Mitochondrial dysfunction in a neural cell model of spinal muscular atrophy

Mutations of the survival motor neuron (SMN) gene in spinal muscular atrophy (SMA) lead to anterior horn cell death. The cause is unknown, but motor neurons depend substantially on mitochondrial oxidative phosphorylation (OxPhos) for normal function. Therefore, mitochondrial parameters were analyzed in an SMA cell culture model using small interfering RNA (siRNA) transfection that decreased Smn expression in NSC‐34 cells to disease levels. Smn siRNA knock‐down resulted in 35% and 66% reduced Smn protein levels 48 and 72 hr posttransfection, respectively. ATP levels were reduced by 14% and 26% at 48 and 72 hr posttransfection, respectively, suggesting decreased ATP production or increased energy demand in neural cells. Smn knock‐down resulted in increased mitochondrial membrane potential and increased free radical production. Changes in activity of cytochrome c oxidase (CcO), a key OxPhos component, were observed at 72 hr with a 26% increase in oxygen consumption. This suggests a compensatory activation of the aerobic pathway, resulting in increased mitochondrial membrane potentials, a condition known to lead to the observed increase in free radical production. Further testing suggested that changes in ATP at 24 hr precede observable indices of cell injury at 48 hr. We propose that energy paucity and increased mitochondrial free radical production lead to accumulated cell damage and eventual cell death in Smn‐depleted neural cells. Mitochondrial dysfunction may therefore be important in SMA pathology and may represent a new therapeutic target. © 2009 Wiley‐Liss, Inc.     

Extracellular heat shock protein 72 protects schwann cells from hydrogen peroxide-induced apoptosis

The cytoprotective role of extracellular heat shock protein (Hsp) 72 has been demonstrated in various cell types, including neuronal cells; however, few studies have investigated the actual role of Hsp72 in the survival of Schwann cells (SCs). In the present study, we investigated the effect of exogenous Hsp72 on Schwann cell apoptotic cell death induced by H₂O₂. We determined that extracellular exposure to Hsp72 reduced cell death in rat SCs in a dose‐dependent manner, with the protection resulting from downregulation of apoptosis induced by H₂O₂ (as shown by TUNEL and annexin V flow cytometry analyses). Moreover, we observed that Hsp72 suppressed caspase‐3 and ?9 activation induced by H₂O₂. This was accompanied by upregulation of the antiapoptotic protein Bcl‐2. These findings indicate that extracellular Hsp72 can afford neuroprotection to peripheral nerves via its ability to inhibit Schwann cell apoptosis and diminish oxidative stress‐mediated injuries. © 2012 Wiley Periodicals, Inc.     

Androgens Selectively Protect Against Apoptosis in Hippocampal Neurones

Androgens can protect neurones from injury, although androgen neuroprotection is not well characterised in terms of either specificity or mechanism. In the present study, we compared the ability of androgens to protect neurones against a panel of insults, empirically determined to induce cell death by apoptotic or non‐apoptotic mechanisms. Three criteria defining but not inclusive of apoptosis are: protection by caspase inhibition, protection by protein synthesis inhibition and the presence of pyknotic nuclei. According to these criteria, β‐amyloid, staurosporine, and Apoptosis Activator II induced cell death involving apoptosis, whereas hydrogen peroxide (H₂O₂), iron, calcium ionophore and 3‐nitropropionic acid induced cell death featuring non‐apoptotic characteristics. Pretreatment of hippocampal neurones with testosterone or dihydrotestosterone attenuated cell death induced by β‐amyloid, staurosporine and Apoptosis Activator II, but none of the other insults. The anti‐oxidant Trolox did not reduce cell death induced by β‐amyloid, staurosporine and Apoptosis Activator II, but did protect against H₂O₂ and iron. Similarly, a supra‐physiological concentration of oestrogen reduced cell death induced by H₂O₂ and iron, an effect not observed with androgens. We also show that activation of oestrogen pathways was not necessary for androgen neuroprotection. These data suggest that androgens directly activate a neuroprotective mechanism specific to inhibition of cell death involving apoptosis. Determining the specificity of androgen neuroprotection may enable the development of androgen compounds for the treatment of neurodegenerative disorders.     

