Browsing by Author "Li, Xuan"

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    Activation of the XBP1s/O-GlcNAcylation Pathway Improves Functional Outcome After Cardiac Arrest and Resuscitation in Young and Aged Mice.
    (Shock (Augusta, Ga.), 2021-11) Li, Ran; Shen, Yuntian; Li, Xuan; Lu, Liping; Wang, Zhuoran; Sheng, Huaxin; Hoffmann, Ulrike; Yang, Wei

    Abstract

    After cardiac arrest (CA) and resuscitation, the unfolded protein response (UPR) is activated in various organs including the brain. However, the role of the UPR in CA outcome remains largely unknown. One UPR branch involves spliced X-box-binding protein-1 (XBP1s). Notably, XBP1s, a transcriptional factor, can upregulate expression of specific enzymes related to glucose metabolism, and subsequently boost O-linked β-N-acetylglucosamine modification (O-GlcNAcylation). The current study is focused on effects of the XBP1 UPR branch and its downstream O-GlcNAcylation on CA outcome. Using both loss-of-function and gain-of-function mouse genetic tools, we provide the first evidence that activation of the XBP1 UPR branch in the post-CA brain is neuroprotective. Specifically, neuron-specific Xbp1 knockout mice had worse CA outcome, while mice with neuron-specific expression of Xbp1s in the brain had better CA outcome. Since it has been shown that the protective role of the XBP1s signaling pathway under ischemic conditions is mediated by increasing O-GlcNAcylation, we then treated young mice with glucosamine, and found that functional deficits were mitigated on day 3 post CA. Finally, after confirming that glucosamine can boost O-GlcNAcylation in the aged brain, we subjected aged mice to 8 min CA, and then treated them with glucosamine. We found that glucosamine-treated aged mice performed significantly better in behavioral tests. Together, our data indicate that the XBP1s/O-GlcNAc pathway is a promising target for CA therapy.
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    Chemogenetics-mediated acute inhibition of excitatory neuronal activity improves stroke outcome.
    (Experimental neurology, 2020-04) Wang, Ya-Chao; Galeffi, Francesca; Wang, Wei; Li, Xuan; Lu, Liping; Sheng, Huaxin; Hoffmann, Ulrike; Turner, Dennis A; Yang, Wei

    Background and purpose

    Ischemic stroke significantly perturbs neuronal homeostasis leading to a cascade of pathologic events causing brain damage. In this study, we assessed acute stroke outcome after chemogenetic inhibition of forebrain excitatory neuronal activity.

    Methods

    We generated hM4Di-TG transgenic mice expressing the inhibitory hM4Di, a Designer Receptors Exclusively Activated by Designer Drugs (DREADD)-based chemogenetic receptor, in forebrain excitatory neurons. Clozapine-N-oxide (CNO) was used to activate hM4Di DREADD. Ischemic stroke was induced by transient occlusion of the middle cerebral artery. Neurologic function and infarct volumes were evaluated. Excitatory neuronal suppression in the hM4Di-TG mouse forebrain was assessed electrophysiologically in vitro and in vivo, based on evoked synaptic responses, and in vivo based on occurrence of potassium-induced cortical spreading depolarizations.

    Results

    Detailed characterization of hM4Di-TG mice confirmed that evoked synaptic responses in both in vitro hippocampal slices and in vivo motor cortex were significantly reduced after CNO-mediated activation of the inhibitory hM4Di DREADD. Further, CNO treatment had no obvious effects on physiology and motor function in either control or hM4Di-TG mice. Importantly, hM4Di-TG mice treated with CNO at either 10 min before ischemia or 30 min after reperfusion exhibited significantly improved neurologic function and smaller infarct volumes compared to CNO-treated control mice. Mechanistically, we showed that potassium-induced cortical spreading depression episodes were inhibited, including frequency and duration of DC shift, in CNO-treated hM4Di-TG mice.

