Methylthioadenosine promotes remyelination by inducing oligodendrocyte differentiation
© The Author(s). 2017
Received: 14 December 2016
Accepted: 7 March 2017
Published: 14 April 2017
Methylthioadenosine is a metabolite of the polyamine pathway that modulates methyltransferase activity, thereby influencing DNA and protein methylation. Since methylthioadenosine produces neuroprotection in models of inflammation, ischemia and epilepsy, we set out to evaluate the role of methylthioadenosine in promoting remyelination, a process that will protect axons in demyelinating diseases and that will aid functional recovery.
The effect of methylthioadenosine in promoting remyelination was tested in mouse cerebellum organotypic cultures that were exposed to lipopolysaccharide to induce neuroinflammation, or lysolecithin to induce chemical demyelination. In addition methylthioadenosine administration was also tested in vivo, using the cuprizone model of demyelination. The molecular pathways involved in this methylthioadenosine activity were evaluated in primary cortical mouse astrocytes.
In models of neuroinflammation or chemical demyelination, methylthioadenosine prevented the loss of myelin and promoted remyelination in vitro by increasing the number of mature myelinating oligodendrocytes. Methylthioadenosine enhanced myelin production in the cuprizone model, in conjunction with a clinical improvement. Methylthioadenosine enhanced STAT-3 phosphorylation in astrocytes in vitro, and the production of ciliary neurotrophic factor (CNTF), a trophic factor known to promote oligodendrocyte maturation and differentiation, as well as remyelination.
The remyelination promoted by methylthioadenosine suggests a role for the polyamine pathway in oligodendrocyte maturation and survival, paving the way for new therapeutic strategies to promote regeneration in Multiple Sclerosis and other demyelinating diseases.
KeywordsMethylthioadenosine Remyelination Oligodendrocyte Neuroprotection Multiple sclerosis Demyelinating diseases Therapy
Multiple Sclerosis (MS) is an autoimmune disease characterized by Central Nervous System (CNS) demyelination, oligodendrocyte damage and axonal loss . Current treatments have focused on preventing inflammation and they have limited efficacy on the progressive phase. Remyelination is considered to be a promising way to protect neurons and restore functionality, a therapeutic strategy for MS that is being actively pursued [2, 3]. The myelin-axon unit is critical to maintain the trophism of long axons, and for the exchange of metabolites and macromolecules like lactate, N-acetyl Aspartate or lipids . This metabolic relationship further reinforces the attractiveness of remyelination as a therapeutic strategy to preventing axon loss in the later stages of the disease . Yet even if remyelination would have been efficient in the early stages of MS its effects might be limited in the long term, particularly given the resistance of adult oligodendrocyte precursor cells (OPCs) to differentiate into myelinating oligodendrocytes (OLs)  and the molecular changes in demyelinated axons that lead to the rejection of myelin (e.g. PSA-NCAM re-expression) .
Methylthioadenosine is a natural metabolite of the polyamine pathway and it serves many functions in cell metabolism: driving the regeneration of adenosine and methionine stocks; modulating gene expression (e.g. activating serine-threonine phosphatase 1 that leads to the dephosphorylation of SR proteins), proliferation, differentiation and apoptosis; or inhibiting protein and DNA methylation by competing with S-adenosylmethionine for methyltransferases [8, 9]. Methylthioadenosine is a selective inhibitor of protein arginine methyltransferase 5 (PRMT5) activity [10, 11], which in turn dampens cRAF methylation and degradation; this cRAF being responsible for increasing MEK1-2 and STAT3 phosphorylation . Methylthioadenosine also exhibits immunomodulatory activity by suppressing the production of pro-inflammatory cytokines and enhancing the production of anti-inflammatory cytokines through an interaction with the nuclear factor kappa B (NFkB) pathway. Interestingly, such activity produces benefits in models of autoimmune diseases like MS [13, 14]. Finally, methylthioadenosine displays neuroprotective activity, preventing neuronal damage in models of ischemia, epilepsy and Parkinson’s disease .
In light of these protective effects of methylthioadenosine, we set out to evaluate the role of methylthioadenosine in promoting OPC maturation and OL myelination in vitro and in vivo using different models of demyelination. We found that this metabolite increases the number of mature myelinating OLs. Thus, methylthioadenosine may prove to be beneficial as a therapeutic agent to manage demyelinating diseases.
In vivo studies
All animals handling was carried out in accordance with the European Council Directive (2010/63/EU) and the Spanish regulations for the procurement and care of experimental animals (1201 RD/2005, October 10). The animal protocols used were approved by the Ethical Committee on Animal Research at the University of Barcelona.
