Plasma metabolomic profiling of patients with transient ischemic attack reveals positive role of neutrophils in ischemic tolerance.

BACKGROUND: Transient ischemic attack (TIA) induces ischemic tolerance that can reduce the subsequent ischemic damage and improve prognosis of patients with stroke. However, the underlying mechanisms remain elusive. Recent advances in plasma metabolomics analysis have made it a powerful tool to investigate human pathophysiological phenotypes and mechanisms of diseases. In this study, we aimed to identify the bioactive metabolites from the plasma of patients with TIA for determination of their prophylactic and therapeutic effects on protection against cerebral ischemic stroke, and the mechanism of TIA-induced ischemic tolerance against subsequent stroke. METHODS: Metabolomic profiling using liquid chromatography-mass spectrometry was performed to identify the TIA-induced differential bioactive metabolites in the plasma samples of 20 patients at day 1 (time for basal metabolites) and day 7 (time for established chronic ischemic tolerance-associated metabolites) after onset of TIA. Mouse middle cerebral artery occlusion (MCAO)-induced stroke model was used to verify their prophylactic and therapeutic potentials. Transcriptomics changes in circulating neutrophils of patients with TIA were determined by RNA-sequencing. Multivariate statistics and integrative analysis of metabolomics and transcriptomics were performed to elucidate the potential mechanism of TIA-induced ischemic tolerance. FINDINGS: Plasma metabolomics analysis identified five differentially upregulated metabolites associated with potentially TIA-induced ischemic tolerance, namely all-trans 13,14 dihydroretinol (atDR), 20-carboxyleukotriene B4, prostaglandin B2, cortisol and 9-KODE. They were associated with the metabolic pathways of retinol, arachidonic acid, and neuroactive ligand-receptor interaction. Prophylactic treatment of MCAO mice with these five metabolites significantly improved neurological functions. Additionally, post-stroke treatment with atDR or 9-KODE significantly reduced the cerebral infarct size and enhanced sensorimotor functions, demonstrating the therapeutic potential of these bioactive metabolites. Mechanistically, we found in patients with TIA that these metabolites were positively correlated with circulating neutrophil counts. Integrative analysis of plasma metabolomics and neutrophil transcriptomics further revealed that TIA-induced metabolites are significantly correlated with specific gene expression in circulating neutrophils which showed prominent enrichment in FoxO signaling pathway and upregulation of the anti-inflammatory cytokine IL-10. Finally, we demonstrated that the protective effect of atDR-pretreatment on MCAO mice was abolished when circulating neutrophils were depleted. INTERPRETATION: TIA-induced potential ischemic tolerance is associated with upregulation of plasma bioactive metabolites which can protect against cerebral ischemic damage and improve neurological functions through a positive role of circulating neutrophils. FUNDING: National Natural Science Foundation of China (81974210), Science and Technology Planning Project of Guangdong Province, China (2020A0505100045), Natural Science Foundation of Guangdong Province (2019A1515010671), Science and Technology Program of Guangzhou, China (2023A03J0577), and Natural Science Foundation of Jiangxi, China（20224BAB216043).

How to use the Surveillance, Epidemiology, and End Results (SEER) data: research design and methodology.

In the United States (US), the Surveillance, Epidemiology, and End Results (SEER) program is the only comprehensive source of population-based information that includes stage of cancer at the time of diagnosis and patient survival data. This program aims to provide a database about cancer incidence and survival for studies of surveillance and the development of analytical and methodological tools in the cancer field. Currently, the SEER program covers approximately half of the total cancer patients in the US. A growing number of clinical studies have applied the SEER database in various aspects. However, the intrinsic features of the SEER database, such as the huge data volume and complexity of data types, have hindered its application. In this review, we provided a systematic overview of the commonly used methodologies and study designs for retrospective epidemiological research in order to illustrate the application of the SEER database. Therefore, the goal of this review is to assist researchers in the selection of appropriate methods and study designs for enhancing the robustness and reliability of clinical studies by mining the SEER database.

Spatial Proteomics Analysis of Soft and Stiff Regions in Human Acute Arterial Thrombus.

