ATF5-regulated Mitochondrial Unfolded Protein Response Attenuates Neuronal Damage in Epileptic Rat by Reducing Endoplasmic Reticulum Stress Through Mitochondrial ROS.

Endoplasmic reticulum (ER) dysfunction caused by excessive ER stress is a crucial mechanism underlying seizures-induced neuronal injury. Studies have shown that mitochondrial reactive oxygen species (ROS) are closely related to ER stress, and our previous study showed that activating transcription factor 5 (ATF5)-regulated mitochondrial unfolded protein response (mtUPR) modulated mitochondrial ROS generation in a hippocampal neuronal culture model of seizures. However, the effects of ATF5-regulated mtUPR on ER stress and the underlying mechanisms remain uncertain in epilepsy. In this study, ATF5 upregulation by lentivirus infection attenuated seizures-induced neuronal damage and apoptosis in a rat model of pilocarpine-induced epilepsy, whereas ATF5 downregulation by lentivirus infection had the opposite effects. ATF5 upregulation potentiated mtUPR by increasing the expression of mitochondrial chaperone heat shock protein 60 (HSP60) and caseinolytic protease proteolytic subunit (ClpP) and reducing mitochondrial ROS generation in pilocarpine-induced seizures in rats. Additionally, upregulation of ATF5 reduced the expression of glucose-regulated protein 78 (GRP78), protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP), suggesting suppression of ER stress; Moreover, ATF5 upregulation attenuated apoptosis-related proteins such as B-cell lymphoma-2 (BCL2) downregulation, BCL2-associated X (BAX) and cleaved-caspase-3 upregulation. However, ATF5 downregulation exerted the opposite effects. Furthermore, pretreatment with the mitochondria-targeted antioxidant mito-TEMPO attenuated the harmful effects of ATF5 downregulation on ER stress and neuronal apoptosis by reducing mitochondrial ROS generation. Overall, our study suggested that ATF5-regulated mtUPR exerted neuroprotective effects against pilocarpine-induced seizures in rats and the underlying mechanisms might involve mitochondrial ROS-mediated ER stress.

Activating Transcription Factor 4-mediated Mitochondrial Unfolded Protein Response Alleviates Hippocampal Neuronal Damage in an In Vitro Model of Epileptiform Discharges.

The mitochondrial unfolded protein response (mtUPR) has been shown to restore protein homeostasis and cell function under stress, and recent studies have confirmed that the activating transcription factor 4 (ATF4) regulates mtUPR. However, the role of ATF4-mediated mtUPR in a hippocampal neuronal culture model of seizures remains unclear. Our results showed that the expression of mtUPR-related proteins (HSP60 and CLpP) increased in primary hippocampal neurons with seizures induced by a magnesium-free solution, suggesting mtUPR activation. Furthermore, ATF4 overexpression by lentiviral vector transfection enhanced the expression of HSP60 and CLpP, whereas ATF4 low expression by lentiviral vector transfection weakened the expression of HSP60 and CLpP. In addition, ATF4 overexpression increased neuronal viability and reduced seizure-induced apoptosis. ATF4 overexpression reduced reactive oxygen species (ROS) production and improved mitochondrial membrane potential damage during seizures. Moreover, ATF4 overexpression reduced the BCL2-associated X protein (Bax) expression and increased the expression of B-cell lymphoma 2 (BCL2). In contrast, ATF4 expression showed the opposite trend. In conclusion, our results showed that ATF4-mediated mtUPR may delay the cascade activation of apoptotic pathways by reducing ROS-mediated oxidative stress, thereby attenuating seizure-induced stress injury.

Single internal carotid cavernous sinus aneurysm presented as bilateral painful ophthalmoplegia: a case report.

