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.

A gonadal gap junction INX-14/Notch GLP-1 signaling axis suppresses gut defense through an intestinal lysosome pathway.

Gap junctions mediate intercellular communications across cellular networks in the nervous and immune systems. Yet their roles in intestinal innate immunity are poorly understood. Here, we show that the gap junction/innexin subunit inx-14 acts in the C. elegans gonad to attenuate intestinal defenses to Pseudomonas aeruginosa PA14 infection through the PMK-1/p38 pathway. RNA-Seq analyses revealed that germline-specific inx-14 RNAi downregulated Notch/GLP-1 signaling, while lysosome and PMK-1/p38 pathways were upregulated. Consistently, disruption of inx-14 or glp-1 in the germline enhanced resistance to PA14 infection and upregulated lysosome and PMK-1/p38 activity. We show that lysosome signaling functions downstream of the INX-14/GLP-1 signaling axis and upstream of PMK-1/p38 pathway to facilitate intestinal defense. Our findings expand the understanding of the links between the reproductive system and intestinal defense, which may be evolutionarily conserved in higher organism.

GABAergic signaling between enteric neurons and intestinal smooth muscle promotes innate immunity and gut defense in Caenorhabditis elegans.

The nervous system is critical for intestinal homeostasis and function, but questions remain regarding its impact on gut immune defense. By screening the major neurotransmitters of C. elegans, we found that γ-aminobutyric acid (GABA) deficiency enhanced susceptibility to pathogenic Pseudomonas aeruginosa PA14 infection. GABAergic signaling between enteric neurons and intestinal smooth muscle promoted gut defense in a PMK-1/p38-dependent, but IIS/DAF-16- and DBL-1/TGF-ß-independent, pathway. Transcriptomic profiling revealed that the neuropeptide, FLP-6, acted downstream of enteric GABAergic signaling. Further data determined that FLP-6 was expressed and secreted by intestinal smooth muscle cells and functioned as a paracrine molecule on the intestinal epithelium. FLP-6 suppressed the transcription factors ZIP-10 and KLF-1 that worked in parallel and converged to the PMK-1/p38 pathway in the intestinal epithelia for innate immunity and gut defense. Collectively, these findings uncover an enteric neuron-muscle-epithelium axis that may be evolutionarily conserved in higher organisms.

The bZIP transcription factor BATF3/ZIP-10 suppresses innate immunity by attenuating PMK-1/p38 signaling.

Innate immunity is the first line of host defense against pathogenic invasion in metazoans. The transcription factor basic leucine zipper transcriptional factor ATF-like 3 (BATF3) plays a crucial role in the development of conventional dendritic cells and the program of CD8 + T cell survival and memory, but the role of BATF3 in innate immune responses remains unclear. Here, we show an evolutionarily conserved basic-region leucine zipper (bZIP) transcription factor BATF3/ZIP-10 suppresses innate immune response through repressing the p38/PMK-1 mitogen-activated protein kinase (MAPK) pathway in vitro and in vivo. The worm mutant lacking the Caenorhabditis elegans homolog BATF3, ZIP-10, exhibited enhanced resistance to PA14 infection, which was completely rescued by transgenic expression of either endogenous zip-10 or mouse or human Batf3 cDNA driven by the worm zip-10 promoter. ZIP-10 expression was inhibited by a microRNA miR-60 that was downregulated upon PA14 infection. Moreover, the level of phosphorylated but not total PMK-1/p38 was attenuated by ZIP-10 and stimulated by miR-60. The human HEK293 cells with Batf3 overexpression or RNA-interference knockdown exhibited a reduction or increase of the cell viability upon Pseudomonas aeruginosa PA14 infection, respectively. The overexpression of either worm ZIP-10 or human BATF3 abolished the activation of p38 and inhibited the expression of antimicrobial peptides and cytokine genes in HEK293 cells. Our findings indicate that the genetic transcriptional program of the evolutionally conserved bZIP transcription factor BATF3/ZIP-10 suppresses innate immunity by attenuating the p38 MAPK signaling activity, which expands our understanding of the pathological mechanisms underlying relevant infectious diseases.

pH as a Key Factor for the Quality Assurance of the Preparation of Gastrodiae Rhizoma Formula Granules.