BACKGROUND: The soft regions of a thrombus tend to be more susceptible to r-tPA (recombinant tissue-type plasminogen activator)-mediated thrombolysis and are more easily removed by mechanical thrombectomy than the stiff counterpart. This study aimed to understand the molecular pathological differences between the soft and stiff regions of human arterial thrombus. METHODS: We developed a spatial proteomic workflow combining proteomics with laser-captured microdissection to analyze human arterial thrombi with Masson trichrome staining to identify stiff and soft regions from 2 independent cohorts of patients with acute myocardial or cerebral infarction. Dysregulated proteins in a C57BL6/J male mouse model of arterial thrombosis were identified by pathway enrichment and pairwise analyses from the common gene ontology enrichment and dysregulated proteins between carotid and coronary arterial thrombi, and validated by immunohistochemistry. RESULTS: Spatial proteomics of the coronary arterial thrombi collected from 7 patients with myocardial infarct revealed 7 common dysregulated proteins in 2 cohorts of patients, and upregulation of TGF-ß1 (transforming growth factor ß1) was the most prominent fibrosis-related protein. Inhibition of TGF-ß1 resulted in delayed arterial thrombosis and accelerated blood flow restoration in mouse model. We further expanded the spatial proteomic workflow to the carotid artery thrombi collected from 11 patients with cerebral infarction. Pairwise proteomic analysis of stiff and soft regions between carotid and coronary arterial thrombi further revealed 5 common gene ontology clusters including features of platelet activation, and a common dysregulated protein COL1A1 (collagen type 1 alpha 1) that was reported to be influenced by TGF-ß1. We also verified the expression in human and mice carotid arterial thrombi. CONCLUSIONS: This study demonstrates the spatially distinct composition of proteins in the stiff and soft regions of human arterial thrombi, and suggests that TGF-ß1 is a key therapeutic target for promoting arterial thrombolysis.

Maf1 is an intrinsic suppressor against spontaneous neural repair and functional recovery after ischemic stroke.

INTRODUCTION: Spontaneous recovery after CNS injury is often very limited and incomplete, leaving most stroke patients with permanent disability. Maf1 is known as a key growth suppressor in proliferating cells. However, its role in neuronal cells after stroke remains unclear. OBJECTIVE: We aimed to investigate the mechanistic role of Maf1 in spontaneous neural repair and evaluated the therapeutic effect of targeting Maf1 on stroke recovery. METHODS: We used mouse primary neurons to determine the signaling mechanism of Maf1, and the cleavage-under-targets-and-tagmentation-sequencing to map the whole-genome promoter binding sites of Maf1 in isolated mature cortical neurons. Photothrombotic stroke model was used to determine the therapeutic effect on neural repair and functional recovery by AAV-mediated Maf1 knockdown. RESULTS: We found that Maf1 mediates mTOR signaling to regulate RNA polymerase III (Pol III)-dependent rRNA and tRNA transcription in mouse cortical neurons. mTOR regulates neuronal Maf1 phosphorylation and subcellular localization. Maf1 knockdown significantly increases Pol III transcription, neurite outgrowth and dendritic spine formation in neurons. Conversely, Maf1 overexpression suppresses such activities. In response to photothrombotic stroke in mice, Maf1 expression is increased and accumulates in the nucleus of neurons in the peripheral region of infarcted cortex, which is the key region for neural remodeling and repair during spontaneous recovery. Intriguingly, Maf1 knockdown in the peri-infarct cortex significantly enhances neural plasticity and functional recovery. Mechanistically, Maf1 not only interacts with the promoters and represses Pol III-transcribed genes, but also those of CREB-associated genes that are critical for promoting plasticity during neurodevelopment and neural repair. CONCLUSION: These findings indicate Maf1 as an intrinsic neural repair suppressor against regenerative capability of mature CNS neurons, and suggest that Maf1 is a potential therapeutic target for enhancing functional recovery after ischemic stroke and other CNS injuries.

Non-enzymatic role of SOD1 in intestinal stem cell growth.

