Multiple Sclerosis (MS) is an inflammatory demyelinating disorder in which remyelination failure contributes to persistent disability. Cholesterol is rate-limiting for myelin biogenesis in the developing CNS; however, whether cholesterol insufficiency contributes to remyelination failure in MS, is unclear. Here, we show the relationship between cholesterol, myelination and neurological parameters in mouse models of demyelination and remyelination. In the cuprizone model, acute disease reduces serum cholesterol levels that can be restored by dietary cholesterol. Concomitant with blood-brain barrier impairment, supplemented cholesterol directly supports oligodendrocyte precursor proliferation and differentiation, and restores the balance of growth factors, creating a permissive environment for repair. This leads to attenuated axon damage, enhanced remyelination and improved motor learning. Remarkably, in experimental autoimmune encephalomyelitis, cholesterol supplementation does not exacerbate disease expression. These findings emphasize the safety of dietary cholesterol in inflammatory diseases and point to a previously unrecognized role of cholesterol in promoting repair after demyelinating episodes.
In demyelinating diseases such as multiple sclerosis (MS), the failure to remyelinate contributes to axonal damage1, a major factor in persistent disability. Remyelination failure can be attributed partially to an insufficient capacity of resident oligodendrocyte precursor cells (OPC) to proliferate, migrate, differentiate and initiate myelin membrane growth2,3. There is now good evidence to implement therapies that combine the established immunosuppressive treatment of MS with compounds that stimulate remyelination and hence may secondarily limit axonal damage4,5. A number of factors that support differentiation of OPCs have been reported recently, some of which are linked to cholesterol metabolism in differentiating oligodendrocytes6,7,8,9.
Patients with MS have disturbed brain lipid metabolism10, but serum lipid profiles are in the normal range11. During active disease and disease progression, total cholesterol levels can rise to the upper limit of the normal range12,13,14,15. Increased dietary intake of cholesterol is assumed to increase serum cholesterol and stimulate immunological responses in inflammatory diseases16. However, it is unclear whether the elevated serum cholesterol in MS patients (i) contributes to disease progression, (ii) is a consequence of acute disease or (iii) reflects an attempt to counterbalance the pathophysiological manifestation of the disease.
We previously showed that cholesterol is rate limiting for CNS myelination17 and that nutritional cholesterol supplementation can stimulate developmental myelination in a mouse model of leukodystrophy18. Here, we investigate the effects of increased dietary cholesterol on disease parameters in three distinct mouse models of MS, that is, on (i) inflammation and demyelination in experimental autoimmune encephalomyelitis (EAE), (ii) remyelination in lysolecithin induced lesions and (iii) demyelination and remyelination in the cuprizone model. High-cholesterol chow does not aggravate clinical symptoms nor inflammatory parameters in EAE or alter demyelination in cuprizone treated animals. Rather, we identify a novel function for cholesterol in myelin repair in adult mice. Dietary cholesterol modulates the profile of growth factors, simultaneously enhancing OPC proliferation and oligodendrocyte differentiation, thereby facilitating remyelination and reducing axonal injury. These data have implications for the treatment of demyelinating diseases.
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.
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).
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.
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).
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).
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).
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.
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.
Cholesterol availability is a prerequisite for myelination17,42,43 and, as we show here, exogenous cholesterol directly increases the rate of OPC differentiation. In agreement with our findings, failure to upregulate expression of sterol synthesis enzymes leads to arrested differentiation of Tcf4 mutant OPCs, which can partially be rescued by cholesterol supplementation44. Further, cholesterol synthesis is enhanced during remyelination in mice29 and statin administration (inhibitors of sterol and isoprenoid synthesis) interferes with remyelination in the cuprizone model45. Nonetheless, monotherapy with statins ameliorates clinical scores in EAE; an effect associated with decreased CNS infiltration and inflammatory activity of T cells, likely reducing demyelination46,47,48. The outcomes of studies using statins in MS patients are contradictory, probably because of the disparate effects of statins on inflammation (beneficial46,47,48) and on remyelination (detrimental45). Accordingly, a recent meta-analysis does not recommend statin treatment for relapsing-remitting MS or clinically isolated syndrome49. Hence, we hypothesize that remyelination failure in MS reflects, at least partially, the inability to locally increase the cholesterol content in demyelinated lesions.
