**7. Impact of phosphorylation and truncation on the abnormal processing of tau protein in AD**

#### **7.1. A neuroprotective mechanism for the phosphorylation of tau protein in the AD brain**

During neurodegeneration in AD, tau protein is abnormally phosphorylated in the prolinerich region at Ser and Thr residues [68], and such phosphorylation sites can be identified us‐ ing highly specific antibodies such as: AT8 (Ser-202/Thr-205) AT100 (Ser-212 and Ser-214), TG3 (Thr-231/Ser-235) and PHF-1 (Ser-396/Ser-404), among others (Fig. 6). However, NFTs are found in viable neurons at late stages of the disease, and they persist in neuronal cells for decades with a significant number of NFTs being found in the cognitively intact elderly [69, 70]. Such NFT-bearing neurons contain normal content and structure of microtubules [68]. The findings from studies in transgenic mice and human data, suggest that tau accu‐ mulation in the somatodendritic compartment may represent the manifestation of a protec‐ tive mechanism or a cellular adaptation that arises with advancing age. An increase in tau phosphorylation in AD brain has been associated with a protective mechanism against oxi‐ dative stress [71]. In another study, intact microtubules were found in NFT-bearing neurons [8], calling into question whether accumulation of phosphorylated tau and destabilization of microtubules are necessarily linked. Although microtubules are depolymerized in neurons with fibrillary degeneration, one study found evidence that the reduction of microtubules in AD is marked and specifically limited to vulnerable pyramidal neurons, and that even these alterations were observed in the absence of PHF [72]. This finding is also consistent with previous work by one of the authors noting a microtubule decrease of nearly 50% in den‐ drites that did not correlate with either PHFs or age [73], suggesting that a proportion of phosphorylated tau protein is associated with microtubules [71]. In animal models, it has been confirmed that axonal transport is not affected by either over-expression or reduction of tau protein *in vivo* [42, 74]. Another study found evidence that axonopathy precedes the formation of NFTs in a transgenic mouse [75]. A transgenic mouse expressing a human tau isoform developed NFTs, neuronal loss and behavioral impairments [58]. After suppression of tau expression, the behavioral deficits stabilized yet NFTs continued to accumulate, sug‐ gesting that NFTs are not sufficient to cause cognitive decline or neuronal death.

neuronal apoptosis [81]. Apoptosis is a process that usually occurs over a period of hours, whereas the accumulation of tangles found in AD brains occurs over a period of years or decades [78]. It has been suggested that hyperphosphorylation of tau protein is a mecha‐ nism used to evade cell death by apoptosis. Cells over-expressing hyperphosphorylated tau

Phosphorylation of Tau Protein Associated as a Protective Mechanism in the Presence of Toxic…

http://dx.doi.org/10.5772/54228

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**8. Participation of hyperphosphorylated and truncated tau species in the**

Despite suggestions of a neuroprotective role for tau protein in AD, links between phos‐ phorylated species (that are presumed to be protective) and the complex assembly of toxic, truncated tau into insoluble PHFs is not clear. In recent years, we have characterized the ear‐ ly stages of tau protein processing in neurons (pre-tangle state) (Fig. 1B, small arrows) and have described accumulations of tau that possess pathological species present in NFT, yet which do not show the presence of assembled structures in PHFs [67, 83]. The pre-tangle (Fig. 1B) is the first step in non-fibrillar aggregation of tau protein in AD and one in which at least 5 different changes take place (Fig 1 C,D). These events include: a) the presence of a Cterminally truncated and toxic tau species (Glu-391); b) a cascade of specific phosphoryla‐ tion of tau protein in the N-terminus; c) C-terminal truncation via the action of caspase-3; d)

oligomerization and aggregation of tau species and e) assembly of tau into PHFs.

further studies to confirm this hypothesis are warranted.