Superoxide dismutase 1 (SOD1) modulates intestinal barrier integrity and intestinal homeostasis as an antioxidant enzyme. Intestinal homeostasis is maintained by the intestinal stem cells (ISCs). However, whether and how SOD1 regulates ISCs is unknown. In this study, we established intestinal organoids from tamoxifen-inducible intestinal epithelial cell-specific Sod1 knockout (Sod1f/f; Vil-creERT2) mice. We found that loss of Sod1 in organoids suppressed the proliferation and survival of cells and Lgr5 gene expression. SOD1 is known for nearly half a century for its canonical role as an antioxidant enzyme. We identified its enzyme-independent function in ISC: inhibition of SOD1 enzymatic activity had no impact on organoid growth, and enzymatically inactive Sod1 mutants could completely rescue the growth defects of Sod1 deficient organoids, suggesting that SOD1-mediated ISC growth is independent of its enzymatic activity. Moreover, Sod1 deficiency did not affect the ROS levels of the organoid, but induced the elevated WNT signaling and excessive Paneth cell differentiation, which mediates the occurrence of growth defects in Sod1 deficient organoids. In vivo, epithelial Sod1 loss induced a higher incidence of apoptosis in the stem cell regions and increased Paneth cell numbers, accompanied by enhanced expression of EGFR ligand Epiregulin (EREG) in the stromal tissue, which may compensate for Sod1 loss and maintain intestinal structure in vivo. Totally, our results show a novel enzyme-independent function of SOD1 in ISC growth under homeostasis.

Mediating bio-fate of polymeric cholecalciferol nanoparticles through rational size control.

Whilst 10-200 nm polymeric nanoparticles hold enormous medical potential, successful clinical translation remains scarce. There is an inadequate understanding of how these nanoparticles could be fabricated with consistent particle architecture in this size range, as well as their corresponding biological performance. We seek to fill this important knowledge gap by employing Design of Experiment (DoE) to examine critical formulation and processing parameters of cholecalciferol (VitD3)-loaded nanoparticles by flash nanoprecipitation (FNP). Based on the regression analysis of the critical processing parameters, six VitD3 nanoparticle formulations with z-average particle sizes between 40 and 150 nm were successfully developed, possessing essentially the same particle shape and zeta potential. To evaluate the effect of particle size on the in vivo performance, not only VitD3 but also its active metabolites (25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3) were assayed in the biodistribution study. Results indicated that VitD3 nanoparticles with sizes ≤110 nm would achieve higher plasma retention. VitD3 nanoparticles with sizes of 40 nm and 150 nm were superior for lung deposition, while particle size had no major role in the brain uptake of VitD3 nanoparticles. The present study demonstrates the value of DoE for generating size-tunable nanoparticles with controlled particle properties in FNP and offers important insights into the particle size effect of nanoparticles <200 nm on their therapeutic potential.

Consensus clustering of gene expression profiles in peripheral blood of acute ischemic stroke patients.

Acute ischemic stroke (AIS) is a primary cause of mortality and morbidity worldwide. Currently, no clinically approved immune intervention is available for AIS treatment, partly due to the lack of relevant patient classification based on the peripheral immunity status of patients with AIS. In this study, we adopted the consensus clustering approach to classify patients with AIS into molecular subgroups based on the transcriptomic profiles of peripheral blood, and we identified three distinct AIS molecular subgroups and 8 modules in each subgroup by the weighted gene co-expression network analysis. Remarkably, the pre-ranked gene set enrichment analysis revealed that the co-expression modules with subgroup I-specific signature genes significantly overlapped with the differentially expressed genes in AIS patients with hemorrhagic transformation (HT). With respect to subgroup II, exclusively male patients with decreased proteasome activity were identified. Intriguingly, the majority of subgroup III was composed of female patients who showed a comparatively lower level of AIS-induced immunosuppression (AIIS). In addition, we discovered a non-linear relationship between female age and subgroup-specific gene expression, suggesting a gender- and age-dependent alteration of peripheral immunity. Taken together, our novel AIS classification approach could facilitate immunomodulatory therapies, including the administration of gender-specific therapeutics, and attenuation of the risk of HT and AIIS after ischemic stroke.

Brain delivering RNA-based therapeutic strategies by targeting mTOR pathway for axon regeneration after central nervous system injury.

