To evaluate the effect of LPS on systemic inflammation and hippocampus neuroinflammation in aged rats, the concentrations of the pro-inflammatory cytokines TNF-α and IL-1β were quantified in both peripheral serum and hippocampal tissues using ELISA. Additionally, the relative mRNA expression levels of TNF-α in the hippocampus were evaluated through quantitative PCR. On the third day following LPS administration, elevated levels of TNF-α and IL-1β were observed in serum, but the change of TNF-α concentration was not statistically significant (TNF-α, t = 2.714, p = 0.0533. IL-1β, t = 2.965, p = 0.0414, unpaired t-test) (Fig. 4A-B).
In the hippocampus, the mRNA expression of TNF-α exhibited significant variations across different days [F(1.057, 4.226) = 30.670, p = 0.0043, two-way ANOVA], treatments [F(1, 4) = 31.740, p = 0.0049, two-way ANOVA], and interaction between days and treatments [F(2, 8) = 35.110, p = 0.0001, two-way ANOVA]. A peak in TNF-α mRNA expression was noted on day three, significantly surpassing levels from day one (q = 12.44, p = 0.0099, Dunnett's post hoc test) (Fig. 4C). The TNF-α mRNA of the hippocampus on day 3 was further detected, which indicated a significant increase in the LPS group (t = 4.360, p = 0.0121, unpaired t-test) (Fig. 4D). For hippocampal pro-inflammatory cytokines, TNF-α concentrations were significantly higher in the LPS group compared to the control group (t = 4.784, p = 0.0087, unpaired t-test), whereas the increase in IL-1β levels did not reach statistical significance (t = 2.653, p = 0.0568, unpaired t-test) (Fig. 4E-F).
LPS-induced myelin damage in the hippocampus of aged rats
Here we examined the effects of LPS on myelin in the hippocampi of aged rats, with a focus on changes observed three days following LPS administration, as the integrity of myelin in the hippocampus is crucially associated with cognitive processes [35]. Although both groups exhibited abnormal myelin in the hippocampus, including irregular and loosely wrapped myelin sheaths at 20000× images, compared with the NC group, the abnormal myelin structure was more severe in the LPS group (Fig. 5A). Additionally, at a magnification of 2,500×, there was a marked decrease in the number of myelinated axons within the LPS-treated group, which was statistically significant (U = 416.000, p < 0.0001, U-test) (Fig. 5B).
The g-ratio, a well-defined ultrastructural parameter, quantifies myelination independently of axon diameter. It is derived by dividing the axon diameter by the sum of the axon and its myelin sheath diameters [36]. This ratio inversely correlates with the thickness of myelin for any given axon diameter. In the group treated with LPS, scatter plots presented in Figs. 5C and 5D show a significant elevation in mean g-ratios when compared to the NC group (U = 24675.000, p < 0.0001, U-test). These findings suggest a reduction in myelin thickness in hippocampal myelinated axons of consistent diameter within the LPS-treated group relative to the NC group. Furthermore, bar graphs demonstrate that g-ratio values rise across axon diameters ranging from 0.1µm to 2.0µm, captured at 10,000× magnification for both experimental and control groups. However, the difference of g-ratio between the two groups became less significant with the increase of axon diameter (0.1µm ~ 0.5µm, t = 7.921, p < 0.0001. 0.5µm ~ 1.0µm, t = 8.397, p < 0.0001. 1.0µm ~ 1.5µm, t = 3.037, p = 0.0130. 1.5µm ~ 2.0µm, t = 1.223, p = 0.7195, Sidak's post hoc test) (Fig. 5E). These results suggest that myelin thickness diminishes as axon diameter increases in the hippocampi of aged rats, with smaller diameter axons exhibiting a more marked response to LPS treatment.
