Cholesterol supplementation does not affect pathology in EAE

To test whether elevated serum cholesterol is a biomarker of acute inflammatory disease, we induced MOG-EAE and determined serum cholesterol at the peak of clinical symptoms, typically 16–20 days after induction. Surprisingly, in acute EAE, total serum cholesterol was reduced to about 75% of normal values (76±2 mg dl−1±s.e.m. cholesterol in EAE mice compared with 103±2 mg dl−1 in untreated controls, n=6–9, P<0.0001, Student’s t-test). Similar reductions were observed during remission at 28 days after immunization (76±1 mg dl−1±s.e.m., n=18, P<0.0001 Student’s t-test).

Next, we asked whether dietary cholesterol supplementation worsens acute inflammatory disease. Unexpectedly, mice on a high-cholesterol chow (5% w/w cholesterol, fat content unchanged) either prophylactically, two weeks before inducing MOG-EAE, or therapeutically with onset of clinical symptoms, showed similar disease onset (normal chow 12.6±0.3 days; cholesterol 12.6±0.4d, n=12–16), mean clinical scores and body weight, as controls, during the 28 days of monitoring (Fig. 1a,b; Supplementary Fig. 1). Moreover, high-cholesterol chow did not correct the reduced serum cholesterol (77±8 mg dl−1, n=6). Correspondingly, at the peak of the clinical symptoms, dietary cholesterol did not influence the level of inflammation: histopathological lesions in the lumbar spinal cord white matter as well as the immune cell infiltration and characteristics of the pro-inflammatory milieu were comparable in extent and composition (Fig. 1c; Supplementary Fig. 2). These findings are in agreement with dietary cholesterol supplementation in the Theiler’s virus model of MS (ref. 19). Nonetheless, inflammation was slightly ameliorated in cholesterol fed animals in remission, 28d after immunization (Fig. 1, Supplementary Fig. 2). Reduced infiltration of T cells and microglia/macrophages was accompanied by attenuated expression of several pro-inflammatory markers, such as interferon-γ (IFNγ), interleukin 17 (IL-17), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor (TNF), and major histocompatibility complex II (MHCII). Taken together, EAE is associated with decreased serum cholesterol that is not restored by supplemented cholesterol. Importantly, cholesterol does not exacerbate disease but even slightly ameliorates inflammation during remission, suggesting it is safe to administer in inflammatory diseases. As cholesterol supplementation promotes developmental myelination18, these data prompted us to examine cholesterol supplementation in a remyelination paradigm.

Figure 1: Dietary cholesterol does not aggravate EAE pathology. (a) Clinical score of mice with MOG-EAE on normal chow or chow supplemented with 5% cholesterol (n=12–16 mice, 2 independent experiments). Start of cholesterol feeding was prophylactic, two weeks before immunization. Arrows illustrate the time points of analyses at the peak of clinical symptoms (16–18 dpi) and at remission (28 dpi). (b) Body weight of experimental animals as in (a) assessed from the day of induction of EAE to the end of monitoring clinical scores (28 days). Data is expressed as mean weight±s.e.m. of n=12–16 animals. Onset of clinical symptoms was paralleled by a drop in body weight, and mice gained weight only after the peak of disease. (c) Lesion characteristics were determined on sections of lumbar spinal cord from mice fed normal chow or cholesterol enriched chow (n=5 animals, representative images on the left, scales 200 μm). Luxol fast blue-periodic acid-Schiff-hematoxylin (LFB/PAS) staining was used to determine the lesion area and number of lesions per section (arrow). Immuno-labeling for myelin basic protein (MBP) was used to determine the per cent of myelinated area within a lesion (defined in the DAPI channel as clusters of >20 nuclei, marked by arrows). On sections immuno-labeled for APP, the number of axonal speroids (arrows) per square mm white matter area was counted, as a readout of axonal damage. In remission, unpaired Student’s t-test revealed significantly less axonal damage in cholesterol fed animals. Sections triple stained for microglia/macrophages, T cells, and astrocytes (Iba1-CD3-GFAP triple immuno-labeling) were used to assess the cellular composition of lesions. Unpaired t-tests revealed significantly reduced densities of microglia/macrophages and T cells in cholesterol fed animals (*, P<0.05). Bars represent mean values with individual data points. Full size image

