Tamoxifen promotes differentiation of oligodendrocyte progenitors in vitro

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TAMOXIFEN PROMOTES DIFFERENTIATION OF OLIGODENDROCYTE PROGENITORS IN VITRO

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H. E. BARRATT, H. C. BUDNICK, R. PARRA, R. J. LOLLEY, C. N. PERRY AND O. NESIC *

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Department of Medical Education, Texas Tech University Health Sciences Center El Paso: Paul L. Foster School of Medicine, United States

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Abstract—The most promising therapeutic approach to finding the cure for devastating demyelinating conditions is the identification of clinically safe pharmacological agents that can promote differentiation of endogenous oligodendrocyte precursor cells (OPCs). Here we show that the breast cancer medication tamoxifen (TMX), with well-documented clinical safety and confirmed beneficial effects in various models of demyelinating conditions, stimulates differentiation of rat glial progenitors to mature oligodendrocytes in vitro. Clinically applicable doses of TMX significantly increased both the number of CNPase-positive oligodendrocytes and protein levels of myelin basic protein, measured with Western blots. Furthermore, we also found that OPC differentiation was stimulated, not only by the pro-drug TMX-citrate (TMXC), but also by two main TMX metabolites, 4-hydroxy-TMX and endoxifen. Differentiating effects of TMXC and its metabolites were completely abolished in the presence of estrogen receptor (ER) antagonist, ICI182780. In contrast to TMXC and 4-hydroxy-TMX, endoxifen also induced astrogliogenesis, but independent of the ER activation. In sum, we showed that the TMX prodrug and its two main metabolites (4-hydroxy-TMX and endoxifen) promote ER-dependent oligodendrogenesis in vitro, not reported before. Given that differentiating effects of TMX were achieved with clinically safe doses, TMX is likely one of the most promising FDA-approved drugs for the possible treatment of demyelinating diseases. Published by Elsevier Ltd. on behalf of IBRO.

Key words: tamoxifen, endoxifen, 4-hydroxy-Tamoxifen, rat glial precursors, oligodendrocyte progenitors, estrogen receptors, astrocytes. 10

*Corresponding author. Address: Texas Tech University Health Science Center Paul L. Foster School of Medicine, 5001 El Paso Dr. El Paso, TX 79905, United States. Tel: +1-915-215-4357. E-mail address: [email protected] (O. Nesic). Abbreviations: 4-OHTMX, 4-hydroxytamoxifen; BBB, blood brain barrier; CNS, central nervous system; DM, differentiation medium; ER, estrogen receptor; EtOH, ethyl alcohol; OPC, oligodendrocyte progenitor cells; PM, proliferation medium; TMX, tamoxifen; TMXC, tamoxifen citrate. http://dx.doi.org/10.1016/j.neuroscience.2016.01.026 0306-4522/Published by Elsevier Ltd. on behalf of IBRO. 1