BACKGROUND: Intracranial aneurysms are the most common vascular cause of painful ophthalmoplegia. Symptoms include retro-orbital pain, diplopia, ophthalmoplegia, trigeminal neuropathy, or a combination of these. Most single aneurysms cause ipsilateral, painful ophthalmoplegia. Here, we report the first, to our knowledge, case of bilateral painful ophthalmoplegia possibly caused by an aneurysm of the cavernous segment of the left internal carotid artery. CASE PRESENTATION: A 62-year-old male patient presented with headache and bilateral ptosis. Laboratory tests revealed hypopituitary function. Computerized tomography angiography showed a large aneurysm in the cavernous sinus segment of the left internal carotid artery. Aneurysm embolization was performed in the Nerve Interventional Department. Four months after surgery, the patient's symptoms returned to normal. CONCLUSIONS: This case suggests that patients with bilateral painful ophthalmoplegia should be screened for aneurysms using computed tomography angiography or magnetic resonance angiography immediately.

Mitochondrial-derived vesicles: Gatekeepers of mitochondrial response to oxidative stress.

Mitochondrial quality control (MQC) mechanisms are a series of adaptive responses that ensure the relative stability of mitochondrial morphology, quantity, and quality to preserve cellular survival and function. While MQC mechanisms range from mitochondrial biogenesis and fusion/fission to mitophagy, mitochondrial-derived vesicles (MDVs) may represent an essential component of MQC. MDVs precede mitochondrial autophagy and serve as the first line of defense against oxidative stress by selectively transferring damaged mitochondrial substances to the lysosome for degradation. In fact, the function of MDVs is dependent on the cargo, the shuttle route, and the ultimate destination. Abnormal MDVs disrupt metabolite clearance and the immune response, predisposing to pathological conditions, including neurodegeneration, cardiovascular diseases, and cancers. Therefore, MDV regulation may be a potential therapeutic for the therapy of these diseases. In this review, we highlight recent advances in the study of MDVs and their misregulation in various diseases from the perspectives of formation, cargo selection, regulation, and transportation.

Autoimmune encephalitis with coexistent LGI1 and GABABR1 antibodies: case report.

BACKGROUND: Autoimmune encephalitis (AE) with multiple auto-antibodies is of great clinical significance because its complex clinical manifestations and atypical imaging increase the difficulty of diagnosis, differential diagnosis and treatment, which may aggravate the disease, increase the recurrence rate and mortality. The coexistence of anti-Leucinie-rich Glioma Inactivated 1 (LGI1) and anti-γ-aminobutyric acid-beta-receptor 1 (GABABR1) has not been published before. CASE PRESENTATION: We herein present the case of a 60-year-old man with slow response, behavioral changes, psychosis and sleep disorders. Laboratory test included serum hyponatremia, positive serum LGI1 and GABABR1 antibodies using transfected cell-based assays. Electroencephalogram exhibited moderate diffusion abnormality. The patient responded well to steroid impulse treatment and sodium supplement therapy, and did not recur during the follow-up. CONCLUSIONS: Here we report the first AE characterized by positive LGI1 and GABABR1 antibodies, as well as summarizing AE with multiple auto-antibodies reported so far, hopefully to provide experience for clinical practice.

Both left upper lobectomy and left pneumonectomy are risk factors for postoperative stroke.

Retrospective studies have found that left upper lobectomy (LUL) may be a new risk factor for stroke, and the potential mechanism is pulmonary vein thrombosis, which more likely develops in the left superior pulmonary vein (LSPV) stump. The LSPV remaining after left pneumonectomy is similar to that remaining after LUL. However, the association between left pneumonectomy, LUL, and postoperative stroke remains unclear. Thus, we sought to analyze whether both LUL and left pneumonectomy are risk factors for postoperative stroke. We prospectively included consecutive patients who underwent resection between November 2016 and March 2018 at our institution with 6 months of follow-up. Baseline demographic and clinical data were taken. A logistic regression model was used to determine independent predictors of postoperative stroke. In our study, 756 patients who underwent an isolated pulmonary lobectomy procedure were screened; of these, 637 patients who completed the 6-month follow-up were included in the analysis. Multivariable logistic regression analysis adjusted for common risk factors showed that the LUL and left pneumonectomy were independent predictors of stroke (odds ratio, 18.12; 95% confidence interval, 2.12-155.24; P = 0.008). Moreover, diabetes mellitus also was a predictor of postoperative stroke. In conclusion, both LUL and left pneumonectomy are significant risk factors for postoperative stroke.

