**4.4 Truncation**

As an intrinsically disordered protein, tau is sensitive to proteolysis. In addition to acetylation-induced auto-proteolysis, tau can be cleaved by a variety of proteases both in vitro and in vivo including a disintegrin and metallopeptidase domain 10 (ADAM-10), asparagine endopeptidase (AEP), Calpain-1, Calpain-2, Caspase-3, Caspase-6, chymotrypsin, thrombin, cathepsins, human high-temperature requirement serine protease A1 (HtrA1) and puromycin-sensitive aminopeptidase (PSA). Besides, many fragments of tau in the brain have been found with undefined proteases (**Table 2**) [74, 93, 94]. In NFTs, at least three site-specific cleavages of tau (N368, E391 and D421) have been identified to be correlated with the progression of Braak stages [95]. Similar cleavage sites are also detected in several mouse models of tauopathies [28]. To date, more than 50 truncated forms of tau have been identified and over 30 are present in AD brains [94].

Truncation of tau plays a crucial role in its pathology. Truncation opens up the "paper clip" tertiary structure of tau protein, increases its site-specific phosphorylation, self-aggregation and affinity to oligomeric tau derived from AD brain (AD O-Tau), and thus promotes tau capture and seeded-aggregation by AD O-Tau [28, 96]. Tau truncation alone is sufficient to trigger hyperphosphorylation and aggregation [97]. Some fragments of tau can spread transcellularly, resulting in the propagation of tau pathology [94]. Additionally, Tau truncation can also induce toxic responses like apoptosis which is independent of its function on aggregates [98].

The characteristics of tau fragments depend on their amino acid composition. The C-terminal truncation increased tau fibrillization in vitro [66], while N-terminal truncations are found more associated with hyperphosphorylated high molecular weight tau oligomers (HMW-tau) isolated from AD brains [74, 93]. Tau fragments containing the aggregation-prone elements (**Table 3**) are prone to assemble the protease-resistant pathological core, which has various compositions in the different tauopathies [99]. Some specific fragments are secreted from the cytosol into the interstitial fluid and further released to the cerebrospinal fluid (CSF) or plasma, making them potentially biomarkers to indicate the progression of AD and other tauopathies [100]. Early study using immunoprecipitation showed that tau in CSF is predominantly the N-terminus fragments with an apparent molecular weight of approximately 20–40 kDa [101]. A 20–22 kDa NH2-truncated form of tau (aa 26–230) identified in CSF is found to be neurotoxic due to its damages to mitochondrial oxidative phosphorylation [102].


**Table 3.**

*Aggregation-prone elements of tau in representative tauopathies [5, 188].*

High-resolution mass spectrometry revealed at least 19 tau fragments in the CSF, of which tau aa 156–224 is the most abundant peptide [67, 68]. Nonetheless, the prionlike seeding activity of CSF tau fragments is quite limited [69].

### **4.5 Other PMTs**

In addition to the above modifications, tau can also be modulated by methylation, SUMOylation, nitration, glycosylation and glycation. The contributions of these PMTs to tau pathology are mostly unelucidated.

Tau methylation mainly occurs on lysine residues in the proline-rich region and the microtubule-binding domain, and a few arginine residues [73]. Up to 11 methylation sites were found in the human brain [70]. Methylated tau is highly concentrated in NFTs particularly in late-stage AD brains [103]. Lysine methylation suppresses tau binding to the microtubule-binding domain, increases abnormal phosphorylation of tau and blocks the UPS-mediated tau degradation. However, the role of methylation on tau self-aggregation is still controversial [84].

K340 residue is the major site of tau that be modified by the small ubiquitin-like modifier protein (SUMO) [104]. SUMO-1 colocalizes with phosphorylated tau in the AD brain. Sumoylation reciprocally stimulates tau phosphorylation at T231 and S262, and competes against K340 ubiquitylation and consequently suppresses degradation [105].

Tau can be nitrated on four Tyr residues, Y18, Y29, Y197 and Y394, which are found in AD and non-AD tauopathies [106]. The effect of nitration on tau assembly depends on the specific nitration sites in vitro. Y18 nitration is reported to be associated with astrocyte activation [107].

In addition, the complexity of PTM is reflected more in the cross-talk between various modifications, not just in the types of modifications. First, a single amino acid residue could be modified by different PMTs. Taking lysine residues as an example, some of these residues in tau protein are competitively modified by ubiquitylation, methylation, acetylation, or SUMOlyation in the AD brain (**Table 2**). The competition between these PMTs will determine whether tau will undergo degradation or pathological transformation [84]. Secondly, PMTs at different sites cross-talk with each other. Many phosphorylation and ubiquitination sites of tau are located within the KXGS motifs in the microtubule-binding domain. Tau hyperphosphorylation is shown to facilitate ubiquitylation of NFT tau [83]. Therefore, further investigations focusing on the cross-talk between tau PMTs are required to reveal and intervene in the pathological changes of tau.

### **5. Propagation of tau aggregation**

In AD brains, the progression of tau aggregation follows a stereotypical pattern of spread (the Braak stages): initiates from the locus coeruleus and transentorhinal cortex (Braak stages I and II), gradually spread to the limbic system (Braak stages III and IV) and eventually to the neocortex (Braak stages V and VI) [108]. The stereotypical transmission of tau pathology is highly correlated with the cognitive impairment in AD [13]. Remarkably, tau pathology can be induced in rodent brains by injecting aggregates isolated from AD brains, and propagating to anatomically connected brain regions, in a similar spreading pattern to that observed in AD patients [80]. Besides, injection of tau aggregates extracted from other neurodegenerative diseases, such as AGD, CBD and PSP, also recapitulated the hallmark lesions of corresponding diseases [109]. A large amount of evidence indicates that the prion-like propagation of misfolded tau may explain the diverse characteristics of tauopathies [10].
