Browsing by Author "Yang, Wei"
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Item Open Access Activation of the ATF6 (Activating Transcription Factor 6) Signaling Pathway in Neurons Improves Outcome After Cardiac Arrest in Mice.(Journal of the American Heart Association, 2021-06-11) Shen, Yuntian; Li, Ran; Yu, Shu; Zhao, Qiang; Wang, Zhuoran; Sheng, Huaxin; Yang, WeiBackground Ischemia/reperfusion injury impairs proteostasis, and triggers adaptive cellular responses, such as the unfolded protein response (UPR), which functions to restore endoplasmic reticulum homeostasis. After cardiac arrest (CA) and resuscitation, the UPR is activated in various organs including the brain. However, the role of the UPR in CA has remained largely unknown. Here we aimed to investigate effects of activation of the ATF6 (activating transcription factor 6) UPR branch in CA. Methods and Results Conditional and inducible sATF6-KI (short-form ATF6 knock-in) mice and a selective ATF6 pathway activator 147 were used. CA was induced in mice by KCl injection, followed by cardiopulmonary resuscitation. We first found that neurologic function was significantly improved, and neuronal damage was mitigated after the ATF6 pathway was activated in neurons of sATF6-KI mice subjected to CA/cardiopulmonary resuscitation. Further RNA sequencing analysis indicated that such beneficial effects were likely attributable to increased expression of pro-proteostatic genes regulated by ATF6. Especially, key components of the endoplasmic reticulum-associated degradation process, which clears potentially toxic unfolded/misfolded proteins in the endoplasmic reticulum, were upregulated in the sATF6-KI brain. Accordingly, the CA-induced increase in K48-linked polyubiquitin in the brain was higher in sATF6-KI mice relative to control mice. Finally, CA outcome, including the survival rate, was significantly improved in mice treated with compound 147. Conclusions This is the first experimental study to determine the role of the ATF6 UPR branch in CA outcome. Our data indicate that the ATF6 UPR branch is a prosurvival pathway and may be considered as a therapeutic target for CA.Item Open Access Activation of the ATF6 branch of the unfolded protein response in neurons improves stroke outcome.(Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 2017-03) Yu, Zhui; Sheng, Huaxin; Liu, Shuai; Zhao, Shengli; Glembotski, Christopher C; Warner, David S; Paschen, Wulf; Yang, WeiImpaired function of the endoplasmic reticulum (ER stress) is a hallmark of many human diseases including stroke. To restore ER function in stressed cells, the unfolded protein response (UPR) is induced, which activates 3 ER stress sensor proteins including activating transcription factor 6 (ATF6). ATF6 is then cleaved by proteases to form the short-form ATF6 (sATF6), a transcription factor. To determine the extent to which activation of the ATF6 UPR branch defines the fate and function of neurons after stroke, we generated a conditional and tamoxifen-inducible sATF6 knock-in mouse. To express sATF6 in forebrain neurons, we crossed our sATF6 knock-in mouse line with Emx1-Cre mice to generate ATF6-KI mice. After the ATF6 branch was activated in ATF6-KI mice with tamoxifen, mice were subjected to transient middle cerebral artery occlusion. Forced activation of the ATF6 UPR branch reduced infarct volume and improved functional outcome at 24 h after stroke. Increased autophagic activity at early reperfusion time after stroke may contribute to the ATF6-mediated neuroprotection. We concluded that the ATF6 UPR branch is crucial to ischemic stroke outcome. Therefore, boosting UPR pro-survival pathways may be a promising therapeutic strategy for stroke.Item Open Access 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, WeiAbstract
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.Item Open Access Aging Is Associated With Impaired Activation of Protein Homeostasis-Related Pathways After Cardiac Arrest in Mice.