Ectopic expression of Miro 1 ameliorates seizures and inhibits hippocampal neurodegeneration in a mouse model of pilocarpine epilepsy.

Epilepsy is a common disease of the central nervous system. This study aimed to investigate the role of mitochondrial Rho (Miro) 1 in epilepsy, using a mouse model of pilocarpine-induced status epilepticus (SE). Intraperitoneal injection of pilocarpine induced epileptic seizures in mice and significantly decreased Miro 1 expression in the hippocampus. Moreover, pilocarpine treatment increased the serum levels of heat shock protein 70 (HSP70) and S100 calcium binding protein B (S100B) and led to hippocampal neuronal injury and apoptosis. The intrinsic apoptotic pathway was activated in the hippocampal neurons following pilocarpine-induced SE, as evidenced by increased levels of cleaved caspase-3 and Bax, downregulation of Bcl-2, and the release of cytochrome c from mitochondria to cytoplasm. By contrast, forced expression of Miro 1 by lateral ventricular administration of adenovirus mitigated pilocarpine-induced epileptic seizures, reduced the elevation of HSP70 and S100B, and inhibited hippocampal neuronal apoptosis by suppressing the intrinsic apoptotic pathway. In summary, our data demonstrates that ectopic expression of Miro 1 alleviated pilocarpine-induced SE and protected hippocampal neurons by inhibiting the intrinsic apoptotic pathway. These findings provide new insights into epileptic disorders and suggest a potential neuroprotective value of Miro 1 in the treatment of epilepsy.

LncRNA SNHG1 promotes α-synuclein aggregation and toxicity by targeting miR-15b-5p to activate SIAH1 in human neuroblastoma SH-SY5Y cells.

Numerous long non-coding RNAs (lncRNAs) have been identified as aberrantly expressed in Parkinson's disease (PD). However, limited knowledge is available concerning the roles of dysregulated lncRNAs and the underlying molecular regulatory mechanism in the pathological process of PD. In this study, we found that lncRNA small nucleolar RNA host gene 1 (SNHG1) and seven in absentia homolog 1 (SIAH1) were upregulated, but microRNA-15b-5p (miR-15b-5p) was downregulated in SH-SY5Y cells pretreated with MPP+, as well as in MPTP-induced mouse model of PD. Overexpression of SIAH1 enhanced cellular toxicity of α-synuclein in SH-SY5Y cells, as indicated by the reduction of cell viability and elevation of LDH release. The percentage of α-synuclein aggregate-positive cells and the number of α-synuclein aggregates per cell were increased in SH-SY5Y cells transfected with pcDNA-SIAH1, while decreased after transfection with short interfering RNA specific for SIAH1 (si-SIAH1). Bioinformatics and luciferase reporter assay revealed that SIAH1 was a direct target of miR-15b-5p. We also found that SNHG1 could directly bind to miR-15-5p and repress miR-15-5p expression. Upregulation of miR-15b-5p alleviated α-synuclein aggregation and apoptosis by targeting SIAH1 in SH-SY5Y cells overexpressing α-synuclein. Overexpression of SNHG1 enhanced, whereas SNHG1 knockdown inhibited α-synuclein aggregation and α-synuclein-induced apoptosis. Moreover, the neuroprotective effect of si-SNHG1 was abrogated by downregulation of miR-15b-5p. In summary, our data suggest that SNHG1, as a pathogenic factor, promotes α-synuclein aggregation and toxicity by targeting the miR-15b-5p/SIAH1 axis, contributing to a better understanding of the mechanisms of Lewy body formation and loss of dopaminergic neurons in PD.

Novel compound heterozygous mutations causing Kufs disease type B.

