**1. Introduction**

Tau inclusions are key components of neurofibrillary tangles (NFTs) a recurring pathological feature for several neurodegenerative diseases including Alzheimer's disease (AD) [1–4]. There are two prevailing hypotheses on the mechanism of Tau pathology linked to protein misfolding and aggregation, both of which are not mutually exclusive [5–7]. One is that Tau has intrinsic aggregation motifs that enable fibrillation, leading to gain-in-toxic function(s) [8–11] exacerbated by the inability of the cellular degradation machinery to remove misfolded or aggregated Tau [12,13]. Another pathological mechanism is that aggregation-promoting Tau accumulation stems from loss-of-normal function(s). Tau is essential for microtubule dynamics and stability; impairment of this function linked to Tau sequestration into aggregates results in neuronal loss [14]. Alterations in protein sequence and structure (such as truncations, mutations, or post-translational modifications) contribute to both Tau loss-of-normal function and gain-in-toxic dysfunction by affecting the protein's ability to bind microtubules or propensity to misfold and aggregate [6,7,11].

Tau is an intrinsically disordered protein (IDP), a class of proteins characterized by a high degree of structural flexibility, conformational heterogeneity and binding promiscuity. Often, these properties not only allow for complex functions involving networks of interactions, but also facilitate dysfunctions as a result of misfolding or aggregation [15–17]. Tau is rich in serine/threonine (S/T) and lysine (K) residues, and is known to undergo post-translational modifications (PTMs), such as phosphorylation, acetylation, ubiquitination, and sumoylation. These PTMs are linked to both Tau function and pathology [18]. Phosphorylation is known to modulate Tau's ability to promote microtubule assembly. Abnormal Tau hyperphosphorylation, however, results in fibrillation, as evidenced by hyperphosphorylated Tau being the primary component of NFTs [1]. At least 20 phosphorylation sites [14] and 23 acetylation sites [19–21] have been reported for Tau.

Tau is a macromolecular polyampholyte consisting of negatively-charged N- and C-terminal domains, and a positively-charged central Proline-rich (P) domain with microtubule binding regions (MTBR, R1–R4; Figure 1A). An increase in negative charge via phosphorylation or removal of lysine positive charge by acetylation can have significant effects on Tau function (microtubule assembly/stabilization) and dysfunction (Tau aggregation). Tau acetylation can be mediated by p300/CREB-binding protein (CBP) HAT [19–23], and reports indicate that Tau, itself, has intrinsic acetyltransferase activity [19,20,22]. In fact, hyperacetylated Tau (Ac-Tau) has been used as a diagnostic marker for AD [20,21,24]. Although the role of hyperphosphorylation in facilitating Tau pathological aggregation is not debated, there are conflicting reports on the role of hyperacetylation in Tau pathology. Several groups have found acetylated Tau in pathological inclusions in vivo [20,21], as well as in co-deposits with hyperphosphorylated Tau [21]. However, Cook et al. report that acetylation at key Tau motifs (K259/353IGS) can be protective through the inhibition of phosphorylation of a nearby serine that otherwise would promote aggregation [23]. In addition, observations from in vitro experiments are contradictory: Cohen et al. report that acetylation accelerates Tau heparin-induced fibrillation [20], whereas others indicate that acetylation inhibits Tau filament assembly [19,23].

Liquid-liquid phase separation (LLPS) has recently gained attention as a physical mechanism for proteins to self-assemble into compartments termed membrane-less organelles [25–28]. The LLPS-mediated enrichment of proteins into membrane-less organelles, such as stress granules [29], provides "hotbeds" or seeds for protein aggregation [30–32]. LLPS was shown to initiate the aggregation of several neurodegenerative disease-associated proteins, such as FUS [30], TDP-43 [31,33], and hnRNPA1 [34]. Recently, Tau LLPS has been implicated in both the functional role of Tau in promoting microtubule assembly [35] and dysfunction in initiating Tau self-interaction and fibrillation [36–38]. Although it has been shown that hyperphosphorylation accelerates Tau LLPS and aggregation, consistent with hyperphosphorylated Tau's abundance in pathological inclusions [36,37], there are no reports on the role of acetylation on Tau phase separation and LLPS-mediated aggregation. Since Tau LLPS is expected to be strongly influenced by electrostatics, here, we investigate the role of acetylation in driving LLPS and determine if this role is consistent with the current hypothesis that LLPS can initiate and mediate Tau aggregation.
