochondrial O(2) consumption is frequently measured in isolated mitochondria, isolated synaptic nerve terminals (synaptosomes), or cultured cells, the absence of mature brain circuitry is a remaining limitation. Here we describe the development of a method that adapts the Seahorse Extracellular Flux Analyzer (XF24) for the microplate-based measurement of hippocampal slice O(2) consumption. As a first evaluation of the technique, we compared whole-slice bioenergetics with previous measurements made with synaptosomes or cultured neurons. We found that mitochondrial respiratory capacity and O(2) consumption coupled to ATP synthesis could be estimated in cultured or acute hippocampal slices with preserved neural architecture. Mouse organotypic hippocampal slices oxidizing glucose displayed mitochondrial O(2) consumption that was well coupled, as determined by the sensitivity to the ATP synthase inhibitor oligomycin. However, stimulation of respiration by uncoupler was modest (<120% of basal respiration) compared with previous measurements in cells or synaptosomes, though enhanced slightly (to ∼150% of basal respiration) by acute addition of the mitochondrial complex I-linked substrate pyruvate. These findings suggest a high basal utilization of respiratory capacity in slices and a limitation of glucose-derived substrate for maximal respiration. The improved throughput of microplate-based hippocampal respirometry over traditional O(2) electrode-based methods is conducive to neuroprotective drug screening. When coupled with cell type-specific pharmacology or genetic manipulations, the ability to measure O(2) consumption efficiently from whole slices should advance our understanding of mitochondrial roles in physiology and neuropathology.

Adaptation of microplate-based respirometry for hippocampal slices and analysis of respiratory capacity.

Cells that exhibit an absolute dependence on the anti-apoptotic BCL-2 protein for survival are termed "primed for death" and are killed by the BCL-2 antagonist ABT-737. Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2 expression. We show here that 1) stable BCL-2 overexpression alone can induce a primed for death state and 2) that an ABT-737-induced loss of functional cytochrome c from the electron transport chain causes a reduction in maximal respiration that is readily detectable by microplate-based respirometry. Stable BCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis. Mitochondria within permeabilized BCL-2 overexpressing cells were selectively vulnerable to ABT-737-induced cytochrome c release compared to those from control-transfected cells, consistent with a primed state. ABT-737 treatment caused a dose-dependent impairment of maximal O(2) consumption in MCF10A BCL-2 overexpressing cells but not in control-transfected cells or in immortalized mouse embryonic fibroblasts lacking both BAX and BAK. This impairment was rescued by delivering exogenous cytochrome c to mitochondria via saponin-mediated plasma membrane permeabilization. An ABT-737-induced reduction in maximal O(2) consumption was also detectable in SP53, JeKo-1, and WEHI-231 B-cell lymphoma cell lines, with sensitivity correlating with BCL-2:MCL-1 ratio and with susceptibility (SP53 and JeKo-1) or resistance (WEHI-231) to ABT-737-induced apoptosis. Multiplexing respirometry assays to ELISA-based determination of cytochrome c redistribution confirmed that respiratory inhibition was associated with cytochrome c release. In summary, cell-based respiration assays were able to rapidly identify a primed for death state in cells with either artificially overexpressed or high endogenous BCL-2. Rapid detection of a primed for death state in individual cancers by "bioenergetics-based profiling" may eventually help identify the subset of patients with chemoresistant but primed tumors who can benefit from treatment that incorporates a BCL-2 antagonist.

Rapid detection of an ABT-737-sensitive primed for death state in cells using microplate-based respirometry.

Polyamidoamine dendrimer nanoparticles (~ 4 nanometers) are inert polymers that can be linked to biologically active compounds. These dendrimers selectively target and accumulate in inflammatory cells upon systemic administration. Dendrimer-linked compounds enable sustained release of therapeutic compounds directly at the site of damage. The purpose of this study was to determine if dendrimers can be used to target the optic nerve (ON) ischemic lesion in our rodent and nonhuman primate models of nonarteritic anterior ischemic optic neuropathy (NAION), a disease affecting >10,000 individuals in the US annually, and for which there currently is no effective treatment.

Zara Mehrabian

Department of Ophthalmology and Visual Sciences

University of Maryland-Baltimore School of Medicine

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

Zara Mehrabian

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Ophthalmology and Visual Sciences

University of Maryland-Baltimore School of Medicine

The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION)

Department of Ophthalmology and Visual Sciences