Roles of Ras-Erk in apoptosis of PC12 cells induced by trophic factor withdrawal or oxidative stress

To understand the role of Ras-MAPK (mitogen-activated protein kinase) in trophic factor withdrawal- and oxidative stress-induced apoptotic cell death processes, undifferentiated rat pheochromocytoma PC12 cells and a PC12 variant cell line stably expressing the Ras dominant-negative mutant (M-M17-26) were subjected to serum withdrawal in the absence or presence of H₂O₂ treatment. The extent of cell death was analyzed by lactate dehydrogenase release, internucleosomal DNA fragmentation, and caspase-3 assays. Both serum with-drawal and H₂O₂ treatment induced apoptotic cell death in PC12 cells, and the extent of cell death was greatly enhanced in M-M17-26 cells. DNA fragmentation induced by serum withdrawal or H₂O₂ treatment was blocked completely by a general caspase inhibitor, Z-VAD-FMK. A selective MAPK kinase inhibitor, U0126, blocked the H₂O₂-induced phosphorylation of Erk1/2 (extracellular signal-regulated kinase) in PC12 cells and increased the levels of active caspase-3 in M-M17-26 under serum withdrawal or H₂O₂ treatment. In addition, the short-term H₂O₂ treatment (5–30 min) was sufficient to cause DNA fragmentation in M-M17-26 cells even though H₂O₂ was removed and cells were incubated in regular growth medium with complete serum for 24 h. However, similar, short-term H₂O₂ treatment of PC12 cells did not induce DNA fragmentation 24 h later. These results suggest that the Ras-Erk pathway is critical in mediating protection against apoptotic cell death induced by either trophic factor withdrawal or increased oxidative stress.     

Formononetin attenuates H2O2-induced cell death through decreasing ROS level by PI3K/Akt-Nrf2-activated antioxidant gene expression and suppressing MAPK-regulated apoptosis in neuronal SH-SY5Y cells

Formononetin is an isoflavone, found in herbs like Trifolium pratense, which executes a variety of physiological activities including anti-neurodegenerative effect. However, the molecular mechanism of formononetin-mediated neuroprotection remains unclear. In this study, we investigated the protective effect of formononetin on hydrogen peroxide (H₂O₂)-induced death of human neuroblastoma SH-SY5Y cells and its underlying molecular mechanism. Formononetin suppressed H₂O₂-induced cytotoxicity. H₂O₂-induced increase in the intracellular reactive oxygen species (ROS) levels was decreased by formononetin, together with the enhanced expression of the antioxidant genes. H₂O₂-induced elevation of the Bax/Bcl-2 ratio and cleaved caspase-3 and caspase-7 levels were lowered by formononetin treatment. Moreover, formononetin repressed H₂O₂-induced phosphorylation of mitogen-activated protein kinases (MAPKs). Nuclear factor erythroid 2-related factor 2 (Nrf2) siRNA decreased antioxidant gene expression and elevated the H₂O₂-induced ROS level in the formononetin-treated cells. Furthermore, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling is involved in the activation of the nuclear translocation of Nrf2. These results indicate that the neuroprotective effect of formononetin against H₂O₂-induced cell death is due to a decrease in the ROS level with the enhanced expression of the antioxidant genes through activation of the PI3K/Akt-Nrf2 signaling. In addition, formononetin suppressed apoptosis through inhibition of phosphorylation of MAPKs in SH-SY5Y cells. Thus, formononetin is a potential therapeutic agent for the treatment of neurodegenerative diseases.     