INTRODUCTION

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A large number of people suffer from demyelinating conditions, but an effective therapy is still lacking. The central nervous system (CNS) responds to demyelinating insults by inducing proliferation and maturation of oligodendrocytes from dividing oligodendrocyte progenitor cells (OPCs; (Fancy et al., 2011). Those progenitors can migrate to demyelinated axons, differentiate to mature oligodendrocytes and remyelinate axons, thus enabling functional recovery. The endogenous OPC population, which comprises upto 8% of the total glial cells in the CNS represents a promising source for the regeneration of oligodendrocytes after demyelinating insults (Franklin and Ffrench-Constant, 2008). However, differentiation of OPCs in demyelinating lesions usually fails, largely due to the failure of the precursor cells to proliferate and differentiate (Wolswijk, 2002; Rosenzweig and Carmichael, 2015). Therefore, the development of therapeutic interventions that can promote the proliferation and differentiation of OPCs is one of the main goals in identifying treatments for demyelinating conditions (Huang et al., 2011). Estrogen facilitates differentiation of OPCs in vitro (Okada et al., 2011), consistent with well-established remyelinating effects of estrogens in multiple sclerosis (Dwosh et al., 2003; Crawford et al., 2010). Despite their neuroprotective effects in various animal models of demyelinating conditions, estrogens have limited therapeutic potential due to their serious systemic adverse effects, such as peripheral feminizing in men, or dangerous pro-proliferative effects in reproductive organs of women (Shao et al., 2012). The cancer drug tamoxifen (TMX) is a selective estrogen receptor (ER) modulator that can mimic the neuroprotective effects of estrogens in the CNS without the adverse systemic effects. TMX inhibits ERs in the breast tissue, and thus remains the most widely used drug by patients with breast cancer and (ER)-positive tumors (Hoskins et al., 2009). Tamoxifen taken orally metabolizes into two main active compounds, 4-hydroxytamoxifen (4-OHTMX) and endoxifen (N-desmethyl-4-hydroxytamoxifen). These metabolites exhibit a 100-fold higher binding affinity to the ER and are more effective in suppressing cancer cell proliferation than TMX (Johnson et al., 2004; Lim et al., 2005). In humans, the conversion from TMX to endoxifen is predominant, and the circulating concentrations of the pro-drug TMX and endoxifen are considerably higher than those of 4-hydroxytamoxifen (Ahmad et al., 2010).

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However, both TMX and its metabolites readily cross the blood–brain barrier (BBB; Zarate et al., 2007; Iusuf et al., 2011), and thus can all significantly affect CNS processes. Although the neuroprotective effects of TMX have been established, the effect of TMX or its metabolites on the differentiation of OPCs has not been studied.

EXPERIMENTAL PROCEDURES

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Oligodendrocyte progenitor cells

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Purified rat glial restricted precursor cells were purchased from MTI-Global Stem (GSC-8040) and plated (3
104 cells/cm2) into poly-L-Ornithine- coated T75 dishes. Cells were kept in proliferation medium (PM) for 2 days: Neural-XÒ serum-free NSC medium with growth factors; PDGF, EGF, and FGF, 2% GS22 Supplement (Global Stem), 1% 100x Glutamax-I Supplement. Cells were split into laminin and poly-L-ornithine coated plates and treated with differentiation medium (DM): NeuralQÒ Basal medium, containing 1% Fetal Bovine Serum and appropriate treatment for 5 days with or without the following treatments.

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Treatments. (1) TMX citrate (TMXC pro-drug; Clayman Chemicals), (2) 4-hydroxytamoxifen (4-OHTMX; Sigma), or (3) endoxifen (Sigma).

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These three TMX forms were tested because 4-OHTMX and endoxifen are the main TMX metabolites, and because 4-OHTMX, endoxifen, and TMX citrate are not only present in high levels in the circulation, but they all cross the blood–brain barrier (Iusuf et al., 2011). In addition to their clinical relevance, 4OHTMX and TMXC are also extensively used in conditional mutagenesis experiments (Jokela and Vainio, 2007). We tested 3 concentrations of each TMX form: 4 lM, 0.8 lM, 0.4 lM. Those concentrations were chosen as clinically relevant, because they closely correspond to the brain concentration (1 lM) of TMX (and its metabolites) in breast cancer patients taking TMX orally (Lien et al., 1991; Iusuf et al., 2011). (4) As a positive control, we used 1 lM triiodothyronine (T3, Sigma) a known inducer of OPC differentiation (Billon et al., 2001). T3 concentration was based on (Deshmukh et al., 2013). (5) ER antagonist, ICI182780 (Santa Cruz Biotechnology), was used as a potent inhibitor of ERa and ERb (Xiao et al., 2012; Karki et al., 2013) at 1 lM ICI18278 alone or in combination with TMXC or TMX metabolites. The concentration of ICI18278 was based on Xiao et al. (2012).

