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Sestrin2 Silencing Exacerbates Cerebral Ischemia/Reperfusion Injury by Decreasing Mitochondrial Biogenesis through the AMPK/PGC-1? Pathway in Rats.

ABSTRACT: Sestrin2 (Sesn2) exerts neuroprotective properties in some neurodegenerative diseases. However, the role of Sesn2 in stroke is unclear. The AMP-activated protein kinase/peroxisome proliferator-activated receptor ? coactivator-1? (AMPK/PGC-1?) pathway plays an important role in regulating mitochondrial biogenesis, which helps prevent cerebral ischemia/reperfusion (I/R) injury. Here, we aimed to determine whether Sesn2 alleviated I/R damage by regulating mitochondrial biogenesis through the AMPK/PGC-1? signaling pathway. To be able to test this, Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) for 1?h with Sesn2 silencing. At 24?h after reperfusion, we found that neurological deficits were exacerbated, infarct volume was enlarged, and oxidative stress and neuronal damage were greater in the Sesn2 siRNA group than in the MCAO group. To explore protective mechanisms, an AMPK activator was used. Expression levels of Sesn2, p-AMPK, PGC-1?, NRF-1, TFAM, SOD2, and UCP2 were significantly increased following cerebral I/R. However, upregulation of these proteins was prevented by Sesn2 small interfering RNA (siRNA). In contrast, activation of AMPK with 5'-aminoimidazole-4-carboxamide riboside weakened the effects of Sesn2 siRNA. These results suggest that Sesn2 silencing may suppress mitochondrial biogenesis, reduce mitochondrial biological activity, and finally aggravate cerebral I/R injury through inhibiting the AMPK/PGC-1? pathway.

SUBMITTER: Li L

PROVIDER: S-EPMC4958997 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Sestrin2 Silencing Exacerbates Cerebral Ischemia/Reperfusion Injury by Decreasing Mitochondrial Biogenesis through the AMPK/PGC-1α Pathway in Rats.

Li Lingyu L Xiao Lina L Hou Yanghao Y He Qi Q Zhu Jin J Li Yixin Y Wu Jingxian J Zhao Jing J Yu Shanshan S Zhao Yong Y

Scientific reports 20160725

Sestrin2 (Sesn2) exerts neuroprotective properties in some neurodegenerative diseases. However, the role of Sesn2 in stroke is unclear. The AMP-activated protein kinase/peroxisome proliferator-activated receptor γ coactivator-1α (AMPK/PGC-1α) pathway plays an important role in regulating mitochondrial biogenesis, which helps prevent cerebral ischemia/reperfusion (I/R) injury. Here, we aimed to determine whether Sesn2 alleviated I/R damage by regulating mitochondrial biogenesis through the AMPK/P ...[more]

PMID: 27453548

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Project description:Background and purposeReperfusion after transient cerebral ischemia causes severe damage to mitochondria; however, little is known regarding the continuous change in mitochondrial biogenesis during reperfusion. Mitochondrial biogenesis causes an increase in the individual mitochondrial mass of neurons and maintains their aerobic set-point in the face of declining function. The aim of this study was to examine mitochondrial biogenesis in the cortex during reperfusion following focal cerebral ischemia.MethodsMale Wistar rats were subjected to transient focal cerebral ischemia. The relative amount of cortical mitochondrial DNA was analyzed using quantitative real-time PCR at 0 h, 24 h, 72 h, and 7 d after reperfusion. Three critical transcriptional regulators of mitochondrial biogenesis were measured by semi-quantitative reverse-transcription PCR. The protein expression of cytochrome C oxidase subunits I and IV was detected by Western blotting.ResultsEvidence of increased mitochondrial biogenesis was observed after reperfusion. The cortical mitochondrial DNA content increased after 24 h, peaked after 72 h, and maintained a high level for 7 d. The cortical expression of three critical genes for the transcriptional regulation of mitochondrial biogenesis, namely, peroxisome proliferator-activated receptor coactivator-1α, nuclear respiratory factor-1, and mitochondrial transcription factor A, also increased at 24 h and 72 h. The expression of peroxisome proliferator-activated receptor coactivator-1α returned to the baseline level at 7 d, but two other factors maintained higher levels compared with the controls. Moreover, the expression of cytochrome C oxidase subunits I and IV was increased in the cortex.ConclusionsThese results indicate that reperfusion increased mitochondrial biogenesis following focal cerebral ischemia, and this tendency was exacerbated as the reperfusion time was extended. Reperfusion-induced mitochondrial biogenesis was mediated through up-regulation of critical transcriptional regulators of mitochondrial biogenesis.

Project description:RationaleAMPK (AMP-activated protein kinase) is a heterotrimeric protein that plays an important role in energy homeostasis and cardioprotection. Two isoforms of each subunit are expressed in the heart, but the isoform-specific function of AMPK remains unclear.ObjectiveWe sought to determine the role of γ2-AMPK in cardiac stress response using bioengineered cell lines and mouse models containing either isoform of the γ-subunit in the heart.Methods and resultsWe found that γ2 but not γ1 or γ3 subunit translocated into nucleus on AMPK activation. Nuclear accumulation of AMPK complexes containing γ2-subunit phosphorylated and inactivated RNA Pol I (polymerase I)-associated transcription factor TIF-IA at Ser-635, precluding the assembly of transcription initiation complexes for rDNA. The subsequent downregulation of pre-rRNA level led to attenuated endoplasmic reticulum (ER) stress and cell death. Deleting γ2-AMPK led to increases in pre-rRNA level, ER stress markers, and cell death during glucose deprivation, which could be rescued by inhibition of rRNA processing or ER stress. To study the function of γ2-AMPK in the heart, we generated a mouse model with cardiac-specific deletion of γ2-AMPK (cardiac knockout [cKO]). Although the total AMPK activity was unaltered in cKO hearts because of upregulation of γ1-AMPK, the lack of γ2-AMPK sensitizes the heart to myocardial ischemia/reperfusion injury. The cKO failed to suppress pre-rRNA level during ischemia/reperfusion and showed a greater infarct size. Conversely, cardiac-specific overexpression of γ2-AMPK decreased ribosome biosynthesis and ER stress during ischemia/reperfusion insult, and the infarct size was reduced.ConclusionsThe γ2-AMPK translocates into the nucleus to suppress pre-rRNA transcription and ribosome biosynthesis during stress, thus ameliorating ER stress and cell death. Increased γ2-AMPK activity is required to protect against ischemia/reperfusion injury. Our study reveals an isoform-specific function of γ2-AMPK in modulating ribosome biosynthesis, cell survival, and cardioprotection.

