Leptin sustains spontaneous remyelination in the adult central nervous system

Demyelination is a common feature of many central nervous system (CNS) diseases and is associated with neurological impairment. Demyelinated axons are spontaneously remyelinated depending on oligodendrocyte development, which mainly involves molecules expressed in the CNS environment. In this study, we found that leptin, a peripheral hormone secreted from adipocytes, promoted the proliferation of oligodendrocyte precursor cells (OPCs). Leptin increased the OPC proliferation via in vitro phosphorylation of extracellular signal regulated kinase (ERK); whereas leptin neutralization inhibited OPC proliferation and remyelination in a mouse model of toxin-induced demyelination. The OPC-specific leptin receptor long isoform (LepRb) deletion in mice inhibited both OPC proliferation and remyelination in the response to demyelination. Intrathecal leptin administration increased OPC proliferation. These results demonstrated a novel molecular mechanism by which leptin sustained OPC proliferation and remyelination in a pathological CNS.


Results
Leptin promotes OPC proliferation. To examine whether leptin promoted OPC proliferation, we first investigated whether OPC expressed leptin receptor. By immunocytochemistry, we confirmed the LepRb expression on PDGFRα -positive OPCs (Fig. 1a). We then investigated whether leptin stimulated OPC proliferation. Treatment with recombinant mouse leptin increased the ratio of Bromodeoxyuridine (BrdU) incorporation into the OPC obtained from brain and spinal cord (Fig. 1b). These results suggest that leptin promoted OPC proliferation. In immune cells, leptin receptors activate intracellular signaling, such as mitogen-activated protein kinase (MAPK) 9 , a well-known cell proliferation signaling 10 ; therefore, we investigated the involvement of MAPK activation in leptin-mediated OPC proliferation. Treatment with MAPK kinase inhibitor U0126 abolished the leptin-mediated increase in BrdU incorporation (Fig. 1c), indicating that ERK phosphorylation was required for leptin-mediated OPC proliferation. We confirmed that leptin treatment enhanced ERK phosphorylation in OPC (Fig. 1d). These data suggested that leptin promoted OPC proliferation by a mechanism that was dependent on ERK phosphorylation.

Leptin neutralization inhibits OPC proliferation and remyelination in vivo.
To assess whether leptin promoted OPC proliferation in vivo, we used the toxin-induced demyelination model, in which the myelin structures were perturbed [11][12][13] (Fig. 2a), but without neuronal damage (Fig. 2b). Leptin protein is expressed in adipose tissue abundantly 14 , which was confirmed in our model (Fig. 2c). We observed an increase in the levels of leptin around the demyelinating lesions after LPC injection (Fig. 2d); the level of leptin mRNA in the spinal cord was unchanged (Fig. 2e). In contrast, LepRb protein expression is detectable in PDGFRα -positive OPC, GFAP-positive astrocyte and NeuN-positive neuron in the spinal cord, but the intensity of LepRb immunoreactivity in these CNS cells was not changed in the response to LPC injection (Fig. 2f).