(Journal of the American Heart Association, 2018-09) Shen, Yuntian; Yan, Baihui; Zhao, Qiang; Wang, Zhuoran; Wu, Jiangbo; Ren, Jiafa; Wang, Wei; Yu, Shu; Sheng, Huaxin; Crowley, Steven D; Ding, Fei; Paschen, Wulf; Yang, WeiBackground The mechanisms underlying worse outcome at advanced age after cardiac arrest ( CA ) and resuscitation are not well understood. Because protein homeostasis (proteostasis) is essential for cellular and organismal health, but is impaired after CA , we investigated the effects of age on proteostasis-related prosurvival pathways activated after CA . Methods and Results Young (2-3 months old) and aged (21-22 months old) male C57Bl/6 mice were subjected to CA and cardiopulmonary resuscitation ( CPR ). Functional outcome and organ damage were evaluated by assessing neurologic deficits, histological features, and creatinine level. CA / CPR -related changes in small ubiquitin-like modifier conjugation, ubiquitination, and the unfolded protein response were analyzed by measuring mRNA and protein levels in the brain, kidney, and spinal cord. Thiamet-G was used to increase O-linked β-N-acetylglucosamine modification. After CA / CPR , aged mice had trended lower survival rates, more severe tissue damage in the brain and kidney, and poorer recovery of neurologic function compared with young mice. Furthermore, small ubiquitin-like modifier conjugation, ubiquitination, unfolded protein response, and O-linked β-N-acetylglucosamine modification were activated after CA / CPR in young mice, but their activation was impaired in aged mice. Finally, pharmacologically increasing O-linked β-N-acetylglucosamine modification after CA improved outcome. Conclusions Results suggest that impaired activation of prosurvival pathways contributes to worse outcome after CA / CPR in aged mice because restoration of proteostasis is critical to the survival of cells stressed by ischemia. Therefore, a pharmacologic intervention that targets aging-related impairment of proteostasis-related pathways after CA / CPR may represent a promising therapeutic strategy.Item Open Access Argon Inhalation for 24 Hours After Onset of Permanent Focal Cerebral Ischemia in Rats Provides Neuroprotection and Improves Neurologic Outcome.(Critical care medicine, 2019-08) Ma, Shuang; Chu, Dongmei; Li, Litao; Creed, Jennifer A; Ryang, Yu-Mi; Sheng, Huaxin; Yang, Wei; Warner, David S; Turner, Dennis A; Hoffmann, UlrikeObjectives
We tested the hypothesis that prolonged inhalation of 70% argon for 24 hours after in vivo permanent or temporary stroke provides neuroprotection and improves neurologic outcome and overall recovery after 7 days.Design
Controlled, randomized, double-blinded laboratory study.Setting
Animal research laboratories.Subjects
Adult Wistar male rats (n = 110).Interventions
Rats were subjected to permanent or temporary focal cerebral ischemia via middle cerebral artery occlusion, followed by inhalation of 70% argon or nitrogen in 30% oxygen for 24 hours. On postoperative day 7, a 48-point neuroscore and histologic lesion size were assessed.Measurements and main results
After argon inhalation for 24 hours immediately following "severe permanent ischemia" induction, neurologic outcome (neuroscore, p = 0.034), overall recovery (body weight, p = 0.02), and infarct volume (total infarct volume, p = 0.0001; cortical infarct volume, p = 0.0003; subcortical infarct volume, p = 0.0001) were significantly improved. When 24-hour argon treatment was delayed for 2 hours after permanent stroke induction or until after postischemic reperfusion treatment, neurologic outcomes remained significantly improved (neuroscore, p = 0.043 and p = 0.014, respectively), as was overall recovery (body weight, p = 0.015), compared with nitrogen treatment. However, infarct volume and 7-day mortality were not significantly reduced when argon treatment was delayed.Conclusions
Neurologic outcome (neuroscore), overall recovery (body weight), and infarct volumes were significantly improved after 24-hour inhalation of 70% argon administered immediately after severe permanent stroke induction. Neurologic outcome and overall recovery were also significantly improved even when argon treatment was delayed for 2 hours or until after reperfusion.Item Open Access Cardiac arrest and resuscitation activates the hypothalamic-pituitary-adrenal axis and results in severe immunosuppression.(Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 2021-05) Zhao, Qiang; Shen, Yuntian; Li, Ran; Wu, Jiangbo; Lyu, Jingjun; Jiang, Maorong; Lu, Liping; Zhu, Minghua; Wang, Wei; Wang, Zhuoran; Liu, Qiang; Hoffmann, Ulrike; Karhausen, Jörn; Sheng, Huaxin; Zhang, Weiguo; Yang, WeiIn patients who are successfully resuscitated after initial cardiac arrest (CA), mortality and morbidity rates are high, due to ischemia/reperfusion injury to the whole body including the nervous and immune systems. How the interactions between these two critical systems contribute to post-CA outcome remains largely unknown. Using a mouse model of CA and cardiopulmonary resuscitation (CA/CPR), we demonstrate that CA/CPR induced neuroinflammation in the brain, in particular, a marked increase in pro-inflammatory