Rapamycin ameliorates brain damage and maintains mitochondrial dynamic balance in diabetic rats subjected to middle cerebral artery occlusion.

To investigate the effect of rapamycin on mitochondrial dynamic balance in diabetic rats subjected to cerebral ischemia-reperfusion injury. Male Sprague Dawley (SD) rats (n = 78) were treated with high fat diet combined with streptozotocin injection to construct diabetic model in rats. Transient middle cerebral artery occlusion (MCAO) of 2 hours was induced and the brains were harvested after 1 and 3 days of reperfusion. Rapamycin was injected intraperitoneally for 3 days prior to and immediately after operation, once a day. The neurological function was assessed, infarct volumes were measured and HE staining as well as immunohistochemistry were performed. The protein of hippocampus was extracted and Western blotting were performed to detect the levels of mTOR, mitochondrial dynamin related proteins (DRP1, p-DRP1, OPA1), SIRT3, and Nix/BNIP3L. Diabetic hyperglycemia worsened the neurological function performance (p < 0.01), enlarged infarct size (p < 0.01) and increased ischemic neuronal cell death (p < 0.01). The increased damage was associated with elevations of p-mTOR, p-S6, and p-DRP1; and suppressions of SIRT3 and Nix/BNIP3L. Rapamycin ameliorated diabetes-enhanced ischemic brain damage and reversed the biomarker alterations caused by diabetes. High glucose activated mTOR pathway and caused mitochondrial dynamics toward fission. The protective effect of rapamycin against diabetes-enhanced ischemic brain damage was associated with inhibiting mTOR pathway, redressing mitochondrial dynamic imbalance, and elevating SIRT3 and Nix/BNIP3L expression.

Neuroprotection by B355252 against Glutamate-Induced Cytotoxicity in Murine Hippocampal HT-22 Cells Is Associated with Activation of ERK3 Signaling Pathway.

Glutamate differentially affects the levels extracellular signal-regulated kinase (ERK)1/2 and ERK3 and the protective effect of B355252, an aryl thiophene compound, 4-chloro-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide, is associated with suppression of ERK1/2. The objectives of this study were to further investigate the impact of B355252 on ERK3 and its downstream signaling pathways affected by glutamate exposure in the mouse hippocampal HT-22 neuronal cells. Murine hippocampal HT22 cells were incubated with glutamate and treated with B355252. Cell viability was assessed, protein levels of pERK3, ERK3, mitogen-activated protein kinase-activated protein kinase-5 (MAPKAPK-5), steroid receptor coactivator 3 (SRC-3), p-S6 and S6 were measured using Western blotting, and immunoreactivity of p-S6 was determined by immunocytochemistry. The results reveal that glutamate markedly diminished the protein levels of p-ERK3 and its downstream targets MK-5 and SRC-3 and increased p-S6, an indicator for mechanistic target of rapamycin (mTOR) activation. Conversely, treatment with B355252 protected the cells from glutamate-induced damage and prevented the glutamate-caused declines of p-ERK3, MK-5 and SRC-3 and increase of p-S6. Our study demonstrates that one of the mechanisms that glutamate mediates its cytotoxicity is through suppression of ERK3 and that B355252 rescues the cells from glutamate toxicity by reverting ERK3 level.

The Involvement of Mitochondrial Biogenesis in Selenium Reduced Hyperglycemia-Aggravated Cerebral Ischemia Injury.

