Our previous work (LaPointe et al., 2009
) established that the N-terminus of tau was required for filamentous tau aggregates to initiate a PP1-GSK3 cascade that led to inhibition of anterograde FAT. Additional studies indicated these effects involved increased exposure of a functional domain we termed the p
omain or PAD. Using phosphorylated tau and pseudophosphorylation tau mutants, we demonstrated that Y18 phosphorylation (a fyn target) prevents the deleterious effects of tau on FAT. Similar experiments using the 6D tau isoform indicated that pseudophosphorylation at Y18 or T17 (and to a lesser extent Y29) prevents FAT inhibition, even when PAD is constitutively exposed. Further, we show that PAD exposure in AD brains occurs before Y18 phosphorylation and that a subset of PAD exposed tau is coincident with this modification, supporting the role of Y18 phosphorylation in mitigating the deleterious effects of PAD exposure in some of the tau pathology in situ
. Together, these data suggest that the toxic effects of pathological tau species on FAT can be regulated via tyrosine kinase-mediated phosphorylation. Such findings have broad implications for the role of tau as a pathogenic factor in AD and other tauopathies.
Impaired axonal transport, as evidenced by numerous indirect measures (e.g. immunohistochemical and ultrastructural analyses of postmortem human tissue), has long been implicated in the pathogenesis of Alzheimer's disease (Dessi et al., 1997
; Praprotnik et al., 1996
; Stokin et al., 2005
; Suzuki and Terry, 1967
). Moreover, an association between FAT defects and pathogenic forms of tau has been implicated in AD and other neurodegenerative diseases (Morfini et al., 2002a
; Morfini et al., 2009a
; Roy et al., 2005
), but the molecular basis was unclear. Studies using in vitro
assays and tau overexpression in cultured cells suggested that tau impairs FAT by physically interfering with the binding of molecular motors to microtubules (Ebneth et al., 1998
; Mandelkow et al., 2003
; Vershinin et al., 2007
). However, supraphysiological levels of tau not known to occur in AD were typically utilized in these studies making their biological relevance unclear. An experimental model was needed to allow precise control of tau levels and quantitative analysis of the effects on FAT. Isolated axoplasm from the squid giant axon provides a unique ex vivo
model system that meets these criteria, and has additional advantages (Morfini et al., 2007a
). These advantages include: 1) the maintenance of functional anterograde and retrograde movement, 2) the maintenance of axoplasm organization and polarity, 3) the absence of protein and gene changes, and 4) molecular machinery that is highly conserved through humans. Using this model, we have shown that disease-associated tau species (e.g. tau filaments, AT8 tau, and an FTD mutant tau) inhibit anterograde FAT by activating a PP1-GSK3 cascade (LaPointe et al., 2009
; Kanaan et al., in preparation, 2011).
Pathological observations in human tissue indicate that phosphorylation events (e.g. AT8) (Braak et al., 1994
) and PAD exposure occur early during tau inclusion formation. Importantly, the abundance of TNT1 positive tau pathology in neuropil threads suggests PAD exposure occurs in both axons and dendrites. While the majority of our findings center around axonal transport, kinesin-based transport is important within both the soma and dendrites of neurons. Thus, the high levels of TNT1 positive tau in the somatodendritic compartment further highlight a role for PAD exposure in anterograde transport impairment in human disease. These findings suggest that the tau that is redistributed to the somatodendritic compartment in disease can negatively affect transport there as well.
Despite the presence of tau pathology, some neurons can survive for 2–3 decades bearing tau inclusions (Morsch et al., 1999
). These findings raise an important question. How can some neurons mitigate the toxic effects of pathogenic tau, while others exhibit progressive pathology and degeneration? These issues imply the existence of regulatory mechanisms that affect the ability of PAD to inhibit FAT in situ
. Tau is a well-known phosphoprotein that aggregates and becomes increasingly phosphorylated in AD and other tauopathies (Iqbal et al., 2005
). In part, this reflects the activation of kinases as an element of AD pathogenesis (LaPointe et al., 2009
; Morfini et al., 2002a
; Pigino et al., 2009
). Multiple disease-associated phosphorylation modifications can reduce tau’s ability to bind microtubules and enhance or reduce the aggregation potential of tau in vitro
(Sun and Gamblin, 2009
). However, the functional implications of these phosphorylation modifications in the disease process have remained unclear in most cases.
Here, we provide direct evidence that phosphorylation within PAD blocks the deleterious effects of pathogenic tau on FAT. Both phosphorylation and pseudophosphorylation at Y18 in full-length WT tau filaments completely abolished their inhibitory effect on anterograde FAT. Furthermore, pseudophosphorylation at Y18 or T17 completely prevented 6D tau from inhibiting FAT. In our studies, the effects of pseudophosphorylation at Y29 on FAT were dependent on the form of tau tested. Tau filaments composed of Y29E tau significantly inhibited anterograde FAT much like WT tau filaments, while Y29E 6D tau partially reduced inhibition. The fact that Y29 is not within PAD, but is close in proximity, may explain this partial effect. Constraints imposed by sequences in full-length tau may explain why Y29E was not effective in preventing inhibition of anterograde FAT within full-length tau filaments.