Injuries to the central nervous system (CNS) such as stroke, brain, and spinal cord trauma often result in permanent disabilities because adult CNS neurons only exhibit limited axon regeneration. The brain has a surprising intrinsic capability of recovering itself after injury. However, the hostile extrinsic microenvironment significantly hinders axon regeneration. Recent advances have indicated that the inactivation of intrinsic regenerative pathways plays a pivotal role in the failure of most adult CNS neuronal regeneration. Particularly, substantial evidence has convincingly demonstrated that the mechanistic target of rapamycin (mTOR) signaling is one of the most crucial intrinsic regenerative pathways that drive axonal regeneration and sprouting in various CNS injuries. In this review, we will discuss the recent findings and highlight the critical roles of mTOR pathway in axon regeneration in different types of CNS injury. Importantly, we will demonstrate that the reactivation of this regenerative pathway can be achieved by blocking the key mTOR signaling components such as phosphatase and tensin homolog (PTEN). Given that multiple mTOR signaling components are endogenous inhibitory factors of this pathway, we will discuss the promising potential of RNA-based therapeutics which are particularly suitable for this purpose, and the fact that they have attracted substantial attention recently after the success of coronavirus disease 2019 vaccination. To specifically tackle the blood-brain barrier issue, we will review the current technology to deliver these RNA therapeutics into the brain with a focus on nanoparticle technology. We will propose the clinical application of these RNA-mediated therapies in combination with the brain-targeted drug delivery approach against mTOR signaling components as an effective and feasible therapeutic strategy aiming to enhance axonal regeneration for functional recovery after CNS injury.

CircOGDH Is a Penumbra Biomarker and Therapeutic Target in Acute Ischemic Stroke.

BACKGROUND: Acute ischemic stroke (AIS) is a leading cause of disability and mortality worldwide. Prediction of penumbra existence after AIS is crucial for making decision on reperfusion therapy. Yet a fast, inexpensive, simple, and noninvasive predictive biomarker for the poststroke penumbra with clinical translational potential is still lacking. We aim to investigate whether the CircOGDH (circular RNA derived from oxoglutarate dehydrogenase) is a potential biomarker for penumbra in patients with AIS and its role in ischemic neuronal damage. METHODS: CircOGDH was screened from penumbra of middle cerebral artery occlusion mice and was assessed in plasma of patients with AIS by quantitative polymerase chain reaction. Magnetic resonance imaging was used to examine the penumbra volumes. CircOGDH interacted with miR-5112 (microRNA-5112) in primary cortical neurons was detected by fluorescence in situ hybridization, RNA immunoprecipitation, and luciferase reporter assay. Adenovirus-mediated CircOGDH knockdown ameliorated neuronal apoptosis induced by COL4A4 (Gallus collagen, type IV, alpha IV) overexpression. Transmission electron microscope, nanoparticle tracking analysis, and Western blot were performed to confirm exosomes. RESULTS: CircOGDH expression was dramatically and selectively upregulated in the penumbra tissue of middle cerebral artery occlusion mice and in the plasma of 45 patients with AIS showing a 54-fold enhancement versus noncerebrovascular disease controls. Partial regression analysis revealed that CircOGDH expression was positively correlated with the size of penumbra in patients with AIS. Sequestering of miR-5112 by CircOGDH enhanced COL4A4 expression to elevate neuron damage. Additionally, knockdown of CircOGDH significantly enhanced neuronal cell viability under ischemic conditions. Furthermore, the expression of CircOGDH in brain tissue was closely related to that in the serum of middle cerebral artery occlusion mice. Finally, we found that CircOGDH was highly expressed in plasma exosomes of patients with AIS compared with those in noncerebrovascular disease individuals. CONCLUSIONS: These results demonstrate that CircOGDH is a potential therapeutic target for regulating ischemia neuronal viability, and is enriched in neuron-derived exosomes in the peripheral blood, exhibiting a predictive biomarker of penumbra in patients with AIS.

Circular RNA circ-FoxO3 attenuates blood-brain barrier damage by inducing autophagy during ischemia/reperfusion.

Blood-brain barrier (BBB) damage can be a result of central nervous system (CNS) diseases and may be a cause of CNS deterioration. However, there are still many unknowns regarding effective and targeted therapies for maintaining BBB integrity during ischemia/reperfusion (I/R) injury. In this study, we demonstrate that the circular RNA of FoxO3 (circ-FoxO3) promotes autophagy via mTORC1 inhibition to attenuate BBB collapse under I/R. Upregulation of circ-FoxO3 and autophagic flux were detected in brain microvessel endothelial cells in patients with hemorrhagic transformation and in mice models with middle cerebral artery occlusion/reperfusion. In vivo and in vitro studies indicated that circ-FoxO3 alleviated BBB damage principally by autophagy activation. Mechanistically, we found that circ-FoxO3 inhibited mTORC1 activity mainly by sequestering mTOR and E2F1, thus promoting autophagy to clear cytotoxic aggregates for improving BBB integrity. These results demonstrate that circ-FoxO3 plays a novel role in protecting against BBB damage, and that circ-FoxO3 may be a promising therapeutic target for neurological disorders associated with BBB damage.

