WWOX Phosphorylation, Signaling, and Role in Neurodegeneration

Abstract

Homozygous null mutation of tumor suppressor WWOX/Wwox gene leads to severe neural diseases, metabolic disorders and early death in the newborns of humans, mice and rats. WWOX is frequently downregulated in the hippocampi of patients with Alzheimer's disease (AD). In vitro analysis revealed that knockdown of WWOX protein in neuroblastoma cells results in aggregation of TRAPPC6AΔ, TIAF1, amyloid β, and Tau in a sequential manner. Indeed, TRAPPC6AΔ and TIAF1, but not tau and amyloid β, aggregates are present in the brains of healthy mid-aged individuals. It is reasonable to assume that very slow activation of a protein aggregation cascade starts sequentially with TRAPPC6AΔ and TIAF1 aggregation at mid-ages, then caspase activation and APP de-phosphorylation and degradation, and final accumulation of amyloid β and Tau aggregates in the brains at greater than 70 years old. WWOX binds Tau-hyperphosphorylating enzymes (e.g., GSK-3β) and blocks their functions, thereby supporting neuronal survival and differentiation. As a neuronal protective hormone, 17β-estradiol (E2) binds WWOX at an NSYK motif in the C-terminal SDR (short-chain alcohol dehydrogenase/reductase) domain. In this review, we discuss how WWOX and E2 block protein aggregation during neurodegeneration, and how a 31-amino-acid zinc finger-like Zfra peptide restores memory loss in mice.

Keywords: 17β-estradiol; TIAF1; TRAPPC6AΔ; WWOX; Zfra; neurodegeneration; sex steroid hormone receptor.

FIGURE 1

Potential role of WWOX and BCD in neuronal death during traumatic injury. (A) Needle insult to the brain was carried out in rats. Post injury for 3 and 24 h, the animals were sacrificed. By immunoelectron microscopy, accumulation of Hyal-2 and WWOX is found in the nuclei of dying neurons in the brain cortex (Hsu et al., 2017). (B) Nuclear accumulation of Hyal-2 and WWOX leads to BCD (Chen et al., 2015). Both schematic graphs and a real-time image are shown. If p53 competes with Hyal-2 to complex with WWOX, both p53/WWOX proteins are retained in the cytoplasm and the extent of Hyal-2/WWOX complex is reduced, no BCD occurs (Hsu et al., 2017). (Data in A is adapted from Hsu et al., 2017, republishing according to the guideline of Oncotarget).

FIGURE 2

WWOX gene and protein expression in human brain. (A) WWOX gene expression was analyzed using the database in the Allen Brain Atlas (http://www.brain-map.org). Detectable signals for WWOX gene expression were found in six postmortem normal individuals (age 42.5 ± 13.4; 3 Caucasians, 2 blacks, 1 Hispanic). Representative WWOX gene expression levels in the brain white matter, pons and hippocampus are shown. Also, see the Supplementary Table S1 for WWOX gene expression in the normal brains (around one-fold changes for all indicated regions). (B,C) In the “Possible AD” and “Traumatic Brain Injury” groups (77–100+ years old), WWOX gene expression levels are shown in the indicated brain areas. (D,E) Expression of wild type WWOX (46 kDa) and isoform WWOX2 (41 kDa) is downregulated in the neurons of AD hippocampi compared with normal controls (a representative set from five immunostains; magnification, 200×; data from Sze et al., 2004). (F) In AD patients, the protein levels for WWOX (n = 8), isoform WWOX2 (n = 8), and pY33-WWOX (n = 6) are significantly downregulated in the hippocampi as determined by Western blotting, compared to age-matched controls (∼32 ± 5% reduction, p < 0.005; data with minor revisions for the art work are adapted from Sze et al., 2004; republishing according to the guideline of the Journal of Biological Chemistry).

