Abstract
Upregulation and activation of developmental axon guidance molecules, such as semaphorins and members of the Eph receptor tyrosine kinase family and their ligands, the ephrins, play a role in the inhibition of axonal regeneration following injury to the central nervous system. Previously we have demonstrated in a knockout model that axonal regeneration following spinal cord injury is promoted in the absence of the axon guidance protein EphA4. Antagonism of EphA4 was therefore proposed as a potential therapy to promote recovery from spinal cord injury. To further assess this potential, two soluble recombinant blockers of EphA4, unclustered ephrin-A5-Fc and EphA4-Fc, were examined for their ability to promote axonal regeneration and to improve functional outcome following spinal cord hemisection in wildtype mice. A 2-week administration of either of these blockers following spinal cord injury was sufficient to promote substantial axonal regeneration and functional recovery by 5 weeks following injury. Both inhibitors produced a moderate reduction in astrocytic gliosis, indicating that much of the effect of the blockers may be due to promotion of axon growth. These studies provide definitive evidence that soluble inhibitors of EphA4 function offer considerable therapeutic potential for the treatment of spinal cord injury and may have broader potential for the treatment of other central nervous system injuries.
Highlights
In addition to inhibitory molecules associated with myelin and astrocytes, including Nogo, myelin-associated glycoprotein and chondroitin sulfate proteoglycans [1,2,3,4,5,6], upregulation of developmental axon guidance molecules, such as semaphorins and members of the Eph receptor tyrosine kinase family, have been shown to play a role in inhibition of axonal regeneration following central nervous system injury [7,8,9,10]
We have previously shown that astrocytes in culture express EphA4 and that the EphA4 is phosphorylated by addition of clustered ephrin-A5-Fc or a number of inflammatory cytokines, including interferon gamma (IFNc) [12]
We first demonstrated that basal EphA4 phosphorylation was blocked by the addition of unclustered ephrin-A5-Fc to the astrocyte cultures, in contrast to clustered ephrin-A5-Fc that has previously been shown to promote EphA4 phosphorylation (Fig. 1a)
Summary
In addition to inhibitory molecules associated with myelin and astrocytes, including Nogo, myelin-associated glycoprotein and chondroitin sulfate proteoglycans [1,2,3,4,5,6], upregulation of developmental axon guidance molecules, such as semaphorins and members of the Eph receptor tyrosine kinase family, have been shown to play a role in inhibition of axonal regeneration following central nervous system injury [7,8,9,10]. EphA4 expression is upregulated following spinal cord injury [11,12,13] and EphA4 null mice show substantially decreased astrocytic gliosis, concomitant with extensive axonal regeneration and recovery of function [12]. Based on the null mouse results, we postulated that blockade of EphA4 function could promote repair following spinal cord injury in wildtype mice. Eph receptors and their ephrin ligands are membrane bound, and activation of the receptor requires clustering within the cell membrane [14]. Clustered ephrin-A5-Fc promotes EphA4 phosphorylation and downstream signaling in astrocytes and in neurons, inhibiting neurite outgrowth [12,17]. If the ephrin-Fc or Eph-Fc proteins are unclustered, they antagonize Eph:ephrin interactions [15,16,18], resulting in enhanced neurite outgrowth when neurons are grown in the presence of EphA4 [7,19]
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