Selenium has been shown to possess antioxidant and neuroprotective effects by modulating mitochondrial function and activating mitochondrial biogenesis. Our previous study has also suggested that selenium protected neurons against glutamate toxicity and hyperglycemia-induced damage by regulating mitochondrial fission and fusion. However, it is still not known whether the mitochondrial biogenesis is involved in selenium alleviating hyperglycemia-aggravated cerebral ischemia reperfusion (I/R) injury. The object of this study is to define whether selenium protects neurons against hyperglycemia-aggravated cerebral I/R injury by promoting mitochondrial biogenesis. In vitro oxygen deprivation plus high glucose model decreased cell viability, enhanced reactive oxygen species production, and meanwhile stimulated mitochondrial biogenesis signaling. Pretreated with selenium significantly decreased cell death and further activated the mitochondrial biogenesis signaling. In vivo 30 min of middle cerebral artery occlusion in the rats under hyperglycemic condition enhanced neurological deficits, enlarged infarct volume, exacerbated neuronal damage and oxidative stress compared with normoglycemic ischemic rats after 24 h reperfusion. Consistent to the in vitro results, selenium treatment alleviated ischemic damage in hyperglycemic ischemic animals. Furthermore, selenium reduced the structural changes of mitochondria caused by hyperglycemic ischemia and further promoted the mitochondrial biogenesis signaling. Selenium activates mitochondrial biogenesis signaling, protects mitochondrial structure integrity and ameliorates cerebral I/R injury in hyperglycemic rats.

Polycomb-like 2 regulates PRC2 components to affect proliferation in glioma cells.

INTRODUCTION: The Polycomb group (PcG) is an important family of transcriptional regulators that controls growth and tumorigenesis. The PcG mainly consists of two complexes, PRC1 and Polycomb Repressive Complex 2 (PRC2). Polycomb-like 2 (PCL2) is known to interact with the PRC2 protein. The role of PCL2 in the development and progression of glioma is unclear. METHODS: We use The Cancer Genome Atlas (TCGA) database to detect the expression of PCL2 in various tumors. 117 cases of clinical glioma (WHOI-IV) were collected, and PCL2 expression and localization were detected by immunohistochemical staining. Glioma cells U87/U251 were infected with overexpressed and interfered PCL2. CCK8 assay, colony formation assay, EdU method, cell cycle and apoptosis were used to detect cell proliferation and apoptosis. Western blot was used to detect the expression of PRC2-related core proteins. After DZNeP intervention, PRC2 protein expression was again measured to discuss the mechanism of PCL2 action. RESULTS: TCGA database results and immunohistochemical staining results suggest that PCL2 is highly expressed in gliomas. We found that the PCL2 gene promoted tumor cell proliferation, enhanced the colony formation ability, and increased S phase in the cell cycle. The overexpression of PCL2 upregulated the expression levels of EZH2 and EED (two core members of PRC2), decreased the expression of SUZ12, increased the level of H3K27 trimethylation (H3K27me3), H3K4 dimethylation (H3K4me2), and decreased H3K9 dimethylation (H3K9me2). The result after interfering with PCL2 was the opposite. CONCLUSIONS: As an important accessory protein of PRC2, PCL2 can not only change the expression of PRC2 components, but also affect the expression level of Histone methylation. Therefore, PCL2 may be an important hub for regulating the synergy among PRC2 members. This study revealed PCL2 as a new target for tumor research and open up a new avenue for future research in glioma.

Mitochondrial dysfunction contributes to Rapamycin-induced apoptosis of Human Glioblastoma Cells - A synergistic effect with Temozolomide.

Mammalian target of rapamycin (mTOR) is upregulated in a high percentage of glioblastomas. While a well-known mTOR inhibitor, rapamycin, has been shown to reduce glioblastoma survival, the role of mitochondria in achieving this therapeutic effect is less well known. Here, we examined mitochondrial dysfunction mechanisms that occur with the suppression of mTOR signaling. We found that, along with increased apoptosis, and a reduction in transformative potential, rapamycin treatment significantly affected mitochondrial health. Specifically, increased production of reactive oxygen species (ROS), depolarization of the mitochondrial membrane potential (MMP), and altered mitochondrial dynamics were observed. Furthermore, we verified the therapeutic potential of rapamycin-induced mitochondrial dysfunction through co-treatment with temzolomide (TMZ), the current standard of care for glioblastoma. Together these results demonstrate that the mitochondria remain a promising target for therapeutic intervention against human glioblastoma and that TMZ and rapamycin have a synergistic effect in suppressing glioblastoma viability, enhancing ROS production, and depolarizing MMP.

Deletion of mitochondrial uncoupling protein 2 exacerbates mitophagy and cell apoptosis after cerebral ischemia and reperfusion injury in mice.