Cuprizone lowers serum cholesterol and affects BBB integrity

We first tested whether serum cholesterol was altered in the cuprizone model of demyelinating disease (see also below). Surprisingly, after 4 weeks on cuprizone, mice had markedly reduced total serum cholesterol (76±3 mg dl−1±s.e.m. in comparison to 103±3 mg dl−1 in controls, n=9–13, P<0.0001, Student’s t-test). Although liver function values were normal (Supplementary Fig. 3), we cannot exclude the possibility that this is due in part to altered liver metabolism20. In contrast to EAE, dietary supplementation with 2% w/w cholesterol normalized total serum cholesterol (106±5 mg dl−1, n=13).

Under physiological conditions, the blood-brain barrier (BBB) prevents the passage of cholesterol from the circulation into the CNS (refs 21, 22). Therefore, we tested whether dietary cholesterol could penetrate the CNS in cuprizone fed mice. Surprisingly, BBB integrity was compromised in mice treated with cuprizone for 4 weeks, as indicated by extravasation of Evans blue dye into the CNS, following systemic administration (Fig. 2a,b). Systematic evaluation revealed increased BBB permeability during the entire treatment period of up to 12 weeks of cuprizone feeding (1.4±0.1 fold, n=4 P<0.05 Student’s t-test).

Figure 2: Increased BBB permeability in cuprizone treated mice. (a) Extravasation of Evans blue on sections of the corpus callosum. In control animals, Evans blue fluorescence is restricted to blood vessels but extravasates in mice on cuprizone (arrows) (scale, 50 μm). (b) BBB permeability was measured by Evans blue (EB) extravasation in brains of animals fed cuprizone (cup) for 5 weeks on normal chow or cholesterol supplemented chow, or in brains of animals with EAE 2d after the peak of clinical symptoms (n=3 animals). All treatment groups were normalized to untreated control animals (n=5) and compared by one way ANOVA (P<0.0001). Nutritional cholesterol did not influence BBB permeability. Bars represent mean±s.e.m. (c) Extravasation of bodipy-cholesterol. Maximum intensity projection of bodipy-cholesterol fluorescence in the corpus callosum (delineated by dashed lines) of mice that were kept on cuprizone for 5 weeks in comparison to untreated mice (control) (scale, 50 μm). (d) Quantification of bodipy-cholesterol extravasation after extraction. Data are expressed as fold changes±s.e.m. in cuprizone treated mice compared with untreated control animals (n=6 mice per group, unpaired Student’s t-test, P<0.0001). Full size image

The extent of extravasation was much smaller than in EAE, likely explaining why previous studies have missed this BBB defect23,24,25,26. Notably, dietary cholesterol did not influence BBB permeability. When tested one week after a single injection of bodipy-cholesterol, the fluorescence from this cholesterol derivative (its biophysical properties are very similar to unmodified cholesterol27) was readily detectable in the corpus callosum of cuprizone fed mice (in contrast to untreated controls) with a pattern typical for an intracellular localization, potentially in glial cells (Fig. 2c). Quantification of extravasated bodipy-cholesterol revealed a ∼3-fold increase in comparison to control mice (Fig. 2d). Thus, in cuprizone fed mice, peripheral cholesterol can cross the BBB.

Cuprizone mediated demyelination is unaltered by cholesterol

Next we tested whether nutritional cholesterol altered histopathology during the demyelination phase of cuprizone treatment (Fig. 3a)25,28. In the corpus callosum, oligodendrocyte loss and demyelination evolved over the same time course in control and cholesterol supplemented mice (Fig. 3b), leading to almost complete depletion of mature oligodendrocytes after four weeks. In addition, oligodendroglial numbers (Olig2, oligodendrocyte lineage transcription factor 2 marks OPCs and oligodendrocytes), astrogliosis (GFAP, glial fibrillary acidic protein) and microgliosis (MAC3, macrophage-3 antigen) steadily increased in a comparable manner in both groups, and axonal damage (APP positive spheroids, Fig. 3c) was similar at all time points tested. Taken together, cholesterol supplementation does not interfere with the cuprizone treatment, and mature oligodendrocytes do not escape the toxic insult.

