WW domain-containing oxidoreductase (WWOX), a putative tumour suppressor, is suggested to be involved in the hyperphosphorylation of Alzheimer's Tau. Tau is a microtubule-associated protein that has an important role in microtubule assembly and stability. Glycogen synthase kinase 3β (GSK3β) has a vital role in Tau hyperphosphorylation at its microtubule-binding domains. Hyperphosphorylated Tau has a low affinity for microtubules, thus disrupting microtubule stability. Bioinformatics analysis indicated that WWOX contains two potential GSK3β-binding FXXXLI/VXRLE motifs. Immunofluorescence, immunoprecipitation and molecular modelling showed that WWOX interacts physically with GSK3β. We demonstrated biochemically that WWOX can bind directly to GSK3β through its short-chain alcohol dehydrogenase/reductase domain. Moreover, the overexpression of WWOX inhibited GSK3β-stimulated S396 and S404 phosphorylation within the microtubule domains of Tau, indicating that WWOX is involved in regulating GSK3β activity in cells. WWOX repressed GSK3β activity, restored the microtubule assembly activity of Tau and promoted neurite outgrowth in SH-SY5Y cells. Conversely, RNAi-mediated knockdown of WWOX in retinoic acid (RA)-differentiated SH-SY5Y cells inhibited neurite outgrowth. These results suggest that WWOX is likely to be involved in regulating GSK3β activity, reducing the level of phosphorylated Tau, and subsequently promoting neurite outgrowth during neuron differentiation. In summary, our data reveal a novel mechanism by which WWOX promotes neuronal differentiation in response to RA.

Numerous investigations support decreased glutamatergic signaling as a pathogenic mechanism of schizophrenia: yet molecular underpinnings for such dysregulation are largely unknown. In the postmortem dorsolateral prefrontal cortex, we found striking decreases in tyrosine phosphorylation of N-methyl-D aspartate (NMDA) receptor subunit 2 (GluN2), which is critical for neuroplasticity. The decreased GluN2 activity in schizophrenia may not be due to downregulation of NMDA receptors since MK-801 binding and NMDA receptor complexes in the PSD were in fact increased in schizophrenia cases. At the post-receptor level, however, we found striking reductions in the protein kinase C, Pyk 2 and Src kinase activity, which in tandem can decrease GluN2 activation. Given that Src serves as a hub of various signaling mechanisms impacting GluN2 phosphorylation, we postulated that Src hypoactivity may result from convergent alterations of various schizophrenia susceptibility pathways and thus mediate their impacts on NMDA receptor signaling. Indeed, the DLPFC of schizophrenia cases exhibit increased PSD-95 and erbB4 and decreased RPTPa and dysbindin-1, each of which reduces Src activity via protein interaction with Src. To test genomic underpinnings for Src hypoactivity, we examined genome wide association study results, incorporating 13,394 cases and 34,676 controls, which yielded no significant association of individual variants of Src and its direct regulators with schizophrenia. However, a wider protein-protein interaction based network centered on Src, showed significant enrichment of gene-level associations with schizophrenia compared to other psychiatric illnesses. Our results together demonstrate striking decreases in NMDA receptor signaling at the post-receptor level and propose Src as a nodal point of convergent dysregulations impacting NMDA receptor pathway via protein-protein associations.