Next, we investigated whether leptin was involved in OPC proliferation after LPC injection. We started intrathecal administration of anti-leptin neutralizing antibodies at 3 days after LPC injection and evaluated the number of PDGFRα -positive OPCs around the demyelinating site. Immunohistochemical analysis revealed that, compared with the control, the mice treated with anti-leptin antibodies showed smaller numbers of BrdU and PDGFRα -double positive proliferating OPCs and GSTπ -positive mature oligodendrocytes in the dorsal spinal cord at 7 days and 14 days after LPC injection, respectively (Fig. 3a). To ask the possibility that endogenous leptin affect OPC differentiation, we compared the change of number of the cells labeled BrdU and PDGFRα 7 days after LPC injection and that of the cells labeled by BrdU and GSTπ 14 days after LPC injection. If endogenous leptin affects OPC differentiation, the number of cells labeled by BrdU and GSTπ 14 days after LPC injection is not comparative level with that of the cells labeled BrdU and PDGFRα 7 days after LPC injection. However, there is no significant difference between the increase of BrdU and PDGFRα -positive cells by anti-leptin antibodies treatment 7 days after LPC injection and that of BrdU and GSTπ -positive cells by anti-leptin treatment 14 days after LPC injection (P = 0.1531317, Fig. 3a), indicating that endogenous leptin does not affect OPC differentiation. Moreover, anti-leptin antibodies treatment did not affect the change of BrdU and Olig2-double positive cells number between 7 days and 14 days after LPC injection (P = 0.1974401, Fig. 3a), indicating that endogenous leptin does not affect the survival of oligodendrocyte lineage cells. Assessment of myelin formation by measurement of myelin basic protein (MBP)-positive area showed that, compared with the control, the mice treated with anti-leptin antibodies demonstrated larger demyelinating area in the spinal cord (Fig. 3b). Anti-leptin antibodies treatment did not affect the number of CD11b-positive microglia/macrophages around the site of LPC lesion (Fig. 3c). Therefore, these results indicated that leptin sustained the increase of OPC proliferation and subsequent remyelination.
Leptin receptors are required for OPC proliferation. Next, we probed whether leptin-mediated OPC proliferation depended on leptin receptor expression in OPCs. Immunohistochemical analysis of the spinal cord revealed the expression of LepRb in PDGFRα -positive OPCs of intact adult mice (Fig. 4a). Real time PCR analysis showed that OPC expressed all the subtypes of leptin receptors mRNA, including LepRb, the main receptor responsible for leptin signaling 15 (Fig. 4b).
Because we observed the LepRb expression on astrocyte and neuron as well as on OPCs (Fig. 2f), we generated conditional knockout mouse with Lepr knockdown in the PDGFRα -positive OPC to investigate the specific the impact for leptin receptors on OPCs. Immunohistochemical analysis confirmed that tamoxifen-inducible Cre-mediated recombination reduced LepRb protein expression in PDGFRα -positive cells in the conditional knockout mice (Pdgfrα -Cre/− :: Lepr flox/flox) compare with control littermate (− /− :: Lepr flox/flox mice) (Fig. 4a). RT-PCR confirmed decreased expression of all types of Leptin receptors mRNA in PDGFRα -positive cells of conditional knockout mice, compared with control littermates (Fig. 4b).
We then conducted LPC injection into the spinal cord of the conditional knockout mice and performed histological analysis to count the number of proliferating OPCs and mature oligodendrocytes in the spinal cord. The number of BrdU and PDGFRα -double positive cells and GSTπ -positive cells in the spinal cord of conditional knockout mice was smaller than that of the control at 7 days and 14 days after LPC injection, respectively (Fig. 4c). The change of BrdU and PDGFRα -double positive cells in conditional knockout mice compared with that in control is comparative to that of BrdU and GSTπ -double positive cells in conditional knockout mice compared with that in control 14 days after LPC injection (Fig. 4c). There were no significant differences in PDGFRα -positive cells or APC-positive cells between the conditional knockout mice and the control mice under intact conditions ( Fig. 4d and e). These data indicate that leptin receptor in OPC is not involved in OPC differentiation. We confirmed that these observations relied on myelin formation by MBP staining at 14 days after LPC injection (Fig. 4f). The number of CD11b-positive cells was not changed between the groups (Fig. 4g). These data suggest that LepRb in OPCs is required for OPC proliferation and remyelination.  g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g g     (Fig. 5a). The number of CD11b-positive cells around the lesion was not changed with or without leptin treatment (Fig. 5b). These data indicated that exogenous leptin treatment may enhance OPC proliferation.

