salubrinal and Brain-Ischemia

Background and Purpose- Ischemic stroke impairs endoplasmic reticulum (ER) function, causes ER stress, and activates the unfolded protein response. The unfolded protein response consists of 3 branches controlled by ER stress sensor proteins, which include PERK (protein kinase RNA-like ER kinase). Activated PERK phosphorylates eIF2α (eukaryotic initiation factor 2 alpha), resulting in inhibition of global protein synthesis. Here, we aimed to clarify the role of the PERK unfolded protein response branch in stroke. Methods- Neuron-specific and tamoxifen-inducible PERK conditional knockout (cKO) mice were generated by cross-breeding Camk2a-CreERT2 with

Topics: Animals; Brain Ischemia; Cinnamates; eIF-2 Kinase; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Infarction, Middle Cerebral Artery; Mice; Mice, Knockout; Neurons; Neuroprotection; Phosphorylation; Protein Biosynthesis; Stroke; Thiourea; Unfolded Protein Response

Ischemic stroke is one of the most important causes of death and disability worldwide. Subroutines underlying cell death after stroke are largely unknown despite their importance in the design of novel therapies for this pathology. Necroptosis, a recently described form of regulated cell death, has been related with inflammation and, in some models, with endoplasmic reticulum (ER) stress. We hypothesize that alleviation of ER stress following a salubrinal treatment will reduce the ischemic-dependent necroptosis. To probe the hypothesis, we measured, at 48 and 72 h after transient global cerebral ischemia in rat, in cerebral cortex and cornu ammonis 1, the main hallmarks of necroptosis: mRNA levels and phosphorylation of mixed lineage kinase domain like pseudokinase as well as receptor interacting serine/threonine protein kinase 3, along the years 2017-2018. Selective neuronal loss after 7 days of the ischemic insult, and other markers related with the inflammatory response were also measured. This study shows that necroptosis in cerebral cortex can be detected after 72 h of the insult and seems to be elicited before 48 h of reperfusion. The type of necroptosis here observed seems to be tumor necrosis factor receptor 1 independent. Necroptotic response is less evident in the cornu ammonis 1 hippocampal area than in cerebral cortex. The treatment with salubrinal administered 1 and 24 h after the ischemia, decreased the necroptotic marker levels and reduced the areas of selective neuronal loss, supporting the presence of ischemic-dependent necroptosis, and the notion that ER stress is involved in the necroptotic response. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

Topics: Animals; Brain Ischemia; Cell Survival; Cerebral Cortex; Cinnamates; Disease Models, Animal; Male; Necroptosis; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Thiourea

Blood reperfusion of the ischemic tissue after stroke promotes increases in the inflammatory response as well as accumulation of unfolded/misfolded proteins in the cell, leading to endoplasmic reticulum (ER) stress. Both Inflammation and ER stress are critical processes in the delayed death of the cells damaged after ischemia. The aim of this study is to check the putative synergic neuroprotective effect by combining anti-inflammatory and anti-ER stress agents after ischemia.. The study was performed on a two-vessel occlusion global cerebral ischemia model. Animals were treated with salubrinal one hour after ischemia and with robenacoxib at 8 h and 32 h after ischemia. Parameters related to the integrity of the blood-brain barrier (BBB), such as matrix metalloproteinase 9 and different cell adhesion molecules (CAMs), were analyzed by qPCR at 24 h and 48 h after ischemia. Microglia and cell components of the neurovascular unit, including neurons, endothelial cells and astrocytes, were analyzed by immunofluorescence after 48 h and seven days of reperfusion.. Pharmacologic control of ER stress by salubrinal treatment after ischemia, revealed a neuroprotective effect over neurons that reduces the transcription of molecules involved in the impairment of the BBB. Robenacoxib treatment stepped neuronal demise forward, revealing a detrimental effect of this anti-inflammatory agent. Combined treatment with robenacoxib and salubrinal after ischemia prevented neuronal loss and changes in components of the neurovascular unit and microglia observed when animals were treated only with robenacoxib.. Combined treatment with anti-ER stress and anti-inflammatory agents is able to provide enhanced neuroprotective effects reducing glial activation, which opens new avenues in therapies against stroke.

Topics: Animals; Blood-Brain Barrier; Brain Ischemia; Cinnamates; Cyclooxygenase 2 Inhibitors; Diphenylamine; Drug Administration Schedule; Drug Therapy, Combination; Endoplasmic Reticulum Stress; Inflammation; Male; Neuroprotective Agents; Phenylacetates; Rats, Sprague-Dawley; Thiourea