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Controls. (a) cells exposed for 2d to the PM, or (b) cells exposed for 5d to the DM

Since TMXC, 4-OHTMX, endoxifen and ICI182780 were dissolved in sterile 100% ethanol (EtOH), control groups contained the corresponding concentration of EtOH that was added to the DM: (c) 1, 0.2 or 0.1 lL/mL EtOH in controls for three different concentrations of TMC, 4-OHTMX or endoxifen (4, 0.8 and 0.4 lM) (d) 0.5 lL/mL EtOH in controls for ICI182780 alone (e) 0.6 lL/mL EtOH in controls for ICI182780 + TMX (TMXC, 4-OHTMX, or endoxifen; 0.4 lM).

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Western blot analysis

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Whole-cell lysates were harvested after 5 days of treatment. Isolated proteins (20–80 lg) were boiled at 90 °C with SDS–Page buffer and 0.5% betamercaptoethanol. Western blot method is described in Guptarak et al. (2014). Antibodies used: mouse antiCNPase (Abcam; 1:300); mouse anti-MBP (Abcam; 1:100); rabbit anti-estrogen receptor a (Abcam; 1:300); rabbit anti-estrogen receptor b (Abcam; 1:300); rabbit anti-GFAP (Invitrogen; 1:500); mouse anti-bactin (Sigma; 1:5000). Mouse anti-NeuN (Millipore; 1:100). NeuN, a neuronal marker, was not detected in Western blots or by immunolabeling, suggesting the absence of neurogenesis in cells exposed to control or other treatments. Final detection was made by incubating membranes with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Goat IgG; BioRad; 1:3000) with Luminol/H2O2. Western blot images were captured and analyzed with ChemiDoc + XRS chemiluminescent imaging system (BioRad) using Quantity-One software. Protein levels of b-actin were used as a loading control in all Western blots.

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Immunofluorescent labeling

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Cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) and blocked with 2% Bovine Serum Albumin, 5% Normal Donkey Serum, and 1
Dulbecco‘s Phosphate-Buffered Saline solution containing Ca2+ and Mg2+. Antibodies used: mouse anti-A2B5 (Invitrogen; 1:500); rabbit anti-NG2 (Millipore; 1:200); mouse anti-CNPase (Abcam; 1:500); rabbit antiestrogen receptor a (Abcam; 1:500); rabbit anti-estrogen receptor b (Abcam; 1:500). Secondary antibodies (goat; Invitrogen; 1:1000) were diluted in blocking solution. To label nuclei, we used 40 ,6-diamidino-2-phenylindole (DAPI; 1:1000). Images were captured the FSX-BFX image capturing software.

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Oligodendrocyte quantification

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Cells were stained with antibody that recognizes 20 ,30 -cyc lic-nucleotide,30 -phosphodiesterase (CNPase), a myelinassociated enzyme expressed in pre-oligodendrocytes or mature oligodendrocytes (Girolamo et al., 2010). Morphological determination of mature oligodendrocytes was based on the method reported by Sperber and McMorris, 2001, and employed in Xiao et al., 2012, which

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yields data consistent with a ‘‘Sholl analysis” (Rajasekharan et al., 2009; Xiao et al., 2012). Only CNPase-positive cells that satisfied the criteria for high levels of morphological complexity (Sperber and McMorris, 2001) were counted: oligodendrocytes with five or more primary processes extending from the cell body and longer than a cell body diameter, with extensive secondary and tertiary processes (that interconnected primary processes and could not be visually separated). A typical cell with a high level of morphological complexity, thus suggesting the latest stages of oligodendrocyte maturation, is presented in Fig. 1F. Cell counting was done in a blind fashion, by a person who did not participate in culturing/treating cells, or in immunolabeling experiments. The percentage of mature oligodendrocytes was determined in three visual fields per experimental group (n = 3 different batches of cells).

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Statistical analysis

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All statistical tests were evaluated at the ‘‘a” level of 0.05, with two-tailed t-test, using the SPSS program. For multiple-group comparisons, data were analyzed using analysis of variance (ANOVA).

