Curcumin promotes oligodendrocyte differentiation and their protection against TNF-α through the activation of the nuclear receptor PPAR-γ

Curcumin is a compound found in the rhizome of Curcuma longa (turmeric) with a large repertoire of pharmacological properties, including anti-inflammatory and neuroprotective activities. The current study aims to assess the effects of this natural compound on oligodendrocyte progenitor (OP) differentiation, particularly in inflammatory conditions. We found that curcumin can promote the differentiation of OPs and to counteract the maturation arrest of OPs induced by TNF-α by a mechanism involving PPAR-γ (peroxisome proliferator activated receptor), a ligand-activated transcription factor with neuroprotective and anti-inflammatory capabilities. Furthermore, curcumin induces the phosphorylation of the protein kinase ERK1/2 known to regulate the transition from OPs to immature oligodendrocytes (OLs), by a mechanism only partially dependent on PPAR-γ. Curcumin is also able to raise the levels of the co-factor PGC1-α and of the cytochrome c oxidase core protein COX1, even when OPs are exposed to TNF-α, through a PPAR-γ-mediated mechanism, in line with the known ability of PPAR-γ to promote mitochondrial integrity and functions, which are crucial for OL differentiation to occur. Altogether, this study provides evidence for a further mechanism of action of curcumin besides its well-known anti-inflammatory properties and supports the suggested therapeutic potential of this nutraceutical in demyelinating diseases.


Results
Effect of curcumin on OP viability and differentiation: role PPAR-γ activation. All experiments were performed in highly purified cultures of rat OPs. At 1 day in vitro (DIV), cells were treated for 24 h, with curcumin at two different concentrations and cell viability evaluated by testing cell metabolic activity (MTT test), damage (LDH test) and apoptosis (Tunel assay). Curcumin markedly decreased OP metabolic activity at the highest concentration (5 μM), while none of the two concentrations affected LDH levels in the supernatants (Fig. 1A). Only at the highest concentration, curcumin induced a slight increase in the number of apoptotic cells (Fig. 1B, C). Tunel quantification data were comparable to those from morphological analysis (Fig. 1C): CTR 0.82 ± 0.08, curc 1 μM 0.93 ± 0.09, curc 5 μM 7.1 ± 0.4, assessed as % of total.
Based on these preliminary experiments, OPs were treated with 1 μM curcumin at 1 DIV. The effects on differentiation were investigated at 2 DIV and 7 DIV, by evaluating markers of specific stages of differentiation, such as O 4 (pre-OL), O 1 (immature OL) and myelin basic protein (MBP) (non-myelinating mature OL). As indicated in Fig. 1D, curcumin induced a drastic change in cell morphology, as depicted by the more complex ramifications of branches as compared to untreated cultures. Along with the morphological variation, curcumin treatment induced an increase in the number of O 4 and O 1 positive cells comparing to untreated cultures (Fig. 1E). Moreover, at 7DIV, the expression of the two isoforms of myelin basic protein (MBP) was increased, further indicating the differentiation-promoting effect of curcumin (Fig. 1F). To verify whether curcumin acted through the activation of the nuclear receptor PPAR-γ, cell differentiation was evaluated in the presence of the PPAR-γ antagonist GW9662 (1 μM). As shown in Fig. 1G, the increased number of O 4 and O 1 -positive cells induced by treatment with curcumin was reduced at control levels by GW9662. Furthermore, curcumin acted as bona fide PPAR-γ agonist, being able to promote the nuclear translocation of PPAR-γ; curcumin-induced PPAR-γ translocation was abolished by the PPAR-γ antagonist GW9662 (Fig. 1H, I).
Curcumin modulates ERK1/2 signaling pathways. Recent studies have shown that, in addition to the induction or repression of target genes, PPAR-γ can perform "non-genomic" actions, including activation or inhibition of several kinases 21 . Among such kinases is the protein kinase ERK (Extracellular Receptor-activated Kinase) 1/2, which is involved in the differentiation of OLs, especially in the early stages 22 .