Methylthioadenosine (Sigma) was dissolved in phosphate buffered saline (PBS) at a concentration of 12 mg/ml and mice received a daily intraperitoneal (i.p.) injection at a dose of 60mg/kg. Placebo animals were injected daily with PBS alone i.p.
Cuprizone demyelinating mouse model
We fed 2-month-old C57BL/6J mice of either sex with 0.2% (wt/wt) cuprizone (Sigma Aldrich) for 4, 5 or 6 weeks (Additional file 1: Figure S1). The animals were then allowed to recover for 7, 8 or 9 weeks before methylthioadenosine (60mg/kg) or the placebo were injected daily (ip) as preventive or curative treatments. Methylthioadenosine dose was selected from previous animal studies in experimental autoimmune encephalomyelitis .
Sensory motor behavior testing
Animals were trained in the rotarod using constant speed (18rpm) 3x per day during 8 days with a rest of 5 min between each trial. Animals were tested during the remyelination phase after cuprizone treatment twice a week.
In vitro studies
Primary cortical mouse astrocytes
Cortical astrocytes were obtained from P3 C57BL6 wild-type mouse pups (of either sex) after dissection of the cortex and removal of the meninges. The tissue obtained was dissociated, plated in 75 cm2 flasks in NM-15 conditioned medium (MEM 1X, Gibco; 15% fetal bovine serum –FBS-, Gibco; 90 mM D-glucose, Sigma) and maintained in an incubator at 37°C in an atmosphere of 5% CO2. After 5 days in vitro (DIV), the cells were purified by agitating in an orbital shaker for 16–18h at 250 rpm, discarding the detached cells and incubating the adherent cells for a further two DIV in fresh medium. Astroglial cells were replated into 6-well plates and cultured to confluence. Prior to performing the experiments, NM-15 medium was replaced with DMEM/F12 (Gibco) serum free medium and the cells were left for 48 h. The cells were then treated with Lipopolysaccharide (LPS) (10 μg/ml, L4391; Sigma) overnight and methylthioadenosine (192μM) or placebo was added the following day for the ELISA and PCR studies (24 h), or it was maintained over the next 5 days for xMAP phosphorylation assays (samples collected 15, 30 min or 1 h after the last treatment was added).
Cerebellar organotypic mouse cultures
Cerebellar organotypic cultures were established as described previously . Parasagittal slices (300 μm) of the postnatal day 7 (P7) C57BL/6 mouse cerebellum (of either sex) were obtained with a tissue slicer (McIIwain), the slices were placed in medium on collagen-coated Millicell-CM culture semiporous inserts (0.4 μm; Millipore, Bedford, MA, USA) and they were kept for up to 3 weeks in an atmosphere of 5% CO2 at 37°C in 50% basal medium containing: Earle´s salts (GIBCO), 25% Hank’s balanced salt solution (GIBCO), 25% heat inactivated horse serum, 5 mg/ml glucose, 0.25 mM l-glutamine, and 25 μg/ml penicillin/streptomycin. To induce neuroinflammation, LPS (15 μg/ml, L4391; Sigma) was added to the organotypic cultures 7 days after preparing the inserts. Methylthioadenosine or a placebo were added 24 h before LPS and the samples were collected 24 h after LPS addition for Myelin Basic Protein (MBP) and Neurofilament (NFL) staining, and 0, 1, 3, 6, 12, 24, 48, 72 and 96 h after the addition of LPS for IL-1β, TNF-α, iNOS and CNPase detection. For chemical demyelination, lysolecithin (0.5 mg/ml, L4129; Sigma) was added 7 days after insert preparation and the cultures were incubated overnight (14 h.) at 37°C. Subsequently, the medium containing lysolecithin was replaced with fresh medium and methylthioadenosine or saline were added the next day and every 2 days thereafter until the tissue was analyzed after 9, 13, 17 and 21 DIV. All control cultures analyzed were time matched with the treated cultures and LLL12 (partial and reversible inhibitor of STAT3 phosphorylation, Ref: 1792–5 Deltaclon) was used at 2.5 μM and 5 μM.
Expression of inducible nitric oxide synthase (iNOS) and 2´,3´-cyclic-nucleotide 3´-phosphodiesterase (CNPase) was assessed in Western blots of mouse cerebellum organotypic slices pre-treated with methylthioadenosine (48 μM or 192 μM) or the placebo, and stimulated with LPS (15 μg/ml) at different time points (0, 1, 3, 6, 12, 24, 48, 72 and 96 h), as described previously . The Western blots were probed with a mouse antibody against CNPase (Abcam) and a rabbit antibody against iNOS (BD Bioscience).
