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Autophagy and apoptosis are differentially induced in neurons and astrocytes treated with an in vitro mimic of the ischemic penumbra.

ABSTRACT: The development of clinical stroke therapies remains elusive. The neuroprotective efficacies of thousands of molecules and compounds have not yet been determined; however, screening large volumes of potential targets in vivo is severely rate limiting. High throughput screens (HTS) may be used to discover promising candidates, but this approach has been hindered by the lack of a simple in vitro model of the ischemic penumbra, a clinically relevant region of stroke-afflicted brain. Recently, our laboratory developed such a mimic (ischemic solution: IS) suitable for HTS, but the etiology of stress pathways activated by this model are poorly understood. The aim of the present study was to determine if the cell death phenotype induced by IS accurately mimics the in vivo penumbra and thus whether our model system is suitable for use in HTS. We treated cultured neuron and astrocyte cell lines with IS for up to 48 hrs and examined cellular energy state ([ATP]), cell and organelle morphology, and gene and molecular profiles related to stress pathways. We found that IS-treated cells exhibited a phenotype of mixed apoptosis/autophagy characteristic of the in vivo penumbra, including: (1) short-term elevation of [ATP] followed by progressive ATP depletion and Poly ADP Ribose Polymerase cleavage, (2) increased vacuole number in the cytoplasm, (3) mitochondrial rupture, decreased mitochondrial and cristae density, release of cytochrome C and apoptosis inducing factor, (4) chromatin condensation, nuclear lamin A and DNA cleavage, fragmentation of the nuclear envelope, and (5) altered expression of mRNA and proteins consistent with autophagy and apoptosis. We conclude that our in vitro model of the ischemic penumbra induces autophagy and apoptosis in cultured neuron and astrocyte cell lines and that this mimic solution is suitable for use in HTS to elucidate neuroprotective candidates against ischemic penumbral cell death.

SUBMITTER: Pamenter ME

PROVIDER: S-EPMC3520810 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Autophagy and apoptosis are differentially induced in neurons and astrocytes treated with an in vitro mimic of the ischemic penumbra.

Pamenter Matthew E ME Perkins Guy A GA McGinness Anelah K AK Gu Xiang Q XQ Ellisman Mark H MH Haddad Gabriel G GG

PloS one 20121212 12

The development of clinical stroke therapies remains elusive. The neuroprotective efficacies of thousands of molecules and compounds have not yet been determined; however, screening large volumes of potential targets in vivo is severely rate limiting. High throughput screens (HTS) may be used to discover promising candidates, but this approach has been hindered by the lack of a simple in vitro model of the ischemic penumbra, a clinically relevant region of stroke-afflicted brain. Recently, our l ...[more]

PMID: 23251543

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Project description:Neuron apoptosis in ischemic penumbra was proved to be involved in ischemic stroke (IS) development and contributed to the poor prognosis of IS. Recent studies showed that aberrant trimethylation of histone H3 lysine 4 (H3K4me3) level was associated with cell apoptosis. This study aimed to explore the underlying mechanism of neuron apoptosis in ischemic penumbra via histone methyltransferase (HMT) mixed lineage leukemia 1 (MLL1) mediated epigenetic pathway. Mouse IS model was established by middle cerebral artery occlusion (MCAO). Mouse primary cortical mixed cells were cultured and treated with oxygen-glucose deprivation (OGD) to simulate IS process. The expressions of apoptosis signal regulating kinase-1 (ASK-1), pASK-1, cleaved caspase-3, ASK-1/serine-threonine kinase receptor-associated protein (STRAP)/14-3-3 complex, ASK-1/tumor necrosis factor-α (TNF-α) complex, and MLL1 in mouse brain tissue and mouse primary cortical mixed cells were analyzed. The function of MLL1 was investigated using small interfering RNA (siRNA) targeting MLL1 and vector overexpressing MLL1. In vivo inhibition of MLL1 was conducted to explore its value as a therapeutic target. The prognostic value of MLL1 was investigated in IS patients. Results showed that the expressions of ASK-1, pASK-1, cleaved caspase-3, ASK-1/TNF-α complex, and MLL1 increased significantly in ischemic penumbra compared to brain tissue from the control group (P < 0.05). MCAO and OGD significantly upregulated the H3K4me3 level in ASK-1 promoter region and promoted the recruitment of MLL1 to this region (P < 0.05). siMLL1 significantly reversed the proapoptosis effects of OGD in primary cortical mixed cells, while MLL1 overexpression induced apoptosis of cells (P < 0.05). In vivo inhibition of MLL1 significantly reduced the infarct volume and the neurological score of MCAO mice (P < 0.05). Serum MLL1 level had a positive association with that in ischemic core and penumbra in mouse model and was positively correlated with the infarct volume and neurological score (P < 0.05). Besides, serum MLL1 level was also significantly correlated with the severity of IS (P < 0.05), and high serum MLL1 level indicated poor prognosis of IS patients (P < 0.05). These results revealed that MLL1 contributed to neuron cell apoptosis in ischemic penumbra after IS onset by promoting the formation of ASK-1/TNF-α complex, and its serum level was associated with poor prognosis of IS.