This hypothesis is supported by the current study. Exogenous cholesterol enters the CNS through an impaired blood-brain barrier, resulting in enhanced repair and an amelioration of the neurological phenotype in two distinct models of remyelination. Our data suggest that cholesterol directly facilitates repair by modulating the profile of growth factor expression, promoting OPC differentiation and, together with the mitogen FGF2, potentiating OPC proliferation. Importantly, cholesterol supplementation does not exacerbate inflammation in EAE.
What could prevent the increase of cholesterol in OPCs in demyelinated lesions? In patients with MS and in models of demyelination, CNS cholesterol homoeostasis is destabilized by a variety of mechanisms. First, expression of enzymes involved in cholesterol synthesis is reduced in demyelinated lesions (our study and refs 9, 29, 50). Second, intercellular cholesterol transport in the CNS is perturbed in patients with MS, because of reduced abundance of relevant proteins such as ApoE (Apolipoprotein E)10; and in mouse mutants with BBB disruption22, by uncontrolled flux of sterols in and out of the brain. BBB disruption has been shown by diffusion MRI in inflammatory diseases of the brain, such as MS (ref. 51) and, as we demonstrate here for the first time, is also a feature in the cuprizone model. Third, the decrease in serum cholesterol in both EAE and cuprizone mouse models, probably contributes to the impairment of CNS cholesterol homoeostasis. Whether patients with MS experience a drop in serum cholesterol during acute demyelinating episodes is unknown, and its analysis complicated by the fact that the standard first-line interferon beta treatment itself reduces total serum cholesterol52. Finally, the OPCs in chronically demyelinated lesions in MS50 and mouse models29, fail to upregulate lipid synthesis and differentiate, potentially as a consequence of an imbalance in signalling, as previously hypothesized4. Here, we demonstrate an imbalance in expression of growth factors in the cuprizone model, in accordance with previous studies28,53,54,55. The growth factor profile associated with cuprizone alone, such as high levels of FGF2 and PDGFa, is predicted to facilitate OPC proliferation but impede efficient remyelination, particularly after chronic demyelination.
Specifically, FGF signalling could critically influence the fate of demyelinated lesions39,53,56,57. FGF signalling comprises a very complex network, including 24 FGF family members and four different receptors, whose signalling outcome depends on various splice isoforms and on the multifaceted crosstalk between different pathways58. Here, we focused on the two major FGF members involved in myelination, FGF1 and FGF2. FGF2 has been implicated in OPC proliferation, migration and inhibition of oligodendrocyte differentiation38,39,41. In the cuprizone model and in patients with MS, FGF2 abundance correlates with the degree of OPC proliferation28,53,56,59. FGF2 is increased in regions of active OPC proliferation and ongoing remyelination, such as active lesions or the rim of demyelinated lesions, while it is downregulated in remyelinated shadow plaques, and it is low in abundance in normal appearing white matter and in the core of demyelinated silent lesions59. We show that cholesterol administration attenuates the overexpression of Fgf2 and other mitogens in the cuprizone model. Surprisingly, this did not restrict proliferation but augmented OPC numbers, likely through synergy with cholesterol (see below).