To further investigate LPS-induced myelin damage in the hippocampus, we assessed the levels of key myelin proteins, including proteolipid protein (PLP) and myelin basic protein (MBP), using both immunofluorescence staining and western blot techniques. Western blot results indicated a significant reduction in MBP expression within the hippocampus of rats treated with LPS (t = 2.796, p = 0.0313, unpaired t-test) (Fig. 6A-B). Immunofluorescence indicated a significant decrease in both MBP (U = 873.000, p < 0.0001, Mann-Whitney U-test) and PLP (U = 985.500, p = 0.0313, Mann-Whitney U-test) coverage in the hippocampus of LPS group rats (Fig. 6C-E).
The glycolysis-related gene Gapdh was downregulated in hippocampus oligodendrocytes of LPS-treated aged rats
To investigate the cellular types and gene expression profiles implicated in hippocampal neuroinflammation induced by intraperitoneal injection of LPS in aged rats, along with the characteristics of hippocampal oligodendrocytes and alterations in gene expression associated with myelin sheath synthesis following LPS exposure, we conducted single-cell RNA sequencing on whole hippocampal tissue from aged rats 3 days post-injection using 10X Genomics technology. Cell clustering analysis identified 18,471 cells from 6 rats (3 treated with LPS) (Fig. 7A). Using prior literature and classical marker genes, we annotated these 15 cell types [37–50] (Fig. 7B). Distribution proportions of various cell types in the hippocampal region of aged rats varied between the NC and LPS groups (Fig. 7C), notably showing a decrease in oligodendrocyte proportion in the LPS group (NC = 9.4%, LPS = 3.5%). Subsequent re-clustering analysis of 1,084 oligodendrocytes identified six clusters (cluster 0 to cluster 5) (Fig. 7D), exhibiting varying distribution proportions between the LPS and NC groups (Fig. 7E). In comparison to the NC group, the proportions of oligodendrocytes cluster 0, cluster 2, cluster 3, and cluster 4 were elevated in the LPS group, with percentage changes of cluster 0 = 28.9%, cluster 2 = 9.4%, cluster 3 = 3.8%, and cluster 4 = 57.7% [the percentage changes=(LPS group proportion - NC group proportion) / NC group proportion], respectively, indicating the most significant increases in cluster 0 and cluster 4. Conversely, the proportions of oligodendrocytes cluster 1 and cluster 5 decreased in the LPS group, with percentage changes of 3.6% and 75.8% for cluster 1 and cluster 5 [the percentage changes=(NC group proportion - LPS group proportion) / NC group proportion], respectively, indicating that the most significant decrease occurred in cluster 5 within the LPS group. Figure 7F illustrates representative marker genes and GO/KEGG functional enrichments of six clusters of oligodendrocytes. Previous studies have summarized various subpopulations of mature oligodendrocytes in humans and mice [51, 52]. In this study, five clusters of hippocampal oligodendrocytes were mature oligodendrocytes, denoted as clusters 0–4.
