Search

Brain injury is the most common cause of death for patients resuscitated from cardiac arrest. Magnesium is an attractive neuroprotective compound which protects neurons from ischemic injury by reducing neuronal calcium overload via NMDA receptor modulation and preventing calcium-induced mitochondrial permeability transition. Intramuscular (IM) delivery of MgSO(4) during CPR has the potential to target these mechanisms within an early therapeutic window. We hypothesize that IM MgSO(4) administrated during CPR could achieve therapeutic serum magnesium levels within 15 min after ROSC and improve neurologic outcomes in a rat model of asphyxial cardiac arrest. Male Long Evans rats were subjected to 8-min asphyxial cardiac arrest and block randomized to receive placebo, 107 mg/kg, 215 mg/kg, or 430 mg/kg MgSO(4) IM at the onset of CPR. Serum magnesium concentrations increased rapidly with IM delivery during CPR, achieving twofold to fourfold increase by 15 min after ROSC in all magnesium dose groups. Rats subjected to cardiac arrest or sham surgery were block randomized to treatment groups for assessment of neurological outcomes. We found that IM MgSO(4) during CPR had no effect on ROSC rate (p > 0.05). IM MgSO(4) treatment had no statistically significant effect on 10-day survival with good neurologic function or hippocampal CA1 pyramidal neuron survival compared to placebo treatment. In conclusion, a single dose IM MgSO(4) during CPR achieves up to fourfold baseline serum magnesium levels within 15 min after ROSC; however, this treatment strategy did not improve survival, recovery of neurologic function, or neuron survival. Future studies with repeated dosing or in combination with hypothermic targeted temperature management may be indicated.

Mitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.

The mitochondrial network continually undergoes events of fission and fusion. Under physiologic conditions, the network is in equilibrium and is characterized by the presence of both elongated and punctate mitochondria. However, this balanced, homeostatic mitochondrial profile can change morphologic distribution in response to various stressors. Therefore, it is imperative to develop a method that robustly measures mitochondrial morphology with high accuracy. Here, we developed a semi-automated image analysis pipeline for the quantitation of mitochondrial morphology for both in vitro and in vivo applications. The image analysis pipeline was generated and validated utilizing images of primary cortical neurons from transgenic mice, allowing genetic ablation of key components of mitochondrial dynamics. This analysis pipeline was further extended to evaluate mitochondrial morphology in vivo through immunolabeling of brain sections as well as serial block-face scanning electron microscopy. These data demonstrate a highly specific and sensitive method that accurately classifies distinct physiological and pathological mitochondrial morphologies. Furthermore, this workflow employs the use of readily available, free open-source software designed for high throughput image processing, segmentation, and analysis that is customizable to various biological models.

Background: Considerable attention has focused on the concept that disruption of mitochondrial structure/function is a determinant of cell death in cardiomyocytes subjected to ischemia-reperfusion (IR). However, the details of this relationship, and the precise mitochondrial event(s) that precipitate lethal IR injury, remain unresolved. Aim: Emerging evidence has revealed that cardiomyocytes subjected to IR display: i) degradation of optic atrophy protein-1 (OPA1), the inner mitochondrial membrane protein responsible for maintaining cristae junction integrity, followed by ii) inappropriate release of OPA1 into the cytosol. Accordingly, our goal was to establish whether degradation of OPA1 plays a causal, mechanistic role in determining cardiomyocyte fate. Methods and Results: In Protocol 1 , HL-1 cardiomyocytes underwent 2.5 hrs of simulated ischemia. This was preceded by either a classic intervention known to attenuate IR injury (ischemic preconditioning: IPC) or a matched control period. Cell viability was quantified at 24 hrs post-R, and release of OPA1 into the cytosol was measured at 30 min post-R. In Controls, IR resulted in ~50% cell death and a >15-fold increase in OPA1 in the cytosol - effects that were both attenuated by IPC (Figure: top). In Protocol 2 : to discern whether these data reflect an association between OPA1 degradation and cell death or cause-and-effect , HL-1 cells were transfected with either siRNA targeting OPA1 (resulting in near-total knockdown of OPA1 expression: data not shown) or scrambled siRNA. Cardiomyocytes then underwent IPC/no intervention and IR as in Protocol 1. If OPA1 disruption contributes to IR injury, we reasoned that OPA1 knockdown would exacerbate cell death in Controls and attenuate IPC-mediated protection. However, OPA1 knockdown had no effect on cell death in either cohort (Figure: bottom). Conclusion: Disruption of OPA1 and loss of cristae junction integrity does not play a causal role in lethal IR injury.