Enzyme-linked immunosorbent assay (ELISA)
Cerebellar organotypic cultures were pre-treated with methylthioadenosine (48 μM or 192 μM) or a placebo at seven DIV and they were then stimulated with LPS (15 μg/ml) at different time points (0, 1, 3, 6, 12, 24, 48, 72 and 96 h). The culture supernatants were collected to quantify interleukin 1β (IL-1β), tumor necrosis factor (TNF-α) and CNTF using mouse enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions (eBioscience, San Diego, CA, USA).
Immunostaining was performed as previously published . After fixation with 4% paraformaldehyde, cerebellar slices were removed gently from the inserts and they were blocked for 2–4 h at room temperature in PBS + 10% goat serum + 1% bovine serum albumin (BSA) + 0.2% Triton X-100. The sections were incubated overnight at 4°C with the primary and secondary antibodies diluted in PBS + 1% goat serum + 1% BSA + 0.2% Triton X-100. After each overnight incubation, the slices were washed at room temperature three times in PBS + 0.05% Triton X-100, each wash lasting 1 h. Finally, the slices were mounted on slides, coverslipped and analyzed on a confocal microscope (LSM 510; Zeiss). Primary antibodies against the following proteins were used for immunostaining: myelin basic protein (MBP, Rat anti-MBP 82–87 antibody; Serotec), Neurofilament light (NF-L, anti-NF-L C28E10; Cell Signaling), glial fibrillary acidic protein (GFAP, rabbit anti-GFAP; DakoCytomation), oligodendrocyte transcription factor 2 (Olig 2, rabbit anti-Olig 2; Santa Cruz Biotechnology) and CNTF (rabbit polyclonal anti-CNTF; Abcam). The secondary antibodies used were: Cy2-linked mouse immunoglobulin G (IgG), Cy3-linked rabbit IgG (from goat, 1:200; GE Healthcare, Freiburg, Germany) and Fluor 488 Goat anti-Rat IgG Alexa (1:200; Molecular Probes, Eugene, OR, USA).
A confocal microscope (Laser Scanning Confocal Microscope Leica SP2) was used to obtain stacks of photographs of MBP and NFH immunolabelling at 1 μm intervals in the organotypic cultures × 40 magnification. Slices thinned after culture to approximately 30μm thickness (depending on the number of days in culture). Myelinated fibers are best observed between a depth of 5 to 20 μm from the upper surface and this is the reason why our results were taken from this level. Myelinated neurofilaments were quantified as the percentage of axons stained with NfL with MBP surrounding sheaths respect to the total number of axons (without MBP sheaths).
Percentage of the area immunoreactive for Olig2 or MBP antibodies was measured in × 40 magnification images acquired by confocal microscopy. The range of the slice was determined using Z-stack imaging at 1-μm intervals and a series of images derived from Z-stack imaging were analyzed. Briefly, binary masks were defined using the same cut-off intensity threshold value for each region of interest, which corresponds to each cell immunostained, defined as the minimum intensity because of specific staining above background values. Then, the percentage of the area occupied by Olig2 or MBP was measured automatically using ImageJ software in each cerebellum organotypic tissue. Results are given by averaging values determined in at least four separate microscopic fields from six different slices. MBP immunoreactivity of the brain stained sections was quantified after obtaining the integrated densities of the midline corpus callosum from the different animals (n = 6 per group).
Cerebellar slices were fixed for 24 h in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M PBS at 4°C, and they were then washed in 0.1 M PBS for 12 h and post-fixed in 2% osmium tetroxide in 0.1 M PBS for 1 h at 4 °C, dehydrated and included in EPON. Ultra-thin sections were stained in a 1% uranyl acetate and lead citrate solution, and visualized with a Tecnai SPIRIT Transmission Electron Microscope (FEI Company, Eindhoven, The Netherlands) working at an acceleration voltage of 120 KV. Electron micrographs were taken from similar regions in the slices. Images were acquired with a Megaview III camera and digitized with the iTEM software (Soft Imaging System). Myelin measurements were obtained with the same program and the g-ratio was determined (axon circumference to myelin circumference). More than 50 randomly selected axons were analyzed per slice, in 3–4 slices per treatment.
