Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Robin JM Franklin ( rjf1000@cam.ac.uk ). This study did not generate new unique reagents.

Isolated OPCs were seeded onto 12mm glass coverslips in 24 well plates (VWR) or into 96 well-plates (InVitro-Sciences) coated with PDL (Sigma). After isolation, OPCs were left to recover in OPC medium (60μg/ml N-Acetyl cysteine (Sigma), 10μg/ml human recombinant insulin (GIBCO), 1mM sodium pyruvate (GIBCO), 50μg/ml apo-transferrin (Sigma), 16.1μg/ml putrescine (Sigma), 40ng/ml sodium selenite (Sigma), 60ng/ml progesterone (Sigma), 330μg/ml bovine serum albumin (Sigma)) supplemented with b-FGF and PDGF (30ng/ml each, Peprotech). OPCs were incubated at 37°C, 5% CO 2 and 5% O 2 . The medium was completely exchanged to OPC medium with 20ng/ml bFGF and PDGF after overnight culture to remove any dead cells. After 3d the cell culture medium was switched to promote further proliferation (OPC medium+20ng/ml bFGF and PDGF) or differentiation (OPCM + 40ng/ml T3). During differentiation or proliferation experiments 66% of the medium were replaced every 48h and growth factors or other small molecules were added fresh to the culture. The culture medium used was 500 μL for cultures in 24 well plate wells and 150 μL for cultures in 96 well plate wells. For differentiation assays the medium was in some instances supplemented with 40ng/ml thyroid-hormone (T3, Sigma), 50nM 9-cis retinoic acid (9cRA, Sigma), 1μM miconazole (Sigma, M3512) or 1.5μM benztropine (Sigma, SML0847). Otherwise used small molecules: 100μM metformin (Sigma), rotenone (Sigma, R8875), 1μM dorsomorphin (LC Laboratories, D-3197).

Adult male and female rats (2-24 months) were decapitated after lethal injection with phenobarbital. The brains were removed quickly and placed into ice-cold isolation medium ( Table S3 ; alternatively Hibernate A Brainbits). The telencephalon and cerebellum were dissected in isolation medium; meninges, and the olfactory bulb were mechanically removed and the brain tissue was mechanically minced into 1mmpieces. The tissue pieces were spun down at 100 g for 1min at RT and the tissue was washed in HBSS- (no Mg2+ and Ca, GIBCO). Each half of the brain was mixed with 5ml of dissociation solution (34U/ml papain (Worthington), 20 μg/ml DNase Type IV (GIBCO) in isolation medium). The brain tissue was dissociated on a shaker (50rpm) for 40 min at 35°C. The digestion was stopped by addition of ice cold HBSS-. The tissue was centrifuged (200 g, 3 min, RT), the supernatant completely aspirated and the tissue resuspended in isolation medium supplemented with 2% B27 and 2mM sodium-pyruvate (trituration solution). The tissue was allowed to sit in this solution for 5min. To obtain a single cell suspension the tissue suspension was triturated 10 times using first a 5ml serological pipette and subsequently three fire polished glass pipettes (opening diameter > 0.5mm). After each trituration step the tissue suspension was allowed to sediment (approximately 1-2 min) and the supernatant (approximately 2ml), containing the cells, was transferred into a fresh tube. After each round of trituration 2ml of fresh trituration solution were added. To remove accidentally transferred undigested tissue bits, the collected supernatant was filtered through 70 μm cell strainers into tubes that contained 90% isotonic Percoll (GE Healthcare, 17-0891-01, in 10xPBS pH7.2 (Lifetech). The final volume was topped up with phenol-red free DMEM/F12 with HEPES (GIBCO) and mixed to yield a homogeneous suspension with a final Percoll concentration of 22.5%. The single cell suspension was separated from remaining debris particles by gradient density centrifugation (800 g, 20min, RT, without break). The myelin debris and all layers without cells were discarded and the brain cell containing phase (last 2ml) and cell pellet were resuspended in HBSS+ and combined in a fresh 15ml tubes and centrifuged (300 g, 5min, RT). The cell pellet was resuspended in red blood cell lysis buffer (Sigma, R7757) and incubated for 1min at RT to remove red blood cells. 10ml of HBSS+ were added to this cell suspension and spun down (300 g, 5min, RT). The cell pellets were resuspended in 0.5ml modified Milteny washing buffer (MWB, 2mM EDTA, 2mM Na-Pyruvate, 0.5% BSA in PBS, pH 7.3) supplemented with 10ng/ml human recombinant insulin (GIBCO). To this cell suspension 2.5μg mouse-anti-rat-A2B5-IgM antibody (Millipore, Table S4 ) were added for every 10 million cells. After 25 min incubation, gently shaking at 4°C, 7ml of MWB were added. The solution was centrifuged (300 g, 5min, RT) and the pellet resuspended in 80 μL MWB supplemented with 20 μL rat-anti-mouse-IgM antibody (Milteny, 130-047-302) per 10 million cells. The cells were incubated for 15 min, slowly shaking at 4°C. The secondary antibody was again washed out with 7ml MWB and the sample was centrifuged (300 g, 5min, RT). The cell pellet was resuspended in 0.5ml and MACS was performed according to the recommendations of the supplier. Briefly, a MS column (Milteny, 130-042-201) were inserted into MiniMACS Separator (Miltenyi; 130-042-102) and pre-wet with 0.5ml MWB. Resuspended cells were put onto one MS column. Subsequently the column was washed three times using 500 μL MWB for each wash. Finally, A2B5 positive cells were flushed out the column with 1ml pre-warmed, COand Opre-equilibrated OPC medium.

