Elsevier

Neurobiology of Disease

Volume 56, August 2013, Pages 131-144
Neurobiology of Disease

Estrogen receptor β ligand therapy activates PI3K/Akt/mTOR signaling in oligodendrocytes and promotes remyelination in a mouse model of multiple sclerosis

https://doi.org/10.1016/j.nbd.2013.04.005Get rights and content

Highlights

  • Decrease in EAE clinical disease with therapeutic estrogen receptor β ligand, DPN

  • DPN improves axon conduction and rotorod motor performance in EAE mice.

  • DPN increases ERβ phosphorylation and activates PI3K/Akt/mTOR signaling pathway.

  • Therapeutic DPN induces remyelination during EAE.

Abstract

The identification of a drug that stimulates endogenous myelination and spares axon degeneration during multiple sclerosis (MS) could potentially reduce the rate of disease progression. Using experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we have previously shown that prophylactic administration of the estrogen receptor (ER) β ligand 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) decreases clinical disease, is neuroprotective, stimulates endogenous myelination, and improves axon conduction without altering peripheral cytokine production or reducing central nervous system (CNS) inflammation. Here, we assessed the effects of therapeutic DPN treatment during peak EAE disease, which represents a more clinically relevant treatment paradigm. In addition, we investigated the mechanism of action of DPN treatment-induced recovery during EAE. Given that prophylactic and therapeutic treatments with DPN during EAE improved remyelination-induced axon conduction, and that ER (α and β) and membrane (m)ERs are present on oligodendrocyte lineage cells, a direct effect of treatment on oligodendrocytes is likely. DPN treatment of EAE animals resulted in phosphorylated ERβ and activated the phosphatidylinositol 3-kinase (PI3K)/serine–threonine-specific protein kinase (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, a pathway required for oligodendrocyte survival and axon myelination. These results, along with our previous studies of prophylactic DPN treatment, make DPN and similar ERβ ligands immediate and favorable therapeutic candidates for demyelinating disease.

Introduction

The development of directly neuroprotective treatments that prevent the loss of oligodendrocytes (OLs) and promote the proliferation and differentiation of progenitors (OLPs) is a treatment goal for multiple sclerosis (MS). Using experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, a significant reduction in clinical symptoms has been shown with estradiol, estriol, and other estrogen receptor (ER) ligand treatments (Crawford et al., 2009a, Crawford et al., 2010, Matejuk et al., 2004, Strigard et al., 1990, Tiwari-Woodruff et al., 2007). The success of estrogens in ameliorating EAE clinical disease within the laboratory setting prompted clinical trials in Europe and the United States using estrogen therapy in human MS patients. However, synthetic estrogen supplementation is associated with increased risk of breast and uterine cancer, heart disease, and stroke (Prentice et al., 2009). Most of these side effects are thought to be mediated through ERα, not ERβ (Caringella et al., 2011). As a result, interest in ERβ as a target for neuroprotective therapy has become desirable.

To this end, we have shown that prophylactic administration of the ERβ ligand 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) decreases clinical features of EAE, is neuroprotective, stimulates endogenous myelination, and improves axon conduction without altering peripheral cytokine production or reducing central nervous system (CNS) inflammation (Crawford et al., 2010, Tiwari-Woodruff et al., 2007). However, DPN treatments were administered prior to disease induction (Crawford et al., 2010, Tiwari-Woodruff and Voskuhl, 2009). Thus, these experiments tested the potential for ERβ ligands to prevent or protect against future axonal damage. Theoretically, estrogen treatment in human relapse-remitting MS (RRMS) patients could protect against a future MS immune attack (relapse). But because treatment would be initiated after initial diagnosis, such treatment would not be considered prophylactic in the strictest sense. Given such limitations, it is important to investigate the therapeutic effects and mechanisms of potential treatments when administered following the onset of MS/EAE symptoms.

Though neuroprotection and remyelination were observed in DPN-treated EAE mice, the mechanism of action of DPN remains unknown. Recent reports have demonstrated an involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway in OL differentiation and CNS myelination during development (Flores et al., 2008, Narayanan et al., 2009). mTOR activation has also been demonstrated to promote OL differentiation (Narayanan et al., 2009). For successful remyelination to occur, activation/recapitulation of developmental myelination pathways, including PI3K/Akt/mTOR, may be required. The observation that DPN treatment during EAE increases the number of OL and axon myelination (Crawford et al., 2010) led us to hypothesize that therapeutic DPN treatment during EAE will be as effective as prophylactic treatment, and that ERβ ligand-induced remyelination/neuroprotection involves the activation of the PI3K/Akt/mTOR pathway. In this report, we verify the involvement of the ERβ ligand DPN in activating these signaling pathways to promote remyelination and repair during EAE. In addition, we show that therapeutic DPN treatment-activated second messenger signaling, which induces remyelination/neuroprotection, is not a rapid response and requires sustained treatment with DPN during EAE.

Section snippets

Animals

The generation, characterization, and genotyping of PLP_EGFP (proteolipid protein-enhanced green fluorescent protein) transgenic mice have been reported (Mallon et al., 2002). Mice were bred in house at the UCLA animal facility. All procedures were conducted in accordance with the NIH and were approved by the Animal Care and Use Committee of the Institutional Guide for the Care and Use of Laboratory Animals at UCLA.

Number of mice

Three different treatment groups (normal, vehicle+EAE, and ERβ ligand+EAE) were

Therapeutic treatment with DPN ameliorates EAE disease

To visualize and characterize the effects of therapeutic DPN treatment on demyelination and axon degeneration, active EAE was induced in PLP_EGFP transgenic C57BL/6 mice (Mallon et al., 2002, Mangiardi et al., 2011). PLP_EGFP mice show no fluorescence in neural cells or axons, but show high levels of green fluorescent protein in oligodendrocytes and myelin throughout the CNS.

Active EAE was induced using MOG 35–55 peptide (Mangiardi et al., 2011, Fig. 1). Groups of mice were treated with DPN (8 

Discussion

This is the first study to show a direct protective effect of therapeutic treatment on OLs, ultimately leading to remyelination and functional axon conduction in an inflammatory demyelinating disease. The increased number of OLs results from a combination of DPN-induced cell protection and generation, as an overall decrease in calpain/caspase/TUNEL activity in combination with an increase in proliferating OLPs was observed. The effectiveness of therapeutic treatment with the ERβ ligand DPN was

Acknowledgments

This work was generously supported by the NMSS grant RG 4538-A-2 and the NIH grant R21NS075198 to STW. We would like to thank Dr. Wendy Macklin and Dr. Terri Wood for helpful discussions and Ms. Anna Khalaj for manuscript editing. We would also like to thank the BRI core facility at UCLA for EM assistance and Sasol, North America for the generous gift of miglyol oil.

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