cytokines, which subsequently activated the hypothalamic-pituitary-adrenal (HPA) axis. Importantly, this activation was associated with a severe immunosuppression phenotype after CA. The phenotype was characterized by a striking reduction in size of lymphoid organs accompanied by a massive loss of immune cells and reduced immune function of splenic lymphocytes. The mechanistic link between post-CA immunosuppression and the HPA axis was substantiated, as we discovered that glucocorticoid treatment, which mimics effects of the activated HPA axis, exacerbated post-CA immunosuppression, while RU486 treatment, which suppresses its effects, significantly mitigated lymphopenia and lymphoid organ atrophy and improved CA outcome. Taken together, targeting the HPA axis could be a viable immunomodulatory therapeutic to preserve immune homeostasis after CA/CPR and thus improve prognosis of post-resuscitation CA patients.Item Open Access Cervical Vagus Nerve Stimulation Improves Neurologic Outcome After Cardiac Arrest in Mice by Attenuating Oxidative Stress and Excessive Autophagy.(Neuromodulation : journal of the International Neuromodulation Society, 2022-04) Duan, Weina; Sun, Qian; Wu, Xiaojing; Xia, Zhongyuan; Warner, David S; Ulloa, Luis; Yang, Wei; Sheng, HuaxinBackground
Cerebral ischemia and reperfusion (I/R) induces oxidative stress and activates autophagy, leading to brain injury and neurologic deficits. Cervical vagus nerve stimulation (VNS) increases cerebral blood flow (CBF). In this study, we investigate the effect of VNS-induced CBF increase on neurologic outcomes after cardiac arrest (CA).Materials and methods
A total of 40 male C57Bl/6 mice were subjected to ten minutes of asphyxia CA and randomized to vagus nerve isolation (VNI) or VNS treatment group. Eight mice received sham surgery and VNI. Immediately after resuscitation, 20 minutes of electrical stimulation (1 mA, 1 ms, and 10 Hz) was started in the VNS group. Electrocardiogram, blood pressure, and CBF were monitored. Neurologic and histologic outcomes were evaluated at 72 hours. Oxidative stress and autophagy were assessed at 3 hours and 24 hours after CA.Results
Baseline characteristics were not different among groups. VNS mice had better behavioral performance (ie, open field, rotarod, and neurologic score) and less neuronal death (p < 0.05, vs VNI) in the hippocampus. CBF was significantly increased in VNS-treated mice at 20 minutes after return of spontaneous circulation (ROSC) (p < 0.05). Furthermore, levels of 8-hydroxy-2'-deoxyguanosine in the blood and autophagy-related proteins (ie, LC-3Ⅱ/Ⅰ, Beclin-1, and p62) in the brain were significantly decreased in VNS mice. Aconitase activity was also reduced, and the p-mTOR/mTOR ratio was increased in VNS mice.Conclusions
Oxidative stress induced by global brain I/R following CA/ROSC leads to early excessive autophagy and impaired autophagic flux. VNS promoted CBF recovery, ameliorating these changes. Neurologic and histologic outcomes were also improved.Item Open Access Characterization of the ubiquitin-modified proteome regulated by transient forebrain ischemia.(Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 2014-03) Iwabuchi, Masahiro; Sheng, Huaxin; Thompson, J Will; Wang, Liangli; Dubois, Laura G; Gooden, David; Moseley, Marthur; Paschen, Wulf; Yang, WeiUbiquitylation is a posttranslational protein modification that modulates various cellular processes of key significance, including protein degradation and DNA damage repair. In animals subjected to transient cerebral ischemia, ubiquitin-conjugated proteins accumulate in Triton-insoluble aggregates. Although this process is widely considered to modulate the fate of postischemic neurons, few attempts have been made to characterize the ubiquitin-modified proteome in these aggregates. We performed proteomics analyses to identify ubiquitylated proteins in postischemic aggregates. Mice were subjected to 10 minutes of forebrain ischemia and 4 hours of reperfusion. The hippocampi were dissected, aggregates were isolated, and trypsin-digested after spiking with GG-BSA as internal standard. K-ɛ-GG-containing peptides were immunoprecipitated and analyzed by label-free quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. We identified 1,664 peptides to 520 proteins containing at least one K-ɛ-GG. Sixty-six proteins were highly ubiquitylated, with 10 or more K-ɛ-GG peptides. Based on selection criteria of greater than fivefold increase and P<0.001, 763 peptides to 272 proteins were highly enriched in postischemic aggregates. These included proteins involved in important neuronal functions and signaling pathways that are impaired after ischemia. Results of this study could serve as an important platform to uncover the mechanisms linking insoluble ubiquitin aggregates to the functions of postischemic neurons.Item Open Access 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, WeiBackground 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.Item Open Access Comparative genomics reveals insights into avian genome evolution and adaptation.