Objective: Uncoupling protein 2 (UCP2) is a member of inner mitochondrial membrane proteins and deletion of UCP2 exacerbates brain damage after cerebral ischemia/reperfusion (I/R). Nevertheless, its functional role during cerebral I/R is not entirely understood. The objective of present study was to explore the influence of UCP2 deletion on mitochondrial autophagy (mitophagy) and mitochondria-mediated cell death pathway after cerebral I/R. Methods: UCP2-/- and wildtype (WT) mice were subjected to 60 min middle cerebral artery occlusion (MCAO) and allowed reperfusion for 24 hours. Infarct volume and histological outcomes were assessed, reactive oxygen species (ROS) and autophagy markers were measured, and mitochondrial ultrastructure was examined. Results: Deletion of UCP2 enlarged infarct volume, increased numbers of necrotic and TUNEL positive cells, and significantly increased pro-apoptotic protein levels in UCP2-/- mice compared with WT mice subjected to the same duration of I/R. Further, deletion of UCP2 increased ROS production, elevated LC3, Beclin1 and PINK1, while it suppressed p62 compared with respective WT ischemic controls. Electron microscopic study demonstrated the number of autophagosomes was higher in the UCP2-/- group, compared with the WT group. Conclusions: It is concluded that deletion of UCP2 exacerbates cerebral I/R injury via reinforcing mitophagy and cellular apoptosis in mice.

Suppression of NLRP3 Inflammasome, Pyroptosis, and Cell Death by NIM811 in Rotenone-Exposed Cells as an in vitro Model of Parkinson's Disease.

BACKGROUND: Parkinson's disease (PD) is characterized by the selective death of dopaminergic neurons in the substantia nigra. Recently, NLRP3 inflammasome and pyroptosis were found to be associated with PD. Cyclosporine A (CsA), an immunosuppressant, reduces neuronal death in PD. However, CsA could hardly pass through the blood-brain barrier (BBB) and high dose is associated with severe side effects and toxicity. N-methyl-4-isoleucine-cyclosporine (NIM811) is a CsA derivate that can pass through the BBB. However, little is known about its effect on PD. OBJECTIVE: The objectives of this study were to explore the mechanism of rotenone-induced cell damage and to examine the protective effects of NIM811 on the neurotoxicity of a Parkinson-like in vitro model induced by rotenone. METHODS: Murine hippocampal HT22 cells were cultured with the mitochondrial complex I inhibitor rotenone, a widely used pesticide that has been used for many years as a tool to induce a PD model in vitro and in vivo and proven to be reproducible. NIM811 was added to the culture media 3 h prior to the rotenone incubation. Cell viability was determined by resazurin assay, reactive oxygen species (ROS) production by dihydroethidine (DHE), and mitochondrial membrane potential by tetramethyl rhodamine methyl ester (TMRM). TUNEL and caspase-1 immunofluorescent double staining was used to detect pyroptosis. NLRP3, caspase-1, pro-caspase-1, GSDMD, and interleukin-18 (IL-18) were measured using Western blotting after 24 h of rotenone incubation. The reactivity of interleukin-1ß (IL-1ß) was determined by ELISA. RESULTS: Our results demonstrated that rotenone caused more than 40% of cell death, increased ROS production, and reduced mitochondrial membrane potential, while NIM811 reversed these alterations. Immunofluorescent double staining showed that rotenone increased the percentage of caspase-1 and TUNEL double-labelled cells, an indication of pyroptosis, after 24 h of incubation. The protein expression of NLRP3, caspase-1, pro-caspase-1, GSDMD, IL-18, and IL-1ß was significantly increased after 24 h of rotenone incubation. NIM811 suppressed rotenone-induced pyroptosis and downregulated the protein expression of NLRP3, caspase-1, pro-caspase-1, GSDMD, IL-1ß, and IL-18. CONCLUSION: These results provide evidence that rotenone activates the NLRP3 inflammomere and induces pyroptosis. NIM811 protects the cell from rotenone-induced damage and inhibits NLRP3 inflammasome and pyroptosis. NIM811 might serve as a potential therapeutic drug in the treatment of PD.

