ABSTRACT
Pyruvate kinase M2 (PKM2) is a key metabolic enzyme. Yet, its role in cerebral ischemia injury remains unclear. In this study we demonstrated that PKM2 expression was increased in the microglia after mouse cerebral ischemia-reperfusion (I/R) injury. We found that microglial polarization-mediated pro-inflammatory effect was mediated by PKM2 after cerebral I/R. Mechanistically, our results revealed that nuclear PKM2 mediated ischemia-induced microglial polarization through association with acetyl-H3K9. Hif-1α mediated the effect of nuclear PKM2/histone H3 on microglial polarization. PKM2-dependent Histone H3/Hif-1α modifications contributed the expression of CCL2 and induced up-regulation of microglial polarization in peri-infarct, resulting in neuroinflammation. Inhibiting nuclear translocation of microglial PKM2 reduced ischemia-induced pro-inflammation and promoted neuronal survival. Together, this study identifies nucleus PKM2 as a crucial mediator for regulating ischemia-induced neuroinflammation, suggesting PKM2 as a potential therapeutic target in ischemic stroke.
ABSTRACT
Neuroinflammation is a secondary response following ischemia stroke. Arginine is a non-essential amino acid that has been shown to inhibit acute inflammatory reaction. In this study we show that arginine treatment decreases neuronal death after rat cerebral ischemia/reperfusion (I/R) injury and improves functional recovery of stroke animals. We also show that arginine suppresses inflammatory response in the ischemic brain tissue and in the cultured microglia after OGD insult. We further provide evidence that the levels of HIF-1α and LDHA are increased after rat I/R injury and that arginine treatment prevents the elevation of HIF-1α and LDHA after I/R injury. Arginine inhibits inflammatory response through suppression of HIF-1α and LDHA in the rat ischemic brain tissue and in the cultured microglia following OGD insult, and protects against ischemic neuron death after rat I/R injury by attenuating HIF-1α/LDHA-mediated inflammatory response. Together, these results indicate a possibility that arginine-induced neuroprotective effect may be through the suppression of HIF-1α/LDHA-mediated inflammatory response in microglia after cerebral ischemia injury.
Subject(s)
Arginine/therapeutic use , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Inflammation/pathology , Neuroprotective Agents/therapeutic use , Reperfusion Injury/drug therapy , Animals , Arginine/pharmacology , Cell Death , Infarction, Middle Cerebral Artery/complications , Infarction, Middle Cerebral Artery/pathology , L-Lactate Dehydrogenase/metabolism , Male , Microglia , Neurons/drug effects , Neurons/metabolism , Neurons/pathology , Neuroprotection , Neuroprotective Agents/pharmacology , Rats, Sprague-Dawley , Recovery of Function , Reperfusion Injury/pathology , Reperfusion Injury/physiopathology , Signal Transduction/drug effectsABSTRACT
l-lysine is a basic amino acid that has been shown to exert neuroprotective effect. However, the underlying mechanism remains to be elucidated. In this study, we investigate how l-lysine exerts its neuroprotective effect in hemin-insulted mouse cortical neurons in vitro and the mouse model of intracerebral hemorrhage (ICH) in vivo. We demonstrate that l-lysine treatment promotes M2 microglial polarization and reduces inflammatory response both in vitro and in vivo, suggesting that l-lysine may play a neuroprotective role in ICH injury. Indeed, we show that l-lysine treatment reduces cortical neuronal death after hemin insult in vitro and decrease the number of degenerating neurons after ICH in vivo. l-lysine also improves the functional recovery of ICH animals in neurobehavioral tests. Consistent with the role of PTEN in regulating inflammatory response, we find that PTEN inhibition promotes M2 microglial polarization and suppresses pro-inflammatory response in mouse ICH injury, which contribute to the neuroprotective effect of l-lysine. Moreover, our results reveal that microRNA-575 directly suppressed PTEN to promote M2 microglial polarization and mediate the neuroprotective effect of l-lysine in ICH injury. Together, our results suggest that l-lysine confers neuroprotection after ICH injury through enhancing M2 microglial polarization and reducing inflammatory response, which is mediated by microRNA-575 upregulation and subsequent PTEN downregulation.
