Specific rescue by ortho-hydroxy atorvastatin of cortical GABAergic neurons from previous oxygen/glucose deprivation: role of pCREB.

The statin atorvastatin (ATV) given as a post-treatment has been reported beneficial in stroke, although the mechanisms involved are not well understood so far. Here, we investigated in vitro the effect of post-treatment with ATV and its main bioactive metabolite ortho-hydroxy ATV (o-ATV) on neuroprotection after oxygen and glucose deprivation (OGD), and the role of the pro-survival cAMP response element-binding protein (CREB). Post-OGD treatment of primary cultures of rat cortical neurons with o-ATV, but not ATV, provided neuroprotection to a specific subset of cortical neurons that were large and positive for glutamic acid decarboxylase (large-GAD(+) neurons, GABAergic). Significantly, only these GABAergic neurons showed an increase in phosphorylated CREB (pCREB) early after neuronal cultures were treated post-OGD with o-ATV. We found that o-ATV, but not ATV, increased the neuronal uptake of glutamate from the medium; this provides a rationale for the specific effect of o-ATV on pCREB in large-GABAergic neurons, which have a higher ratio of synaptic (pCREB-promoting) vs extrasynaptic (pCREB-reducing) N-methyl-D-aspartate (NMDA) receptors (NMDAR) than that of small-non-GABAergic neurons. When we pharmacologically increased pCREB levels post-OGD in non-GABAergic neurons, through the selective activation of synaptic NMDAR, we observed as well long-lasting neuronal survival. We propose that the statin metabolite o-ATV given post-OGD boosts the intrinsic pro-survival factor pCREB in large-GABAergic cortical neurons in vitro, this contributing to protect them from OGD.

The effect of simvastatin on the proteome of detergent-resistant membrane domains: decreases of specific proteins previously related to cytoskeleton regulation, calcium homeostasis and cell fate.

Cell death induced by over-activation of glutamate receptors occurs in different neuropathologies. Cholesterol depletors protect from neurotoxic over-activation of glutamate receptors, and we have recently reported that this neuroprotection is associated with a reduction of the N-methyl-D-aspartate subtype of glutamate receptors in detergent-resistant membrane domains (DRM). In the present study we used comparative proteomics to further identify which proteins, besides the N-methyl-D-aspartate receptor, change its percentage of association to DRM after treatment of neurons with simvastatin. We detected 338 spots in neuronal DRM subjected to 2-DE; eleven of these spots changed its intensity after treatment with simvastatin. All 11 differential spots showed reduced intensity in simvastatin-treated samples and were identified as adipocyte plasma membrane associated protein, enolase, calretinin, coronin 1a, f-actin capping protein alpha1, f-actin capping protein alpha2, heat shock cognate protein 71, malate dehydrogenase, n-myc downregulated gene 1, prohibitin 2, Rab GDP dissociation inhibitor, translationally controlled tumor protein and voltage dependent anion selective channel protein 1. The proteins tested colocalized with the lipid raft marker caveolin-1. Interestingly, the proteins we have identified in the present study had been previously reported to play a role in cell fate and, thus, they might represent novel targets for neuroprotection.

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

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.

Simvastatin reduces the association of NMDA receptors to lipid rafts: a cholesterol-mediated effect in neuroprotection.

BACKGROUND AND PURPOSE: Excess brain extracellular glutamate induced by cerebral ischemia leads to neuronal death, mainly through overactivation of N-methyl-D-aspartate (NMDA) receptors. The cholesterol-lowering drugs statins have been reported to protect from NMDA-induced neuronal death but, so far, the mechanism underlying this protection remains unclear. Because NMDA receptors have been reported to be associated with the cholesterol-rich membrane domains known as lipid rafts, we have investigated the effect of treatments that deplete cholesterol levels on excitotoxicity and on association of NMDA receptors to lipid rafts. METHODS: Primary neuronal cultures were pretreated with inhibitors of cholesterol synthesis and cholesterol, and NMDA-induced cell death was determined by measuring release of lactate dehydrogenase. Lipid raft fractions were isolated and Western blots were performed. RESULTS: Treatment with the inhibitors of cholesterol synthesis simvastatin, which inhibits the first step of cholesterol synthesis, or AY9944, which inhibits the last step of cholesterol synthesis, protected neurons from NMDA-induced neuronal death by 70% and 54%, respectively. Treatment with these compounds reduced neuronal cholesterol levels by 35% and 13%, respectively. Simvastatin and AY9944 reduced the association of the subunit 1 of NMDA receptors (NMDAR1) to lipid rafts by 42% and 21%, respectively, and did not change total expression of NMDAR1. Addition of cholesterol reduced neuroprotection by statins and AY9944, and partially reverted the effect of simvastatin on the association of NMDAR1 to lipid rafts. CONCLUSIONS: These data demonstrate that reduction of cholesterol levels protects from NMDA-induced neuronal damage probably by reducing the association of NMDA receptors to lipid rafts.

Amyloidogenic properties of the prion protein fragment PrP(185-208): comparison with Alzheimer's peptide Abeta(1-28), influence of heparin and cell toxicity.

Amyloid fibrils are a hallmark of Alzheimer's and prion diseases. In both pathologies fibrils are found associated to glycosaminoglycans, modulators of the aggregation process. Amyloid peptides and proteins with very poor sequence homologies originate very similar aggregates. This implies the possible existence of a common formation mechanism. A homologous structural motif has recently been described for the Alzheimer's peptide Abeta(1-28) and the prion protein fragment PrP(185-208). We have studied the influence histidine residues and heparin on the aggregation process of both peptides and determined the possible amyloid characteristics of PrP(185-208), still unknown. The results show that PrP(185-208) forms amyloid aggregates in the presence of heparin. Histidines influence the aggregation kinetics, as in Abeta(1-28), although to a lesser extent. Other spectroscopic properties of the PrP(185-208) fragment are shown to be equivalent to those of other amyloid peptides and PrP(185-208) is shown to be cytotoxic using a neuroblastoma cell line.

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

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.