Sulfiredoxin‐1 protects PC12 cells against oxidative stress induced by hydrogen peroxide

Oxidative stress results in protein oxidation and is implicated in cerebral disease, such as Parkinson's disease, Alzheimer's disease, and ischemic stroke. Sulfiredoxin‐1 (Srxn1) is an endogenous antioxidant protein that has neuroprotective effects. The mechanisms of Srxn1 in oxidative stress have not been well studied, however. This study used 180 μM H₂O₂ exposure for 24 hr to model oxidative stress. This experimental design allowed us to explore the protective effects and underlying mechanisms of Srxn1 in PC12 cells. To investigate Srxn1's role in oxidative stress protection, transient knockdowns of Srxn1 in PC12 cells were performed prior to treatment with 180 μM H₂O₂ for 24 hr. Knockdown of Srxn1 resulted in decreased cell viability and increased cellular damage as determined by 3‐(4,5‐dimethylthiazol‐2‐yl)?2,5‐diphenyl tetrazolium bromide and lactate dehyrogenase analysis, respectively. Intracellular superoxide dismutase and glutathione are important indexes of oxidative stress; these were reduced in Srxn1 knockdown PC12. We further found that the decreased Srxn1 correlated with a reduction in 2‐Cys Prdxs activity. Moreover, 2‐Cys Prdxs protein levels were increased in the H₂O₂‐dosed cells, as measured by RT‐PCR and immunoblot analysis. These results suggested that Srxn1 can protect PC12 cells from H₂O₂‐induced oxidative stress and are involve in Prdxs activity. Srxn1 play a protective role against oxidative injury and demonstrates potential as a target for neuroprotective intervention in oxidative stress. © 2013 Wiley Periodicals, Inc.     

Role of Paraoxonase-1 in the Protection of Hydrogen Sulfide-Donating Sildenafil (ACS6) Against Homocysteine-Induced Neurotoxicity

ACS6, a novel hydrogen sulfide (H₂S)-releasing sildenafil, has been demonstrated to inhibit superoxide formation through donating H₂S. We have previously found that ACS6 antagonizes homocysteine-induced apoptosis and cytotoxicity. The aim of the present study is to explore the molecular mechanisms underlying ACS6-exerted protective action against the neurotoxicity of homocysteine. In the present work, we used PC12 cells to explore whether paraoxonase-1 (PON-1) is implicated in ACS6-induced neuroprotection against homocysteine neurotoxicity. We show that ACS6 treatment results in prevention of homocysteine-caused neurotoxicity and overproduction of reactive oxygen species (ROS). Homocysteine downregulates the expression and activity of PON-1; however, this effect is significantly blocked by co-treatment with ACS6. The specific inhibitor of PON-1 2-hydroxyquinoline reverses the inhibitory effect of ACS6 on homocysteine-induced neurotoxicity and intracellular ROS accumulation. These results indicate that ACS6 protects PC12 cells against homocysteine-induced neurotoxicity by upregulating PON-1 and suggest a promising role of PON-1 as a novel therapeutic strategy for homocysteine-induced toxicity.     

Overexpression of amyloid precursor protein induces susceptibility to oxidative stress in human neuroblastoma SH-SY5Y cells

Summary. In Alzheimer’s disease amyloid β peptide (Aβ) produced from amyloid precursor protein (APP) is considered to induce cell death. To clarify the molecular mechanism underlying Aβ neurotoxicity, we established the cell line overexpressing wild or mutant (His684Arg) APP in human SH-SY5Y cells. This paper presents that overexpression of wild-APP in the cells (SH/w-APP) increased the levels of APP and Aβ_1–40 but not Aβ_1–42, and reduced Bcl-2 level and proteasome activity with increased susceptibility to oxidative stress. The intracellular levels of reactive oxygen species in SH/w-APP increased significantly by H₂O₂ treatment. The level of Bcl-2 protein, but not mRNA, was markedly decreased in SH/w-APP cells, which was inversely correlated with APP expression among subcloned SH/w-APP cells. These results indicate that increased expression of wild type APP renders neuronal cells more vulnerable to oxidative stress leading to cell death.     