Tyrosine 18 is of particular interest because it is a well characterized phosphorylation site for fyn (Lee et al., 2004
) in a region of tau with significant divergence between primates and other organisms. In addition, pathologically aggregated tau is phosphorylated at Y18 in AD brains (Lee et al., 2004
), and fyn kinase levels increase in neurons where fyn co-localizes with neurofibrillary tangles in AD (Ho et al., 2005
; Shirazi and Wood, 1993
). Fyn is a member of the src family of non-receptor tyrosine kinases that includes src, lyn and lck (Robinson et al., 2000
), and tau is a substrate for many of these as well as other non-receptor tyrosine kinases (e.g. c-abl and syk) (Lebouvier et al., 2009
; Lee, 2005
). Collectively, these data suggest that non-receptor tyrosine kinases may play a role in reducing the toxic capacity of PAD exposure during disease pathogenesis ().
Fig. 8 Schematic diagram of the proposed role for N-terminal phosphorylation of tau in cargo delivery and aberrant inhibition of anterograde FAT in disease. (A) Normally, tau localizes to microtubules where, in addition to stabilizing microtubules, it is capable (more ...)
Highlighting the relevance of results obtained from studies in squid axoplasm, we found that Y18 phosphorylation occurs after PAD exposure in human brain tissue, and is mainly found in seemingly more mature tau inclusions in control brains. As AD progresses, both PAD exposure and phosphorylation at Y18 increase; however, the extent of phosphorylation at Y18 does not match the amount of tau pathology with PAD exposed. These data suggest that while Y18 phosphorylation may represent a compensatory mechanism aimed at rendering tau aggregates non-toxic in the brain, this mechanism is only partially effective. Additional mechanisms to reduce tau-mediated toxicity likely include modifications that block PAD by altering tau conformation (Jeganathan et al., 2006
; Jeganathan et al., 2008
) and/or N-terminal cleavage events that eliminate PAD (Horowitz et al., 2004
The ability of fyn kinase to modify Y18 may be affected by a variety of factors. We show that fyn has a reduced ability to phosphorylate Y18 in pathological forms of tau including tau filaments, AT8 tau and an FTD mutant tau. Previously, a reduction in binding between fyn and tau on surface plasmon resonance assays was seen when 3 repeat tau was pseudophosphorylated at S199 and S202; however, pseudophosphorylated 4 repeat tau exhibited increased fyn binding (Bhaskar et al., 2005
). Perhaps the additional modification at T205 reduces the interaction between 4 repeat tau and fyn leading to reduced Y18 phosphorylation in the current studies. Reduced levels of Y18 phosphorylation in Δ144–273 tau can be explained by the fact that all seven putative SH3 binding domains for docking fyn kinase are in the deleted region. Results from in vitro
phosphorylation experiments here suggest that tau filaments and some disease-associated modifications of tau may impair phosphorylation at Y18 and prevent inactivation of PAD.
Our data, and those recently published (Ittner et al., 2010
), suggest a complex relationship between fyn and the N-terminus of tau. Ittner and coworkers (2010)
demonstrated that the N-terminus of tau is responsible for mediating the distribution of fyn kinase into dendritic spines. Interestingly, redistribution of tau, and subsequently fyn, to the soma reduced amyloid-β induced memory deficits and seizures in AD transgenic mice (Ittner et al., 2010
). In light of our results, there are likely parallel functions of fyn that may depend upon cellular distribution and/or disease state. Although fyn may be localized to dendritic spines in a tau-dependent fashion where it can regulate synaptic function and excitotoxicity, fyn may also directly inactivate PAD-mediated FAT disruption as tau accumulates during disease progression (). Our data also provide insight into a potential role for Y18 phosphorylation by fyn (and other tyrosine kinases) in modulating PAD-mediated signaling under normal conditions for local cargo delivery (). Further investigations are required to understand functional relationships between tau and non-receptor tyrosine kinases more fully.
Identification of a mechanism for the toxic gain-of-function exhibited by tau filaments suggests that novel therapeutic approaches for AD and other tauopathies are feasible. Modulating the activities of PP1 and GSK3 is an obvious choice, but these enzymes are important in multiple pathways affecting many cellular processes (Avila and Hernandez, 2007
; Cohen, 2002
). The identification of PAD within the N-terminus of tau as a critical component in this gain of toxicity may provide a more specific target. Phosphorylation in this region (e.g. Y18) does not affect microtubule binding (Lee et al., 2004
) or increase the aggregation potential of tau (Reynolds et al., 2005
), which makes it an attractive site for intervention. Enhancing N-terminal phosphorylation and/or physically blocking or binding PAD could provide protection against the deleterious effects of pathogenic tau on FAT.