Mei Cui

Amelioration of Ischemic Mitochondrial Injury and Bax‐Dependent Outer Membrane Permeabilization by Mdivi‐1

Disturbance of the balance between mitochondrial fission and fusion has been implicated in cerebral ischemia and several neurodegenerative diseases, whereas the underlying mechanisms remain poorly understood. In the present study, we attempted to investigate the role of dynamin‐related protein 1 (Drp1), a key mitochondrial fission protein, in the pathogenesis of cerebral ischemia.

Decreased Extracellular Adenosine Levels Lead to Loss of Hypoxia-Induced Neuroprotection after Repeated Episodes of Exposure to Hypoxia

Achieving a prolonged neuroprotective state following transient ischemic attacks (TIAs) is likely to effectively reduce the brain damage and neurological dysfunction associated with recurrent stroke. HPC is a phenomenon in which advanced exposure to mild hypoxia reduces the stroke volume produced by a subsequent TIA. However, this neuroprotection is not long-lasting, with the effects reaching a peak after 3 days. Therefore, in this study, we investigated the use of multiple episodes of hypoxic exposure at different time intervals to induce longer-term protection in a mouse stroke model. C57BL/6 mice were subjected to different hypoxic preconditioning protocols: a single episode of HPC or five identical episodes at intervals of 3 days (E3d HPC) or 6 days (E6d HPC). Three days after the last hypoxic exposure, temporary middle cerebral artery occlusion (MCAO) was induced. The effects of these HPC protocols on hypoxia-inducible factor (HIF) regulated gene mRNA expression were measured by quantitative PCR. Changes in extracellular adenosine concentrations, known to exert neuroprotective effects, were also measured using in vivo microdialysis and high pressure liquid chromatography (HPLC). Neuroprotection was provided by E6d HPC but not E3d HPC. HIF-regulated target gene expression increased significantly following all HPC protocols. However, E3d HPC significantly decreased extracellular adenosine and reduced cerebral blood flow in the ischemic region with upregulated expression of the adenosine transporter, equilibrative nucleoside transporter 1 (ENT1). An ENT1 inhibitor, propentofylline increased the cerebral blood flow and re-established neuroprotection in E3d HPC. Adenosine receptor specific antagonists showed that adenosine mainly through A1 receptor mediates HPC induced neuroprotection. Our data indicate that cooperation of HIF-regulated genes and extracellular adenosine is necessary for HPC-induced neuroprotection.

The Parkinson's disease-associated gene PINK1 protects neurons from ischemic damage by decreasing mitochondrial translocation of the fission promoter Drp1

Our previous study has shown that PTEN‐induced novel kinase 1 (PINK1) knocking down significantly induced mitochondrial fragmentation. Although PINK1 is proved to be associated with autosomal recessive parkinsonism and its function in this chronic pathological process is widely studied, its role in acute energy crisis such as ischemic stroke is poorly known. In this study by employing an oxygen–glucose deprivation (OGD) neuronal model, we explored the function of PINK1 in cerebral ischemia. Human PINK1, two PINK1 mutants W437X and K219M, or Pink1 shRNA were transduced before OGD using lentiviral delivery. Our results showed that over‐expression of wild‐type PINK1 significantly ameliorated OGD induced cell death and energy disturbance including reduced ATP generation and collapse of mitochondrial membrane potential. PINK1 over‐expression also reversed OGD increased mitochondrial fragmentation, and suppressed the translocation of the mitochondrial fission protein dynamin‐related protein 1 (Drp1) from the cytosol to the mitochondria. Transduction of the mutant PINK1 failed to provide any protective effect, while knockdown of Pink1 significantly increased the severity of OGD‐induced neuronal damage. Importantly, inhibition of Drp1 reversed the effects of knocking down Pink1 on neuronal death and ATP production in response to OGD. This study demonstrates that PINK1 prevents ischemic damage in neurons by attenuating mitochondrial translocation of Drp1, which maintains mitochondrial function and inhibits ischemia‐induced mitochondrial fission. These novel findings implicate a pivotal role of PINK1 regulated mitochondrial dynamics in the pathology of ischemic stroke.

Mdivi-1 Protects Against Ischemic Brain Injury via Elevating Extracellular Adenosine in a cAMP/CREB-CD39-Dependent Manner