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RESULTS

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Rat glial progenitor cells maintained in PM for 2d were labeled with A2B5, which recognizes gangliosides in oligodendrocyte progenitors (Abney et al., 1983; Baracskay et al., 2007), and showed a typical progenitor morphology with two distinct processes (Fig. 1A; n = 3). However, 5d treatment with TMXC (4 lM; n = 14;

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Fig. 1B) changed the morphology and phenotype of OPCs: cells began to show morphologies with much longer and more highly branched processes. Neural/glial antigen-2 (NG2; n = 6/group) labeled all OPCs in both control- and TMXC-treated cells, including cells resembling mature oligodendrocytes with richly arborized processes (labeled with the white arrow), which were found predominantly in TMXC-treated OPCs. Furthermore, TMXC (4 lM, 5d) induced differentiation of progenitors to CNPase-expressing oligodendrocytes (Girolamo et al., 2010). We quantified only CNPase-positive cells with ‘‘high morphological complexity” (Sperber and McMorris, 2001), similar to a cell presented in Fig. 1E, and oligodendrocytes at the latest stages of maturation. Five- day treatment of progenitors with TMXC doubled the number of CNPase- labeled oligodendrocytes with highly ramified processes (n = 3; p = 0.003; Fig. 1F). This result was confirmed by the CNPase Western blots (n = 3; Fig. 1G). CNPase levels were markedly elevated in the presence of TMXC compared to control (C), but even more in the presence of the 5-times lower TMXC dose (4 lM [1
] vs. 0.8 lM [0.2
]; Fig. 1G). Interestingly, CNPase levels were more than two-fold higher in TMXC-treated than in T3-treated cells (n = 3), suggesting high differentiating potency of TMXC. Increased TMXC-induced oligodendrocyte differentiation was also confirmed by measuring levels of myelin basic protein (MBP), the most abundant protein produced by mature oligodendrocytes. The lowest tested TMXC concentration (0.4 lM) induced the most robust increases in MBP levels (Fig. 2A), consistent with CNPase Western blots (Fig. 1G). Quantitative analyses of MBP levels in Western blots (Fig. 2B; n = 4),

Fig. 1. (A) Glia progenitors cultured in PM showed typical progenitor morphology and were all labeled by A2B5. Calibration: 8 lm. (B) NG2-labeled cells with TMXC treatment (4 lM; 5d). OPCs at different stages of maturation, including cells with the morphology of a mature oligodendrocyte (white arrow). Calibration: 107 lm. (C) CNPase labeling of control cells: DM + 0.4 lL/mL ETOH. Calibration: 153 lm. (D) Cells treated with TMXC (4 lM, 5d) showed markedly increased abundance of the CNPase-labeled OPCs. Calibration: 153 lm. (E) A representative CNPase-labeled mature oligodendrocyte. Calibration line: 149 lm. (F) Quantitative analysis of the oligodendrocyte abundance in the control- and TMXC-treated cells (4 lM; 5d; 3 visual field per slide; n = 3 per experimental group). (G) A representative example of the CNPase Western blot (40 kDa) in control-, and in TMXC-treated cells (4 lM, 2 lM; 5d). T3 (1 lM) also significantly increased CNPase levels, but less than 0.2 lM TMXC. Please cite this article in press as: Barratt HE et al. Tamoxifen promotes differentiation of oligodendrocyte progenitors in vitro. Neuroscience (2016), http://dx.doi.org/10.1016/j.neuroscience.2016.01.026