Pharmacological preconditioning by TERT inhibitor BIBR1532 confers neuronal ischemic tolerance through TERT-mediated transcriptional reprogramming.

After a sublethal ischemic preconditioning (IPC) stimulus, the brain has a remarkable capability of acquiring tolerance to subsequent ischemic insult by establishing precautionary self-protective mechanism. Understanding this endogenous mechanism would reveal novel and effective neuroprotective targets for ischemic brain injury. Our previous study has implied that telomerase reverse transcriptase (TERT) is associated with IPC-induced tolerance. Here, we investigated the mechanism of TERT-mediated ischemic tolerance. Preconditioning was modeled by oxygen-glucose deprivation (OGD) and by TERT inhibitor BIBR1532 in primary neurons. We found that ischemic tolerance was conferred by BIBR1532 preconditioning. We used the Cleavage-Under-Targets-And-Tagmentation approach, a recently developed method with superior signal-to-noise ratio, to comprehensively map the genomic binding sites of TERT in primary neurons, and showed that more than 50% of TERT-binding sites were located at the promoter regions. Mechanistically, we demonstrated that under normal conditions TERT physically bound to many previously unknown genomic loci in neurons, whereas BIBR1532 preconditioning significantly altered TERT-chromatin-binding profile. Intriguingly, we found that BIBR1532-preconditioned neurons showed significant up-regulation of promoter binding of TERT to the mitochondrial anti-oxidant genes, which were correlated with their elevated expression. Functional analysis further indicated that BIBR1532-preconditioning significantly reduced ROS levels and enhanced tolerance to severe ischemia-induced mitochondrial oxidative stress in neurons in a TERT-dependent manner. Together, these results demonstrate that BIBR1532 confers neuronal ischemic tolerance through TERT-mediated transcriptional reprogramming for up-regulation of mitochondrial anti-oxidation gene expression, suggesting the translational potential of BIBR1532 as a therapeutic agent for the treatment of cerebral ischemic injury and oxidative stress-induced neurological disorders.

Rifampicin Suppresses Amyloid-ß Accumulation Through Enhancing Autophagy in the Hippocampus of a Lipopolysaccharide-Induced Mouse Model of Cognitive Decline.

BACKGROUND: Alzheimer's disease (AD) is characterized by amyloid-ß (Aß) deposition. The metabolism of Aß is critically affected by autophagy. Although rifampicin is known to mediate neuroinflammation, the underlying mechanism by which rifampicin regulates the cognitive sequelae remains unknown. OBJECTIVE: Based on our previous findings that rifampicin possesses neuroprotective effects on improving cognitive function after neuroinflammation, we aimed to examine in this study whether rifampicin can inhibit Aß accumulation by enhancing autophagy in a mouse model of lipopolysaccharide (LPS)-induced cognitive impairment. METHODS: Adult C57BL/6 mice were intraperitoneally injected with rifampicin, chloroquine, and/or LPS every day for 7 days. Pathological and biochemical assays and behavioral tests were performed to determine the therapeutic effect and mechanism of rifampicin on the hippocampus of LPS-induced mice. RESULTS: We found that rifampicin ameliorated cognitive impairments in the LPS-induced mice. In addition, rifampicin attenuated the inhibition of autophagosome formation, suppressed the accumulation of Aß1-42, and protected the hippocampal neurons against LPS-induced damage. Our results further demonstrated that rifampicin improved the neurological function by promoting autophagy through the inhibition of Akt/mTOR/p70S6K signaling pathway in the hippocampus of LPS-induced mice. CONCLUSION: Rifampicin ameliorates cognitive impairment by suppression of Aß1-42 accumulation through inhibition of Akt/mTOR/p70S6K signaling and enhancement of autophagy in the hippocampus of LPS-induced mice.