(Science, 2014-12-12) Zhang, Guojie; Li, Cai; Li, Qiye; Li, Bo; Larkin, Denis M; Lee, Chul; Storz, Jay F; Antunes, Agostinho; Greenwold, Matthew J; Meredith, Robert W; Ödeen, Anders; Cui, Jie; Zhou, Qi; Xu, Luohao; Pan, Hailin; Wang, Zongji; Jin, Lijun; Zhang, Pei; Hu, Haofu; Yang, Wei; Hu, Jiang; Xiao, Jin; Yang, Zhikai; Liu, Yang; Xie, Qiaolin; Yu, Hao; Lian, Jinmin; Wen, Ping; Zhang, Fang; Li, Hui; Zeng, Yongli; Xiong, Zijun; Liu, Shiping; Zhou, Long; Huang, Zhiyong; An, Na; Wang, Jie; Zheng, Qiumei; Xiong, Yingqi; Wang, Guangbiao; Wang, Bo; Wang, Jingjing; Fan, Yu; da Fonseca, Rute R; Alfaro-Núñez, Alonzo; Schubert, Mikkel; Orlando, Ludovic; Mourier, Tobias; Howard, Jason T; Ganapathy, Ganeshkumar; Pfenning, Andreas; Whitney, Osceola; Rivas, Miriam V; Hara, Erina; Smith, Julia; Farré, Marta; Narayan, Jitendra; Slavov, Gancho; Romanov, Michael N; Borges, Rui; Borges, Rui; Machado, João Paulo; Khan, Imran; Springer, Mark S; Gatesy, John; Hoffmann, Federico G; Opazo, Juan C; Håstad, Olle; Sawyer, Roger H; Kim, Heebal; Kim, Kyu-Won; Kim, Hyeon Jeong; Cho, Seoae; Li, Ning; Huang, Yinhua; Bruford, Michael W; Zhan, Xiangjiang; Dixon, Andrew; Bertelsen, Mads F; Derryberry, Elizabeth; Warren, Wesley; Wilson, Richard K; Li, Shengbin; Ray, David A; Green, Richard E; O'Brien, Stephen J; Griffin, Darren; Johnson, Warren E; Haussler, David; Ryder, Oliver A; Willerslev, Eske; Graves, Gary R; Alström, Per; Fjeldså, Jon; Mindell, David P; Edwards, Scott V; Braun, Edward L; Rahbek, Carsten; Burt, David W; Houde, Peter; Zhang, Yong; Yang, Huanming; Wang, Jian; Avian Genome Consortium; Jarvis, Erich D; Gilbert, M Thomas P; Wang, JunBirds are the most species-rich class of tetrapod vertebrates and have wide relevance across many research fields. We explored bird macroevolution using full genomes from 48 avian species representing all major extant clades. The avian genome is principally characterized by its constrained size, which predominantly arose because of lineage-specific erosion of repetitive elements, large segmental deletions, and gene loss. Avian genomes furthermore show a remarkably high degree of evolutionary stasis at the levels of nucleotide sequence, gene synteny, and chromosomal structure. Despite this pattern of conservation, we detected many non-neutral evolutionary changes in protein-coding genes and noncoding regions. These analyses reveal that pan-avian genomic diversity covaries with adaptations to different lifestyles and convergent evolution of traits.Item Open Access 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, WeiBackground 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.Item Open Access Ectopic Expression of OsJAZs Alters Plant Defense and Development.(International journal of molecular sciences, 2022-04) Sun, Baolong; Shang, Luyue; Li, Yang; Zhang, Qiang; Chu, Zhaohui; He, Shengyang; Yang, Wei; Ding, XinhuaA key step in jasmonic acid (JA) signaling is the ligand-dependent assembly of a coreceptor complex comprising the F-box protein COI1 and JAZ transcriptional repressors. The assembly of this receptor complex results in proteasome-mediated degradation of JAZ repressors, which in turn bind and repress MYC transcription factors. Many studies on JAZs have been performed in Arabidopsis thaliana, but the function of JAZs in rice is largely unknown. To systematically reveal the function of OsJAZs, in this study, we compared the various phenotypes resulting from 13 OsJAZs via ectopic expression in Arabidopsis thaliana and the phenotypes of 12 AtJAZs overexpression (OE) lines. Phylogenetic analysis showed that the 25 proteins could be divided into three major groups. Yeast two-hybrid (Y2H) assays revealed that most OsJAZ proteins could form homodimers or heterodimers. The statistical results showed that the phenotypes of the OsJAZ OE plants were quite different from those of AtJAZ OE plants in terms of plant growth, development, and immunity. As an example, compared with other JAZ OE plants, OsJAZ11 OE plants exhibited a JA-insensitive phenotype and enhanced resistance to Pst DC3000. The protein stability after JA treatment of OsJAZ11 emphasized the specific function of the protein. This study aimed to explore the commonalities and characteristics of different JAZ proteins functions from a genetic perspective, and to screen genes with disease resistance value. Overall, the results of this study provide insights for further functional analysis of rice JAZ family proteins.Item Open Access High-speed widefield photoacoustic microscopy of small-animal hemodynamics.