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Fig. 2. (A) A representative example of the MBP Western blot, showing several MBP bands (20 kDa; described in Guptarak et al., 2014) in cells treated with three TMXC concentrations: 0.4 lM (0.1
); 2 lM (0.5
) and 4 lM (1
). MBP bands were faint in the control sample (C = DM + 0.4 lL/mL EtOH). MBP was not detected in OPCs treated with DM + 0.1 lL/mL or 0.2 lL/mL EtOH (not shown). (B) Quantitative analysis of MBP Western blots (n = 4). The cumulative intensity of all MBP bands was first normalized to b-actin levels, and then to the average control MBP value = 1. (C) Representative examples of MBP immunolabeling of cells treated with either vehicle (control) or TMXC (0.4 lM) for 5d. Calibration line: 153 lM. Yellow arrows mark MBP-labeled oligodendrocytes. High magnification image (calibration line: 56 lM) depicts one MBP-labeled oligodendrocytes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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indicated a significant 3.4-fold increase (p = 0.003) in 0.4 lM TMXC-treated cells. We also demonstrated a markedly increased number of MBP-labeled cells in TMXC-treated cultures (Fig. 2C). MBP-positive cells that were >100 lm in diameter (encompassing all processes) demonstrated typical morphology of mature MBP-labeled oligodendrocytes in vitro (Kawai et al., 2009; Schoemans et al., 2010; Xiao et al., 2012). Although richly arborized processes of mature oligodendrocytes were more clearly identified with the CNPase antibody (Fig. 1E), MBP- and CNPase labeled cells were similar in size and shape, suggesting that ‘‘highly ramified” CNP-ase-positive cells (Fig. 1E, F) were most likely mature MBP-expressing oligodendrocytes. Markedly increased presence of MBPpositive oligodendrocytes in TMXC-treated cultures (Fig. 2C) was consistent with the results of our MBP Western blot analyses (Fig. 2B). In sum, our data presented in Figs. 1 and 2 suggest that TMXC promoted differentiation of progenitors toward oligodendrocytes, and that 5d after TMXC treatment cells were at different stages of oligodendrocyte maturation, including a subset of mature oligodendrocytes expressing MBP. To test if the differentiating effect of TMXC was ER-dependent, the presence of ERs in OPCs was

confirmed using immunolabeling and Western blots, and MBP levels were quantified in the presence of 1 lM of the pan-ER antagonist, ICI182780. Both ERs, a and b were expressed in TMX-treated cells (n = 3/group; Fig. 3A) and in control OPCs (not shown), consistent with previous findings that showed abundant and strong ERs expression in OPCs (Xiao et al., 2012), and in mature oligodendrocytes (Platania et al., 2003; Zhang et al., 2004; Khalaj et al., 2013). We confirmed the expression of both ER forms in TMX-treated cells using Western blots (Fig. 3B; n = 2). Protein levels for ERs were detectable in two control samples: OPCs exposed only to PM for 2d (C0; Fig. 3B) or to vehicle (DM + 0.1 ll/ml EtOH) for 5d (C1; Fig. 3B), suggesting that glia progenitors during proliferation and differentiation (without TMX) expressed measurable levels of ERs, consistent with Xiao et al. (2012), and with results of our immunolabeling experiments. Western blot analyses also showed marked 2-fold increases in protein levels of both ER forms in cells treated, not only with TMXC (0.4 lM), but also with two main TMX metabolites, endoxifen (E; 0.4 lM) and 4-hydroxy TMX (4OH; 0.4 lM), not reported before. The presence of pan-ER antagonist, 1 lM ICI182780 (TMXc + A; Fig. 3C) completely abolished the effect of TMXC (0.4 lM) on MBP levels (n = 3). Cells were