Prevention of post-ischemic seizure by rapamycin is associated with deactivation of mTOR and ERK1/2 pathways in hyperglycemic rats.

Pre-ischemic hyperglycemia increases the occurrence of post-ischemic seizures both in experimental and clinical settings. The underlying mechanisms are not fully delineated; however, activation of mammalian target of rapamycin (mTOR) has been shown to be engaged in the pathogenesis of epilepsy, in which seizures are a regular occurrence. Therefore, we wanted to explore specifically the capacity of an mTOR inhibitor, rapamycin, in preventing post-ischemic seizures in hyperglycemic rats and to explore the underlying molecular mechanisms. The results showed that none of the rats in the sham control, EG ischemic, or within 3 h of I/R in hyperglycemic ischemic groups experienced seizures. Generalized tonic-clonic seizures were observed in all 8/8 of hyperglycemic ischemic rats at 16 h of I/R. Treatment with rapamycin successfully blocked post-ischemic seizures in 7/8 hyperglycemic ischemic animals. Rapamycin also lessened the neuronal death extraordinarily in hyperglycemic ischemic animals as revealed by histopathological studies. Protein analysis revealed that transient ischemia resulted in increases in p-mTOR and p-S6, especially in the hippocampi of the hyperglycemic ischemic rats. Rapamycin treatment completely blocked mTOR activation. Furthermore, hyperglycemic ischemia induced a much prominent rise of p-ERK1/2 both in the cortex and the hippocampi compared with EG counterparts; whereas rapamycin suppressed it. We conclude that the development of post-ischemic seizures in the hyperglycemic animals may be associated with activations of mTOR and ERK1/2 pathways and that rapamycin treatment inhibited the post-ischemic seizures effectively by suppressing the mTOR and ERK1/2 signaling.

Coenzyme Q10 Protects Astrocytes from Ultraviolet B-Induced Damage Through Inhibition of ERK 1/2 Pathway Overexpression.

Overexpression of extracellular signal-regulated kinase ½ (ERK ½) signaling pathway leads to overproduction of reactive oxygen species (ROS) which induces oxidative stress. Coenzyme Q10 (CoQ10) scavenges ROS and protects cells against oxidative stress. The present study was designed to examine whether the protection of Coenzyme Q10 against oxidative damage in astrocytes is through regulating ERK 1/2 pathway. Ultraviolet B (UVB) irradiation was chosen as a tool to induce oxidative stress. Murine astrocytes were treated with 10 µg/ml and 25 µg/ml of CoQ10 for 24 h prior to UVB and maintained during UVB and 24 h post-UVB. Cell viability was evaluated by counting viable cells and MTT conversion assay. ROS production was measured using fluorescent probes. Levels of p-ERK 1/2, ERK 1/2, p-PKA, PKA were detected using immunocytochemistry and/or Western blotting. The results showed that UVB irradiation decreased the number of viable cells. This damaging effect was associated with accumulation of ROS and elevations of p-ERK 1/2 and p-PKA. Treatment with CoQ10 at 25 µg/ml significantly increased the number of viable cells and prevented the UVB-induced increases of ROS, p-ERK 1/2, and p-PKA. It is concluded that suppression of the PKA-ERK 1/2 signaling pathway may be one of the important mechanisms by which CoQ10 protects astrocytes from UVB-induced oxidative damage.

Rapamycin treatment increases hippocampal cell viability in an mTOR-independent manner during exposure to hypoxia mimetic, cobalt chloride.