    Conclusions

    Our data demonstrate that acute inhibition of a subset of excitatory neurons after ischemic stroke can prevent brain injury and improve functional outcome. This study, together with the previous work in optogenetic neuronal modulation during the chronic phase of stroke, supports the notion that targeting neuronal activity is a promising strategy in stroke therapy.
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    Development and Evaluation of a Novel Mouse Model of Asphyxial Cardiac Arrest Revealed Severely Impaired Lymphopoiesis After Resuscitation.
    (J Am Heart Assoc, 2021-05-20) Wang, Wei; Li, Ran; Miao, Wanying; Evans, Cody; Lu, Liping; Lyu, Jingjun; Li, Xuan; Warner, David S; Zhong, Xiaoping; Hoffmann, Ulrike; Sheng, Huaxin; Yang, Wei
    Background Animal disease models represent the cornerstone in basic cardiac arrest (CA) research. However, current experimental models of CA and resuscitation in mice are limited. In this study, we aimed to develop a mouse model of asphyxial CA followed by cardiopulmonary resuscitation (CPR), and to characterize the immune response after asphyxial CA/CPR. Methods and Results CA was induced in mice by switching from an O2/N2 mixture to 100% N2 gas for mechanical ventilation under anesthesia. Real-time measurements of blood pressure, brain tissue oxygen, cerebral blood flow, and ECG confirmed asphyxia and ensuing CA. After a defined CA period, mice were resuscitated with intravenous epinephrine administration and chest compression. We subjected young adult and aged mice to this model, and found that after CA/CPR, mice from both groups exhibited significant neurologic deficits compared with sham mice. Analysis of post-CA brain confirmed neuroinflammation. Detailed characterization of the post-CA immune response in the peripheral organs of both young adult and aged mice revealed that at the subacute phase following asphyxial CA/CPR, the immune system was markedly suppressed as manifested by drastic atrophy of the spleen and thymus, and profound lymphopenia. Finally, our data showed that post-CA systemic lymphopenia was accompanied with impaired T and B lymphopoiesis in the thymus and bone marrow, respectively. Conclusions In this study, we established a novel validated asphyxial CA model in mice. Using this new model, we further demonstrated that asphyxial CA/CPR markedly affects both the nervous and immune systems, and notably impairs lymphopoiesis of T and B cells.
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    Increasing O-GlcNAcylation is neuroprotective in young and aged brains after ischemic stroke.
    (Experimental neurology, 2021-05) Wang, Zhuoran; Li, Xuan; Spasojevic, Ivan; Lu, Liping; Shen, Yuntian; Qu, Xingguang; Hoffmann, Ulrike; Warner, David S; Paschen, Wulf; Sheng, Huaxin; Yang, Wei
    Spliced X-box binding protein-1 (XBP1s) together with the hexosamine biosynthetic pathway (HBP) and O-GlcNAcylation forms the XBP1s/HBP/O-GlcNAc axis. Our previous studies have provided evidence that activation of this axis is neuroprotective after ischemic stroke and critically, ischemia-induced O-GlcNAcylation is impaired in the aged brain. However, the XBP1s' neuroprotective role and its link to O-GlcNAcylation in stroke, as well as the therapeutic potential of targeting this axis in stroke, have not been well established. Moreover, the mechanisms underlying this age-related impairment of O-GlcNAcylation induction after brain ischemia remain completely unknown. In this study, using transient ischemic stroke models, we first demonstrated that neuron-specific overexpression of Xbp1s improved outcome, and pharmacologically boosting O-GlcNAcylation with thiamet-G reversed worse outcome observed in neuron-specific Xbp1 knockout mice. We further showed that thiamet-G treatment improved long-term functional recovery in both young and aged animals after transient ischemic stroke. Mechanistically, using an analytic approach developed here, we discovered that availability of UDP-GlcNAc was compromised in the aged brain, which may constitute a novel mechanism responsible for the impaired O-GlcNAcylation activation in the aged brain after ischemia. Finally, based on this new mechanistic finding, we evaluated and confirmed the therapeutic effects of glucosamine treatment in young and aged animals using both transient and permanent stroke models. Our data together support that increasing O-GlcNAcylation is a promising strategy in stroke therapy.
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    MCC950, a selective NLPR3 inflammasome inhibitor, improves neurologic function and survival after cardiac arrest and resuscitation.
    (Journal of neuroinflammation, 2020-08-31) Jiang, Maorong; Li, Ran; Lyu, Jingjun; Li, Xuan; Wang, Wei; Wang, Zhuoran; Sheng, Huaxin; Zhang, Weiguo; Karhausen, Jörn; Yang, Wei