Intracellular protein phosphorylation assays by xMAP technology
The phosphorylation state of different intracellular proteins was evaluated in xMAP assays (Luminex). Mouse cerebellar organotypic cultures were collected in lysis buffer and tested with different antibodies against: total Akt/PKB, total p38/SAPK, total STAT3, total extracellular signal-regulated kinase (ERK)/MAPK, total P70 S6Kinase, total JNK/SAPK1, total activating transcription factor-2 (ATF-2), phosphorylated Akt/PKB (Ser473), phosphorylated Akt/PKB (Thr 308), phosphorylated p38/SAPK (Thr180/Tyr182), phosphorylated STAT3 (Tyr705/Ser727), phosphorylated ERK/MAPK (Thr185/Tyr187), phosphorylated P70 S6Kinase (Thr412), phosphorylated JNK/SAPK1 (Thr183/Tyr185) and phosphorylated ATF-2 (Thr69/71). Control beads were used to verify calibration and optical integrity of the system. Moreover, a positive and negative antibody control was used in each experiment.
RNA was extracted from the mouse cerebellar organotypic slices after different treatment times using the RNeasy kit, according to the manufacturer´s instructions (Qiagen). Using the RNA (1 μg) as a template, cDNAs were synthesized (Applied Biosystems) and the following genes were assayed by real-time PCR (Applied Biosystems): Brain derived neurotrophic factor (BDNF), Neurotrophin 3 (NT3), leukemia inhibitory factor (LIF), hairy and enhancer of split 5 (Hes5), CNTF, fibroblast growth factor 2 (FGF-2), mammalian target of rapamycin (mTOR), and nerve growth factor (NGF). To quantify gene expression the comparative CT method was used where the ΔCT value was obtained by subtracting the CT value of an endogenous gene (GAPDH for mouse samples, 18S for human samples) from the CT value of the gene of interest, in each case using a mean value of three reactions.
The data is expressed as the mean ± SEM and statistical analyses were performed with SPSS 20.0 software. The data were tested using the paired sample Student t-test or with an one-way analysis of variance (ANOVA) and the Bonferroni’s t-test for multiple comparisons. The relevant statistical results are indicated in the figure legends.
Methylthioadenosine promotes remyelination in organotypic cerebellar cultures
Methylthioadenosine increases the number of mature myelinating oligodendrocytes in cerebellar organotypic cultures
Methylthioadenosine induces remyelination and functional recovery in vivo in the cuprizone model
Methylthioadenosine promotes STAT-3 phosphorylation and CNTF release in cerebellar cultures
A physiological response of the CNS to damage is the release of trophic factors that promote cell survival and restore the CNS microenvironment . For this reason, we performed real-time PCR to measure the expression of genes encoding different trophic factors that could protect OPCs or OLs against damage when the mouse cerebellar slices were challenged with lysolecithin. Of the trophic factors associated with myelin maintenance tested (CNTF, LIF, BDNF, NT3, NGF and FGF-2), only CNTF expression had increased significantly in methylthioadenosine treated slices by day 17 DIV. We also quantified Hes5 expression, a transcription factor associated with the Notch/Jagged pathway that is implicated in the inhibition of remyelination, and mTOR, a serine-protein kinase that regulates several pathways involved in cell growth, and survival. We found that neither Hes5 nor mTOR expression changed significantly in cultures exposed to methylthioadenosine (Additional file 2: Figure S2).
Role of astrocytes in Methylthioadenosine mediated remyelination
In this study, we evaluated the ability of methylthioadenosine, a natural metabolite, to promote remyelination and protect against demyelination in models of inflammatory and chemical demyelination. Intermediate metabolism plays a critical role in maintaining cell homeostasis and in the response to stress. For example, an intermediate metabolite of the Kreb’s cycle, fumarate acid, displays anti-oxidant properties by increasing the expression of Nrf2, a transcription factor that induces the expression of anti-oxidant enzymes . Kynurenines or the methylthioadenosine precursor S-Adenosyl-Methionine are other examples, both able to modulate cell proliferation and survival in different pathological situations [22–24]. In this study we provide in vitro and in vivo evidence that methylthioadenosine promotes remyelination and protects against demyelination in two different model of demyelination. First, methylthioadenosine promotes remyelination and prevents demyelination in mouse cerebellar cultures exposed to LPS (a model of inflammatory demyelination) and promotes remyelination when exposed to lysolecithin (a model of chemical demyelination), increasing the number of mature myelinating OLs. In the in vivo model of demyelination with cuprizone, methylthioadenosine was able to prevent demyelination and also enhanced remyelination 2 weeks after starting the treatment, as reflected by a clinical improvement in locomotion in this period of time. In this study we did not use the experimental autoimmune encephalitis model because the immunomodulatory activity of methylthioadenosine would prevent the emergence of its effect in demyelination/remyelination.
