Dephosphorylation Targeting Chimaera Facilitates Tau Dephosphorylation And Degradation In Alzheimer’s Disease

【Background】Alzheimer disease（AD）is a destructive neurodegenerative disease.Its two major pathological features are the deposition of extracellualr amyloid beta（Aβ）and Neurofibrillary tangles（NFTs）in neurons.The main component of NFTs is the hyperphosphorylated microtubule-associated protein tau.Early studies have shown that tau accounts for more than 80%of all microtubule-related proteins,which is essential for the initiation,elongation and microtubule stability of microtubule assembly.Tau’s main function is to promote microtubule assembly and maintain a balance between microtubule assembly and dissociation.During normal development,tau undergoes a variety of post-translational modifications,including phosphorylation,glycosylation,ubiquitination,nitration,etc.The normal level of tau phosphorylation is very important to maintain its function.Excessive hyperphosphorylation of tau destructs the structure of microtubules and impaires neuron’s axonal transport.Eventually,it leads to structural damage and dysfunction of neurons.The level of tau phosphorylation is closely related with the activity of protein kinases to promotet tau phosphorylation and protein phosphatases to induce tau dephosphorylation.Protein phosphatase 2A（PP2A）is considered to play a very important role to determine the level of tau phosphorylation.【Objective】To design and synthesize a novel dephosphorylation targeting chimaera（DEPTAC）peptide to link tau to protein phosphatase 2A（PP2A）.The DEPTAC exhibited high specificity and efficiency in dephosphorylating and eliminating tau proteins.【Methods】Based on the principle of tau PROTACs by specifically recruiting E3 ligase or proteolysis-relevant chaperones to tau,instead,we here designed and synthesized a novel DEPhosphorylation TArgeting Chimaera（termed as DEPTAC）which recruits PP2A,meanwhile specifically combines with tau protein.The DEPTAC(～4.4 k Da,C₁₈₅H₃₁₄N₇₂O₅₆)is consisted of 38 amino acid residues from N-to C-terminals with four functional motifs:（1）YQQYQDATADEQG（β-tubulin 422–434）,for recognizing and binding tau.（2）GSGS,a linker for increasing the peptide flexibility.（3）KKVAVVRTPPKSP,for recruiting PP2A-Bα,and（4）RRRRRRRR,for cell penetrating.For tracing the chimaera,a fluorescein FITC was conjugated at the K-18 residual.A peptide with mutations in both the tau-binding and PP2A-Bαrecruiting domains（YQQYQAATAAAQG-GSGS-K（FITC）AVAVVRTPPASP-RRRRRRRR）was used as a control.The binding ability of DEPTAC to tau and PP2A was detected by Fluorescence polarization assay and co-immunoprecipitation.Primary hippocampal neurons were treated with DEPTAC,and DEPTAC was delivered through a guiding cannula implanted into the lateral ventricle of Tau368 mice.The efficiency of tau dephosphorylation and degradation was detected by western blot.Effects of DEPTAC on synaptic and microtubule structures were observed by electron microscopy.The effects of DEPTAC on hippocampal dependence memory and spatial learning memory were tested by behavioral experiments such as Object-place and novel-object recognition and Morris water maze.【Results】1.DEPTAC binds to tau and recruits PP2A-Bα,and shows high neuron penetrability;2.DEPTAC downregulated tau phosphorylation at multiple AD-related sites,including the Ser199,Thr205,Ser396,Ser404 and the AT8 epitope（Ser199/Ser202/Thr205）. DEPTAC also reduced total tau probed by Tau5 antibody;3.DEPTAC ameliorates phospho-tau accumulation in Tau368 mice;4.DEPTAC improves synaptic plasticity and alleviates h Tau-induced microtubules depolymerization in Tau368 mice5.DEPTAC improved hippocampus-dependent cognition in Tau368 mice.【Conclusion】The DEPTAC exhibited high specificity and efficiency in dephosphorylating tau thus to eliminate abnormal tau accumulation.Importantly,the DEPTAC facilitated microtubule assembly,improved synaptic plasticity and ameliorated learning and memory deficits in human tau transgenic mice.These findings provide a promising tool for selectively promoting tau dephosphorylation and degradation,which sheds new light for AD therapy.