Subject(s)
Cerebral Hemorrhage/metabolism , Inflammation/drug therapy , Lysine/pharmacology , MicroRNAs/metabolism , Neuroprotective Agents/pharmacology , PTEN Phosphohydrolase/metabolism , Signal Transduction/drug effects , Animals , Cell Polarity/drug effects , Cerebral Hemorrhage/complications , Disease Models, Animal , Down-Regulation/drug effects , Inflammation/etiology , Inflammation/metabolism , Lysine/therapeutic use , Mice , Microglia/drug effects , Microglia/metabolism , Neuroprotection/drug effects , Neuroprotective Agents/therapeutic use , Up-Regulation/drug effectsABSTRACT
Traumatic brain injury (TBI) is a major cause of motor and cognitive impairment in young adults. It is associated with high mortality rates and very few effective treatment options. Bisperoxovanadium (pyridine-2-carboxyl) [bpV(pic)] is an commercially available inhibitor of Phosphatase and tensin homolog (PTEN). Previous studies have shown that bpV(pic) has protective effects in central nervous system. However, the role of bpV(pic) in TBI is unclear. In this study we aimed to investigate the neuroprotective role of bpV(pic) in rat TBI model. We found that injection of bpV(pic) significantly reduces brain edema and neurological dysfunction after TBI and this is mediated by AKT pathway. TBI is known to promote the M1 pro-inflammatory phenotype of microglial polarization and this effect is inhibited by bpV(pic) treatment which, instead promotes M2 microglial polarization in vivo and in vitro. We also found evidence of bpV(pic)-regulated neuroinflammation mediated by AKT activation and NF-κB p65 inhibition. BpV(pic) treatment also suppressed microglia in the peri-TBI region. MCP-1 is known to recruit monocytes and macrophages to promote inflammation, we show that bpV(pic) can inhibit TBI-induced up-regulation of MCP-1 via the AKT/NF-κB p65 signaling pathway. Taken together, our findings demonstrate that bpV(pic) plays a neuroprotective role in rat TBI, which may be achieved by inhibiting M1 microglia polarization and MCP-1 expression by modulating AKT/NF-κB p65 signaling pathway.
Subject(s)
Brain Injuries, Traumatic/drug therapy , Brain Injuries, Traumatic/metabolism , Chemokine CCL2/metabolism , Microglia/drug effects , Neuroprotection/drug effects , Organometallic Compounds/pharmacology , Signal Transduction/drug effects , Animals , Disease Models, Animal , Inflammation/drug therapy , Inflammation/metabolism , Macrophages/drug effects , Macrophages/metabolism , Male , Microglia/metabolism , Monocytes/drug effects , Monocytes/metabolism , NF-kappa B/metabolism , Neurons/drug effects , Neurons/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Glycine is a simple nonessential amino acid known to have neuroprotective properties. Treatment with glycine results in reduced infarct volume of the brain, neurologic function scores, and neuronal and microglial death in ischemic stroke injury. Neuroinflammation has been considered a major contributor to cerebral ischemia-induced brain damage. However, the role of glycine in neuroinflammation following ischemic stroke is unclear. The present study aimed to determine whether neuroinflammation is involved in the neuroprotective effects of glycine in cerebral ischemia injury. Ischemic stroke promotes M1 microglial polarization. Interestingly, we found that the injection of glycine in rats after injury can inhibit ischemia-induced inflammation and promote M2 microglial polarization in vivo (Sprague-Dawley rats) and in vitro (cortical microglia and BV-2 cells). We show that glycine suppresses Hif-1α by inhibiting the upregulation of NF-κB p65 after ischemia-reperfusion injury, resulting in the inhibition of proinflammatory activity. The activation of AKT mediates the inhibition of NF-κB p65/Hif-1α signaling by glycine. Moreover, we confirm that glycine-regulated AKT activation is mediated by the inhibition of PTEN in a PTEN depletion cell line, U251 cells. Glycine modulates microglial polarization after ischemic stroke, which indirectly inhibits ischemia-induced neuronal death and functional recovery. Taken together, our findings provide a new understanding of glycine in neuroprotection by inhibiting M1 microglial polarization and promoting anti-inflammation by suppressing NF-κB p65/Hif-1α signaling.