In OP cultures exposed to 1 μM curcumin, ERK1/2 phosphorylation was induced in a time-dependent manner with maximum levels of p-ERK1/2 at 60 min, as determined either by immunofluorescence and subsequent quantification of mean fluorescence intensity (MFI) or by western blot ( Fig. 2A, C). Interestingly, the PPAR-γ antagonist GW9662 significantly prevented the phosphorylation of ERK1/2 induced by curcumin (Fig. 2B), suggesting that the effect of curcumin on p-ERK1/2 is, at least in part, PPAR-γ-dependent. The results were confirmed by western blot experiments (Fig. 2D). As expected, phosphorylation of ERK1/2 induced by curcumin was abolished by 10 μM PD98059, a selective, cell-permeable inhibitor of the MEK/ERK pathway (Fig. 2E).
Curcumin induces the expression of PGC1-α and COX1. One of the outcomes of PPAR-γ activation is the promotion of mitochondrial functions, as already shown in a variety of cells, including OPs. To confirm the capability of curcumin-induced PPAR-γ activation to impinge on mitochondrial functions, we studied the expression of two genes: (i) the peroxisome proliferator-activated co-activator 1-alpha (PGC1-α), known to promote the biogenesis of mitochondria and the defenses against oxidative stress, whose expression is induced by PPAR-γ; (ii) cytochrome C oxidase subunit 1 (COX1), known as a core protein of the cytochrome C oxidase (Complex IV) of the mitochondrial respiratory chain, whose expression is regulated by PGC1-α.
After 24 h of treatment, curcumin increased the levels of PGC1-α and COX1, an effect prevented by the presence of GW9662, confirming, also in this case, the effect of curcumin to be PPAR-γ-dependent (Fig. 3). Regarding ERK1/2 activation, the reduced expression of PGC1-α by PD98059 suggests that ERK1/2 is probably implicated in the modulation of the expression of PGC1-α. However, the strong reduction of COX1 expression of by PD98059 even in CTR condition does not allow conclusive speculation on the involvement of ERK1/2 in curcumin-dependent induction of COX1 (Fig. 3).
Curcumin prevents TNF-α-induced harmful effects. It is known that TNF-α is capable of influencing proliferation, differentiation, and death in several cell types [23][24][25] , including OLs 18,26 . In this study, we wanted to determine the capability of curcumin to counteract the effect of TNF-α on OP differentiation and mitochondrial functions. We demonstrated that curcumin could prevent the adverse effects of TNF-α on viability (Fig. 4A)     Besides, we demonstrated that TNF-α treatment induced a time-dependent increase of p-ERK, peaking at 5 min and then decreasing to a steady level that remained higher than in unstimulated cells up to 120 min, as revealed by MFI and by western blot analyses (Fig. 4E, F and H). Pretreatment with PD98059 was capable of counteracting OP differentiation induced by curcumin, either in the presence or absence of TNF-α, (Fig. 4I), suggesting that activation of ERK1/2 is involved in the processes triggered by both PPAR-γ and TNF-α.
In agreement with the literature, we observed that TNF-α caused a mild, although significant, decrease of the levels of both PGC1-α and COX1 proteins, accentuating the toxic effect of TNF-α on mitochondria. When TNF-α was co-applied with curcumin, which alone increased COX1 and PGC1-α, the levels of the two proteins remained higher than in control as shown by immunofluorescence and western blot analyses (Fig. 5A, B, respectively). Together, the protective effect of curcumin on metabolic activity (MTT), PGC1-α and complex IV protein COX1 levels, support a protective effect of curcumin on mitochondrial functions.

Discussion
In the present study, we tested if curcumin, acting as a PPAR-γ agonist, could favor the differentiation of OPs and their recovery from the damage caused by the pro-inflammatory cytokine TNF-α. Our final goal was to find experimental evidence to support the suggested therapeutic use of curcumin in myelin diseases, such as multiple sclerosis 27,28 .
We observed that curcumin favors the differentiation of OPs, as indicated by the increased expression of markers typically associated with different development stages. In particular, we assessed O 4 and O 1 , two developmental markers whose presence is related to postmitotic commitment/initiation of terminal OL differentiation, and myelin basic protein (MBP), a late marker of OL maturation. Furthermore, cell morphology was more complex in curcumin-treated than in untreated cells, in keeping with cell maturation.
Curcumin-induced differentiation is consistent with previous studies showing curcumin action on neuronal differentiation and neurite outgrowth of PC-12 cells in combination with NGF 29 , and on the expression of differentiation genes in cells of thyroid carcinoma inducing G2/M arrest, apoptosis, and inhibition of NF-κB 30 .
