Multi-Omics Profiling Identifies Microglial Annexin A2 as a Key Mediator of NF-κB Pro-inflammatory Signaling in Ischemic Reperfusion Injury.

Cerebral stroke is one of the leading causes of mortality and disability worldwide. Restoring the cerebral circulation following a period of occlusion and subsequent tissue oxygenation leads to reperfusion injury. Cerebral ischemic reperfusion (I/R) injury triggers immune and inflammatory responses, apoptosis, neuronal damage, and even death. However, the cellular function and molecular mechanisms underlying cerebral I/R-induced neuronal injury are incompletely understood. By integrating proteomic, phosphoproteomic, and transcriptomic profiling in mouse hippocampi after cerebral I/R, we revealed that the differentially expressed genes and proteins mainly fall into several immune inflammatory response-related pathways. We identified that Annexin 2 (Anxa2) was exclusively upregulated in microglial cells in response to cerebral I/R in vivo and oxygen-glucose deprivation and reoxygenation (OGD/R) in vitro. RNA-seq analysis revealed a critical role of Anxa2 in the expression of inflammation-related genes in microglia via the NF-κB signaling. Mechanistically, microglial Anxa2 is required for nuclear translocation of the p65 subunit of NF-κB and its transcriptional activity upon OGD/R in BV2 microglial cells. Anxa2 knockdown inhibited the OGD/R-induced microglia activation and markedly reduced the expression of pro-inflammatory factors, including TNF-α, IL-1ß, and IL-6. Interestingly, conditional medium derived from Anxa2-depleted BV2 cell cultures with OGD/R treatment alleviated neuronal death in vitro. Altogether, our findings revealed that microglia Anxa2 plays a critical role in I/R injury by regulating NF-κB inflammatory responses in a non-cell-autonomous manner, which might be a potential target for the neuroprotection against cerebral I/R injury.

Identification of Vigilin as a Potential Ischemia Biomarker by Brain Slice-Based Systematic Evolution of Ligands by Exponential Enrichment.

Stroke is one of the leading causes of disability and death among adults worldwide and results in numerous biochemical alterations. However, few efficient biomarkers are clinically available to diagnose stroke because of the limitations of biomarkers and their probes. In this work, we utilized frozen brain slices of middle cerebral artery occlusion (MCAO) in a mouse model of ischemia to select a specific binding aptamer, termed LCW17, by tissue-based SELEX (systematic evolution of ligands by exponential enrichment). LCW17 was enhanced in binding in ischemic brain slices compared to sham control. We identified the binding target of LCW17 as vigilin. Vigilin is increased in ischemia brain slices and exhibits enhanced release from cultured hippocampal neurons after oxygen glucose deprivation in vitro. Taken together, ischemic brain slice-based aptamer selection will enable identification of more probes and potential target molecules for diagnosis and therapy of ischemic stroke. Aptamer LCW17 and vigilin may potentially be applied to define the molecular mechanism underlying ischemic stroke, as well as its diagnosis.

Intracellular energy controls dynamics of stress-induced ribonucleoprotein granules.

Energy metabolism and membraneless organelles have been implicated in human diseases including neurodegeneration. How energy deficiency regulates ribonucleoprotein particles such as stress granules (SGs) is still unclear. Here we identified a unique type of granules induced by energy deficiency under physiological conditions and uncovered the mechanisms by which the dynamics of diverse stress-induced granules are regulated. Severe energy deficiency induced the rapid formation of energy deficiency-induced stress granules (eSGs) independently of eIF2α phosphorylation, whereas moderate energy deficiency delayed the clearance of conventional SGs. The formation of eSGs or the clearance of SGs was regulated by the mTOR-4EBP1-eIF4E pathway or eIF4A1, involving assembly of the eIF4F complex or RNA condensation, respectively. In neurons or brain organoids derived from patients carrying the C9orf72 repeat expansion associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the eSG formation was enhanced, and the clearance of conventional SGs was impaired. These results reveal a critical role for intracellular energy in the regulation of diverse granules and suggest that disruptions in energy-controlled granule dynamics may contribute to the pathogenesis of relevant diseases.

Derivation of Stem Cell-like Cells From Spherical Culture of Astrocytes for Enhanced Neural Repair After Middle Cerebral Artery Occlusion.