BACKGROUND: Cobalt chloride (CoCl2) induces chemical hypoxia through activation of hypoxia-inducible factor-1 alpha (HIF-1α). Mammalian target of rapamycin (mTOR) is a multifaceted protein capable of regulating cell growth, angiogenesis, metabolism, proliferation, and survival. In this study, we tested the efficacy of a well-known mTOR inhibitor, rapamycin, in reducing oxidative damage and increasing cell viability in the mouse hippocampal cell line, HT22, during a CoCl2-simulated hypoxic insult. RESULTS: CoCl2 caused cell death in a dose-dependent manner and increased protein levels of cleaved caspase-9 and caspase-3. Rapamycin increased viability of HT22 cells exposed to CoCl2 and reduced activation of caspases-9 and -3. Cells exposed to CoCl2 displayed increased reactive oxygen species (ROS) production and hyperpolarization of the mitochondrial membrane, both of which rapamycin successfully blocked. mTOR protein itself, along with its downstream signaling target, phospho-S6 ribosomal protein (pS6), were significantly inhibited with CoCl2 and rapamycin addition did not significantly lower expression further. Rapamycin promoted protein expression of Beclin-1 and increased conversion of microtubule-associated protein light chain 3 (LC3)-I into LC3-II, suggesting an increase in autophagy. Pro-apoptotic protein, Bcl-2 associated × (Bax), exhibited a slight, but significant decrease with rapamycin treatment, while its anti-apoptotic counterpart, B cell lymphoma-2 (Bcl-2), was to a similar degree upregulated. Finally, the protein expression ratio of phosphorylated mitogen-activated protein kinase (pMAPK) to its unphosphorylated form (MAPK) was dramatically increased in rapamycin and CoCl2 co-treated cells. CONCLUSIONS: Our results indicate that rapamycin confers protection against CoCl2-simulated hypoxic insults to neuronal cells. This occurs, as suggested by our results, independent of mTOR modification, and rather through stabilization of the mitochondrial membrane with concomitant decreases in ROS production. Additionally, inhibition of caspase-9 and -3 activation and stimulation of protective autophagy reduces cell death, while a decrease in the Bax/Bcl-2 ratio and an increase in pMAPK promotes cell survival during CoCl2 exposure. Together these results demonstrate the therapeutic potential of rapamycin against hypoxic injury and highlight potential pathways mediating the protective effects of rapamycin treatment.

B355252, A Novel Small Molecule, Confers Neuroprotection Against Cobalt Chloride Toxicity In Mouse Hippocampal Cells Through Altering Mitochondrial Dynamics And Limiting Autophagy Induction.

Cerebral hypoxia as often occurs in cases of stroke, hemorrhage, or other traumatic brain injuries, is one of the leading causes of death worldwide and a main driver of disabilities in the elderly. Using a chemical mimetic of hypoxia, cobalt chloride (CoCl2), we tested the ability of a novel small molecule, 4-chloro-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide (B355252), to alleviate CoCl2-induced damage in mouse hippocampal HT22 cells. A dose-dependent decrease in cell viability was observed during CoCl2 treatment along with increases in mitochondrial membrane potential and generation of reactive oxygen species (ROS). B355252 conferred protection against these changes. We further found that mitochondrial dynamics, the balance between mitochondrial fusion and fission, were perturbed by CoCl2 treatment. Mitochondrial fusion, which was assessed by measuring the expression of proteins optic atrophy protein 1 (OPA1) and mitofusin 2 (Mfn2), declined due to CoCl2 exposure, but B355252 addition was able to elevate Mfn2 expression while OPA1 expression was unchanged. Mitochondrial fission, measured by phosphorylated dynamin-related protein 1 (p-DRP1) and fission protein 1 (FIS1) expression, also decreased following CoCl2 exposure, and was stabilized by B355252 addition. Finally, autophagy was assessed by measuring the conversion of cytosolic microtubule-associated protein 1A/1B-light chain three-I (LC3-I) to autophagosome-bound microtubule-associated protein 1A/1B-light chain three-II (LC3-II) and was found to be increased by CoCl2. B355252 addition significantly reduced autophagy induction. Taken together, our results indicate B355252 has therapeutic potential to reduce the damaging effects caused by CoCl2 and should be further evaluated for applications in cerebral ischemia therapy.

Mitochondrial biogenesis in neurodegeneration.