Figure 3: Cholesterol does not affect cuprizone mediated demyelination. (a) Scheme depicting the time course of demyelination/remyelination during 6 week cuprizone feeding (upper panel, based on own results and on other studies25,28) to the treatment paradigm. To assess the influence of high-cholesterol feeding on demyelination, mice on normal chow or high-cholesterol chow additionally received cuprizone in the diet for between 2 and 5 weeks (black bars) after which mice were analysed histologically. (b) Representative pictures of the corpus callosum of untreated control mice and mice after 5 weeks on cuprizone with the corresponding quantification on the right. Assessed were myelination (Gallyas silver impregnation), the number of mature oligodendrocytes (CAII), the number of oligodendrocyte lineage cells (Olig2), activated microglia (MAC3) and astrocytes (GFAP). Each bar represents the mean value for 3–5 (week 2–4) or 9–10 (week 5; untreated controls, ctrl) animals per condition with individual data points (scale 100 μm). (c) APP positive spheroids per mm2 in the corpus callosum at the end of 2–5 weeks of cuprizone with or without cholesterol supplementation (n=3–4 animals at 2 and 3 weeks, n=4–5 at week 4, n=6 untreated controls, n=9–10 at week 5). Full size image

Cholesterol facilitates remyelination and motor learning

Next, we tested the hypothesis that dietary cholesterol supplementation enhances adult remyelination. When mice are continuously exposed to cuprizone, an episode of spontaneous repair occurs in the sixth week, resulting in marked remyelination (Fig. 4a)25,29. At this time point, cholesterol neither influenced oligodendrocyte numbers, remyelination nor glial responses (Fig. 4b,c). However, the density of APP positive axonal spheroids in cholesterol fed animals was reduced, suggesting attenuated axonal damage (Fig. 4d).

Figure 4: Cholesterol facilitates remyelination after chronic cuprizone exposure. (a) Scheme depicting the time course of demyelination/remyelination during cuprizone feeding to the treatment paradigm. To assess the influence of high-cholesterol feeding on spontaneous remyelination, mice received cuprizone in normal chow or chow supplemented with cholesterol for 5, 6 or 12 weeks (black bars) after which mice were analysed histologically. (b) Evaluation of disease in the corpus callosum of mice that were treated with cuprizone for 5, 6 or 12 weeks on normal chow or chow enriched with cholesterol. Corresponding representative pictures of the 12 weeks treatment cohort are on the left. Assessed were myelination (Gallyas silver impregnation), the number of mature oligodendrocytes (CAII), the number of oligodendrocyte lineage cells (Olig2), activated microglia (MAC3) and astrocytes (GFAP). Each bar represents the mean value of 4 (week 12) or 8–10 (week 5, 6) animals per condition with individual data points (scale 100 μm). (c) Myelinated axons per 10 μm2 in the corpus callosum at the end of 6 and 12 weeks of cuprizone with or without cholesterol supplementation (n=4 animals, Two-way ANOVA and Sidak’s post test). (d) APP positive spheroids per mm2 in the corpus callosum at the end of 6 and 12 weeks of cuprizone with or without cholesterol supplementation (n=3–8 animals, Two-way ANOVA and Sidak’s post test). Asterisks represent significant differences with *P<0.05; **P<0.01; ****P<0.0001. Full size image

After chronic cuprizone exposure (12 weeks), a second episode of weak and transient remyelination (up to 20% of full myelination) occurs (Fig. 4a). However, even if cuprizone is withdrawn at this point, repair is very limited30. Thus, despite a considerable density of OPCs and mature oligodendrocytes, remyelination is marginal and astrogliosis substantial (Fig. 4b, blue bars at 12 weeks). Remarkably, cholesterol supplementation increased remyelination ∼1.6-fold as assessed in Gallyas silver impregnated sections (Fig. 4b) and in electron micrographs of the corpus callosum (Fig. 4c, Supplementary Fig. 4). Coupled to this, a similar increase in OPCs and in mature oligodendrocytes was observed (Fig. 4b, 12 weeks). In addition, the positive influence of cholesterol was associated with increased body weight (Supplementary Fig. 5). Thus, in the context of recurrent depletion of mature oligodendrocytes, cholesterol supplementation enhances tissue repair.