Benzothiazole Aniline Tetra(ethylene glycol) and 3-Amino-1,2,4-triazole Inhibit Neuroprotection against Amyloid Peptides by Catalase Overexpression in Vitro

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Regulation of Human Brain Microvascular Endothelial Cell Adhesion and Barrier Functions by Memantine

Oxidative stress plays a critical role in neuronal injury and is associated with various neurological diseases. Here, we explored the potential protective effect of neuroserpin against oxidative stress in primary cultured hippocampal neurons. Our results show that neuroserpin inhibits H₂O₂-induced neurotoxicity in hippocampal cultures as measured by WST, LDH release, and TUNEL assays. We found that neuroserpin enhanced the activation of AKT in cultures subjected to oxidative stress and that the AKT inhibitor Ly294002 blocked this neuroprotective effect. Neuroserpin increased the expression of the anti-apoptotic protein BCL-2 and blocked the activation of caspase-3. Neuroserpin did not increase the level of neuroprotection over levels seen in neurons transduced with a BCL-2 expression vector, and an inhibitor of Trk receptors, K252a, did not block neuroserpin’s effect. Taken together, our study demonstrates that neuroserpin protects against oxidative stress-induced dysfunction and death of primary cultured hippocampal neurons through the AKT-BCL-2 signaling pathway through a mechanism that does not involve the Trk receptors and leads to inhibition of caspase-3 activation.     

Physiologically normal 5% O2 supports neuronal differentiation and resistance to inflammatory injury in neural stem cell cultures

Recent studies have demonstrated that neural stem cell (NSC) culture at physiologically normoxic conditions (2–5% O₂) is advantageous in terms of neuronal differentiation and survival. Neuronal differentiation is accompanied by a remarkable shift to mitochondrial oxidative metabolism compared with preferentially glycolytic metabolism of proliferating cells. However, metabolic changes induced by growth in a normoxic (5%) O₂ culture environment in NSCs have been minimally explored. This study demonstrates that culturing under 5% O₂ conditions results in higher levels of mitochondrial oxidative metabolism, decreased glycolysis, and reduced levels of reactive oxygen species in NSC cultures. Inflammation is one of the major environmental factors limiting postinjury NSC neuronal differentiation and survival. Our results show that NSCs differentiated under 5% O₂ conditions possess better resistance to in vitro inflammatory injury compared with those exposed to 20% O₂. The present work demonstrates that lower, more physiologically normal O₂ levels support metabolic changes induced during NSC neuronal differentiation and provide increased resistance to inflammatory injury, thus highlighting O₂ tension as an important determinant of cell fate and survival in various stem cell therapies. © 2015 Wiley Periodicals, Inc.     

17β-Estradiol Impedes Bax-Involved Mitochondrial Apoptosis of Retinal Nerve Cells Induced by Oxidative Damage via the Phosphatidylinositol 3-Kinase/Akt Signal Pathway

Oxidative stress leading to retinal nerve cells (RNCs) apoptosis is a major cause of neurodegenerative disorders of the retina. 17β-Estradiol (E2) has been suggested to be a neuroprotective agent in the central nervous system; however, at present, the underlying mechanisms are not well understood, and the related research on the RNCs is less reported. Here, in order to investigate the protective role and mechanism of E2 against oxidative stress-induced damage on RNCs, the transmission electron microscopy and annexin V-FITC/propidium iodide assay were applied to detect the RNCs apoptosis. Western blot and real-time PCR were used to determine the expression of the critical molecules in Bcl-2 and caspase family associated with apoptosis. The transmission electron microscopy results showed that H₂O₂ could induce typical features of apoptosis in RNCs, including formation of the apoptosome. E2 could, however, suppress the H₂O₂-induced morphological changes of apoptosis. Intriguingly, we observed E2-mediated phagocytic scavenging of apoptosome. In response to H₂O₂-induced apoptosis, Bax, acting as one of the pivotal pro-apoptotic members of Bcl-2 family, increased significantly, which directly resulted in an increased ratio of Bax to anti-apoptotic protein Bcl-2 (Bax/Bcl-2). Additionally, caspases 9 and 3, which are the critical molecules of the mitochondrial apoptosis pathway, were activated by H₂O₂. In contrast, E2 exerted anti-apoptotic effects by reducing the expression of Bax to decrease the ratio of Bax/Bcl-2 and impeded the caspases 9/3 activation. Moreover, LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, could sharply block the effect of E2 in reducing the percentage of apoptotic cells resistance to H₂O₂. And the attenuation of Bax, the reduced activities of caspases 9/3 and the impeded release of mitochondrial cytochrome c mediated by E2 resistance to H₂O₂ damage were significantly retrieved by LY294002 administration. Taken together, E2 protects the RNCs against H₂O₂-induced apoptosis by significantly inhibiting the Bax-involved mitochondrial apoptosis via the activation of PI3K/Akt signal pathway.     