Previous studies from our group have shown that PPAR-γ is constitutively expressed in OPs 14,20 and that its activation by specific agonists promotes OP differentiation to mature OLs. Since curcumin is known to induce the activation of PPAR-γ in numerous cellular systems 31-33 , we hypothesized that similarly, the effects induced by curcumin on OPs could be mediated by PPAR-γ activation. In keeping with the hypothesis, we confirmed that curcumin activates PPAR-γ in OPs, by showing curcumin-dependent nuclear translocation of PPAR-γ, which was inhibited by the specific PPAR-γ antagonist GW9662.
The mechanism of action of PPAR-γ is mainly related to its ability to act as a ligand-dependent transcription factor. However, in recent years it has been highlighted how PPAR-γ can also act through "non-genomic" mechanisms. Once activated, PPAR-γ can physically interact with some proteins among which protein kinases, thereby modulating their activities. Among the protein kinases regulated by PPAR-γ is ERK1/2 21,34 .
ERK1/2 control the differentiation processes of different cell types. In the OL lineage activation of ERK1/2 is crucial for the transition from OPs to immature OLs 22,35 . Recently, we have shown that both synthetic (pioglitazone) and natural (the omega-3 PUFA DHA) PPAR-γ agonists induce ERK1/2 phosphorylation and that the activation of this molecular signaling contributes to OL differentiation and survival 17 . Here we show that curcumin induces the phosphorylation of ERK1/2, although with different kinetics as compared to those observed for pioglitazone and DHA 17 . ERK1/2 phosphorylation was partially inhibited by GW9662 pretreatment, demonstrating that the effect of curcumin is at least partly dependent on the activation of PPAR-γ. Indeed, a direct activation, i.e. phosphorylation, of ERK 1/2 by curcumin could also occur, as previously shown 36 .  www.nature.com/scientificreports/ Another goal of our study was to test whether curcumin could protect OP cultures from an inflammatory insult, as shown for other PPAR-γ agonists 16,17 . We mimicked the inflammatory condition by treating OPs with a subtoxic dosage of TNF-α (10 ng/ml) 18 , a pro-inflammatory cytokine involved in demyelinating diseases such as multiple sclerosis 26 . The addition of TNF-α in OP cultures inhibits their differentiation, probably through an adverse action on mitochondrial functions and Ca 2+ signals 18,37 .
Among its numerous effects, TNF-α has been shown to regulate the activity of PPAR-γ negatively 38,39 . We observed that in the presence of TNF-α, curcumin induces the translocation of PPAR-γ and counteracts in a PPAR-γ-dependent manner, the maturational arrest of OPs triggered by the cytokine.
Both curcumin and TNF-α induced ERK1/2 phosphorylation, but with different kinetics, being faster for TNF-α than for curcumin (maximum level after 5 min and 60 min, respectively). The different kinetics of ERK1/2 phosphorylation 40 , could probably explain the opposite effects on OP differentiation by TNF-α and curcumin.
A large body of evidence supports the need for a proper mitochondrial function for myelination to occur. These organelles play fundamental roles in OL differentiation 16,18,37 . They are a source of energetic supply and a site where peculiar synthetic activities take place, such as part of the synthesis of cholesterol, one of the major Figure 5. Curcumin protects from the effect of TNF-α on PGC1-α and COX1 expression. PGC1-α and COX1 expression levels were evaluated in cells treated for 24 h with curcumin (curc) and/or TNF-α. Representative photomicrographs are shown (A). Nuclei were stained using the Hoechst 33258 nuclear fluorochrome (blue, H). Representative western blot of PCG1-α or COX1 and β-actin (used as an internal control) are shown (B). The intensities of bands corresponding to PCG1-α or COX1 and β-actin were measured by densitometric analysis and the ratio PCG1-α/β-actin or COX1/β-actin is given as percentage on untreated cells. Data are means ± SEM of three independent experiments *p < 0.05 versus CTR, **p < 0.005 versus curc + TNF-α. Scale bar 10 µm. Image Lab 4.0 software (Bio-Rad, http://www.bio-rad.com/en-us/sku/17096 90-image -lab-softw are) and Leica Application Suite Software (260RI, https: //www.leica -micro syste ms.com/produ cts/micro scope -softw are/detai ls/ produ ct/leica -las-x-ls/) were used respectively for Wb and IF image acquisitions. www.nature.com/scientificreports/ constituents of myelin. We focused our attention on two proteins particularly significant in mitochondrial biogenesis: PGC1-α, which participates in the control of the transcription of genes involved in mitochondrial biogenesis, cholesterol synthesis and MBP gene