To specifically determine the effect of cholesterol during remyelination, we exposed mice to cuprizone for four weeks to achieve complete demyelination, then withdrew cuprizone to induce remyelination (‘induced remyelination’) (Fig. 5a). Mice fed normal chow during the first 7 days after cuprizone withdrawal demyelinated further and had only slightly increased oligodendrocyte densities (Fig. 5b, compare blue bars 4 and 4+1). In contrast, cholesterol supplementation following cuprizone withdrawal dramatically increased OPC proliferation and augmented Olig2 positive cell density 1.5-fold (Fig. 5b,c). Densities of newly differentiated TCF4+ PCNA− (TCF4, also called TCF7L2, transcription factor 7-like 2; PCNA, proliferating cell nuclear antigen) oligodendrocytes were also increased by cholesterol (Fig. 5d, Supplementary Fig. 6), similarly as found in actively repairing lesions from patients with MS31,32,33. The resulting 2.7-fold increase in mature oligodendrocytes (Fig. 5b, time point 4+1) led to a 1.8-fold increase in myelin content on Gallyas silver impregnated sections and on electron micrographs (Fig. 5e; Supplementary Fig. 4). Cholesterol supplementation also altered the glial response, leading to a ∼30% increase in astrocytes and ∼50% reduction in microglial cells (Fig. 5b, 4+1). Axonal damage was attenuated to ∼70% in cholesterol fed animals of controls (Fig. 5f). These histological signs of repair were associated with a net gain in body weight, occurring within 7 days of cholesterol supplementation and contrasting with weight maintenance in mice fed normal chow (Supplementary Fig. 5). The beneficial effect of cholesterol persisted, leading to a robust increase in mature oligodendrocytes and myelin content at 2 weeks after cuprizone withdrawal (Fig. 5b, 4+2); a result that was confirmed on electron micrographs (Fig. 5e).

Figure 5: Cholesterol facilitates remyelination after cuprizone withdrawal. (a) Scheme depicting the time course of demyelination/remyelination during cuprizone feeding (remyelination after cuprizone withdrawal in purple). The influence of cholesterol on remyelination was assessed by feeding mice cuprizone in normal chow for 4 weeks (4, black bars) followed by ‘induced remyelination’ after cuprizone withdrawal for 1 (4+1) or 2 (4+2) weeks on normal chow or cholesterol supplemented chow. (b) Representative pictures of the corpus callosum of mice after one week (4+1) remyelination. Corresponding quantification is on the right also including values for 2 weeks remyelination (4+2). Assessed were myelination (Gallyas silver impregnation), the number of mature oligodendrocytes (CAII), the number of oligodendrocyte lineage cells (Olig2), activated microglia (MAC3), and astrocytes (GFAP). Each bar represents the mean value from n=4–5 (4 and 4+2) or n=7 (4+1) animals (scale, 100 μm; Two-way ANOVA and Sidak’s post test). (c) Quantification of proliferating OPCs (PCNA positive Olig2 positive) in the corpus callosum of mice after 4+1 treatment paradigm (4+1) or after 12 weeks (12) of cuprizone. Each bar represents the mean of n=6–7 (week 4+1), or n=4 (week 12) animals (Student’s t-test). (d) Quantification of newly differentiated postmitotic oligodendrocytes (TCF4 positive, PCNA negative) in the corpus callosum treated as in c). Each bar represents the mean of n=6–7 (week 4+1), or n=4 (week 12) animals (Student’s t-test). (e) Myelinated axons per 10 μm2 in the corpus callosum at the end of the 4+1 (n=7) and 4+2 (n=4) treatment paradigm (two-way ANOVA and Sidak’s post test). (f) APP positive spheroids per mm2 in the corpus callosum (4+1 n=7; 4+2 n=3–4 animals, two-way ANOVA and Sidak’s post test). (g) Motor learning as assessed by maximum velocity (Vmax) on a complex wheel (n=6 animals), expressed as per cent of the Vmax on a training wheel (mean of the last 7 days before changing to a complex wheel). Statistical evaluation of Vmax was done by Two-way ANOVA (cholesterol effect P<0.0001) and Sidak’s post tests. Asterisks represent significant differences with *P<0.05; **P<0.01; ***P<0.001. Full size image