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Fig. 3. (A) Immunocytochemical localization of ERa and b revealed ubiquitous expression of both forms in control- (not shown) and TMXC-treated cells (0.4 lM 5d). Calibration: 22 lm. High magnification images showed punctate ERa and b labeling in cell bodies, in processes and, more intensely, in nuclei, consistent with Xiao et al., 2012. Calibration: 14 lm. (B) A representative Western blot analysis of ERs levels in two control samples (proliferating cells and differentiating cells treated with vehicle) and in cells treated with: TMXC (0.4 lM), Endoxifen (E; 0.4 lM) and 4HydroxyTMX (4OH; 0.4 lM). ERa band of expected MW (60KDa) was detected in all samples. The intensity of ERa band markedly increased (2 fold) in TMX-treated cells. We detected ERb band (55KDa; REF), whose intensity also increased in TMX-treated cells. (C) A representative Western blot showing MBP, GFAP and b-actin bands in: C1: DM + 0.1 lL/mL EtOH. C2: DM + 0.6 lL/mL EtOH. TMXC: 0.4 lM. TMXC + A: TMXC (0.4 lM) + ICI182780 (0.1 lM). E: Endoxifen 0.4 lM. E + A: Endoxifen (0.4 lM) + ICI182780 (0.1 lM). 4-OH: 4-OHTMX 0.4 lM. 4-OH + A: 4-OHTMX (0.4 lM) + ICI182780 (0.1 lM). (D) Quantitative analyses of MBP Western blots (n = 3) in OPCs treated with: (a) TMXC or endoxifen (E), and normalized to control MBP levels (see C1 in Fig. 3C); (b) TMXC + ICI182780 (TMXC + A); or endoxifen + ICI182780 (E + A), normalized to control MBP levels (see C2 in Fig. 3C). (E) Quantitative analyses of GFAP levels (as described in Fig. 3D), with a representative GFAP Western blot. Treatment groups are described in Fig. 3C, with the addition of a C0 sample (cells exposed only to the PM for 2d). (F) A representative example of GFAP immunolabeling of endoxifen (0.4 lM, 5d)—treated cells showed the presence of GFAP-positive cells with a typical morphology of mature astrocytes. Calibration: 153 lM. High-magnification image of one GFAP-labeled astrocyte is presented below: Calibration: 22 lM. 283 284 285 286 287 288 289 290 291 292 293 294

treated with 1 lM ICI182780 + TMXC for 5d. Similar to TMXC, endoxifen (E; 0.4 lM; Fig. 3C) and 4-OHTMX (0.4 lM) also induced increases in MBP levels 5d after treatment (Fig. 3C, D; E: n = 3; 4-OHTMX: n = 5). TMXC was the most potent, and endoxifen was the least potent stimulator of the oligodendrogenesis (TMXC > 4-OHTMX > endoxifen; p < 0.05). In the presence of ICI182780 (‘‘A”; Fig. 3D) TMXC- and endoxifen-induced increases in MBP levels were reduced to control MBP levels (#p < 0.01; C = 100%; n = 3), indicating that all three TMX forms stimulate ER-dependent oligodendrogenesis.

Since glial-restricted precursors generate both oligodendrocytes and astrocytes, we also tested if TMX stimulates astrogliogenesis by measuring protein levels of glial fibrillary acidic protein (GFAP). Neither TMXC nor 4-OHTMX (0.4 lM; 5d) affected GFAP levels (n = 3; Figs. 3C, E). However, endoxifen (0.4 lM; 5d) induced significant increases in GFAP levels (3.3-fold; p = 0.04; n = 3; Fig. 3C) compared to C1 levels, which was not significantly changed in the presence of 1 lM ICI182780 (Fig. 3C, D), suggesting ER-independent astrogliogenesis. We did not detect MBP (not shown) or GFAP bands in C0-treated cells (cells exposed only to

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PM for 2d; Fig. 3E), reflecting an absence of differentiated oligodendrocytes or astrocytes during the proliferation phase, and thus confirming that endoxifen stimulated astrogliogenesis (Fig. 3C, E), and not the de-differentiation of progenitors, which can also express GFAP (Codega et al., 2014). Immunocytochemical experiments (Fig. 3F) confirmed the presence of GFAP-positive cells in endoxifen-treated OPCs (n = 3). High-magnification images of GFAP-labeled cells (Fig. 3F, image below) revealed a typical morphology of mature astrocytes. Interestingly, GFAP levels were increased by 60% (#p < 0.05; Fig. 3E) in cells exposed to TMXC + ICI182780, suggesting that the suppression of ER signaling may redirect TMXC-induced differentiation of progenitors toward astrocytes.

