Recently, we demonstrated that WT tau aggregates inhibit anterograde FAT by activating a PP1–GSK3 signaling cascade through a mechanism requiring the N terminus of tau (LaPointe et al., 2009
). Extending those observations, we have identified a tau domain comprised of amino acids 2–18 that is both necessary and sufficient to activate the PP1–GSK3 cascade and inhibit anterograde FAT. These effects suggest a novel function for tau and define a new functional motif in tau, the PAD. Inhibition of anterograde FAT by the 6D and 6P isoforms of tau and a synthetic PAD peptide demonstrates that this effect does not require tau binding to microtubules or tau aggregation. Our results demonstrate that exposure of PAD is a critical factor mediating the ability of pathogenic forms of tau to inhibit FAT. Consistent with this hypothesis, two disease-associated tau modifications that increase PAD exposure inhibited anterograde FAT as soluble monomers. Further supporting the role of PAD exposure as an important disease-related event, the novel PAD-specific antibody TNT1 selectively labeled both soluble and insoluble tau isolated from AD brains, but not that from controls. Moreover, qualitative observations of TNT1 immunoreactivity in human AD tissue sections suggested that PAD exposure represents an early pathological event that closely associates in time with AT8 phosphorylation. These data have important implications regarding the role of tau in disease pathogenesis and provide a mechanism through which disease-associated modifications and/or mutations in tau lead to a toxic gain-of-function in AD and other tauopathies.
The demonstration that PAD is a region within tau that can modulate the PP1–GSK3 cascade has significant implications for tau pathology and suggests a novel biological role for tau regulating the activity of phosphotransferases and anterograde FAT (). Tau has been shown to target PP1 to microtubules and can directly interact with PP1 (Liao et al., 1998
). Amino acids 5–8, located within PAD, comprise one of the proposed PP1 binding sites in tau (Liao et al., 1998
). Additionally, tau and GSK3β have been copurified as part of a high-molecular-weight complex in association with purified microtubules (Sun et al., 2002
). Thus, in addition to microtubule stabilization, tau may bind and target phosphotransferases to the vicinity of microtubules (Shahani and Brandt, 2002
) and regulate FAT through modulation of these enzymes (), a functional scheme that may be disrupted in disease ().
Proposed role of PAD in anterograde fast axonal transport regulation and in axonal transport dysfunction in disease
Deficits in FAT have previously been implicated in the neuronal dysfunction associated with dysferopathies such as AD, non-AD tauopathies, and other neurodegenerative diseases (Morfini et al., 2002a
; Roy et al., 2005
). In AD brains, dystrophic neurites, synaptic loss, and protein mislocalization (Scheff et al., 1990
; Dessi et al., 1997
) are all consistent with FAT deficits. Accordingly, studies in various animal models of AD and tauopathies have reported alterations in axonal transport (e.g., axonal swellings, synaptic loss, and impaired vesicle transport) (for review, see Higuchi et al., 2006
). Interestingly, tau appears to play a critical role in amyloid-β-mediated axonal transport disruption, as genetically removing tau mitigates the effects of treating neurons with amyloid-β oligomers (Vossel et al., 2010
). These studies suggested a link among tau, axonal dysfunction, and neurodegenerative disease; however, until this report the molecular mechanisms linking deficits in FAT to disease-related modifications of tau had remained unclear (Morfini et al., 2002a
Several studies on the role for tau in axonal transport dysfunction proposed a mechanism involving direct interference of tau with the binding of conventional kinesin to microtubules (Ebneth et al., 1998
; Seitz et al., 2002
; Vershinin et al., 2007
). However, overexpression of human tau in transgenic mice did not affect FAT rates in the optic nerve (Yuan et al., 2008
), and levels of soluble WT tau 20-fold greater than endogenous levels of tau had no effect on FAT in squid axoplasm (Morfini et al., 2007
). Studies on aggregated tau (LaPointe et al., 2009
) and the current data provide evidence for an alternative mechanism independent of microtubule binding that does not require aggregation. Specifically, many of the tau species examined in these studies do not effectively bind microtubules (e.g., tau aggregates, 6D/6P tau, PAD peptide, and FTD Δ144–273 tau), and many were used as monomers (e.g., 6D/6P tau, PAD peptide, FTD Δ144–273 tau, and AT8 tau). Moreover, we have established that tau inhibits anterograde FAT by activating the PP1–GSK3 cascade with nearly all of the tau species tested thus far. Together, these data clearly demonstrate that tau-mediated disruption of anterograde FAT via the PP1–GSK3 cascade is independent of microtubule binding and does not require aggregation.
