Specific rescue by ortho‐hydroxy atorvastatin of cortical <scp>GABA</scp>ergic neurons from previous oxygen/glucose deprivation: role of <scp>pCREB</scp>

Martí-Sistac

Ponce

et al. 2018

Redox Biology

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Despite transferrin being the main circulating carrier of iron in body fluids, and iron overload conditions being known to worsen stroke outcome through reactive oxygen species (ROS)-induced damage, the contribution of blood transferrin saturation (TSAT) to stroke brain damage is unknown. The objective of this study was to obtain evidence on whether TSAT determines the impact of experimental ischemic stroke on brain damage and whether iron-free transferrin (apotransferrin, ATf)-induced reduction of TSAT is neuroprotective. We found that experimental ischemic stroke promoted an early extravasation of circulating iron-loaded transferrin (holotransferrin, HTf) to the ischemic brain parenchyma. In vitro, HTf was found to boost ROS production and to be harmful to primary neuronal cultures exposed to oxygen and glucose deprivation. In stroked rats, whereas increasing TSAT with exogenous HTf was detrimental, administration of exogenous ATf and the subsequent reduction of TSAT was neuroprotective. Mechanistically, ATf did not prevent extravasation of HTf to the brain parenchyma in rats exposed to ischemic stroke. However, ATf in vitro reduced NMDA-induced neuronal uptake of HTf and also both the NMDA-mediated lipid peroxidation derived 4-HNE and the resulting neuronal death without altering Ca2+-calcineurin signaling downstream the NMDA receptor. Removal of transferrin from the culture media or blockade of transferrin receptors reduced neuronal death. Together, our data establish that blood TSAT exerts a critical role in experimental stroke-induced brain damage. In addition, our findings suggest that the protective effect of ATf at the neuronal level resides in preventing NMDA-induced HTf uptake and ROS production, which in turn reduces neuronal damage.

Section: Discussionmentioning

confidence: 87%

Iron-loaded transferrin (Tf) is detrimental whereas iron-free Tf confers protection against brain ischemia by modifying blood Tf saturation and subsequent neuronal damage

Martí-Sistac

Ponce

et al. 2018

Redox Biology

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“…The impact of excess extracellular glutamate on neuronal survival is more than that on the cystine/glutamate exchanger normalXnormalc−. In fact, most of the effect is produced by glutamate binding to specific glutamate receptors present in cell membranes of several cell types and, especially in neurons, N -methyl- D -aspartate (NMDA) subtype of glutamate receptors (NMDARs) (Guirao et al, 2017) that are coupled to an intracellular downstream complex of signaling effectors, which are known to initiate excitotoxic neuronal death following ischemia.…”

Section: The Ferroptotic Component Of Stroke-induced Neurodegenerationmentioning

confidence: 99%

Deciphering the Iron Side of Stroke: Neurodegeneration at the Crossroads Between Iron Dyshomeostasis, Excitotoxicity, and Ferroptosis

2019

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In general, iron represents a double-edged sword in metabolism in most tissues, especially in the brain. Although the high metabolic demands of brain cells require iron as a redox-active metal for ATP-producing enzymes, the brain is highly vulnerable to the devastating consequences of excessive iron-induced oxidative stress and, as recently found, to ferroptosis as well. The blood–brain barrier (BBB) protects the brain from fluctuations in systemic iron. Under pathological conditions, especially in acute brain pathologies such as stroke, the BBB is disrupted, and iron pools from the blood gain sudden access to the brain parenchyma, which is crucial in mediating stroke-induced neurodegeneration. Each brain cell type reacts with changes in their expression of proteins involved in iron uptake, efflux, storage, and mobilization to preserve its internal iron homeostasis, with specific organelles such as mitochondria showing specialized responses. However, during ischemia, neurons are challenged with excess extracellular glutamate in the presence of high levels of extracellular iron; this causes glutamate receptor overactivation that boosts neuronal iron uptake and a subsequent overproduction of membrane peroxides. This glutamate-driven neuronal death can be attenuated by iron-chelating compounds or free radical scavenger molecules. Moreover, vascular wall rupture in hemorrhagic stroke results in the accumulation and lysis of iron-rich red blood cells at the brain parenchyma and the subsequent presence of hemoglobin and heme iron at the extracellular milieu, thereby contributing to iron-induced lipid peroxidation and cell death. This review summarizes recent progresses made in understanding the ferroptosis component underlying both ischemic and hemorrhagic stroke subtypes.

“…In brief, pregnant rats we anesthetized with 4% isoflurane in a 30% O 2 /70% N 2 O mixture, fetuses rapidly and carefully excised for further processing, and rats euthanized by decapitation. Primary cultures of rat cortical neurons were prepared, as we previously described [13,14], from E18 fetuses obtained from pregnant Sprague-Dawley rats (Envigo/Harlan, Barcelona, Spain). Neurons in culture were grown in Neurobasal™ medium (Gibco, Alcobendas, Spain) supplemented with 2% B-27 (Life Technologies, Alcobendas, Spain), 0.5 µM L-glutamine and 40 µg/mL gentamicin and kept in an incubator (Galaxy RX, RS Biotech, Irvine, UK) at 37 • C in 95% air with 5% CO 2 .…”

Section: Primary Culture Of Cortical Neuronsmentioning

confidence: 99%

“…Cell death by OGD or NMDA was determined by measuring the incorporation of propidium iodide along 24 h. We used a Varioskan flash reader (Thermo Fisher Scientific, Alcobendas, Spain) and followed the method by Rudolph and col. [15], adapted in our lab [13,14]. Ferroptosis induced by erastin produced delayed neuronal death that was assessed by measuring the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as a result of metabolic activity in living cells.…”

Section: Assessment Of Neuronal Viabilitymentioning

confidence: 99%

Comparative Proteomics Unveils LRRFIP1 as a New Player in the DAPK1 Interactome of Neurons Exposed to Oxygen and Glucose Deprivation

Guirao

Ponce

et al. 2020

Antioxidants

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Death-associated protein kinase 1 (DAPK1) is a pleiotropic hub of a number of networked distributed intracellular processes. Among them, DAPK1 is known to interact with the excitotoxicity driver NMDA receptor (NMDAR), and in sudden pathophysiological conditions of the brain, e.g., stroke, several lines of evidence link DAPK1 with the transduction of glutamate-induced events that determine neuronal fate. In turn, DAPK1 expression and activity are known to be affected by the redox status of the cell. To delineate specific and differential neuronal DAPK1 interactors in stroke-like conditions in vitro, we exposed primary cultures of rat cortical neurons to oxygen/glucose deprivation (OGD), a condition that increases reactive oxygen species (ROS) and lipid peroxides. OGD or control samples were co-immunoprecipitated separately, trypsin-digested, and proteins in the interactome identified by high-resolution LC-MS/MS. Data were processed and curated using bioinformatics tools. OGD increased total DAPK1 protein levels, cleavage into shorter isoforms, and dephosphorylation to render the active DAPK1 form. The DAPK1 interactome comprises some 600 proteins, mostly involving binding, catalytic and structural molecular functions. OGD up-regulated 190 and down-regulated 192 candidate DAPK1-interacting proteins. Some differentially up-regulated interactors related to NMDAR were validated by WB. In addition, a novel differential DAPK1 partner, LRRFIP1, was further confirmed by reverse Co-IP. Furthermore, LRRFIP1 levels were increased by pro-oxidant conditions such as ODG or the ferroptosis inducer erastin. The present study identifies novel partners of DAPK1, such as LRRFIP1, which are suitable as targets for neuroprotection.