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DISCUSSION

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In our previous study (Guptarak et al., 2014), we have shown that TMX reverses loss of oligodendrocytes and myelin in a rat model of spinal cord injury. We also found that the beneficial effect of TMX corresponds to significant improvement of motor recovery, indicating that TMX not only restores myelin levels, but also promotes functional remyelination in injured spinal cords. Neuroprotective effects of TMX have also been demonstrated in a number of other demyelinating conditions, such as multiple sclerosis (Bebo et al., 2009), traumatic injury to the central and peripheral nervous system, and stroke (Arevalo et al., 2011), although the underlying mechanism responsible for these protective effects is not fully understood. Despite well-documented effects of TMX on oligodendrocytes and myelin in various neuropathological conditions, possible effects of TMX on OPCs have not been investigated in vivo or in vitro. Here we show that clinically applicable doses of TMX stimulate differentiation of OPCs. Clinical safety of TMX has been well established in the past four decades, not only in women but also in men (Day et al., 1999; Yildiz et al., 2008). Moreover, TMX is FDA-approved as a prophylactic treatment of women, with increased risk of breast cancer (Waters et al., 2010), further supporting its clinical safety. The translational potential of TMX as a neuroprotective intervention is additionally reinforced by its penetrability across the BBB (O’Brian et al., 1985, 1988; Zarate et al., 2007). As reported in three recent Nature publications (Deshmukh et al., 2013; Mei et al., 2014; Najm et al., 2015), the search for FDA-approved drugs that can facilitate OPC differentiation, and thus be re-used as remyelinating agents, holds the promise of finding the cure for demyelinating diseases. In the high-throughput study by Najm et al. (2015), TMX is identified as one of 22 FDA-approved drugs- that can be potential remyelinating treatments, whose potency to induce differentiation of OPCs is higher than the potency of T3, consistent with our findings (Fig. 1G). A large body of evidence firmly supports the role of thyroid hormone T3 in inducing oligodendrocyte differentiation in vitro and in vivo (Barres et al., 1994; Billon et al., 2001; Tokumoto et al., 2001; Baas et al.,

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2002; Calza et al., 2002; Deshmukh et al., 2013). However, because of its many physiological effects, T3 is not an attractive therapeutic agent. The desirable features of a promising therapeutic agent for demyelinating conditions would be both clinical safety and differentiating potency that is even higher than T3, which are both characteristics of TMX. Najm et al. (2015) study tested a relatively high TMX dose (5 lM), which showed poorer differentiating potency than lower TMX doses in our experiments (0.4 lM > 0.8 lM > 4 lM). Moreover, this TMX dose is less clinically relevant, because currently prescribed oral doses of TMX (20 mg/day) result in brain concentrations several fold lower than 5 lM (Lien et al., 1991; Iusuf et al., 2011). Therefore, our study suggests that TMX is a more promising therapeutic candidate, than suggested in Najm et al. (2015) study. The mechanism underlying lower differentiation potencies of higher TMX doses remains to be investigated. In addition, our data suggest that TMX metabolites also exert differentiating effect on OPCs (4-OHTMX > E), not reported before. Therefore, differentiating effects of systemically administered TMX on endogenous OPCs must take into consideration contributing effects of TMX metabolites, especially because they all cross the blood–brain barrier (Iusuf et al., 2011). The comparison of translational potentials of TMX and two top potential remyelinating agents identified by Najm et al. (2015) underscores clinically relevant advantages of TMX. In contrast to two identified drugs, miconazole (antifungal topical agent) and clobetasol (topical corticosteroid), systemic administration of TMX has a long history of clinical safety, and its neuroprotective effects in various models of different demyelinating conditions, including MS, have been confirmed in independent studies. However, TMX, miconazole, and clobetasol may have something in common: they activate similar signaling mechanisms that likely contribute to their differentiating effects on OPCs. Najm et al. (2015) shows that miconazole exerts its differentiating effect via extracellular signal-regulated kinases ERK1 and ERK2, which are also affected by TMX (Zheng et al., 2007). Furthermore, Najm et al. (2015) report that clobetasol significantly upregulates ER signaling, which not only involves ERK-dependent pathways in the brain (Fernandez et al., 2008; Boulware et al., 2014), but is also the main target for TMX. TMX and its metabolites bind to ERs and induce cell-specific changes (Brzozowski et al., 1997; Paige et al., 1999). Our data strongly suggest that TMXC and 4-OHTMX generate oligodendrocytes via ER-dependent process, consistent with estrogen-induced oligodendrogenesis (Okada et al., 2008, 2010). Therefore, our and other cited studies taken together strongly suggest that activation of ER-mediated pathways in OPCs is likely the most promising therapeutic target for demyelinating diseases. Interestingly, we also found that OPCs treated with TMXC and two TMX analogs induced marked upregulation of both ER forms. It has been shown that estradiol increases expression of ERs in oligodendroglial cell lines (Guzman et al., 2005), but the effect of TMX on ERs in OPCs or oligodendrocytes has not been

