Distinct age and differentiation-state dependent metabolic profiles of oligodendrocytes under optimal and stress conditions

Abstract

Within the microenvironment of multiple sclerosis lesions, oligodendrocytes are subject to metabolic stress reflecting effects of focal ischemia and inflammation. Previous studies have shown that under optimal conditions in vitro, the respiratory activity of human adult brain-derived oligodendrocytes is lower and more predominantly glycolytic compared to oligodendrocytes differentiated in vitro from post natal rat brain oligodendrocyte progenitor cells. In response to sub-lethal metabolic stress, adult human oligodendrocytes reduce overall energy production rate impacting the capacity to maintain myelination. Here, we directly compare the metabolic profiles of oligodendrocytes derived from adult rat brain with oligodendrocytes newly differentiated in vitro from oligodendrocyte progenitor cells obtained from the post natal rat brain, under both optimal culture and metabolic stress (low/no glucose) conditions. Oxygen consumption and extracellular acidification rates were measured using a Seahorse extracellular flux analyzer. Our findings indicate that under optimal conditions, adult rat oligodendrocytes preferentially use glycolysis whereas newly differentiated post natal rat oligodendrocytes, and the oligodendrocyte progenitor cells from which they are derived, mainly utilize oxidative phosphorylation to produce ATP. Metabolic stress increases the rate of ATP production via oxidative phosphorylation and significantly reduces glycolysis in adult oligodendrocytes. The rate of ATP production was relatively unchanged in newly differentiated post natal oligodendrocytes under these stress conditions, while it was significantly reduced in oligodendrocyte progenitor cells. Our study indicates that both age and maturation influence the metabolic profile under optimal and stressed conditions, emphasizing the need to consider these variables for in vitro studies that aim to model adult human disease.

Fig 1. Morphology and survival of cell cultures under optimal and stress conditions.

Panels A-C: Adult brain derived OLs–illustration of expression of OL lineage markers O4(red)/Olig2(green) (Panel A) and O4(red)/MBP (green) (Panel B) under optimal conditions (N1) at day 1, and MBP (green) expression on day 12 (Panel C) post isolation. MBP immunoreactivity in early cultures (day 1 Panel B) is mainly punctate in cell bodies whereas immunoreactivity is observed in the cell processes that are extended during the culture period (panel C). Panels D-E: Post natal OLs differentiated in vitro (Panel D) at day 8 post-isolation express MBP; MBP+ cells ~ 70% and OPCs (Panel E) express O4 at day 4 post isolation under optimal conditions (N1); O4+ cells ~ 85%, All cultures were also stained with the nuclear marker, Hoechst. Scale Bar = 10μM. Panels F-H: Cell death under optimal (N1), LG and NG conditions were measured with TUNEL assay for adult OLs (Panel F, N1 vs. NG *p<0.05); post natal OLs differentiated in vitro (Panel G, N1 vs. LG *p<0.05, N1 vs. NG ***p<0.001 and LG vs. NG ***p<0.001); and post natal OPCs (Panel H, N1 vs. LG **p<0.01, N1 vs. NG ***p<0.001).

Fig 2. Differences in energy utilization by adult and newly differentiated post natal OLs.

(A) The resting OCR of adult OLs is lower than post natal OLs under optimal conditions. (** p < 0.001) (B) Adult OLs preferentially produce ATP through glycolysis. (* p < 0.05, ** p < 0.01) (C) The resting ECAR of adult OLs is higher than post natal OLs. (** p < 0.01) (D) Adult OLs have a higher glycolytic ECAR compared to post natal OLs. (*** p < 0.001).

Fig 3. Differences in energy utilization by post natal OPCs and newly differentiated OLs.

(A) The OCR of post natal OPCs is higher than post natal OLs under resting conditions and in presence of mitotoxins. (*** p < 0.001) (B) Post natal OPCs have a higher rate of ATP production compared to post natal OLs. (* p < 0.05, ** p < 0.01) (C) The resting ECAR of post natal OPCs is higher than post natal OLs under resting conditions and in presence of mitotoxins. (*** p < 0.001) (D) Post natal OPCs have a higher ECAR rate compared to post natal OLs. (* p < 0.05).

Fig 4. Differential respiration profiles in metabolic stress conditions in adult and newly differentiated post natal OLs.

(A) NG but not LG increases the resting OCR of adult OLs. (NG vs. N1, * p < 0.05) (B) NG but not LG increases the OCR of post natal OLs, under resting conditions (*** p < 0.001) and after the addition of FCCP (** p < 0.01) (C) NG but not LG reduces the resting ECAR of adult OLs. (*** p < 0.001) (D) Neither NG or LG changes the ECAR of post natal OLs. (E) Adult OLs maintain their rate of ATP production under NG and LG conditions. Under NG condition there is a significant increase of OXPHOS compared to N1. (* p < 0.05, ** p < 0.01) (F) Post natal OLs increase their rate of ATP production under NG conditions (NG vs. N1, * p < 0.05) (G) NG reduces ECAR in adult OLs. (NG vs. N1, *** p < 0.001) (H) Neither NG or LG changes ECAR in post natal OLs.

Fig 5. Differential respiration profiles in metabolic stress conditions in post natal newly differentiated OLs and OPCs.

(A) NG but not LG increases the OCR of post natal OLs, under resting conditions (*** p < 0.001) and after the addition of FCCP (** p < 0.01) (B) NG and LG decrease OCR in resting conditions and in presence of mitotoxins in post natal OPCs compared to N1 conditions. (*** p < 0.001) (C) Neither NG or LG changes the ECAR of post natal OLs. (D) NG and LG reduce the ECAR of post natal OPCs compared to N1 conditions. (*** p < 0.001) (E) Post natal OLs increase their rate of ATP production under NG conditions (NG vs. N1, * p < 0.05) (F) NG and LG reduce the rate of ATP production in post natal OPCs compared to N1 conditions. (* p < 0.05, ** p < 0.01, *** p < 0.001) (G) Neither NG or LG changes ECAR in post natal OLs. (H) NG and LG reduce the ECAR in post natal OPCs compared to N1 conditions. (* p < 0.05, ** p < 0.01, *** p < 0.001).