Biochemical Society Transactions

The Biology of Tau and its Role in Tauopathies

Tau cleavage and tau aggregation in neurodegenerative disease

Diane P. Hanger, Selina Wray


Deposition of highly phosphorylated tau in the brain is the most significant neuropathological and biochemical characteristic of the group of neurodegenerative disorders termed the tauopathies. The discovery of tau fragments in these diseases suggests that tau cleavage and tau phosphorylation, both of which induce conformational changes in tau, could each have roles in disease pathogenesis. The identities of the proteases responsible for degrading tau, resulting in the appearance of truncated tau species in physiological and pathological conditions, are not known. Several fragments of tau are reported to have pro-aggregation properties, but the lack of disease-relevant cell models of tau aggregation has hampered investigation of the effects of tau aggregation on normal cellular functioning. In the present paper, we describe our findings of N-terminally truncated tau in the brain in a subgroup of the tauopathies in which tau isoforms containing four microtubule-binding domains predominate. We also discuss the evidence for the involvement of proteases in the generation of tau pathology in neurodegenerative disease, since these enzymes warrant further investigation as potential therapeutic targets in the tauopathies.

  • dementia
  • phosphorylation
  • protein aggregation
  • proteolysis
  • tau
  • tauopathy

Tau deposition and tau fragmentation in the tauopathies

Changes in tau conformation could be induced by post-translational modifications including phosphorylation and truncation [1]. Of these, tau phosphorylation has been the most actively investigated, but in the present paper, we consider tau truncation and the significance of tau fragments as potential pathogenic mediators in neurodegenerative disease.

One of the major functions of tau is to bind to microtubules, regulating their growth and shrinkage, and hence the dynamic instability of the neuronal cytoskeleton [2]. Tau usually exists as a highly soluble protein in brain; however, in several neurodegenerative disorders, tau becomes characteristically insoluble and aggregated, probably due to changes in its secondary structure that lead to conformational alterations. Highly phosphorylated and aggregated tau is a major component of the intracellular NFTs (neurofibrillary tangles) observed in AD (Alzheimer's disease) and of the related tau lesions that characterize several other human tauopathies [3]. The presence of tau inclusions in neurons and/or glia in the tauopathies indicates that this protein probably has a role in disease pathogenesis. Indeed, the overexpression of mutant and wild-type human tau in mice results in biochemical and behavioural phenotypes that recapitulate features of human tauopathies. Despite intensive research, the mechanism underlying tau deposition remains largely unknown; however, tau phosphorylation and tau truncation, both of which introduce conformational changes into the polypeptide, are likely to be key factors in disease-associated tau aggregation and accumulation.

Truncated tau has been reported in AD, and these fragments appear to lack both N- and C-termini of tau (reviewed in [4]). Specifically, a caspase-cleaved tau species, termed Δtau, in which the 20 most C-terminal amino acids of tau have been removed, has previously been reported. This form of tau is recognized by an antibody specific to Δtau which labels NFTs in the CA1 layer of the hippocampus in AD [5]. Another specific antibody has shown that tau is truncated at Glu391 in AD-affected brain, and this event occurs at a late stage of NFT evolution [6]. Co-staining of tissue sections with Alz50 and MC1, monoclonal antibodies that recognize specific conformational epitopes on tau, suggests that, together with the adoption of an abnormal conformation, cleavage of tau to the Δtau form may be an early event in the pathogenesis of AD [7,8]. The likely sequence of events is that tau takes on the Alz50 conformation, after which Δtau appears, followed by a further structural alteration that generates the MC1 tau conformation.

Tau is a highly phosphorylated protein and is a substrate for multiple kinases, including PKA (protein kinase A) [9] and GSK3 (glycogen synthase kinase 3) [10]. Caspase degradation of tau can be inhibited by phosphorylation of residues adjacent to the caspase cleavage site. Thus mutation of Ser422 in full-length tau to glutamate (S422E), to mimic a permanent state of phosphorylation, prevents caspase cleavage at Asp421 [11]. Furthermore, tau phosphorylation at this site has been observed in sections of brain affected by both AD and PSP (progressive supranuclear palsy) [7,12]. Thus phosphorylation of tau at Ser422 may be another early event that is common to both disorders, but further abnormal processing in these diseases appears to diverge subsequently with respect to caspase cleavage.