To examine the generality of this response, we investigated whether dietary cholesterol enhanced remyelination in another, completely distinct in vivo model of remyelination that is accompanied by confined BBB disruption. Localized injection of lysolecithin into the ventral-lateral spinal cord of adult mice was used to produce focal demyelination. As in the cuprizone model, demyelination was associated with a reduction in serum cholesterol to about 70% of untreated controls. Further, dietary cholesterol (2% w/w for 14 days) increased serum cholesterol slightly (79±3 mg dl−1±s.e.m. in cholesterol fed mice compared with 72±6 mg dl−1 in chow fed controls, n=3–5), enhanced remyelination and significantly increased the density of oligodendroglial cells within the lesion (Fig. 6a–c). The beneficial effect of cholesterol was also reflected in significantly increased body weight, relative to chow fed mice (Fig. 6d).

Figure 6: Cholesterol supports remyelination in the lysolecithin model. (a) Representative images of spinal cord sections 14 days post lesion (dpl) with 1 μl 1% lysolecithin in the ventral spinal cord with quantification of n=5 (cholesterol chow) and n=6 (normal chow) animals. Student’s t-tests revealed significantly more Olig2 positive oligodendroglial cells within the lesion area (P<0.0001), and significantly more MBP positive area (P=0.027; scales, 100 μm). (b,c) Representative electron micrographs (scale 1 μm) and quantification of myelin sheath thickness and the portion of remyelinated axons in control and cholesterol fed mice at 14 dpl by g-ratio analysis (n=3 animals per group). (d) Body weight of experimental animals assessed at the day of lesion (day 0), after 7d and after 14d at the end of the experiment. Shown are the means±s.e.m. of n=9 (chol chow) to 10 (normal chow) animals. Two-way ANOVA with Sidaks post tests revealed a significant influence of cholesterol feeding at both time points (7 dpi P<0.0003, 14 dpi P=0.0362). Full size image

To investigate whether the histopathological improvements in cholesterol fed animals was associated with improved clinical measures, we returned to the ‘induced remyelination’ paradigm in the cuprizone model (for a scheme of experimental paradigm, see Supplementary Fig. 7), measuring the maximum running velocity (Vmax) on a running wheel. First, a training wheel with regularly spaced rungs was placed into the cages to improve cardiopulmonary and musculoskeletal strength. One week after cuprizone withdrawal, the training wheel was replaced by a complex wheel with irregularly spaced rungs to measure bilateral sensorimotor coordination34 (Supplementary Fig. 7). The Vmax of mice remyelinated on normal chow dropped to about 40% of levels on the training wheel, and did not improve above 75% (Fig. 5g). In contrast, mice receiving cholesterol supplementation showed a less severe drop in Vmax (to 63%), followed by a steady increase that reached the velocity achieved on the training wheel after two weeks. Importantly, in control mice (without cuprizone) cholesterol supplementation, did neither influence performance on the training wheel nor motor learning (Vmax, run duration, number of runs and running distance on the complex wheel) (Supplementary Fig. 7 and not shown). Hence, cholesterol supplementation enhances repair after demyelination and improves neurological outcomes by supporting oligodendrocyte proliferation and differentiation, promoting remyelination, decreasing microgliosis, and attenuating axonal damage in a permissive environment (‘induced remyelination’ after cuprizone withdrawal).

