The T3-induced gene KLF9 regulates oligodendrocyte differentiation and myelin regeneration

Abstract

Hypothyroidism is a well-described cause of hypomyelination. In addition, thyroid hormone (T3) has recently been shown to enhance remyelination in various animal models of CNS demyelination. What are the ways in which T3 promotes the development and regeneration of healthy myelin? To begin to understand the mechanisms by which T3 drives myelination, we have identified genes regulated specifically by T3 in purified oligodendrocyte precursor cells (OPCs). Among the genes identified by genomic expression analyses were four transcription factors, Kruppel-like factor 9 (KLF9), basic helix-loop-helix family member e22 (BHLHe22), Hairless (Hr), and Albumin D box-binding protein (DBP), all of which were induced in OPCs by both brief and long term exposure to T3. To begin to investigate the role of these genes in myelination, we focused on the most rapidly and robustly induced of these, KLF9, and found it is both necessary and sufficient to promote oligodendrocyte differentiation in vitro. Surprisingly, we found that loss of KLF9 in vivo negligibly affects the formation of CNS myelin during development, but does significantly delay remyelination in cuprizone-induced demyelinated lesions. These experiments indicate that KLF9 is likely a novel integral component of the T3-driven signaling cascade that promotes the regeneration of lost myelin. Future analyses of the roles of KLF9 and other identified T3-induced genes in myelination may lead to novel insights into how to enhance the regeneration of myelin in demyelinating diseases such as multiple sclerosis.

Introduction

Myelination has evolved in vertebrates to insulate axons and thereby promote rapid, energy efficient action potential propagation. Loss of myelin sheaths produces a wide variety of neurological symptoms, as observed in multiple sclerosis and other demyelinating diseases. In the central nervous system (CNS), myelin sheaths are produced by oligodendrocytes (OLs). The location and timing of CNS myelination is controlled largely by regulating the onset of OL differentiation, as the expression of markers of OL maturity is rapidly followed by the initiation of myelination in proximal axon tracts (Baumann and Pham-Dinh, 2001). Therefore, better understanding of the mechanisms that regulate OL differentiation should provide novel insight into how myelin formation is regulated in vivo, both during development and also during remyelination of demyelinated lesions produced by disease or injury.

Interestingly, OL differentiation can be stimulated by distinct pathways. Mitogen signaling by trophic factors such as platelet derived growth factor (PDGF) and fibroblast growth factor (FGF) maintains OL precursor cells (OPCs) in a proliferative, undifferentiated state, and mitogen withdrawal leads to rapid OL differentiation (Barres et al., 1993, Baumann and Pham-Dinh, 2001). Alternatively, thyroid hormone (T3) can override mitogen signaling to promote OL differentiation even in the presence of saturating amounts of mitogens (Barres et al., 1994, Temple and Raff, 1986). These different environmental cues apparently induce OL differentiation through distinct molecular pathways, as evidenced by differential regulation of several cell cycle control genes in response to T3 versus mitogen withdrawal (Tokumoto et al., 2001). The importance of T3 in myelin development is illustrated by the reduced CNS myelination observed in hypothyroid rodents (Leung et al., 1992, Malone et al., 1975, Walters and Morell, 1981) and human patients (Gupta et al., 1995, Jagannathan et al., 1998, Mussa et al., 2001). Hypothyroidism also results in the reduced expression of several myelin genes in vivo (Barradas et al., 2001, Ibarrola and Rodriguez-Pena, 1997, Noguchi and Sugisaki, 1984, Pombo et al., 1998, Rodriguez-Pena et al., 1993). Conversely, increasing T3 levels in vivo accelerates both myelin gene expression and myelination during development (Figueiredo et al., 1993, Marta et al., 1998, Pombo et al., 1998, Walters and Morell, 1981), and also myelin regeneration after demyelination (Calza et al., 2005, Fernandez et al., 2004, Franco et al., 2008, Harsan et al., 2008).

To better understand how T3 promotes myelination, we wanted to identify candidate downstream genes that could transduce the pro-myelinating effects of T3. Utilizing genomics, we identified a select set of genes that are immediately induced by T3 in OPCs. To begin to characterize these genes, we focused on the most robustly induced of these, KLF9. Our finding that KLF9 is induced by T3 in OPCs is consistent with previous reports looking at KLF9 regulation in the CNS (Denver et al., 1999, Furlow and Kanamori, 2002, Hoopfer et al., 2002, Martel et al., 2002). KLF9 has previously been found to regulate development in a number of different tissues, including the uterus, intestine, adipocytes, and myocytes (Mitchell and DiMario, 2010, Pei et al., 2011, Simmen et al., 2004, Simmen et al., 2007, Velarde et al., 2005). Consistent with a pro-differentiation role, KLF9 levels are reduced in endometrial tumors, and KLF9 expression can induce glioblastoma tumor cell differentiation (Simmen et al., 2010, Ying et al., 2011). We found that KLF9 is both sufficient to induce OL differentiation and required for normal T3-induced OL differentiation in vitro. Interestingly, loss of KLF9 in vivo negligibly impacts the initial development of myelin, but does significantly disrupt CNS remyelination in cuprizone-induced demyelinated lesions. Cumulatively, these data support the hypothesis that KLF9 is a novel integral component of the T3-driven signaling cascade that regulates the timing of OL differentiation and myelination regeneration. These experiments indicate that KLF9, and therefore potentially T3, may play a more crucial role in promoting remyelination after injury or disease than in the initial development of myelin, and support the concept of targeting the T3-signaling pathway to promote remyelination.