**5.1 Tau aggregation**

The term "prion", originally defined by Prusiner, refers to a 'proteinaceous infectious particle' that causes degeneration of the CNS [110]. In addition to prion, other misfolded proteins, most notably Aβ, tau, α-synuclein and TAR DNA-binding protein 43 (TDP43), also act as proteopathic seeds to template the physiological alterations of the same protein, transmit between cells and spread to neuroanatomical connected regions, described as the 'prion-like property' [110].

The natively unfolded and highly soluble tau monomer contains a minimal content of ordered secondary structure, which shows little tendency to undergo aggregation [111]. An initial disruption likely changes tau conformation to obtain more β-sheet structures required for the interaction between monomers [94]. Two hexapeptide motifs of tau, 275VQIINK280 and 306VQIVYK311, are crucial for the conformational switch and filament assembly [112]. As the intrinsic cause of fibrillation, the aggregation-prone sequence elements are found in multiple tauopathies (**Table 3**) [10]. Once the partially folded tau monomers are stimulated, they may sequentially aggregate to form dimmers, soluble oligomers, and eventually the insoluble PHFs and NTFs [113]. The microtubule-binding domains that contain β-sheet structures assemble into the rigid core of PHFs, while the N- and C-terminus of tau protein form a 'fuzzy coat' surrounding the core [99]. All six isoforms of CNS tau are detected in AD PHFs [114]. Oligomeric tau and PHF-tau isolated from AD brains can serve as prion-like seeds to induce the aggregation of normal tau both in vitro and in vivo, while monomeric heat-stable and straight filament (SF)-tau showed limited prion-like properties. However, AD O-tau is the most potent toxic species to induce pathological tau aggregation and propagation in vivo [80, 96, 115–117].

Tau fibrils isolated from the brains of patients with different tauopathies show disease-specific folding. In some cases, even in the same disease, the protofilaments of tau pack in distinct ways to form different polymorphs although the conformation of tau monomer is relatively preserved [10, 118]. Tau strains from different sources exhibit variant ability in inducing aggregation in vivo. For example, both the structures of PHF and SF in AD contain eight β-sheets in a C-shaped fold, but the intermolecular organization between the two kinds of protofilament is different [119]. PHF-tau, but not SF-tau, dramatically seeds tau aggregation in vitro and triggers the propagation of tau pathology in vivo [116]. Furthermore, the induced pathology in specific cell types is related to the origin of tau species. Injection of AD pathological tau to mouse brain induces pathology only in neurons, while injection of CBD or PSP pathological tau gives rise to pathology in neurons as well as astrocytes and oligodendrocytes [120].

Truncation and hyperphosphorylation are two major PMTs that contribute to tau aggregation. Truncation of tau may expose the microtubule-binding repeats that are responsible for aggregation. Tau truncation alone is sufficient to induce site-specific phosphorylation and self-aggregates [97]. Among various truncations of tau,

deletion of the first 150 aa and the last 50 aa (tau151–391) promotes its pathological characteristics most significantly. Compared with full-length tau, tau151–391 is more prone to phosphorylation, self-aggregation and seeded aggregation by AD O-tau [96]. Hyperphosphorylation alone is not sufficient to induce tau aggregation, but only when it occurs together with truncation [121]. Phosphorylation at several sites (e.g. pT231, pS235 and pS262) flanking the microtubule-binding domains inhibits tau-mediated microtubule assembly and facilitates tau aggregation into PHFs [122]. Interestingly, the phosphorylation levels of these sites are positively correlated to the seeding capacity of tau species isolated from AD patients. Notably, the effect of phosphorylation on tau seeding activity seems to be site-specific. For instance, phosphorylation levels at pS198/S199/S202 and pS400/T403/S404 show a negative correlation with tau seeding activity [72].

Mutation, dysregulation of tau alternative splicing and local contextual factors are also risks for tau aggregation [22]. Moreover, some chemical factors with strong negative charges (e.g. heparin, RNA, dextran sulphate and arachidonic acid) can induce tau aggregation in vitro [10]. Nevertheless, it should be pointed out that none of the fibrils synthesized in vitro resemble patient-derived fibrils so far, because the structures of chemical-induced tau filaments are quite different from those in diseases [118]. In addition to neurons, microglias also take up both soluble and insoluble tau [123] and are involved in promoting the spread of tau pathology [124].

### **5.2 Propagation of tau pathology**

Once formed in the coeruleus/subcoeruleus region, the prion-like tau seeds are transmitted from a "donor cell" to a "recipient cell" to template more aggregation, and progressively spread the pathology along synaptically connected neurons to large-scale human brain networks. Propagation of tau pathology involves the following steps: 1) uptake of tau seeds, 2) seeded aggregation, 3) secretion of new seeds and 4) transcellular transmission of the toxic seeds [125].

The proteopathic seeds released from neurons can be engulfed by interconnected neurons at the somatodendritic compartment and the axon terminals, and are transported both anterogradely and retrogradely [126]. Endocytosis is the primary pathway for the internalization of proteopathic seeds, although the specific mechanisms vary [120]. It has been reported that pathological tau could be taken up by macropinocytosis, heparan sulfate proteoglycans (HSPGs)-dependent endocytosis, clathrin-mediated endocytosis, phagocytosis and tunneling nanotubes (TNT)-dependent direct intercellular transport, of which HSPGs-mediated endocytosis is the most predominant way [127]. The mechanisms of tau uptake are mainly similar for both vesicle-bound and free proteins [126]. Diverse tau strains display different uptake efficiency. Tau trimers are the minimal fragment that can be spontaneously internalized by primary neurons, while no clear upper limit is observed [128]. However, the soluble HMW-tau isolated from AD brain is the most efficiently internalized species [129].

After internalization, tau seeds are transported to the endo-lysosomal system, and some of them are degraded in the lysosomes [130]. Due to age-related dysfunction or other unknown mechanisms [131], a part of the seeds disrupt the endosomal vesicle and enter the cytoplasm, where they template amplification of new fibrils [132]. The danger receptor galectin-8 could protect against the release of seeds by monitoring endomembrane integrity and activating autophagy [133].

Tau contains no signal peptides. But it is secreted into the culture medium, interstitial fluid or CSF in a monomeric and/or truncated non-phosphorylated form lacking the C-terminal portion in physiological conditions [125], implying a potential physiologic role. Nonetheless, the transmission of tau monomer is unlikely to mediate transcellular propagation of tau pathology [134].

The proteopathic tau seeds are released from neurons through multiple pathways, including exocytosis [135], exosome [136], synaptic vesicles [137], presynapse membrane penetration [138] or even direct translocation across neurons [139]. Truncation and hyperphosphorylation of tau or increased synaptic activity facilitate its secretion [94]. The released proteopathic seeds are taken up by postsynaptic neurons and subsequently propagate tau pathology in the interconnected cells.
