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

Altres ajuts: The group has received funding from 'la Caixa Foundation' CI15-00009, from the European Institute of Innovation and Technology (EIT) PoC-2016-SPAIN-04, which receives support from the European Union's Horizon 2020 research and innovation program, and from the 'Fundación para la Innovación y la Prospectiva en Salud en España (FIPSE)' program 3594-18.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

Preclinical Characterization of Antioxidant Quinolyl Nitrone QN23 as a New Candidate for the Treatment of Ischemic Stroke

Nitrones are encouraging drug candidates for the treatment of oxidative stress-driven diseases such as acute ischemic stroke (AIS). In a previous study, we found a promising quinolylnitrone, QN23, which exerted a neuroprotective effect in neuronal cell cultures subjected to oxygen–glucose deprivation and in experimental models of cerebral ischemia. In this paper, we update the biological and pharmacological characterization of QN23. We describe the suitability of intravenous administration of QN23 to induce neuroprotection in transitory four-vessel occlusion (4VO) and middle cerebral artery occlusion (tMCAO) experimental models of brain ischemia by assessing neuronal death, apoptosis induction, and infarct area, as well as neurofunctional outcomes. QN23 significantly decreased the neuronal death and apoptosis induced by the ischemic episode in a dose-dependent manner and showed a therapeutic effect when administered up to 3 h after post-ischemic reperfusion onset, effects that remained 11 weeks after the ischemic episode. In addition, QN23 significantly reduced infarct volume, thus recovering the motor function in a tMCAO model. Remarkably, we assessed the antioxidant activity of QN23 in vivo using dihydroethidium as a molecular probe for radical species. Finally, we describe QN23 pharmacokinetic parameters. All these results pointing to QN23 as an interesting and promising preclinical candidate for the treatment of AIS.This work was supported by the Instituto de Salud Carlos III and co-financed by the European Development Regional Fund (FEDER) through grants PI18/00255, RD16/0019/0006, and RD21/0006/0019 to J.M. and A.A., and RD16/0019/0008 and RD21/0006/0014 to J.B.S.; the MINECO grant SAF2015-65586-R to J.M.-C.; and the Comunidad de Madrid Neurocentro project B2017/BMD-3760 to D.G.-N

Evaluación del potencial neuroprotector del modulador selectivo de los receptores estrogénicos, Bazedoxifeno, en un modelo animal de ictus con reperfusión: Influencia de la diabetes

El ictus es una de las primeras causas de muerte y discapacidad permanente, y la diabetes mellitus (DM) es uno de los factores de riesgo principales en esta patología. Actualmente el tratamiento establecido para el ictus isquémico consiste en la restauración del flujo sanguíneo cerebral mediante disolución farmacológica o retirada mecánica del trombo. Sin embargo, sólo un pequeño porcentaje de pacientes es susceptible de recibir estos tratamientos endovasculares, lo que refuerza la necesidad de contar con fármacos neuroprotectores. En este contexto se enmarcan los “moduladores selectivos de los receptores estrogénicos”, específicamente Bazedoxifeno (BZA) como potencial terapia neuroprotectora coadyudante en el ictus isquémico. La hipótesis planteada en este estudio es: “BZA es un neuroprotector tan eficaz como estradiol (E2), pero con perfil farmacológico más favorable, en el ictus isquémico agudo con reperfusión cuando se administra simultáneamente con la eliminación mecánica del coágulo oclusor. Este efecto neuroprotector se mantiene incluso en una situación más desfavorable como es aquella en la que concurre la DM”. Los objetivos y resultados obtenidos son: 1) Análisis de la influencia de la DM sobre la perfusión cerebrocortical así como sobre la fisiología básica de la isquemia-reperfusión, en el modelo de oclusión transitoria de la arteria cerebral media (tMCAO) mediante la técnica del filamento intraluminal en rata. Los resultados demuestran que la DM no altera la función microvascular ni la fisiología básica en la isquemia-reperfusión. 2) Análisis de la influencia de la DM sobre el daño cerebral inducido por isquemia-reperfusión en términos de valoración neurofuncional y volúmenes de infarto y edema cerebrales. Los resultados demuestran que la DM influye negativamente sobre el daño cerebral isquémico. 3) Evaluación comparativa de los efectos de BZA y E2 sobre el daño cerebral inducido por isquemia-reperfusión en condiciones de normoglucemia (NG) y DM. Los resultados ponen de manifiesto el efecto neuroprotector de BZA en condiciones de NG y DM, mientras que E2 posee un efecto menos consistente en condiciones de NG. 4) Análisis del posible efecto de BZA y E2 sobre la expresión de receptores estrogénicos ERα, ERβ y GPER en condiciones de NG y DM. Los resultados indican que ERα y ERβ, pero no GPER, están implicados en el efecto neuroprotector de BZA y E2. 5) Análisis del posible efecto de BZA y E2 sobre la actividad de las vías de supervivencia celular, MAPK/ERK1/2 y PI3K/Akt, en condiciones de NG y DM. Los resultados demuestran que la vía MAPK/ERK1/2, pero no la vía PI3K/Akt, está implicada en los efectos neuroprotectores de BZA y E2. El hecho de que BZA ejerza efecto neuroprotector en el ictus isquémico incluso bajo condiciones de diabetes, aumenta la traslacionalidad de nuestros resultados y refuerza su consideración como una alternativa eficaz pero más segura que E2 en el tratamiento coadyudante del ictus isquémico