Neural precursor cells (NPCs) tend to aggregate and develop into three-dimensional (3D) spheres, which in turn help maintain the stemness of the cells. This close relationship between spherical environments and cell stemness direct us to assume that 3D spheres of astrocytes (ASTs) may facilitate the acquisition of stem cell-like features and generate sufficient seed cells for the regeneration of neurons. In vitro results confirmed that mouse ASTs cultured on agarose surfaces spontaneously formed cell spheres and exhibited molecular features similar to stem cells, particularly capable of further differentiating into neurons and forming functional synaptic networks with synchronous burst activities. RNA-sequencing results revealed the similarity between AST-derived stem cells (A-iSCs) and NPCs in global gene expression profiles. The potency of A-iSCs in repairing neural injuries was evaluated in a mouse model of middle cerebral artery occlusion. It was observed that the transplanted A-iSCs expressed a series of markers related to neural differentiation, such as NeuN, Tuj1, and Map2, indicating the conversion of the transplanted A-iSCs into neurons in the scenario. We also found that the injured mice injected with A-iSCs exhibited significant improvements in sensorimotor functions after 8 weeks compared with the sham and control mice. Taken together, mouse ASTs form cell spheres on agarose surfaces and acquire stem cell-associated features; meanwhile, the derived A-iSCs possess the capacity to differentiate into neurons and facilitate the regeneration of damaged nerves.

Enolase2 and enolase1 cooperate against neuronal injury in stroke model.

Stroke is one of the leading causes of death in adults worldwide. However, the mechanism causing neuronal death remains poorly understood. Our previous report showed that enolase1 (ENO1), a key glycolytic enzyme, alleviates cerebral ischemia-induced neuronal injury. It remained unclear whether enolase2 (ENO2) affects neuronal injury in stroke models. Here, we examined the effects of ENO2 in several stroke models. The results showed that the expression level of ENO2 was downregulated after 3 h of cerebral ischemia by middle cerebral artery occlusion (MCAO) in the mouse model. ENO2 was expressed in mouse brain and cultured hippocampus neurons. Overexpression of ENO2 in cultured hippocampus neurons did not affect neuronal injury in our oxygen-glucose deprivation (OGD) model. Interestingly, double knock-down (KD) of ENO1 and ENO2 increased neuronal injury while either KD of ENO1 or ENO2 failed to increase neuronal injury in OGD. Deletion of ENO1 did not affect anoxia-starvation (AS)-induced worm death in C. elegans. These findings demonstrated that ENO2 and ENO1 work together against neuronal injury in these stroke models.

Enolase1 Alleviates Cerebral Ischemia-Induced Neuronal Injury via Its Enzymatic Product Phosphoenolpyruvate.

Stroke is a leading cause of disability and the second leading cause of death among adults worldwide, while the mechanisms underlying neuronal death and dysfunction remain poorly understood. Here, we investigated the differential proteomic profiles of mouse brain homogenate with 3 h of middle cerebral artery occlusion (MCAO) ischemia, or sham, using Coomassie Brilliant Blue staining, followed by mass spectrometry. We identified enolase1 (ENO1), a key glycolytic enzyme, as a potential mediator of neuronal injury in MCAO ischemic model. Reverse transcription polymerase chain reaction and western blotting data showed that ENO1 was ubiquitously expressed in various tissues, distinct regions of brain, and different postnatal age. Immunohistochemical analysis revealed that ENO1 is localized in neuronal cytoplasm and dendrites. Interestingly, the expression level of ENO1 was significantly increased in the early stage, but dramatically decreased in the late stage, of cerebral ischemia in vivo. This dynamic change was consistent with our finding in cultured hippocampal neurons treated with oxygen/glucose deprivation (OGD) in vitro. Importantly, ENO1 overexpression in cultured neurons alleviated dendritic and spinal loss caused by OGD treatment. Furthermore, the enzymatic product of ENO1, phosphoenolpyruvate (PEP), was also synchronously changed along with the dynamic ENO1 level. The neuronal injury caused by OGD treatment in vitro or ischemia in vivo was mitigated by the application of PEP. Taken together, our data revealed that ENO1 plays a novel and protective role in cerebral ischemia-induced neuronal injury, highlighting a potential of ENO1 as a therapeutic target of neuronal protection from cerebral ischemia.