Microtubule assembly

hTau40AT8*+AT100+PHF1 hTau40wt

Tubulin

Figure 3.10: Microtubule assembly of phosphomimetic mutant hTau40AT8*+AT100+PHF1: Microtubule polymerization in the presence of hTau40wt and hTau40AT8*+AT100+PHF1. Both proteins reach similar final levels, but the pseudo-phosphorylated protein shows a somewhat longer lag time. The bottom curve (blue line) shows that there is no assembly of tubulin without tau.

In contrast, the microtubule polymerizing ability of hTau40AT8*+AT100+PHF1 showed no difference in final level and only a small retardation on assembly rate was observed (Fig. 3.10). This is consistent with the notion that the AD-like phosphorylation in the flanking domains has no or only a small effect on tau-microtubule interactions.

3.2.2 Pseudophosphorylation at AT8 and PHF1 epitopes induces compaction and generates a pathological (Alz-50 and MC1) conformation

The cleavage of the C-terminal tail by caspases is believed to favor tau aggregation (Gamblin et al., 2003). The deletion of the C-terminal tail allows the N-terminal domain to approach the repeats (similar to the compaction by double arm phosphorylation at AT8* + AT100 + PHF1, Jeganathan et al., 2008). We created a tau mutant lacking amino acids 422-441 (hTau∆CT), and then we tested the reactivity of the mutant hTau∆CT with antibodies MC1 and Alz-50 (Fig. 3.11 A and C). There was a clear increase in signal for the triple-site mutant (AT8* + AT100 + PHF1) hTau40/N-RAT8*+AT100+PHF1. Remarkably, the increase became very pronounced with the C-terminal truncated mutant of hTau40∆CT/N-RAT8*+AT100+PHF1, compared with the unphosphorylated protein hTau40∆CT/N-R. Both these observations suggest that the compaction of the molecule observed by FRET resembles the “pathological conformation” detected by the MC1 and Alz-50 antibodies. The bar diagrams shows the blot intensities for MC1 and Alz-50.

The most exciting aspect of the results is the relationship between Alzheimer-like phosphorylation (at AT8*, AT100, and PHF1) epitopes, the compaction of the paperclip

Gel Intensity

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hTau Del CT/N-R hTau Del CT/ N-R

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T8*+AT100 hTau Del CT/ N-R

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T8*+AT100+PHF1 hTau AT8*+PHF1

Tau Constructs

hTau40

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Gel Intensity

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hTau Del CT/N-R hTau Del CT/ N-R

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Figure 3.11: MC1 and Alz-50 reactivity of phosphomimic mutants of tau: (A and C) Western blots showing reactivity of MC1and Alz-50 (conformational antibodies whose discontinuous epitope comprises residues from the N-terminal and repeat domain, detecting the folding of the protein) with phosphomimic mutants of tau. Note that double arm pseudo-phosphorylation causes an increased MC1 and Alz-50 reactivity. (B and D) shows the quantification of blots for MC1 and Alz-50.

conformation and the reactivity of tau with antibodies that report on the pathological conformation of tau in early stages of AD, such as MC1, Alz-50, and others. This reaction is best observed in brain tissue and has been difficult to reproduce with tau in vitro. However, the fact that the most compact conformations are the ones showing the highest reactivity with MC1 and Alz-50 (Fig. 3.11 A and C) argues that the compaction of the paper clip conformation reflects the pathological state. The increase in MC1 and Alz-50 reactivity becomes more pronounced when the C-terminal tail is absent, which argues that this tail normally opposes close approach between the N-terminus and the repeats (consistent with the paperclip model).

3.2.3 Aggregation propensity of hTau40AT8*+AT100+PHF1 and C-terminus deletion mutants expressed in neuroblastoma cell2 (N2a)

The phosphorylation abnormality in tau from patients with AD appears to be an increased stoichiometry and a decreased turnover of phosphate incorporated at selected sites.