Insight into the molecular sex dimorphism of ischaemic stroke in rat cerebral cortex: Focus on neuroglobin, sex steroids and autophagy

Including sex is of paramount importance in preclinical and clinical stroke researches, and molecular studies dealing in depth with sex differences in stroke pathophysiology are needed. To gain insight into the molecular sex dimorphism of ischaemic stroke in rat cerebral cortex, male and female adult rats were subjected to transient middle cerebral artery occlusion. The expression of neuroglobin (Ngb) and other functionally related molecules involved in sex steroid signalling (oestrogen and androgen receptors), steroidogenesis (StAR, TSPO and aromatase) and autophagic activity (LC3B-II/LC3B-I ratio, UCP2 and HIF-1α) was assessed in the ipsilateral ischaemic and contralateral non-ischaemic hemispheres. An increased expression of Ngb was detected in the injured female cerebral cortex. In contrast, increased expression of oestrogen receptor α, GPER, StAR, TSPO and UCP2, and decreased androgen receptor expression were detected in the injured male cortex. In both sexes, the ischaemic insult induced an upregulation of LC3B-II/-I ratio, indicative of increased autophagy. Therefore, the cerebral cortex activates both sex-specific and common molecular responses with neuroprotective potential after ischaemia–reperfusion, which globally results in similar stroke outcome in both sexes. Nonetheless, these different potential molecular targets should be taken into account when neuroprotective drugs aiming to reduce brain damage in ischaemic stroke are investigated

Insight into the molecular sex dimorphism of ischaemic stroke in rat cerebral cortex: Focus on neuroglobin, sex steroids and autophagy

Including sex is of paramount importance in preclinical and clinical stroke researches, and molecular studies dealing in depth with sex differences in stroke pathophysiology are needed. To gain insight into the molecular sex dimorphism of ischaemic stroke in rat cerebral cortex, male and female adult rats were subjected to transient middle cerebral artery occlusion. The expression of neuroglobin (Ngb) and other functionally related molecules involved in sex steroid signalling (oestrogen and androgen receptors), steroidogenesis (StAR, TSPO and aromatase) and autophagic activity (LC3B-II/LC3B-I ratio, UCP2 and HIF-1α) was assessed in the ipsilateral ischaemic and contralateral non-ischaemic hemispheres. An increased expression of Ngb was detected in the injured female cerebral cortex. In contrast, increased expression of oestrogen receptor α, GPER, StAR, TSPO and UCP2, and decreased androgen receptor expression were detected in the injured male cortex. In both sexes, the ischaemic insult induced an upregulation of LC3B-II/-I ratio, indicative of increased autophagy. Therefore, the cerebral cortex activates both sex-specific and common molecular responses with neuroprotective potential after ischaemia–reperfusion, which globally results in similar stroke outcome in both sexes. Nonetheless, these different potential molecular targets should be taken into account when neuroprotective drugs aiming to reduce brain damage in ischaemic stroke are investigated

Brain Cell Senescence: A New Therapeutic Target for the Acute Treatment of Ischemic Stroke.

Aging is a major risk factor for cerebral infarction. Since cellular senescence is intrinsic to aging, we postulated that stroke-induced cellular senescence might contribute to neural dysfunction. Adult male Wistar rats underwent 60-minute middle cerebral artery occlusion and were grouped according to 3 reperfusion times: 24 hours, 3, and 7 days. The major biomarkers of senescence: 1) accumulation of the lysosomal pigment, lipofuscin; 2) expression of the cell cycle arrest markers p21, p53, and p16INK4a; and 3) expression of the senescence-associated secretory phenotype cytokines interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and interleukin-1β (IL-1β) were investigated in brain samples. Lipofuscin accumulation was scarce at the initial stage of brain damage (24 hours), but progressively increased until it reached massive distribution at 7 days post-ischemia. Lipofuscin granules (aggresomes) were mainly confined to the infarcted areas, that is parietal cortex and adjacent caudate-putamen, which were equally affected. The expression of p21, p53, and p16INK4a, and that of IL-6, TNF-α, and IL-1β, was significantly higher in the ischemic hemisphere than in the non-ischemic hemisphere. These data indicate that brain cell senescence develops during acute ischemic infarction and suggest that the acute treatment of ischemic stroke might be enhanced using senolytic drugs

