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Cerebral ischemia/reperfusion (I/R) can lead to serious brain function impairments. Long noncoding RNA (lncRNA) CCAAT enhancer binding protein α antisense RNA 1 ( CEBPA-AS1 ) was shown to be upregulated in human ischemic stroke. This study investigated the function and mechanism of CEBPA-AS1 in I/R. An oxygen-glucose deprivation/reperfusion (OGD/R) model was used to induce I/R injury in SH-SY5Y cells in vitro . RT-qPCR examined the expression of CEBPA-AS1 , microRNA 24-3p (miR-24-3p), and Bcl-2-related ovarian killer (Bok). The cell viability, apoptosis, oxidative stress in OGD/R-treated cells were detected using CCK-8, flow cytometry, Western blotting, and enzyme-linked immunosorbent assays. The relationship among genes was tested by RNA pulldown and luciferase reporter assays. We found that OGD/R upregulated CEBPA-AS1 expression in SH-SY5Y cells. Functionally, CEBPA-AS1 depletion ameliorated OGD/R-induced apoptosis and oxidative stress in SH-SY5Y cells by reducing reactive oxygen species production and superoxide dismutase and glutathione. Mechanistic investigations indicated that CEBPA-AS1 acts as a sponge for miR-24-3p, and miR-24-3p binds to BOK. Moreover, miR-24-3p upregulation or BOK downregulation antagonized the protective role of CEBPA-AS1 depletion in SH-SY5Y cells exposed to OGD/R. Overall, downregulation of CEBPA-AS1 exerts protective functions against OGD/R-induced injury by targeting the miR-24-3p/BOK axis.

Regulation of cell life and death by members of the BCL-2 family of proteins occurs at the mitochondria. Large portions of the mitochondria's outer membrane are found in tight approximation with the endoplasmic reticulum (ER), known as mitochondria-associated membranes (MAMs) or mitochondria-ER contact sites (MERCs). We recently reported that BOK is present within MAMs where it regulates Ca 2+ transfer from the ER to the mitochondria, appropriate MAM components and MERC structure, and apoptosis., (© 2021 Taylor & Francis Group, LLC.)

Myeloid cell leukemia 1 ( MCL1 ) gene amplification occurs in a wide range of human cancers and protein overexpression associates with malignant cell growth and evasion of apoptosis. We recently reported that disrupting the interaction between the transmembrane domains of MCL1 and BCL-2 related ovarian killer (BOK) induces cell death, thereby suggesting a new target site for anti-tumorigenic strategies., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Bioactive sphingolipids including ceramides are involved in a variety of pathophysiological processes by regulating cell death and survival. The objective of the current study was to examine ceramide metabolism in preeclampsia, a serious disorder of pregnancy characterized by oxidative stress, and increased trophoblast cell death and autophagy. Maternal circulating and placental ceramide levels quantified by tandem mass spectrometry were elevated in pregnancies complicated by preeclampsia. Placental ceramides were elevated due to greater de novo synthesis via high serine palmitoyltransferase activity and reduced lysosomal breakdown via diminished ASAH1 expression caused by TGFB3-induced E2F4 transcriptional repression. SMPD1 activity was reduced; hence, sphingomyelin degradation by SMPD1 did not contribute to elevated ceramide levels in preeclampsia. Oxidative stress triggered similar changes in ceramide levels and acid hydrolase expression in villous explants and trophoblast cells. MALDI-imaging mass spectrometry localized the ceramide increases to the trophophoblast layers and syncytial knots of placentae from pregnancies complicated by preeclampsia. ASAH1 inhibition or ceramide treatment induced autophagy in human trophoblast cells via a shift of the BOK-MCL1 rheostat toward prodeath BOK. Pharmacological inhibition of ASAH1 activity in pregnant mice resulted in increased placental ceramide content, abnormal placentation, reduced fetal growth, and increased autophagy via a similar shift in the BOK-MCL1 system. Our results reveal that oxidative stress-induced reduction of lysosomal hydrolase activities in combination with elevated de novo synthesis leads to ceramide overload, resulting in increased trophoblast cell autophagy, and typifies preeclampsia as a sphingolipid storage disorder.

Autophagy is the catabolic degradation of cellular cytoplasmic constituents via the lysosomal pathway that physiologically elicits a primarily cytoprotective function, but can rapidly be upregulated in response to stressors thereby inducing cell death. We have reported that the balance between the BCL2 family proteins BOK and MCL1 regulates human trophoblast cell fate and its alteration toward cell death typifies preeclampsia. Here we demonstrate that BOK is a potent inducer of autophagy as shown by increased LC3B-II production, autophagosomal formation and lysosomal activation in HEK 293. In contrast, using JEG3 cells we showed that prosurvival MCL1 acts as a repressor of autophagy via an interaction with BECN1, which is abrogated by BOK. We found that MCL1-cleaved products, specifically MCL1c157, trigger autophagy while the splicing variant MCL1S has no effect. Treatment of JEG3 cells with nitric oxide donor SNP resulted in BOK-MCL1 rheostat dysregulation, favoring BOK accumulation, thereby inducing autophagy. Overexpression of MCL1 rescued oxidative stress-induced autophagy. Of clinical relevance, we report aberrant autophagy levels in the preeclamptic placenta due to impaired recruitment of BECN1 to MCL1. Our data provided the first evidence for a key role of the BOK-MCL1 system in regulating autophagy in the human placenta, whereby an adverse environment as seen in preeclampsia tilts the BOK-MCL1 balance toward the build-up of isoforms that triggers placental autophagy.