Co-ultramicronized Palmitoylethanolamide/Luteolin Promotes the Maturation of Oligodendrocyte Precursor Cells

Oligodendrocytes have limited ability to repair the damage to themselves or to other nerve cells, as seen in demyelinating diseases like multiple sclerosis. An important strategy may be to replace the lost oligodendrocytes and/or promote the maturation of undifferentiated oligodendrocyte precursor cells (OPCs). Recent studies show that a composite of co-ultramicronized N-palmitoylethanolamine (PEA) and luteolin (co-ultramicronized PEA/luteolin, 10:1 by mass) is efficacious in improving outcome in experimental models of spinal cord and traumatic brain injuries. Here, we examined the ability of co-ultramicronized PEA/luteolin to promote progression of OPCs into a more differentiated phenotype. OPCs derived from newborn rat cortex were placed in culture and treated the following day with 10 μM co-ultramicronized PEA/luteolin. Cells were collected 1, 4 and 8 days later and analyzed for expression of myelin basic protein (MBP). qPCR and Western blot analyses revealed a time-dependent increase in expression of both mRNA for MBP and MBP content, along with an increased expression of genes involved in lipid biogenesis. Ultramicronized PEA or luteolin, either singly or in simple combination, were ineffective. Further, co-ultramicronized PEA/luteolin promoted morphological development of OPCs and total protein content without affecting proliferation. Co-ultramicronized PEA/luteolin may represent a novel pharmacological strategy to promote OPC maturation.

. Co-ultramicronized PEA/luteolin promotes the morphological development of cortical oligodendrocyte precursor cells. One day after plating OPCs were treated with 10 μ M co-ultramicronized PEA/luteolin as detailed in Methods. Following a further 4 days of incubation the cultures were photographed under phase contrast microscopy. Note what appears to be a more complex morphology and greater extent of branching in cells treated with co-ultramicronized PEA/luteolin (b) compared to untreated cells (a). Scale bar: 30 μ m. Table S1), a well-accepted proliferation marker 33,34 . Conceivably, this effect might also reflect an increased tissue mass or cell hypertrophy of the cultures.
Co-ultramicronized PEA/luteolin regulates mRNA expression of myelin genes in differentiating OPCs. Based on these initial observations, we next examined the ability of co-ultramicronized PEA/luteolin to induce the expression of markers characteristic of a more mature oligodendrocyte phenotype. Incubation of OPCs with co-ultramicronized PEA/luteolin (10 μ M), commencing the day after cell plating led to a time-dependent rise in the expression of MBP and PLP mRNA levels by quantitative PCR, which was significantly greater compared to vehicle-treated cells (Fig. 3a,b). The effect of co-ultramicronized PEA/luteolin was concentration-dependent, increasing MBP gene expression after 4 days by 0.90 ± 0.08, 1.37 ± 0.22 and 1.85 ± 0.02-fold at 0.1, 1 and 10 μ M, respectively (mean ± s.e.m., n = 3, p < 0.001 vs vehicle at 10 μ M). This was accompanied by a corresponding increase in the cellular content of both the 18 kDa and 24 kDa isoforms of MBP up to 8 days (Fig. 4). The membrane-anchored myelin enzyme 2′ ,3′ -cyclic nucleotide 3′ -phosphodiesterase (CNPase) 1 , which is believed to mediate process outgrowth in oligodendrocytes 35 and play a critical role in the events leading up to myelination 36 also displayed elevated gene levels in OPCs treated with co-ultramicronized PEA/luteolin over this time, as compared to vehicle alone (Fig. 3c). Coincidentally with this up-regulated expression of myelin protein genes, platelet-derived growth factor receptor alpha (PDGFRα ), a recognized marker of OPCs but not premyelinating or myelinating oligodendrocytes 1,31 , fell by about 95% by day 4 and remained so until day 8, and was not influenced by incubation with co-ultramicronized PEA/luteolin at any time point ( Supplementary Fig. S2). Interestingly, the magnitude of this drop in PDGFRα expression matches the percentage of differentiating immunocyochemically characterized oligodendrocytes in these cultures, based on our earlier study (see Methods,ref. 32).