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reported. Although increases in ERs levels may solely reflect the OPC differentiation and higher ERs expression levels in oligodendrocytes, this explanation alone seems unlikely, since all three TMX forms induced similar increases in ER levels, in contrast to the variable differentiating effects of TMXC, endoxifen and 4OHTMX. It is also possible that ER binding affinities of TMXC, E and 4OHTMX are different. Although binding affinities of ERs in OPCs are not known, it has been shown that 4OHTMX and endoxifen have 100-times higher affinity for ERs than TMXC in cancer cells (Johnson et al., 2004; Lim et al., 2005), which would suggest that 4OHTMX and endoxifen should stimulate OPC differentiation more potently than TMXC, in contrast to our results. However, it is possible that upregulation of ERs in the presence of TMX precedes and boosts TMX-induced differentiation of OPCs, and thus contributes to the considerable differentiating potency of TMX (that exceeded the effectiveness of T3). Endoxifen, the least potent oligodendroglial differentiating agent in our experiments, also stimulated astrogliogenesis, but in an ER-independent fashion, not reported before. Differential effects of endoxifen vs. TMXC and 4-OHTMX have already been reported in cancer cells. For example, endoxifen, in contrast to other TMX forms, degrades ER in cancer cells (Wu et al., 2009), which could explain why endoxifen stimulated modest oligodendrogenesis, but it is unlikely occurring in OPCs, given that our Western blot analyses showed similar levels of ERs in cells treated by three TMX forms. However, the ER-independent effect of TMXC has also been reported: in mature astrocytes (and in cancer cells) TMX activates the G Proteincoupled Receptor 30 (Carmeci et al., 1997; Karki et al., 2013), suggesting that the transactivation of growth factor receptors in OPCs might be an ER-independent pathway underlying endoxifen-induced astrogliogenesis, which can also be activated by TMXC if ER is suppressed. Our data indicate that TMX-induced activation of classical ERs in glial progenitors generates oligodendrocytes, while their suppression (and activation of alternative pathways) generates astrocyte. However, systemically administered TMX suppresses astrogliosis (Arevalo et al., 2011; Guptarak et al., 2014), suggesting a predominant effect of TMX and its metabolites on oligodendrogenesis. It is possible that TMX-induced ER activation may have a role in switching the differentiation of endogenous progenitors from astrogliogenesis to oligodendrogenesis, which may significantly contribute to the neuroprotective effects of TMX, but has not yet been studied. Although signaling mechanisms that mediate effects of TMX and its metabolites on glial progenitors remain to be elucidated in vivo and in vitro, our data suggest that TMX is likely one of the most promising FDAapproved drugs that should be considered for the treatment of demyelinating diseases. Acknowledgment—This work was in part supported by the 2014 PLFSOM Individual Investigator-Initiated Seed Grant Program (P.I.O.N). The authors would like to thank Dr. Ruth Perez (PLFSOM) for the generous help with reagents and lab equipment.

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(Accepted 13 January 2016) (Available online xxxx)

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