Regulation of PLP alternative splicing by GSK3b in oligodendrocytes in vivo

Glycogen synthase kinase 3 (GSK3) is a serine-threonine kinase which phosphorylates multiple substrates and is regulated by signal transduction pathways linked to cell proliferation, differentiation and human disease 1. Of the two isoforms, GSK3b is highly expressed in brain and is a negative regulator of oligodendrocyte (OL) differentiation in both development and myelin repair 2-4. Thus, interventions that inhibit GSK3b activity may have potential therapeutic applications. We have discovered a novel role of GSK3b in the regulation of myelin proteolipid protein (PLP) alternative splicing in OL. Pharmacological inhibition and genetic ablation of GSK3b increase the PLP/DM20 ratio in OL in vitro. GSK3b is a unique kinase in that it is constitutively active and is inhibited by upstream signals 5. Inhibitory phosphorylation of GSK3b Serine 9 (S9) by Akt, through activation of phosphoinositol-3-kinase (PI3K), results in decreased kinase activity and hyposphorylation of GSK3b downstream targets 1,6. Importantly, PI3K and Akt play pivotal roles in OL survival and differentiation 7-9, thus linking GSK3b regulation of alternative splicing with a signaling pathway critical for OL cell biology. Phosphorylation of GSK3b S9 is developmentally regulated in OL in vivo, suggesting that changes in S9 phosphorylation may regulate GSK3b activity and downstream targets involved in alternative splicing in OL (Fig. 2). One of these targets is a multifunctional splicing factor, the polypyrimidine binding protein associated-factor (PSF) 10-11. We have discovered that PSF regulates the PLP/DM20 ratio (Fig. 1B and unpublished results). siRNA-mediated knock down of PSF increases the PLP/DM20 ratio, similarly to chemical inhibition and knock down of GSK3b (Fig. 1A, B). Simultaneous knock down of PSF and GSK3b did not cause a greater increase in the PLP/DM20 ratio than the individual factor's knock down (Fig. 1E), suggesting that PSF is a downstream target of GSK3b involved in the regulation of PLP/DM20 ratio. Collectively, our data support the hypothesis that a GSK3b-PSF dependent pathway regulates the alternative splicing of PLP in OL.