Activation of caspase-9 and -3 during H2O2-induced apoptosis of PC12 cells independent of ceramide formation

Abstract

The treatment of PCI2 cells with H₂0₂ (100-500 µM) resulted in typical apoptotic changes including fragmentation and condensation of nuclei, and DINA fragmentation observed as DNA ladder. H₂O₂-induced apoptosis was associated with activation of caspase-3 as assessed by cleavage of specific fluorogenic substrate peptide and processing of procaspase-3 and poly(ADP-ribose) polymerase. However, formation of ceramide, which often locates upstream of caspase-3, was not observed. The inhibitory peptide relatively specific for caspase-3, z-DEVD-FMK and non-selective caspase inhibitor z-VAD-FMK inhibited activation of caspase-3 and apoptotic cell death. However, the relatively specific inhibitors, Ac-YVKD for caspase-1 and Ac-IETD for caspase-8/6, did not affect the occurrence of apoptotic cell death. As an upstream activation of caspase-3, induction of cytochrome c release followed by processing of procaspase-9 was observed by Western blotting, although the formation of intracellular ceramide was not observed. On the other hand, in PCI2 cells overexpressing Bcl-2, the number of apoptotic cells was markedly decreased and activation of both caspases-9 and -3 was prevented. These results suggest that cytochrome c and caspase-9 initiate the activation of executor caspase-3 in H₂O₂-treated PCI2 cells, and that Bcl-2 inhibits H₂O₂-induced release of cytochrome c from mitochondria and then proteolytic processing of procaspase-9. [Neurol Res 2000; 22: 556-564]     

Conditioned Media of Choroid Plexus Epithelial Cells Induces Nrf2-Activated Phase II Antioxidant Response Proteins and Suppresses Oxidative Stress-Induced Apoptosis in PC12 Cells

Based on the critical role of the choroid plexus (CP) in detoxification processes in the central nervous system (CNS), herein we investigated the effect of choroid plexus epithelial cells conditioned media (CPECs-CM) under oxidative conditions. CPECs were isolated from rat brains, cultured, and the conditioned media were collected. Then pheochromocytoma neuron-like cells (PC12) were treated simultaneously with CPECs-CM and H₂O₂ as the oxidative stressor. Next, the effect of CPECs-CM on neurite outgrowth and cell differentiation in the presence of H₂O₂ was determined. Our results showed that CPECs-CM improved the expansion of neurites and differentiation in PC12 cells under oxidative stress conditions. Changes in apoptotic factors, nuclear factor erythroid 2-related factor 2 (Nrf2) and γ-glutamylcysteine synthetase as the highlighted pathway in the antioxidant defense system were determined by western blot. Also, the activity of antioxidant enzymes and lipid peroxidation level were determined. CPECs-CM-treated PC12 cells could survive after exposure to H₂O₂ by reduction of caspase-3 cleavage and Bax level and elevation of anti-apoptotic factor Bcl2. Our data also revealed that Nrf2 activation, and consequently its downstream protein levels, increased in the presence of CPECs-CM. Based on our data, we can conclude that CPECs-CM protects PC12 cells against oxidative stress and apoptosis. It seems that CPECs secrete antioxidative agents and neurotrophic factors that have a role in the health of the CNS.     

5-Hydroxymethylfurfural from wine-processed Fructus corni inhibits hippocampal neuron apoptosis