Phosphorylation also appears to influence tau aggregate formation in cultured cells. Exogenous expression of full-length tau, with or without GSK3, in CHO (Chinese-hamster ovary) cells, results in the appearance of tau in the soluble fraction, corresponding to the cytosolic compartment. Transfection of Δtau alone also causes it to be expressed in the cytosol, whereas, in contrast, co-transfection with Δtau together with GSK3 leads to the inclusion of Δtau in sarkosyl-insoluble, thioflavin-S positive tau aggregates [13].

It has been suggested that the generation of Δtau is restricted to neuronal lesions and thus, although it is readily detectable in AD, Δtau is less apparent in PSP, a disease exhibiting significant glial pathology [12]. Tau processing might be dependent on the cell type and/or the specific isoforms that are expressed and hence certain forms of truncated tau could be associated with different tauopathies, which could contribute towards their related, but distinct, neuropathological signatures.

Together with the caspases, the activities of the calcium-activated proteases (calpains) are likely to be an important factor in disease pathogenesis, particularly in AD. Thus Aβ (amyloid β-peptide), the major constituent of amyloid plaques in AD, is capable of inducing tau cleavage and this is thought to be mediated by both caspases and calpains [14]. Pre-aggregated Aβ has also been reported to cause the appearance of a 17 kDa tau fragment before tau phosphorylation, possibly due to the activation of both calpain and caspase 3 [15]. Tau fragments of this size induce cell death when transfected into CHO cells and have also been detected during induced apoptosis in cerebellar neurons [16]. However, to date, the 17 kDa calpain-cleaved tau species has not been identified in human tauopathy-affected brain.

We and others have found that specific fragments of cleaved tau are characteristic of tauopathies in which there is an overproduction of tau isoforms harbouring four microtubule-binding repeat domains [1719]. This subgroup of the tauopathies includes PSP, corticobasal degeneration and the majority of the FTDP-17Ts (frontotemporal dementias with parkinsonism linked to tau mutations on chromosome 17), but not Pick's disease (Figure 1). MS sequencing of a prominent tau fragment, of approx. 35 kDa, enriched from PSP-affected brain, determined that this cleavage product contained four microtubule-binding repeats and an intact C-terminus [19]. Hence this disease-related tau fragment differs from the truncated tau species identified in AD-affected brain and suggests that distinct proteolytic enzymes may be involved in the abnormal tau processing observed in different tauopathies, a factor that may underlie their differing pathologies. Shorter tau species extracted from the insoluble compartment of PSP-affected brain have also been detected with antibodies against various tau epitopes, including a band at 37 kDa [20], which may correspond to the truncated tau detected in other studies [18,19].

Figure 1 Tau fragments associated with four-repeat tauopathies

Insoluble tau extracts from PSP, corticobasal degeneration (CBD), frontotemporal dementia (FTD) with tau silent mutation N296N, AD and Pick's disease (PiD) were analysed on Western blots with an antibody recognizing the C-terminus of tau (TP70). The migration of some of the intact tau species (60–68 kDa) is indicated on the right of the blots. A 35 kDa truncated tau species was detected only in disorders in which there was an excess of tau isoforms containing four (4R), rather than three (3R), microtubule-binding repeats. The tau isoforms present in each disorder are indicated below each panel.

In addition to tau fragments in post-mortem human brain, a 33 kDa N-terminal fragment of tau has been reported to be significantly reduced in cerebrospinal fluid taken from PSP patients, compared with AD patients and controls, suggesting that such tau fragments could have potential as biomarkers for disease progression [21]. Although the identification of specific fragments of tau in PSP suggests the intracellular processing of tau may differ between disorders, the enzymes responsible for creating these specific fragments are not yet known.

Tau proteases

Tau can be cleaved by a variety of proteolytic enzymes including caspases [16], calpains [22], thrombin [23], cathepsins [24] and PSA (puromycin-sensitive aminopeptidase) [25], each of which is considered below.


