PKM2 Nuclear Translocation Promotes Glial Cell Activation and Aggravates the Brain Injury of Intracerebral Hemorrhage.

BACKGROUND: The purpose of this study was to investigate the potential involvement of pyruvate kinase M2 (PKM2), an enzyme acting as a rate-limiting enzyme in the final phase of glycolysis, in the regulation of glial activation and brain damage of intracerebral hemorrhage (ICH). METHODS: Western blotting and immunofluorescence were performed to investigate PKM2 expression, terminal deoxynucleotidyl transferase deoxyurinary triphosphate (dUTP) nick end labeling staining, hematoxylin and eosin staining, and behavioral tests were employed to evaluate the brain damage of ICH mice, and RNA-seq and bioinformatic analyses were performed to detect gene expression changes in ICH mice treated with TEPP-46. RESULTS: Increased PKM2 levels in perihematomal brain tissue were found starting from 3 days following ICH and peaked at 5 and 7 days post ICH. The increased expression of PKM2 was mainly co-localized with glial fibrillary acidic protein (GFAP)+ astrocytes and ionized calcium binding adaptor molecule-1 (IBA-1)+ microglia. Furthermore, we observed a notable increase in the nuclear translocation of PKM2 in glial cells following ICH. TEPP-46 treatment significantly reduced PKM2 nuclear translocation, and effectively attenuated glial activation and brain injury, and improved functional recovery of mice with ICH. RNA-seq data indicated that 91.1% (205/225) of differentially expressed genes (DEGs) were down-regulated in the TEPP-46 treated groups compared with the vehicle-treated groups in ICH brains. Furthermore, bioinformatic analyses revealed that these down-regulated DEGs were involved in a variety of biological processes, including autophagy and metabolic processes. In addition, the majority of these downregulated DEGs had a primary high expression in neurons, with subsequent expression seen in endothelial cells, microglia, and astrocytes. CONCLUSIONS: These results indicate that increased PKM2 nuclear translocation promotes the activation of glial cells after ICH, hence aggravating ICH-induced brain damage, and aggravates the brain injury induced by ICH. This highlights a potential therapeutic target for inhibiting glial activation to attenuate brain injury after ICH.

Deep Succinylproteomics of Brain Tissues from Intracerebral Hemorrhage with Inhibition of Toll-Like Receptor 4 Signaling.

It is unclear how Toll-like receptor (TLR) 4 signaling affects protein succinylation in the brain after intracerebral hemorrhage (ICH). Here, we constructed a mouse ICH model to investigate the changes in ICH-associated brain protein succinylation, following a treatment with a TLR4 antagonist, TAK242, using a high-resolution mass spectrometry-based, quantitative succinyllysine proteomics approach. We characterized the prevalence of approximately 6700 succinylation events and quantified approximately 3500 sites, highlighting 139 succinyllysine site changes in 40 pathways. Further analysis showed that TAK242 treatment induced an increase of 29 succinyllysine sites on 28 succinylated proteins and a reduction of 24 succinyllysine sites on 23 succinylated proteins in the ICH brains. TAK242 treatment induced both protein hypersuccinylations and hyposuccinylations, which were mainly located in the mitochondria and cytoplasm. GO analysis showed that TAK242 treatment-induced changes in the ICH-associated succinylated proteins were mostly located in synapses, membranes and vesicles, and enriched in many cellular functions/compartments, such as metabolism, synapse, and myelin. KEGG analysis showed that TAK242-induced hyposuccinylation was mainly linked to fatty acid metabolism, including elongation and degradation. Moreover, a combined analysis of the succinylproteomic data with previously published transcriptome data revealed that most of the differentially succinylated proteins induced by TAK242 treatment were mainly distributed throughout neurons, astrocytes, and endothelial cells, and the mRNAs of seven and three succinylated proteins were highly expressed in neurons and astrocytes, respectively. In conclusion, we revealed that several TLR4 signaling pathways affect the succinylation processes and pathways in mouse ICH brains, providing new insights on the ICH pathophysiological processes. Data are available via ProteomeXchange with identifier PXD025622.

Metabolomics Analysis of Electroacupuncture Pretreatment Induced Neuroprotection on Mice with Ischemic Stroke.