In mediating these effects on myelination, methylthioadenosine appears to employ a complex and pleiotropic mechanism of action, even though it is a well-documented anti-oxidant [9, 13, 25]. In this study, we show that methylthioadenosine dampens the expression of iNOS and it enhances the phosphorylation of ATF2, a factor that protects against oxidative stress . Indeed, this finding agrees with previous studies showing that methylthioadenosine can counteract oxidative stress in other cell types and settings [9, 13, 25]. Here, we also show that methylthioadenosine can down-regulate microglia/astrocytes activation, inhibiting the release of pro-inflammatory cytokines like TNF-α and IL-1β. Microglia/astrocytes activation apparently plays a critical role in amplifying brain damage and has been considered an interesting therapeutic strategy in many brain diseases .
Methylthioadenosine also promotes STAT-3 phosphorylation in the mouse cerebellum organotypic cultures exposed to lysolecithin, a signaling protein previously associated with neurite outgrowth , axonal regeneration and survival . It should be noted that STAT-3 expression and activation in the CNS is very specific given that it can fulfill a number of different roles both in the developing and adult brain, many related to neuroprotection . The enhanced STAT-3 phosphorylation induced by methylthioadenosine in the CNS was associated with the release of CNTF by astrocytes, as demonstrated using a partial and reversible chemical inhibitor of STAT3 phosphorylation in organotypic cultures. However, understanding the mechanisms by which methylthioadenosine promotes STAT-3 phosphorylation and the release of CNTF will require further studies.
CNTF promotes remyelination and it enhances the survival and differentiation of CNS-derived oligodendroglia [30–33]. The identification of pathways that control OPC differentiation and myelination has paved the way for the development of new approaches to achieve remyelination . CNTF is produced by microglia and astrocytes following brain injury [35, 36], and it enhances the survival and differentiation of OLs [30–33]. Finally, methylthioadenosine is signalling through adenosine receptors , and activation of adenosine receptor in OPCs promote myelination . Therefore, methylthioadenosine might contribute to myelin repair through activation of adenosine pathway as well. Further studies are required to understand the role of each pathway involved in the remyelinating activity we have observed.
Based on these results, we propose that methylthioadenosine may offer a natural means to obtain cell protection and to achieve repair in circumstances of CNS damage. Methylthioadenosine therapy may recapitulate such responses and also provides benefits by limiting brain damage in neuroinflammation. The results of this study provide the opportunity to test methylthioadenosine as a regenerative therapy for neuroinflammatory diseases like MS. Accordingly, methylthioadenosine could be integrated in a combination therapy for MS taking advantage of the immunomodulatory activity of current drugs along with methylthioadenosine’s immunomodulatory and regenerative effects. Indeed, methylthioadenosine has already proven to be safe in previous phase I studies , a critical consideration when developing combination therapies for autoimmune diseases.
One-way analysis of variance
Brain derived neurotrophic factor
Bovine serum albumin
Central Nervous System
Ciliary neurotrophic factor
Days in vitro
Fibroblast growth factor 2
Glial fibrillary acidic protein
Hairy and enhancer of split 5
Inducible nitric oxide synthase
Leukemia inhibitory factor
Myelin basic protein
Mammalian target of rapamycin
Nuclear factor kappa B
Nerve growth factor
Oligodendrocyte precursor cells
Protein arginine methyltransferase 5
We would like to thank Mark Sefton for the English revision.
This work was supported by grants PI12/01823 and RD07/60/001 to PV from the Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitivity (Spain) and CERCA Programme / Generalitat de Catalunya. Funding agency has no role in the design, analysis and writing of the article.
Availability of data and material
Raw data is available from authors upon request
PV and BM designed the research plan, analyzed the results and wrote the manuscript. BM, AdP, GV, RV, OE, NE and BF performed the organotypic culture assays, and the mechanism of action studies. AM and JA performed the astrocyte assays. All authors read and approved the final manuscript.
PV and BM hold patent rights on the use of methylthioadenosine for the treatment of MS and other neurological diseases. PV has received consultancy honoraria from Digna Biotech (Pamplona, Spain).
Consent for publication
The animal protocols used were approved by the Ethical Committee on Animal Research at the University of Barcelona.
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