For metformin treatment 15 months old female SD rats that were fed ad libitum received metformin (Glucophage) in their drinking water (300mg/kg bodyweight per day). Metformin treatment was interrupted for two days before and three days after surgery and then commenced to the end of the study (21 days after lesion induction). Fluid consumption was continuously monitored to adapt dosages. All animals were randomly allocated to experimental groups.

For alternate day fasting (ADF) 12 months old female SD rats were restricted from food every other day. ADF animals had access to food on Tuesday, Thursday, Saturday and Sunday and all food was removed from their cages on Monday, Wednesday and Friday. The food was removed and returned in the mornings. The weight of each animal was weekly monitored. The fasting paradigm was interrupted for the first three days after surgery when all animals had free access to food.

All animal procedures were performed in compliance with United Kingdom Home Office regulations. The animals were housed under standard laboratory conditions on a 12 h light/dark cycle with constant access to food and water. All animals were housed in pairs or groups of up to 4 animals.

Method Details

Induction of white matter lesions and assessment of remyelination Woodruff and Franklin, 1999 Woodruff R.H.

Franklin R.J.M. Demyelination and remyelination of the caudal cerebellar peduncle of adult rats following stereotaxic injections of lysolecithin, ethidium bromide, and complement/anti-galactocerebroside: a comparative study. For studies involving demyelination, female Sprague Dawley rats (Harlan Laboratories) 18 months of age were used. The rats were anesthetized with buprenorphine (0.03mg/kg, s.c.) and 2.5% isoflurane. Demyelination was induced by stereotaxic injection of 4μl of 0.01% ethidium bromide (EB) into the caudal cerebellar peduncles (CCPs), as previously described (). EB was delivered at a rate of 1 μl/min. After EB delivery, the injection needle remained in position for additional 4 min. To assess remyelination the rats were transcardially perfused with 4% glutaraldehyde and 0.4 mM CaCl 2 in PBS. The cerebellum was cut in to transverse 1mm thick sections. The tissue was fixed in 2% osmium-tetroxide at 4°C overnight, dehydrated through a series of washes in ethanol and propylene-oxide and embedded in resin. From the resin blocks 1μm thick sections were cut and stained with 1% toluidine blue. To compare the extent of remyelination in different experimental groups we first used a blinded ranking analysis. Single blocks from each animal from which sections containing the largest area of lesion were identified and used for subsequent analysis. In resin sections, remyelinated axons can be readily distinguished from normally myelinated axons outside the lesion by the thinness of the myelin sheath. Within the lesion, remyelinated axons can be distinguished from demyelinated axons because the former have myelin sheaths recognizable as a dark staining rim around the axon. Sections from each animal was examined by an observer blind to the experimental group form which the animal came. The highest rank was given to the animal exhibiting the highest proportion of remyelinated axons. If it was not possible to differentiate two animals using this method then they were given the same rank. In this method, no attempt is made to assign a value to the proportion of remyelination, but simply to establish how a section from an individual animal ranks relative to others. Additional analysis was undertaken by counting the number of remyelinated and demyelinated axons within the lesion by electron microscopy, where remyelinated axons can be identified using the same morphological criteria used in semi-thin section light microscopy. For electron microscopy (EM), ultrathin sections of lesion sites were cut and transferred onto copper grids. The sections were stained with uranyl acetate and imaging was performed using a Hitachi-H600 Transmission Electron Microscope. G ratio was measured with open source software Fiji (ImageJ, https://imagej.net/Fiji ) on transverse electron micrographs at 4,000 – 6,500 magnification with internal calibration. The perimeters of each axon and the myelin sheath were measured with freehand tool on Fiji by tracing the outer surfaces of each structure, then converted the perimeters into hypothetical diameters assuming their circular morphology. The G ratio was calculated as the ratio of the diameter of axon over that of myelin on the same axons, which is inversely correlated to myelin thickness, that a maximum G ratio of 1 results from unmyelinated or demyelinated axons.