Introduction: Brain injury is the most common cause of death for patients resuscitated from cardiac arrest. Magnesium is an attractive neuroprotective therapy that has the potential to reduce neuronal calcium overload via NMDA receptor modulation and prevent mitochondrial permeability transition. Intramuscular (IM) delivery of MgSO4 during CPR has the potential to target these mechanisms within their presumed therapeutic window. Hypothesis: IM MgSO4 administrated during CPR will achieve therapeutic serum magnesium levels, which is ≥ 2 times of baseline, within 15 minutes after ROSC and improve neurologic outcomes. Methods: Male Long Evans rats were subjected to 8-minute asphyxial cardiac arrest. For the dose-finding study (n = 4/group), rats were block randomized to receive placebo, 107 mg/kg, 215 mg/kg, or 430 mg/kg MgSO4 IM at the onset of CPR. Serial blood samples were collected at baseline, 15 min, 30 minutes, 1 hour and 2 hours after return of spontaneous circulation (ROSC) and analyzed for serum magnesium concentration. For the long-term outcome study (n = 9/group), rats subjected to cardiac arrest were blindly block randomized to the same treatment groups with the addition of a sham-operated group. Post-cardiac arrest care included maintenance of normothermia (36.7 °C - 37.3 °C) for 72 hours. Serial blood samples were collected at baseline, 15 min and 1 hour after ROSC and neurologic function score (NFS) was performed daily for 10 days. Good neurologic function was predefined as NFS ≥ 450. Results: IM MgSO4 during CPR had no effect on ROSC rate (p > 0.05). The serum magnesium concentrations at 15 min after ROSC of 107 mg/kg group, 215 mg/kg group and 430 mg/kg group were 3.9 ± 0.9, 6.2 ± 1.8, and 7.6 ± 1.8 mg/dl, which were 2.0 fold, 3.1 fold and 4.2 fold of baseline respectively. IM MgSO4 had no statistically significant effect on 10-day survival or 10-day survival with good neurologic function. Conclusion: Single dose IM MgSO4 during CPR achieves up to 4-fold baseline magnesium levels within 15 min after ROSC in a rat model of asphyxia cardiac arrest. This treatment strategy did not improve survival or recovery of neurologic function. Future studies with repeated dosing or in combination with hypothermic targeted temperature management may be indicated.

Aim: High-dose valproic acid (VPA) improves the survival and neurologic outcomes after asphyxial cardiac arrest (CA) in rats. We characterized the pharmacokinetics, pharmacodynamics, and safety of high-dose VPA in a swine CA model to advance clinical translation. Methods: After 8 ​min of untreated ventricular fibrillation CA, 20 male Yorkshire swine were resuscitated until return of spontaneous circulation (ROSC). They were block randomized to receive placebo, 75 ​mg/kg, 150 ​mg/kg, or 300 ​mg/kg VPA as 90-min intravenous infusion (n ​= ​5/group) beginning at ROSC. Animals were monitored for 2 additional hours then euthanized. Experimental operators were blinded to treatments. Results: The mean(SD) total CA duration was 14.8(1.2) minutes. 300 ​mg/kg VPA animals required more adrenaline to maintain mean arterial pressure ≥80 ​mmHg and had worse lactic acidosis. There was a strong linear correlation between plasma free VPA Cmax and brain total VPA (r2 ​= ​0.9494; p ​