(Biomedical optics express, 2018-10) Lan, Bangxin; Liu, Wei; Wang, Ya-Chao; Shi, Junhui; Li, Yang; Xu, Song; Sheng, Huaxin; Zhou, Qifa; Zou, Jun; Hoffmann, Ulrike; Yang, Wei; Yao, JunjieOptical-resolution photoacoustic microscopy (OR-PAM) has become a popular tool in small-animal hemodynamic studies. However, previous OR-PAM techniques variously lacked a high imaging speed and/or a large field of view, impeding the study of highly dynamic physiologic and pathophysiologic processes over a large region of interest. Here we report a high-speed OR-PAM system with an ultra-wide field of view, enabled by an innovative water-immersible hexagon-mirror scanner. By driving the hexagon-mirror scanner with a high-precision DC motor, the new OR-PAM has achieved a cross-sectional frame rate of 900 Hz over a 12-mm scanning range, which is 3900 times faster than our previous motor-scanner-based system and 10 times faster than the MEMS-scanner-based system. Using this hexagon-scanner-based OR-PAM system, we have imaged epinephrine-induced vasoconstriction in the whole mouse ear and vascular reperfusion after ischemic stroke in the mouse cortex in vivo, with a high spatial resolution and high volumetric imaging speed. We expect that the hexagon-scanner-based OR-PAM system will become a powerful tool for small animal imaging where the hemodynamic responses over a large field of view are of interest.Item Open Access 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, WeiSpliced 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.Item Open Access 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, WeiBackground
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.Item Open Access Neuron-specific SUMO knockdown suppresses global gene expression response and worsens functional outcome after transient forebrain ischemia in mice.(Neuroscience, 2017-02) Zhang, Lin; Liu, Xiaozhi; Sheng, Huaxin; Liu, Shuai; Li, Ying; Zhao, Julia Q; Warner, David S; Paschen, Wulf; Yang, WeiSmall ubiquitin-like modifier (SUMO) conjugation (SUMOylation) plays key roles in neurologic function in health and disease. Neuronal SUMOylation is essential for emotionality and cognition, and this pathway is dramatically activated in post-ischemic neurons, a neuroprotective response to ischemia. It is also known from cell culture studies that SUMOylation modulates gene expression. However, it remains unknown how SUMOylation regulates neuronal gene expression in vivo, in the physiologic state and after ischemia, and modulates post-ischemic recovery of neurologic function. To address these important questions, we used a SUMO1-3 knockdown (SUMO-KD) mouse in which a Thy-1 promoter drives expression of 3 distinct microRNAs against SUMO1-3 to silence SUMO expression specifically in neurons. Wild-type and SUMO-KD mice were subjected to transient forebrain ischemia. Microarray analysis was performed in hippocampal CA1 samples, and neurologic function was evaluated. SUMOylation had opposite effects on neuronal gene expression before and after ischemia. In the physiological state, most genes regulated by SUMOylation were up-regulated in SUMO-KD compared to wild-type mice. Brain ischemia/reperfusion significantly modulated the expression levels of more than 400 genes in wild-type mice, with a majority of those genes upregulated. The extent of this post-ischemic transcriptome change was suppressed in SUMO-KD mice. Moreover, SUMO-KD mice exhibited significantly worse functional outcome. This suggests that suppression of global gene expression response in post-ischemic brain due to SUMO knockdown has a negative effect on post-ischemic neurologic function. Together, our data provide a basis for future studies to mechanistically link SUMOylation to neurologic function in health and disease.Item Open Access Neuron-specific Sumo1-3 knockdown in mice impairs episodic and fear memories.