Suppression of Tau-tubulin kinase 1 mitigates tau phosphorylation, and Aβ mediated neurotoxicity via altered kinase network.

Tau-tubulin kinase-1 (TTBK1) is a neuron-specific serine/threonine tau kinase that is highly expressed in the entorhinal and hippocampal regions and phosphorylates microtubule-associated protein tau at sites relevant to Alzheimer's disease (AD), thereby contributing to early tau pathology. Targeting TTBK1 can be a promising therapeutic approach for AD, although how suppression of TTBK1 may change neuronal functions is poorly understood. Here we show the proteome and phosphoproteome analysis of murine primary cortical neurons transduced with adeno-associated virus (AAV) expressing short hairpin RNA (shRNA) targeting murine Ttbk1, using tandem mass tag and data independent acquisition liquid chromatography/mass-spectrometry. Suppression of TTBK1 expression by shRNA transduction increases expression of neuronal intermediate filament proteins and V1-domain subunits of the vacuolar H+-ATPase, suggesting the regulatory roles of TTBK1 on cytoskeletal dynamics and lysosomal activity. Silencing of Ttbk1 significantly reduces phosphorylation of TTBK1 and MAP1A as well as increases phosphorylation of neuronal intermediate filament proteins, indicating the regulation of microtubule-related proteins and axonal stabilization. Phosphosite-specific signature analysis identifies the kinases regulated by TTBK1 including AD-related cyclin-dependent kinase 5 (CDK5) and AKT1. Silencing of Ttbk1 significantly reduces CDK5 activator p25/p35 ratio and pT231 phospho-tau level, consistent with the reduced activity of CDK5. Finally, silencing of Ttbk1 reduces amyloid-β-induced neurotoxicity in vitro. These results suggest a close functional relationship or coordinated regulation among TTBK1 and other kinases in maintaining axonal homeostasis via phosphorylation of microtubule-associated proteins, and mediating amyloid-β toxicity, thus emphasize TTBK1 as an emerging therapeutic target of AD.