The Use of Myelinating Cultures as a Screen of Glycomolecules for CNS Repair

Abstract

In vitro cell-based assays have been fundamental in modern drug discovery and have led to the identification of novel therapeutics. We have developed complex mixed central nervous system (CNS) cultures, which recapitulate the normal process of myelination over time and allow the study of several parameters associated with CNS damage, both during development and after injury or disease. In particular, they have been used as a reliable screen to identify drug candidates that may promote (re)myelination and/or neurite outgrowth. Previously, using these cultures, we demonstrated that a panel of low sulphated heparin mimetics, with structures similar to heparan sulphates (HSs), can reduce astrogliosis, and promote myelination and neurite outgrowth. HSs reside in either the extracellular matrix or on the surface of cells and are thought to modulate cell signaling by both sequestering ligands, and acting as co-factors in the formation of ligand-receptor complexes. In this study, we have used these cultures as a screen to address the repair potential of numerous other commercially available sulphated glycomolecules, namely heparosans, ulvans, and fucoidans. These compounds are all known to have certain characteristics that mimic cellular glycosaminoglycans, similar to heparin mimetics. We show that the N-sulphated heparosans promoted myelination. However, O-sulphated heparosans did not affect myelination but promoted neurite outgrowth, indicating the importance of structure in HS function. Moreover, neither highly sulphated ulvans nor fucoidans had any effect on remyelination but CX-01, a low sulphated porcine intestinal heparin, promoted remyelination in vitro. These data illustrate the use of myelinating cultures as a screen and demonstrate the potential of heparin mimetics as CNS therapeutics.

Operation spinal cord regeneration: Patterning information residing in extracellular matrix glycosaminoglycans

“…Most importantly, injecting a bacterial enzyme that degrades chondroitin sulfate promotes axonal regeneration in a variety of animal models (Alilain, Horn, Hu, Dick, & Silver, ; Bradbury et al, ; Carter et al, ; Rosenzweig et al, ), which represents a feasible approach for human therapy in the near future. In addition to chondroitin sulfate, heparan sulfate glycosaminoglycans also play important roles in neuronal development and axonal regeneration (Inatani et al, ; Lander, Stipp, & Ivins, ; McCanney et al, ; Poulain & Yost, ).…”

“…However, Barnett's laboratory has developed myelinating cultures (Sorensen, Moffat, Thomson, & Barnett, ) to test the role of heparan sulfate mimetics in remyelination and/or neurite outgrowth to study such aspects of SCIs (McCanney et al, ). Their results showed that N‐sulfated heparan sulfate mimetics promote myelination whereas O‐sulfated heparan sulfate mimetics do not affect myelination but promote neurite outgrowth (McCanney et al, ). Again, these findings demonstrated that different sulfation patterns in heparan sulfate play different roles for axonal regeneration.…”

“…Furthermore, N‐sulfated heparan sulfate mimetics promote myelination. In contrast, O‐sulfated heparan sulfate mimetics do not affect myelination but promote neurite outgrowth (McCanney et al, ). Thus, both chondroitin sulfate and heparan sulfate glycosaminoglycans play important roles in axonal regeneration.…”

Validation of Recombinant Heparan Sulphate Reagents for CNS Repair

“…We have shown that a panel of non-anticoagulant HS mimetics (mHep) generated by selective desulphation of commercial heparin have beneficial effects on CNS repair mechanisms [ 8 ]. Mixed neural cell cultures generated from dissociated embryonic rat spinal cords develop robust myelinated fibers separated by nodes of Ranvier (termed myelinating cultures) [ 9 , 10 ]. Using these cultures, we have demonstrated that low-sulphated mHeps (LS-mHeps) promote many facets of CNS repair [ 11 , 12 , 13 ].…”

“…Using these cultures, we have demonstrated that low-sulphated mHeps (LS-mHeps) promote many facets of CNS repair [ 11 , 12 , 13 ]. We have used modifications of these cultures to mimic CNS injury, which has allowed us to examine factors that regulate the process of de novo myelination, remyelination and neurite outgrowth after injury [ 8 , 10 , 14 , 15 ]. We have previously shown that as many as 108 factors are released after CNS injury that may inhibit repair.…”