Isolation of adult oligodendrocyte progenitor cells 3 pieces. The tissue pieces were spun down at 100 g for 1min at RT and the tissue was washed in HBSS- (no Mg2+ and Ca2+, GIBCO). Each half of the brain was mixed with 5ml of dissociation solution (34U/ml papain (Worthington), 20 μg/ml DNase Type IV (GIBCO) in isolation medium). The brain tissue was dissociated on a shaker (50rpm) for 40 min at 35°C. The digestion was stopped by addition of ice cold HBSS-. The tissue was centrifuged (200 g, 3 min, RT), the supernatant completely aspirated and the tissue resuspended in isolation medium supplemented with 2% B27 and 2mM sodium-pyruvate (trituration solution). The tissue was allowed to sit in this solution for 5min. To obtain a single cell suspension the tissue suspension was triturated 10 times using first a 5ml serological pipette and subsequently three fire polished glass pipettes (opening diameter > 0.5mm). After each trituration step the tissue suspension was allowed to sediment (approximately 1-2 min) and the supernatant (approximately 2ml), containing the cells, was transferred into a fresh tube. After each round of trituration 2ml of fresh trituration solution were added. To remove accidentally transferred undigested tissue bits, the collected supernatant was filtered through 70 μm cell strainers into tubes that contained 90% isotonic Percoll (GE Healthcare, 17-0891-01, in 10xPBS pH7.2 (Lifetech). The final volume was topped up with phenol-red free DMEM/F12 with HEPES (GIBCO) and mixed to yield a homogeneous suspension with a final Percoll concentration of 22.5%. The single cell suspension was separated from remaining debris particles by gradient density centrifugation (800 g, 20min, RT, without break). The myelin debris and all layers without cells were discarded and the brain cell containing phase (last 2ml) and cell pellet were resuspended in HBSS+ and combined in a fresh 15ml tubes and centrifuged (300 g, 5min, RT). The cell pellet was resuspended in red blood cell lysis buffer (Sigma, R7757) and incubated for 1min at RT to remove red blood cells. 10ml of HBSS+ were added to this cell suspension and spun down (300 g, 5min, RT). The cell pellets were resuspended in 0.5ml modified Milteny washing buffer (MWB, 2mM EDTA, 2mM Na-Pyruvate, 0.5% BSA in PBS, pH 7.3) supplemented with 10ng/ml human recombinant insulin (GIBCO). To this cell suspension 2.5μg mouse-anti-rat-A2B5-IgM antibody (Millipore; 2 and O 2 pre-equilibrated OPC medium. Adult male and female rats (2-24 months) were decapitated after lethal injection with phenobarbital. The brains were removed quickly and placed into ice-cold isolation medium ( Table S3 ; alternatively Hibernate A Brainbits). The telencephalon and cerebellum were dissected in isolation medium; meninges, and the olfactory bulb were mechanically removed and the brain tissue was mechanically minced into 1mmpieces. The tissue pieces were spun down at 100 g for 1min at RT and the tissue was washed in HBSS- (no Mg2+ and Ca, GIBCO). Each half of the brain was mixed with 5ml of dissociation solution (34U/ml papain (Worthington), 20 μg/ml