A model to accommodate the observations are represented schematically in Fig. 7. In this model, the first event to occur would be the emergence of tau oligomers or a PHF subunit (Fig. 7 A,B). The mechanism whereby this is initiated is unknown, but its toxicity and high affinity for binding to intact tau molecules would trigger an immediate need for the cell to protect itself. That would be reflected by hyperphosphorylation of the molecule in a failed attempt to hide the PHF and prevent the capture of further intact tau molecules (Fig. 7 B,C). In AD, this protective function of the phosphorylated species favors more molecules becom‐ ing available for sequestration and formation into PHFs (Fig. 7D). Gradually, phosphorylat‐ ed tau will be affected by exogenous proteolysis to re-expose the PHF-core (Fig. 7E). These steps follow as a molecular consequence of the catastrophic fragmentation of the microtu‐ bule, synaptic dysfunction, oxidative stress and post-translational modifications of tau. This model emphasizes that polymerization and neuroprotective mechanisms are both involved in the development of PHFs. The phosphorylated species of tau protein play a role in the initial protective response of the neuron to prevent the assembly of these filaments [35]. Thus NFTs, in which externally available tau is hyperphosphorylated, represents a mecha‐ nism whereby the neuron may try to protect itself from neurofibrillary degeneration and

appear to avoid the apoptotic process [82].

**8.1. Model for the mechanism of assembly**

**early formation of PHFs**

Within NFTs, different species of tau protein associated with phosphorylation are observed, but the neurodegenerating neurons still appear to be functional [75]. These observations suggest that the cytoplasmic accumulation of hyperphosphorylated tau protein is non-toxic, similar to the accumulation of lipofuscin that does not alter cellular metabolism[68]. It is generally assumed that disintegration of microtubules is associated with an imbalance be‐ tween kinase and phosphatase activities, which lead to an alteration in the stability of micro‐ tubules, disruption of cell function and culminate in neuronal death. The data, however, suggest that, at least, a subpopulation of hyperphosphorylated NFTs may be not toxic. This is controversial, given the fact that the hyperphosphorylation of tau and NFTs are consid‐ ered to be toxic. However, the ability of tau protein fractions, purified from AD brains, to alter microtubule assembly, *in vitro,* has been attributed to sequestration of normal tau mole‐ cules [18]. Alonso and colleagues [28] demonstrated that recombinant hyperphosphorylated tau, *in vitro*, decreases the breakdown of the recombinant microtubule when assembled into small aggregates [19, 28]. On this basis, the authors suggested that hyperphosphorylated tau protein plays a protective role against the disintegration of the microtubule.

Tau that has been hyperphosphorylated with GSK3-β kinase becomes immunoreactive with mAbs AT8, PHF1 and TG-3, antibodies whose epitopes are very closely related to AD [29, 76]. The fact that GSK3-β is capable of creating epitopes considered pathological in AD sug‐ gests that there are other participants that require to be considered. These data suggest the possible existence of a toxic species of non-phosphorylated tau protein, which would be re‐ sponsible for capturing further molecules of tau in PHFs, yet would not be exposed on the filament [7, 57, 61]. It is possible that, by hiding the toxic form in PHFs could protect the neuron [77]. It is important to note that the presumed "intermediaries" are present in the cy‐ toplasm of the neuron when it is still viable. Another study showed that NFTs (and presum‐ ably tau oligomers) could remain in the cytoplasm of the neuron for decades [78], an observation that would further argue against a primary toxicity of phosphorylated tau pro‐ tein in AD.

#### **7.2. Hyperphosphorylation of tau protein protecting neurons from apoptosis**

It is also proposed that apoptosis plays an important role in neuronal damage in AD. This proposal is based on the detection of fragmented DNA and expression of apoptosis signal‐ ing proteins such as caspases 3, 6, 8 and 9, Bax, Fas and Fas-L, in the cortex and hippocam‐ pus, in postmortem brain tissue [79, 80] and observations that amyloid-β can induce neuronal apoptosis [81]. Apoptosis is a process that usually occurs over a period of hours, whereas the accumulation of tangles found in AD brains occurs over a period of years or decades [78]. It has been suggested that hyperphosphorylation of tau protein is a mecha‐ nism used to evade cell death by apoptosis. Cells over-expressing hyperphosphorylated tau appear to avoid the apoptotic process [82].
