Figure 4.

Metformin Restores the Ability of Aged OPCs to Differentiate in an AMPK-Dependent Manner

(A) Representative images of differentiation assays. Newly formed oligodendrocytes are identified as MBP⁺/Olig2⁺ cells. Scale bar, 50 μm for all images. Prior to differentiation, OPCs were isolated from aged animals (≥18 months) and cultured in the presence of growth factors for 5 days. Some cells were treated with 100 μM metformin with each medium change during the first 5 days (days 2 and 4).

(B) Quantification of the differentiation assay as the proportion of MBP⁺ cells among all lineage cells (Olig2⁺). All data are presented as mean ± SD (n = 3 biological replicates, one-way ANOVA with Dunnett’s multiple comparisons test against aged OPCs culture in the absence of growth factors [aged no GF]).

(C) Schematic illustration.

(D) Western blots for AMPK pathway proteins from aged OPCs treated with DMSO, 100 μM metformin, or 100 μM metformin and 1 μM dorsomorphin (labeled Dorso) for 3 days in vitro. P, phosphorylated (n = 2 biological repeats for each group.)

(E) Representative images of differentiation assay findings. Aged OPCs were treated with metformin alone or with metformin and dorsomorphin during the first 5 days of culture, similar as described in Figure S5G. Newly formed oligodendrocytes are identified as MBP⁺/Olig2⁺ cells. Scale bars, 50 μm.

(F) Proportion of Olig2⁺ in the differentiation assay that are differentiated (MBP⁺/Olig2⁺). All data are presented as mean ± SD (n = 3 biological replicates, one-way ANOVA with Dunnett’s multiple comparisons test against “+met”).

(G) Schematic for experiments to knock out Prkaa2 (AMPK) in aged OPCs using CRISPR/Cas9.

(H) qPCR confirming knockout of prkaa2 mRNA after CRISPR with guide RNAs (gRNAs) targeting Prkaa2 (n = 3 biological repeats).

(I) Representative images of differentiation assays after CRISPR-mediated knockout of Prkaa2. Newly formed oligodendrocytes are identified as MBP⁺/Olig2⁺ cells. Scale bars, 50 μm.

(J) Quantification of the differentiation assay in (H) (n = 3 biological repeats, all data are mean ± SD, two-tailed t test).

met, metformin; dorso, dorsomorphin. Error bars represent SDs. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ns, p > 0.05.

See also Figures S4 and S6.