Oligomer Formation of Tau Protein Hyperphosphorylated in Cells

Abstract

Background: The causal relationship between Tau hyperphosphorylation and aggregation in neuropathology is still under debate.Results: Tau highly phosphorylated in cells increases oligomerization without pronounced aggregation. Oligomers cause reduction of dendritic spines but not cell death.Conclusion: Hyperphosphorylation does not drive Tau fibrillization but contributes to synaptotoxicity.Significance: Pathways and effects of Tau hyperphosphorylation are distinct from those of aggregation.

Tau protein liquid–liquid phase separation can initiate tau aggregation

“…Surprisingly, we found that the addition of heparin or RNA to phosphorylated p‐tau441 led to spontaneous formation of p‐tau441 droplets, even in the absence of PEG (Fig 5A). We confirmed that an extended incubation of p‐tau441 with heparin (data not shown), or even without (Tepper et al , 2014), supports the formation of fibrillary tau aggregates in vitro . Non‐phosphorylated tau441 did not show LLPS with heparin and RNA at the same low protein concentration (2 μM) and in the same buffer conditions.…”

“…We decided to describe the conditions for tau LLPS in more detail and produced recombinant full‐length human tau (p‐tau441, aa 1–441; Fig 2A) in SF9 insect cells, which are able to introduce PTMs including phosphorylation into recombinant tau (Tepper et al , 2014). Previously, the phosphorylation sites found in p‐tau441 by mass spectrometry were reported to be similar to the phosphorylation of tau extracted from AD brains (Mair et al , 2016; Fig EV1G), with phosphorylation in the repeat domain (R1–R4), in the proline‐rich region (P1 + P2), and some in the N‐terminal insets (N1 and N2) of tau441 (Fig 2A).…”

“…To evaluate the role of phosphorylation for tau LLPS, we compared the ability of tau proteins with different degrees of phosphorylation to undergo phase separation. We tested two different p‐tau441 constructs for LLPS (Fig 3A and B): (i) p‐tau441 produced in SF9 insect cells (Tepper et al , 2014; Mair et al , 2016), which carries an average of ≈12 phosphate groups, and for which we confirmed the presence of most reported phospho‐sites by Western blot (Fig EV1G), (ii) alkaline phosphatase digested p‐tau441, deP‐tau441, with an average of ≈4–5 phosphates. (Suggested remaining sites: pT153, pT181, pS214, pS217, pT231; Mair et al , 2016.)…”

“…The in vitro aggregation of tau into amyloid‐like fibrils has been manifold described by several authors, mostly with emphasis on aggregation kinetics and aggregate structure (von Bergen et al , 2000; Tepper et al , 2014). Most of these aggregation studies have been performed on non‐phosphorylated recombinant tau (tau441), which needs the addition of polyanionic co‐factors such as heparin (Goedert et al , 1996) or RNA (Kampers et al , 1996) in order to form aggregates.…”

“…This process is considered a normal physiological function of tau phosphorylation that is necessary to regulate MT stability. However, hyperphosphorylation of tau often coexists with tau aggregation in the diseased brain (Augustinack et al , 2002; Iqbal et al , 2005), but the data for a causal relationship of tau aggregation to tau phosphorylation are conflicting (Kumar et al , 2014; Tepper et al , 2014; Šimić et al , 2016). It has been reported though that tau phosphorylation, for example, by GsK3β at multiple sites, may precede the aggregation of tau in AD (Iqbal et al , 2005; Stoothoff & Johnson, 2005; Mondragón‐Rodríguez et al , 2008).…”