3-(2-4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3h)-quinazolinone and Brain-Ischemia

This study aimed to examine whether the neuroprotective effects of Mdivi-1 are attributable to extracellular ATP and adenosine. Mdivi-1 was administered prior to or post middle cerebral artery occlusion (MCAO). The extracellular adenosine was measured by in vivo microdialysis and high-pressure liquid chromatography (HPLC) in MCAO mouse model. Western blot was done to determine the influence of Mdivi-1 on the expression of CD39 and CREB phosphorylation both in vivo and in the cultured astrocytes. Intracellular cAMP and protein kinase A (PKA) activity were detected in primary astrocytes. Results showed that Mdivi-1 significantly reduced infarct volume and neurological scores when administered either prior to or post MCAO. Interestingly, pretreatment with Mdivi-1 resulted in marked increase of extracellular adenosine and concomitant decrease in ATP. The expression of CD39, but not CD73, was upregulated by Mdivi-1, which was associated with the elevated phosphorylated cAMP response element-binding protein (CREB), a transcription factor potentially regulating CD39 expression. In primary astrocytes, Mdivi-1 treatment induced increases in intracellular cAMP, PKA activity and CREB phosphorylation, and PKA-specific inhibitor completely reversed Mdivi-1-induced CD39 expression. Our results demonstrate that Mdivi-1 protects against ischemic brain injury through increasing extracellular adenosine, a process involving elevated CD39 expression that is likely modulated by cAMP/PKA/CREB cascade. Figure Potential mechanisms by which Mdivi-1 mediates the neuroprotection on cerebral ischemic stroke. Results from the present study indicate that Mdivi-1 protects against ischemic brain injury through increasing extracellular adenosine, a process involving elevated CD39 expression that is likely modulated by the cAMP/PKA/CREB cascades.

Topics: Adenosine; Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Astrocytes; Blood-Brain Barrier; Brain Ischemia; Cells, Cultured; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Dynamins; Extracellular Space; Hydrolysis; Male; Mice, Inbred C57BL; Neuroprotective Agents; Quinazolinones; Receptor, Adenosine A1; Recovery of Function; Signal Transduction; Up-Regulation

The resistance of CA3 neurons to ischaemia and the ischaemic tolerance conferred by ischaemic preconditioning (IPC) are two well-established endogenous neuroprotective mechanisms. Elucidating the molecules involved may help us find new therapeutic targets. Thus, we determined whether dynamin-related protein 1 (Drp-1) is involved in these processes.. In vivo, we subjected rats to either 10 min severe global ischaemia using a four-vessel occlusion (4-VO) model or 2 min IPC before the onset of 4-VO. In vitro, we performed oxygen glucose deprivation (OGD) studies in rat hippocampal neurons. Drp-1 was silenced or inhibited by siRNA or pharmacological inhibitor Mdivi1. To assess whether mitochondrial Drp-1 alters neuronal vulnerability to ischaemic injury, various approaches were used including western blot, immunohistochemistry, immunofluorescence staining and electron microscopy. Hippocampal function was assessed using an open-field test.. Mitochondrial dynamin-related protein 1 (mtDrp-1) was selectively induced by ischaemia in hippocampal CA3 neurons. In hippocampal CA1 neurons, mtDrp-1 was not affected by ischaemia but significantly up-regulated by IPC. Suppression of Drp-1 increased the vulnerability of cells to OGD and global ischaemia. Inhibition of Drp-1 in vivo resulted in loss of acquisition and encoding of spatial information, and also prevented ischaemia-induced mitophagy in CA3. Thus mitochondrial-mediated injury was amplified and resistance to ischaemic injury lost.. Our findings that Drp-1 increases the resistance of neurons of hippocampal CA3 affected by global ischaemia and contributes to the tolerance conferred by IPC highlight Drp-1 as a potential therapeutic target for brain ischaemic stroke.