Myelin is also dependent on its characteristic lipid contents, which provide critical building blocks of this membrane structure 37 . Therefore, it is important to assess as well the potential regulatory effects of co-ultramicronized PEA/luteolin on lipogenesis. We measured the mRNA levels of UDP glycosyltransferase 8 (Ugt-8), which catalyzes the final step in the biosynthesis of cerebrosides 38 , a major component of myelin membranes up-regulated during oligodendrocyte differentiation 39 . Here also, OPCs incubated with co-ultramicronized PEA/luteolin (10 μ M) showed a time-dependent increase in gene expression in comparison to vehicle-only cultures (Fig. 5). In addition, genes involved in cholesterol synthesis (HMG-CoA reductase, HMGCR; isopentenyl-diphosphate delta isomerase 1, IDI1) and fatty acid synthesis (stearoyl CoA desaturase-1, SCD1) were analyzed. As Fig. 6 shows, co-ultramicronized PEA/luteolin (10 μ M) up-regulated mRNA expression of IDI1 (left panel) and HMGCR (right panel) in differentiating OPCs already after 1 day of treatment, and remained above control through 8 days -in contradistinction to the more delayed rise in the genes for MBP and PLP. Co-ultramicronized PEA/ luteolin significantly up-regulated also mRNA levels for SCD1 when examined after 4 days of treatment (Fig. 7d) Supplementary Fig. S3) were unaltered, suggesting that co-ultramicronized PEA/luteolin effects may not reflect a general induction of transcription.
The effects of co-ultramicronized PEA/luteolin on OPC development were not mimicked by either ultramicronized PEA (10 μ M), luteolin (1 μ M), or the combined treatment with ultramicronized PEA (10 μ M) plus luteolin (1 μ M). This was the case for expression of genes involving both myelin proteins and pathway components of lipogenesis. Figure 7 describes several examples for illustrative purposes, for cells analysed after 4 days of treatment. The effective concentrations of co-ultramicronized PEA/luteolin in the present study, and the superior efficacy of the co-ultramicronized composite in comparison to  its constituent molecules are consistent with prior reports [25][26][27] . Increasing the luteolin concentration to 10 μ M was found, following a 4-day incubation to compromise cell vitality. Ultramicronized PEA alone, up to 20 μ M was without effect; above this concentration one encounters potential vehicle issues and solution behaviour. mTOR and the OPC-differentiating action of co-ultramicronized PEA/luteolin. Emerging evidence now points to mammalian target of rapamycin (mTOR) as playing a key role in oligodendrocyte  differentiation and myelin [40][41][42][43][44][45] . Rapamycin, an established, selective inhibitor of mTOR was used to interrogate a role for this kinase in OPC differentiation. When added the day after cell plating, 2 nM rapamycin (effective concentrations as described by others 40,45 ) reduced the expression levels of mRNA for MBP and Ugt-8 in differentiating OPCs when analysed 4 days later (Fig. 8, left and right panels,  respectively). Co-ultramicronized PEA/luteolin appeared able to partially restore, at least to control levels, MBP and Ugt-8 gene transcripts. Analogous results were obtained for PLP gene expression (data not shown). Further, OPC cultures treated with 2 nM rapamycin displayed an attenuated morphological maturation and lower protein content after 4 days, which co-ultramicronized PEA/luteolin (10 μ M) was able to overcome, in part (Fig. 9).  Cultures of OPCs were pre-treated 30 min the day after plating with 2 nM rapamycin, followed by addition of co-ultramicronized PEA/luteolin ('PEALut' , 10 μ M final) or not. Cultures were harvested 4 days later and protein content measured (expressed as μ g/ml cell lysate). Data are means ± s.e.m. (n = 6). *p < 0.05 for rapamycin + co-ultramicronized PEA/luteolin vs CTRL; ***p < 0.001 for rapamycin vs vehicle (CTRL, 0.02% Pluronic F-68); § § § p < 0.001for rapamycin vs rapamycin + co-ultramicronized PEA/luteolin.

Discussion
Oligodendrocytes, the myelin-producing cells of the CNS, are the main target in chronic immunological diseases such as MS, the latter's pathophysiological pattern characterized by inflammatory cell infiltration, demyelination, axonal damage, astrogliosis, and neurodegenerative processes 4 . Currently, approved disease-modifying therapies are based on the use of immunomodulatory agents, several of which can be administered orally 46 . However, they do not improve disease outcome after degeneration occurs, and therefore are insufficient to treat chronic neurological disability in patients with progressive MS. A promising perspective for future disease therapy is the regeneration of lesions with replacement of the damaged oligodendrocytes [14][15][16][17]47,48 . As one step in this direction, we identify a novel property of co-ultramicronized PEA/luteolin in enhancing the maturation of differentiating cortical OPCs in vitro by stimulating the expression of myelin-specific genes (MBP, CNPase, and PLP) as well as cellular content of MBP. In addition, of co-ultramicronized PEA/luteolin increased the expression of genes involved in lipogenesis, including Ugt-8 which catalyzes the final step in the biosynthesis of cerebrosides 38 .