USP8 protects against lipopolysaccharide-induced cognitive and motor deficits by modulating microglia phenotypes through TLR4/MyD88/NF-κB signaling pathway in mice.

Ubiquitin-specific protease 8 (USP8) regulates inflammation in vitro; however, the mechanisms by which USP8 inhibits neuroinflammation and its pathophysiological functions are not completely understood. In this study, we aimed to determine whether USP8 exerts neuroprotective effects in a mouse model of lipopolysaccharide (LPS)-induced cognitive and motor impairment. We commenced intracerebroventricular USP8 administration 7 days prior to i.p. injection of LPS (750 µg/kg). All treatments and behavioral experiments were performed once per day for 7 consecutive days. Behavioral tests and pathological/biochemical assays were performed to evaluate LPS-induced hippocampal damage. USP8 attenuated LPS-induced cognitive and motor impairments in mice. Moreover, USP8 downregulated several pro-inflammatory cytokines [nitric oxide (NO), tumor necrosis factor α (TNF-α), prostaglandin E2 (PGE2), and interleukin-1ß (IL-1ß)] in the serum and brain, and the relevant protein factors [inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2)] in the brain. Furthermore, USP8 upregulated the anti-inflammatory mediators interleukin (IL)-4 and IL-10 in the serum and brain, and promoted a shift from pro-inflammatory to anti-inflammatory microglial phenotypes. The LPS-induced microglial pro-inflammatory phenotype was abolished by TLR4 inhibitor and in TLR4-/- mice; these effects were similar to those of USP8 treatment. Mechanistically, we found that USP8 increased the expression of neuregulin receptor degradation protein-1 (Nrdp1), potently downregulated the expression of TLR4 and myeloid differentiation primary response protein 88 (MyD88) protein, and inhibited the phosphorylation of IκB kinase (IKK) ß and kappa B-alpha (IκBα), thereby reducing nuclear translocation of p65 by inhibiting the activation of the nuclear factor-kappaB (NF-κB) signaling pathway in LPS-induced mice. Our results demonstrated that USP8 exerts protective effects against LPS-induced cognitive and motor deficits in mice by modulating microglial phenotypes via TLR4/MyD88/NF-κB signaling.

Identification of Blood Circular RNAs as Potential Biomarkers for Acute Ischemic Stroke.

Many hospitals lack facilities for accurate diagnosis of acute ischemic stroke (AIS). Circular RNA (circRNA) is highly expressed in the brain and is closely associated with stroke. In this study, we examined whether the blood-borne circRNAs could be promising candidates as adjunctive diagnostic biomarkers and their pathophysiological roles after stroke. We profiled the blood circRNA expression in mice subjected to experimental focal cerebral ischemia and validated the selected circRNAs in AIS patients. We demonstrated that 128, 198, and 789 circRNAs were significantly altered at 5 min, 3 h, and 24 h after ischemic stroke, respectively. Our bioinformatics analysis revealed that the circRNA-targeted genes were associated with the Hippo signaling pathway, extracellular matrix-receptor interaction, and fatty acid metabolism at 5 min, 3 h and 24 h after ischemic stroke, respectively. We verified that many of these circRNAs existed in the mouse brain. Furthermore, we found that most of the predicted circRNA-miRNA interactions apparently exhibited functional roles in terms of regulation of their target gene expression in the brain. We also verified that many of these mouse circRNAs were conserved in human. Finally, we found that circBBS2 and circPHKA2 were differentially expressed in the blood of AIS patients. These results demonstrate that blood circRNAs may serve as potential biomarkers for AIS diagnosis and reveal the pathophysiological responses in the brain after ischemic stroke.

Inhibition of PDE1-B by Vinpocetine Regulates Microglial Exosomes and Polarization Through Enhancing Autophagic Flux for Neuroprotection Against Ischemic Stroke.