DNase Type IV (GIBCO) in isolation medium). The brain tissue was dissociated on a shaker (50rpm) for 40 min at 35°C. The digestion was stopped by addition of ice cold HBSS-. The tissue was centrifuged (200 g, 3 min, RT), the supernatant completely aspirated and the tissue resuspended in isolation medium supplemented with 2% B27 and 2mM sodium-pyruvate (trituration solution). The tissue was allowed to sit in this solution for 5min. To obtain a single cell suspension the tissue suspension was triturated 10 times using first a 5ml serological pipette and subsequently three fire polished glass pipettes (opening diameter > 0.5mm). After each trituration step the tissue suspension was allowed to sediment (approximately 1-2 min) and the supernatant (approximately 2ml), containing the cells, was transferred into a fresh tube. After each round of trituration 2ml of fresh trituration solution were added. To remove accidentally transferred undigested tissue bits, the collected supernatant was filtered through 70 μm cell strainers into tubes that contained 90% isotonic Percoll (GE Healthcare, 17-0891-01, in 10xPBS pH7.2 (Lifetech). The final volume was topped up with phenol-red free DMEM/F12 with HEPES (GIBCO) and mixed to yield a homogeneous suspension with a final Percoll concentration of 22.5%. The single cell suspension was separated from remaining debris particles by gradient density centrifugation (800 g, 20min, RT, without break). The myelin debris and all layers without cells were discarded and the brain cell containing phase (last 2ml) and cell pellet were resuspended in HBSS+ and combined in a fresh 15ml tubes and centrifuged (300 g, 5min, RT). The cell pellet was resuspended in red blood cell lysis buffer (Sigma, R7757) and incubated for 1min at RT to remove red blood cells. 10ml of HBSS+ were added to this cell suspension and spun down (300 g, 5min, RT). The cell pellets were resuspended in 0.5ml modified Milteny washing buffer (MWB, 2mM EDTA, 2mM Na-Pyruvate, 0.5% BSA in PBS, pH 7.3) supplemented with 10ng/ml human recombinant insulin (GIBCO). To this cell suspension 2.5μg mouse-anti-rat-A2B5-IgM antibody (Millipore; Table S4 ) were added for every 10 million cells. After 25 min incubation, gently shaking at 4°C, 7ml of MWB were added. The solution was centrifuged (300 g, 5min, RT) and the pellet resuspended in 80 μL MWB supplemented with 20 μL rat-anti-mouse-IgM antibody (Milteny, 130-047-302) per 10 million cells. The cells were incubated for 15 min, slowly shaking at 4°C. The secondary antibody was again washed out with 7ml MWB and the sample was centrifuged (300 g, 5min, RT). The cell pellet was resuspended in 0.5ml and MACS was performed according to the recommendations of the supplier. Briefly, a MS column (Milteny, 130-042-201) were inserted into MiniMACS Separator (Miltenyi; 130-042-102) and pre-wet with 0.5ml MWB. Resuspended cells were put onto one MS column. Subsequently the column was washed three times using 500 μL MWB for each wash. Finally, A2B5 positive cells were flushed out the column with 1ml pre-warmed, COand Opre-equilibrated OPC medium.