FGF1 is reduced in active MS lesions60, and increased expression is only found in remyelinated lesions56. In contrast to FGF2, FGF1 is not mitogenic for OPCs (our study and refs 39, 56). Rather, FGF1 accelerates myelination in vitro56, to an extent remarkably similar to what we observed in cholesterol treated cultures. FGF1 might support CNS repair by inducing lipid synthesis and secretion by astrocytes61. The crosstalk between FGF signalling and regulation of cholesterol metabolism is supported by the presence of sterol responsive elements (consensus sequences for the SREBF2 transcription factor that increases cholesterol synthesis) in the Fgf1 and Fgf2 promoters (https://www.genomatix.de/). Altered cellular cholesterol levels can modulate signalling pathways, as shown for wnt signalling; the cholesterol content regulates the recruitment of different sets of scaffolding/adaptor proteins to the plasma membrane62. We demonstrate that cholesterol induces the expression of Fgf1 in astrocytes; however, the affected signalling remains enigmatic. In addition to astrocytes, other cells such as microglia, OPCs, neurons and vascular cells likely participate in growth factor synthesis. In cholesterol treated mice, the expression profile of growth factors was altered such that the mitogens FGF2 and PDGFα were attenuated and differentiating cues such as FGF1 and Shh were enhanced. Other factors that are unaffected by cholesterol probably also contributed to the repair process.
Our data reveal a previously unknown function of nutritional cholesterol in adult remyelination (Fig. 8 shows a working model). In response to cuprizone-mediated demyelination in mice, the secreted mitogens and growth factors favour the proliferation and oppose the differentiation of OPCs, which slows and ultimately impairs remyelination. Dietary supplementation increases cholesterol availability within the demyelinated CNS and this is associated with rebalancing of growth factor expression. The altered profile of growth factors in cholesterol treated mice simultaneously facilitates OPC proliferation and oligodendrocyte differentiation in vivo. Thus arrested repair can be overcome by increasing the local availability of cholesterol which we achieved by nutritional supplementation.
We envision that moderate concentrations of the mitogen FGF2, when in synergy with cholesterol, potentiate OPC proliferation, and at the same time opens a window for OPC differentiation that is also enhanced by increased levels of pro-differentiation factors. In addition, cholesterol might directly facilitate oligodendrocyte differentiation, by relieving cells of the burden of establishing the complex time- and energy-intensive anabolic cholesterol pathway. Supplemented cholesterol can directly support myelination by incorporation into myelin membranes, as shown previously in a leukodystrophy model18. The current study suggests that cholesterol provides a ‘fast track’ to remyelination and repair.
In contrast to the beneficial effect on remyelination, high-cholesterol chow (2% cholesterol) has no effect on demyelination and oligodendrocyte survival in the cuprizone model, likely because oligodendrocyte loss is induced by direct cuprizone-mediated damage, and is concomitant with a low-grade inflammatory cascade involving T cells, astrocytes and microglia63. Consistent with cholesterol supplementation not exacerbating demyelination in the cuprizone model, high-cholesterol chow (5% cholesterol) did not aggravate disease in EAE. Moreover, cholesterol supplementation attenuated axonal damage in both models; during active remyelination in the cuprizone model and during remission in EAE. Likely, this is secondary to ameliorated disease states and balanced expression of growth factors and pro-inflammatory factors. We found that dietary cholesterol slightly ameliorated inflammation in EAE, while, in contrast, a high-fat chow aggravates EAE symptoms64,65. Medium and long chain fatty acids of the high-fat chow probably modulate T cell differentiation66. Whether fatty acids contribute to the increased disease activity in some MS patients with elevated serum cholesterol remains unclear12. Importantly, feeding cholesterol appears to be safe in mice with inflammatory disease.
Taken together, our data show that demyelinating disease destabilizes peripheral and CNS cholesterol homoeostasis. Dietary cholesterol supplementation supports cholesterol metabolism in the CNS and has the remarkable potential to ameliorate disease by facilitating several repair mechanisms, leading to improved remyelination and neurological outcome. This study highlights the safety of dietary cholesterol and might have implications for the management of demyelinating diseases, but further studies, especially in combination with immune suppressive drugs, are required to determine its feasibility for patients.
All animal studies were performed in compliance with the animal policies of the Max Planck Institute of Experimental Medicine, and were approved by the German Federal State of Lower Saxony. Adult male C57BL/6N mice (8–10 weeks of age) were taken for all analyses. Animals were randomly assigned to an experimental group. Mice were fed normal chow (V1124 ssniff Spezialdiäten GmbH, Germany) or chow supplemented with either 2% w/w (cuprizone and lysolecithin experiments) or 5% w/w cholesterol (EAE experiments).