Cluster 0 was characterized by markers of mature oligodendrocytes, including Ptgds [53], and genes associated with myelin sheath and oligodendrocyte maturation, such as Mal, Olig1, Pllp [54, 52, 11], and Htra1 [55]. Functional enrichment analysis suggested that this cluster represented mature oligodendrocytes involved in myelination. Cluster 1 was a subcluster involved in migration to injury sites and participated in remyelination processes, which expressed markers including Cd9, Olig2, Sh3gl3, and Opalin, along with genes related to cell adhesion such as Mcam [56], cell migration-related gene Npr1 [57], development of oligodendrocyte lineage and myelin stability-related gene Tspan2 [58], and genes associated with cytoskeleton, organelle assembly, and motility (Clmn) [59]. Cluster 2 was characterized by markers of mature oligodendrocytes, such as S100b, Klk6, Apod, Gjb1, and Anln, along with genes involved in lipid synthesis, cell-cell interactions, and regeneration, such as Fabp7, Celsr2, and Hopx. Functional enrichment analysis suggested that this subcluster is the metabolically active mature oligodendrocyte cluster. Cluster 3 was also a mature oligodendrocyte subcluster, of which the marker genes included the mature oligodendrocyte marker Anln, genes involved in glucose metabolism such as Adpgk [60], Glul [61], injury-related gene Slc24a2 [62], cytoskeletal proteins encoding gene Cdc42bpa [63], gene related to cell differentiation and migration Etv1 [64], and genes involved in complement-mediated cell lysis and lymphocyte signaling Cd59 [65]. Functional enrichment suggested that this cluster is involved in cytoskeletal organization, regulation of cell adhesion assembly, modulation of cell morphology, intracellular substance transport, cell migration, and adhesive pathways. Cluster 4 marker genes suggested that it was an immune-related subcluster of mature oligodendrocytes, characterized by high expression of mature oligodendrocyte markers such as Cst3 [53], as well as disease-associated markers Apoe, Cd74 [66], and Ctss. Cd74 and Ctss are crucial genes for MHC-II-mediated responses [51]. Additionally, this cluster exhibited high expression of complement-related genes (C1qa, C1qb, C1qc) and chemokine genes (Ccl3, Ccl4), as well as Trem2, which promotes microglial phagocytosis of apoptotic cells [67] and facilitates inflammatory myeloid cell infiltration [68]. Cluster 5 contains highly expressed genes associated with oligodendrocyte development, such as Atrn [69] and Slc12a2 [70], the postnatal myelination and normal structural integrity gene Nfasc [71, 72], and genes involved in maintaining myelin membrane lipid structure (Scd2, Abca2) [73, 74]. Functional enrichment suggested that this subcluster was a group of oligodendrocytes with potential functional alterations.
To explore the changes in myelin-related functions of oligodendrocytes in the hippocampus under LPS stimulation, we further compared the expression differences of representative genes involved in myelin synthesis and regeneration between the NC and LPS groups in each cluster. In subclusters associated with myelin synthesis, repairment, and regeneration (cluster 0 ~ cluster 2) of the LPS group, the expression levels of genes related to myelin synthesis (Mbp, Plp1, Sox10), regeneration (Cd81, Ptn), and membrane structural assembly (Mapt) were slightly decreased compared with those in the NC group (Fig. 8A). Moreover, the expression levels of genes associated with ribosome assembly (Rpl37a, Rpl41, Rpl30) and protein elongation (Eef1a1, Eef2) were significantly decreased in the hippocampal oligodendrocytes of the LPS group compared with those of the NC group (FDR p < 0.05) (Fig. 8B), indicating that LPS might induce a decrease in the translation efficiency of proteins related to myelin synthesis.
Protein translation and elongation are energy-consuming processes. Compared with oxidative phosphorylation, mature oligodendrocytes tend to rely on glycolysis to produce ATP [27, 75]. To investigate the effect of LPS on glycolytic energy supply in hippocampal oligodendrocytes of aged rats, we compared the expression levels of representative genes involved in different steps of glycolysis (Aldoa, Gapdh, Aldh2) in each cluster (Fig. 8C). The expression levels of glycolysis-related genes were generally decreased in the LPS group, among which the expression level of Gapdh was significantly reduced across all clusters (FDR p < 0.0001) (Fig. 8D-E).
TNF-α induced apoptosis in mature oligodendrocytes and decreases the production of MBP and GAPDH
To explore the impact of TNF-α on myelin synthesis in mature oligodendrocytes, we stimulated mature oligodendrocytes with 50 ng/mL TNF-α or PBS for 18 h. The TNF-α concentration and time exploring are shown in Supplementary Material 1. The expression of the glycolytic key enzymes GAPDH and MBP and cell apoptosis in both groups of mature oligodendrocytes were examined. Immunofluorescence staining revealed that the mean fluorescence intensity of TNF receptor 1 (TNFR1) in mature oligodendrocytes was significantly higher after 18 h of stimulation with 50 ng/mL TNF-α compared to the PBS control group (t = 6.529, p < 0.0001, unpaired-t test). Additionally, the fluorescence intensity levels of GAPDH (t = 7.573, p < 0.0001, unpaired-t test) and MBP (t = 7.041, p < 0.0001, unpaired-t test) were notably decreased in the TNF-α treated group when compared to controls (Fig. 9A-D). QPCR and western blot results analyses further confirmed that the GAPDH mRNA (t = 6.395, p = 0.0031, unpaired-t test) and protein content (t = 4.087, p = 0.0064, unpaired-t test) were significantly lower in the TNF-α group than in the PBS controls (Fig. 9E-G).