Culture of adult oligodendrocyte progenitor cells Isolated OPCs were seeded onto 12mm glass coverslips in 24 well plates (VWR) or into 96 well-plates (InVitro-Sciences) coated with PDL (Sigma). After isolation, OPCs were left to recover in OPC medium (60μg/ml N-Acetyl cysteine (Sigma), 10μg/ml human recombinant insulin (GIBCO), 1mM sodium pyruvate (GIBCO), 50μg/ml apo-transferrin (Sigma), 16.1μg/ml putrescine (Sigma), 40ng/ml sodium selenite (Sigma), 60ng/ml progesterone (Sigma), 330μg/ml bovine serum albumin (Sigma)) supplemented with b-FGF and PDGF (30ng/ml each, Peprotech). OPCs were incubated at 37°C, 5% CO 2 and 5% O 2 . The medium was completely exchanged to OPC medium with 20ng/ml bFGF and PDGF after overnight culture to remove any dead cells. After 3d the cell culture medium was switched to promote further proliferation (OPC medium+20ng/ml bFGF and PDGF) or differentiation (OPCM + 40ng/ml T3). During differentiation or proliferation experiments 66% of the medium were replaced every 48h and growth factors or other small molecules were added fresh to the culture. The culture medium used was 500 μL for cultures in 24 well plate wells and 150 μL for cultures in 96 well plate wells. For differentiation assays the medium was in some instances supplemented with 40ng/ml thyroid-hormone (T3, Sigma), 50nM 9-cis retinoic acid (9cRA, Sigma), 1μM miconazole (Sigma, M3512) or 1.5μM benztropine (Sigma, SML0847). Otherwise used small molecules: 100μM metformin (Sigma, PHR1084-500MG), rotenone (Sigma, R8875), 1μM dorsomorphin (LC Laboratories, D-3197).

Immunofluorescence for tissue sections Schindelin et al., 2012 Schindelin J.

Arganda-Carreras I.

Frise E.

Kaynig V.

Longair M.

Pietzsch T.

Preibisch S.

Rueden C.

Saalfeld S.

Schmid B.

et al. Fiji: an open-source platform for biological-image analysis. Rats received a lethal dose of pento-barbitol and were transcardially perfused with 4% paraformaldehyde (PFA) in PBS. The brains were removed and post-fixed for 2h at RT with 4% PFA. After a rinse in PBS the tissue was incubated in 20% sucrose solution (in PBS) overnight. The tissue was then imbedded in OCT- medium (TissueTek) and stored at −80°C. 12 μm sections were obtained using a cryostat. Tissue sections were air-dried and stored at −80°C. Cryostat cut sections were dried for 45 min at RT. For antigen-retrieval the slides were submerged in preheated citrate buffer pH 6.0 (Sigma) in a water bath at 95°C for 15 min. The slides were washed three times with PBS (5min, RT) and blocked in 0.3% PBST with 10%NDS for 1h at RT. Primary antibodies ( Table S4 ) were diluted in 0.1% PBST with 5%NDS and incubated overnight at 4°C. The slides were washed 3 times for 10min with PBS. Next, secondary antibodies in blocking solution were applied at a concentration of 1:500 for 2h at RT. Slides were washed 3 times with PBS for 10 min each, whereby the first wash contained Hoechst 33342 nuclear stain (2 μg/ml,). The slides were mounted with coverslips using FluoSave (CalBiochem). Image acquisition was performed using a Leica-SP5 microscope (Leica) and LAS software (Leica) or a Zeiss Observer A1 inverted microscope (Zeiss) and Zeiss Axivision software. Further image processing and analysis was performed using the ImageJ software package ().

Immunofluorescence for cells Schindelin et al., 2012 Schindelin J.

Arganda-Carreras I.

Frise E.

Kaynig V.

Longair M.

Pietzsch T.

Preibisch S.

Rueden C.

Saalfeld S.