BACKGROUND: Kufs disease type B (also termed CLN13), an adult-onset form of neuronal ceroid lipofuscinosis (NCL), is genetically heterogeneous and challenging to diagnose. Recently, mutations in cathepsin-F have been identified as the causative gene for autosomal recessive Kufs disease type B. RESULTS: Here, we report a sporadic case of Kufs disease type B with novel compound heterozygous mutations, a novel missense mutation c.977G>T (p.C326F) and a novel nonsense mutation c.416C>A (p.S139X), in the cathepsin-F gene. The magnetic resonance imaging findings were consistent with those demonstrated in adult neuronal ceroid lipofuscinosis: diffuse cortical atrophy, mild hyperintensity and reduction of the deep white matter on T2-weighted images. A skin biopsy was negative for abnormalities. CONCLUSIONS: Altogether, our findings broaden the mutation database in relation to the neuronal ceroid lipofuscinosis, and the clinical diagnosis of Kufs disease type B was confirmed.

A Multicenter Clinical Study on Treating Post-Dural Puncture Headache with an Intravenous Injection of Aminophylline.

BACKGROUND: Post-dural puncture headache (PDPH) is the most common complication of lumbar puncture. Aminophylline has been reported to be effective in the prevention of PDPH in some clinical studies, but its efficacy for the treatment of PDPH has been unproven. OBJECTIVE: To evaluate the efficacy and safety of an intravenous (IV) injection of aminophylline on PDPH. STUDY DESIGN: The study was a multicenter, open-label study to assess the effectiveness and safety of aminophylline on PDPH. SETTING: The First Affiliated Hospital of Zhengzhou University, The Fifth Affiliated Hospital of Zhengzhou University, and Henan Province Hospital of Traditional Chinese Medicine. METHODS: Thirty-two PDPH patients received an IV injection of aminophylline. The primary and secondary endpoints were the degree of headache and the patient's overall response to the treatment, respectively. Treatment safety was evaluated based on the occurrence of adverse reactions. RESULTS: Thirty-one patients completed the study. Before the initial aminophylline administration, the visual analog scale (VAS) score was 7.72 ± 1.65. The VAS scores at 30 minutes, one hour, 8 hours, one day, and 2 days post-treatment were 4.84 ± 2.53, 3.53 ± 2.06, 2.38 ± 1.96, 1.44 ± 1.87, and 0.81 ± 1.79, respectively, and were statistically significantly different (P < 0.05) compared with those before treatment. More than 50% (17/32) of the patients reported that they were "very much improved" or "much improved" 30 minutes after the initial treatment, increasing to 93.8% (30/32) at 2 days post-treatment. One patient experienced mild allergic reaction after treatment. LIMITATIONS: Although this study had the largest sample size among current studies on treating PDPH with theophylline drugs, the sample size was still relatively small and the method employed was not compared with a placebo or other current clinical treatments for PDPH. CONCLUSION: An IV injection of aminophylline may be an effective and safe early-stage treatment for PDPH.

ß-Arrestin 2 Promotes Hepatocyte Apoptosis by Inhibiting Akt Protein.

Recent studies reveal that multifunctional protein ß-arrestin 2 (Arrb2) modulates cell apoptosis. Survival and various aspects of liver injury were investigated in WT and Arrb2 KO mice after bile duct ligation (BDL). We found that deficiency of Arrb2 enhances survival and attenuates hepatic injury and fibrosis. Following BDL, Arrb2-deficient mice as compared with WT controls displayed a significant reduction of hepatocyte apoptosis as demonstrated by the TUNEL assay. Following BDL, the levels of phospho-Akt and phospho-glycogen synthase kinase 3ß (GSK3ß) in the livers were significantly increased in Arrb2 KO compared with WT mice, although p-p38 increased in WT but not in Arrb2-deficient mice. Inhibition of GSK3ß following BDL decreases hepatic apoptosis and decreased p-p38 in WT mice but not in Arrb2 KO mice. Activation of Fas receptor with Jo2 reduces phospho-Akt and increases apoptosis in WT cells and WT mice but not in Arrb2-deficient cells and Arrb2-deficient mice. Consistent with direct interaction of Arrb2 with and regulating Akt phosphorylation, the expression of a full-length or N terminus but not the C terminus of Arrb2 reduces Akt phosphorylation and coimmunoprecipates with Akt. These results reveal that the protective effect of deficiency of Arrb2 is due to loss of negative regulation of Akt due to BDL and decreased downstream GSK3ß and p38 MAPK signaling pathways.