For MOG-EAE, mice purchased from Charles River were immunized subcutaneously with 200 μg myelin oligodendrocyte glycoprotein peptide 35–55 (MOG35–55) in complete Freund’s adjuvant (M. tuberculosis at 3.75 mg ml−1) and i.p. injected twice with 500 ng pertussis toxin as described67. Animals were examined daily and scored for clinical signs of the disease. If disease did not start within 15 days after induction or the clinical score rose above 4, animals were excluded from the analysis. The clinical score was: 0 normal; 0.5 loss of tail tip tone; 1 loss of tail tone; 1.5 ataxia, mild walking deficits (slip off the grid); 2 mild hind limb weakness, severe gait ataxia, twist of the tail causes rotation of the whole body; 2.5 moderate hind limb weakness, cannot grip the grid with hind paw, but able to stay on a upright tilted grid; 3 mild paraparesis, falls down from a upright tiled grid; 3.5 paraparesis of hind limbs (legs strongly affected, but move clearly); 4 paralysis of hind limbs, weakness in forelimbs; 4.5 forelimbs paralyzed; 5 moribund/dead. Mice received 5% cholesterol chow commencing either two weeks before immunization defined as prophylactic regimen or at the first appearance of EAE symptoms defined as therapeutic regimen and continued until day 28.
For cuprizone experiments, mice were fed 0.2% w/w cuprizone (Sigma-Aldrich Inc., Germany) in powder chow with or without cholesterol for ‘demyelination’ (2–5 weeks) and ‘chronic cuprizone’ (6 and 12 weeks) paradigms. For ‘induced remyelination’ experiments, mice were fed cuprizone in standard chow for 4 weeks, followed by cuprizone withdrawal and feeding mice standard chow with or without cholesterol supplementation. Mice were fed three times a week an exceeding amount of chow by dispenser. Food intake and animal weight was monitored. Age-matched untreated controls were fed standard powder chow.
Focal spinal cord demyelinating lesions were induced under anaesthesia by stereotactic injection of 1 μl lysolecithin (1%, from egg yolk, alpha-lysophosphatidylcholine, Sigma) into the ventro-lateral funiculus at Th10 of 8-week old animals, as previously described68. The injection was performed with a 10 μl Hamilton syringe, fitted with a thin tapered glass tip, at a rate of ∼1 μl min−1. This procedure created fusiform demyelinating lesions, 5–6 mm in length. At the day of injection, mice were randomly assigned to normal or 2% cholesterol chow for 14 days, after which the animals were killed, and the spinal cord processed for histology.
Bodipy-cholesterol injections were done as described18. Briefly, bodipy-cholesterol (Topflour, Avanti Polar Lipids) was injected i.p. (16 μg g−1 body weight). After one week, mice were perfused, and bodipy-cholesterol fluorescence was analysed on vibratome sections using a custom made two-photon laser scanning microscope equipped with a titanium-sapphire laser and a × 20 water immersion objective (NA 1.0). Z-stacks of 100-μm depth were obtained and processed to maximum intensity projections. For tracer quantification of bodipy-cholesterol (5 μg g−1 body weight, i.p. injection 7d circulation time) or Evans blue (50 μg g−1 body weight, i.v. injection, 4 h circulation time) animals were perfused with PBS to remove tracer from the circulatory system. Brains were dissected and immediately frozen on dry ice, weighed and stored at −80 °C for further processing. Tissue was lyophilized (Christ LMC-1 BETA 1-16) at –36 °C for 24 h under vacuum of 0.2 mBar. For tracer extraction, hemispheres were incubated shaking in 10 μl formamide per mg brain at 57 °C for 24 h. Integrated density of tracer fluorescence was determined in triplicates on a fluorescent microscope (Observer Z2, Zeiss, Germany), equipped with an AxioCam MRc3, × 1 Camera Adaptor and the ZEN 2012 blue edition software recorded at × 10 magnification (Plan-Apochromat × 10/0.45 M27). Tracer concentration was calculated using a standard curve and normalized to matched controls (set to 1).