Schmid B.

et al. Fiji: an open-source platform for biological-image analysis. Cultured cells were rinsed with PBS before fixation with 4% PFA (10 min, RT). Subsequently, the cells were washed three times with PBS (5 min, RT, shaking). If permeabilisation was required, the cells were incubated with PBST (0.1% Triton X-100 in PBS) for 20 min at RT. The samples were then blocked in PBS supplemented with 10% normal donkey serum (NDS). Primary antibodies ( Table S4 ) were diluted in PBS with 5% NDS and incubated overnight at 4◦C in a humidified chamber. Excess antibodies were washed off with three washes in PBS (10 min, RT, shaking). The primary antibodies were then labeled with secondary antibodies ( Table S4 ) diluted in PBS with 5% normal donkey serum. Again, excess antibody was washed off with three washes PBS (10 min, RT, shaking). If visualization of nuclei was required the first wash contained 2 μg/ml Hoechst 33342 (Sigma). If coverslips were used, they were mounted onto Polysine glass slides (VWR) in a drop of Fluosave (Calbiochem) and the slides were dried for at least 3h at RT in the dark. Images were taken with a Axio-Vision (Zeiss), Leica-SP5 (Leica) or Nikon microscope. For 96 well plate assays cells were kept in PBS after staining. Further image processing and analysis was performed using the ImageJ software package ().

Comet assay Olive and Banáth (2006) Olive P.L.

Banáth J.P. The comet assay: a method to measure DNA damage in individual cells. Collins, 2004 Collins A.R. The comet assay for DNA damage and repair: principles, applications, and limitations. For comet assays, a single cell gel electrophoresis based assay for detecting DNA damage, approximately 5000 OPCs were resuspended in 100μl PBS and mixed with 300 μl 1% low melting point agarose (37°C). Alternatively, when OPCs were cultured prior to the assay, the cells were detached using TrypLE 1x Select (GIBCO) for 8 min at 37°C. The comet assay was then performed as described in. Briefly, OPCs were centrifuged at 300 g for 5 min. at room temperature and the cell pellet was resuspended with 100μl PBS and then mixed with 300μl molten low-melting point agarose pre-incubated at 37°C. The cell-agarose suspension was then applied gently onto polysine slides that were pre-treated with 1% agarose and allowed to solidify at 4°C. The slides were submersed in alkaline cell lysis buffer (0.3M NaOH, 100mM EDTA, 0.1% (w/v) N-Lauroylsarcosine (Sigma, 61745), 1.2M NaCl in ddH2O) for 16 h at 4°C in the dark. The slides were then electrophoresed in alkaline electrophoresis buffer (0.03M NaOH, 2mM EDTA, pH > 12.3, pre-chilled at 4°C) for 25 min at RT with 1V/cm, whereby cm represents the distance between the electrodes. Finally, electrophoresed and propidium iodide stained DNA was visualized using a Zeiss Axiovision Fluorescence microscope (Carl Zeiss), and 50-100 nuclei per animal were visually scored according to published protocols (). Statistical significance was determined comparing respective damage categories between experimental groups by a two-tailed unpaired t test. A significant result was assumed for p < 0.05.

RNA sequencing and downstream analysis Kim et al., 2013 Kim D.

Pertea G.

Trapnell C.

Pimentel H.

Kelley R.

Salzberg S.L. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Love et al., 2014 Love M.I.

Huber W.

Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Marques et al. (2016) Marques S.

Zeisel A.

Codeluppi S.

van Bruggen D.

Mendanha Falcão A.

Xiao L.

Li H.

Häring M.

Hochgerner H.