Moreover, a significant increase in the proportion of TUNEL-positive cells was observed in the TNF-α treated mature oligodendrocytes compared to the control group (t = 9.871, p < 0.0001, Unpaired-t test) (Fig. 9H-I).
Gapdh overexpression ameliorated glycolysis and oxidative phosphorylation in TNF-α treated mature oligodendrocytes
Oligodendrocytes prefer glycolysis to meet their high ATP demand [24, 27, 76]. However, research has also found that mitochondrial function is crucial for oligodendrocytes [75, 77]. Inhibiting glycolysis and mitochondrial oxidative phosphorylation hinders oligodendrocyte differentiation and maturation, whereas enhancing glycolysis and mitochondrial oxidative phosphorylation promotes remyelination induced by cuprizone [78]. Thus, although oligodendrocytes rely on glycolysis for energy supply, oxidative phosphorylation is equally important.
To further explore the role of the key gene Gapdh in oxidative phosphorylation and glycolysis, the two energy synthesis pathways during TNF-α damaging in mature oligodendrocytes, we employed the Seahorse XFe96 analyzer. This tool enabled us to measure alterations in the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR), which respectively reflect the capacities for oxidative phosphorylation and glycolysis in these cells.
Figure 10A, B, and C demonstrate the ECAR and glycolytic capacity of the four groups of cells. From the real-time ECAR curves (Fig. 10A), it can be observed that compared with the OE-NC + PBS group, the ECAR of mature oligodendrocytes in the OE-GAPDH + PBS group increased. The ECAR curve of mature oligodendrocytes in the OE-NC + TNF-α group shifted downward compared with that in the OE-NC + PBS group, whereas the ECAR of mature oligodendrocytes in the OE-GAPDH + TNF-α group shifted downward compared with that in the OE-GAPDH + PBS group, indicating that TNF-α decreases the glycolytic capacity of mature oligodendrocytes.
ECAR before adding oligomycin reflects the basal glycolytic capacity of the cells, whereas after complete inhibition of oxidative phosphorylation by adding oligomycin, ECAR increases to its maximum value, reflecting the maximum glycolytic capacity of the cells. There were significant differences in the basal glycolytic capacity [F (3, 56) = 48.540, p < 0.0001, one-way ANOVA] and maximum glycolytic capacity [F (3, 56) = 39.280, p < 0.0001, one-way ANOVA] between the groups. Compared with the OE-NC + PBS group, the basal glycolytic capacity of mature oligodendrocytes in the OE-GAPDH + PBS group was significantly increased (q = 7.586, p < 0.0001, Turkey's post hoc test), but the increase in maximum glycolytic capacity was not significant (q = 3.726, p = 0.0516, Turkey's post hoc test). In the OE-NC + TNF-α group, there was a significant reduction in both the basal and maximum glycolytic capacities of mature oligodendrocytes compared to the OE-NC + PBS group (basal: q = 8.833, p < 0.0001. maximum: q = 11.01, p < 0.0001, Turkey's post hoc test). Similarly, in the OE-GAPDH + TNF-α group, a significant decline was observed in both basal and maximum glycolytic capacities relative to the OE-GAPDH + PBS group (basal: q = 11.76, p < 0.0001. maximum: q = 6.973, p < 0.0001, Tukey's post hoc test). However, Gapdh overexpression protected the decrease in basal and maximum glycolytic capacity of mature oligodendrocytes when treated with TNF-α, compared to OE-NC + TNF-α group (basal glycolysis q = 4.662, p = 0.0090, maximum glycolysis q = 7.759, p < 0.0001) (Fig. 10B-C).
