Low sulfated heparins target multiple proteins for central nervous system repair

Abstract

The lack of endogenous repair following spinal cord injury (SCI) accounts for the frequent permanent deficits for which effective treatments are absent. Previously, we demonstrated that low sulfated modified heparin mimetics (LS‐mHeps) attenuate astrocytosis, suggesting they may represent a novel therapeutic approach. mHeps are glycomolecules with structural similarities to resident heparan sulfates (HS), which modulate cell signaling by both sequestering ligands, and acting as cofactors in the formation of ligand–receptor complexes. To explore whether mHeps can affect the myelination and neurite outgrowth necessary for repair after SCI, we created lesioned or demyelinated neural cell co‐cultures and exposed them with a panel of mHeps with varying degrees and positions of their sulfate moieties. LS‐mHep7 enhanced neurite outgrowth and myelination, whereas highly sulfated mHeps (HS‐mHeps) had attenuating effects. LS‐mHeps had no effects on myelination or neurite extension in developing, uninjured myelinating cultures, suggesting they might exert their proregenerating effects by modulating or sequestering inhibitory factors secreted after injury. To investigate this, we examined conditioned media from cultures using chemokine arrays and conducted an unbiased proteomics approach by applying TMT‐LC/MS to mHep7 affinity purified conditioned media from these cultures. Multiple protein factors reported to play a role in damage or repair mechanisms were identified, including amyloid betaA4. Amyloid beta peptide (1–42) was validated as an important candidate by treating myelination cultures and shown to inhibit myelination. Thus, we propose that LS‐mHeps exert multiple beneficial effects on mechanisms supporting enhanced repair, and represent novel candidates as therapeutics for CNS damage.

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

“…The same panel of heparosans (detailed in Figure 2a) was used to treat MC-Dev and were found to have no effect on developmental myelination or on neurite density, similar to results previously published by us using sulphated polysaccharides [10] (Figure 3a,b). This suggests that the beneficial effect on myelination following K5 NS treatment in the MC-Inj screen is unlikely to be a direct effect on the compounds ability to promote oligodendrocyte precursor cell (OPC) differentiation and/or the process of oligodendrocyte ensheathment of axons.…”

“…Previously, we demonstrated using a panel of mHeps that the level of sulphation effected their potential to promote re/myelination and neurite outgrowth as well as decreasing characteristics of astrogliosis with only low sulphated mHeps promoting repair [11,12,13]. Moreover, their mechanism-of-action appeared to function via sequestering molecules that inhibited the repair process [10]. This was substantiated by other studies, which demonstrated that heparin promotes PNS myelination in DRG via the specific inhibition of the soluble immunoglobulin (Ig)-containing isoforms of neuregulin 1 (i.e., NRG1 types I and II), which negatively regulate myelination [43].…”

“…MC-Demy cultures were generated based on our previously published methods [9,10]. At 24 DIV, myelinating cultures were demyelinated by overnight incubation with the Z2 antibody (100 ng/mL Hybridoma, a kind gift from Christopher Linington, University of Glasgow), which recognises myelin oligodendrocyte glycoprotein (MOG) and a rabbit complement (100 μg/mL, Millipore, Watford, UK) at 37 °C.…”

“…Lastly, we used these cultures to mimic diseases in which demyelination occurs, such as multiple sclerosis (MS). Axons were demyelinated using complement and the myelin oligodendrocyte glycoprotein (MOG) antibody, and the resulting cultures could be used to examine factors that promote remyelination [9,10]; we have termed these myelinating culture-demyelination (MC-Demy) (Figure 1). These cultures can be easily generated in large numbers, allowing us to screen several therapeutics simultaneously [1].…”

“…We, therefore, applied the panel of mHeps with varying sulphation patterns to all three types of myelinating cultures. Although the mHeps had no effect on developmental myelination (MC-Dev), we found that low sulphated (LS) mHeps could promote myelination and neurite outgrowth and act via sequestering inhibitors of repair in MC-Demy and MC-Inj [10]. This led us to propose that LS-mHeps exert multiple beneficial effects on mechanisms supporting enhanced repair, and represent novel candidates as therapeutics for CNS damage.…”