Project description:ImportanceWhether endovascular thrombectomy (EVT) efficacy for patients with acute ischemic stroke and large cores varies depending on the extent of ischemic injury is uncertain.ObjectiveTo describe the relationship between imaging estimates of irreversibly injured brain (core) and at-risk regions (mismatch) and clinical outcomes and EVT treatment effect.Design, setting, and participantsAn exploratory analysis of the SELECT2 trial, which randomized 352 adults (18-85 years) with acute ischemic stroke due to occlusion of the internal carotid or middle cerebral artery (M1 segment) and large ischemic core to EVT vs medical management (MM), across 31 global centers between October 2019 and September 2022.InterventionEVT vs MM.Main outcomes and measuresPrimary outcome was functional outcome-90-day mRS score (0, no symptoms, to 6, death) assessed by adjusted generalized OR (aGenOR; values >1 represent more favorable outcomes). Benefit of EVT vs MM was assessed across levels of ischemic injury defined by noncontrast CT using ASPECTS score and by the volume of brain with severely reduced blood flow on CT perfusion or restricted diffusion on MRI.ResultsAmong 352 patients randomized, 336 were analyzed (median age, 67 years; 139 [41.4%] female); of these, 168 (50%) were randomized to EVT, and 2 additional crossover MM patients received EVT. In an ordinal analysis of mRS at 90 days, EVT improved functional outcomes compared with MM within ASPECTS categories of 3 (aGenOR, 1.71 [95% CI, 1.04-2.81]), 4 (aGenOR, 2.01 [95% CI, 1.19-3.40]), and 5 (aGenOR, 1.85 [95% CI, 1.22-2.79]). Across strata for CT perfusion/MRI ischemic core volumes, aGenOR for EVT vs MM was 1.63 (95% CI, 1.23-2.16) for volumes ≥70 mL, 1.41 (95% CI, 0.99-2.02) for ≥100 mL, and 1.47 (95% CI, 0.84-2.56) for ≥150 mL. In the EVT group, outcomes worsened as ASPECTS decreased (aGenOR, 0.91 [95% CI, 0.82-1.00] per 1-point decrease) and as CT perfusion/MRI ischemic core volume increased (aGenOR, 0.92 [95% CI, 0.89-0.95] per 10-mL increase). No heterogeneity of EVT treatment effect was observed with or without mismatch, although few patients without mismatch were enrolled.Conclusion and relevanceIn this exploratory analysis of a randomized clinical trial of patients with extensive ischemic stroke, EVT improved clinical outcomes across a wide spectrum of infarct volumes, although enrollment of patients with minimal penumbra volume was low. In EVT-treated patients, clinical outcomes worsened as presenting ischemic injury estimates increased.Trial registrationClinicalTrials.gov Identifier: NCT03876457.

Dataset Information

Autophagy and apoptosis are differentially induced in neurons and astrocytes treated with an in vitro mimic of the ischemic penumbra.

Publications

Autophagy and apoptosis are differentially induced in neurons and astrocytes treated with an in vitro mimic of the ischemic penumbra.

Similar Datasets

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