Introduction: Post-cardiac arrest brain injury is a major cause of mortality and morbidity. Insulin has well-established CNS neuroprotecive properties mediated by the AKT survival signaling pathway, and known transnasal transport mechanisms make it possible to rapidly achieve therapeutic brain insulin levels after intranasal delivery. Hypothesis: We hypothesize that high-dose intranasal insulin (HD-IN-I) administered during CPR, could improve neurologic outcomes in a rat model of asphyxial cardiac arrest. Methods: Male Long Evans rats were subjected to 8-minute asphyxial cardiac arrest then block randomized to HD-IN-I (1.92 U/g brain wet weight) or placebo (PL) given at the onset of CPR by an experimental operator blinded to treatment. Sham operated rats were used as uninjured controls. For the long-term outcome study (n = 14/group), rats with return of spontaneous circulation (ROSC) were maintained at 37.0 ±0.5 °C for 72 hours and neurologic function was assessed on days 7 - 10. For the mechanistic study (n = 6/group), rats with ROSC were euthanized 30 minutes after ROSC and regional brain homogenates were analyzed by western blot for AKT phosporylation. Results: HD-IN-I during CPR had no statistically significant effect on ROSC rate (93% (13/14) HD-IN-I vs. 86% (12/14) PL, p > 0.05) or 10-day survival (71% (10/14) HD-INI vs. 43% (6/14) PL, p > 0.05). HD-IN-I had no significant impact on serum glucose concentrations. At 10 days post-ROSC, rats in the HD-IN-I group had significantly improved performance on rotarod (latency to fall 87±27 sec HD-IN-I vs. 41±18 sec PL, p < 0.05), Barnes maze (latency to escape box 27±15 sec HD-IN-I vs. 298±5 sec PL, p < 0.05), and passive avoidance (latency to re-enter shock chamber 300±0 sec HD-IN-I vs. 142±46 sec PL, p < 0.05) testing. Hippocampal phosph-AKT/total AKT ratio increased 2-fold in the placebo group and 5.7-fold in the HD-IN-I group relative to shams (p < 0.05). Conclusions: HD-IN-I administered during CPR causes rapid activation of brain AKT survival signaling and improves recovery of neurologic function in a rat cardiac arrest model. Additional studies are warranted to determine dose optimization, therapeutic window and effectiveness in large animal models to advance this novel therapy toward clinical trials.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

The interaction of light with biological tissue has been successfully utilized for multiple therapeutic purposes. Previous studies have suggested that near infrared light (NIR) enhances the activity of mitochondria by increasing cytochrome c oxidase (COX) activity, which we confirmed for 810 nm NIR. In contrast, scanning the NIR spectrum between 700 nm and 1000 nm revealed two NIR wavelengths (750 nm and 950 nm) that reduced the activity of isolated COX. COX-inhibitory wavelengths reduced mitochondrial respiration, reduced the mitochondrial membrane potential (ΔΨm), attenuated mitochondrial superoxide production, and attenuated neuronal death following oxygen glucose deprivation, whereas NIR that activates COX provided no benefit. We evaluated COX-inhibitory NIR as a potential therapy for cerebral reperfusion injury using a rat model of global brain ischemia. Untreated animals demonstrated an 86% loss of neurons in the CA1 hippocampus post-reperfusion whereas inhibitory NIR groups were robustly protected, with neuronal loss ranging from 11% to 35%. Moreover, neurologic function, assessed by radial arm maze performance, was preserved at control levels in rats treated with a combination of both COX-inhibitory NIR wavelengths. Taken together, our data suggest that COX-inhibitory NIR may be a viable non-pharmacologic and noninvasive therapy for the treatment of cerebral reperfusion injury.

Cellular mechanisms involved in multiple neurodegenerative diseases converge on mitochondria to induce overproduction of reactive oxygen species, damage to mitochondria, and subsequent cytochrome c release. Little is currently known regarding the contribution mitochondrial dynamics play in cytochrome c release following oxidative stress in neurodegenerative disease. Here we induced oxidative stress in the HT22 cell line with glutamate and investigated key mediators of mitochondrial dynamics to determine the role this process may play in oxidative stress induced neuronal death. We report that glutamate treatment in HT22 cells induces increase in reactive oxygen species (ROS), release of the mitochondrial fusion protein Opa1 into the cytosol, with concomitant release of cytochrome c. Furthermore, following the glutamate treatment alterations in cell signaling coincide with mitochondrial fragmentation which culminates in significant cell death in HT22 cells. Finally, we report that treatment with the antioxidant tocopherol attenuates glutamate induced-ROS increase, release of mitochondrial Opa1 and cytochrome c, and prevents cell death.

