Desert hedgehog is a mediator of demyelination in compression neuropathies

Highlights

• Chronic nerve compression injuries were created in both WT and dhh knockout mice.

• dhh KO mice underwent more rapid and severe CNC-induced changes as compared to WT.

• dhh is one of the essential mediators to myelination in vitro and can be rescued via exogenous dhh.

• Exogenous dhh rescues CNC-mediated demyelination in an in vitro model.

• dhh is a potential therapeutic target in treating compressive neuropathies.

Abstract

The secreted protein desert hedgehog (dhh) controls the formation of the nerve perineurium during development and is a key component of Schwann cells that ensures peripheral nerve survival. We postulated that dhh may play a critical role in maintaining myelination and investigated its role in demyelination‐induced compression neuropathies by using a post-natal model of a chronic nerve injury in wildtype and dhh^−/− mice. We evaluated demyelination using electrophysiological, morphological, and molecular approaches. dhh transcripts and protein are down-regulated early after injury in wild-type mice, suggesting an intimate relationship between the hedgehog pathway and demyelination. In dhh^−/− mice, nerve injury induced more prominent and severe demyelination relative to their wild-type counterparts, suggesting a protective role of dhh. Alterations in nerve fiber characteristics included significant decreases in nerve conduction velocity, increased myelin debris, and substantial decreases in internodal length. Furthermore, in vitro studies showed that dhh blockade via either adenovirus-mediated (shRNA) or pharmacological inhibition both resulted in severe demyelination, which could be rescued by exogenous Dhh. Exogenous Dhh was protective against this demyelination and maintained myelination at baseline levels in a custom in vitro bioreactor to applied biophysical forces to myelinated DRG/Schwann cell co-cultures. Together, these results demonstrate a pivotal role for dhh in maintaining myelination. Furthermore, dhh signaling reveals a potential target for therapeutic intervention to prevent and treat demyelination of peripheral nerves in compression neuropathies.

Introduction

Compression neuropathies are highly prevalent, debilitating conditions with variable functional recovery following treatment. Evidence suggests that Schwann cells are the primary mediators of the disease process in compression neuropathies and undergo concurrent proliferation and apoptosis early (Gupta and Steward, 2003). These proliferating Schwann cells down-regulate myelin proteins, such as myelin-associated glycoprotein, leading to both demyelination and remyelination of the axon as well as axonal sprouting (Gupta et al., 2006). Interestingly, these early changes occur in the absence of both morphological and electrophysiological evidence of axonal damage (Pham and Gupta, 2009). Contrastingly, acute neural injuries, such as transection or crush injuries, are characterized by axonal injury followed by the ensuing Wallerian degeneration. Alternatively, compression neuropathies are characterized by a local demyelination in the absence of Wallerian degeneration (Gupta et al., 2012).

Central and peripheral nerve demyelination remains a major challenge for multiple neural conditions ranging from multiple sclerosis to compression neuropathies. After demyelination in the peripheral nervous system (PNS), Schwann cells remyelinate axons with a thinner layer of myelin, resulting in decreased conduction velocities and impulse propagation (Iwashita and Blakemore, 2000, Ludwin and Maitland, 1984, Schröder, 1972, Sherman and Brophy, 2005). Multiple cellular pathways have been implicated in demyelination and remyelination, including neuregulin (NRG), ERK/MAPK, Notch, and Sonic hedgehog signaling (Napoli et al., 2012, Tang et al., 2010, Taveggia et al., 2005, Woodhoo et al., 2009). Furthermore, in the context of the peripheral nervous system, all of these signaling cascades have been shown to originate from the Schwann cell (Hashimoto et al., 2008, Napoli et al., 2012, Woodhoo et al., 2009). After injury, high concentrations of NRG isoforms at the site of injury inhibit myelination and induce myelin degradation and Schwann cell proliferation and migration (Guertin et al., 2005, Zanazzi et al., 2001). Furthermore, following demyelinating injuries, there is a rapid and robust activation of ERK signaling that is critical for controlling myelin thickness (Fyffe-Maricich et al., 2013, Ishii et al., 2012, Napoli et al., 2012). Interestingly, high levels of ERK activity have also been observed in Schwann cell models of various hereditary and infectious peripheral neuropathies (Stoll et al., 2002, Suter and Scherer, 2003). While there has been much work directed at mechanisms involved with hereditary, infectious, and lysolecithin-induced demyelination, acquired compression demyelinating neuropathies have not been as widely studied.

Desert hedgehog (dhh) is a signaling molecule produced by Schwann cells that ensures axonal survival and is required to regulate myelination in the PNS (Sharghi-Namini et al., 2006). dhh induces formation of nerve perineurium when bound to its receptor patched, ptc, on peripheral nerves (Parmantier et al., 1999). While mice lacking dhh exhibit normal function and gross phenotype, they exhibit abnormal perineurium development and mini-fascicle formation. Moreover, recent data has shown that Sox10 activates dhh expression in Schwann cells via an enhancer and thereby increases dhh levels to promote formation of the perineurial sheath (Küspert et al., 2012). Decreased dhh has also been linked to diabetic neuropathies, as treatment via Sonic hedgehog (Shh) fusion proteins has shown recovery of conduction velocities (Calcutt et al., 2003). Interestingly, the three hedgehog signaling proteins, Sonic, Indian, and desert have all been well studied and shown to act through the same receptor and signaling cascade (Hahn et al., 1999, Parmantier et al., 1999, Stone et al., 1996, Varjosalo and Taipale, 2008, Xie et al., 1998). When Schwann cells are stimulated by the addition of dhh or smoothened agonists, formation of myelin segments occurs; however, in their absence, myelin segments were lacking (Yoshimura and Takeda, 2012).

As a proof of principle for using dhh for therapeutic interventions with demyelinating neuropathies, we investigated the role of dhh signaling in demyelination by using an acquired form of demyelination with a compression-induced injury. We investigated the effects of dhh deletion on the myelination of peripheral nerves in both in vivo and in vitro models of CNC injury and identify de-activation of dhh expression being the pivotal event in demyelination. Furthermore, we show that Dhh has a vital role against mechanical stimuli in our in vitro model of compression neuropathies. Finally, loss of dhh with demyelination provides evidence for considering the protein as a regulator of myelination as well as a future therapeutic target for compressive neuropathies and possibly other demyelinating diseases.