Topics: Animals; Brain Ischemia; Dynamins; Male; Mitochondria; Quinazolinones; Rats; Rats, Sprague-Dawley; Stroke

Dynamin-related protein 1 (Drp1) is a mitochondrial fission protein that, upon phosphorylation at serine 616 (p-Drp1(Ser616)), plays a pivotal role in neuronal death after ischemia. In the present study, we hypothesized that peroxisome proliferator-activated receptor-gamma (PPARγ)-dependent pathway can reduce the expression of p-Drp1(Ser616) and ameliorate hippocampal injury induced by global ischemia in rats.. We found that pretreatment of the rats with Mdivi-1, a selective Drp1 inhibitor, decreased the level of transient global ischemia (TGI)-induced p-Drp1(Ser616) and reduced cellular contents of oxidized proteins, activated caspase-3 expression as well as the extent of DNA fragmentation. Delivery of siRNA against Drp1 attenuated the expression of p-Drp1(Ser616) that was accompanied by alleviation of the TGI-induced protein oxidation, activated caspase-3 expression and DNA fragmentation in hippocampal proteins. Exogenous application of pioglitazone, a PPARγ agonist, reduced the p-Drp1(Ser616) expression, decreased TGI-induced oxidative stress and activated caspase-3 expression, lessened the extents of DNA fragmentation, and diminished the numbers of TUNEL-positive neuronal cells; all of these effects were reversed by GW9662, a PPARγ antagonist.. Our findings thus indicated that inhibition of TGI-induced p-Drp1(Ser616) expression by Drp1 inhibitor and Drp1-siRNA can decrease protein oxidation, activated caspase-3 expression and neuronal damage in the hippocampal CA1 subfield. PPARγ agonist, through PPARγ-dependent mechanism and via decreasing p-Drp1(Ser616) expression, can exert anti-oxidative and anti-apoptotic effects against ischemic neuronal injury.

Topics: Animals; Brain Ischemia; CA1 Region, Hippocampal; Dynamins; Male; Phosphorylation; PPAR gamma; Quinazolinones; Rats; Rats, Sprague-Dawley; Signal Transduction

When given prior to brain ischemia, mitochondrial division inhibitor-1 (mdivi-1) attenuates the brain damage caused by ischemia. Here, we investigated the potential effects of post-ischemia mdivi-1 treatment (1mg/kg, i.p., administered immediately after 2h of ischemia and prior to reperfusion) using a MCAO rat model. Mdivi-1 treatment decreased infarct volume and improved neurological function. In addition, cytochrome C release was attenuated, and neuronal apoptosis was decreased. The mitochondrial fission protein dynamin-related protein 1 (Drp1) was decreased in the mitochondrial fraction but increased in the cytosolic fraction. Mdivi-1 treatment augmented the increases in the mRNA expression of peroxisome proliferator-activated receptor coactivator-1α, nuclear respiratory factor-1, and mitochondrial transcriptional factor A. In conclusion, when given after ischemia and prior to reperfusion, mdivi-1 can protect against brain damage by inhibiting the mitochondria-mediated apoptosis induced by mitochondrial fission. Post-ischemia mdivi-1 treatment might promote I/R-induced mitochondrial biogenesis.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Cytochromes c; Disease Models, Animal; Dynamins; Male; Mitochondria; Mitochondrial Dynamics; Neurons; Neuroprotective Agents; Organelle Biogenesis; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Quinazolinones; Rats; Rats, Wistar; Reperfusion Injury

