Mitochondria are in a constant balance of fusing and dividing in response to cellular cues. Fusion creates healthy mitochondria, whereas fission results in removal of non-functional organelles. Changes in mitochondrial dynamics typify several human diseases. However, the contribution of mitochondrial dynamics to preeclampsia, a hypertensive disorder of pregnancy characterized by placental cell autophagy and death, remains unknown. Herein, we show that the mitochondrial dynamic balance in preeclamptic placentae is tilted toward fission (increased DRP1 expression/activation and decreased OPA1 expression). Increased phosphorylation of DRP1 (p-DRP1) in mitochondrial isolates from preeclamptic placentae and transmission electron microscopy corroborated augmented mitochondrial fragmentation in cytotrophoblast cells of PE placentae. Increased fission was accompanied by build-up of ceramides (CERs) in mitochondria from preeclamptic placentae relative to controls. Treatment of human choriocarcinoma JEG3 cells and primary isolated cytrophoblast cells with CER 16:0 enhanced mitochondrial fission. Loss- and gain-of-function experiments showed that Bcl-2 member BOK, whose expression is increased by CER, positively regulated p-DRP1/DRP1 and MFN2 expression, and localized mitochondrial fission events to the ER/MAM compartments. We also identified that the BH3 and transmembrane domains of BOK were vital for BOK regulation of fission. Moreover, we found that full-length PTEN-induced putative kinase 1 (PINK1) and Parkin, were elevated in mitochondria from PE placentae, implicating mitophagy as the process that degrades excess mitochondria fragments produced from CER/BOK-induced fission in preeclampsia. In summary, our study uncovered a novel CER/BOK-induced regulation of mitochondrial fission and its functional consequence for heightened trophoblast cell autophagy in preeclampsia.

IntroductionGestational diabetes mellitus (GDM), a common pregnancy disorder, increases the risk of fetal overgrowth and later metabolic morbidity in the offspring. The placenta likely mediates these sequelae, but the exact mechanisms remain elusive. Mitochondrial dynamics refers to the joining and division of these organelles, in attempts to maintain cellular homeostasis in stress conditions or alterations in oxygen and fuel availability. These remodeling processes are critical to optimize mitochondrial function, and their disturbances characterize diabetes and obesity.Methods and resultsHerein we show that placental mitochondrial dynamics are tilted toward fusion in GDM, as evidenced by transmission electron microscopy and changes in the expression of key mechanochemical enzymes such as OPA1 and active phosphorylated DRP1. In vitro experiments using choriocarcinoma JEG-3 cells demonstrated that increased exposure to insulin, which typifies GDM, promotes mitochondrial fusion. As placental ceramide induces mitochondrial fission in pre-eclampsia, we also examined ceramide content in GDM and control placentae and observed a reduction in placental ceramide enrichment in GDM, likely due to an insulin-dependent increase in ceramide-degrading ASAH1 expression.ConclusionsPlacental mitochondrial fusion is enhanced in GDM, possibly as a compensatory response to maternal and fetal metabolic derangements. Alterations in placental insulin exposure and sphingolipid metabolism are among potential contributing factors. Overall, our results suggest that GDM has profound impacts on placental mitochondrial dynamics and metabolism, with plausible implications for the short-term and long-term health of the offspring.

Temporal and spatial expression of AMOT in the developing human placenta. We first characterized the spatial and temporal expression patterns of AMOT in the human placenta during early gestation. Expressed as 2 isoforms, the 80-kDa product (AMOT 80) has been implicated in endothelial cell migration, whereas the 130-kDa product (AMOT 130) is involved in changes in endothelial cell shape due to its distinguishing N-terminal extension that promotes its association with the F-actin cytoskeleton (24, 25). Protein analysis revealed that both AMOT 130 and 80 isoforms are expressed in the human placenta as early as 5 weeks of gestation, and their expression significantly increased after 10 weeks (Figure 1A), a critical time point when changes in oxygen tension and TGF-β signaling are known to occur (10, 26). Quantitative PCR analysis revealed that AMOT mRNA expression is higher in placentae from 10 to 15 weeks of gestation, compared with placentae from 5 to 9 weeks of gestation (Figure 1B). These analyses were performed on whole placenta samples, thus encompassing a heterogenous mixture of trophoblasts. Analysis of AMOT expression in distinct trophoblast subpopulations isolated through laser capture microdissection (LCM) (27) demonstrated AMOT expression in syncytiotrophoblasts (STs) and CTs and proximal (PC) and distal column (DC) trophoblasts (Figure 1C). However, with advancing gestation, AMOT expression only increased in the ST/ CT layer, where trophoblast cells are undergoing active fusion, and more importantly in the DC, where migratory and invasive EVTs reside (Figure 1C). This was corroborated by immunohistochemical analysis of AMOT in first-trimester placentae sections, which revealed (a) a striking localization of AMOT to the cell boundaries of EVTs comprising the anchoring column, particularly restricted to the intermediate and distal regions of the EVT column and absent in the proximal region; and (b) AMOT localization to the underlying, proliferative CTs, as well as in the overlying, multinucleated ST layer with advancing gestation (Figure 1D). During placenta development, critical cellular events, including trophoblast migration, rely on tightly regulated changes in oxygen tension. Hence, we examined the effect of low oxygen on AMOT expression levels. Exposure of trophoblast-derived JEG3 cells to 3% oxygen significantly decreased AMOT 130 and 80 protein levels compared with normoxic 21% oxygen (Figure 1E). TGF-β regulates AMOT expression, subcellular localization, and interaction with PAR6. During the early events of trophoblast differentiation, low oxygen tension via HIF-1α has been demonstrated to upregulate levels of TGF-β3 (10). Further, we have shown a TGF-β-dependent regulation of polarity protein PAR6 in guiding trophoblast cell migration (28). Considering that AMOT is a scaffolding protein implicated in cell polarity, we next investigated if there was a TGF-β-dependent regulation of AMOT utilizing JEG3 cells. Treatment of JEG3 cells with 10 ng/ml TGF-β1/3 ligand for 24 hours resulted in a significant