The fatty acid amide signalling molecule PEA has been shown in a number of studies to display anti-neuroinflammatory and neuroprotective actions [21][22][23] . Following these reports, more recent investigations indicate that a composite containing PEA co-ultramicronized with the flavonoid luteolin may possess an enhanced pharmacological profile in these experimental models [25][26][27]49 . Flavonoids like luteolin are polyphenolic phytochemicals with potent antioxidant capacity and exhibit neuroprotective/anti-inflammatory actions 50 . Among the family of flavonoids, luteolin is claimed to possess memory-improving 51 and anxiolytic 52 effects, and not wholly explained by its antioxidant capacity. Treatment of differentiating OPCs with co-ultramicronized PEA/luteolin resulted in a time-dependent increase in mRNA levels for the major myelin proteins MBP and PLP, together with the genes for constituent enzymes involved in lipid biosynthesis. The timing of gene expression was neither synchronous nor sustained in all cases: for example, IDI1 and HMGCR mRNAs were already elevated after 1 day of incubation with co-ultraPEA/Lut, and remained at that level between 4 and 8 days; Ugt-8 mRNA was first increased at 4 days, and remained significantly elevated at 8 days of co-ultramicronized PEA/Lut treatment. This is perhaps not surprising, as the differentiation of OPCs is a rather complex process requiring the coordinated expression of genes to achieve maturation. Importantly, the rise in MBP mRNA was accompanied by a marked and sustained increase in the cellular content of MBP protein. These effects of co-ultramicronized PEA/luteolin are not necessarily the result of a general induction of transcription, as other genes (e.g. PDGFRα , Ki-67, cannabinoid receptors 1 and 2, SOD2) were unaltered. These observations with the composite of PEA and luteolin, and ineffectiveness of its constituent molecules are consistent with other studies [25][26][27]49 . The mechanistic basis underlying the actions of co-ultramicronized PEA/luteolin remains to be investigated. PEA actions are known to be pleiotropic in nature and involve interaction with a number of targets, including peroxisome proliferator-activated receptor alpha, the transient receptor potential vanilloid type 1 receptor and GPR55 22 .
Oligodendrocytes have the highest rate of oxidative metabolic activity in the brain, a likely consequence of the energy consumption needed for biosynthesis of lipid-rich myelin 53 . This may result in the generation of large amounts of reactive oxygen species, the latter playing a role in inflammation and inflammatory demyelinating disorders, such as MS and experimental autoimmune encephalomyelitis 54,55 . As oligodendrocytes are the brain's predominant iron-containing cells, this fact may also contribute to their susceptibility to oxidative injury -coupled with low glutathione levels 56,57 . In particular, one major contributor to oxidative damage is H 2 O 2 , which is converted from superoxide that leaks from the mitochondria. Catalase and superoxide dismutase ameliorate the damaging effects of H 2 O 2 and superoxide, respectively, by converting these compounds into oxygen and H 2 O 2 (which is later converted to water) 58 . Interestingly, incubation of oligodendrocytes at day 6 in culture with 10 μ M co-ultramicronized PEA/luteolin for 24 h produced a two-fold increase in catalase mRNA level (Supplementary Fig. S3). Co-ultramicronized PEA/luteolin at a lower concentration (1 μ M) was ineffective; neither concentration altered expression of SOD2 ( Supplementary Fig. S3).
mTOR is a serine/threonine protein kinase that controls cell growth, proliferation and survival. A number of studies support a role for mTOR signalling in oligodendrocyte differentiation and myelination [40][41][42][43][44][45] . Rapamycin (also known as sirolimus), a potent and selective inhibitor of mTOR, regulated the expression of myelin genes in differentiating OPCs (Fig. 8), consistent with previous reports 40 . In addition, rapamycin blunted the ability of co-ultramicronized PEA/luteolin to promote the biochemical maturation of the differentiating OPCs, suggesting that co-ultramicronized PEA/luteolin may act via a mTOR-dependent molecular pathway. Further studies will be needed to elucidate this possibility.