SUMOylation is an essential posttranslational modification and regulates many cellular processes. Dysregulation of SUMOylation plays a critical role in metastasis, yet how its perturbation affects this lethal process of cancer is not well understood. We found that SUMO-2/3 modification is greatly up-regulated in metastatic breast cancer cells compared with nonmetastatic control cells. To identify proteins differentially modified by SUMO-2/3 between metastatic and nonmetastatic cells, we established a method in which endogenous SUMO-2/3 conjugates are labeled by stable isotope labeling by amino acids in cell culture (SILAC), immunopurified by SUMO-2/3 monoclonal antibodies and epitope-peptide elution, and analyzed by quantitative mass spectrometry. We identified 66 putative SUMO-2/3-conjugated proteins, of which 15 proteins show a significant increase/decrease in SUMO-2/3 modification in metastatic cells. Targets with altered SUMOylation are involved in cell cycle, migration, inflammation, glycolysis, gene expression, and SUMO/ubiquitin pathways, suggesting that perturbations of SUMO-2/3 modification might contribute to metastasis by affecting these processes. Consistent with this, up-regulation of PML SUMO-2/3 modification corresponds to an increased number of PML nuclear bodies (PML-NBs) in metastatic cells, whereas up-regulation of global SUMO-2/3 modification promotes 3D cell migration. Our findings provide a foundation for further investigating the effects of SUMOylation on breast cancer progression and metastasis.

Gene looping juxtaposes the promoter and terminator regions of RNA polymerase II-transcribed genes in yeast and mammalian cells. Here we report an activator-dependent interaction of transcription initiation and termination factors during gene looping in budding yeast. Chromatin analysis revealed that MET16, INO1, and GAL1p-BUD3 are in a stable looped configuration during activated transcription. Looping was nearly abolished in the absence of transcription activators Met28, Ino2, and Gal4 of MET16, INO1, and GAL1p-BUD3 genes, respectively. The activator-independent increase in transcription was not accompanied by loop formation, thereby suggesting an essential role for activators in gene looping. The activators did not facilitate loop formation directly because they did not exhibit an interaction with the 3′ end of the genes. Instead, activators physically interacted with the general transcription factor TFIIB when the genes were activated and in a looped configuration. TFIIB cross-linked to both the promoter and the terminator regions during the transcriptionally activated state of a gene. The presence of TFIIB on the terminator was dependent on the Rna15 component of CF1 3′ end processing complex. Coimmunoprecipitation revealed a physical interaction of Rna15 with TFIIB. We propose that the activators facilitate gene looping through their interaction with TFIIB during transcriptional activation of genes.

Annulate lamellae are cytoplasmic organelles containing stacked sheets of membranes embedded with pore complexes. These cytoplasmic pore complexes at annulate lamellae are morphologically similar to nuclear pore complexes at the nuclear envelope. Although annulate lamellae has been observed in nearly all types of cells, their biological functions are still largely unknown. Here we show that SUMO1-modification of the Ran GTPase-activating protein RanGAP1 not only target RanGAP1 to its known sites at nuclear pore complexes but also to annulate lamellae pore complexes through interactions with the Ran-binding protein RanBP2 and the SUMO-conjugating enzyme Ubc9 in mammalian cells. Furthermore, upregulation of annulate lamellae, which decreases the number of nuclear pore complexes and concurrently increases that of annulate lamellae pore complexes, causes a redistribution of nuclear transport receptors including importin α/β and the exportin CRM1 from nuclear pore complexes to annulate lamellae pore complexes and also reduces the rates of nuclear import and export. Moreover, our results reveal that importin α/β-mediated import complexes initially accumulate at annulate lamellae pore complexes upon the activation of nuclear import and subsequently disassociate for nuclear import through nuclear pore complexes in cells with upregulation of annulate lamellae. Lastly, CRM1-mediated export complexes are concentrated at both nuclear pore complexes and annulate lamellae pore complexes when the disassembly of these export complexes is inhibited by transient expression of a Ran GTPase mutant arrested in its GTP-bound form, suggesting that RanGAP1/RanBP2-activated RanGTP hydrolysis at these pore complexes is required for the dissociation of the export complexes. Hence, our findings provide a foundation for further investigation of how upregulation of annulate lamellae decreases the rates of nuclear transport and also for elucidation of the biological significance of the interaction between annulate lamellae pore complexes and nuclear transport complexes in mammalian cells.