Motor skill performance was assessed essentially as described34. Mice were randomly divided into two treatment and two control groups (n=6–13) and housed in individual cages that allow computer-controlled recording of wheel rotation as a function of time (MatLab-based custom software). The axis of each wheel was attached to a rotation sensor with a resolution of 16 per turn. One wheel revolution comes up to a running distance of 35.5 cm. The running wheel revolutions were recorded continuously at a sampling rate of 1/0.48 s by a customized recording device and software (Boenig & Kallenbach oHG, Dortmund, Germany). Mice in treatment groups were treated as in the ‘induced remyelination’ paradigm (feeding 4 weeks cuprizone in normal chow followed by withdrawal of cuprizone and feeding normal chow or cholesterol supplemented chow). Control animals received normal chow for the entire experiment or were switched to cholesterol chow after 4 weeks. One week after the start of the experiment training wheels with regularly spaced rungs were placed into the cages for adaptation of cardiopulmonary and musculoskeletal strength. One week after the switch of diets (week 5 of experiment), wheels were replaced by complex wheels with irregularly spaced rungs to assess the bilateral sensorimotor coordination that likely involves the cerebellum and motor cortex and connecting white matter such as the corpus callosum. Specifically, we measured maximum running velocity (Vmax), in addition to the total maximum run duration (Dmax), accumulative distance in metres (Distac) and the number of individual runs (Nrun). Parameters were logged once daily (12 am).
Animals were fasted for 4 h, blood was collected from the retroorbital sinus, and serum was prepared after clotting by centrifugation. Cholesterol measurements were done with the architectII system (Abbott Diagnostics).
Cell isolation and flow cytometry
Single-cell suspensions from spinal cords were obtained via mechanical dissociation on a cell strainer. Immune cells were separated over a two-phase Percoll-density gradient. Staining of αβTCR/CD4+ T cells, αβTCR/CD8+ T cells and CD45/CD11b cells (macrophages/microglia) was performed using the following antibodies in a 1:200 dilution: Anti-CD3e (clone 145-2C11), BioLegend; anti-CD4 (clone GK 1.5), BD; anti-CD8 (clone 53-6.7), BD; anti-CD8 (clone 53–6.7), BD; anti-CD11b (clone M1/70), BioLegend; anti-CD45.2 (clone 104), BioLegend. The addition of Calibrite APC beads (BD) allowed for cell quantification. Flow cytometry was performed using a FACSCalibur operated by Cell Quest software (Becton Dickinson).
Anesthetized mice were perfused with 4% formaldehyde (PFA). Brain samples of cuprizone treated animals were cut at Bregma 1.58 for comparable pathology because the extent of cuprizone mediated demyelination strongly depends on the rostral/caudal position69. Tissue was postfixed overnight, embedded in paraffin and cut into 5 μm sections (HMP 110, MICROM). Gallyas silver impregnation was done as described18. For immunohistological analyses, sections were deparaffinized followed by antigen-retrieval in sodium citrate buffer (0.01 M, pH 6.0). For immunofluorescence, sections were blocked with serum free protein block (Dako). Primary antibodies were diluted in 2% bovine serum albumin (BSA)/PBS and incubated for 48 h followed by fluorophor coupled secondary antibodies. For immunohistochemistry, endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Sections were then blocked (20% goat serum in BSA/PBS) and incubated with primary antibodies. Detection was done with the LSAB2 kit (Dako, Hamburg, Germany) or the Vector Elite ABC kit (Vector Labs). HRP substrate 3,3′-Diaminobenzidine (DAB) was applied by using the DAB Zytomed Kit (Zytomed Systems GmbH). Haematoxylin stain was done to label nuclei. Sections were dehydrated before mounting (Eukitt). Specimens were analysed on