Activated microglia and macrophages play a key role in driving demyelination during MS 59 . Mast cells, also a component of the innate immune system, are undoubtedly major contributors to autoimmune disease, as well 60 . Given the likelihood that microglia, mast cells and oligodendrocytes interact with each other 61,62 , approaching MS as both an inflammatory and neurodegenerative disease has important implications for treatment, with remyelination of axons to protect neurons from damage being necessary in addition to controlling inflammation 63 . The findings reported here encourage the view that co-ultramicronized PEA/luteolin may represent a potentially novel avenue in the treatment of inflammatory demyelinating disorders, and merits further investigation in appropriate animal models as a next step. Of consequence, neither PEA 24 not luteolin 64  Methods Materials. Tissue culture media, N2 supplement, antibiotics and fetal calf serum (FCS) and NP40 cell lysis buffer (10×) were obtained from Life Technologies (San Giuliano Milanese, Italy); poly-D-lysine hydrobromide (mol wt 70,000-150,000), poly-L-lysine hydrobromide (mol wt 70,000-150,000), cytosine β -D-arabinoside, 3,3′ ,5-triiodo-L-thyronine, L-thyroxine, rapamycin, luteolin, papain, DNase I (bovine pancreas), trypsin inhibitor, protease inhibitor cocktail, Pefabloc ® SC (100 mM), Pluronic F68 and all other biochemicals were purchased from Sigma-Aldrich (Milan, Italy) unless noted otherwise; Falcon tissue culture plasticware was purchased from BD Biosciences (SACCO srl, Cadorago (CO), Italy). Sterilin petri plastic dishes (10 cm Ø) were from Sarstedt (Verona, Italy). Co-ultramicronized PEA/luteolin (10:1 mass ratio) and ultramicronized PEA were kindly provided by Epitech S.p.A., Saccolongo (PD), Italy.
Primary culture of oligodendrocyte precursors. Experiments were performed in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and those of the Italian Ministry of Health (D.L. 116/92). The University of Padua Institutional Animal Care and Use Committee approved the experimental protocols used in this study. Mixed glial cell cultures from cortex were prepared from postnatal day 1 rat pups (strain: CD) as previously described 32,65 . Briefly, tissue dissociates were plated in 75-cm 2 poly-L-lysine-coated (10 μ g/ml) tissue culture flasks at a density of 1.5 brains and grown in high-glucose Dulbecco's modified Eagle's medium with 2 mM glutamine, 100 units/ml penicillin/50 μ g/ml streptomycin, 50 μ g/ml gentamicin and 10% FCS. Medium was changed after 24 h. Upon reaching confluence (approximately 7-8 days later) microglia adhering to the astroglial monolayer were dislodged by shaking the flask for 1 h at 200 rpm in a rotatory shaker (37 °C). This medium was discarded and the flasks re-fed with fresh medium and returned to the incubator for another 2 days. These flasks were subjected to a second cycle of rotary shaking (6 h); the culture supernatant was subsequently transferred to plastic Petri dishes (Sterilin) and incubated for 30 min at 37 °C (5% CO 2 /95% air) to allow differential adhesion of any remaining microglia. The final cell suspension (characterized previously as greater than 93% A2B5 negative and positive for O1 antigen, CNPase and galactocerebroside 32 ) was collected and centrifuged (200g, 5 min). The resulting cell pellet was re-suspended in Sato's medium (high-glucose Dulbecco's modified Eagle's medium supplemented to contain 400 ng/mL 3,3′ ,5-triiodo-L-thyronine, 400 ng/ml L-thyroxine, 2 mM L-glutamine, 50 U/ml penicillin and 50 μ g/ml streptomycin, 5 ml N2 supplement) and 0.5% (v/v) FCS. Cortical OPCs were seeded in poly-D-lysine-coated (10 μ g/ml) 24-well plates at a density of 450,000 cells per well, or in 96 well microplates (72,000 cells per well) in Sato's medium and maintained at 37 °C in a 5% CO 2 /95% air incubator. After 24 h cytosine β -D-arabinoside (10 μ M; to inhibit growth of any residual astrocytes) was added.
