Abstract
BackgroundThere is currently no effective treatment for promoting regeneration of injured nerves in patients who have sustained injury to the central nervous system such as spinal cord injury. Chondroitinase ABC is an enzyme, which promotes neurite outgrowth and regeneration. It has shown considerable promise as a therapy for these conditions. The aim of the study is to determine if targeting chondroitinase ABC expression to the neuronal axon can further enhance its ability to promote axon outgrowth. Long-distance axon regeneration has not yet been achieved, and would be a significant step in attaining functional recovery following spinal cord injury.Methodology/Principal findingsTo investigate this, neuronal cultures were transfected with constructs encoding axon-targeted chondroitinase, non-targeted chondroitinase or GFP, and the effects on neuron outgrowth and sprouting determined on substrates either permissive or inhibitory to neuron regeneration. The mechanisms underlying the observed effects were also explored. Targeting chondroitinase to the neuronal axon markedly enhances its ability to promote neurite outgrowth. The increase in neurite length is associated with an upregulation of β-integrin staining at the axonal cell surface. Staining for phosphofocal adhesion kinase, is also increased, indicating that the β-integrins are in an activated state. Expression of chondroitinase within the neurons also resulted in a decrease in expression of PTEN and RhoA, molecules which present a block to neurite outgrowth, thus identifying two of the pathways by which ChABC promotes neurite outgrowth.Conclusions / SignificanceThe novel finding that targeting ChABC to the axon significantly enhances its ability to promote neurite extension, suggests that this may be an effective way of promoting long-distance axon regeneration following spinal cord injury. It could also potentially improve its efficacy in the treatment of other pathologies, where it has been shown to promote recovery, such as myocardial infarction, stroke and Parkinson’s disease.
Highlights
There is currently no effective treatment for promoting regeneration of injured nerves in patients following brain trauma or spinal cord injury (SCI)
Cultures of SH-SY5Y cells transfected with targeted Chondroitinase ABC (ChABC) and plated onto CSA, had significantly longer neurites when compared to cells transfected with GFP, plated onto CSA (MWUtest: P
Comparison of neurite lengths of cells transfected with targeted ChABC plated on laminin with those plated onto CSA, shows that the inhibitory effect of CSA on neurite outgrowth is reversed, as there is no significant difference in neurite length between the two groups P>0.05, Unexpectedly, cultures expressing targeted ChABC have longer neurites when plated on laminin, compared to cells transfected with GFP and plated onto laminin (MWU- test P
Summary
There is currently no effective treatment for promoting regeneration of injured nerves in patients following brain trauma or spinal cord injury (SCI). It functions by removing growth-inhibitory CSPGs at the lesion site and this promotes neural plasticity by dissolution of perineuronal nets [4, 5,6]. This latter action results in the formation of new synaptic connections by intact, undamaged neurons, with the beneficial consequence of allowing spared axons to replace the function of damaged neurons. This is of particular importance for promoting recovery following SCI, as most injuries are not complete and spared axons remain. Long-distance axon regeneration has not yet been achieved, and would be a significant step in attaining functional recovery following spinal cord injury.
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