Figure 10D, E, and F display the OCR and oxidative phosphorylation capacities of the four groups of cells. According to the OCR curves depicted in Fig. 10D, there was no notable increase in OCR within the OE-GAPDH + PBS group compared with the OE-NC + PBS group. The OCR curve of mature oligodendrocytes in the OE-NC + TNF-α group decreased compared with that in the OE-NC + PBS group. Similarly, the OCR curve of the OE-GAPDH + TNF-α group is shifted downward compared with the OE-GAPDH + PBS group.
The decrease in OCR after adding oligomycin reflects the basal oxidative phosphorylation oxygen consumption rate of the cells, whereas OCR after adding FCCP reflects the maximum respiratory oxygen consumption rate of the cells. There were significant differences in the basal oxidative phosphorylation capacity [F (3, 32) = 80.820, p < 0.0001, one-way ANOVA] and maximum respiratory capacity [F (3, 32) = 11.360, p < 0.0001, one-way ANOVA] between the groups. In the OE-GAPDH + PBS group, there were no notable alterations observed in the basal oxygen consumption rate (q = 2.207, p = 0.4149, ,Tukey's post hoc test) or the peak respiratory oxygen consumption rate (q = 0.856, p = 0.9298, Tukey's post hoc test) when compared to the OE-NC + PBS group. However, compared with the OE-NC + PBS group, both the basal oxidative phosphorylation oxygen consumption rate (q = 19.520, p < 0.0001, Turkey's post hoc test) and maximum respiratory oxygen consumption rate (q = 7.340, p < 0.0001, Turkey's post hoc test) were significantly decreased in mature oligodendrocytes of the OE-NC + TNF-α group. In addition, compared with the OE-GAPDH + PBS group, only the basal oxidative phosphorylation oxygen consumption rate significantly decreased (q = 9.339, p < 0.0001, Turkey's post hoc test) in mature oligodendrocytes of the OE-GAPDH + TNF-α group, with no significant change in the maximum respiratory oxygen consumption rate (q = 0.303, p = 0.9964, Turkey's post hoc test). Compared with the OE-NC + TNF-α group, there was no significant change in the basal oxidative phosphorylation oxygen consumption rate (q = 0.771, p = 0.9472, Turkey's post hoc test) in mature oligodendrocytes of the OE-GAPDH + TNF-α group, but maximum respiratory oxygen consumption rates (q = 6.181, p = 0.0007, Turkey's post hoc test) were significantly increased (Fig. 10E-F).
Cellular ATP is primarily derived from glycolysis and oxidative phosphorylation. Using an ATP content detection kit, the effect of Gapdh on ATP synthesis in TNF-α-damaged mature oligodendrocytes was observed. Lactic acid is mainly derived from glycolysis. Using a lactate detection kit, the changes in glycolysis function of four groups of mature oligodendrocytes could be reflected. The ATP content detection results showed that ATP production was significantly different between the groups [F (3, 8) = 36.500, p < 0.0001, one-way ANOVA]. There was a significant reduction in ATP content in mature oligodendrocytes of the OE-NC + TNF-α group compared to the OE-NC + PBS group (q = 8.600, p = 0.0013, Turkey's post hoc test). Similarly, the OE-GAPDH + TNF-α group exhibited a significantly lower ATP content than the OE-GAPDH + PBS group (q = 6.810, p = 0.0058, Turkey's post hoc test). Conversely, ATP content in mature oligodendrocytes was significantly higher in the OE-GAPDH + PBS group relative to the OE-NC + PBS group (q = 6.130, p = 0.0107, Turkey's post hoc test). Additionally, the ATP content in the OE-GAPDH + TNF-α group was markedly higher than that in the OE-NC + TNF-α group (q = 7.920, p = 0.0023, Turkey's post hoc test) (Fig. 10G).
