**4. Post-translational modifications (PTMs) of tau**

Tau is post-translationally modified by multiple mechanisms, including phosphorylation, ubiquitination, acetylation, methylation, glycosylation, glycation, nitration, lipoperoxidation, sumoylation and truncation [22]. Recently, up to 95 PTMs have been identified in human brains by high-resolution quantitative proteomics (**Table 2**) [70]. In neurodegenerative diseases, tau undergoes a series of pathological changes, such as PTMs alterations and the prion-like seeding and propagation, many of which not just accompany the diseases and indicate the pathology progression and individual heterogeneity, but are also the driving force of diseases.

### **4.1 Phosphorylation**

Tau is a phosphoprotein. Theoretically, 80 serine/threonine and 5 tyrosine residues of the longest CNS tau isoform-tau441 can potentially be phosphorylated. In the brains of normal elderly, tau contains 2–3 mol phosphate/mole of the protein [71]. Phosphorylation sites like pT231, pT181, pS404 are found with high frequency (> 50%) [70]. However, tau phosphorylation increases 2–3 times in AD brains, i.e. hyperphosphorylation [71]. At least 55 phosphorylation sites are detected on pathological tau (**Table 2**) [70]. Hyperphosphorylation of soluble, oligomeric and seed-competent tau all exhibit substantial patient-to-patient heterogeneity, even though certain hot spots are present in AD brains, including pT181, pT217, pT231/S235, pS262, pS396 and pS400/T403/S404 [70, 72]. Among these sites, the phosphorylation levels of pT231/ S235 and pS262 are positively correlated to the seeding capacity of tau species [72].

Rodent tau exhibits approximately 90% homology with human tau. Profiling of PTMs showed modifications on up to 63 sites on tau protein in the wild-type mouse brain, of which 27 were phosphorylation sites [73]. Phosphorylation of tau at specific


### **Table 2.**

*Tau PMTs found in AD brains [50, 66–69].*

sites was different in the brains of human and AD model mice. For example, 3 × TG-AD mice, the commonly-used AD model which contains three mutations associated with familial AD (APP Swedish, MAPT P301L, and PSEN1 M146V), are not significantly hyperphosphorylated at pS199, pS214, pS396/S400 and pS422 as in AD brains, implying a limitation of a mouse model in studying human tau pathology [74].

Abnormal hyperphosphorylation of tau is a pivotal step in neurofibrillary degeneration in AD and other tauopathies [71]. Taking the AD brain as an example, tau can be separated into three pools according to the phosphorylation state and solubility: (i) non-hyperphosphorylated normal tau (AD-tau), (ii) hyperphosphorylated tau (AD P-tau) and (iii) polyubiquitinated, hyperphosphorylated and aggregated tau in the insoluble PHFs (PHF-tau) [75]. Hyperphosphorylation of tau induces pathology through multiple mechanisms. First, hyperphosphorylation reduces tau affinity for microtubules. Natural tau forms a "paper clip" structure, with the N- and C-terminus fold over the microtubule-binding domain to prevent self-aggregation [76]. Hyperphosphorylation changes the net charge of tau protein and alters tau conformation to expose the microtubule-binding domain, thereby facilitating self-oligomerization and aggregation. Hyperphosphorylated and/or aggregated tau detach from the microtubules and lose their ability to stabilize microtubules [71]. Besides normal, AD P-tau captures microtubule-associated proteins other than tau, such as MAP1 and MAP2 [77], leading to further disruption of microtubules. Second, hyperphosphorylated tau redistributes from axons to the somatodendritic compartment and impairs synaptic function (see Section 3.3) [16, 54]. Third, phosphorylation may change the interaction of tau with other regulatory proteins [28]. Recently, 75 proteins specifically bound to phosphorylated tau in NFTs have been identified by quantitative proteomics coupled with affinity purification-mass spectrometry; most enriches in the protein ubiquitination pathway and phagosome maturation [78]. Whether hyperphosphorylation of tau alters its affinity to these proteins and directly leads to the damage of relevant pathways deserves more extensive investigation.