The Ran GTPase activating protein RanGAP1 plays an essential role in nuclear transport by stimulating RanGTP hydrolysis in the cytoplasmic compartment. In mammalian cells, unmodified RanGAP1 is predominantly cytoplasmic, whereas modification by small ubiquitin-related modifier protein (SUMO) targets RanGAP1 to the cytoplasmic filaments of nuclear pore complex (NPC). Although RanGAP1 contains nine putative nuclear export signals and a nuclear localization signal, little is known if RanGAP1 shuttles between the nuclear and cytoplasmic compartments and how its primary localization in the cytoplasm and at the NPC is regulated. Here we show that inhibition of CRM1-mediated nuclear export using RNAi-knockdown of CRM1 and inactivation of CRM1 by leptomycin B (LMB) results in nuclear accumulation of RanGAP1. LMB treatment induced a more robust redistribution of RanGAP1 from the cytoplasm to the nucleoplasm compared to CRM1 RNAi and also uniquely triggered a decrease or loss of RanGAP1 localization at the NPC, suggesting that LMB treatment is more effective in inhibiting CRM1-mediated nuclear export of RanGAP1. Our time-course analysis of LMB treatment reveals that the NPC-associated RanGAP1 is much more slowly redistributed to the nucleoplasm than the cytoplasmic RanGAP1. Furthermore, LMB-induced nuclear accumulation of RanGAP1 is positively correlated with an increase in levels of SUMO-modified RanGAP1, suggesting that SUMOylation of RanGAP1 may mainly take place in the nucleoplasm. Lastly, we demonstrate that the nuclear localization signal at the C-terminus of RanGAP1 is required for its nuclear accumulation in cells treated with LMB. Taken together, our results elucidate that RanGAP1 is actively transported between the nuclear and cytoplasmic compartments, and that the cytoplasmic and NPC localization of RanGAP1 is dependent on CRM1-mediated nuclear export.

Gene looping, defined as the interaction of the promoter and the terminator regions of a gene during transcription, requires transcription factor IIB (TFIIB). We have earlier demonstrated association of TFIIB with the distal ends of a gene in an activator-dependent manner (El Kaderi, B., Medler, S., Raghunayakula, S., and Ansari, A. (2009) J. Biol. Chem. 284, 25015–25025). The presence of TFIIB at the 3′ end of a gene required its interaction with cleavage factor 1 (CF1) 3′ end processing complex subunit Rna15. Here, employing affinity chromatography and glycerol gradient centrifugation, we show that TFIIB associates with poly(A) polymerase and the entire CF1 complex in yeast cells. The factors required for general transcription such as TATA-binding protein, RNA polymerase II, and TFIIH are not a component of the TFIIB complex. This holo-TFIIB complex was resistant to MNase digestion. The complex was observed only in the looping-competent strains, but not in the looping-defective sua7-1 strain. The requirement of Rna15 in gene looping has been demonstrated earlier. Here we provide evidence that poly(A) polymerase (Pap1) as well as CF1 subunits Rna14 and Pcf11 are also required for loop formation of MET16 and INO1 genes. Accordingly, cross-linking of TFIIB to the 3′ end of genes was abolished in the mutants of Pap1, Rna14, and Pcf11. We further show that in sua7-1 cells, where holo-TFIIB complex is not formed, the kinetics of activated transcription is altered. These results suggest that a complex of TFIIB, CF1 subunits, and Pap1 exists in yeast cells. Furthermore, TFIIB interaction with the CF1 complex and Pap1 is crucial for gene looping and transcriptional regulation.

Brain ischemia/reperfusion injury results in death of vulnerable neurons and extensive brain damage. It is well known that mitochondrial release of cytochrome c (cyto c) is a hallmark of neuronal death, however the molecular events underlying this release are largely unknown. We tested the hypothesis that cyto c release is regulated by breakdown of the cristae architecture maintenance protein, optic atrophy 1 (OPA1), located in the inner mitochondrial membrane. We simulated ischemia/reperfusion in isolated primary rat neurons and interrogated OPA1 release from the mitochondria, OPA1 oligomeric breakdown, and concomitant dysfunction of mitochondrial dynamic state. We found that ischemia/reperfusion induces cyto c release and cell death that corresponds to multiple changes in OPA1, including: (i) translocation of the mitochondrial fusion protein OPA1 from the mitochondria to the cytosol, (ii) increase in the short isoform of OPA1, suggestive of proteolytic processing, (iii) breakdown of OPA1 oligomers in the mitochondria, and (iv) increased mitochondrial fission. Thus, we present novel evidence of a connection between release of cyto c from mitochondria and disruption of the mitochondrial fusion.