    Background

    Cardiac arrest (CA) is associated with high morbidity and mortality, even after spontaneous circulation is re-established. This dire situation is partly due to post-CA syndrome for which no specific and effective intervention is available. One key component of post-CA syndrome is sterile inflammation, which affects various organs including the brain. A major effector of sterile inflammation is activated NLRP3 inflammasome, which leads to increased release of interleukin (IL)-1β. However, how NLRP3 inflammasome impacts neuroinflammation and neurologic outcome after CA is largely undefined.

    Methods

    Mice were subjected to a potassium-based murine CA and cardiopulmonary resuscitation (CPR) model. MCC950 was used to suppress activation of NLRP3 inflammasome after CA/CPR. Levels of protein and mRNA were examined by Western blotting and quantitative PCR, respectively. Immunologic changes were assessed by measuring cytokine expression and immune cell compositions. CA outcomes, including neurologic deficits, bacterial load in the lung, and survival rate, were evaluated.

    Results

    Using our CA/CPR model, we found that NLRP3 inflammasome was activated in the post-CA brain, and that pro-inflammatory cytokine levels, including IL-1β, were increased. After treatment with MCC950, a potent and selective NLRP3 inflammasome inhibitor, mice exhibited improved functional recovery and survival rate during the 14-day observational period after CA/CPR. In line with these findings, IL-1β mRNA levels in the post-CA brain were significantly suppressed after MCC950 treatment. Interestingly, we also found that in MCC950- vs. vehicle-treated CA mice, immune homeostasis in the spleen was better preserved and bacterial load in the lung was significantly reduced.