Gastrodiae rhizoma (GR) formula granules and preparations have been used as a popular traditional Chinese medicine for clinical treatment since they have good pharmacological activity to treat nervous system diseases. Gastrodin and parishins have been the main active components in aqueous extracts for GR formula granules, but their pharmacological activities and metabolism are different. For quality control of the extracts, the extraction conditions should be investigated to accurately control the contents of two kinds of components. In this paper, the transfer rate of six index components (including gastrodin, p-hydroxybenzyl alcohol, parishin A, parishin B, parishin C, and parishin E) obtained by HPLC were used as indicators to investigate the effect of pH on the GR extraction process. The results demonstrated that pH is a key factor for preventing transforming parishins into gastrodin and maintaining high content of parishins in the extracts. It can be concluded that the weak acid environment could improve the transfer rate of parishins, thus ensuring the gastrodin and parishins consistency between GR raw materials and its aqueous extracts. Therefore, pH is an essential condition for accurate quality control of the extracts.

GABAergic Neuromuscular Junction Suppresses Intestinal Defense of Caenorhabditis elegans by Attenuating Muscular Oxidative Phosphorylation.

Innate immunity is an ancient and evolutionarily conserved system that constitutes the first line of host defense against invading microbes. We previously determined that the GABAergic neuromuscular junction (NMJ) suppresses intestinal innate immunity via muscular insulin signaling. Here, we found that a muscular mitochondrial oxidative phosphorylation pathway of Caenorhabditis elegans is involved in GABAergic NMJs-mediated intestinal defense. Deficiency in GABAergic neurotransmission increases reactive oxygen species (ROS) abundance and inhibits the nuclear translocation of SKN-1, whereas exogenous GABA administration represses it. SKN-1 is an important transcription factor involved in oxidative stress and the innate immune response. Moreover, deficiency in GABAergic postsynaptic UNC-49/GABAAR robustly promotes the mitochondrial function of GABAergic postsynaptic muscle cells, which may contribute to the muscular ROS decrease and intestinal SKN-1 suppression, ultimately inhibiting the intestinal defense of C. elegans. Our findings reveal a potential role of muscle mitochondrial ROS in intestinal defense in vivo and expand our understanding of mechanisms of intestinal innate immunity.

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.

Phosphoproteome Analysis Identifies a Synaptotagmin-1-Associated Complex Involved in Ischemic Neuron Injury.

Cerebral stroke is one of the leading causes of death in adults worldwide. However, the molecular mechanisms of stroke-induced neuron injury are not fully understood. Here, we obtained phosphoproteomic and proteomic profiles of the acute ischemic hippocampus by LC-MS/MS analysis. Quantitative phosphoproteomic analyses revealed that the dysregulated phosphoproteins were involved in synaptic components and neurotransmission. We further demonstrated that phosphorylation of Synaptotagmin-1 (Syt1) at the Thr112 site in cultured hippocampal neurons aggravated oxygen-glucose deprivation-induced neuronal injury. Immature neurons with low expression of Syt1 exhibit slight neuronal injury in a cerebral ischemia model. Administration of the Tat-Syt1T112A peptide protects neurons against cerebral ischemia-induced injury in vitro and in vivo. Surprisingly, potassium voltage-gated channel subfamily KQT member 2 (Kcnq2) interacted with Syt1 and Annexin A6 (Anxa6) and alleviated Syt1-mediated neuronal injury upon oxygen-glucose deprivation treatment. These results reveal a mechanism underlying neuronal injury and may provide new targets for neuroprotection after acute cerebral ischemia onset.

The bZIP Transcription Factor ZIP-11 Is Required for the Innate Immune Regulation in Caenorhabditis elegans.

Innate immunity is the first line of host defense against pathogen infection in metazoans. However, the molecular mechanisms of the complex immune regulatory network are not fully understood. Based on a transcriptome profiling of the nematode Caenorhabditis elegans, we found that a bZIP transcription factor ZIP-11 was up-regulated upon Pseudomonas aeruginosa PA14 infection. The tissue specific RNAi knock-down and rescue data revealed that ZIP-11 acts in intestine to promote host resistance against P. aeruginosa PA14 infection. We further showed that intestinal ZIP-11 regulates innate immune response through constituting a feedback loop with the conserved PMK-1/p38 mitogen-activated protein signaling pathway. Intriguingly, ZIP-11 interacts with a CCAAT/enhancer-binding protein, CEBP-2, to mediate the transcriptional response to P. aeruginosa PA14 infection independently of PMK-1/p38 pathway. In addition, human homolog ATF4 can functionally substitute for ZIP-11 in innate immune regulation of C. elegans. Our findings indicate that the ZIP-11/ATF4 genetic program activates local innate immune response through conserved PMK-1/p38 and CEBP-2/C/EBPγ immune signals in C. elegans, raising the possibility that a similar process may occur in other organisms.

