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The transcription factor Sox11 is expressed at high levels in developing sensory neurons and injured adult neurons but little is known about its transcriptional targets and function. In this study we examined the role of Sox11 using Neuro2a neuroblastoma cells and cultured mouse dorsal root ganglia (DRG) neurons. Results show Sox11 has an essential role in regulation of neuron survival and neurite outgrowth in Neuro2a cells and primary sensory neurons. Neuro2a cells increase expression of Sox11 as they differentiate in culture. Following addition of 20μM retinoic acid (RA), a stimulus for differentiation that enhances neurite growth and differentiation, Sox11 level rises. RNAi-mediated knockdown of Sox11 in RA-differentiated Neuro2a cells caused a decrease in neurite growth and an increase in the percent of apoptotic cells. RNA expression analysis showed that Sox11 knockdown modulated the level of mRNAs encoding several genes related to cell survival and death. Further validation in the Neuro2a model showed Sox11 knockdown increased expression of the proapoptotic gene BNIP3 (Bcl2 interacting protein 1 NIP3) decreased expression of the anti-apoptotic gene TANK (TRAF family member-associated NFκB activator). Cultured primary DRG neurons also express Sox11 and treatment with Sox11 siRNA caused a significant decrease in neurite growth and branching and a decrease in mRNA encoding actin-related protein complex 3 (Arpc3), an actin organizing protein that may be involved in axon growth. The percent of apoptotic neurons also increased in cultures of DRG neurons treated with Sox11 siRNA. Similar to Neuro2a cells, a decrease in TANK gene expression occurred, suggesting at least some overlap in Sox11 transcriptional targets in Neuro2a and DRG neurons. These data are consistent with a central role for Sox11 in regulating events that promote neurite growth and neuron survival.

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 RNAiinjected 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.

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