Lactate detection results showed significant differences between the groups [F (3, 8) = 36.500, p < 0.0001, one-way ANOVA]. Mature oligodendrocytes in the OE-NC + TNF-α group exhibited a significant reduction in lactate content compared to those in the OE-NC + PBS group (q = 4.790, p = 0.0386, Turkey's post hoc test). Similar to the OE-GAPDH + TNF-α group, when compared with the OE-GAPDH + PBS group, the lactate content decreased significantly (q = 6.290, p = 0.0092, Turkey's post hoc test). In the OE-GAPDH + PBS group, mature oligodendrocytes exhibited a marked increase in lactate content compared to the OE-NC + PBS group (q = 7.120, p = 0.0044, Turkey's post hoc test). The lactate content of the OE-GAPDH + TNF-α group increased significantly (q = 5.620, p = 0.0173, Turkey's post hoc test) compared with the OE-NC + TNF-α group (Fig. 10H).
The above results indicate that TNF-α reduces the synthesis of ATP by damaging both glycolysis and oxidative phosphorylation in mature oligodendrocytes.
Gapdh overexpression improved MBP production in TNF-α treated mature oligodendrocytes
To identify whether Gapdh is a critical part of myelin basic protein synthesis of mature oligodendrocyte during TNF-α damaging, we detected the expression of MBP mRNA and protein, as well as MYRF, a regulator of Mbp transcription [79]. QPCR results showed that there were differences in MYRF mRNA [F (3, 8) = 11.860, p < 0.0001, one-way ANOVA] and MBP mRNA [F (3, 8) = 6.480, p < 0.0001, one-way ANOVA] between groups. Compared with the OE-NC + PBS group, the relative expression level of mature oligodendrocyte MYRF mRNA significantly decreased in the OE-NC + TNF-α group (q = 5.709, p = 0.0158, Turkey's post hoc test). In the OE-GAPDH + TNF-α group, there was a notable reduction in MYRF mRNA expression relative to the OE-GAPDH + PBS group (q = 4.796 p = 0.0384, Turkey's post hoc test). Conversely, when compared with the OE-NC + TNF-α group, the OE-GAPDH + TNF-α group exhibited a modest elevation in MYRF mRNA levels, although this difference did not reach statistical significance (q = 3.248, p = 0.1779, Turkey's post hoc test) (Fig. 11A). As for MBP mRNA, only a significant decrease between the OE-NC + PBS group and OE-NC + TNF-α group was detected (q = 5.747, p = 0.0153, Turkey's post hoc test). The OE-GAPDH + TNF-α group exhibited elevated levels of MBP mRNA relative to the OE-NC + TNF-α group. However, this increase did not reach statistical significance (q = 3.701, p = 0.1142, Turkey's post hoc test) (Fig. 11B).
The relative MBP protein content indicated that there was a difference between the groups [F (3, 8) = 20.520, p = 0.0004, one-way ANOVA]. TNF-α significantly lower the MBP protein expression in mature oligodendrocytes of OE-NC + TNF-α group compared with the OE-NC + PBS group (q = 10.560, p = 0.0003, Turkey's post hoc test), whereas the change was not significant between the OE-GAPDH + TNF-α group and OE-GAPDH + PBS group (q = 2.219, p = 0.4450, Turkey's post hoc test). In addition, overexpression of Gapdh improved the relative expression of MBP in the OE-GAPDH + TNF-α group compared with the OE-NC + TNF-α group (q = 6.013, p = 0.0119, Turkey's post hoc test), indicating that Gapdh is the key to the inhibitory effect of TNF-α on MBP synthesis in mature oligodendrocytes (Fig. 11C-D).