expression 41 , and COX1, a core subunit of cytochrome C oxidase (Complexx IV) of the respiratory chain, whose expression is controlled by PPAR-γ through PGC1-α 42 . As for other PPAR-γ agonists or partial agonists, curcumin was able to increase their expression, by a PPAR-γ-dependent mechanism. Inhibition of ERK1/2 phosphorylation partially inhibited the effect of curcumin on PGC1-α, but not that of COX1. As expected, TNF-α caused a moderate decrease of the expression levels of both PGC1-α 10,18,24,43 and COX1 18,25,44 , which was counteracted by curcumin. A protective effect of curcumin against mitochondrial injury and OL/neuronal apoptosis has been shown in experimental autoimmune encephalomyelitis (EAE), an animal model of MS 6 , suggesting that a similar mechanism could occur also in TNF-α treated OPs. In summary, our study highlights the capability of curcumin of promoting the differentiation of OPs, even in an inflammatory context, through multiple mechanisms involving PPAR-γ and ERK1/2 activation, possibly through mechanisms affecting mitochondria (Fig. 6).
Despite the wide range of beneficial effects of curcumin, its therapeutic use is limited by its poor water solubility, poor absorption from gastrointestinal mucosa, low stability in blood and rapid hepatic metabolism 45 . Several approaches have been taken to enhance its bioavailability, including the use of adjuvants (such as piperine‚ quercetin or silibinin), of complexes of curcumin with phospholipids, polysaccharides or proteins, and, more recently encapsulation in nanoparticles 46 .
Promising in vivo studies and clinical trials support the use of curcumin nanoparticle (nanocurcumin) in CNS diseases. Polymerized nanocurcumin was effective in controlling inflammation in EAE. Moreover, curcumin-loaded nanoparticles promoted myelin repair in a model of demyelination induced by lysolecithin in the rat corpus callosum 47 . Of note, nanocurcumin was recently shown to reduce neuroinflammation and restore dysregulated Treg functions in a clinical trial enrolling 50 relapsing-remitting MS subjects, after six months of treatment 48,49 .
The picture emerging is of growing interest for curcumin, with the possibility of expanding the use of an old natural compound through new and more efficient formulations in different pathological situations 50 .
The present study, based on a well-established in vitro model, provides evidence for a further mechanism of action of curcumin, by showing its ability to activate PPAR-γ in OPs and promote their differentiation to OLs and, in turn, myelin formation. Further studies on more complex models of myelination will deepen our knowledge of curcumin and provide mechanistic evidence besides its well-known anti-inflammatory properties in support of the proposed utilization of curcumin in demyelinating pathologies 23,27 .

Methods
All the experiments have been carried out on purified cell cultures. The procedure for obtaining purified cell Cell cultures. Purified cultures of OPs were obtained as previously described 14 . Forebrains from postnatal day 1 Wistar rats (RRID: RGD_13508588; from Charles River, Lecco, Italy) were mechanically dissociated, and the cells obtained were grown for 10 days in Dulbecco's modified Eagle's medium (DMEM with high glucose), supplemented with 10% foetal bovine serum (Gibco, ThermoFisher Scientific, Inc.) on poly-L-lysine coated 60-mm-diameter culture dishes. OPs growing on top of mixed glial monolayers were mechanically detached from the mixed cultures. A mechanical detachment was followed by 1 h incubation at 37 °C in culture flasks to remove adhering cells and thus minimize contamination by microglia. Then cells were seeded at the density of 6 × 10 4 cells/cm 2 into poly-L-lysine-coated 35-mm diameter plastic culture dishes or 96 well plates. After 2 h, the culture medium was replaced with a chemically defined serum-free medium consisting of DMEM/ www.nature.com/scientificreports/ HamF12 (4:1) supplemented with 5.6 mg/ml glucose, 5 µg/mL insulin, 100 µg/mL human transferrin, 100 µg/ mL bovine serum albumin, 0.06 ng/mL progesterone, 40 ng/mL sodium selenite, 16 µg/mL putrescine, 50 U/mL penicillin, 50 µg/mL streptomycin, 2 mmol/L glutamine,10 ng/ml human recombinant PDGF-AA and 10 ng/ml human recombinant bFGF (PeproTech EC, Ltd, UK). Immunocytochemical analysis with LB 1 and O 4 antibodies revealed a pure population (around 98%) of cells of the OL lineage. Cell culture reagents were from BioWest (Fla, USA) or Invitrogen (Milan, Italy); other chemicals were from Sigma-Aldrich Italia (Milan, Italy). The cells were treated at 1DIV after seeding, and the experiments were done at 2DIV (if not otherwise stated in the figure legend).
