Sox11 transcription factor modulates peripheral nerve regeneration in adult mice

Abstract

The ability of adult peripheral sensory neurons to undergo functional and anatomical recovery following nerve injury is due in part to successful activation of transcriptional regulatory pathways. Previous in vitro evidence had suggested that the transcription factor Sox11, a HMG-domain containing protein that is highly expressed in developing sensory neurons, is an important component of this regenerative transcriptional control program. To further test the role of Sox11 in an in vivo system, we developed a new approach to specifically target small interfering RNAs (siRNAs) conjugated to the membrane permeable molecule Penetratin to injured sensory afferents. Injection of Sox11 siRNAs into the mouse saphenous nerve caused a transient knockdown of Sox11 mRNA that transiently inhibited in vivo regeneration. Electron microscopic level analysis of Sox11 RNAi-injected nerves showed that regeneration of myelinated and unmyelinated axons was inhibited. Nearly all neurons in ganglia of crushed nerves that were Sox11 immunopositive showed colabeling for the stress and injury-associated activating transcription factor 3 (ATF3). In addition, treatment with Sox11 siRNAs in vitro and in vivo caused a transcriptional and translational level reduction in ATF3 expression. These anatomical and expression data support an intrinsic role for Sox11 in events that underlie successful regeneration following peripheral nerve injury.

Introduction

The SRY-box containing gene 11 (Sox11) transcription factor is a member of the group C high mobility group transcription factor family, which also includes Sox4 and Sox12 (Azuma et al., 1999, Dy et al., 2008, Hargrave et al., 1997, Wright et al., 1993). Group C proteins are expressed throughout the developing nervous system and comprise one of seven sequence homology defined subgroups in the Sox family (Bergsland et al., 2006, Wegner and Stolt, 2005). Sox gene expression is regulated both spatially and temporally during development (Gubbay et al., 1990, Wilson and Koopman, 2002, Wright et al., 1993) and all appear to have critical roles in embryonic growth (Wegner and Stolt, 2005). Sox factors may activate or repress transcription of target genes and in many cases overlap in expression. For example, in developing mice both Sox11 and Sox4 are required for expression of the pan-neuronal gene Tuj1 (Bergsland et al., 2006). This overlap, coupled with embryonic or perinatal lethality in gene deletion models, has made detailed study of the functional roles of Sox factors challenging (Cheung et al., 2000, Sock et al., 2004).

In addition to their role in development, some Sox proteins have been found to modulate adult injury responses as well. For example, increased Sox18 expression in epithelial cells correlates with capillary sprouting after wounding (Darby et al., 2001). Similarly, Sox15 knockout mice display disrupted muscle regeneration (Meeson et al., 2007) and increased expression of Sox 5, 6 or 9 is important for healing of bone fractures (Uusitalo et al., 2001). Whether Sox11 has a similar role in adult tissues has not been directly tested. Sox11 is expressed at high levels in developing sensory neurons and is hypothesized to regulate neuronal maturation (Hargrave et al., 1997). Its expression is significantly reduced during late phases of gestation and normally remains at low levels in adult neurons. A robust induction occurs however, in adult dorsal root sensory neurons following axotomy (Jankowski et al., 2006, Tanabe et al., 2003), suggesting a regulatory role in nerve regeneration. In support of this possibility, cultured adult DRG neurons treated with Sox11 siRNAs exhibit a significant decrease in regeneration as indicated by reduced neurite length and branching index (Jankowski et al., 2006).

Elements that regulate regeneration in the peripheral nervous system (PNS) following nerve injury are of significant interest because, in contrast to the central nervous system, axon regeneration in the PNS can occur quite successfully (Cajal, 1928, Silver and Miller, 2004). Transcription factors such as c-Jun, a component of the AP-1 transcription factor complex, and activated transcription factor 3 (ATF3), a member of the ATF/cAMP-responsive element binding protein (CREB) family, may underlie part of this dichotomy in regenerative ability. Both genes are normally expressed at low levels in adult DRG neurons and rise significantly following peripheral axotomy (Lindwall et al., 2004, Tsujino et al., 2000, Raivich et al., 2004) or after dissociation and culture (Seijffers et al., 2006). For ATF3, the increase in expression is hypothesized to facilitate expression of survival and axon growth related genes (Lindwall and Kanje, 2005, Seijffers et al., 2006). Indeed, constitutive expression of a Thy-1.2 ATF3 transgene in neurons of transgenic mice enhanced PNS regeneration (Seijffers et al., 2007). Because Sox11 is similarly upregulated following axotomy, we tested its role in vivo using a newly developed RNAi nerve injection delivery system. Results indicate that Sox11 has an important role in axon growth that may involve interaction with ATF3.