an Axio Imager.Z1 (Zeiss) equipped with an AxioCam MRc3, × 0.63 Camera Adaptor and the ZEN 2012 blue edition software using × 10 objective (Plan Apochromat × 10/0.45 M27) or × 20 objective (Plan-Apochromat × 20/0.8) and evaluated with Image J software. Quantification of areas (Gallyas, GFAP, MAC3) were done by applying semi-automated ImageJ software macro to threshold (variable threshold in case of Gallyas and fixed threshold for antibody stainings) and colour deconvolute the images of the corpus callosum above the fornix (Bregma 1.58). Three to five sections per animal were analysed. Quantification of EAE lumbar spinal cord lesions was done on two to four quadruple stained sections (Iba1 (induction of brown adipocytes 1), CD3, GFAP, DAPI) per animal recorded with tile region setup and shading correction. Lesion area was defined by focal accumulation of at least 20 DAPI positive cells, the presence of microglia and infiltration of CD3 positive cells. Lesion area, number of Iba1 and CD3 positive cells and GFAP positive area were evaluated. Quantification of cuprizone treated animals: cell number (CAII (carbonic anhydrase 2), Olig-2, TCF4, PCNA), APP positive spheroids and area (Gallyas, GFAP, MAC3) was done in the corpus callosum above the fornix (Bregma 1.58). Three to five sections per animal were analysed. Microscope settings are listed in Supplementary Table 4.
Electron microscopic analysis was done as previously described17. Briefly, tissue was fixed in 4% PFA, 2.5% Glutaraldehyde, 0.1 M Phosphate buffer and sagittal sections were cut on a vibratome (Leica VT1200, 300 μm). The corpus callosum with adjacent tissue (−0.04 mm lateral) was punched with a 2 mm diameter punching tool and embedded in epon (EMTP, Leica). At least 15 digital pictures (× 12,000 magnification, TRS, Moorenweis) of uranyl acetate contrasted ultrathin sections were taken with the Zeiss EM900.
The following antibodies were used: APP (Chemicon MAB348), CAII (Said Ghandour); CD3 (Serotec MCA1477);CD3e (Biolegend clone 145-2C11), CD4 (Becton Dickinson clone GK1.5), CD8 (Becton Dickinson clone 53-6.7), CD11b (Biolegend clone M1/70), CD45.2 (Biolegend clone 104), CNP (2′,3′-Cyclic-nucleotide 3′-phosphodiesterase, Sigma C5922), GFAP (Chemicon MAB3402), Iba1 (Wako 019-19741), MAC3 (Pharmigen 01781D); MBP (Serotec MCA409S), Olig2 (Prof Charles Stiles/ Dr. John Alberta, DF308), PCNA (Abcam ab29), SMI31 (Covance SMI-31P), TCF4 (Millipore 04-1080).
For the characterization of the proinflammatory milieu in EAE mice, RNA from total spinal cord lysates was isolated using Trizol (Thermo Fisher). complementary DNA (cDNA) was synthesized using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher) according to the manufacturer's protocol. Quantitative RT-PCR was performed using a StepOnePlus Real-Time PCR System operated by StepOnePlus Software v2.0. Target-specific FAM- and TAMRA-labeled TaqMan probes were used in all cases. Measurements were performed in independent duplicates. Gene expression was normalized to β-actin. Relative changes in gene expression were analysed via the 2ΔΔC(T) method.
For expression analyses on brain sections, mice were killed by cervical dislocation and brains were quickly cooled and sliced coronally using a brain matrix (Asi-Instruments). The corpus callosum was dissected from Bregma +1.10 to −2.46 and RNA was extracted using RNeasy Mini (Qiagen). The concentration and quality of RNA was evaluated using a NanoDrop spectrophotometer and RNA Nano (Agilent). cDNA was synthesized with Superscript III (Invitrogen) and quantitative PCRs were done in triplicates with the GoTaq master mix (Promega) on a 7500 Fast Real-Time PCR System (Applied Biosystems). Expression values were normalized to the geometric mean of two housekeeping genes, Hprt (Hypoxanthin-Phosphoribosyl-Transferase 1) and Rplp0 (60S acidic ribosomal protein P) and analysed by the ΔΔCt method.