Tau phosphorylation is regulated by both proline-directed [GSK-3β, cyclindependent-like kinase-5 (CDK5), dual-specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A) and extracellular signal-related protein kinase (Erk)] and non-proline-directed protein kinases [calcium/calmodulin activated protein kinase II (CaMKII), protein kinase A (PKA), casein kinase 1 (CK1) and microtubule affinity-regulated kinase 110 (MARK p110)] in vivo, which are activated and/ or overexpressed in AD brains [22]. On the contrary, tau is dephosphorylated by protein phosphatases, in particular protein phosphatases 2A (PP2A), which is responsible for over 70% of the total tau phosphatase activity in the human brain [79]. Dephosphorylation of AD P-tau with PP2A restores tau activity in promoting microtubule assembly in vitro and diminishes AD P-tau-induced propagation of tau pathology in mouse brain [80]. In disease conditions, phosphatase activity against tau is reduced to half, further increasing the imbalance between kinase and phosphatase activities, eventually resulting in excessive phosphorylation of tau [79]. It should be noted that tau is rapidly dephosphorylated during postmortem in a site-specific manner, suggesting timely dissection and proper cooling of the brain tissues [81].

### **4.2 Ubiquitination**

Ubiquitination, a PTM that covalently conjugated ubiquitin (a highly conserved 76 amino acid protein) to the ε-amino group of target lysine residues in a protein, is usually involved in cellular protein degradation as well as non-degradative pathways including cell signaling, mitochondrial homoeostasis and DNA damage responses [82]. Ubiquitin positive pathological aggregates are present in AD, FTD, PD, CBD and other neurodegenerative diseases [83]. Quantitative analysis of ubiquitylome in AD brain reveals 28 ubiquitination sites in tau protein, which are the most abundant PTMs except phosphorylation (**Table 2**) [83]. Most of these ubiquitylation sites are located in the proline-rich region and the microtubule-binding domain.

Ubiquitination of tau is catalyzed by various ubiquitin ligases (E3 ligases), for instance, the C-terminus of the Hsc70-interacting protein (CHIP), TNF receptorassociated factor 6 (TRAF6) and axotrophin/MARCH7 [22]. High molecular weight (HMW) tau extracted from AD brain is shown to be polyubiquitinated, likely through K6-, K11- or K48-linkages [83], while PHF-tau is mostly monoubiquitylated, making it insufficient to trigger the ubiquitin-proteasome system (UPS) mediated proteolysis [84]. Both polyubiquitylation and monoubiquitylation of tau contribute to the formation of insoluble protein inclusions [85, 86].

As a natively unfolded protein, tau is degraded by ATP/ubiquitin-independent 20S proteasome in physiological conditions [87]. Misfolded tau is typically ubiquitylated and is sent to the proteasome for degradation [88]. However, misfolded oligomers and aggregates cannot be fully degraded by the proteasome, but also directly damage the proteasome activity [89]. The autophagy-lysosome system provides a more potent pathway to degrade tau aggregates, which also relies on ubiquitination modification for recognition [88]. Therefore, activating ubiquitin degradation for toxic tau species is considered as one of the potential therapeutic strategies for the treatment of AD and related tauopathies.

### **4.3 Acetylation**

Acetylation is a co- or post-translational modification that utilizes acetyl-CoA as the acetyl source to modify the N-termini or specific lysine residues in proteins [84]. Tau acetylation is catalyzed by the histone acetyltransferase p300 (EP300) or CREB-binding protein (CBP), and removed by sirtuin 1 (SIRT1) and histone deacetylase 6 (HDAC6) [22]. Tau is also able to catalyze self-acetylation by using cysteine residues C291 and C322 in the R2 and R3 repeats, respectively. 4R-tau displays the higher activity of autoacetylation than 3R-tau because the latter lacks the R2 repeat [90]. 19 distinct acetylation sites have been mapped in tau protein isolated from AD brains, most of which are located within the microtubule-binding repeats and the flanking region (**Table 2**) [70].

The pathological effect of tau acetylation depends on specific modification sites. For example, high levels of tau acetylation are found at Lys163, Lys174, Lys180, Lys274, Lys280, Lys281 and Lys369 in AD brains, which may be related to the impairment of tau function [84]. Acetylation at these sites could prevent the polyubiquitylation and degradation of hyperphosphorylated tau, thus accelerating the accumulation of phosphorylated tau and promoting NFTs formation, accompanied by increased cognitive impairment. Acetylated tau also mislocalizes to the somatodendritic compartment and disrupts cytoskeleton dynamics, postsynaptic protein localization and receptor trafficking, consequently giving rise to synaptic plasticity deficits and memory loss [91]. Moreover, auto-acetylation of tau in C291 and C322 is coupled to its auto-proteolysis at K281-L282 and K340-S341 [90]. On the contrary, Lys259, Lys290, Lys321 and Lys353 within the KXGS motifs, which are found hypoacetylated in the AD brain, are normally acetylated to inhibit tau phosphorylation and aggregation [92].
