Tumor necrosis factor-α impairs oligodendroglial differentiation through a mitochondria-dependent process

Abstract

Mitochondrial defects, affecting parameters such as mitochondrial number and shape, levels of respiratory chain complex components and markers of oxidative stress, have been associated with the appearance and progression of multiple sclerosis. Nevertheless, mitochondrial physiology has never been monitored during oligodendrocyte progenitor cell (OPC) differentiation, especially in OPCs challenged with proinflammatory cytokines. Here, we show that tumor necrosis factor alpha (TNF-α) inhibits OPC differentiation, accompanied by altered mitochondrial calcium uptake, mitochondrial membrane potential, and respiratory complex I activity as well as increased reactive oxygen species production. Treatment with a mitochondrial uncoupler (FCCP) to mimic mitochondrial impairment also causes cells to accumulate at the progenitor stage. Interestingly, AMP-activated protein kinase (AMPK) levels increase during TNF-α exposure and inhibit OPC differentiation. Overall, our data indicate that TNF-α induces metabolic changes, driven by mitochondrial impairment and AMPK activation, leading to the inhibition of OPC differentiation.

Figure 1

(a(i)) Differentiation assay in control (CTRL) and TNF-α-pretreated OPCs induced to differentiate by 5 days of exposure to the thyroid hormone T3. The proportions of different markers were determined on oligodendrocytes expressing the marker OSP. Percentages (mean±S.E.M.) of NG2⁺ cells (progenitors): CTRL, 13.2±1.87% TNF-α, 18.43±2.64% O4⁺ cells (intermediate stage): CTRL, 25.14±4.99% TNF-α, 18.85±6.04% and MBP⁺ cells (adult): CTRL, 19.21±4.73% TNF-α 12.51±3.39%. Representative images of cells expressing different markers are shown in the right panel. * Significant at P<0.05; line, mean; bars, maximum and minimum values. The boxes envelop the 25th to the 75th percentile of the assayed population, with n=6 independent cultures. (a(ii)) Percentage variation in proportions (mean±S.E.M.) within assayed cultures between CTRL and TNF-α-treated cells: NG2⁺: 41.71±14.67% O4+: −30.61±19.06% and MBP⁺: −36.78±8.63%. Representative images of cells expressing different markers are shown in the lower panel. (b) Expression levels of the adult oligodendrocyte marker MBP in OPCs after 5 days of differentiation induction. (c) Proportions (mean±S.E.M.) of TUNEL-positive OPCs after 5 days of differentiation in the CTRL (6.23±3.697%), 10 ng/ml TNF-α (7.43±1.85%), 50 ng/ml TNF-α (11.143±3.89%), and 100 ng/ml TNF-α (14.76±3.24%) groups. *Significant at P<0.05; bars=S.E.M. from three independent cultures

Figure 2

Expression levels of markers of cell types within the mixed glial population (NG2 for OPCs, β-tubulin IIII for neurons, and MBP for adult oligodendrocytes) and of the apoptotic marker PARP (whole and cleaved forms) (a) in control (CTRL) and TNF-α-exposed cells (b). Immunostaining for MBP and OSP in a mixed glial culture and relative quantification of the proportion of MBP⁺ cells among all OSP⁺ cells. The values are expressed as the percentage variation compared with the CTRL (mean±S.E.M.: TNF-α, 64.2±4.98%). (c) Maximum intensity projection (top) and 3D volume rendering (bottom) of co-cultured glia and OPCs stained for MBP (myelin marker) and β-tubulin III (axonal marker) during induction of the differentiation of CTRL and TNF-α-exposed cells by the thyroid hormone T3. The myelination activity was quantified as the proportion of β-tubulin III signal colocalizing with MBP signal (mean±S.E.M.: CTRL, 26.6±3.15% TNF-α, 14.9±2.82%). *Significant at P<0.05; bars, S.E.M. from four independent cultures

