An Electrophysiological and Proteomic Analysis of the Effects of the Superoxide Dismutase Mimetic, MnTMPyP, on Synaptic Signalling Post-Ischemia in Isolated Rat Hippocampal Slices.

Metabolic stress and the increased production of reactive oxygen species (ROS) are two main contributors to neuronal damage and synaptic plasticity in acute ischemic stroke. The superoxide scavenger MnTMPyP has been previously reported to have a neuroprotective effect in organotypic hippocampal slices and to modulate synaptic transmission after in vitro hypoxia and oxygen-glucose deprivation (OGD). However, the mechanisms involved in the effect of this scavenger remain elusive. In this study, two concentrations of MnTMPyP were evaluated on synaptic transmission during ischemia and post-ischemic synaptic potentiation. The complex molecular changes supporting cellular adaptation to metabolic stress, and how these are modulated by MnTMPyP, were also investigated. Electrophysiological data showed that MnTMPyP causes a decrease in baseline synaptic transmission and impairment of synaptic potentiation. Proteomic analysis performed on MnTMPyP and hypoxia-treated tissue indicated an impairment in vesicular trafficking mechanisms, including reduced expression of Hsp90 and actin signalling. Alterations of vesicular trafficking may lead to reduced probability of neurotransmitter release and AMPA receptor activity, resulting in the observed modulatory effect of MnTMPyP. In OGD, protein enrichment analysis highlighted impairments in cell proliferation and differentiation, such as TGFß1 and CDKN1B signalling, in addition to downregulation of mitochondrial dysfunction and an increased expression of CAMKII. Taken together, our results may indicate modulation of neuronal sensitivity to the ischemic insult, and a complex role for MnTMPyP in synaptic transmission and plasticity, potentially providing molecular insights into the mechanisms mediating the effects of MnTMPyP during ischemia.

The effects of the superoxide dismutase mimetic, MnTMPyP, post hypoxia and oxygen glucose deprivation in isolated rat hippocampal slices.

The contributions of hypoxia, oxygen glucose deprivation (OGD) and oxidative stress, to the pathophysiology of acute ischemic stroke (AIS) are well established and can lead to disruptions in synaptic signaling. Antioxidant compounds have previously been shown to have a preconditioning and neuroprotective effect against an ischemic insult. Therefore, in this study we explored the effects of the reactive oxygen species (ROS) scavenger, MnTMPyP, on synaptic transmission in two models, hypoxia and oxygen glucose deprivation (OGD), in isolated rat hippocampal slices using electrophysiological techniques and organotypic hippocampal slice cultures. We report a novel modulatory effect of MnTMPyP on synaptic transmission post hypoxia and OGD, an effect specific to the CA1 region of the hippocampus. This reduction of the fEPSP by MnTMPyP post hypoxia in the CA1 was attenuated through the co-application of the adenosine A1 receptor antagonist, DPCPX (200 nM), and the NMDA receptor antagonists, AP-5 (10 µM) and DCKA (5 µM). These effects were not observed in the OGD model. Our organotypic data demonstrated a protective role for MnTMPyP, where slices had significantly less cell death in the CA1 region post hypoxia and OGD, compared to controls. Taken together, our results suggest a complex role for MnTMPyP on both synaptic signaling in an hypoxic environment and cell viability. Whether this SOD mimetic will play an important role in ischemia still remains to be determined.

MnTMPyP Reduces Paraquat-Induced Lung Epithelial-Like Cell Injury by Inhibiting Oxidative Stress and Endoplasmic Reticulum Stress / 中国医科大学学报

Objective To investigate the protective effect and underlying mechanism of the superoxide dismutase mimic, manganese (Ⅲ) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP), on paraquat (PQ) -induced lung epithelial-like cell injury. Methods Alveolar epithelial-like cells (A549) were pretreated with 10 μmol/L of MnTMPyP for 1.5 h and then cultured with or without PQ (750 μmol/L) for 24 h. Cell survival was determined using the MTT assay. Reactive oxygen species (ROS) production and Ca2+ levels were measured using flow cytometry. Glutathione reductase (GR) activity was determined using spectrophotometry. Expressions of the endoplasmic reticulum (ER) stress proteins, glucose regulatory protein 78 (Grp78) and C/EBP homologous protein (CHOP), were measured using Western blotting. Results Cell viability and GR activity were decreased, but ROS production, cytoplasmic Ca2+ levels, and expressions of Grp78 and CHOP were all increased in the PQ group compared to those in the control group. There were no statistically significant changes in the MnTMPyP group. Cell viability and GR activity were increased, while ROS production, cytoplasmic Ca2+ levels, and expressions of Grp78 and CHOP were all significantly reduced in the MnTMPyP group compared to those in the PQ group. Conclusion MnTMPyP effectively reduced PQ-induced lung epithelial-like cell injury, and the underlying mechanism is related to antagonism of PQ-induced ER stress and oxidative stress.