Exosomes contribute to cell-cell communications. Emerging evidence has shown that microglial exosomes may play crucial role in regulation of neuronal functions under ischemic conditions. However, the underlying mechanisms of microglia-derived exosome biosynthesis are largely unknown. Herein, we reported that the microglial PDE1-B expression was progressively elevated in the peri-infarct region after focal middle cerebral artery occlusion. By an oxygen-glucose-deprivation (OGD) ischemic model in cells, we found that inhibition of PDE1-B by vinpocetine in the microglial cells promoted M2 and inhibited M1 phenotype. In addition, knockdown or inhibition of PDE1-B significantly enhanced the autophagic flux in BV2 cells, and vinpocetine-mediated suppression of M1 phenotype was dependent on autophagy in ischemic conditions. Co-culture of BV2 cells and neurons revealed that vinpocetine-treated BV2 cells alleviated OGD-induced neuronal damage, and treatment of BV2 cells with 3-MA abolished the observed effects of vinpocetine. We further demonstrated that ischemia and vinpocetine treatment significantly altered microglial exosome biogenesis and release, which could be taken up by recipient neurons and regulated neuronal damage. Finally, we showed that the isolated exosome per se from conditioned BV2 cells is sufficient to regulate cortical neuronal survival in vivo. Taken together, these results revealed a novel microglia-neuron interaction mediated by microglia-derived exosomes under ischemic conditions. Our findings further suggest that PDE1-B regulates autophagic flux and exosome biogenesis in microglia which plays a crucial role in neuronal survival under cerebral ischemic conditions.

Prostaglandin E1 Alleviates Cognitive Dysfunction in Chronic Cerebral Hypoperfusion Rats by Improving Hemodynamics.

Compensatory vascular mechanisms can restore cerebral blood flow (CBF) but fail to protect against chronic cerebral hypoperfusion (CCH)-mediated neuronal damage and cognitive impairment. Prostaglandin E1 (PGE1) is known as a vasodilator to protect against ischemic injury in animal models, but its protective role in CCH remains unclear. To determine the effect of PGE1 on cerebral hemodynamics and cognitive functions in CCH, bilateral common carotid artery occlusion (BCCAO) was used to mimic CCH in rats, which were subsequently intravenously injected with PGE1 daily for 2 weeks. Magnetic resonance imaging, immunofluorescence staining and Morris water maze (MWM) were used to evaluate CBF, angiogenesis, and cognitive functions, respectively. We found that PGE1 treatment significantly restored CBF by enhancing vertebral artery dilation. In addition, PGE1 treatment increased the number of microvascular endothelial cells and neuronal cells in the hippocampus, and decreased the numbers of astrocyte and apoptotic cells. In the MWM test, we further showed that the escape latency of CCH rats was significantly reduced after PGE1 treatment. Our results suggest that PGE1 ameliorates cognitive dysfunction in CCH rats by enhancing CBF recovery, sustaining angiogenesis, and reducing astrocyte activation and neuronal loss.

HMG-CoA Reductase Inhibitors Attenuate Neuronal Damage by Suppressing Oxygen Glucose Deprivation-Induced Activated Microglial Cells.

Ischemic stroke is usually followed by inflammatory responses mediated by microglia. However, the effect of statins on directly preventing posthypoxia microglia inflammatory factors to prevent injury to surrounding healthy neurons is unclear. Atorvastatin and rosuvastatin, which have different physical properties regarding their lipid and water solubility, are the most common HMG-CoA reductase inhibitors (statins) and might directly block posthypoxia microglia inflammatory factors to prevent injury to surrounding neurons. Neuronal damage and microglial activation of the peri-infarct areas were investigated by Western blotting and immunofluorescence after 24 hours in a middle cerebral artery occlusion (MCAO) rat model. The decrease in neurons was in accordance with the increase in microglia, which could be reversed by both atorvastatin and rosuvastatin. The effects of statins on blocking secretions from posthypoxia microglia and reducing the secondary damage to surrounding normal neurons were studied in a coculture system in vitro. BV2 microglia were cultured under oxygen glucose deprivation (OGD) for 3 hours and then cocultured following reperfusion for 24 hours in the upper wells of transwell plates with primary neurons being cultured in the bottom wells. Inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and cyclooxygenase-2 (COX2), which are activated by the nuclear factor-kappa B (NF-κB) signaling pathway in OGD-induced BV2 microglia, promoted decreased release of the anti-inflammatory cytokine IL-10 and apoptosis of neurons in the coculture systems according to ELISA and Western blotting. However, pretreatment with atorvastatin or rosuvastatin significantly reduced neuronal death, synaptic injury, and amyloid-beta (Aß) accumulation, which might lead to increased low-density lipoprotein receptors (LDLRs) in BV2 microglia. We concluded that the proinflammatory mediators released from postischemia damage could cause damage to surrounding normal neurons, while HMG-CoA reductase inhibitors prevented neuronal apoptosis and synaptic injury by inactivating microglia through blocking the NF-κB signaling pathway.