Mitochondria play a key role in energy production, calcium homeostasis, cell survival, and death. Adverse stimulations including neurodegenerative diseases may result in mitochondrial dynamic imbalance, free radical production, calcium accumulation, intrinsic cell death pathway activation and eventually cell death. Therefore, preserving or promoting mitochondrial function is a potential therapeutic target for the treatment of neurodegenerative disorders. Mitochondrial biogenesis is a process by which new mitochondria are produced from existing mitochondria. This biogenesis process is regulated by Peroxisome proliferator-activated receptor-gamma (PPARγ) coactivator-1alpha (PGC-1α). Once being activated by either phosphorylation or de-acetylation, PGC-1α activates nuclear respiratory factor 1 and 2 (NRF1 and NRF2), and subsequently mitochondrial transcription factor A (Tfam). The activation of this PGC-1α - NRF -Tfam pathway leads to synthesis of mitochondrial DNA and proteins and generation of new mitochondria. © 2017 Wiley Periodicals, Inc.

Selenium suppresses glutamate-induced cell death and prevents mitochondrial morphological dynamic alterations in hippocampal HT22 neuronal cells.

BACKGROUND: Previous studies have indicated that selenium supplementation may be beneficial in neuroprotection against glutamate-induced cell damage, in which mitochondrial dysfunction is considered a major pathogenic feature. However, the exact mechanisms by which selenium protects against glutamate-provoked mitochondrial perturbation remain ambiguous. In this study glutamate exposed murine hippocampal neuronal HT22 cell was used as a model to investigate the underlying mechanisms of selenium-dependent protection against mitochondria damage. RESULTS: We find that glutamate-induced cytotoxicity was associated with enhancement of superoxide production, activation of caspase-9 and -3, increases of mitochondrial fission marker and mitochondrial morphological changes. Selenium significantly resolved the glutamate-induced mitochondria structural damage, alleviated oxidative stress, decreased Apaf-1, caspases-9 and -3 contents, and altered the autophagy process as observed by a decline in the ratio of the autophagy markers LC3-I and LC3-II. CONCLUSION: These findings suggest that the protection of selenium against glutamate stimulated cell damage of HT22 cells is associated with amelioration of mitochondrial dynamic imbalance.

Water-Soluble Coenzyme Q10 Reduces Rotenone-Induced Mitochondrial Fission.

Parkinson's disease is a neurodegenerative disorder characterized by mitochondrial dysfunction and oxidative stress. It is usually accompanied by an imbalance in mitochondrial dynamics and changes in mitochondrial morphology that are associated with impaired function. The objectives of this study were to identify the effects of rotenone, a drug known to mimic the pathophysiology of Parkinson's disease, on mitochondrial dynamics. Additionally, this study explored the protective effects of water-soluble Coenzyme Q10 (CoQ10) against rotenone-induced cytotoxicity in murine neuronal HT22 cells. Our results demonstrate that rotenone elevates protein expression of mitochondrial fission markers, Drp1 and Fis1, and causes an increase in mitochondrial fragmentation as evidenced through mitochondrial staining and morphological analysis. Water-soluble CoQ10 prevented mitochondrial dynamic imbalance by reducing Drp1 and Fis1 protein expression to pre-rotenone levels, as well as reducing rotenone treatment-associated mitochondrial fragmentation. Hence, water-soluble CoQ10 may have therapeutic potential in treating patients with Parkinson's disease.

Suppression of Inner Mitochondrial Membrane Peptidase 2-Like (IMMP2L) Gene Exacerbates Hypoxia-Induced Neural Death Under High Glucose Condition.

It is known that diabetes hyperglycemia enhances cerebral ischemia and reperfusion induced damage. We have previously shown that mutation of inner mitochondrial membrane peptidase 2-like (IMMP2L) increases brain damage caused by transient cerebral ischemia. In this study, we attempt to examine the impact of IMMP2L deficiency on an in vitro model that mimics the diabetic hypoxic conditions. Normal IMMP2L wild type and IMMP2L gene deleted HT22 cells were cultured. Hypoxia was induced under high glucose and acidic conditions with 4 h of oxygen deprivation. Cell viability was assessed by CCK-8 assay and cell death was determined using Annexin V/7-AAD assay. Superoxide production was measured using dihydroethidium staining and mitochondrial membrane potential was detected using JC-1 probe. Suppression of IMMP2L reduced the cell viability, increased the ROS production and decreased the mitochondrial membrane potential. In conclusion, our study demonstrated that deficiency of IMMP2L in cells, cultured under hypoxia, high glucose and acidic conditions, exacerbated neuronal death under a condition that mimics in vivo cerebral ischemia in diabetic condition.