The GABAB receptor mediates neuroprotection by coupling to G13.

G protein­coupled receptors (GPCRs) activate various mitogen-activated protein kinase (MAPK) pathways to regulate critical cell functions. ß-Arrestins mediate this mechanism for most GPCRs but not the GABAB receptor (GABABR). When coupled to the G protein Gi/o, GABABR phosphorylates the kinases ERK1 and ERK2. Here, we uncovered a distinct ß-arrestin­independent mechanism of MAPK pathway activation by GABABR. We found that GABABR also phosphorylated the kinase JNK downstream of activation of the small guanosine triphosphatases (GTPases) RhoA and Rac1 in primary mouse neurons. However, instead of Gi/o proteins, activation of this RhoA/Rac1-JNK pathway was mediated by G13. This pathway promoted the phosphorylation and accumulation of the postsynaptic scaffolding protein PSD95 and GABABR-mediated neuroprotection in granule neurons. In addition, this pathway synergized with a previously reported GABABR-mediated neuroprotection mediated by a Gi/o-dependent mechanism. GABABR agonists activated G13 with slower kinetics and lower potency than with which they activated Gi/o. Our findings reveal distinct, ß-arrestin­independent, context-specific synergistic mechanisms of MAPK activation by G protein­mediated GPCR signaling.

Extracellular Matrix Muscle Arm Development Defective Protein Cooperates with the One Immunoglobulin Domain Protein To Suppress Precocious Synaptic Remodeling.

Synaptic remodeling plays important roles in health and neural disorders. Although previous studies revealed that several transcriptional programs control synaptic remodeling in the nematode Caenorhabditis elegans, the molecular mechanisms of the dorsal D-type (DD) synaptic remodeling are poorly understood. Here we show that extracellular matrix molecule muscle arm development defective protein-4 (MADD-4) cooperates with the one immunoglobulin domain protein-1 (OIG-1) to defer precocious DD synaptic remodeling. Specifically, loss of MADD-4 exhibited the precocious DD synaptic remodeling. The long isoform MADD-4L is dynamically expressed while the short isoform MADD-4B is persistently expressed in DD neurons of L1 stage. In the unc-30 mutant lacking the Pitx-type homeodomain transcription factor UNC-30, the expression levels of both MADD-4B and -L isoforms were dramatically downregulated in DD neurons of the L1 stage. Our further data showed that MADD-4B and -L isoforms physically interact with OIG-1 and madd-4 acts in the oig-1 genetic pathway to modulate the DD synaptic remodeling. Our findings demonstrated that the extracellular matrix plays a novel role in synaptic plasticity.

GABAergic synapses suppress intestinal innate immunity via insulin signaling in Caenorhabditis elegans.

GABAergic neurotransmission constitutes a major inhibitory signaling mechanism that plays crucial roles in central nervous system physiology and immune cell immunomodulation. However, its roles in innate immunity remain unclear. Here, we report that deficiency in the GABAergic neuromuscular junctions (NMJs) of Caenorhabditis elegans results in enhanced resistance to pathogens, whereas pathogen infection enhances the strength of GABAergic transmission. GABAergic synapses control innate immunity in a manner dependent on the FOXO/DAF-16 but not the p38/PMK-1 pathway. Our data reveal that the insulin-like peptide INS-31 level was dramatically decreased in the GABAergic NMJ GABAAR-deficient unc-49 mutant compared with wild-type animals. C. elegans with ins-31 knockdown or loss of function exhibited enhanced resistance to Pseudomonas aeruginosa PA14 exposure. INS-31 may act downstream of GABAergic NMJs and in body wall muscle to control intestinal innate immunity in a cell-nonautonomous manner. Our results reveal a signaling axis of synapse-muscular insulin-intestinal innate immunity in vivo.

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.

The Ig-like domain of Punctin/MADD-4 is the primary determinant for interaction with the ectodomain of neuroligin NLG-1.

