Absence of a Role for Phosphorylation in the Tau Pathology of Alzheimer's Disease

Abstract

Alzheimer's disease is characterized by redistribution of the tau protein pool from soluble to aggregated states. Aggregation forms proteolytically stable core polymers restricted to the repeat domain, and this binding interaction has prion-like properties. We have compared the binding properties of tau and tubulin in vitro using a system in which we can measure binding affinities for proteins alternated between solid and aqueous phases. The study reveals that a phase-shifted repeat domain fragment from the Paired Helical Filament core contains all that is required for high affinity tau-tau binding. Unlike tau-tubulin binding, tau-tau binding shows concentration-dependent enhancement in both phase directions due to an avidity effect which permits one molecule to bind to many as the concentration in the opposite phase increases. Phosphorylation of tau inhibits tau-tau binding and tau-tubulin binding to equivalent extents. Tau-tau binding is favoured over tau-tubulin binding by factors in the range 19-41-fold, irrespective of phosphorylation status. A critical requirement for tau to become aggregation-competent is prior binding to a solid-phase substrate, which induces a conformational change in the repeat domain permitting high-affinity binding to occur even if tau is phosphorylated. The endogenous species enabling this nucleation event to occur in vivo remains to be identified. The findings of the study suggest that development of disease-modifying drugs for tauopathies should not target phosphorylation, but rather should target inhibitors of tau-tau binding or inhibitors of the binding interaction with as yet unidentified endogenous polyanionic substrates required to nucleate tau assembly.

Keywords: Alzheimer’s disease; phosphorylation; protein aggregation; tau protein.

Figure 1

Immunoreactivity profile for tau antibodies. (a) The mAb 7/51 recognised tau proteins isolated from the brain of neonatal rat both before (lane 1; NT) and after (lane 2; NTP) hyperphosphorylation in vitro; (b) The mAb AT8 recognised endogenously phosphorylated tau (lane 1), the extent of which is increased following further in vitro phosphorylation (lane 2); (c) The mAb 21/D10 detected a phosphorylation-dependent epitope which is absent (lane 1) in endogenously phosphorylated neonatal tau (lane 1), but present following in vitro hyperphosphorylation (lane 2), and is also present in sarkosyl-insoluble tau protein isolated from AD brain tissue (lane 4); (d) The human tau-specific mAb 27/499 did not recognise neonatal tau isolated from rat before (lane 1) or after phosphorylation in vitro (lane 2), but recognised tau protein isolated from the brain of normal adult human (lane 3) and in the sarkosyl-insoluble tau extract from AD brain (lane 4), and the recombinant human tau both before (lane 5; T40) and after (lane 6; T40P) hyperphosphorylation in vitro; (e) In contrast, mAb 27/342 recognised neonatal rat tau both before (lane 1) and after (lane 2) hyperphosphorylation in vitro, but otherwise had a similar immunoreactive profile to mAb 27/499; (f,g) Non-Pronase treated PHFs from AD brain tissue were immunodecorated by mAb 21/10, as visualized by gold particles. The latter immunoreactivity was abolished by prior treatment of PHFs with alkaline phosphatase (AP).

Figure 2

Tau-tau binding through the repeat domain. The truncated repeat domain dGA tau fragment was adsorbed (“ads.”) tothe solid phase at the concentrations indicated and binding of full-length T40 in the aqueous-phase (“aq.”) measured immunochemically using mAb 27/499. (a) For any fixed concentration of dGA-ads., binding of T40-aq. could be approximated by a family of Langmuir curves (Equation (1) in text). Systematic variation in the apparent B_max (B_m1, (b)) and K_d (K_d1, (c)) values depended on the concentration of dGA-ads. and could be approximated by the empirical relationships described by Equations (2) and (3) in the text. Conversely, for any fixed concentration of T40 in the aqueous-phase, binding could be approximated by a corresponding set of Langmuir curves (d) (Equation (4) in text). In this case, systematic variation in the apparent B_max (B_m2, (e)) and K_d (K_d2, (f)) values depended on the concentration of T40 in the aqueous-phase and could be approximated by the empirical relationships described by Equations (5) and (6) in the text. Correlation coefficients for all approximations exceeded 0.94.

Figure 3

Non-specific binding of dGA (a) or full-length tau (b) to the solid-phase PVC matrix. Binding was detected using mAbs 7/51 or 499, respectively. In both instances, saturation of non-specific binding sites occurred at plating concentrations greater than 20 nM.

Figure 4

Intrinsic inhibitory effects of phosphorylation. Binding of T40 and T40P in the aqueous-phase to adsorbed neonatal tau (“NT”; (a,b)) or adsorbed depolymerised bovine tubulin (c,d) at the plating concentrations indicated. Although NT in the aqueous-phase did not bind to solid-phase dGA (data not shown), binding of T40 (a) to NT was indistinguishable from that seen with dGA in the solid-phase. Binding was inhibited by a factor of 24-fold following in vitro hyperphosphorylation of recombinant tau (“T40P”; (b)). Binding of recombinant human tau to depolymerised bovine tubulin (c) was likewise inhibited by a factor of 24-fold following hyperphosphorylation of tau in vitro (d). Tau binding was detected using mAb 27/499.

Figure 5

Induced inhibitory effects of tau phosphorylation. (a) Representative binding curves for nonphosphorylated T40 to adsorbed neonatal NT or NTP. Binding data is shown only for the case in which adsorbed neonatal tau was present at saturating concentration (>0.54 μM) but, for calculation of the K_d values shown here and in Table 2, binding was measured over the full range of NT concentrations shown in Figure 4. Prior hyperphosphorylation of neonatal tau (adsorbed NTP) inhibited binding of T40 in the aqueous-phase by a factor of 36-fold; (b) Representative curves for binding of adsorbed NT at saturating concentrations of T40 or T40P (>654 μM and >4362 μM, respectively). The corresponding solid-phase binding constants calculated from data obtained over the full range of aqueous-phase tau concentrations are shown. The effect of hyperphosphorylation of tau in the aqueous-phase is to reduce the binding affinity of the adsorbed species by a factor of 3-fold.

Figure 6

Schematic representation of tau-tau binding. (i) Tau is initially captured by binding to a solid-phase substrate. The identity of the endogenous substrate in the aging brain is unknown, but may be formed from macromolecular complexes which escape normal endosomal/lysosomal processing with advancing age; (ii) The tau bound to a solid-phase substrate is able to capture further full-length tau through a tau-tau binding interaction which has greater affinity (lower energy) than the physiological tau-tubulin binding interaction; (iii) This locks the repeat domain into a proteolytically stable configuration such that proteolytic cleavage of the N- and C-termini leaves behind a characteristic tau fragment restricted to the repeat domain, the core tau unit of the PHF; (iv) The oligomeric form of truncated tau is able to propagate capture and proteolytic processing of further tau, and repeated cycles of binding and proteolysis result in accumulation of PHF-core tau; (v) It is only at a later stage that non-proteolysed, full-length tau molecules are added and these become secondarily phosphorylated. The optimal points of pharmaceutical intervention are therefore blocking the binding of tau to the initiating endogenous substrate or blocking the propagation of tau aggregation cascade with tau aggregation inhibitors.