    Conclusions

    Our data demonstrate that activation of NLRP3 inflammasome could be a key event shaping the post-CA immuno- and neuro-pathology, and identify this pathway as a unique and promising therapeutic target to improve outcomes after CA/CPR.
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    PERK (Protein Kinase RNA-Like ER Kinase) Branch of the Unfolded Protein Response Confers Neuroprotection in Ischemic Stroke by Suppressing Protein Synthesis.
    (Stroke, 2020-05) Wang, Ya-Chao; Li, Xuan; Shen, Yuntian; Lyu, Jingjun; Sheng, Huaxin; Paschen, Wulf; Yang, Wei
    Background and Purpose- Ischemic stroke impairs endoplasmic reticulum (ER) function, causes ER stress, and activates the unfolded protein response. The unfolded protein response consists of 3 branches controlled by ER stress sensor proteins, which include PERK (protein kinase RNA-like ER kinase). Activated PERK phosphorylates eIF2α (eukaryotic initiation factor 2 alpha), resulting in inhibition of global protein synthesis. Here, we aimed to clarify the role of the PERK unfolded protein response branch in stroke. Methods- Neuron-specific and tamoxifen-inducible PERK conditional knockout (cKO) mice were generated by cross-breeding Camk2a-CreERT2 with Perkf/f mice. Transient middle cerebral artery occlusion was used to induce stroke. Short- and long-term stroke outcomes were evaluated. Protein synthesis in the brain was assessed using a surface-sensing-of-translation approach. Results- After tamoxifen-induced deletion of Perk in forebrain neurons was confirmed in PERK-cKO mice, PERK-cKO and control mice were subjected to transient middle cerebral artery occlusion and 3 days or 3 weeks recovery. PERK-cKO mice had larger infarcts and worse neurological outcomes compared with control mice, suggesting that PERK-induced eIF2α phosphorylation and subsequent suppression of translation protects neurons from ischemic stress. Indeed, better stroke outcomes were observed in PERK-cKO mice that received postischemic treatment with salubrinal, which can restore the ischemia-induced increase in phosphorylated eIF2α in these mice. Finally, our data showed that post-treatment with salubrinal improved functional recovery after stroke. Conclusions- Here, we presented the first evidence that postischemic suppression of translation induced by PERK activation promotes recovery of neurological function after stroke. This confirms and further extends our previous observations that recovery of ER function impaired by ischemic stress critically contributes to stroke outcome. Therefore, future research should include strategies to improve stroke outcome by targeting unfolded protein response branches to restore protein homeostasis in neurons.
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    Post-ischemia common carotid artery occlusion worsens memory loss, but not sensorimotor deficits, in long-term survived stroke mice.
    (Brain research bulletin, 2022-06) Yang, Zhong; Li, Xuan; Cao, Zhipeng; Wang, Peng; Warner, David S; Sheng, Huaxin
    Ischemic stroke in rodents is usually induced by intraluminal occlusion of the middle cerebral artery (MCA) via the external carotid artery (ECA) or the common carotid artery (CCA). The latter route requires permanent CCA occlusion after ischemia, and here, we assess its effects on long-term outcomes. Transient occlusion of MCA and CCA was performed at normal body temperature. After 90 min of ischemia, mice were randomized to permanent CCA occlusion or no occlusion (control group). Body weight, and motor and sensory functions, ie, pole test, adhesive tape removal, and elevated plus maze, were evaluated at 24 h, and at 7 and 28 days after stroke. Infarct volume, apoptosis, and activation of astrocytes and microglia were assessed at 4 weeks by an investigator blinded to groups. The Morris water maze test was performed at 3 weeks in the second experiment. One mouse died at 4 days, and the other mice survived with persistent neurologic deficits. CCA-occluded mice exhibited delayed turn on the pole at 24 h and decreased responses to the von Frey filament, and spent more time on the pole at 7 and 28 days than the control group. Infarction, hemispheric atrophy, glial activation, and apoptotic neuronal death were present in all mice, and no intra-group difference was found. However, CCA-occluded mice had a significantly poorer performance in the Morris water maze compared to the control group, which showed an adverse effect of post-ischemia CCA occlusion on cognition. Thus, the model selection should be well considered in preclinical efficacy studies on stroke-induced vascular dementia and stroke with Alzheimer's disease.
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    Single-cell transcriptomic analysis of the immune cell landscape in the aged mouse brain after ischemic stroke.
    (Journal of neuroinflammation, 2022-04-07) Li, Xuan; Lyu, Jingjun; Li, Ran; Jain, Vaibhav; Shen, Yuntian; Del Águila, Ángela; Hoffmann, Ulrike; Sheng, Huaxin; Yang, Wei

    Background

    Ischemic stroke is a medical emergency that primarily affects the elderly. A complex immune response in the post-stroke brain constitutes a key component of stroke pathophysiology. This study aimed to determine how stroke affects immune cell populations in the aged brain based on molecular profiles of individual cells.

    Methods

    Single-cell RNA sequencing and a new transient ischemic stroke mouse model with late reperfusion were used.

    Results

    We generated, for the first time, a composite picture of immune cell populations in the stroke aged brain at single-cell resolution. We discovered at least 6 microglial subsets in the stroke aged brain, including a potentially stroke-specific subtype. Moreover, we identified major cell subpopulations formed by infiltrated myeloid cells after stroke, and revealed their unique molecular profiles.

    Conclusions

    This study provided the first scRNA-seq data set for immune cells in the stroke aged brain, and offered novel insights into post-stroke immune cell heterogeneity.