Punctin/MADD-4, a member of the ADAMTSL extracellular matrix protein family, was identified as an anterograde synaptic organizer in the nematode Caenorhabditis elegans. At GABAergic neuromuscular junctions, the short isoform MADD-4B binds the ectodomain of neuroligin NLG-1, itself a postsynaptic organizer of inhibitory synapses. To identify the molecular bases of their partnership, we generated recombinant forms of the two proteins and carried out a comprehensive biochemical and biophysical study of their interaction, complemented by an in vivo localization study. We show that spontaneous proteolysis of MADD-4B first generates a shorter N-MADD-4B form, which comprises four thrombospondin (TSP) domains and one Ig-like domain and binds NLG-1. A second processing event eliminates the C-terminal Ig-like domain along with the ability of N-MADD-4B to bind NLG-1. These data identify the Ig-like domain as the primary determinant for N-MADD-4B interaction with NLG-1 in vitro We further demonstrate in vivo that this Ig-like domain is essential, albeit not sufficient per se, for efficient recruitment of GABAA receptors at GABAergic synapses in C. elegans The interaction of N-MADD-4B with NLG-1 is also disrupted by heparin, used as a surrogate for the extracellular matrix component, heparan sulfate. High-affinity binding of heparin/heparan sulfate to the Ig-like domain may proceed from surface charge complementarity, as suggested by homology three-dimensional modeling. These data point to N-MADD-4B processing and cell-surface proteoglycan binding as two possible mechanisms to regulate the interaction between MADD-4B and NLG-1 at GABAergic synapses.

[Progress in the mechanisms of response to different oxygen concentrations in Caenorhabditis elegans].

Oxygen levels are unequal in different living geographical locations of human and related to normal physiology of health. The reduction of oxygen level in the body can lead to a variety of diseases, such as stroke caused by cerebral ischemia and hypoxia. In the recent years, many studies have elucidated the molecular and cellular mechanisms of organism response to different oxygen concentrations by using the nematode Caenorhabditis elegans (C. elegans) as model organism. C. elegans can escape hypoxia or hyperoxia and adapt to the ambient oxygen environments, and there are different response and regulation mechanisms in different degrees of hypoxia environment. In this paper, recent advances in the reaction of nematodes to different oxygen concentrations and the underlying mechanism were reviewed.

Programmable pH-Responsive DNA Nanosensors for Imaging Exocytosis and Retrieval of Synaptic Vesicles.

Exocytosis and retrieval of synaptic vesicles (SVs) are vital steps during neurotransmitter propagation between neurons. Visualization of this dynamics of SVs is significant for elucidating the mechanisms underlying synaptic transmission but remains challenging without an efficient, reliable, and biocompatible labeling method. In this work, we developed pH-responsive ratiometric DNA tetrahedral nanoprobes (pHadtnps) that could specifically label recycling SVs with high stability and effective background suppression. On the basis of the luminal pH alternation during the recycling of SVs, pHadtnps were able to illustrate their exocytosis and retrieval in real time. Moreover, with the high programmability of DNA nanotechnology, these nanoprobes could be flexibly equipped with different functional moieties, holding great promise for developing various versatile tools for studying communication in neuronal networks.

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.

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.

Isotopic graphene-isolated-Au-nanocrystals with cellular Raman-silent signals for cancer cell pattern recognition.

For cancer diagnosis, technologies must be capable of molecular recognition, and they must possess a built-in pattern recognition component for efficient imaging and discrimination of targeted cancer cells. Surface enhanced Raman scattering (SERS) tags based on plasmonically active nanoparticles hold promise for accurate and efficient cancer cell recognition, owing to ultra-narrow peak and sensitive optical properties. However, a complex fingerprint spectrum increases data analysis difficulty, making it necessary to develop multicolor SERS tags with a simple fingerprint spectrum. To address this, we herein fabricated SERS-encoded nanoparticles (NPs) with stable and simple fingerprint spectrum through synthesis of isotopic cellular Raman-silent graphene-isolated-Au-nanocrystals (GIANs) and conjugation with phospholipid-polyethylene glycol-linked aptamers to target proteins overexpressed on the cancer cell surface. GIANs, which possess the properties of graphitic nanomaterials, such as super-stable optical properties and high Raman cross-section, showed enhanced SERS signals. The 2D-band Raman shift of GIAN, which located in the cellular Raman-silent region, was easily regulated through fabrication of isotopic GIANs without changing their molecular structure. Such GIAN tags demonstrated multiplexed Raman imaging capability, both in vivo and in vitro, with low background interference. Moreover, cell membrane protein (nucleolin, mucin and epithelial cell adhesion molecule)-specific, aptamer-conjugated isotopic GIANs were fabricated and feasibly applied to built-in coding for rapid imaging and pattern recognition of targeted cancer cells. Such isotopic GIAN-aptamer-encoders show high potential for efficient cancer cell identification and diagnosis.