Discussion
We found that leptin sustained OPC proliferation and contributed to remyelination in the adult CNS. The mechanism of remyelination has been investigated by focusing on the molecules in the CNS microenvironment; therefore, our findings provided the possibility that in pathological states of the CNS, the peripheral environment may also contribute to remyelination. In this context, we found that leptin promoted OPC proliferation and contributed to remyelination. The association of leptin with oligodendrocyte development has been pointed out by reports that leptin-deficient ob/ob mice brain had a significantly lower amount of myelin compared with that of control (+ /+ ) mice 16 . During brain development, leptin receptors are not expressed in OPC, but are detected in the late-phase oligodendrocyte progenitors 17,18 . Therefore, leptin-mediated myelination may be strongly supported by the promotion of the late phase of oligodendrocyte development, such as differentiation of oligodendrocyte progenitors into mature oligodendrocytes and/or increase in myelin-associated protein expression. Meanwhile, we detected that OPCs expressed leptin receptors that contributed to OPC proliferation in response to demyelination in a pathological adult CNS. Therefore, the function of leptin on oligodendrocyte development may differ between normal development and pathological conditions; one possible mechanism that may explain this difference is the change in leptin receptor expression. It was reported that leptin receptor expression in rodents was increased by several pathological stimuli, such as hypoxia 19 , injury 8 , and cytokines (IGF-1) 20 . Additional experiments that will clarify the changes in leptin receptor expression under pathological CNS conditions may enable further understanding of the role of leptin in oligodendrocyte development after CNS damage.
We observed the LepRb expression on astrocyte and neuron as well as on OPCs. This observation raises the possibility that leptin act on non-OPC cells resulting in increase of the OPC proliferating factor production and contribute to OPC proliferation. At present, it is not clarified that leptin-stimulated astrocyte and neuron increase the production of well-known OPC proliferation factor. However, there is no significant difference between the increase of BrdU and PDGFRα -positive cells by anti-leptin antibodies treatment after LPC injection and that of BrdU and PDGFRα -positive cells by LepRb deletion after LPC injection (Pint = 0.442048), highlighting that leptin have direct action to OPC.
Our observations suggested that leptin treatment promoted CNS remyelination, which is a regenerative process that is associated with recovery from neurological deficits. Therefore, we speculated that leptin treatment may be beneficial for treating demyelinating diseases. However, we should note that leptin has a proinflammatory immune response 21 . A previous report suggested that leptin reduces the number of immunosuppressive regulatory T cells in an experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis 22 . Moreover, leptin neutralization inhibits T cell proliferation and changes the T cell profile, which is associated with improvement in clinical score and prevention of disease progression in EAE 23 . Therefore, leptin therapy for CNS pathologies may not guarantee the absence of detrimental effects.
Investigation on leptin-mediated OPC-specific signal transduction may develop the leptin-associated remyelination method. Among the various intracellular signal transduction processes mediated by leptin, we focused on the involvement of ERK activation, which previously pointed out the role of myelination 24 . However, because ERK is almost universally expressed in many cells, we cannot indicate OPC-specific leptin-mediated signaling. OPC has cell type-specific signal transduction 25 ; therefore, if we identify OPC-specific signal transduction by leptin, these mechanisms will enhance the understanding of the molecular biology of leptin and may enable development of therapies for demyelinating diseases.