B355252 Suppresses LPS-Induced Neuroinflammation in the Mouse Brain.

B355252 is a small molecular compound known for potentiating neural growth factor and protecting against neuronal cell death induced by glutamate in vitro and cerebral ischemia in vivo. However, its other biological functions remain unclear. This study aims to investigate whether B355252 suppresses neuroinflammatory responses and cell death in the brain. C57BL/6j mice were intraperitoneally injected with a single dosage of lipopolysaccharide (LPS, 1 mg/kg) to induce inflammation. B355252 (1 mg/kg) intervention was started two days prior to the LPS injection. The animal behavioral changes were assessed pre- and post-LPS injections. The animal brains were harvested at 4 and 24 h post-LPS injection, and histological, biochemical, and cytokine array outcomes were examined. Results showed that B355252 improved LPS-induced behavioral deterioration, mitigated brain tissue damage, and suppressed the activation of microglial and astrocytes. Furthermore, B355252 reduced the protein levels of key pyroptotic markers TLR4, NLRP3, and caspase-1 and inhibited the LPS-induced increases in IL-1ß, IL-18, and cytokines. In conclusion, B355252 demonstrates a potent anti-neuroinflammatory effect in vivo, suggesting that its potential therapeutic value warrants further investigation.

N6022 attenuates cerebral ischemia/reperfusion injury-induced microglia ferroptosis by promoting Nrf2 nuclear translocation and inhibiting the GSNOR/GSTP1 axis.

Stroke poses a significant risk of mortality, particularly among the elderly population. The pathophysiological process of ischemic stroke is complex, and it is crucial to elucidate its molecular mechanisms and explore potential protective drugs. Ferroptosis, a newly recognized form of programmed cell death distinct from necrosis, apoptosis, and autophagy, is closely associated with the pathophysiology of ischemic stroke. N6022, a selective inhibitor of S-nitrosoglutathione reductase (GSNOR), is a "first-in-class" drug for asthma with potential therapeutic applications. However, it remains unclear whether N6022 exerts protective effects in ischemic stroke, and the precise mechanisms of its action are unknown. This study aimed to investigate whether N6022 mitigates cerebral ischemia/reperfusion (I/R) injury by reducing ferroptosis and to elucidate the underlying mechanisms. Accordingly, we established an oxygen-glucose deprivation/reperfusion (OGD/R) cell model and a middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model to mimic cerebral I/R injury. Our data, both in vitro and in vivo, demonstrated that N6022 effectively protected against I/R-induced brain damage and neurological deficits in mice, as well as OGD/R-induced BV2 cell damage. Mechanistically, N6022 promoted Nrf2 nuclear translocation, enhancing intracellular antioxidant capacity of SLC7A11-GPX4 system. Furthermore, N6022 interfered with the interaction of GSNOR with GSTP1, thereby boosting the antioxidant capacity of GSTP1 and attenuating ferroptosis. These findings provide novel insights, showing that N6022 attenuates microglial ferroptosis induced by cerebral I/R injury through the promotion of Nrf2 nuclear translocation and inhibition of the GSNOR/GSTP1 axis.

Asialo-rhuEPO as a Potential Neuroprotectant for Ischemic Stroke Treatment.