Zinc (Zn(2+)) is considered to be one of the factors aggravating brain damage after cerebral ischemia. Since Zn(2+) activates microglia, immune cells in the brain, this metal is proposed to modulate neuroinflammatory responses in the post-ischemic brain. Interleukin (IL)-23 is a heterodimeric cytokine composed of the p19 subunit unique to IL-23 and the p40 subunit common to IL-12. IL-23 has been shown to play a critical role in the progression of ischemic brain injury. However, whether Zn(2+) participates in the expression of IL-23 in microglia remains unknown. In this study, we examined the effect of Zn(2+) on IL-23 p19 mRNA expression using rat immortalized microglia HAPI cells. Exposure to Zn(2+) dose- and time-dependently induced the expression of IL-23 p19 mRNA in HAPI cells. Inhibitors of MAPK and NF-κB pathways failed to suppress this induction. Interestingly, we found that Zn(2+) stimulated the phosphorylation of eIF2α and promoted the nuclear accumulation of activating transcription factor 4 (ATF4). Treatment with salubrinal, an eIF2α dephosphorylation inhibitor, enhanced Zn(2+)-induced ATF4 accumulation and IL-23 p19 mRNA expression. In addition, reporter assay using the IL-23 p19 promoter region revealed that ATF4 directly transactivated IL-23 p19 promoter and that dominant-negative ATF4 suppressed Zn(2+)-induced activation of IL-23 p19 promoter. Taken together, these findings suggest that Zn(2+) up-regulates expression of the IL-23 p19 gene via the eIF2α/ATF4 axis in HAPI cells.

Topics: Activating Transcription Factor 4; Animals; Brain Ischemia; Cinnamates; Eukaryotic Initiation Factor-2; Gene Expression Regulation; Humans; Interleukin-23 Subunit p19; Microglia; Neuroimmunomodulation; Promoter Regions, Genetic; Rats; RNA, Messenger; Thiourea; Zinc

To investigate whether endoplasmic reticulum (ER) stress participates in the neuroprotective effects of ischemic preconditioning (IPC)-induced neuroprotection and autophagy activation in rat brains.. The right middle cerebral artery in SD rats was occluded for 10 min to induce focal cerebral IPC, and was occluded permanently 24 h later to induce permanent focal ischemia (PFI). ER stress inhibitor salubrinal (SAL) was injected via intracerebral ventricle infusion 10 min before the onset of IPC. Infarct volume and motor behavior deficits were examined after the ischemic insult. The protein levels of LC3, p62, HSP70, glucose-regulated protein 78 (GRP 78), p-eIF2α and caspase-12 in the ipsilateral cortex were analyzed using immunoblotting. LC3 expression pattern in the sections of ipsilateral cortex was observed with immunofluorescence.. Pretreatment with SAL (150 pmol) abolished the neuroprotective effects of IPC, as evidenced by the significant increases in mortality, infarct volume and motor deficits after PFI. At the molecular levels, pretreatment with SAL (150 pmol) significantly increased p-eIF2α level, and decreased GRP78 level after PFI, suggesting that SAL effectively inhibited ER stress in the cortex. Furthermore, the pretreatment with SAL blocked the IPC-induced upregulation of LC3-II and downregulation of p62 in the cortex, thus inhibiting the activation of autophagy. Moreover,SAL blocked the upregulation of HSP70, but significantly increased the cleaved caspase-12 level, thus promoting ER stress-dependent apoptotic signaling in the cortex.. ER stress-induced autophagy might contribute to the neuroprotective effect of brain ischemic preconditioning.

Topics: Animals; Apoptosis; Autophagy; Brain; Brain Ischemia; Cinnamates; Endoplasmic Reticulum Stress; Ischemic Preconditioning; Male; Rats; Rats, Sprague-Dawley; Signal Transduction; Thiourea

Recent studies have suggested that autophagy plays a prosurvival role in ischemic preconditioning (IPC). This study was taken to assess the linkage between autophagy and endoplasmic reticulum (ER) stress during the process of IPC. The effects of IPC on ER stress and neuronal injury were determined by exposure of primary cultured murine cortical neurons to 30 min of OGD 24 h prior to a subsequent lethal OGD. The effects of IPC on ER stress and ischemic brain damage were evaluated in rats by a brief ischemic insult followed by permanent focal ischemia (PFI) 24 h later using the suture occlusion technique. The results showed that both IPC and lethal OGD increased the LC3-II expression and decreased p62 protein levels, but the extent of autophagy activation was varied. IPC treatment ameliorated OGD-induced cell damage in cultured cortical neurons, whereas 3-MA (5-20 mM) and bafilomycin A 1 (75-150 nM) suppressed the neuroprotection induced by IPC. 3-MA, at the dose blocking autophagy, significantly inhibited IPC-induced HSP70, HSP60 and GRP78 upregulation; meanwhile, it also aggregated the ER stress and increased activated caspase-12, caspase-3 and CHOP protein levels both in vitro and in vivo models. The ER stress inhibitor Sal (75 pmol) recovered IPC-induced neuroprotection in the presence of 3-MA. Rapamycin 50-200 nM in vitro and 35 pmol in vivo 24 h before the onset of lethal ischemia reduced ER stress and ischemia-induced neuronal damage. These results demonstrated that pre-activation of autophagy by ischemic preconditioning can boost endogenous defense mechanisms to upregulate molecular chaperones, and hence reduce excessive ER stress during fatal ischemia.

Topics: Adenine; Animals; Apoptosis; Autophagy; Brain Ischemia; Caspase 12; Caspase 3; Cells, Cultured; Cerebral Cortex; Cinnamates; Cytoprotection; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Glucose; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Ischemic Preconditioning; Male; Mice; Neurons; Oxygen; Rats; Rats, Sprague-Dawley; Sirolimus; Thiourea; Transcription Factor CHOP