Expression of the following genes was measured: Abca1 (ATP-binding cassette transporter A1), Actb (beta actin), Aif1 (allograft inflammatory factor 1), Apoe (apolipoprotein E), Bdnf (Brain-derived neurotropic factor), Bmp2 and Bmp4 (Bone morphogenic protein 2 and 4), Car2 (carbonic anhydrase 2), Ch25h (Cholesterol 25-Hydroxylase ), Cntf (ciliary neurotrophic factor), Cyp27a1 (Sterol 27-hydroxylase ), Cyp46a1 (Cholesterol 24-hydroxylase), Cyp51a1 (Sterol 14 alpha-demethylase), Dhcr24 (24-Dehydrocholesterol reductase), Egf (epidermal growth factor), Fdft1 (Farnesyl-Diphosphate Farnesyltransferase 1), members of the Fgf (fibroblast growth factor) gene family, Gfap (glial fibrillary acidic protein), Gmcsf (granulocyte-macrophage colony-stimulating factor, CSF2), H2-DMb2 (MHCII, major histocompatibility complex class II), Hmgcr (3-Hydroxy-3-Methylglutaryl-CoA Reductase), Hmgcs1 (3-Hydroxy-3-methylgutaryl-CoA synthase 1), Ifng (interferon gamma), Igf1 (insulin-like growth factor 1), Il2 (interleukin 2), Il17, Inhba (inhibin beta-A, also called activin beta-A), Ldlr (Low density lipoprotein receptor), Lrp1 (Low density lipoprotein receptor-related protein 1), Mvk (Mevalonate kinase), Ntf3 (Neurotrophin 3), Ngf (Nerve growth factor ), Nr1h3 (Liver X receptor alpha, LXR alpha), Nr1h2 (Liver X receptor beta, LXR beta), Olig2 (oligodendrocyte lineage transcription factor 2), Pdgfa (platelet derived growth factor alpha), Plp1 (proteolipid protein 1), Ptn (Pleiotrophin), Rxrg (Retinoic X receptor gamma, RXR gamma), S100b (S100 calcium-binding protein B), Shh (sonic hedgehog), Srbf2 (sterol regulatory element binding transcription factor 2), Tnf (tumour necrosis factor), Vldlr (Very low density lipoprotein receptor). All primer sequences are listed in Supplementary Table 3.
For primary oligodendrocyte cultures, dissected cortices of newborn mice or rats were digested in 0.25 mg ml−1 Trypsin/EDTA for 10 min followed by triturating and plating in plating media (DMEM 4.5 g l−1 Glucose, 10% fetal calf serum containing about 300 μg ml−1 cholesterol, GlutaMAX, penicillin/streptomycin). About 14 days after plating, OPCs were isolated by differential shaking and lectin panning, and plated in differentiating Sato media (DMEM 4.5 g l−1 glucose, 4 mM glutamine, 5 μg ml−1 insulin, 16 μg ml−1 putrescine, 6.2 ng ml−1 progesterone, 5 ng ml−1 sodium selenite, 400 ng ml−1 L-thyroxine, 400 ng ml−1 triiodothyroxine, 50 μg ml−1 holo-transferrin, penicillin/streptomycin, lacking any cholesterol source). Cell purity was routinely determined by immune stainings and always exceeded 95%. Cholesterol (10 μg ml−1) was added from a 10 mg ml−1 stock solution in ethanol. Control cultures received 0.1% ethanol. In case of growth factor supplementation assays, cells were allowed to adhere for 2 h before treatment with EdU (5-ethynyl-2′-deoxyuridine, 10 μM, Invitrogen) and growth factors (FGF1, FGF2; Peprotech) at 100 ng ml−1 for proliferation experiments or 45 ng ml−1 FGF1 plus 80 ng ml−1 FGF2 or 90 ng ml−1 FGF1 plus 35 ng ml−1 FGF2 for differentiation experiments. Cultures were fixed with PFA and permeabilized with 0.5% Triton X100.