Project description:Ischemic stroke is an acute and severe neurological disease with high mortality and disability rates worldwide. Polymerase I and transcript release factor (PTRF) plays a pivotal role in regulating cellular senescence, glucose intolerance, lipid metabolism, and mitochondrial bioenergetics, but its mechanism, characteristics, and functions in neuronal cells following the cerebral ischemia-reperfusion (I/R) injury remain to be determined. Methods: Transcription factor motif analysis, chromatin immunoprecipitation (ChIP), luciferase and co-Immunoprecipitation (co-IP) assays were performed to investigate the mechanisms of PTRF in neuronal cells after I/R injury. Lentiviral-sgRNA against PTRF gene was introduced to HT22 cells, and adeno-associated virus (AAV) encoding a human synapsin (hSyn) promoter-driven construct was transduced a short hairpin RNA (shRNA) against PTRF mRNA in primary neuronal cells and the cortex of the cerebral I/R mice for investigating the role of PTRF in neuronal damage and PLA2G4A change induced by the cerebral I/R injury. Results: Here, we reported that neuronal PTRF was remarkably increased in the cerebral penumbra after I/R injury, and HIF-1α and STAT3 regulated the I/R-dependent expression of PTRF via binding to its promoter in neuronal cells. Moreover, overexpression of neuronal PTRF enhanced the activity and stability of PLA2G4A by decreasing its proteasome-mediated degradation pathway. Subsequently, PTRF promoted reprogramming of lipid metabolism and altered mitochondrial bioenergetics, which could lead to oxidative damage, involving autophagy, lipid peroxidation, and ferroptosis via PLA2G4A in neuronal cells. Furthermore, inhibition of neuronal PTRF/PLA2G4A-axis markedly reduced the neurological deficits, cerebral infarct volumes, and mortality rates in the mice following cerebral I/R injury. Conclusion: Our results thus identify that the STAT3/HIF-1α/PTRF-axis in neurons, aggravating cerebral I/R injury by regulating the activity and stability of PLA2G4A, might be a novel therapeutic target for ischemic stroke.

Project description:BackgroundTo study the protective effects of delayed remote ischemic preconditioning (RIPC) against spinal cord ischemia-reperfusion injury (SCIRI) in mice and determine whether SIRT3 is involved in this protection and portrayed its upstream regulatory mechanisms.MethodsIn vivo, WT or SIRT3 global knockout (KO) mice were exposed to right upper and lower limbs RIPC or sham ischemia. After 24 h, the abdominal aorta was clamped for 20 min, then re-perfused for 3 days. The motor function of mice, number of Nissl bodies, apoptotic rate of neurons, and related indexes of oxidative stress in the spinal cord were measured to evaluate for neuroprotective effects. The expression and correlation of SIRT3 and NMDAR were detected by WB and immunofluorescence. In vitro, primary neurons were exacted and OGD/R was performed to simulate SCIRI in vivo. Neuronal damage was assessed by observing neuron morphology, detecting LDH release ratio, and flow cytometry to analyze the apoptosis. MnSOD and CAT enzyme activities, GSH and ROS level were also measured to assess neuronal antioxidant capacity. NMDAR-AMPK-PGC-1α signaling was detected by WB to portray upstream regulatory mechanisms of RIPC regulating SIRT3.ResultsCompared to the SCIRI mice without RIPC, mice with RIPC displayed improved motor function recovery, a reduced neuronal loss, and enhanced antioxidant capacity. To the contrary, the KO mice did not exhibit any effect of RIPC-induced neuroprotection. Similar results were observed in vitro. Further analyses with spinal cord tissues or primary neurons detected enhanced MnSOD and CAT activities, as well as increased GSH level but decreased MDA or ROS production in the RIPC + I/R mice or NMDA + OGD/R neurons. However, these changes were completely inhibited by the absence of SIRT3. Additionally, NMDAR-AMPK-PGC-1α signaling was activated to upregulate SIRT3 levels, which is essential for RIPC-mediated neuroprotection.ConclusionsRIPC enhances spinal cord ischemia tolerance in a SIRT3-dependent manner, and its induced elevated SIRT3 levels are mediated by the NMDAR-AMPK-PGC-1α signaling pathway. Combined therapy targeting SIRT3 is a promising direction for treating SCIRI.

Dataset Information

Sestrin2 Silencing Exacerbates Cerebral Ischemia/Reperfusion Injury by Decreasing Mitochondrial Biogenesis through the AMPK/PGC-1? Pathway in Rats.

Publications

Sestrin2 Silencing Exacerbates Cerebral Ischemia/Reperfusion Injury by Decreasing Mitochondrial Biogenesis through the AMPK/PGC-1α Pathway in Rats.

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