Mammalian-produced chondroitinase AC mitigates axon inhibition by chondroitin sulfate proteoglycans.
Chondroitin sulfate proteoglycans (CSPGs) are up-regulated following spinal cord injury and are partly responsible for failed regeneration. Experimental paradigms in vivo that degrade chondroitin sulfate glycosaminoglycan chains with the bacterial enzyme, chondroitinase, greatly enhance the ability of axons to regenerate through the glial scar. Unfortunately, enthusiasm for this treatment paradigm is diminished by the lack of a minimally invasive and sustained delivery method. To address these deficits, we have engineered a Tet-On adenoviral vector encoding chondroitinase AC and have characterized its enzymatic function in vitro. U373 human astrocytoma cells were transduced with adenovirus and subsequently induced with doxycycline to secrete enzymatically active chondroitinase as detected by western blot and kinetic analyses. Enzymatic activity demonstrated biological relevance in studies where neurite outgrowth into and across CSPG-adsorbed regions pre-treated with conditioned media from chondroitinase secreting astrocytes was significantly increased compared with untreated controls (p < 0.0001). We also measured important parameters of enzyme activity including: pH, temperature, and enzyme stability that are fundamental to harnessing the true therapeutic potential of this approach. The use of resident cells for continuous secretion of CSPG-degrading enzymes at the site of the glial scar promises to be of greater clinical relevance than contemporary methods.
Curinga
Gabrielle M
GM
Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky 40536-0509, USA.
Snow
Diane M
DM
Mashburn
Charles
C
Kohler
Katharina
K
Thobaben
Rebecca
R
Caggiano
Anthony O
AO
Smith
George M
GM
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001369:Axons / Q000502:physiology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002645:Chickens; D002808:Chondroitin Lyases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002809:Chondroitin Sulfates / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D003001:Cloning, Molecular; D004318:Doxycycline / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation; D009416:Nerve Regeneration / Q000187:drug effects; D009475:Neurons, Afferent / Q000187:drug effects; D011509:Proteoglycans / Q000037:antagonists & inhibitors* / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D013696:Temperature
D011509:Proteoglycans; D002809:Chondroitin Sulfates; D002808:Chondroitin Lyases; D004318:Doxycycline