Methods
Mice. This study was approved by the institutional committee of Osaka University. C57BL/6 J mice were obtained from Charles River Japan or Japan SLC. B6N.Cg-Tg (Pdgfra-cre/ERT) 467Dbe/J (stock no. 018280) and B6.129P2-Leprtm1Rck/J (stock no. 008327) were purchased from the Jackson Laboratory. The experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Graduate School of Medicine Osaka University (no. 24-067-055). 7 days (left panels) and 14 days (right panels) after LPC injection. BrdU was administrated during 3-7 days after LPC injection; the graph shows quantification (n = 5-8). Primary culture of OPC and BrdU incorporation assay. Primary culture of OPC was isolated from brain and spinal cord of C57BL/6 J mice at postnatal day 1 (ref. 26). Tissues were dissected in ice-cold phosphate-buffered saline (PBS) and dissociated into single-cell suspensions using 0.25% trypsin solution. Single cell suspension was treated with anti-PDGFRα antibodies-conjugated microbeads (Miltenyi-Biotec). Isolated cells (OPCs) were plated on poly-L-lysine-coated 96-well plates (Greiner Bio-One) at a density of 1.5 × 10 4 cells/ well. The cells were maintained at 37 °C with 5% CO 2 and cultured in Dulbecco's modified Eagles medium (DMEM) supplemented with 4 mM L-glutamine, 1 mM sodium pyruvate (Sigma), 0.1% bovine serum albumin (BSA, Sigma), 50 μ g/ml apo-transferrin (Sigma), 5 μ g/ml insulin (Sigma), 30 nM sodium selenite (Sigma), 10 nM biotin (Sigma), 10 nM hydrocortisone (Sigma), 10 ng/ml platelet-derived growth factor (PDGF)-AA (PeproTech), and 10 ng/ml basic-fibroblast growth factor (FGF) (PeproTech). The ratio of BrdU incorporation was evaluated by using the Cell Proliferation ELISA and BrdU (colorimetric) kit (Roche). Cells were incubated 24 h after BrdU addition. To inhibit MAPK kinase, cells were pre-treated with 20 μ M of U0126 (9903, Cell Signaling Technology) for 15 min before the start of recombinant leptin treatment.
Immunocytochemistry. Cultures were fixed with 4% paraformaldehyde (PFA) in PBS for 30 min at room temperature. Cells were permeabilized with PBS containing 0.3% Triton X-100 and 10% goat serum (Sigma), followed by overnight treatment with primary antibodies at 4 °C. Cells were then incubated with fluorescent-labeled secondary antibody for 1 h at room temperature. The following antibodies were used: rat anti-mouse PDGFRα (1:500, 558774, BD Biosciences); chicken anti-rat LepRb (1:100, CH14104, Neuromics); Alexa Fluor 488-conjugated donkey antibody against chicken IgG and Alexa Fluor 568-conjugated goat antibody against rat IgG. Immunofluorescence images were captured with an Olympus BX60 fluorescence microscope equipped with a cooled CCD camera (DP80; Olympus).
Conditional knockout mice. The OPC-specific leptin receptor deletion mice were obtained by crossing the Lepr flox mice with the Pdgfrα -cre/ERT mice. Cre recombination in the generated mice was induced by administering tamoxifen (75 mg/kg, i.p.; Sigma-Aldrich) on each of the 11 consecutive days. To assess the efficiency of Lepr deletion at mRNA level, OPC was obtained from the brains of Cre/− ::flox/flox mice (conditional knockout mice) and − /− ::flox/flox mice (control littermates) using PDGFRα -specific antibody-coated magnetic beads (Milteny-Biotech). The relative Lepr expressions in the isolated OPCs were assessed by real time PCR. To confirm reduction of leptin receptor protein expression in OPCs, spinal cord sections were obtained from conditional knockout mice and control littermates. The sections were immunostained with antibodies against LepRb and PDGFRα , and the fluorescence intensity of LepRb in PDGFRα -positive cells was measured by ImageJ software. The relative fluorescent intensity of LepRb was calculated by the using of value obtained from control littermates.
Quantitative RT-PCR. RNA was isolated using TRIzol reagent (Invitrogen) and was purified using a RNA Clean & Concentrator-5 column (Zymo Research). cDNA synthesis was performed on 2 μ g of total RNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Samples for the Taqman assays consisted of 1× final concentration of Taqman gene expression master mix (Applied Biosystems), 500 nM of gene-specific primers, and 250 nM of Taqman probe. PCR conditions included one cycle at 50 °C for 2 min and 95 °C for 10 min; and 40 cycles at 95 °C for