Ubisol-Q10 Prevents Glutamate-Induced Cell Death by Blocking Mitochondrial Fragmentation and Permeability Transition Pore Opening

Santosh Kumari, Suresh L Mehta, Gaolin Z Milledge, Xinyu Huang, Haining Li, P Andy Li, Santosh Kumari, Suresh L Mehta, Gaolin Z Milledge, Xinyu Huang, Haining Li, P Andy Li

Abstract

Mitochondrial dysfunction and oxidative stress are the major events that lead to the formation of mitochondrial permeability transition pore (mPTP) during glutamate-induced cytotoxicity and cell death. Coenzyme Q10 (CoQ10) has widely been used for the treatment of mitochondrial disorders and neurodegenerative diseases. Comparing to traditional lipid-soluble CoQ10, water soluble CoQ10 (Ubisol-Q10) has high intracellular and intra-mitochondrial distribution. The aims of the present study are to determine the neuroprotective effects of Ubisol-Q10 on glutamate-induced cell death and to explore its functional mechanisms. HT22 neuronal cells were exposed to glutamate. Cell viability was measured and mitochondrial fragmentation was assessed by mitochondrial imaging. The mPTP opening was determined by mitochondrial membrane potential and calcium retention capacity. The results revealed that the anti-glutamate toxicity effects of Ubisol-Q10 was associated with its ability to block mitochondrial fragmentation, to maintain calcium retention capacity and mitochondrial membrane potential, and to prevent mPTP formation, AIF release, and DNA fragmentation. We concluded that Ubisol-Q10 protects cells from glutamate toxicity by preserving the integrity of mitochondrial structure and function. Therefore, adequate CoQ10 supplementation may be beneficial in preventing cerebral stroke and other disorders that involve mitochondrial dysfunction.

Keywords: AIF; Coenzyme Q10; calcium retention.; glutamate toxicity; mitochondrial fragmentation; neuronal cell death.

Conflict of interest statement

Competing Interests: The authors declare that no competing interest exists.