Preparation of co-ultramicronized PEA/luteolin solutions. Co-ultramicronized PEA/luteolin was prepared as a 5 mM stock solution in 10% (w/v) Pluronic F-68. Concentration was calculated based on the molecular weight of PEA (the co-ultramicronized PEA/luteolin composite contains PEA and luteolin in a 10:1 mass ratio). The co-ultramicronized PEA/luteolin solution was sonicated for 20 min in a Elmasonic S (Singen, Germany) sonicating water bath. The co-ultramicronized PEA/luteolin solution was then diluted into Sato's medium at 100x the desired final concentration, and added (10 μ l/1 ml) directly to the cell cultures without exchange of medium. Stock solutions (5 mM) of ultramicronized PEA and luteolin were prepared/sonicated in the same manner; from these the co-respective working solutions were prepared. The concentration of Pluronic F-68 was maintained constant at 0.02%, and added to the control culture wells also. In experiments where ultramicronized PEA and luteolin were added together the final concentration of Pluronic F-68 was 0.022%; this had no effect of cell behaviours. In those experiments where rapamycin was introduced (Figs 8 and 9), the compound was first prepared as a 20 μ M stock solution in dimethylsulfoxide (DMSO) and then diluted 100-fold in Sato's medium (0.2 μ M working solution, 1% DMSO). The latter was added to the cultures (10 μ l/1 ml) to give a final concentration of 2 nM rapamycin (0.01% DMSO final). After 30 min incubation co-ultramicronized PEA/luteolin was added, as above. All cultures (including control) contained a final concentration of 0.02% Pluronic F-68 and 0.01% DMSO.

Real Time-Polymerase Chain Reaction (RT-PCR).
The day following plating, OPCs were incubated with: co-ultramicronized PEA/luteolin (1-10 μ M final concentration), 10 μ M ultramicronized PEA, 1 μ M luteolin, or 10 μ M ultramicronized PEA and 1 μ M luteolin added individually. At various times (as indicated in the corresponding figure legend) total RNA was extracted from cells by TRIzol (Invitrogen), according to the manufacturer's instructions. RNA integrity and quantity were determined by RNA 6000 Nano assay in an Agilent BioAnalyser. RT was performed with Superscript III reverse transcriptase (Invitrogen). The RT-PCR reaction was performed as described previously 66 . Primer sequences are listed in Table 1. Amounts of each gene product were calculated using linear regression analysis from standard curves, demonstrating amplification efficiencies ranging from 90 to 100%. Dissociation curves were generated for each primer pair, showing single product amplification. Data are normalized to β -actin mRNA level.
Immunocytochemistry. OPCs were cultured on poly-D-lysine-coated 8-well chamber slides (Labtek). After allowing 1 h for cells to attach co-ultramicronized PEA/luteolin was added to a final Scientific RepoRts | 5:16676 | DOI: 10.1038/srep16676 concentration of 10 μ M. Following 1 day and 4 days of incubation the cells were fixed with 4% paraformaldehyde for 30 min at 4 °C, and washed 4 × 5 min with phosphate-buffered saline (PBS)/0.05% Triton X-100, and blocked with PBS/10% FCS for 1 h at room temperature. The cells were then processed for immunostaining with primary antibodies against MBP (mouse monoclonal, Santa Cruz Biotechnology, Heidelberg, Germany, 1:400), or PLP (Abcam, rabbit polyclonal, 1:200). Cells were then washed 5 × 5 min with PBS, and incubated for 1 h at room temperature with goat anti-rabbit AlexaFluor555 (red) or goat anti-mouse AlexaFluor488 (green) secondary antibody (1:500, Invitrogen). Chamber slides were mounted beneath glass slides using Fluoromount-G (Southern Biotech, USA), and images were acquired on a Leica DMI4000 B microscope equipped for immunofluorescence (Leica Microsystems GmbH, Wetzlar, Germany) using a Leica DFC 480 digital camera.
Western blot. The day following plating, OPCs were treated with: co-ultramicronized PEA/luteolin  with one of the following primary antibodies: MBP (Santa Cruz Biotechnology, Heidelberg, Germany, 1:200); β -actin (Sigma-Aldrich, 1:25000). The membranes were washed and then incubated for 1 h with the appropriate secondary antibody (goat anti-rabbit (Bio-Rad) or goat anti-mouse (Millipore) conjugated to horseradish peroxidase at a dilution of 1:3000. Development was performed using an enhanced chemiluminescence substrate (Sigma-Aldrich). Immunreactivity was visualized using the VersaDoc Imaging System (Bio-Rad) and protein expression normalized to β -actin for band density quantification.
Statistics. Data are given as mean ± s.e.m. Statistical analyses to determine group differences were performed either by two-sample equal variance Student's t-test, or by one-way analysis of variance, followed by Dunnett's or Bonferroni's post-hoc tests for comparisons involving more than two data groups.
Significance was taken at p < 0.05.