Romanov R.A.

et al. Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system. RNA was isolated from freshly purified young adult (2-3 months) and aged (20-24 months) OPCs using QIAGEN RNAeasy Micro kit (QIAGEN) and RNA was stored at −80°C. RNA quality was assessed by Qubit measurement and posterior RNA nanochip/picochip Bioanalyzer. Ribosomal RNA was depleted with rat-specific oligos (InDA-C technology). Sequencing libraries were prepared using 10-100ng total RNA and the Nugen Ovation RNA-Seq Systems 1–16 for Model Organisms Kit (0349-32). Sequencing was performed on the Illumina HiSeq4000 in a pair-end 150 base pair format. Adaptor sequences were removed and reads were quality-trimmed using TrimGalore. Trimmed reads were aligned to the rat reference genome (RGSC6.0/rn6) by using TopHat2 () ( http://ccb.jhu.edu/software/tophat , version: 2.0.13) guided by Ensembl gene models. Raw counts per gene regions were obtained by featureCounts. Replicates were evaluated, counts were normalized and differential expression of transcripts was evaluated by the R Bioconductor DESeq2 package (). Expression levels were further normalized by transcript length (per kB). Transcript annotations were based on Ensembl (Release 82). GO term analysis was performed using the goseq package. For ingenuity pathway analysis we used differentially expressed genes with an adjusted p value cutoff (p.adj < 0.05). The OPC gene dataset for Figure 2 A was taken from Table S1 from Raw data files from RNaseq were made available at the NCBI GEO database (accession number GSE134765 ).

RNA isolation and qRT-PCR RNA was isolated from freshly purified OPCs or from cultured OPCs according to the Directzol RNA MicroPrep Kit (Zymo Research; R2061). All RNA samples were stored at −80°C prior to further processing. cDNA was generated using the QuantiTect Reverse Transcription Kit’s according to the instructions of the manufacturer (QIAGEN; 205310). For RT-qPCR, primers (see Table S5 ) were used at a concentration of 400μM. The efficiency of each primer was greater than ∼95% as determined for each primer pair by serial dilutions of OPC cDNA. cDNA, primers, and the Syber Green Master Mix (QIAGEN; 204141) were mixed as instructed by the manufacturer, and RT-qPCR and melting curve analysis were performed on Life Technologies’ Quantstudio 6 Flex Real-Time PCR System. Fold changes in gene expression were calculated using the delta delta Ct method in Microsoft Excel. Statistical significance was determined using two-tailed unpaired t tests assuming equal variances.

ATP measurements For the comparison of freshly isolated cell populations (young, aged and ADF aged) cells were spun down (300 g, 5min). The supernatant was removed and the cell pellets were frozen down at −80°C and stored until analysis. The relative ATP content was measured using the ATPlite luminescence Assay System (Perkin Elmer). Cell pellets were resuspended in 100μl OPC medium, alternatively freshly cultured cells in 100μl OPC medium were used. To this suspension 100μl of lysis buffer were added and the samples shaken at 600rpm for 5min on a horizontal shaker. 50μl of substrate solution were added and the samples were shaken at 600rpm in a horizontal shaker for another 5min. The luminescence signal was recorded using an Infinite pro 200 Tecan plate reader (Tecan) and the Magellan software. The luminescence was measured for 1 s intervals. The counts were normalized to cell numbers.

Oxygen Consumption rate measurements OPCs or pre-oligodendrcoytes were seeded onto PDL coated Seahorse cell culture plates (Agilent). The oxygen consumption rate was recorded using the manufacturers standard protocol for mitochondrial stress tests (Agilent). For the comparison of young and aged cells or OPCs and POLs, the cells were cultured overnight prior to the assay. For the comparison of aged cells treated with metformin and dorsomorphin the cells were cultured for 5d in the presence of the drugs. The cells were cultured 1h before the assay in modified assay medium (1.5mM sodium pyruvate, 2mM L-Glutamine, 2mM Glucose, 1% SATO in XF base medium, pH 7.4). The OCR measurements were carried out using a Seahorse XFp or a Seahorse XF96 analyzer. The basal OCR was calculated as the difference between the average of the measurements taken under untreated conditions and the average of the measurements taken after the injection of rotenone and antimycin A. All OCR values were then normalized to the cell number. The final OCR values were normalized to one treatment group and the results are represented as the relative basal oxygen consumption rate between the groups. The concentrations of the small molecules in the assay were: oligomycin (1μM), FCCP (0.5μM) rotenone (0.5μM) and antimycin A (0.5μM)