Based on our prior studies (LaPointe et al., 2009
), it was unclear why monomeric WT tau failed to affect FAT even though the PAD motif was present. Tau was originally thought to exist in an extended random coil state based on spectroscopic studies (Mandelkow et al., 2007
). However, fluorescence resonance energy transfer studies indicated that soluble monomeric WT tau adopts a folded paperclip conformation (Jeganathan et al., 2006
), that may prevent PAD from activating the PP1–GSK3 cascade. Conversely, conformations of tau that increase exposure of PAD should promote inhibition of anterograde FAT.
We tested this hypothesis using numerous forms of tau in which the accessibility of PAD is increased. In tau aggregates, both termini of tau are believed to maintain a random coil structure extending from the filament core formed by the MTBRs (Barghorn et al., 2004
). Evidence also suggests aggregated tau favors an Alz50-type conformation (Carmel et al., 1996
), in which the N terminus is in close contact with the MTBRs. However, the results presented here suggest that the extreme N terminus of tau remains accessible in aggregated tau. The 6D and 6P isoforms of tau lack the MTBRs and C terminus (Luo et al., 2004
) necessary for the paperclip conformation, leaving PAD constitutively available, and the PAD peptide is composed of only amino acids 2–18. Similarly, a recombinant pseudophosphorylated AT8 tau exhibits reduced folding of the N terminus into the paperclip conformation (Jeganathan et al., 2008
). Finally, deletion of amino acids 144–273 in the FTD tau construct should dramatically reduce or eliminate the ability of the N terminus to fold into the paperclip, as this deletion removes the proline-rich hinge region involved in N-terminal folding. Consistent with our hypothesis, all of these “PAD-exposed” tau species inhibited anterograde FAT; thus, exposure of PAD provides a common mechanism of toxicity for biochemically heterogeneous forms of pathogenic tau.
Highlighting the relevance of results obtained from studies in isolated squid axoplasm, a PAD-specific antibody (TNT1) that preferentially recognizes disease-related forms of tau confirmed the importance of PAD exposure in human disease pathogenesis. Data obtained from immunostaining studies in human brain tissue indicated that increased PAD exposure occurs very early in the process of tau inclusion formation and that AT8 is closely associated with PAD exposure during AD progression. Importantly, quantitative analyses are required to confirm our observations and conclusively determine the time course of PAD exposure in relation to other tau modifications during the progression of disease in humans. However, in combination with our squid axoplasm data, it is reasonable to assume that AT8 modification abundantly found in AD (Braak et al., 1994
) and other tauopathies (Takahashi et al., 2002
; Sakai et al., 2006
; Shiarli et al., 2006
) may facilitate PAD exposure and tau-mediated FAT dysfunction in situ
. Additionally, our data suggest a cyclical relationship between PAD exposure and AT8 phosphorylation in which the AT8 modification might result, at least in part, from the increased GSK3 activation triggered by abnormally exposed PAD, since S199, S202, and T205 are GSK3β phosphorylation sites.
Since neurons are thought to maintain function for years while bearing tau inclusions (Morsch et al., 1999
), it is reasonable to assume the existence of compensatory mechanisms that render pathogenic forms of tau innocuous. Phosphorylation of tyrosine 18 (within PAD) by kinases such as fyn (Lee et al., 2004
) or N-terminal cleavage of tau (Horowitz et al., 2004
; Sengupta et al., 2006
) may represent such protective mechanisms. Jeganathan et al. (2008)
demonstrated that combinations of phosphorylation events have different effects on tau folding. While AT8 alone reduces N-terminal folding, AT8 in combination with the AT100 (T212/S214) and PHF-1 (S396/S404) phosphorylation epitopes causes tau to fold into a more compact paperclip conformation. Regulation of tau conformation through protein–protein or intraprotein interactions as well as through modifications like phosphorylation or proteolysis might be part of a complex set of events that regulate tau function, including microtubule binding, modulation of phosphotransferase-based signaling pathways, and anterograde FAT.
Although tau has been recognized as a potential therapeutic target in AD and tauopathies, choosing therapeutic targets and testable outcome measures has proven difficult without an understanding of the mechanisms through which tau can influence disease progression. The identification of PAD and its effects on phosphotransferase activity and FAT regulation allow us to propose specific targets for intervention. For instance, reducing the activities of PP1 and/or GSK3 may reduce the toxic potential of pathogenic forms of tau. Alternatively, therapeutic strategies aimed at directly blocking PAD exposure and preventing it from activating the PP1–GSK3 cascade may provide yet another viable point of intervention.