Experimental design. Purified oligodendrocyte precursors (OPs) were maintained in a chemically defined serum-free medium for all experiments. At 1 DIV, 24 h after plating, OPs were stimulated by adding different drugs, without changing the medium. Curcumin (1 or 5 μM) was added alone or in the presence of TNF-α (10 ng/ml). Depending on the parameter to be studied, the experiments were carried out for short time (from 5 to 120 min), or for longer times for 24 h (2 DIV) or 7 DIV (see details in the results section and the legends). When GW9662 (1 μM) or PD98059 (10 μM) were used, the OPs were pretreated for 30 min before the addition of other drugs. After the experimental time, cells were treated according to the different procedure, as described later. Conditioned media were collected, centrifuged, filtered, and stored at − 80 °C until tested.
Briefly, MTT was added at a final concentration of 0.25 mg/ml during the last 4 h of incubation. The medium was then removed, and 100 μl DMSO added to each well to dissolve the dark blue crystals. The plates were then read on a microplate reader, using a test wavelength of 570 nm and a reference wavelength of 630 nm. The release of the cytosolic enzyme lactate dehydrogenase (LDH) was used to evaluate the integrity of the membrane, by verifying its presence in the culture supernatant using a colorimetric cytotoxicity assay kit (Roche Diagnostics MI, Italy). Apoptotic cells were evaluated observing the changes of nuclei morphology. Cells were exposed to 1 µg/mL of Hoechst 33258 for 60 min to 37 °C, rinsed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde. Nuclear morphology was visualized using a fluorescence microscope. To detect and quantify, at the single-cell level, DNA strand breaks generated during apoptosis we used terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-fluorescein nick end-labeling (TUNEL) staining. An in-situ cell death detection kit was used (Boehringer Mannheim, Indianapolis, IN) and TUNEL reaction was analyzed by fluorescence light microscopy.
Immunofluorescence. Cell differentiation was evaluated by immunofluorescence for membrane surface antigens, using the monoclonal immunoglobulin M, O 4 and O 1 , as previously described 17 (IgM, hybridoma supernatants, custom made, or Millipore, Italia; 1:5 or 1:100, (Cat#MAB344 clone 59 Lot#2343576), respectively). For immunofluorescence of intracellular antigens, cells were fixed in 4% paraformaldehyde for 10 min at room temperature (RT). After fixation and permeabilization with 0.2% Triton X-100 for 10 min at RT, the cells were pre-incubated with 3% BSA in 0.1% Triton X-100/PBS solution for 1 h at RT and then incubated overnight at 4 °C with rabbit polyclonal anti-COX1 (i. www.nature.com/scientificreports/ This procedure was performed for at least 40 cells per condition for each independent experiment (n = 3 independent experiments). Alternatively, the cells were counted in 10 microscopic fields of 0.18-mm 2 per coverslip prepared in duplicate for each condition from at least 3 independent experiments.
Statistical analysis. Data were expressed as means ± SEM. Statistical significance was evaluated using Student's t-test or one-way ANOVA (Microsoft Windows Excel Office 365 and confirmed in Origin 6.0, Origin-Lab, Northampton, MA). Numbers of independent experiments are indicated in the figure legends; p < 0.05 was accepted as statistically significant. The experimental procedures and statistical analysis of the data presented in this study followed the methodologies and standards generally used in in vitro studies. In all experiments, the sources of variability, as well as any residuals deriving from random errors of the replicates, were well controlled, as demonstrated by the low SEM. Bonferroni correction was adopted to account for multiple comparisons. Normality of distribution was assessed by the Shapiro Wilk test (Microsoft Windows Excel Office 365 and confirmed by online software http://www.stats kingd om.com) 53 .