Cholesterol changes the expression profile of growth factors

To obtain insight into the mechanism by which cholesterol supports the simultaneous expansion of OPC and oligodendrocyte densities, we monitored differentiation of cultured primary oligodendrocytes in defined Sato media, with or without cholesterol supplementation. Oligodendrocytes differentiated significantly faster in the presence of cholesterol, as indicated by expression of differentiation markers and morphological changes (Fig. 7a, Supplementary Fig. 8a). However, the final stage of maturation after 5d in culture was unchanged, as shown previously18. Similarly, the rate of myelination as measured by MBP (myelin basic protein) positive area per axonal area (SMI31, phosphorylated axonal neurofilaments), was increased in spinal cord co-cultures differentiated in the presence of cholesterol (Fig. 7b, Supplementary Fig. 8b); neither the final degree of neurite outgrowth35 nor myelination, were influenced by cholesterol. These findings suggest that external cholesterol directly facilitates oligodendrocyte differentiation and the synthesis of myelin membranes.

Figure 7: Cholesterol alters the expression profile of growth factors. (a) Differentiation time course of OPCs in oligodendroglial enriched cultures in the presence or absence of cholesterol supplementation (bars represent mean of n=4 cultures with individual data points). Drawings illustrate chosen categories of oligodendrocyte differentiation. In each category, significance was assessed by two-way ANOVA and Sidak’s post tests. (b) Myelination at 20–28 days in vitro (DIV) in myelinating cocultures in the presence or absence of cholesterol (n=5–9 cultures). Myelin segments and axons were counted (see Supplementary Fig. 8b; two-way ANOVA and Sidak’s post tests). (c–h) Quantitative RT-PCR analysis on dissected corpus callosi from mice after ‘induced remyelination’ (4+1 weeks) and controls determining the expression of oligodendrocyte and myelin related genes (c; Car2, Plp1, Olig2), marker genes for microglia (Aif1) and astrocytes (Gfap) (d), genes involved in cholesterol synthesis (e; Hmgcr, Fdft1, Srebf2) and uptake (f; Ldlr, Lrp1), and growth factors downregulated (g; Pdgfa, Fgf2) and upregulated by cholesterol supplementation (h; Fgf1, Fgf9, Fgf12, Shh, Fgf17, Fgf22). Bars represent the means (n=4 animals) with individual data points (Student’s t tests) normalized to untreated control mice (set to 1, grey line). (i) Differentiation of rat oligodendroglial cells in cultures supplemented with FGF1 and FGF2 (concentrations in ng per ml as indicated) in the presence or absence of cholesterol. Bars represent mean percentage of cells in each category of n=3 cultures (two-way ANOVA with Sidak’s post test). (j) Proliferation of OPCs in response to growth factors and cholesterol. OPCs were cultured in the presence or absence of the growth factors (100 ng ml−1) FGF1 or FGF2 with or without cholesterol for 24 h. Data are mean EdU positive cells of all oligodendroglial cells±s.e.m. (n=13 (no GF, FGF2) or n=7 (FGF1) cultures of individual rats; Student’s t-tests). (k) Quantitative RT-PCR on primary astrocytes treated with cuprizone (cup) with or without cholesterol (chol) supplementation. Bars represent the mean of n=3 independent experiments with individual data points compared with untreated cultures (set to 1, grey line; one-way ANOVA with Sidak’s post tests. Asterisks represent significant differences with *P<0.05; **P<0.01; ***P<0.001. Full size image

In principle, a substantial induction of OPC differentiation could be unfavourable, if it occurs at the expense of OPC numbers. Indeed, gradual depletion of OPCs was observed in cholesterol supplemented oligodendroglial cultures (Fig. 7a, left panel). Thus, the expansion of proliferative OPCs in vivo (Fig. 5c) is likely an indirect consequence of additional factors from the local environment. To identify factors that mediate cholesterol dependent OPC proliferation, we analysed another cohort of mice in the ‘induced remyelination’ treatment paradigm (4+1 weeks), using quantitative RT-PCR on dissected corpus callosi. In agreement with our histological data, oligodendrocyte related genes were (i) strongly downregulated in cuprizone fed mice in comparison to untreated controls (grey line) and (ii) significantly enhanced in cholesterol fed animals in comparison to chow fed animals (Fig. 7c, compare Fig. 5, Supplementary Table 1). Similarly, the astrogliosis (Gfap) and diminished microgliosis (Aif1, allograft inflammatory factor 1) were also reflected in the expression levels of respective marker genes (Fig. 7d). Surprisingly, cholesterol supplementation did not lead to feedback inhibition of cholesterol synthesis, but rather, increased the expression of genes involved in cholesterol synthesis and uptake (Fig. 7e,f), likely indicating enhanced remyelination. In contrast, expression of LXR family genes, which influence OPC differentiation36, was not affected by cholesterol (Supplementary Table 1).