MnTMPyP inhibits paraquat-induced pulmonary epithelial-like cell injury by inhibiting oxidative stress.

OBJECTIVE: To investigate the protective effect and underlying mechanism of the superoxide dismutase mimic, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP), on paraquat (PQ)-induced lung alveolar epithelial-like cell injury. METHODS: Lung alveolar epithelial-like cells (A549) were pretreated with 10 µM MnTMPyP for 1.5 hr and then cultured with or without PQ (750 uM) for 24 hr. Cell survival was determined using the MTT assay. Apoptosis, mitochondrial transmembrane potential, reactive oxygen species (ROS) production, and Ca2+ levels were measured using flow cytometry. Glutathione reductase activity (GR activity) and caspase-3 activation were determined using spectrophotometry. Expression of the apoptosis proteins, Bcl-2 and Bax, and the endoplasmic reticulum (ER) stress proteins, glucose regulatory protein 78 (Grp78) and C/EBP homologous protein (CHOP), was measured using Western blot analysis. RESULTS: Cell viability, mitochondrial membrane potential, GR activity, and Bcl-2 expression were decreased, but apoptosis, ROS production, caspase-3 activity, cytoplasmic Ca2+ levels, and Bax, Grp78 and CHOP expression were all increased in the PQ group compared to the control group. There were no statistically significant changes in the MnTMPyP group. Cell viability, GR activity, mitochondrial membrane potential, and Bcl-2 protein expression were all increased, while apoptosis, ROS production, cytoplasmic Ca2+ levels, caspase-3 activity, and Bax, Grp78 and CHOP expression were all significantly reduced in the MnTMPyP group compared to PQ group. CONCLUSION: MnTMPyP effectively reduced PQ-induced lung epithelial-like cell injury, and the underlying mechanism is related to antagonism of PQ-induced oxidative stress.

A sensitive impedimetric platform biosensing protein: Insoluble precipitates based on the biocatalysis of manganese(III) meso-tetrakis (4-N-methylpyridiniumyl)-porphyrinin in HCR-assisted dsDNA.

In this study, a sensitive biosensing interface for protein was reported based on nonconductive insoluble precipitates (IPs) by the biocatalysis of manganese(III) meso-tetrakis (4-N-methylpyridiniumyl)-porphyrin (MnTMPyP), which was intercalated into formed double-strand DNA (dsDNA) scaffold triggered by hybridization chain reaction (HCR). In the proposed impedimetric aptasensor, carcinoembryonic antigen (CEA) and its aptamer were used as testing model. PtPd nanowires (PtPdNWs) with large surface area and superior conductivity were employed as nanocarriers to greatly immobilize biomolecules (e.g. CEA aptamers). Then, two DNA hairpins H1 and H2 were introduced to trigger HCR with the assistance of DNA initiator, resulting in the formation of a long dsDNA scaffold. Meanwhile, mimicking enzyme MnTMPyP molecules were embedded into the resultant dsDNA, in situ generating the complex MnTMPyP-dsDNA with peroxidase-like activity. Under the biocatalysis of MnTMPyP-dsDNA, 3,3-diaminobenzidine (DAB) was oxidized to form nonconductive IPs. As a result, the electron transfer between electrode interface and redox probe was vastly hindered, leading to the significant amplification of electrochemical impedimetric signal. So, greatly improved analytical performances of the proposed aptasensor were achieved with a detection limit as low as 0.030pgmL(-1). And the successful assay of CEA in human serum samples enabled the developed biosensing platform to have promising potential in bioanalysis.

Cyclosporine A and MnTMPyP Alleviate α-Synuclein Expression and Aggregation in Cypermethrin-Induced Parkinsonism.

