Eva-Maria Mandelkow, of the Max-Planck Institute for Structural and Molecular Biology in Hamburg, provided interesting information on the pathway of how tau stabilizes microtubules and mediates axonal transport. In AD, tau disassembles from microtubules (MTs) and forms paired helical filaments (PHFs), but it is unclear whether these are directly toxic to the neuron. It is possible that MT-bound tau can be harmful, too. Proline-directed phosphorylation yields epitopes that are recognized by the most commonly used AD-diagnostic antibodies, but these phosphorylations barely affect tau binding to MTs. MT disassembly occurs after phosphorylation of the C-terminal KXGS motifs catalyzed by MARK kinases, for example. In fact, MARK2 induces neurite outgrowth, and dominant-negative forms of MARK2 inhibit neurite outgrowth in N2A cells (Biernat et al., 2002). Interestingly, MARK2 tagged with a HA epitope co-localizes with F-actin, and phospho-tau (12E8) localizes to phalloidin-positive actin filaments. Physiologically, KXGS site phosphorylation allows for dynamic microtubules as exist in growth cones, for example.
What, then, is the effect of increased binding of tau to MT? Apparently, tau inhibits plus-end directed transport by competing with kinesin for the same binding site on MT. When tau is overexpressed, more of it binds to MTs, increasingly hindering both antero- and retrograde transport. Mandelkow measured this by determining the run length and velocity of individual vesicles. The velocity does not change, but the run length is shortened in both directions by tau. Since tau also interferes with the binding of kinesin, but not dynein, to MT, its net effect is that retrograde transport becomes dominant. MARK influences this process by causing the removal of the tau obstacle. In conclusion, tau inhibits and MARK facilitates the transport of active mitochondria, influencing synaptic energy production. Tau40 overexpression also inhibits the transport of AβPP vesicles. Tau thus leads to accumulation in the cell body of axonal transport cargoes (synaptic vesicles, mitochondria, etc.) with imaginable adverse effects including, for example, increased sensitivity to H2O2.
Continuing along similar lines, Eckhard Mandelkow stated that tau is a natively unfolded and highly soluble protein, whose fibril formation accelerates in the presence of additional factors such as polyanions (heparin, polyglutamine, DNA). Indeed, phosphorylation prevents aggregation, and it is therefore surprising that PHF can form despite tau being phosphorylated. The VQIVYK sequence in the tau protein tends to form β-structures, and it associates with this motif in other tau proteins to nucleate further assembly, leading to classical amyloid. The FTDP-17 mutations have little effect on MT stability. Instead, P301L and _K280 FTDP-17 mutations in tau accelerate PHF assembly, perhaps by favoring β-strand conformation
Tryptophan mutation scans and autofluorescence measurements performed to analyze the vicinity of domains within PHFs reveal that the hexapeptide motifs that form the aggregation nucleus are buried in the PHFs. Interestingly, PHFs in vitro have low intrinsic stability; they are easily denatured by guanidine as monitored by tryptophan fluorescence. Screening of PHF inhibitor compounds is based on thioflavin S fluorescence. Actually, tau bound to MT induces thioflavin S fluorescence like in PHFs, and overloading of tau on MTs leads to some filament-like structures on the surface of microtubules. (See also Barghorn et al., 2002.)