The expression of growth factors involved in OPC survival, proliferation, migration or differentiation28, including Igf1 (insulin-like growth factor), Cntf (ciliary neurotrophic factor), Inhba (inhibin beta-A, also called activin beta-A) and Egf (epidermal growth factor) was strongly increased (2–20 fold) by cuprizone, but was not further regulated by cholesterol supplementation (Supplementary Table 1). A set of genes whose products are known to inhibit differentiation of OPCs, such as Fgf2 (fibroblast growth factor 2) and Pdgfa (platelet derived growth factor alpha)37,38,39, was also strongly upregulated by cuprizone (8–12 fold higher than untreated controls). Strikingly, the expression of these mitogens was attenuated in cholesterol fed animals to levels only 3–8 fold higher than in untreated controls (Fig. 7g). Moreover, in comparison to untreated controls, expression of another set of factors, some of which are known to facilitate differentiation of oligodendrocytes39,40, such as Fgf1 and Shh (sonic hedgehog), was reduced by cuprizone, but strongly elevated by cholesterol supplementation (Fig. 7h). Expression of FGF receptors (1–3) was not influenced by cholesterol (data not shown). Demonstrating the generality of these findings (Supplementary Table 2), cholesterol influenced the profile of growth factor expression in a similar manner in mice treated chronically with cuprizone (12 weeks, see Fig. 4). In contrast to the ‘induced remyelination’ paradigm, expression of enzymes involved in cholesterol synthesis was reduced in this cohort, suggesting feedback inhibition after remyelination is accomplished (Supplementary Table 2).

To determine whether the growth factor expression profile observed in cholesterol treated mice might be causally related to the enhanced repair, we tested whether these growth factor combinations directly enhance OPC differentiation in vitro, a surrogate for remyelination in vivo. Indeed, differentiation was enhanced when OPCs were cultured for 3 days in media supplemented with 90 ng ml−1 FGF1, 35 ng ml−1 FGF2 and cholesterol (exemplifying cuprizone+cholesterol chow), in comparison to 45 ng ml−1 FGF1 and 80 ng ml−1 FGF2 (exemplifying cuprizone+normal chow) (Fig. 7i). These data suggest the changes in growth factor expression are directly contributing to the improved repair.

Next, we cultured OPCs for 24 h in the presence of EdU (5-ethynyl-2′-deoxyuridine), a marker of cells in S-phase of the cell cycle, to determine whether the proliferative effect of growth factors was modified by cholesterol. Compared with vehicle treated controls, FGF2 doubled the number of EdU positive cells (as expected41), while FGF2 plus cholesterol elicited a threefold increase in this population (Fig. 7j), suggesting that cholesterol potentiates the effects of FGF2. Indeed, cholesterol alone only slightly increased the proportion of EdU+ cells in these cultures (Fig. 7j). We speculate that, despite attenuated Fgf2 expression (compare Fig. 7g), potentiated FGF2 signalling contributes to the expansion of proliferating OPCs in cholesterol fed animals (compare Fig. 5c).

As only relatively few microglial cells are present in the corpus callosum of cholesterol fed mice in the ‘induced remyelination’ paradigm (4+1 weeks) and in the ‘chronic cuprizone’ paradigm (12 weeks), we hypothesized that astrocytes contributed principally to the altered profile of growth factors. Indeed, while primary astrocytes downregulated Fgf1 expression in response to cuprizone, its expression was upregulated in response to cholesterol, irrespective of cuprizone (Fig. 7k), correlating with our in vivo data. Taken together, in the cuprizone model, cholesterol supplementation modulates the expression profile of growth factors, rebalancing proliferative and differentiation signals creating a permissive environment for repair.