Curcumin Improves Amyloid ß-Peptide (1-42) Induced Spatial Memory Deficits through BDNF-ERK Signaling Pathway.

Curcumin, the most active component of turmeric, has various beneficial properties, such as antioxidant, anti-inflammatory, and antitumor effects. Previous studies have suggested that curcumin reduces the levels of amyloid and oxidized proteins and prevents memory deficits and thus is beneficial to patients with Alzheimer's disease (AD). However, the molecular mechanisms underlying curcumin's effect on cognitive functions are not well-understood. In the present study, we examined the working memory and spatial reference memory in rats that received a ventricular injection of amyloid-ß1-42 (Aß1-42), representing a rodent model of Alzheimer's disease (AD). The rats treated with Aß1-42 exhibited obvious cognitive deficits in behavioral tasks. Chronic (seven consecutive days, once per day) but not acute (once a day) curcumin treatments (50, 100, and 200 mg/kg) improved the cognitive functions in a dose-dependent manner. In addition, the beneficial effect of curcumin is accompanied by increased BDNF levels and elevated levels of phosphorylated ERK in the hippocampus. Furthermore, the cognition enhancement effect of curcumin could be mimicked by the overexpression of BDNF in the hippocampus and blocked by either bilateral hippocampal injections with lentiviruses that express BDNF shRNA or a microinjection of ERK inhibitor. These findings suggest that chronic curcumin ameliorates AD-related cognitive deficits and that upregulated BDNF-ERK signaling in the hippocampus may underlie the cognitive improvement produced by curcumin.

Puerarin protects against ß-amyloid-induced microglia apoptosis via a PI3K-dependent signaling pathway.

Puerarin extracted from Radix puerariae is well-known for its anti-oxidative and neuroprotective activities. In this study, we investigated the protective effect of puerarin on amyloid-ß protein (Aß)-induced cytotoxicity and its potential mechanisms in BV-2 and primary microglial cells. We found that pretreatment with puerarin afforded protection against Aß-induced cytotoxicity through inhibiting apoptosis in BV-2 and primary microglial cells. This result was also confirmed by the activated caspase-3 assay. Phospho-Akt and Bcl-2 expression increased after pretreatment with puerarin in BV-2 and primary microglial cells exposed to Aß, whereas Bax expression and cytochrome c release decreased. In addition, puerarin treatment prevented the loss of mitochondrial membrane potential and reactive oxygen species production. Interestingly, these effects of puerarin against Aß insult were abolished by LY294002, an inhibitor of PI3K phosphorylation. Taken together, these findings suggest that puerarin prevents Aß-induced microglial apoptosis via the activation of PI3K/Akt signaling pathway, and might be a potential preventive or therapeutic agent for Alzheimer's disease.

Adenosine monophosphate-activated protein kinase and peroxisome proliferator-activated receptor gamma coactivator 1α signaling provides neuroprotection in status epilepticus in rats.

Status epilepticus (SE) can cause severe neuronal loss and oxidative damage. Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) can be neuroprotective by inducing the antioxidant system, so we evaluated the role of PGC-1α in SE. The expression of PGC-1α and one of its target genes, uncoupling protein 2 (UCP2), was upregulated after SE, which may represent an endogenous neuroprotective mechanism. Furthermore, pretreatment with an adenosine monophosphate-activated protein kinase (AMPK) inhibitor significantly attenuated both AMPK and PGC-1α activation, followed by downregulation of UCP2 and enhanced oxidative stress and hippocampal neuronal damage. AMPK/PGC-1α may be neuroprotective in SE-induced brain damage, at least in part via UCP2.

Diazoxide preconditioning against seizure-induced oxidative injury is via the PI3K/Akt pathway in epileptic rat.