Previous studies have shown that 5-hydroxymethylfurfural, a compound extracted from wine- processed Fructus corni, has a protective effect on hippocampal neurons. The present study was designed to explore the related mechanisms. Our study revealed that high and medium doses （10, 1 μmol/L） of 5-hydroxymethylfurfural could improve the morphology of H2O2-treated rat hippocampal neurons as revealed by inverted phase-contrast microscopy and transmission electron microscopy. MTT results showed that incubation with high and medium doses of 5-hydroxymethylfurfural caused a significant increase in the viability of neuronal cells injured by H2O2. Flow cytometry assays con- firmed that H2O2 could induce cell apoptosis, while high and medium doses of 5-hydroxymethylfurfural had a visible protective effect on apoptotic rat hippocampal neurons. Real-time PCR and western blot analysis showed that high and medium doses of 5-hydroxymethylfurfural prevented H2O2-induced up-regulation of p53, Bax and caspase-3 and an- tagonized the down-regulation of Bcl-2 induced by H2O2 treatment. These results suggested that 5-hydroxymethylfurfural could inhibit apoptosis of cultured rat hippocampal neurons injured by H2O2 via increase in Bcl-2 levels and decrease in p53, Bax and caspase-3 protein expression levels.     

Involvement of ERK1/2 Pathway in Neuroprotection by Salidroside Against Hydrogen Peroxide-Induced Apoptotic Cell Death

Salidroside is isolated from Rhodiola rosea L., a traditional Chinese medicinal plant, and has a potent antioxidant property. The aim of this study was to investigate the effects of salidroside on hydrogen peroxide (H₂O₂)-induced cell apoptosis in nerve growth factor (NGF)-differentiated PC12 cells and the possible involvement of the extracellular signal-related protein kinase 1/2 (ERK1/2) signaling pathway. MTT assay, Hoechst 33342 staining, and TdT-mediated dUTP-biotin nick end labeling assay collectively showed that pretreatment with salidroside alleviated, in a dose-dependent manner, cell viability loss and apoptotic cell death induced by H₂O₂ stimulation in cultured NGF-differentiated PC12 cells. According to Western blot analysis, pretreatment with salidroside transiently caused the activation of ERK1/2 pathway; a selective inhibitor of the mitogen-activated protein kinase kinase (MAPKK, MEK) blocked salidroside-activated ERK pathway and thus attenuated the influences of salidroside on H₂O₂-induced increase in the level of cleaved caspase-3, a chief executant of apoptosis cascades. Morphological analysis further indicated that in the presence of the MEK inhibitor, the neuroprotective effect of salidroside against H₂O₂-evoked cell apoptosis was significantly abrogated. Taken together, the results suggest that the neuroprotective effects of salidroside might be modulated by ERK signaling pathway, especially at the level or upstream of the caspase-3 activation.     

IMM-H004, a Novel Coumarin Derivative Compound,Inhibits H2O2-Induced Neurotoxicity via Antioxidant and Antiapoptosis in PC12 Cells

IMM-H004 [7-hydroxy-5-methoxy-4-methyl-3-(4-methylpiperazin-1-yl)-coumarin] is a novel derivative of coumarin, which played neuroprotective roles in brain ischemia in rats in previous studies. Although antiapoptosis and improving synapsis structure were proved, the effects and mechanisms of IMM-H004 in brain ischemia need further study. In this paper, the effect of IMM-H004 on H₂O₂-induced neurotoxicity in pheochromocytoma (PC12) cells was researched. Morphological observation, MTT method and PI/Hoechst staining were used to indicate cell viability and apoptosis. JC-1 and DCFH-DA were used to test mitochondrial membrane potential (MMP) and reactive oxygen species (ROS), respectively. The antioxidative activity was detected by Glutathione (GSH) and Total Antioxidant Capacity (TAC) Assay kits. Western blot was used to test apoptosis related proteins. Our results showed that treatment with 1-10 μM IMM-H004 markedly increased cell viability and decreased cell apoptosis induced by H₂O₂. Moreover, 1-10 μM IMM-H004 could enhance MMP and protect mitochondrial function. 1-10 μM IMM-H004 also could lower the ROS and raise the GSH and TAC level. Furthermore, 1-10 μM IMM-H004 could decrease the ratio of Bax/Bcl-2 and increase the ratio of p-AKT/AKT, which were related to apoptosis and survival. All these indicated that IMM-H004 protects PC12 cells against H₂O₂-induced neurotoxicity. Antioxidative and antiapoptosis may be the mechanisms of IMM-H004 in brain ischemia. These studies indicate that IMM-H004 might be a potential drug for treatment brain ischemia.