There are three consensus sites in tau for caspase cleavage, namely residues Asp22–Asp25, Asp345–Asp348 and Asp418–Asp421, of which Asp421 is the preferred cleavage site, as is evident from mutagenesis [5,26]. The consensus sequence at Asp421 is recognized by caspase 3 and caspase 7 and mutation of this residue to glutamate inhibits caspase-mediated tau proteolysis [27]. Transfection of cells with the fragment of tau corresponding to residues 1–421 (Δtau) resulted in an increase in cell death when compared with full-length tau [27]. Interestingly, cell death was increased further when neuronal hippocampal cultures were transfected with a tau fragment corresponding to Δtau and harbouring the tau mutation N279K, which is associated with frontotemporal dementia [28]. In addition to cleavage at Asp421, caspase 6 cleaves tau close to its N-terminus, at the semi-canonical caspase consensus site, Asp13, at least in vitro. This may be physiologically significant because N-terminal tau epitopes are lost during NFT evolution in AD [29].


The calcium-activated cysteine proteases, calpains 1 and 2, have been shown to cleave tau in vitro [22]. Epitope mapping has indicated that tau digested with calpain retains its N-terminus [30]. Similarly to caspases, the susceptibility of tau to cleavage by calpain is reduced by tau phosphorylation. Soluble tau extracted from human post-mortem brain exhibits a lower phosphorylation state than the insoluble tau that comprises the NFTs in AD-affected brain. Such insoluble human tau is also less susceptible to calpain degradation than soluble brain tau [31,32], suggesting that phosphorylation may be linked to tau cleavage in vivo. Tau harbouring mutations associated with the development of dementia is also more resistant to calpain degradation, although a notable exception to this is the FTDP-17T-associated tau mutation G389R that displays increased turnover by calpain [33].

Treating hippocampal neurons with Aβ leads to calpain activation and the generation of a 17 kDa tau fragment. Of the nine potential sites of calpain action that are present in tau, cleavage at two of these sites (Leu43 and Val229) would generate a tau fragment of the correct size. In vitro cleavage of tau by calpain is prevented by mutation of Leu43 and Val229, and transfection of CHO cells and hippocampal neurons with a construct expressing tau residues 45–230 results in increased apoptosis [15], suggesting that this 17 kDa tau fragment has inherent neurotoxic properties.

Interestingly, there is an increase in calpain activity in AD-affected brain compared with controls [34]. Furthermore, the endogenous calpain inhibitor calpastatin is depleted in AD, and this reduction is mediated by a combination of the activities of caspases 1 and 3 and calpains [35].


Thrombin is an extracellular serine protease that induces neurite retraction and modulates morphological changes in glial cells [36]. In vitro, thrombin degrades tau in a stepwise manner at sites including Arg155, Arg209, Arg230, Lys257 and Lys340, with the initial cleavage occurring at Arg155 to produce a tau fragment of 37 kDa [37]. This truncated tau polypeptide of 286 amino acids is then cleaved further at Arg230 to generate a 25 kDa tau fragment [38]. In brain lysates incubated with different protease inhibitors, specific inhibition of thrombin was able to completely repress tau degradation [23]. With respect to a role in disease, both thrombin and its precursor prothrombin, are expressed by neurons and glia, and both proteins accumulate in NFTs in AD [39].

As with several of the other enzymes cleaving tau, phosphorylation of tau appears to increase its resistance to thrombin cleavage, both for recombinant tau phosphorylated in vitro and for insoluble aggregated tau from AD-affected brain, which became more susceptible to thrombin degradation following treatment with alkaline phosphatase [23]. Phosphorylation of tau by GSK3 inhibited all but the initial cleavage of tau at Arg155, and phosphorylation by PKA also induced a resistance to thrombin cleavage [40], supporting the view that phosphorylation may be a mechanism that protects tau from proteolysis.


Lysosomal dysfunction has been demonstrated in aged brain, leading to leakage into the cytoplasm of lysosomal enzymes, including the protease cathepsin D [41]. The amount of this enzyme is elevated in neurons vulnerable to pathology in AD [42]. Cathepsin D has been shown to cleave tau in vitro with the primary cleavage event occurring between amino acids 200 and 257 of tau and resulting in the generation of a 29 kDa product [24]. In human neuroblastoma cells inducibly expressing tau, disruption of lysosomes with chloroquine resulted in inhibition of tau degradation and the appearance of tau aggregates; this study also indicated the involvement of additional cathepsins B and L [43]. Thus dysfunction of lysosomal protease activity could be responsible for the appearance of tau aggregates in cells.