(Journal of psychiatry & neuroscience : JPN, 2014-07) Wang, Liangli; Rodriguiz, Ramona M; Wetsel, William C; Sheng, Huaxin; Zhao, Shengli; Liu, Xiaozhi; Paschen, Wulf; Yang, WeiBACKGROUND:Growing evidence suggests that small ubiquitin-like modifier (SUMO) conjugation plays a key role in brain plasticity by modulating activity-dependent synaptic transmission. However, these observations are based largely on cell culture experiments. We hypothesized that episodic and fear memories would be affected by silencing SUMO1-3 expression. METHODS:To investigate the role of SUMO conjugation in neuronal functioning in vivo, we generated a novel Sumo transgenic mouse model in which a Thy1 promoter drives expression of 3 distinct microRNAs to silence Sumo1-3 expression, specifically in neurons. Wild-type and Sumo1-3 knockdown mice were subjected to a battery of behavioural tests to elucidate whether Sumoylation is involved in episodic and emotional memory. RESULTS:Expression of Sumo1-3 microRNAs and the corresponding silencing of Sumo expression were particularly pronounced in hippocampal, amygdala and layer V cerebral cortex neurons. The Sumo knockdown mice displayed anxiety-like responses and were impaired in episodic memory processes, contextual and cued fear conditioning and fear-potentiated startle. LIMITATIONS:Since expression of Sumo1-3 was silenced in this mouse model, we need to verify in future studies which of the SUMO paralogues play the pivotal role in episodic and emotional memory. CONCLUSION:Our results indicate that a functional SUMO conjugation pathway is essential for emotionality and cognition. This novel Sumo knockdown mouse model and the technology used in generating this mutant may help to reveal novel mechanisms that underlie a variety of neuropsychiatric conditions associated with anxiety and impairment of episodic and emotional memory.Item Open Access Novel Modification of Potassium Chloride Induced Cardiac Arrest Model for Aged Mice.(Aging and disease, 2018-02) Liu, Huaqin; Yu, Zhui; Li, Ying; Xu, Bin; Yan, Baihui; Paschen, Wulf; Warner, David S; Yang, Wei; Sheng, HuaxinExperimental cardiac arrest (CA) in aging research is infrequently studied in part due to the limitation of animal models. We aimed to develop an easily performed mouse CA model to meet this need. A standard mouse KCl-induced CA model using chest compressions and intravenous epinephrine for resuscitation was modified by blood withdrawal prior to CA onset, so as to decrease the requisite KCl dose to induce CA by decreasing the circulating blood volume. The modification was then compared to the standard model in young adult mice subjected to 8 min CA. 22-month old mice were then subjected to 8 min CA, resuscitated, and compared to young adult mice. Post-CA functional recovery was evaluated by measuring spontaneous locomotor activity pre-injury, and on post-CA days 1, 2, and 3. Neurological score and brain histology were examined on day 3. Brain elF2α phosphorylation levels were measured at 1 h to verify tissue stress. Compared to the standard model, the modification decreased cardiopulmonary resuscitation duration and increased 3-day survival in young mice. For aged mice, survival was 100 % at 24 h and 54% at 72 h. Neurological deficit was present 3 days post-CA, although more severe versus young mice. Mild neuronal necrosis was present in the cortex and hippocampus. The modified model markedly induced elF2α phosphorylation in both age groups. This modified procedure makes the CA model feasible in aged mice and provides a practical platform for understanding injury mechanisms and developing therapeutics for elderly patients.Item Open Access O-linked β-N-acetylglucosamine modification of proteins is activated in post-ischemic brains of young but not aged mice: Implications for impaired functional recovery from ischemic stress.(Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, 2016-02) Liu, Shuai; Sheng, Huaxin; Yu, Zhui; Paschen, Wulf; Yang, WeiTo evaluate the effect of age on the response of brains to an ischemic challenge, we subjected young and aged mice to transient forebrain ischemia, and analyzed the heat shock response and unfolded protein response, ubiquitin conjugation and SUMO conjugation, and O-linked β-N-acetylglucosamine modification of proteins (O-GlcNAcylation). The most prominent age-related difference was an inability of aged mice to activate O-GlcNAcylation. Considering many reports on the protective role of O-GlcNAcylation in various stress conditions including myocardial ischemia, this pathway could be a promising target for therapeutic intervention to improve functional recovery of aged patients following brain ischemia.Item Open Access 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, WeiBackground 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.