Neuronal Metabolism and Neuroprotection: Neuroprotective Effect of Fingolimod on Menadione-Induced Mitochondrial Damage

Abstract

Imbalance in the oxidative status in neurons, along with mitochondrial damage, are common characteristics in some neurodegenerative diseases. The maintenance in energy production is crucial to face and recover from oxidative damage, and the preservation of different sources of energy production is essential to preserve neuronal function. Fingolimod phosphate is a drug with neuroprotective and antioxidant actions, used in the treatment of multiple sclerosis. This work was performed in a model of oxidative damage on neuronal cell cultures exposed to menadione in the presence or absence of fingolimod phosphate. We studied the mitochondrial function, antioxidant enzymes, protein nitrosylation, and several pathways related with glucose metabolism and glycolytic and pentose phosphate in neuronal cells cultures. Our results showed that menadione produces a decrease in mitochondrial function, an imbalance in antioxidant enzymes, and an increase in nitrosylated proteins with a decrease in glycolysis and glucose-6-phosphate dehydrogenase. All these effects were counteracted when fingolimod phosphate was present in the incubation media. These effects were mediated, at least in part, by the interaction of this drug with its specific S1P receptors. These actions would make this drug a potential tool in the treatment of neurodegenerative processes, either to slow progression or alleviate symptoms.

Keywords: fingolimod phosphate; glycolytic pathway; mitochondrial damage; neuroprotection; pentose phosphate pathway; redox homeostasis; sphingosine-1-phosphate receptor analogue.

Figure 1

Study of neuronal mitochondrial function after treatment of a cell with menadione (VitK3) in the absence or presence of fingolimod phosphate (FP). The oxygen consumption rate was assessed in (a) basal after 2 h of incubation with VitK3; (b) ATP production, measured after addition of oligomycin; (c) proton leak, considered as the residual oxygen consumption after oligomycin addition; (d) spare respiratory capacity (SRC), as the difference between maximal respiration and basal respiration; and (e) maximal respiration, obtained as the oxygen consumption after subsequent addition of oligomycin and carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP). The sequence of mitochondrial toxins added was oligomycin, 1 µM; FCCP, 0.5 µM; rotenone/antimycin A, 0.5/0.5 µM. The inclusion of rotenone/antimycin A served to measure respiration by non-mitochondrial processes as these compounds abolish mitochondrial respiration, and it was subtracted in all oxygen consumption rate (OCR) values obtained. Values represent mean and SD of at least five experiments per situation performed in triplicate. (* p < 0.05 versus CO, ^$ p < 0.05 versus VitK3).

Figure 2

FP recovered the glycolytic damage induced by VitK3 on neuronal SN4741 cells. (a) Time course of extracellular acidification rate (ECAR) and glycolytic function after incubation with VitK3 in the presence or absence of FP (blue line: CO; red line: VitK3; green line: VitK3+FP; purple line: VitK3+FP+W). To uncouple mitochondrial respiration from ATP synthesis, we used 2,4-dinitrophenol (2,4-DNP) 100 µM; 2-deoxy-D-glucose (2-DG) 100 mM was used to inhibit glycolysis and rotenone 1 µM was used to inhibit NADH hydrogenase/complex I. (b) Basal glycolysis after 2 h of treatment with VitK3 in the presence or absence of FP. (c) Glycolytic capacity obtained after incubation with 2,4-DNP and rotenone. (d) Glycolytic reserve obtained as the difference between the maximal glycolytic capacity and basal. In all situations, the non-glycolytic ECAR, obtained after addition of rotenone, was subtracted. Values represent mean and SD of at least five experiments per situation performed in triplicate. (* p < 0.05 versus CO, $ p < 0.05 versus VitK3, # p < 0.05 versus VitK3+FP, & p < 0.05 versus VitK3+FP+W).

Figure 3

FP recovered the enzyme aldolase from the inhibition induced by VitK3. Aldolase was measured in a homogenate of neurons after 2 hours of incubation with VitK3 in the presence or absence of FP. Values represent mean and SD of at least five experiments per situation performed in triplicate. (* p < 0.05 versus CO, $ p < 0.05 versus VitK3, # p < 0.05 versus VitK3+FP, & p < 0.05 versus VitK3+FP+W).

Figure 4

FP was necessary in the medium to maintain its actions. Time course of ECAR after 2 h of incubation with VitK3 in the presence or absence of FP. After 2 h of incubation, VitK3 was removed from the media and cells were incubated for 4 additional hours in the following conditions: two groups were incubated with media (VitK3 group (red line) and CO group (blue line)), one with FP (VitK3+FP group (green line)), one with FP+W (VitK3+FP+W group (purple line)), and one with FP (FP group (orange line). Values represent mean and SD of at least four experiments per situation performed in triplicate.

Figure 5

FP increased the enzyme glucose-6-phosphate-dehydrogenase (G-6-PDH) reduced by VitK3. G-6-PDH was measured in a homogenate of neurons after 2 hours of incubation with VitK3 in the presence or absence of FP. Values represent mean and SD of at least five experiments per situation performed in triplicate. (* p < 0.05 versus CO, $ p < 0.05 versus VitK3, # p < 0.05 versus VitK3+FP, & p < 0.05 versus VitK3+FP+W).

Figure 6

Immunocytochemistry of S1P1 receptor in SN4741 neuronal cells. Image were acquired using an Olympus BX51 epifluorescence microscope at 40× magnification.