Cerebroprotective Effect of 17β-Estradiol Replacement Therapy in Ovariectomy-Induced Post-Menopausal Rats Subjected to Ischemic Stroke: Role of MAPK/ERK1/2 Pathway and PI3K-Independent Akt Activation

Despite the overwhelming advances in the understanding of the pathogenesis of stroke, a devastating disease affecting millions of people worldwide, currently there are only a limited number of effective treatments available. Preclinical and clinical studies show that stroke is a sexually dimorphic disorder, affecting males and females differently. Strong experimental evidence indicates that estrogen may play a role in this difference and that exogenous 17β-estradiol (E2) is neuroprotective against stroke in both male and female rodents. However, the molecular mechanisms by which E2 intervenes in ischemia-induced cell death, revealing these sex differences, remain unclear. The present study was aimed to determine, in female rats, the molecular mechanisms of two well-known pro-survival signaling pathways, MAPK/ERK1/2 and PI3K/Akt, that mediate E2 neuroprotection in response to acute ischemic stroke. E2 pretreatment reduced brain damage and attenuated apoptotic cell death in ovariectomized female rats after an ischemic insult. Moreover, E2 decreased phosphorylation of ERK1/2 and prevented ischemia/reperfusion-induced dephosphorylation of both Akt and the pro-apoptotic protein, BAD. However, MAPK/ERK1/2 inhibitor PD98059, but not the PI3K inhibitor LY294002, attenuated E2 neuroprotection. Thus, these results suggested that E2 pretreatment in ovariectomized female rats modulates MAPK/ERK1/2 and activates Akt independently of PI3K to promote cerebroprotection in ischemic stroke. A better understanding of the mechanisms and the influence of E2 in the female sex paves the way for the design of future successful hormone replacement therapies

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

Altres ajuts: The group has received funding from 'la Caixa Foundation' CI15-00009, from the European Institute of Innovation and Technology (EIT) PoC-2016-SPAIN-04, which receives support from the European Union's Horizon 2020 research and innovation program, and from the 'Fundación para la Innovación y la Prospectiva en Salud en España (FIPSE)' program 3594-18.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

Molecular mechanisms mediating the neuroprotective role of the selective estrogen receptor modulator, bazedoxifene, in acute ischemic stroke: A comparative study with 17β-estradiol

As the knowledge on the estrogenic system in the brain grows, the possibilities to modulate it in order to afford further neuroprotection in brain damaging disorders so do it. We have previously demonstrated the ability of the selective estrogen receptor modulator, bazedoxifene (BZA), to reduce experimental ischemic brain damage. The present study has been designed to gain insight into the molecular mechanisms involved in such a neuroprotective action by investigating: 1) stroke-induced apoptotic cell death; 2) expression of estrogen receptors (ER) ERα, ERβ and the G-protein coupled estrogen receptor (GPER); and 3) modulation of MAPK/ ERK1/2 and PI3K/Akt signaling pathways. For comparison, a parallel study was done with 17β-estradiol (E2)- treated animals. Male Wistar rats subject to transient right middle cerebral artery occlusion (tMCAO, intraluminal thread technique, 60 min), were distributed in vehicle-, BZA- (20.7 ± 2.1 ng/mL in plasma) and E2- (45.6 ± 7.8 pg/mL in plasma) treated groups. At 24 h from the onset of tMCAO, RT-PCR, Western blot and histochemical analysis were performed on brain tissue samples. Ischemia-reperfusion per se increased apoptosis as assessed by both caspase-3 activity and TUNEL-positive cell counts, which were reversed by both BZA and E2. ERα and ERβ expression, but not that of GPER, was reduced by the ischemic insult. BZA and E2 had different effects: while BZA increased both ERα and ERβ expression, E2 increased ERα expression but did not change that of ERβ. Both MAPK/ERK1/2 and PI3K/Akt pathways were stimulated under ischemic conditions. While BZA strongly reduced the increased p-ERK1/2 levels, E2 did not. Neither BZA nor E2 modified ischemia-induced increase in p-Akt levels. These results show that modulation of ERα and ERβ expression, as well as of the ERK1/2 signaling pathway accounts, at least in part, for the inhibitory effect of BZA on the stroke-induced apoptotic cell death. This lends mechanistic support to the consideration of BZA as a potential neuroprotective drug in acute ischemic stroke treatment.Supported in part by Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (RETICS networks INVICTUS (RD12/0014/ 0004) and INVICTUS+ (RD16/0019/0008), as well as grant PI12/ 00145) and by Conselleria d' Educació, Investigació, Cultura i Esport – GVA (grant GV/2015/133).Peer reviewe