The brain metabolic changes caused by the interruption of blood supply are the initial factors of brain injury in ischemic stroke. Electroacupuncture (EA) pretreatment has been shown to protect against ischemic stroke, but whether its neuroprotective mechanism involves metabolic regulation remains unclear. Based on our finding that EA pretreatment significantly alleviated ischemic brain injury in mice by reducing neuronal injury and death, we performed a gas chromatography-time of flight mass spectrometry (GC-TOF/MS) to investigate the metabolic changes in the ischemic brain and whether EA pretreatment influenced these changes. First, we found that some glycolytic metabolites in the normal brain tissues were reduced by EA pretreatment, which may lay the foundation of neuroprotection for EA pretreatment against ischemic stroke. Then, 6[Formula: see text]h of cerebral ischemia-induced brain metabolic changes, especially the enhanced glycolysis, were partially reversed by EA pretreatment, which was manifested by the brain levels of 11 of 35 up-regulated metabolites and 18 of 27 down-regulated metabolites caused by cerebral ischemia significantly decreasing and increasing, respectively, due to EA pretreatment. A further pathway analysis showed that these 11 and 18 markedly changed metabolites were mainly involved in starch and sucrose metabolism, purine metabolism, aspartate metabolism, and the citric acid cycle. Additionally, we found that EA pretreatment raised the levels of neuroprotective metabolites in both normal and ischemic brain tissues. In conclusion, our study revealed that EA pretreatment may attenuate the ischemic brain injury by inhibiting glycolysis and increasing the levels of some neuroprotective metabolites.

Full-length transcriptome analysis reveals the mechanism of acupuncture at PC6 improves cardiac function in myocardial ischemia model.

BACKGROUND: The pathological process of myocardial ischemia (MI) is very complicated. Acupuncture at PC6 has been proved to be effective against MI injury, but the mechanism remains unclear. This study investigated the mechanism that underlies the effect of acupuncture on MI through full-length transcriptome. METHODS: Adult male C57/BL6 mice were randomly divided into control, MI, and PC6 groups. Mice in MI and PC6 group generated MI model by ligating the left anterior descending (LAD) coronary artery. The samples were collected 5 days after acupuncture treatment. RESULTS: The results showed that treatment by acupuncture improved cardiac function, decreased myocardial infraction area, and reduced the levels of cTnT and cTnI. Based on full-length transcriptome sequencing, 5083 differential expression genes (DEGs) and 324 DEGs were identified in the MI group and PC6 group, respectively. These genes regulated by acupuncture were mainly enriched in the inflammatory response pathway. Alternative splicing (AS) is a post-transcriptional action that contributes to the diversity of protein. In all samples, 8237 AS events associated with 1994 genes were found. Some differential AS-involved genes were enriched in the pathway related to heart disease. We also identified 602 new genes, 4 of which may the novel targets of acupuncture in MI. CONCLUSIONS: Our findings suggest that the effect of acupuncture on MI may be based on the multi-level regulation of the transcriptome.

Succinylation profiles of brain injury after intracerebral hemorrhage.

Protein posttranslational modifications (PTMs) regulate the biological processes of human diseases by genetic code expansion and cellular pathophysiology regulation; however, system-wide changes in PTM levels in the intracerebral hemorrhage (ICH) brain remain poorly understood. Succinylation refers to a major PTM during the regulation of multiple biological processes. In this study, according to the methods of quantitative succinyllysine proteomics based on high-resolution mass spectrometry, we investigated ICH-associated brain protein succinyllysine modifications and obtained 3,680 succinylated sites and quantified around 3,530 sites. Among them, 25 succinyllysine sites on 23 proteins were upregulated (hypersuccinylated), whereas 13 succinyllysine sites on 12 proteins were downregulated (hyposuccinylated) following ICH. The cell component enrichment analysis of these succinylproteins with significant changes showed that 58.3% of the hyposuccinylated proteins were observed in the mitochondria, while the hyper-succinylproteins located in mitochondria decreased in the percentage to about 35% in ICH brains with a concomitant increase in the percentage of cytoplasm to 30.4%. Further bioinformatic analysis showed that the succinylproteins were mostly mitochondria and synapse-related subcellular located and involved in many pathophysiological processes, like metabolism, synapse working, and ferroptosis. Moreover, the integrative analysis of our succinylproteomics data and previously published transcriptome data showed that the mRNAs matched by most differentially succinylated proteins were especially highly expressed in neurons, endothelial cells, and astrocytes. Our study uncovers some succinylation-affected processes and pathways in response to ICH brains and gives us novel insights into understanding pathophysiological processes of brain injury caused by ICH.