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.
Subject(s)
Arthrogryposis/complications , Demyelinating Diseases/etiology , Demyelinating Diseases/metabolism , Gene Expression Regulation/physiology , Hedgehog Proteins/deficiency , Hedgehog Proteins/metabolism , Hereditary Sensory and Motor Neuropathy/complications , Animals , Animals, Newborn , Axons/pathology , Cell Culture Techniques , Disease Models, Animal , Embryo, Mammalian , Functional Laterality/genetics , Ganglia, Spinal/cytology , Hedgehog Proteins/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myelin Sheath/metabolism , Myelin Sheath/pathology , Neural Conduction/genetics , Neurons/physiology , Peripheral Nerves/pathology , Peripheral Nerves/ultrastructure , Rats , Rats, Sprague-Dawley , Schwann Cells/physiologyABSTRACT
OBJECTIVE: Traumatic peripheral nerve injuries often produce permanent functional deficits despite optimal surgical and medical management. One reason for the impaired target organ reinnervation is degradation of motor endplates during prolonged denervation. Here we investigate the effect of preserving agrin on the stability of denervated endplates. Because matrix metalloproteinase 3 (MMP3) is known to degrade agrin, we examined the changes in endplate structure following traumatic nerve injury in MMP3 knockout mice. METHODS: After creation of a critical size nerve defect to preclude reinnervation, we characterized receptor area, receptor density, and endplate morphology in denervated plantaris muscles in wild-type and MMP3 null mice. The level of agrin and muscle-specific kinase (MuSK) was assessed at denervated endplates. In addition, denervated muscles were subjected to ex vivo stimulation with acetylcholine. Finally, reinnervation potential was compared after long-term denervation. RESULTS: In wild-type mice, the endplates demonstrated time-dependent decreases in area and receptor density and conversion to an immature receptor phenotype. In striking contrast, all denervation-induced changes were attenuated in MMP3 null mice, with endplates retaining their differentiated form. Agrin and MuSK were preserved in endplates from denervated MMP3 null animals. Furthermore, denervated muscles from MMP3 null mice demonstrated greater endplate efficacy and reinnervation. INTERPRETATION: These results demonstrate a critical role for MMP3 in motor endplate remodeling, and reveal a potential target for therapeutic intervention to prevent motor endplate degradation following nerve injury.