AbstractThis study aimed to examine whether the neuroprotective effects of Mdivi-1 are attributable to extracellular ATP and adenosine. Mdivi-1 was administered prior to or post middle cerebral artery occlusion (MCAO). The extracellular adenosine was measured by in vivo microdialysis and high-pressure liquid chromatography (HPLC) in MCAO mouse model. Western blot was done to determine the influence of Mdivi-1 on the expression of CD39 and CREB phosphorylation both in vivo and in the cultured astrocytes. Intracellular cAMP and protein kinase A (PKA) activity were detected in primary astrocytes. Results showed that Mdivi-1 significantly reduced infarct volume and neurological scores when administered either prior to or post MCAO. Interestingly, pretreatment with Mdivi-1 resulted in marked increase of extracellular adenosine and concomitant decrease in ATP. The expression of CD39, but not CD73, was upregulated by Mdivi-1, which was associated with the elevated phosphorylated cAMP response element-binding protein (CREB), a transcription factor potentially regulating CD39 expression. In primary astrocytes, Mdivi-1 treatment induced increases in intracellular cAMP, PKA activity and CREB phosphorylation, and PKA-specific inhibitor completely reversed Mdivi-1-induced CD39 expression. Our results demonstrate that Mdivi-1 protects against ischemic brain injury through increasing extracellular adenosine, a process involving elevated CD39 expression that is likely modulated by cAMP/PKA/CREB cascade. FigurePotential mechanisms by which Mdivi-1 mediates the neuroprotection on cerebral ischemic stroke. Results from the present study indicate that Mdivi-1 protects against ischemic brain injury through increasing extracellular adenosine, a process involving elevated CD39 expression that is likely modulated by the cAMP/PKA/CREB cascades.

Tissue kallikrein protects SH-SY5Y neuronal cells against oxygen and glucose deprivation-induced injury through bradykinin B2 receptor-dependent regulation of autophagy induction

Previous studies have demonstrated that tissue kallikrein (TK) protects against cerebral ischemia injury mainly through inhibition of apoptosis via bradykinin B2 receptor (B2R). In this study, we proposed that autophagy induction contributed to the neuroprotective mechanism of TK. To validate this hypothesis, we investigated TK‐induced autophagy and its signaling mechanisms in human SH‐SY5Y cells exposed to oxygen and glucose deprivation (OGD). We found that TK treatment enhanced autophagy induction, reflected by augmented LC3 conversion and Beclin1 expression, decreased p62 levels and increased monomeric red fluorescent protein‐LC3 puncta formation. Green fluorescent protein‐monomeric red fluorescent protein‐LC3 adenovirus assay indicated that TK maintained autophagic flux. Moreover, bafilomycin A1 (Baf.A1) caused obvious LC3‐II accumulation either in the presence or absence of TK. Autophagy inhibition by Beclin1 knockdown or Baf.A1 treatment abrogated the neuroprotective effects of TK. Mitogen‐activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal‐regulated kinase (ERK)1/2 and AMP‐activated protein kinase (AMPK)/tuberous sclerosis complex 2 (TSC2)/mammalian target of rapamycin (mTOR) signaling were induced by OGD stress and enhanced by TK. MEK/ERK inhibitor U0126 alone elevated autophagy in OGD conditions, but impaired TK‐induced autophagy. Blockade of AMPK/TSC2/mTOR signaling by AMPK inhibitor compound C and shRNA mediated the knockdown of AMPK α1 and TSC2 but abolished autophagy in SH‐SY5Y cells exposed to OGD treated either with or without TK. Moreover, B2R expression was up‐regulated by OGD exposure. B2R knockdown attenuated autophagy and suppressed MEK1/2/ERK1/2 and AMPK/TSC2/mTOR signaling in OGD conditions in either the presence or absence of TK. In sum, we revealed the significance of B2R‐mediated MEK/ERK and AMPK signaling in autophagy induction under OGD stress, and proposed novel mechanisms involved in the neuropotective function of TK through B2R‐dependent regulation of autophagy.

Ischemic preconditioning with a ketogenic diet improves brain ischemic tolerance through increased extracellular adenosine levels and hypoxia-inducible factors

Ischemic tolerance reduces brain damage and neurological dysfunction after brain ischemia. A ketogenic diet (KD) has disease-modifying effects in several neurodegenerative disorders. In this study, we fed mice with a KD for three weeks and performed reversible middle cerebral artery occlusion (MCAO) in the animals. KD-fed mice had a significantly reduced infarct volume, increased regional cerebral blood flow (rCBF) and extracellular adenosine levels in both the ischemic and the reperfusion phases. In vitro and in vivo experiments revealed that the KD-induced neuroprotection was mediated through the adenosine A1 receptor. The KD increased Akt and ERK1/2 phosphorylation via A1R activation. Besides, the KD also upregulated robustly HIF-1α/HIF-2α and HIF regulated genes, such as VEGF and EPO. A three-week preconditioning period with a KD improved ischemic tolerance in mice with MCAO. The underlying mechanisms might include elevated extracellular adenosine levels, and increased Akt and ERK1/2 phosphorylation via A1 adenosine receptor activation, together with upregulated HIFs and HIF-regulated genes.