Subject(s)
Brain/drug effects , Glycine/pharmacology , Microglia/drug effects , Neuroprotective Agents/pharmacology , Stroke/immunology , Animals , Brain/immunology , Brain/pathology , Brain Ischemia/immunology , Brain Ischemia/metabolism , Brain Ischemia/pathology , Female , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Male , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Stroke/metabolism , Stroke/pathology , Transcription Factor RelA/metabolismABSTRACT
DJ-1 (also called PARK7) is a multifunctional redox-sensitive protein that is protective against oxidative stress-induced cell death. TAR DNA-binding protein 43 (TDP-43) is a major protein component of pathological inclusions in amyotrophic lateral sclerosis and frontotemporal dementia. Reducing aberrant aggregation of TDP-43 is a potential approach to prevent cell death. To investigate whether DJ-1 might inhibit TDP-43 aggregation to exert a protective effect in oxidative stress-induced injury, we tested the protein level and subcellular localization of TDP-43 and DJ-1 in SH-SY5Y cells transfected with wild-type DJ-1, DJ-1 mutant (L166P) cDNA, or DJ-1 siRNA. We show that oxidative stress induced by paraquat leads to the formation of cytosolic TDP-43 aggregation in SH-SY5Y cells. DJ-1 overexpression decreases paraquat-induced cytoplasmic accumulation of TDP-43 in SH-SY5Y cells and protects against paraquat-induced cell death. Transfection of DJ-1 L166P mutant or DJ-1 siRNA leads to increased cytosolic aggregation of TDP-43 in paraquat-treated SH-SY5Y cells and promotes cell death. These data suggest that DJ-1 may protect against oxidative stress-induced cell death through the suppression of cytoplasmic TDP-43 aggregation.
Subject(s)
DNA-Binding Proteins/metabolism , Neurons/metabolism , Oxidative Stress/physiology , Protein Deglycase DJ-1/genetics , Amyotrophic Lateral Sclerosis/metabolism , Cell Line, Tumor , Humans , Neurons/drug effects , Oxidative Stress/drug effects , Paraquat/pharmacology , Phosphorylation , Protein Deglycase DJ-1/metabolismABSTRACT
PURPOSE: To investigate the effects of estrogen G protein-coupled receptor 30 (GPR30) agonist G1 on hippocampal neuronal apoptosis and microglial polarization in rat traumatic brain injury (TBI). METHODS: Male SD rats were randomly divided into sham group, TBI + vehicle group, TBI + G1 group. Experimental moderate TBI was induced using Feeney's weigh-drop method. G1 (100µg/kg) or vehicle was intravenously injected from femoral vein at 30 min post-injury. Rats were sacrificed at 24 h after injury for detection of neuronal apoptosis and microglia polarization. Neuronal apoptosis was assayed by immunofluorescent staining of active caspase-3. M1 type microglia markers (iNOS and IL-1ß) and M2 type markers (Arg1 and IL-4) were examined by immunoblotting or ELISA. Total protein level of Akt and phosphorylated Akt were assayed by immunoblotting. RESULTS: G1 significantly reduced active caspase-3 positive neurons in hippocampus. Meanwhile G1 increased the ratio of Arg1/iNOS. IL-1ß production was decreased but IL-4 was increased after G1 treatment. G1 treatment also increased the active form of Akt. CONCLUSIONS: GPR30 agonist G1 inhibited neuronal apoptosis and favored microglia polarization to M2 type.
Subject(s)
Apoptosis/drug effects , Brain Injuries, Traumatic/drug therapy , Microglia/drug effects , Neurons/drug effects , Receptors, G-Protein-Coupled/agonists , Animals , Brain Injuries, Traumatic/pathology , Cell Polarity , Hippocampus/drug effects , Interleukin-1beta/biosynthesis , Male , Proto-Oncogene Proteins c-akt/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Bisperoxovanadium (pyridine-2-carboxyl) [bpV(pic)] is a commercially available PTEN inhibitor. Previous studies from us and others have shown that bpV(pic) confers neuroprotection in cerebral ischemia injury. We set up to determine whether ERK 1/2 activation plays a role in bpV(pic)-induced neuroprotective effect in cerebral ischemia injury. We found that the phosphorylation levels of Akt (p-AKT) and ERK1/2 (p-ERK 1/2) were down-regulated after cerebral ischemia-reperfusion injury. The injection of bpV(pic) after injury not only increased the level of p-AKT but also the level of p-ERK 1/2. While the inhibition of PTEN mediated the up-regulatation of p-AKT and p-ERK 1/2 by bpV(pic). Interestingly, the ERK 1/2 activation induced by bpV(pic) was also independent of the inhibition of PTEN. Our results indicate that bpV(pic) protects against OGD-induced neuronal death and promotes the functional recovery of stroke animals through PTEN inhibition and ERK 1/2 activation, respectively. This study suggests that the effect of bpV(pic) on ERK 1/2 signaling should be considered while using bpV(pic) as a PTEN inhibitor.