To test the ability of the pseudo-phosphorylated mutant hTau40AT8*+AT100+PHF1, we transiently transfected this mutant or hTau40_wt as a control into the inducible neuroblastoma cell line N2a,

and the constructs were expressed by addition of doxycyclin. The presence of tau aggregates was tested by sarkosyl extraction of the cells and analysis of soluble and insoluble components.

The resulting proteins were separated by SDS-gel electrophoresis and blotted with tau specific antibody K9JA. Figure 3.12A illustrates that after three days of protein expression the control protein hTau40 remains mostly in soluble fraction. The expression of hTau40AT8*+AT100+PHF1

leads to noticeable accumulation of material in the sarkosyl-insoluble pellet representing tau aggregation. 0.5% of aggregated hTau40AT8*+AT100+PHF1 compared tohTau40wt confirms moderate increase in the aggregation. This result is in complete agreement with the in vitro result shown above (Fig.3.9A).

hTau40

(E- AT8*,AT100,PHF1) + + hTau40 wt + +

S P S P

hTau40

( C∆ E- AT8*,AT100,PHF1) + +

hTau40 C∆ + +

S P S P

hTau40

(E- AT8*,AT100,PHF1) + + hTau40 wt + +

S P S P

A B C

Figure 3.12: Aggregation propensity of pseudo-phosphorylation mutants expressed in N2a cells:

(A) Aggregation propensity of hTau40wt and hTau40AT8*+AT100+PHF1 (pseudo-phosphorylated at three epitopes) in full length in neuroblastoma N2a cell line; blot analysis with K9JA (independent of phosphorylation). (B) Aggregation propensity of hTau40∆CT421 and hTau40∆CT421AT8*+AT100+PHF1

(pseudo-phosphorylated at three epitopes) in hTau40 truncated at D421 in neuroblastoma N2a cell line;

blot analysis with K9JA (independent of phosphorylation). (C) The blot with MC1 conformational antibody performed on soluble and insoluble protein fractions on the level of hTau40wt and hTau40AT8*+AT100+PHF1 molecules. The analysis shows stronger signal coming from soluble and insoluble fractions of E-mutated hTau40AT8*+AT100+PHF1. In the case of hTau40wt only a weak MC1 signal in the soluble fraction was observed.

In the N2a cells we have to do with endogenous phosphorylation of hTau40wt resulting in a combination of E-mutated hTau40AT8*+AT100+PHF1 epitopes with some additional phospho-sites.

Figure 3.12B illustrates soluble and insoluble protein fractions on the level of hTau40molecules truncated at D421 (hTau40∆CT421wt and hTau40∆CT421AT8*+AT100+PHF1). In both cases the proteins were nearly entirely soluble. Some traces of insoluble material are visible in the case of hTau40∆CT421 and hTau40∆CT421AT8*+AT100+PHF1. The mutant of hTau40∆CT421AT8*+AT100+PHF1 molecule aggregated much more efficiently than hTau40∆CT421_wt. Figure 3.12C shows the blot analysis with MC1 conformational antibody performed on soluble and insoluble protein fractions on the level of hTau40wt and

hTau40AT8*+AT100+PHF1 molecules. The analysis shows stronger signal coming from soluble and insoluble fractions of E-mutated hTau40AT8*+AT100+PHF1. In the case of hTau40wt we observed only a weak MC1 signal in soluble fraction, exclusively.

This result is accompanied by a strong increase in the reaction with conformation-dependent antibodies Alz-50 and MC1. There was a clear increase in signal for the triple-site mutant Tau/N-RAT8*+AT100+PHF1. Remarkably, the increase in Alz-50 and MC1 reactivity becomes more pronounced when the C-terminal truncated version of the protein Tau∆CT/N-RAT8*+AT100+PHF1, compared with the unphosphorylated protein Tau∆CT/N-R (Fig. 3.11A and B). Both these observations suggest that the compaction of the molecule observed by FRET resembles the

“pathological conformation” detected by Alz-50 and MC1 antibodies (Jeganathan et al., 2008).

The data provide a framework for the global folding of tau dependent on proline-directed phosphorylation in the domains flanking the repeats and the consequences for pathological properties of tau.