Diazoxide (DZ), a highly selective opener of the mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel, has neuroprotective effects against neuronal cell death by reducing oxidative stress. However, the mechanism of DZ protecting hippocampal neurons against seizure-induced oxidative injury is unknown. In this study, we investigated DZ attenuating neuronal loss caused by pilocarpine-induced seizures in rat hippocampus. DZ attenuated oxidative stress injury by upregulating superoxide dismutase (SOD) activity and downregulating malondialdehyde (MDA) level, which could be abolished with 5-hydroxydecanoic acid, an inhibitor of mitoK(ATP). In addition, wortmannin, an inhibitor of phosphatidylinositol-3-kinase (PI3K), attenuated the changes in MDA and SOD levels after seizures. DZ could reduce oxidative injury induced by seizures by suppressing the activity of MDA and increasing the level of SOD in part by the PI3K/Akt pathway.

Role of PI3K/Akt in diazoxide preconditioning against rat hippocampal neuronal death in pilocarpine-induced seizures.

Diazoxide (DZ), a highly selective opener of the mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel, has neuroprotective effects. However, the mechanism of DZ protecting hippocampal neurons against cell death in pilocarpine-induced seizures is unknown. In this study, we investigated DZ attenuating neuronal loss caused by pilocarpine-induced seizures in rat hippocampus. DZ inhibited seizure-induced change in phospho-Akt expression, translocation of apoptosis-inducing factor (AIF), release of cytochrome c (CytC) and caspase-3 activation, which could be abolished by preincubation with 5-hydroxydecanoic acid, an inhibitor of mitoK(ATP). In addition, wortmannin, an inhibitor of phosphatidylinositol-3-kinase (PI3K), attenuated the translocation of AIF, CytC release and caspase-3 activation after seizures. DZ could reduce neuronal death induced by seizures in hippocampus by suppressing the translocation of AIF, CytC release and the activation of caspase-3 via the PI3K/Akt pathway.

Glycogen synthase kinase-3 and p38 MAPK are required for opioid-induced microglia apoptosis.

Opioids have been widely applied in clinics as one of the most potent pain relievers for centuries, but their abuse has deleterious physiological effects beyond addiction. We previously reported that opioids inhibit cell growth and trigger apoptosis in lymphocytes. However, the underlying mechanism by which microglia apoptosis in response to opioids is not yet known. In this study, we show that morphine induces microglia apoptosis and caspase-3 activation in an opioid-receptor dependent manner. Morphine decreased the levels of microglia phosphorylated Akt (p-Akt) and p-GSK-3ß (glycogen synthase kinase-3 beta) in an opioid-receptor dependent manner. More interestingly, GSK-3ß inhibitor SB216763 significantly increases morphine-induced apoptosis in both BV-2 microglia and mouse primary microglial cells. Moreover, co-treatment of microglia with SB216763 and morphine led to a significant synergistic effect on the level of phospho-p38 mitogen-activated protein kinase (MAPK). In addition, inhibition of p38 MAPK by its specific inhibitor SB203580 significantly inhibited morphine-induced apoptosis and caspase-3 activation. Taken together, our data clearly demonstrates that morphine-induced apoptosis in microglial cells, which is mediated via GSK-3ß and p38 MAPK pathways.

Mitochondrial DNA damage and the involvement of antioxidant defense and repair system in hippocampi of rats with chronic seizures.

In this study, we demonstrated a decreased level of mitochondrial DNA (mtDNA) with a large number of oxidized bases in hippocampi of rats with epilepsy induced by pilocarpine. In order to verify the underlying mechanism of mtDNA impairment, we detected the response of antioxidant defense system and mitochondrial base excision repair (mtBER) pathway. Superoxide dismutase2 (SOD-2) and glutathione (GSH) were significantly decreased in the experimental group, manifesting a decreased capacity of scavenging free radicals. Mitochondrial base excision repair (mtBER) pathway, which is the main repair pathway for the removal of oxidative base modifications, displayed unbalanced expression in epileptic group. DNA polymerasegamma (polgamma) increased, while apurinic/apyrimidinic endonuclease (APE1), one of mtBER initiators, decreased in mitochondria in the chronic phase of epileptogenesis. In conclusion, mtDNA was impaired during chronic recurrent seizures, whereas the endogenous antioxidants and the mtBER pathway failed to respond to the elevated mtDNA damage.