Figures

Figure 1
Figure 1
Ubisol-Q10 blocks glutamate toxicity-induced cell death. (A) Concentration-dependent effect of water soluble Ubisol-Q10 on HT22 cell viability alone and with 4 mM glutamate. Ubisol-Q10 was added to the cell media prior to the glutamate exposure and cell viability was estimated 24 h after addition. Ubisol-Q10 alone upto 25 μg had no effect on cell viability whereas glutamate exposure significantly reduced the survival of cells to nearly 20 % of control. Pretreatment of cells with Ubisol-Q10 resisted the effect of glutamate toxicity thereby showed significant reduction in cell mortality. Ubisol-Q10 concentration of 15 μg showed maximum protection to glutamate induced cell death in HT222 cells. (B) Representative photomicrograph of HT22 cells treated with Ubisol-Q10, glutamate and both. Data are the representation of 3 or more independent experiments conducted in triplicate. Values are means±SD and analyzed by one-way ANOVA followed by Tukey's multiple comparison test. Significant levels is shown by ***=p<0.001 vs. respective group. Glu=glutamate, Q10=Ubisol-Q10, Glu+Q10=glutamate +Ubisol-Q10 and Glu+Q10-15 to 25 = glutamate (4 mM) +Ubisol-Q10 (15-25 µg).
Figure 2
Figure 2
Ubisol-Q10 prevents glutamate induced ROS production and mitochondrial potential loss. (A) ROS production were measured using DHE in Ubisol-Q10 pretreated HT22 cells exposed to glutamate. Ubisol-Q10 normalized the glutamate exposure increased levels of ROS. (B) Micrograph of HT22 cells shows potential loss after glutamate exposure (arrow). Ubisol-Q10 pretreatment prevented glutamate induced depolarization of mitochondrial membrane potential. Mitochondrial potential measured with JC-1, shows red staining as normal potential whereas green staining represents depolarization and potential loss. (C) Summarized graph shows the effect of Antioxidant Trolox and Ubisol-Q10 on glutamate-induced cell death. Data are the representation of 3 or more independent experiments conducted in triplicate. Values are means±SD and analyzed by one-way ANOVA followed by Tukey's multiple comparison test. Significant levels is shown by *=p<0.05 and ***=p<0.001 vs. respective group. Glu=glutamate, Q10=Ubisol-Q10 and Glu+Q10 = glutamate+Ubisol-Q10.
Figure 3
Figure 3
Ubisol-Q10 restores glutamate reduced mitochondrial calcium retention capacity. Figures show cytosolic calcium uptake by mitochondrial in permeabilized HT22 cells. The addition of calcium (cytosolic) indicated by an arrow (25 µM of calcium pulse each) results in a rapid increase in signal that subsequently decline due to calcium uptake by mitochondria. Result shows that glutamate exposure reduced the capacity of mitochondrial to uptake cytosolic excessive calcium. Ubisol-Q10 pretreatment in contrast improved the mitochondrial calcium uptake to normal levels, thereby maintained the mitochondrial calcium uptake capacity. Cont=Control,
Figure 4
Figure 4
Ubisol-Q10 inhibits glutamate-induced change mitochondrial fragmentation markers. Representative Western blots and quantitative analysis of the protein band of mitochondrial fission protein Drp1, pDrp1 and Fis1. Glutamate exposure significantly increased the levels of mitochondrial fragmentation markers Drp1, pDrp1 and Fis1 without affecting the levels of VDAC and cytochrome c release after 18 h. Ubisol-Q10, in contrast blunted the corresponding elevation of Drp1, pDrp1 and Fis1 in these cells, thereby Ubisol-Q10 significantly prevented the glutamate-induced increase in these markers. Data are the representation of 3 or more independent experiments conducted in triplicate. Values are means±SD and analyzed by one-way ANOVA followed by Tukey's multiple comparison test. Significant levels is shown by *=p<0.05, **=p<0.01 and ***=p<0.001 vs. respective group. Glu=glutamate and Q10=Ubisol-Q10.
Figure 5
Figure 5
Ubisol-Q10 preserved mitochondrial morphology. (A) Photomicrograph shows mitochondrial morphology in HT22 cells treated with glutamate and Ubisol-Q10. Mitochondrial morphology was studied using MitoTracker Red with confocal microscopy. Images were processed and analyzed for structural alterations. Glutamate exposure (18 h) decreased the mitochondrial reticular network resulting into small round shaped broken mitochondria. Ubisol-Q10 pretreatment restored the mitochondrial tubular network in glutamate-exposed cells. (B) Figure shows mitochondrial perimeter and form factor (branching) analyzed from the mitochondrial micrograph. Glutamate exposure not only lowered the mitochondrial perimeter but also reduced the mitochondrial branching. Ubisol-Q10 pretreatment blocked these effects of glutamate and thereby restored normal mitochondrial morphology. Data are the representation of 3 or more independent experiments conducted in triplicate. Values are means±SD and analyzed by one-way ANOVA followed by Tukey's multiple comparison test. Significant levels is shown by *=p<0.05 and **=p<0.01 vs. respective group. Glu=glutamate and Q10=Ubisol-Q10.
Figure 6
Figure 6
Ubisol-Q10 prevented glutamate-induced AIF release. (A) Representative Western blots and quantitative analysis of the protein bands of AIF in mitochondrial and nuclear fractions. AIF levels were relatively similar in HT22 cells treated with glutamate and Ubisol-Q10 in mitochondrial fraction. In contrast, glutamate exposure significantly increased AIF nuclear translocation. Ubisol-Q10 pretreatment blocked the AIF nuclear translocation in cell exposed to glutamate. (B) Photomicrograph and quantitation of AIF positive cells. Cells shows peripheral punctuated staining in control untreated and Ubisol-Q10 only treated cells. AIF translocates to the nucleus following glutamate exposure as shown by diffused staining. Ubisol-Q10 blocked AIF nuclear translocation. Data are the representation of 3 or more independent experiments conducted in triplicate. Values are means±SD and analyzed by one-way ANOVA followed by Tukey's multiple comparison test. Significant levels is shown by **=p<0.01 and ***=p<0.001 vs. respective group. Cont=Control, Glu=glutamate and Q10=Ubisol-Q10.
Figure 7
Figure 7
Ubisol-Q10 prevented DNA damage in cells treated with glutamate. Photomicrograph and quantitation of cells with DNA fragmentation determined with TUNEL staining. Glutamate increased the number of cells positive for TUNEL measure after 18 h of exposure. Ubisol-Q10 in contrast prevented the DNA fragmentation in cells exposed to glutamate. Data are the representation of 3 or more independent experiments conducted in triplicate. Values are means±SD and analyzed by one-way ANOVA followed by Tukey's multiple comparison test. Significance is shown by *=p<0.05 vs. respective group. Cont=Control, Glu=glutamate and Q10=Ubisol-Q10.

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