Western Blot Cells were lysed in IP lysis buffer (Thermo Scientific) supplemented with 1% Halt protease inhibitor (Thermo Scientific, 87786) for 10 min on ice. The lysates were spun down for 10min at 4°C and 10, 000 g in a table top centrifuge. The supernatant was stored at −80°C. Protein quantification was carried out using Pierce BCA protein assay kit (Thermo Scientific) measured with a Nanodrop2000. Equal amounts of protein (15-20μg) were loaded mixed with 4X Bolt® LDS Sample Buffer (Thermo Fisher; B0007) and 10X Bolt reduction agent and boiled to 70°C for 10min. Protein was run on Bolt 4%–12% Bis-Tris Plus Gels (Thermo Fisher; NW04120BOX) in Bolt MOPS SDS running buffer (Thermo Scientific, B0001) for 32min at 200V. Protein was transferred for 60min at 20V to a nitrocellulose membrane (Immobilon FL 0.45μm pore size, Millipore) membrane using the Mini Blot module (Thermo Scientific, B1000) and Bolt transfer buffer with 10% methanol and 1% Bolt antioxidant (Thermo Scientific, BT005) according to the manufacturer’s instructions. Membranes were blocked in 50% Odyseey blocking buffer TBS (Li-Cor, 927-50100) in TBS. All primary antibodies were used in a dilution of 1:1000 in 0.1%TBS-Tween with 50% Odyseey blocking buffer (TBS) in TBS. The membranes were incubated shaking in antibody solution overnight at 4°C. The membranes were washed twice in 0.1% TBS-Tween. Secondary antibodies were added in a concentration of 1:10000 in 50% Odyssey blocking buffer in 0.1% TBS-Tween. Secondary antibodies were incubated at room temperature for 1h in the dark. The membranes were washed three times in 0.1% TBS Tween. Fluorescent antibody signal was detected using the Odyssey (Li-Cor) and Image Studio v4.0 software. For luminescent signal detection the membranes were incubated with ECL solution (Amersham ECL Western Blot analysis system, GE Healthcare) and the signal was detected as described for fluorescent signals. For PGC1a detection we used a secondary antibody anti-mouse-IgG coupled to HRP (CST) and the

Fluorescence Activated Cell Sorting Freshly isolated OPCs were fixed in ice-cold 4% PFA for 10min and washed in FACS buffer (0.5% BSA in PBS). The cells were stained with primary antibodies (Anti-A2B5-PE, anti-MOG-biotin and anti-rat-Cd11b-PerCP-Cy5.5 and appropriate isotype controls; see Table S4 ) for 30min at 4°C. With exception of A2B5 staining, which was carried out for 10min at 4°C. Cells were washed with FACS buffer and stained with secondary antibody (Streptavidin-BV421) for 15min at 4°C in the dark. Cells were washed and resuspended in FACS buffer. Cells were analyzed using an Attune-NXT (Thermo Scientific) equipped with 405, 488 and 561 lasers. For compensation, beads (OneComp) were used for single stains for each fluorophore. The compensation matrix was automatically calculated and applied by the Attune software. Gates for the quantification of A2B5, Cd11b and MOG positive cells were set according to appropriate FMOs. A minimum of 30,000 cell singlets were recorded and used for quantification with FlowJo software (v10).

Oil-Red-O staining 12μm cryostat cut sections were dried for 45 min at RT. Tissue sections were dehydrated in 100% 1,2 propanediol (Sigma) for 2 times 5 min. The slides were stained at 60C in prewarmed 0.5% Oil Red O solution (ORO, Sigma). Then, the samples were placed into 85% 1,2 propanediol (v/v in distilled water) for 8 min to differentiate the staining. The slides were then rinsed three times with distilled water and mounted with jelly mounting media. Image acquisition was performed with a Nikon microscope. Digital images were converted into 8-bit greyscale images. The pixel values were inverted so that a more intense staining corresponded to higher pixel values. The staining was quantified using ImageJ software, measuring the mean gray value for the lesion area.