Gapdh overexpression improves mitochondrial oxidative stress induced by TNF-α in mature oligodendrocytes
Mitochondrial calcium (Ca2+) is essential for regulating mitochondrial metabolic processes and sustaining the production of ATP within mitochondria. [80]. An increase in mitochondrial Ca2+ concentration indicates mitochondrial dysfunction [81]. Rhod-2 AM is a fluorescent probe that specifically binds to mitochondrial Ca2+. Fluorescence intensity is proportional to mitochondrial Ca2+ concentration. In this study, we investigated the influence of TNF-α on mitochondrial function in mature oligodendrocytes, focusing particularly on the role of Gapdh. Utilizing the Rhod-2 AM fluorescent probe, we monitored alterations in mitochondrial Ca2+ levels within these cells. Our findings revealed marked variations in mitochondrial Ca2+ concentrations among different groups, indicating significant disparities. [F (3, 44) = 202.000, p < 0.0001, one-way ANOVA]. Compared with the OE-NC + PBS group, the Ca2+ concentration in the mitochondria of mature oligodendrocytes in the OE-NC + TNF-α group increased significantly (q = 26.300, p < 0.0001, Turkey's post hoc test). As for the OE-GAPDH + TNF-α group, when compared with the OE-GAPDH + PBS group, the Ca2+ concentration increased significantly (q = 22.400, p < 0.0001, Turkey's post hoc test). However, the Ca2+ concentration of the OE-GAPDH + TNF-α group significantly lower than that of the OE-NC + TNF-α group (q = 4.990, p = 0.0053, Turkey's post hoc test) (Fig. 12A-B). These results indicate that overexpression of Gapdh could significantly alleviate the increase in mitochondrial Ca2+ concentration induced by TNF-α stimulation in mature oligodendrocytes, but it was still higher than the normal level.
Inflammatory factors may disrupt the balance between antioxidant and free radicals, elevating reactive oxygen species levels, which in turn impair mitochondrial function. This mitochondrial dysfunction manifests as decreased ATP production and disruption of mitochondrial membrane potential. MitoSOX Red, a specific fluorescent probe, binds to mitochondrial superoxide, with its fluorescence intensity reflecting the concentration of superoxide within the mitochondria directly. We used the high selectivity of MitoSOX Red to detect superoxide concentrations in mature oligodendrocyte mitochondria. The changes in mitochondrial superoxide levels were similar to Ca2+ concentration, that was TNF-α significantly elevated the mitochondrial superoxide content in mature oligodendrocytes (OE-NC + PBS vs. OE-NC + TNF-α, q = 20.700, p < 0.0001. OE-GAPDH + PBS vs. OE-GAPDH + TNF-α, q = 4.820, p = 0.0074, Turkey’s post hoc test). However, the mitochondrial superoxide content of the OE-GAPDH + TNF-α group significantly lower than that of the OE-NC + TNF-α group (q = 15.700, p < 0.0001, Turkey's post hoc test) (Fig. 12A and C). These findings suggested that Gapdh overexpression has a protective effect on mitochondrial oxidative stress stimulated by TNF-α.
The collapse in the mitochondrial membrane potential can reflect mitochondrial dysfunction. To investigate how overexpression of Gapdh influences TNF-α induced damage to the mitochondrial membrane potential in mature oligodendrocytes, we employed the JC-1 assay kit to evaluate alterations in their mitochondrial membrane potential. The mitochondrial membrane potential of mature oligodendrocytes significantly declined when treated with TNF-α (q = 9.240, p < 0.0001, Turkey's post hoc test), but Gapdh overexpression protected the mitochondrial membrane potential, showing no significant differences in mitochondrial membrane potential between OE-GAPDH + TNF-α group and OE-NC + TNF-α group (q = 1.020, p = 0.8886, Turkey's post hoc test), whereas there was a significantly higher mitochondrial membrane potential in OE-GAPDH + TNF-α compared to OE-NC + TNF-α group (q = 5.800, p = 0.0010, Turkey's post hoc test) (Fig. 12D-E).