Thr175-phosphorylated tau induces pathologic fibril formation via GSK3β-mediated phosphorylation of Thr231 in vitro

We have previously shown that amyotrophic lateral sclerosis with cognitive impairment can be characterized by pathologic inclusions of microtubule-associated protein tau (tau) phosphorylated at Thr175 (pThr175) in association with GSK3β activation. We have now examined whether pThr175 induces GSK3β activation and whether this leads to pathologic fibril formation through Thr231 phosphorylation. Seventy-two hours after transfection of Neuro2A cells with pseudophosphorylated green fluorescent protein-tagged 2N4R tau (Thr175Asp), phosphorylated kinase glycogen synthase kinase 3 beta (active GSK3β) levels were significantly increased as was pathologic fibril formation and cell death. Treatment with each of 4 GSK3β inhibitors or small hairpin RNA knockdown of GSK3β abolished fibril formation and prevented cell death. Inhibition of Thr231 phosphorylation (Thr231Ala) prevented pathologic tau fibril formation, regardless of Thr175 state, whereas Thr231Asp (pseudophosphorylated at Thr231) developed pathologic tau fibrils. Ser235 mutations did not affect fibril formation, indicating an unprimed mechanism of Thr231 phosphorylation. These findings suggest a mechanism of tau pathology by which pThr175 induces GSK3β phosphorylation of Thr231 leading to fibril formation, indicating a potential therapeutic avenue for amyotrophic lateral sclerosis with cognitive impairment.