Subject(s)
Brain Injuries/drug therapy , Brain Ischemia/drug therapy , MAP Kinase Signaling System/drug effects , Reperfusion Injury/drug therapy , Vanadium Compounds/pharmacology , Animals , Disease Models, Animal , Male , Neurons/drug effects , Neurons/metabolism , Neuroprotection/drug effects , Neuroprotective Agents/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Rats, Sprague-Dawley , Recovery of Function/drug effectsABSTRACT
Glioblastoma (GBM) is the most aggressive glioma in the brain. Recurrence of GBM is almost inevitable within a short term after tumor resection. In a retrospective study of 386 cases of GBM collected between 2013 and 2016, we found that recurrence of GBM mainly occurs in the deep brain regions, including the basal ganglia, thalamus, and corpus callosum. But the mechanism underlying this phenomenon is not clear. Previous studies suggest that neuroligin-3 (NLGN3) is necessary for GBM growth. Our results show that the levels of NLGN3 in the cortex are higher than those in the deep regions in a normal human brain, and similar patterns are also found in a normal mouse brain. In contrast, NLGN3 levels in the deep brain regions of GBM patients are high. We also show that an increase in NLGN3 concentration promotes the growth of U251 cells and U87-MG cells. Respective use of the cortex neuron culture medium (C-NCM) and basal ganglia neuron culture medium (BG-NCM) with DMEM to cultivate U251, U87-MG and GBM cells isolated from patients, we found that these cells grew faster after treatment with C-NCM and BG-NCM in which the cells treated with C-NCM grew faster than the ones treated with BG-NCM group. Inhibition of NLGN3 release by ADAM10i prevents NCM-induced cell growth. Together, this study suggests that increased levels of NLGN3 in the deep brain region under the GBM pathological circumstances may contribute to GBM recurrence in the basal ganglia, thalamus, and corpus callosum.
ABSTRACT
Glycine has been shown to protect against ischemic stroke through various mechanisms. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) which antagonize Akt-dependent cell survival has been linked to neuronal damage. However, whether glycine has a neuroprotective property in intracerebral hemorrhage (ICH) was unknown. This study aimed to determine the protective effect of glycine in rats ICH. Adult male Sprague-Dawley (SD) rats were subjected to left striatum infusion of autologous blood. ICH animals received glycine (0.2-3â¯mg/kg, icv) at 1â¯h after ICH with or without pre-injection of Akt Inhibitor IV (100⯵M, 2⯵l, icv) 0.5â¯h prior to glycine treatment. Our results showed that in the perihematomal area PTEN was up-regulated in the early stage after ICH. However, glycine treatment decreased PTEN protein level and increased the phosphorylation level of AKT (p-AKT) in the perihematomal area. With the administration of glycine, neuronal death was significantly reduced and Evans blue leakage was alleviated as well as the brain edema after ICH. Moreover, hematoma volume was decreased and neurobehavioral outcome was improved. Nevertheless, Akt Inhibitor IV abolished the neuroprotective effects of glycine after ICH. Together, our findings demonstrate, for the first time, the protective role of glycine on ICH rats, and suggest that the neuroprotective effect of glycine was mediated through PTEN/Akt signal pathway.
Subject(s)
Cerebral Hemorrhage/drug therapy , Cerebral Hemorrhage/metabolism , Glycine/pharmacology , Neuroprotection/drug effects , PTEN Phosphohydrolase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Animals , Benzimidazoles/pharmacology , Benzothiazoles/pharmacology , Brain Edema/drug therapy , Brain Edema/metabolism , Brain Edema/pathology , Cell Death/drug effects , Cerebral Hemorrhage/pathology , Disease Models, Animal , Male , Neurons/drug effects , Neurons/pathology , Neuroprotective Agents/pharmacology , PTEN Phosphohydrolase/antagonists & inhibitors , Phosphorylation , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effectsABSTRACT
Lysophosphatidic acid (LPA), an extracellular signaling molecule, influences diverse biological events, including the pathophysiological process induced after ischemic brain injury. However, the molecular mechanisms mediating the pathological change after ischemic stroke remain elusive. Here we report that asparagine endopeptidase (AEP), a lysosomal cysteine proteinase, is regulated by LPA during stroke. AEP proteolytically cleaves tau and generates tauN368 fragments, triggering neuronal death. Inhibiting the generation of LPA reduces the expression of AEP and tauN368, and alleviates neuronal cell death. Together, this evidence indicates that the LPA-AEP pathway plays a key role in the pathophysiological process induced after ischemic stroke. Inhibition of LPA could be a useful therapeutic for treating neuronal injury after stroke.