Figure 3

(a) Aequorin-based measurements of mitochondrial Ca²⁺ uptake (mean±S.E.M.) induced in OPCs by stimulation with 500 μM CCh in control (CTRL; 53.64±6.73 μM) and TNF-α-pretreated (29.35±9.85 μM) cells. (b) Measurements of [Ca²⁺]_c based on cytAEQ in OPCs (mean±S.E.M.: CTRL, 1.82±0.08 μM; TNF-α, 1.72±0.09 μM) after stimulation with 500 μM CCh. (c) Maximum intensity projection of the mitochondrial network in OPCs expressing mtGFP. The network morphology was analyzed by measuring the total mitochondrial volume (mean±S.E.M.: CTRL, 40837±12213.19 voxels; TNF-α, 43501±6463.32 voxels) and the average mitochondrial volume (CTRL, 1021.35±183.17 voxels; TNF-α, 922.69±183.17 voxels). (d) ΔΨ_m measurements in OPCs exposed to TNF-α for 24 h and in CTRL OPCs before and after treatment with 1 μM FCCP (for baseline detection), with relative quantification (mean±S.E.M.: CTRL, 123.882±21.52 A.F.U.; TNF-α, 93.574±9.85 A.F.U.). Representative pseudocolored images are shown on the right. *Significant at P<0.05; bars=S.E.M. from at least three independent experiments

Figure 4

(a) Representative pseudocolored images of MitoSOX-stained OPCs and measurement of superoxide production in control (CTRL) cells (mean±S.E.M.: CTRL, 277.87±86.99A.F.U.; TNF-α, 333.454±101.08A.F.U.). (b) Expression levels of different respiratory complex subunits in CTRL and TNF-α-treated OPCs. (c) Relative activity of respiratory complex I, measured as integrated density in a blue native gel and expressed as a percentage of the CTRL (mean±S.E.M.: TNF-α, 77.77±5.25 A.U.). *Significant at P<0.05; bars, S.E.M. from at least three independent experiments

Figure 5

(a) Representative pseudocolored images of MitoSOX-stained mature OLs and measurements of superoxide production in control (CTRL) cells (mean±S.E.M.: CTRL, 347.88±178.6A.F.U.; TNF-α, 676.93±268A.F.U.). (b) Single-cell measurement and relative quantification of [Ca²⁺]_m in OLs expressing mtD3cpv (mean±S.E.M.: CTRL, 1.75±0.085A.F.U.; TNF-α, 1.46±0.1A.F.U.). (c) Expression levels of different respiratory complex subunits in CTRL and TNF-α-treated OLs. *Significant at P<0.05; bars, S.E.M. from at least three independent experiments

Figure 6

Differentiation assay in OPCs pretreated with control (CTRL) treatment, 10 ng/ml TNF-α, 10 nM Rot, or 100 nM AA and then treated with the thyroid hormone T3 for 5 days. The proportions of cells expressing different markers were counted among the cells that were positive for the O4 OSP. Percentages (mean±S.E.M.) of NG2⁺ cells (progenitors, a(i)): CTRL, 10.9±1.638% TNF-α, 22.325±8.46% AA, 38.72±8.46% Rot, 26.13±2.47%. Percentages (mean±S.E.M.) of MBP⁺ cells (adult, a(ii)): CTRL 39.57±2.17% TNF-α, 27.4±4.62% AA, 3.18±0.63% Rot, 25.36±3.84%. *Significant at P<0.05, **significant at P<0.01, and ***significant at P<0.005; line, mean; bars, maximum and minimum values. The boxes envelop the 25th to the 75th percentile of the assayed population, including n=3 independent cultures. (b) Expression levels of AMPK alpha and its phosphorylated form, as monitored by western blotting in control (CTRL) and TNF-α-exposed OPCs. (c) LC3 conversion, as monitored by western blotting in CTRL and TNF-α-exposed OPCs. (d) Respiration rate in CTRL OPCs and OPCs exposed to TNF-α at 10 ng/ml and monitored by time-resolved fluorimetry using the O₂-sensitive probe MitoXpress-Intra. The values are expressed as normalized lifetimes (mean±S.E.M.: CTRL, 0.56±0.038; TNF-α 0.42±0.044). *Significant at P<0.05; line, mean; bars, maximum and minimum values. The boxes envelop the 25th to the 75th percentile of the assayed population, including n=3 independent experiments. (e) Lactate concentration in media from CTRL OPCs and OPCs exposed to TNF-α at 10 ng/ml and monitored by spectroscopy of the colorimetric conversion of a lactate-sensitive substrate (mean±S.E.M: CTRL, 1.93±0.098 mM; TNF-α, 2.21±0.13 mM). *Significant at P<0.05; line, mean; bars, maximum and minimum values. The boxes envelop the 25th to the 75th percentile of the assayed population, and each dot represents an assayed culture, with n=4 independent experiments