Using the acetylated K280 tau specific polyclonal antibody, we thoroughly evaluated diverse CNS regions in a large number of Alzheimer's disease, progressive supranuclear palsy and corticobasal degeneration cases with a detailed examination of Alzheimer's disease hippocampus throughout various Braak stages of disease. We demonstrate that acetylated K280 immunoreactivity is a significant marker of tau pathology in 4R tauopathies including Alzheimer's disease, corticobasal degeneration and progressive supranuclear palsy. Significantly, the acetylated K280 tau epitope was consistently found in the tau pathology of all cases studied, and it followed a similar distribution and regional severity to that of hyperphosphorylated tau epitopes. Moreover, we determined a potential temporal sequence for acetylation at K280 of tau in the process of tangle development. These findings provide insight into the role of acetylation in tangle formation.
Comparison of the acetylated K280-immunoreactive tau pathology burden to phosphorylated tau-immunoreactive pathology within cases generally showed similarity in more severely affected areas. Assuming that less severely affected areas would have a predominance of early pathology; tau hyperphosphorylation may precede acetylation at the K280 residue, mirroring our data from examination of early Braak stages. Interestingly, most phosphorylation sites occur in regions flanking the microtubule-binding repeat (Buee et al., 2000
), in which K280 is located. Perhaps, hyperphosphorylation renders this residue available for subsequent acetylation, which would further impair microtubule binding and/or promote tau aggregation as well as further drive pathological alterations of tau.
Additionally, the minimal pre-tangle reactivity and weak co-localization of acetylated K280 immunoreactivity in neuropil threads labelled by phosphorylation-dependent monoclonal antibodies support this observation, since neuropil threads are thought to emerge earlier than neurofibrillary tangles (Ghoshal et al., 2002
). This should be interpreted with the caveat that the potential tau de-acetylase enzyme HDAC-6, (Cohen et al., 2011
) is noted to be highly expressed in the cytoplasm of neurons in Alzheimer's disease cases (Miki et al., 2011
). Thus, the minimal neuritic staining observed here with acetylated K280 immunoreactivity may be a reflection of increased de-acetylase activity in neuronal processes that could abolish this epitope.
The most striking feature of acetylated K280 immunoreactivity in Alzheimer's disease was the prominent detection of intracellular tangles. This finding was evidenced by the almost exclusive co-localization of acetylated K280 immunoreactivity with thioflavin--S-positive neurofibrillary tangles, but not in neuropil threads detected by thioflavin-S or multiple other anti-tau antibodies examined here. Indeed, acetylated K280 was mostly associated with intracellular neurofibrillary tangles compared to pre-tangles or extracellular ghost tangles throughout all Braak stages. Similar findings have been reported for conformational and truncation tau epitopes that are thought to represent intermediate stages of tangle progression (Garcia-Sierra et al., 2003
; Guillozet-Bongaarts et al., 2005
). However, unlike these epitopes, acetylated K280 also co-localized with N- and C-terminal specific anti-tau epitopes (Supplementary Fig. 4
), indicating it is present in neurofibrillary tangles prior to subsequent tau truncation. This is supported by our observations that acetylated K280 immunoreactivity did not co-localize well with the truncation-specific tau epitope, MN423 and detected less glial fibrillary acidic protein-immunoreactive ghost tangles. Interestingly, the variability in acetylated K280 immunoreactive ghost tangles suggests that de-acetylation of K280 could occur when neurofibrillary tangles are released into the extracellular space from dying tangle bearing neurons. Another possibility is masking of the epitope in the paired-helical filament core, although prolonged antigen retrieval steps did not reveal additional extracellular tangle staining. In this regard, acetylated K280 was similar to the early Alz50 epitope, in that both antibodies did not detect many extracellular ghost tangles; however, there was only partial co-localization with Alz50, especially for early pre-tangle structures, and acetylated K280 immunoreactivity outnumbered Alz50 in neurofibrillary tangles in cases with severe Alzheimer's disease as this epitope was lost due to truncation events.
These data suggest that acetylation of K280 may be an intermediate step in tangle formation from threads and pre-tangle structures, which predominate in Alz50-immunoreactivity, is most associated with thioflavin-S-positive intracellular neurofibrillary tangles, and lost prior to the emerge of the majority of extracellular ghost tangles detected by MN423-immunoreactivity and glial fibrillary acidic protein-immunoreactivity.
Although pathological tau lesions in corticobasal degeneration and progressive supranuclear palsy do not react with amyloid-binding dyes (Dickson, 2004
) such as thioflavin-S, and are thought to contain less post-translational modifications than in Alzheimer's disease (Arai et al., 2003
), they were robustly positive for acetylated K280. These tauopathies have minimal extracellular tau pathology, and several late tau epitopes are not present in corticobasal degeneration and progressive supranuclear palsy cases (Berry et al., 2004
; Guillozet-Bongaarts et al., 2005
), further suggesting that acetylated K280 may precede tau-amyloid formation and late truncation events. The minimal acetylated K280 immunoreactivity in neuronal lesions in superficial layers of corticobasal degeneration cortical sections is intriguing, and may suggest alternative pathological cascades of tau modifications in differing cell types. Indeed, others have also shown a dissociation of tau epitope expression between cell types in these tauopathies (Guillozet-Bongaarts et al
Our data presented here suggest that acetylation of K280 in tau could play a mechanistic role in driving tau polymerization into neurofibrillary pathology and tau mediated neurodegeneration. We also previously identified three other potential tau acetylation sites, two of which are also in the microtubule-binding repeat (Cohen et al., 2011
). Further examination of these epitopes may also suggest a potential dynamic interplay between acetylation and phosphorylation at multiple sites that may act synergistically in the pathogenesis of tau fibrillization. Thus, a better understanding of the relationship of these post-translational modifications could be crucial to identify potential targets for therapy in Alzheimer's disease and other tauopathies, as well as biomarker development using acetylation-specific antibodies such as the acetylated K280 polyclonal antibody studied here. Although further work is required in cell and animal models to elucidate a possible functional role of tau acetylation in the pathogenesis of neurofibrillary tangles, such insights will advance efforts to test whether disruption of this process could prevent cell death and alter disease progression.