CNS lesions induce production of ECM molecules that inhibit axon regeneration. One major inhibitory family is the chondroitin sulfate proteoglycans (CSPGs). Reduction of their glycosaminoglycan (GAG) chains with chondroitinase ABC leads to increased axon regeneration that does not extend well past the lesion. Chondroitinase ABC, however, is unable to completely digest the GAG chains from the protein core, leaving an inhibitory "stub" carbohydrate behind. We used a newly designed DNA enzyme, which targets the mRNA of a critical enzyme that initiates glycosylation of the protein backbone of PGs, xylosyltransferase-1. DNA enzyme administration to TGF--stimulated astrocytes in culture reduced specific GAG chains. The same DNA enzyme applied to the injured spinal cord led to a strong reduction of the GAG chains in the lesion penumbra and allowed axons to regenerate around the core of the lesion. Our experiments demonstrate the critical role of PGs, and particularly those in the penumbra, in causing regeneration failure in the adult spinal cord.
We have addressed the question of whether a family of axon growth-promoting molecules known as the laminins may play a role during axon regeneration in the CNS. A narrow sickle-shaped region containing a basal lamina-independent form of laminin exists in and around the cell bodies and proximal portion of the apical dendrites of CA3 pyramidal neurons of the postnatal hippocampus. To understand the possible function of laminin in axon regeneration within this pathway, we have manipulated laminin synthesis at the mRNA level in a slice culture model of the lesioned mossy system. In this model early postnatal mossy fibers severed near the hilus can regenerate across the lesion and elongate rapidly within strata lucidum and pyramidale. In slice cultures of the postnatal day 4 hippocampus, 2 d before lesion and then continuing for 1-5 d after lesion, translation of the gamma1 chain product of laminin was reduced by using antisense oligodeoxyribonucleotides and DNA enzymes. In the setting of the lesioned organotypic hippocampal slice, astroglial repair of the lesion and overall glial patterning were unperturbed by the antisense or DNA enzyme treatments. However, unlike controls, in the treated, lesioned slices the vast majority of regenerating mossy fibers could not cross the lesion site; those that did were very much shorter than usual, and they took a meandering course. In a recovery experiment in which the DNA enzyme or antisense oligos were washed away, laminin immunoreactivity returned and mossy fiber regeneration resumed. These results demonstrate the critical role of laminin(s) in an axon regeneration model of the CNS.
The failure of CNS regeneration and subsequent motor and sensory loss remain major unsolved questions despite massive accumulation of experimental observations and results. The sheer volume of data and the variety of resources from which these data are generated make it difficult to integrate prior work to build new hypotheses. To address these challenges we developed a prototypic suite of computer programs to extract protein names from relevant publications and databases and associated each of them with several general categories of biological functions in nerve regeneration. To illustrate the usefulness of our data mining approach, we utilized the program output to generate a hypothesis for a biological function of CD44 interaction with osteopontin (OPN) and laminin in axon outgrowth of CNS neurons. We identified CD44 expression in retinal ganglion cells and when these neurons were plated on poly-L-lysine 3% of them initiated axon growth, on OPN 15%, on laminin-111 (1·) 41%, on laminin-111 (0.5·) 56%, and on a mixture of OPN and laminin (1·) 67% of neurons generated axon growth. With the aid of a deoxyribozyme (DNA enzyme) to CD44 that digests the target mRNA, we demonstrated that a reduction of CD44 expression led to reduced axon initiation of retinal ganglion cells on all substrates. We suggest that such an integrative, applied systems biology approach to CNS trauma will be critical to understand and ultimately overcome the failure of CNS regeneration.
In the injured spinal cord, proteoglycans (PGs) within scar tissue obstruct axon growth through their glycosaminoglycan (GAG)-side chains. The formation of GAG-side chains (glycosylation) is catalysed by xylosyltransferase-1 (XT-1). Here, we knocked down XT-1 mRNA using a tailored deoxyribozyme (DNAXTas) and hypothesized that this would decrease the amount of glycosylated PGs and, consequently, promote axon growth in the adult rat spinal cord. A continuous 2-week delivery of DNAXTas near the rostral border of a peripheral nerve graft bridging the transected dorsal columns in the thoracic spinal cord resulted in an 81% decrease in XT-1 mRNA, an average of 1.4-fold reduction in GAG-side chains of chondroitin sulphate or heparan sulphate-PGs and 2.2-fold reduction in neurocan and brevican core proteins in scar tissue. Additionally, compared to control deoxyribozyme, the DNAXTas treatment resulted in a 9-fold increase in length and a 4-fold increase in density of ascending axons growing through the nerve graft and scar tissue present at the rostral spinal cord. Together our data showed that treatment with a deoxyribozyme against XT-1 mRNA decreased the amount of glycosylated PGs and promoted axon growth through scar tissue in the injured spinal cord. The deoxyribozyme approach may become a contributing factor in spinal cord repair strategies.
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