Ketogenic Diet Improves Brain Ischemic Tolerance and Inhibits NLRP3 Inflammasome Activation by Preventing Drp1-Mediated Mitochondrial Fission and Endoplasmic Reticulum Stress

Background: Neuroprotective effects of ketogenic diets (KD) have been reported in stroke models, and nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome has also been implicated in the pathogenesis of stroke. This study aimed to investigate the effects of KD on NLRP3 inflammasome and explore the potential molecular mechanisms. Methods: In in vivo study, mice were fed with KD for 3 weeks and then subjected to middle cerebral artery occlusion/reperfusion (MCAO/R)-injury. In in vitro study, SH-SY-5Y cells were treated with β-hydroxybutyrate (BHB) followed by oxygen–glucose deprivation/reoxygenation (OGD/R). NLRP3 inflammasome activation and related regulatory mechanisms were evaluated. Results: Mice fed with KD had increased tolerance to MCAO/R. KD inhibited endoplasmic reticulum (ER) stress and suppressed TXNIP/NLRP3 inflammasome activation in the brain. The in vitro study showed BHB (10 mM) prevented the mitochondrial translocation of dynamin-related protein 1 (Drp1) to inhibit mitochondrial fission. Furthermore, BHB decreased reactive oxygen species (ROS) generation, inhibited ROS-NLRP3 pathway in OGD/R-treated cells, and suppressed ER stress-induced NLRP3 inflammasome activation. Conclusions: KD may suppress ER stress and protect mitochondrial integrity by suppressing the mitochondrial translocation of Drp1 to inhibit NLRP3 inflammasome activation, thus exerting neuroprotective effects. Our findings provide evidence for the potential application of KD in the prevention of ischemic stroke.

Clinical features of seizures after cerebral venous sinus thrombosis and its effect on outcome among Chinese Han population

Background and purpose Clinical features of epileptic seizures after cerebral venous sinus thrombosis (CVST) among Chinese patients are not known, and it is still controversial whether seizures would affect the outcome of CVST. Methods In a Chinese hospital-based study of consecutive patients with CVST between 2003 and 2015, we described the clinical features of seizures and determined the predictors of seizure onset using multivariable logistic regression analysis. We also compared the in-hospital case-fatality and short-term functional outcome (modified Rankin Scale (mRS) at discharge) in patients with versus without seizures using ordinal regression analysis. Results Among 151 patients with CVST, 52 (34.4%) presented seizures, of which 42 (80.8%) were generalised seizures. Male gender (OR 6.32, 95% CI 2.06 to 19.35, p=0.001), motor deficits (OR 4.89, 95% CI 1.52 to 15.68, p=0.008), intracerebral haemorrhage (OR 3.93, 95% CI 1.16 to 13.26, p=0.027), cerebral infarction (OR 3.78, 95% CI 1.15 to 12.36, p=0.029) and superior sagittal sinus thrombosis (OR 3.38, 95% CI 91.16 to 9.86, p=0.026) were independent predictors for seizures. The overall in-hospital case-fatality rate was 2.0% (3/151), and 21 (13.9%) had mRS >2 at discharge. Compared with patients without seizures, patients with seizures were more likely to have a worse outcome (p=0.02) at discharge, independent of age, gender, clinical presentation, clot burden and presence of parenchymal lesions. Conclusions In Chinese Han patients, compared with patients without seizures, patients with seizures after CVST had a worse outcome. Risk factors such as male gender, paresis, parenchymal lesion and superior sagittal sinus thrombosis were independently associated with seizure onset after CVST. Generalised seizure was the main form of seizures after CVST, which was obviously different to seizures after strokes of arterial origin.

Tissue kallikrein promotes survival and β‑catenin degradation in SH‑SY5Y cells under nutrient stress conditions via autophagy

Previous studies by our group showed that tissue kallikrein (TK) exerts neuroprotective effects during cerebral ischemia. Autophagy is an important adaptive response to cellular stress during nutrient deprivation, and β-catenin in known to repress autophagy. The present study investigated the possible involvement of autophagy and β-catenin signaling in the protective effects of TK under nutrient deprivation-induced stress conditions. TK was shown to promote the survival and inhibit the death of SH-SY5Y cells under serum starvation and enhanced autophagic activity in a concentration-dependent manner, as indicated by augmented light chain (LC)3-II levels and Beclin-1 expression. The autophagy inhibitors 3-methyladenine and NH4Cl abolished the protective effects of TK. Of note, although serum starvation alone and TK treatment increased p62 protein levels and mRNA expression, incubation with the lysosome inhibitor NH4Cl increased the accumulation of LC3-II and p62 protein, indicating normal autophagic flux. It was also observed that β-catenin expression was significantly downregulated by TK treatment. TK stimulated the interaction between LC3 and β-catenin, and NH4Cl abolished the effects of TK on β-catenin levels in serum-starved cells, suggesting the autophagic degradation of β-catenin, which may have led to the enhancement of autophagy. In conclusion, the findings of the present study demonstrated that TK promoted cell survival and β-catenin degradation in serum-starved SH-SY5Y cells via increasing autophagy, which indicated the therapeutic potential of TK under nutrient deprivation-associated stress conditions.