Mitochondrial ferritin alleviates apoptosis by enhancing mitochondrial bioenergetics and stimulating glucose metabolism in cerebral ischemia reperfusion

Abstract

Oxidative stress and deficient bioenergetics are key players in the pathological process of cerebral ischemia reperfusion injury (I/R). As a mitochondrial iron storage protein, mitochondrial ferritin (FtMt) plays a pivotal role in protecting neuronal cells from oxidative damage under stress conditions. However, the effects of FtMt in mitochondrial function and activation of apoptosis under cerebral I/R are barely understood. In the present study, we found that FtMt deficiency exacerbates neuronal apoptosis via classical mitochondria-depedent pathway and the endoplasmic reticulum (ER) stress pathway in brains exposed to I/R. Conversely, FtMt overexpression significantly inhibited oxygen and glucose deprivation and reperfusion (OGD/R)-induced apoptosis and the activation of ER stress response. Meanwhile, FtMt overexpression rescued OGD/R-induced mitochondrial iron overload, mitochondrial dysfunction, the generation of reactive oxygen species (ROS) and increased neuronal GSH content. Using the Seahorse and O2K cellular respiration analyser, we demonstrated that FtMt remarkably improved the ATP content and the spare respiratory capacity under I/R conditions. Importantly, we found that glucose consumption was augmented in FtMt overexpressing cells after OGD/R insult; overexpression of FtMt facilitated the activation of glucose 6-phosphate dehydrogenase and the production of NADPH in cells after OGD/R, indicating that the pentose-phosphate pathway is enhanced in FtMt overexpressing cells, thus strengthening the antioxidant capacity of neuronal cells. In summary, our results reveal that FtMt protects against I/R-induced apoptosis through enhancing mitochondrial bioenergetics and regulating glucose metabolism via the pentose-phosphate pathway, thus preventing ROS overproduction, and preserving energy metabolism.

Keywords: G6PDH; Glucose metabolism; Ischemic stroke; Mitochondrial bioenergetics; Mitochondrial ferritin.

Fig. 1

FtMt ablation aggravates cerebral I/R-induced apoptosis. Apoptotic cell death was assessed by DAPI and TUNEL staining. (A) Representative photographs of uninjured and injured cortices of the mouse brains (left panels). The DAPI-stained, TUNEL-positive cells were counted (right panel). (B–I) Western blot and densitometric analyses of (B) the ratio of Bcl-2 to Bax, (C) the amount of cleaved caspase 3, and (D) the ratio of p-Erk1/2 to Erk1/2, (E) GRP78, (F) p-EIF2α, (G) ATF4, (H) CHOP, and (I) ATF6 in wild-type (WT) and Ftmt-knockout (KO) mice after MCAO (90 min) and subsequent reperfusion (24 h). I/R, penumbral area in the cortex of the injured hemisphere; Con, corresponding area in the cortex of the uninjured hemisphere. The results are presented as the mean ± SEM. n = 3, *^/$P < 0.05, **^/$$P < 0.01.

Fig. 2

Overexpression of FtMt decreases OGD/R injury-induced apoptosis. SH-SY5Y cells (WT), stable FtMt-overexpressing SH-SY5Y cells (FtMt) and pcDNA3.1 (−) empty vector-transfected cells (Vector) were subjected to OGD/R insult. (A) Apoptosis, as measured by flow cytometry. The right panel shows the proportion of apoptotic cells in the different groups. The levels of (B) GRP78, (C) p-EIF2α, (D) ATF4, and (E) ATF6, as assessed by Western blot (n = 4). The results are presented as the mean ± SEM. *^/$P < 0.05, **^/$$P < 0.01.

Fig. 3

FtMt deletion promotes ROS generation and decreases mitochondrial bioenergetics during cerebral I/R. (A) ROS generation in brain slices from wild-type mice and Ftmt-knockout mice after I/R treatment (MCAO, 90 min followed by 24 h reperfusion), as determined by dihydroethidium (DHE) staining. (B) Quantitative evaluation of DHE fluorescence intensity (n = 3). Wild-type and Ftmt-knockout mice were subjected to I/R. Next, the oxygen consumption rate (OCR) of the Con and I/R groups was determined using an O2K analyzer. OCR was recorded at baseline and after the sequential injection of oligomycin (1 μM), FCCP (4 μM) and a mixture of rotenone and antimycin A (1 μM). (C) Basal respiration, (D) ATP -linked OCR, (E) maximal respiration and (F) spare respiratory capacity were calculated (n = 4). The results are presented as the mean ± SEM. ^$P < 0.05, **^/$$P < 0.01.

Fig. 4

FtMt overexpression restricts ROS generation and enhances mitochondrial bioenergetics during OGD/R injury. (A) Representative images showing co-localization of MitoSOX Red fluorescence and Mito Tracker Green fluorescence in SH-SY5Y cells (WT), stable FtMt-overexpressing SH-SY5Y cells (FtMt) and pcDNA3.1 (−) empty vector-transfected cells (Vector) after OGD/R treatment. (B) Quantitative evaluation of MitoSOX fluorescence intensity (n = 3). (C) MDA and (D) GSH contents, as quantified using commercially available assays (n = 3). The oxygen consumption rate (OCR) of the Con and OGD/R groups, as determined using a Seahorse XF analyzer. (E) OCR was recorded at baseline and after the sequential injection of oligomycin (2 μM), FCCP (8 μM) and a mixture of rotenone and antimycin A (1 μM) (n = 4). (F) Basal respiration, (G) ATP -linked OCR, (H) quantitative results of the levels of ATP (I) maximal respiration, and (J) spare respiratory capacity. The results are presented as the mean ± SEM. *^/$P < 0.05, **^/$$P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Overexpression of FtMt enhances glucose metabolism in neuronal cells under OGD/R insult. SH-SY5Y (WT) cells, empty vector transfectants (Vector) and FtMt-overexpressing transfectants (FtMt) were subjected to OGD/R insult. Next, the ECAR of the Con and OGD/R groups was assessed with a Seahorse XF analyzer. (A) ECAR was recorded after the sequential injection of glucose (10 mM), oligomycin (2 μM), and 2-deoxy-glucose (50 mM). (B) Basal ECAR, (C) maximal ECAR and (D) the reserve capacity were calculated. The results are presented as the mean ± SEM, n = 4, *P < 0.05, **^/$$P < 0.01.

Fig. 6

Overexpression of FtMt enhances the pentose-phosphate pathway in neuronal cells under OGD/R insult. SH-SY5Y cells (WT), empty vector transfectants (vector) and FtMt-overexpressing transfectants (FtMt) were subjected to OGD/R insult. (A) NADPH content and (B) G6DPH activity were quantified using a commercially available assay. The results are presented as the mean ± SEM, n = 4, *^/$P < 0.05, **^/$$P < 0.01.

Fig. 7

Schematic representation of the proposed antioxidative mechanism of FtMt in cerebral I/R. Ischemic stroke causes mitochondrial dysfunction and ROS overproduction, resulting in mitochondria dependent apoptosis and ER stress-associated apoptosis in the brain. FtMt reduces cerebral I/R-induced oxidative stress by maintaining mitochondrial bioenergetics and regulating glucose metabolism via the pentose-phosphate pathway, thus inhibiting ROS overproduction, and preventing neuronal deficits in I/R.