FIGURE 3

WWOX limits Tau hyperphosphorylation and aggregation. The C-terminal SDR domain of WWOX physically binds GSK3β preventing hyperphosphorylation of Tau (Sze et al., 2004; Wang et al., 2012). Also, the first WW domain binds JNK, thereby preventing Tau hyperphosphorylation (Sze et al., 2004). The first WW domain binds ERK (Huang et al., 2016). Tau protein supports polymerization of tubulin monomers to assemble microtubules, which are needed for neurite outgrowth (Wang et al., 2012).

FIGURE 4

TPC6AΔ and TIAF1 in a cascade of protein aggregation and WWOX blocks the aggregation. (A) Representative human AD hippocampal tissue sections were pre-stained with Bielschowsky stain, followed by staining with specific antibody against TIAF1 (green), and Aβ (red) and DAPI for nuclei. A representative confocal image of a plaque is shown (Lee et al., 2010). (B) Shown is a TIAF1-containing plaque from a hippocampal section of a 9-month-old APP/PS1 transgenic mouse, containing Aβ aggregates in the center (Lee et al., 2010). (C) In representative human brain cortical tissue sections from AD patients and age-matched controls, a pS35-TPC6AΔ-containing plaque is shown. In negative controls, the immunizing peptide blocks the immunoreactivity (Chang et al., 2015). (D,E) Presence of pS35-TPC6AΔ and pT181-Tau aggregates is shown in the cortex and hippocampus of 3-week-old Wwox knockout mice (Chang et al., 2015). (F) Endogenous TPC6A and TPC6AΔ shuttle between nucleoli and mitochondria. Ser35 phosphorylation supports shuttling from the nucleus to the nucleolus, and Tyr112 phosphorylation is needed for translocation from the nucleolus to the mitochondrion (Chang and Chang, 2015). (G) Upon WWOX downregulation, a sequential protein aggregation cascade occurs. When WWOX level is reduced, pS35-TPC6AΔ starts to polymerize and recruit pS37-TIAF1 for further polymerization and accumulation in the outer membrane of mitochondria (Chang and Chang, 2015; Chang et al., 2015). The aggregated pS35-TPC6AΔ and pS37-TIAF1 cause caspase 3 activation and cytochrome c release. The activated caspase 3 leads to APP degradation and formation of Aβ and amyloid fibrils and Tau tangles. SH3GLB2 aggregation (Lee et al., 2017) occurs probably right after that of pS37-TIAF1. (All data are adapted with revisions in art work from Lee et al., 2010; Chang and Chang, 2015; Chang et al., 2015, under the guidelines of the publishers).

FIGURE 5

Role of E2/ER/WWOX in initiating protective pathways. The pathways include: Route I, E2/ER-mediated upregulation of antiapoptotic Bcl-2 family proteins, and downregulation of proapoptotic Bcl-2 family members (Yao et al., 2007) (see the route in yellow line). Route II, Activation of the pro-survival ERK/WWOX and PI3K/Akt signaling cascades to block the pro-apoptotic JNK signaling and protect the neural tissues from damages (Tang et al., 2014) (route in blue). Route III, E2 activates PI3K via ERα and mERs, followed by activating Akt to phosphorylate GSK-3β at Ser9 for functional inactivation (Ruiz-Palmero et al., 2013) (route in green). Route IV, The SDR domain of WWOX binds and limits GSK-3β activity for neuroprotection (Wang et al., 2012) (route in light blue). Route V, Suppression of GSK-3β (e.g., by WWOX) leads to a reduced β-catenin degradation, which is regulated by E2 through the ERα/PI3K/AKT/GSK-3β signaling pathway (Perez-Alvarez et al., 2012) (route in purple). Route VI, In the Wnt/Frizzled signaling pathway, Wnt protein induces the activation of Dvl to block the activity of GSK-3β. Without Wnt, β-catenin is subjected to destruction by the complex of axin, APC, CK1α, and GSK-3β (Bouteille et al., 2009). Transiently overexpressed WWOX binds Dvl to suppress the Wnt signaling (Bouteille et al., 2009) (route in orange).