Cypermethrin induces the mitochondrial dysfunction and oxidative damage to the nigrostriatal dopaminergic neurons leading to Parkinsonism in rats. Despite α-synuclein aggregation is reported to be critical in Parkinson's disease, its role and alliance with the mitochondrial dysfunction and oxidative damage leading to cypermethrin-induced Parkinsonism have not yet been deciphered. The present study aimed to examine the effect of cypermethrin on the expression and aggregation of α-synuclein and its subsequent connection with oxidative damage and mitochondrial dysfunction leading to the nigrostriatal dopaminergic neurodegeneration in the presence or absence of a mitochondrial membrane transition pore opening inhibitor, cyclosporine A and a superoxide dismutase/catalase mimetic, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP). The expression of α-synuclein, 3-nitrotyrosine (3-NT), 4-hydroxynonenal (4-HNE)-modified proteins, mitochondrial dysfunction-dependent apoptotic proteins, nitrite content, lipid peroxidation (LPO) and number of tyrosine hydroxylase (TH)-positive neurons were estimated in the substantia nigra and dopamine content in the striatum of control and treated rats employing standard procedures. Cypermethrin augmented the expression of α-synuclein, 3-NT, 4-HNE-modified proteins, caspase-3, mitochondrial Bax and cytosolic cytochrome-c along with nitrite and LPO and reduced the expression of cytosolic Bax, mitochondrial cytochrome-c, dopamine and number of TH-positive neurons. Cyclosporine A or MnTMPyP alleviated the expression and aggregation of α-synuclein along with indicators of the mitochondrial dysfunction, oxidative damage and dopaminergic neurodegeneration. The results demonstrate that cypermethrin induces α-synuclein expression and aggregation while cyclosporine A or MnTMPyP rescues from α-synuclein over-expression and aggregation along with the mitochondrial dysfunction and oxidative damage leading to Parkinsonism in rats.

The effects of superoxide dismutase mimetic MnTMPyP on the altered blood-brain barrier integrity in experimental preeclampsia with or without seizures in rats.

We investigated the effects of a cell-permeable superoxide dismutase mimetic, manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) on blood-brain barrier (BBB) integrity following pentylenetetrazole (PTZ)-induced seizures in experimental preeclampsia symptoms induced by N(omega)-nitro-l-arginine methyl ester (l-NAME) in pregnant rats. To show the functional and morphological alterations in BBB integrity, quantitative analysis of sodium fluorescein (NaFlu) extravasation, immunohistochemistry and electron microscopic assessment of horseradish peroxidase (HRP) permeability were performed. Varying degrees of proteinuria were seen and arterial blood pressure increased in l-NAME-treated pregnant rats (p<0.01). MnTMPyP pretreatment and convulsive PTZ challenge significantly decreased the immunoreactivity of occludin in hippocampal capillaries in l-NAME-treated pregnant rats (p<0.01). BBB permeability to NaFlu significantly increased in pregnant rats treated with l-NAME plus PTZ (p<0.01), but MnTMPyP pretreatment did not significantly decrease NaFlu penetration into the brain parenchyma in these animals. Ultrastructurally, frequent vesicles containing HRP reaction products were observed in the capillary endothelial cells in the cerebral cortex and hippocampus of pregnant rats treated with l-NAME and l-NAME plus PTZ with the abundance being more in the latter group. MnTMPyP pretreatment caused a marked reduction in the frequency of HRP reaction product containing vesicles in both experimental settings. In conclusion, the results of the present study provide evidence that MnTMPyP plays an important role in limiting the enhanced vesicle-mediated transcellular transport in BBB endothelium in a rat model of preeclampsia and the differences in the way of transports of NaFlu and HRP might be responsible for the different effects of MnTMPyP on the BBB permeability to these two tracers.

Aging-dependent changes in rat heart mitochondrial glutaredoxins--Implications for redox regulation.

Clinical and animal studies have documented that hearts of the elderly are more susceptible to ischemia/reperfusion damage compared to young adults. Recently we found that aging-dependent increase in susceptibility of cardiomyocytes to apoptosis was attributable to decrease in cytosolic glutaredoxin 1 (Grx1) and concomitant decrease in NF-κB-mediated expression of anti-apoptotic proteins. Besides primary localization in the cytosol, Grx1 also exists in the mitochondrial intermembrane space (IMS). In contrast, Grx2 is confined to the mitochondrial matrix. Here we report that Grx1 is decreased by 50-60% in the IMS, but Grx2 is increased by 1.4-2.6 fold in the matrix of heart mitochondria from elderly rats. Determination of in situ activities of the Grx isozymes from both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria revealed that Grx1 was fully active in the IMS. However, Grx2 was mostly in an inactive form in the matrix, consistent with reversible sequestration of the active-site cysteines of two Grx2 molecules in complex with an iron-sulfur cluster. Our quantitative evaluations of the active/inactive ratio for Grx2 suggest that levels of dimeric Grx2 complex with iron-sulfur clusters are increased in SSM and IFM in the hearts of elderly rats. We found that the inactive Grx2 can be fully reactivated by sodium dithionite or exogenous superoxide production mediated by xanthine oxidase. However, treatment with rotenone, which generates intramitochondrial superoxide through inhibition of mitochondrial respiratory chain Complex I, did not lead to Grx2 activation. These findings suggest that insufficient ROS accumulates in the vicinity of dimeric Grx2 to activate it in situ.