PSA has recently been identified as being protective against neurodegeneration. PSA was up-regulated in the cerebellum of transgenic mice expressing P301L mutant human tau, and modulation of PSA expression in a Drosophila model of tauopathy showed that its up-regulation reduced the rate of tau-induced neurodegeneration [44]. Furthermore, in human brain tissue from FTDP-17T patients, expression of PSA was highest in the cerebellum, which is relatively spared from pathology in these disorders [44]. PSA can digest recombinant tau as well as tau purified from brain tissue in vitro, although insoluble tau from AD-affected brain is more resistant to cleavage, supporting the view that post-translational modifications of tau influence its degradation by proteases [25].

Tau cleavage in animal models of tauopathy

Fragments of tau have been used to model aggregation in cells and animals, providing further evidence that proteolytic processing of tau could be important for NFT formation. Exogenous expression of the microtubule-binding repeat region of tau containing a pro-aggregation tau mutation (deletion of Lys280) in N2a neuroblastoma cells, results in the formation of tau aggregates that are also labelled with thioflavin-S [45]. Furthermore, conditional expression of the same mutant tau fragment in mice induces the formation of NFT-like structures which also incorporate endogenous mouse tau. Interestingly, endogenous mouse tau continues to accumulate in the NFTs after the expression of the fragment of mutant tau is terminated [46]. It is worth noting, however, that, although deletion of Lys280 in tau is associated with frontotemporal dementia [47], a tau fragment corresponding to this region of tau with Lys280 deleted has yet to be detected in human brain and therefore, although significant, these findings may not be directly relevant to disease pathogenesis in the tauopathies.

Transgenic rats have been produced that overexpress a tau fragment comprising residues Ile151–Glu391 and, as mentioned above, tau terminating at Glu391 has been isolated previously from AD-affected brain [6]. These animals develop AD-like neurofibrillary pathology, and the sarkosyl-insoluble brain fraction exhibits a biochemical profile similar to that of insoluble tau from AD-affected brain [48]. Interestingly, these transgenic rats also display a progressive neurobehavioural impairment [49]. The detection of truncated tau in several lines of transgenic mice engineered to overexpress mutant forms of tau, with resultant NFT formation, suggests that cleavage of tau may be part of the mechanism involved in such neurodegenerative processes [50,51].


The identities of the proteolytic enzymes responsible for degrading tau under both physiological and pathological conditions have not been determined completely. Fragments of tau are present in the brain in several tauopathies, and some tau fragments have been demonstrated to possess an increased propensity for aggregation, thus suggesting a possible link between tau cleavage and tau deposition. Truncation of tau could induce subsequent conformational changes that cause it to aggregate in affected cells, and there is evidence to suggest that sites of tau cleavage, and hence the activity of tau proteases, may be specific to individual or subgroups of tauopathies. Since several cleaved tau species have been shown to exert neurotoxic influences, targeting tau degradation could provide a new therapeutic approach for the tauopathies.


Work in this laboratory is supported by the Medical Research Council, the Wellcome Trust, the PSP Association, the Alzheimer's Society and the Henry Smith Charity.


  • The Biology of Tau and its Role in Tauopathies: A Biochemical Society Focused Meeting held at Robinson College, Cambridge, U.K., 7–8 January 2010. Organized and Edited by Amrit Mudher (Southampton, U.K.) and Makis Skoulakis (Alexander Fleming Research Center, Greece).

Abbreviations: Aβ, amyloid β-peptide; AD, Alzheimer's disease; CHO, Chinese-hamster ovary; FTDP-17T, frontotemporal dementia with parkinsonism linked to tau mutations on chromosome 17; GSK3, glycogen synthase kinase 3; NFT, neurofibrillary tangle; PKA, protein kinase A; PSA, puromycin-sensitive aminopeptidase; PSP, progressive supranuclear palsy


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