Myelinating co-cultures were established as described70 with minor modifications. Briefly, six E13 embryonic spinal cords per culture were digested in 0.125% Trypsin solution in HBSS (without Ca+2 and Mg+2) at 37 °C for 20 min. After stopping the digestion with 1 ml plating media (DMEM, 25% horse serum, 25% HBSS, 50 μg ml−1 DNAse) the tissue was homogenized by gentle trituration and centrifuged for 5 min. 150,000 cells were plated per poly-L-lysine coated coverslip; 3 coverslips per 35 mm Petri dish. After cell attachment in plating media, differentiation media was added (low glucose DMEM, 10 μg ml−1 insulin, 10 ng ml−1 biotin, 50 nM hydrocortisone, 0.5% N1-mix). N1 mix was 1 mg ml−1 apo-transferrin, 20 mM putrescine, 4 μM progesterone, and 6 μM sodium selenite. 50% media change was performed every 24–48 h with differentiation media. After 12 days, insulin was removed from differentiation media. Coverslips were fixed with PFA after 20, 24 and 28 days in culture and permeabilized with −20 °C methanol for 10 min.
Fixed and permeabilized cells were blocked with 10% horse serum in PBS and incubated with primary antibodies in blocking solution followed by secondary antibodies together with click-it kit for detection of EdU and DAPI for nuclear staining. Coverslips were mounted on slides with aqua polymount. On five randomly chosen visual fields of primary oligodendrocyte cultures (× 10 magnification), stainings were evaluated. For differentiation of oligodendrocytes, CNP and MBP positive cells were categorized according to morphological criteria (see Fig. 7a). New oligodendrocytes (new OL) were CNP-positive MBP-negative cells with complex processes. MBP+OL cells contained few MBP-positive intracellular spots but did not form sheaths. Mature oligodendrocytes were CNP- and MBP-positive with sheaths. In myelinating co-cultures, the axonal (SMI31) area and the area with myelin sheaths (MBP) of seven randomly chosen visual fields of myelinating co-cultures (× 10 magnification) was measured after binarization of thresholded images. Specimens were analysed on an Axiophot observer.Z1 (Zeiss) equipped with an AxioCam MRm and the ZEN 2012 blue edition software and evaluated with Image J software. Microscope settings are listed in Supplementary Table 4.
Statistical evaluation was done by unpaired Student’s t-test for pairwise comparisons or by ANOVA for comparisons of more than two groups as stated in the figure legends. Two-way ANOVA was combined with a post test to evaluate individual groups. For all statistical tests, significance was measured against an alpha value of 0.05. All error bars show s.e.m. P values are shown as *P<0.05; **P<0.01; ***P<0.001. No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in previous publications25,28. Data analysis was performed blind to the experimental groups.
All data generated or analysed during this study are included in this published article (and its supplementary information files) or available from the authors on request.
How to cite this article: Berghoff, S. A. et al. Dietary cholesterol promotes repair of demyelinated lesions in the adult brain. Nat. Commun. 8, 14241 doi: 10.1038/ncomms14241 (2017).
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We are grateful to Klaus-Armin Nave for constant support. We cordially thank Annette Fahrenholz, Tanja Freerck, Boguslawa Sadowski and Lennart Wiegand for technical support. We thank Prof Charles Stiles, Dr John Alberta and Prof Said Ghandour for generous gifts of antibodies. This work was funded by the Deutsche Forschungsgemeinschaft (SA 2014/2-1 to G.S.). J.E. and W.M. were funded by an ERC Advanced grant awarded to Klaus-Armin Nave.
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Gene expression in oligodendrocytes during remyelination reveals cholesterol homeostasis as a therapeutic target in multiple sclerosis
Proceedings of the National Academy of Sciences (2019)