A balancing act: mTOR integrates nutrient signals to regulate redox-dependent growth and survival through SOD1.

Maintaining cellular redox is critical for growth, metabolism and survival in response to changing environments. How nutrients regulate this process is a long-standing fundamental question in cell biology. Our recent study revealed a conserved mechanism by which eukaryotes, particularly cancer cells, couple nutrient signaling to dynamically regulate redox homeostasis. Abbreviations: ATP: adenosine triphosphate; Ala: alanine; C6H12O6: glucose; OH-: hydroxyl radical; Glu: glutamate; mRNA: messenger RNA; mTOR: mechanistic/mammalian target of rapamycin; OXYPHOS: oxidative phosphorylation; Ser: serine; ROS: reactive oxygen species; O2 -: superoxide; SOD1: superoxide dismutase 1; Thr: threonine.

SOD1 Phosphorylation by mTORC1 Couples Nutrient Sensing and Redox Regulation.

Nutrients are not only organic compounds fueling bioenergetics and biosynthesis, but also key chemical signals controlling growth and metabolism. Nutrients enormously impact the production of reactive oxygen species (ROS), which play essential roles in normal physiology and diseases. How nutrient signaling is integrated with redox regulation is an interesting, but not fully understood, question. Herein, we report that superoxide dismutase 1 (SOD1) is a conserved component of the mechanistic target of rapamycin complex 1 (mTORC1) nutrient signaling. mTORC1 regulates SOD1 activity through reversible phosphorylation at S39 in yeast and T40 in humans in response to nutrients, which moderates ROS level and prevents oxidative DNA damage. We further show that SOD1 activation enhances cancer cell survival and tumor formation in the ischemic tumor microenvironment and protects against the chemotherapeutic agent cisplatin. Collectively, these findings identify a conserved mechanism by which eukaryotes dynamically regulate redox homeostasis in response to changing nutrient conditions.

Neuroprotective Mechanisms of Lycium barbarum Polysaccharides Against Ischemic Insults by Regulating NR2B and NR2A Containing NMDA Receptor Signaling Pathways.

Glutamate excitotoxicity plays an important role in neuronal death after ischemia. However, all clinical trials using glutamate receptor inhibitors have failed. This may be related to the evidence that activation of different subunit of NMDA receptor will induce different effects. Many studies have shown that activation of the intrasynaptic NR2A subunit will stimulate survival signaling pathways, whereas upregulation of extrasynaptic NR2B will trigger apoptotic pathways. A Lycium barbarum polysaccharide (LBP) is a mixed compound extracted from Lycium barbarum fruit. Recent studies have shown that LBP protects neurons against ischemic injury by anti-oxidative effects. Here we first reported that the effect of LBP against ischemic injury can be achieved by regulating NR2B and NR2A signaling pathways. By in vivo study, we found LBP substantially reduced CA1 neurons from death after transient global ischemia and ameliorated memory deficit in ischemic rats. By in vitro study, we further confirmed that LBP increased the viability of primary cultured cortical neurons when exposed to oxygen-glucose deprivation (OGD) for 4 h. Importantly, we found that LBP antagonized increase in expression of major proteins in the NR2B signal pathway including NR2B, nNOS, Bcl-2-associated death promoter (BAD), cytochrome C (cytC) and cleaved caspase-3, and also reduced ROS level, calcium influx and mitochondrial permeability after 4 h OGD. In addition, LBP prevented the downregulation in the expression of NR2A, pAkt and pCREB, which are important cell survival pathway components. Furthermore, LBP attenuated the effects of a NR2B co-agonist and NR2A inhibitor on cell mortality under OGD conditions. Taken together, our results demonstrated that LBP is neuroprotective against ischemic injury by its dual roles in activation of NR2A and inhibition of NR2B signaling pathways, which suggests that LBP may be a superior therapeutic candidate for targeting glutamate excitotoxicity for the treatment of ischemic stroke.