Neuroprotective drugs to protect the brain against cerebral ischemia and reperfusion (I/R) injury are urgently needed. Mammalian cell-produced recombinant human erythropoietin (rhuEPOM) has been demonstrated to have excellent neuroprotective functions in preclinical studies, but its neuroprotective properties could not be consistently translated in clinical trials. The clinical failure of rhuEPOM was thought to be mainly due to its erythropoietic activity-associated side effects. To exploit its tissue-protective property, various EPO derivatives with tissue-protective function only have been developed. Among them, asialo-rhuEPO, lacking terminal sialic acid residues, was shown to be neuroprotective but non-erythropoietic. Asialo-rhuEPO can be prepared by enzymatic removal of sialic acid residues from rhuEPOM (asialo-rhuEPOE) or by expressing human EPO gene in glycoengineered transgenic plants (asialo-rhuEPOP). Both types of asialo-rhuEPO, like rhuEPOM, displayed excellent neuroprotective effects by regulating multiple cellular pathways in cerebral I/R animal models. In this review, we describe the structure and properties of EPO and asialo-rhuEPO, summarize the progress on neuroprotective studies of asialo-rhuEPO and rhuEPOM, discuss potential reasons for the clinical failure of rhuEPOM with acute ischemic stroke patients, and advocate future studies needed to develop asialo-rhuEPO as a multimodal neuroprotectant for ischemic stroke treatment.

Immp2l Mutation Induces Mitochondrial Membrane Depolarization and Complex III Activity Suppression after Middle Cerebral Artery Occlusion in Mice.

OBJECTIVE: We previously reported that mutations in inner mitochondrial membrane peptidase 2-like (Immp2l) increase infarct volume, enhance superoxide production, and suppress mitochondrial respiration after transient cerebral focal ischemia and reperfusion injury. The present study investigated the impact of heterozygous Immp2l mutation on mitochondria function after ischemia and reperfusion injury in mice. METHODS: Mice were subjected to middle cerebral artery occlusion for 1 h followed by 0, 1, 5, and 24 h of reperfusion. The effects of Immp2l+/- on mitochondrial membrane potential, mitochondrial respiratory complex III activity, caspase-3, and apoptosis-inducing factor (AIF) translocation were examined. RESULTS: Immp2l+/- increased ischemic brain damage and the number of TUNEL-positive cells compared with wild-type mice. Immp2l+/- led to mitochondrial damage, mitochondrial membrane potential depolarization, mitochondrial respiratory complex III activity suppression, caspase-3 activation, and AIF nuclear translocation. CONCLUSION: The adverse impact of Immp2l+/- on the brain after ischemia and reperfusion might be related to mitochondrial damage that involves depolarization of the mitochondrial membrane potential, inhibition of the mitochondrial respiratory complex III, and activation of mitochondria-mediated cell death pathways. These results suggest that patients with stroke carrying Immp2l+/- might have worse and more severe infarcts, followed by a worse prognosis than those without Immp2l mutations.

Selenium preserves mitochondrial function, stimulates mitochondrial biogenesis, and reduces infarct volume after focal cerebral ischemia.

BACKGROUND: Mitochondrial dysfunction is one of the major events responsible for activation of neuronal cell death pathways during cerebral ischemia. Trace element selenium has been shown to protect neurons in various diseases conditions. Present study is conducted to demonstrate that selenium preserves mitochondrial functional performance, activates mitochondrial biogenesis and prevents hypoxic/ischemic cell damage. RESULTS: The study conducted on HT22 cells exposed to glutamate or hypoxia and mice subjected to 60-min focal cerebral ischemia revealed that selenium (100 nM) pretreatment (24 h) significantly attenuated cell death induced by either glutamate toxicity or hypoxia. The protective effects were associated with reduction of glutamate and hypoxia-induced ROS production and alleviation of hypoxia-induced suppression of mitochondrial respiratory complex activities. The animal studies demonstrated that selenite pretreatment (0.2 mg/kg i.p. once a day for 7 days) ameliorated cerebral infarct volume and reduced DNA oxidation. Furthermore, selenite increased protein levels of peroxisome proliferator-activated receptor-γ coactivator 1alpha (PGC-1α) and nuclear respiratory factor 1 (NRF1), two key nuclear factors that regulate mitochondrial biogenesis. Finally, selenite normalized the ischemia-induced activation of Beclin 1 and microtubule-associated protein 1 light chain 3-II (LC3-II), markers for autophagy. CONCLUSIONS: These results suggest that selenium protects neurons against hypoxic/ischemic damage by reducing oxidative stress, restoring mitochondrial functional activities and stimulating mitochondrial biogenesis.