Mitochondrial fission is predominantly controlled by the activity of dynamin-related protein1 (Drp1), which has been reported to be involved in mitochondria apoptosis pathways. However, the role of Drp1 in a rat model of cardiac arrest remains unknown. In this study, we found that activation of Drp1 in the mitochondria was increased after cardiac arrest and inhibition of Drp1 by 1.2 mg/kg of mitochondrial division inhibitor-1 (Mdivi-1) administration after the restoration of spontaneous circulation (ROSC) significantly protected against cerebral ischemic injury, shown by the increased 72-h survival rate and improved neurological function. Moreover, the increase of the vital neuron and the reduction of cytochrome c (CytC) release, apoptosis-inducing factor (AIF) translocation and caspase-3 activation in the brain indicate that this protection might result from the suppression of neuron apoptosis. Altogether, these results indicated that Drp1 is activated after cardiac arrest and the inhibition of Drp1 is protective against cerebral ischemic injury in a rat of cardiac arrest model via inhibition of the mitochondrial apoptosis pathway.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Disease Models, Animal; Dose-Response Relationship, Drug; Dynamins; Heart Arrest; Male; Mitochondria; Neurons; Neuroprotective Agents; Quinazolinones; Random Allocation; Rats, Sprague-Dawley

Cerebral ischemia-reperfusion (I-R) is a complex pathological process. Although autophagy can be evoked by ischemia, its involvement in the reperfusion phase after ischemia and its contribution to the fate of neurons remains largely unknown. In the present investigation, we found that autophagy was activated in the reperfusion phase, as revealed in both mice with middle cerebral artery occlusion and oxygen-glucose deprived cortical neurons in culture. Interestingly, in contrast to that in permanent ischemia, inhibition of autophagy (by 3-methyladenine, bafilomycin A 1, Atg7 knockdown or in atg5(-/-) MEF cells) in the reperfusion phase reinforced, rather than reduced, the brain and cell injury induced by I-R. Inhibition of autophagy either with 3-methyladenine or Atg7 knockdown enhanced the I-R-induced release of cytochrome c and the downstream activation of apoptosis. Moreover, MitoTracker Red-labeled neuronal mitochondria increasingly overlapped with GFP-LC3-labeled autophagosomes during reperfusion, suggesting the presence of mitophagy. The mitochondrial clearance in I-R was reversed by 3-methyladenine and Atg7 silencing, further suggesting that mitophagy underlies the neuroprotection by autophagy. In support, administration of the mitophagy inhibitor mdivi-1 in the reperfusion phase aggravated the ischemia-induced neuronal injury both in vivo and in vitro. PARK2 translocated to mitochondria during reperfusion and Park2 knockdown aggravated ischemia-induced neuronal cell death. In conclusion, the results indicated that autophagy plays different roles in cerebral ischemia and subsequent reperfusion. The protective role of autophagy during reperfusion may be attributable to mitophagy-related mitochondrial clearance and inhibition of downstream apoptosis. PARK2 may be involved in the mitophagy process.

Topics: Adenine; Animals; Apoptosis; Autophagy; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Brain Ischemia; Cytochromes c; Cytoprotection; Glucose; Hypoxia; Male; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Mitochondria; Mitophagy; Neurons; Quinazolinones; Rats; Reperfusion Injury; Ubiquitin-Protein Ligases

Mitochondrial division inhibitor (mdivi-1) is a derivative of quinazolinone that acts as a selective inhibitor of a mitochondrial fission protein Drp1. A previous study demonstrated that as a selective inhibitor of Drp1, mdivi-1 has a protective effect in an experimental model of heart ischemia/reperfusion injury. In this study, we investigated the protective effects of mdivi-1 on cerebral ischemia/reperfusion injury in a middle cerebral artery occlusion mouse model. We found that mdivi-1 (1.2mg/kg) significantly reduced cerebral damage induced by ischemia/reperfusion. This neuroprotective effect was dose-dependent. Mdivi-1 treatment blocked apoptotic cell death in cerebral ischemia/reperfusion injury, and significantly decreased the expression of Drp1 and Cytochrome C. These results suggest that mdivi-1 exerts neuroprotective effects against nerve injury after cerebral ischemia/reperfusion, and the underlying mechanism may be through the prevention of Cytochrome C release and suppression of the mitochondrial apoptosis pathway.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Cytochromes c; Dynamins; Infarction, Middle Cerebral Artery; Male; Neurons; Neuroprotective Agents; Quinazolinones; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger