GSK-3β inhibition promotes oligodendroglial differentiation and remyelination after spinal cord injury

Abstract

Spinal cord injury (SCI) results in extensive demyelination, leading to deleterious axon degeneration and inability of functional recovery. Remyelination has become a part of the fundamental strategy for SCI repair. Endogenous neural progenitor cells (NPCs) respond to SCI producing progenies and provide a possible source of regenerated oligodedrocytes for remyelination. During development of the central nervous system, glycogen synthase kinase-3 isoform beta (GSK-3β) is involved in multiple pathways that regulate oligodendrocyte differentiation and myelination, and thus may also play an important part in remyelination after SCI. This study aims to investigate (1) the role of GSK-3β in the differentiation of adult spinal cord derived-neural progenitor cells (ASC-NPCs); (2) whether AR-A014418 as a GSK-3β inhibitor, can promote oligodendroglial differentiation of ASC-NPCs; (3) the effect of LiCl, another GSK-3β inhibitor, on functional recovery after SCI; (4) the effects of LiCl on the myelin and axonal preservation after SCI.

Neurosphere culture from adult mouse spinal cord was performed to test the effect of GSK-3β inhibitors, LiCl and AR-A014418, on differentiation of ASC-NPCs. Phenotyping of differentiated ASC-NPCs by immunocytochemistry (ICC) was performed to identify oligodendroglia progenitor cells (OPCs) at different stages. It was shown that LiCl (1 mM) and AR-A014418 (5 μM) promoted differentiation of OPCs as labeled by oligodendrocyte lineage-specific markers: PDGFR-α, NG2 and O4, while AR-A014418 was more potent in the OPC differentiation. Moreover, preliminary data from western blot confirmed that ARA014418 (5 μM) treatment increased the expression level of pGSK (inactive form of GSK-3) in differentiated ASC-NPCs. This suggests a possible strategy to modulate endogenous NPC response to SCI: to induce the preferential differentiation of NPCs into oligodendrocyte lineage by inhibiting GSK-3β activity and thus leading to enhanced remyelination by the differentiated oligodendrocytes.

Basso Mouse Scale (BMS) open field test was used to evaluate the locomotive function of the spinal cord injured mice. The result showed that LiCl (4 mM, 200 μl) administration delivered locally at the lesion site by osmotic pump for 2 weeks improved functional recovery after SCI. Furthermore, immunohistochemistry (IHC) analyses revealed that LiCl treatment inhibited GSK-3β activity in the 〖Olig2〗^+ OPCs/oligodendrocytes, confirming LiCl as a GSK-3β inhibitor in vivo. Moreover, LiCl treatment better preserved myelin and axons detected by myelin basic protein (MBP) immunostaining and neurofilment-200 (NF-200) immunostaining respectively in the injured spinal cords. All together, the data from our in vitro and in vivo experiments suggested that LiCl treatment after spinal cord injury is beneficial for functional recovery by preventing the loss of myelin and axons after SCI and this effect is mediated via GSK-3β inhibition

This study provided evidence for the involvement of GSK-3β in the regulation of OPC differentiation and the subsequent remyelination in the injured adult spinal cord. We propose GSK-3β as an important therapeutic target for SCI repair, LiCl as a potential candidate for SCI clinical treatment and the possibility to manipulate endogenous NPCs after SCI to enhance oligodendrocyte differentiation, remyelination, and ultimately better functional recovery..