A novel DNA enzyme reduces glycosaminoglycan chains in the glial scar and allows microtransplanted dorsal root ganglia axons to regenerate beyond lesions in the spinal cord

Abstract

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-beta-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.

Figure 2.

The histogram shows the numbers of microtransplanted DRG axon fragments proximal and distal to the lesion in DNA enzyme-treated and untreated spinal cords.

Figure 1.

Confocal photomicrographs of TGF-β-stimulated primary astrocyte cultures treated with the DNA enzyme (A) or control DNA enzyme (B) or left untreated (C) and stained with the CS-56 antibody. Uptake of the biotin-streptavidin-labeled DNA enzyme can be seen in the astrocytes (D). Scale bars, 10 μm. E, F, RT-PCR-Southern blot of primary astrocyte cultures for XT-1 (E) and RT-PCR for GAPDH and cyclophilin (F) of the same mRNA. G, Dot blot of primary astrocyte cultures treated with the DNA enzyme or control DNA enzyme and stained with the CS-56 antibody. H, Restaining of the same dot blot with GFAP. I, RT-PCR-Southern blot for the XT-1 in a DNA enzyme- and control DNA enzyme-treated spinal cord.

Figure 3.

Confocal photomicrographs of treated and untreated spinal cords. Red/green (A) and green (A′) channels show the green DRGs and their axons passing the distinct borders of the lesion cores (red CS-56 staining) in a DNA enzyme-treated spinal cord. Axons that enter the lesion core become dystrophic (A, A′, arrows). Red/green (B) and green (B′) channels show an untreated spinal cord with the green DRG and red CS-56 staining. Note the struggling axon in the heart of the lesion. Scale bar, 50 μm. C, Diffusion of a labeled DNA enzyme in vivo 7 d after treatment. Scale bar, 100 μm. D, GFAP-stained section of the lesion area in a different animal. Note the disrupted astrocyte alignment in the core and borders of the lesion (L) and the aligned astrocytes (arrows) in the region where axons regenerate.