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

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

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‐

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

gesting that NFTs are not sufficient to cause cognitive decline or neuronal death.

protein plays a protective role against the disintegration of the microtubule.

**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

tein in AD.

98 Understanding Alzheimer's Disease

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‐

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.

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 further studies to confirm this hypothesis are warranted.

**Author details**

José Luna-Muñoz1

Benjamin Floran-Garduño1

Jesús Avila3

Aberdeen, USA

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**Figure 7.** Scheme illustrating the early steps of aggregation and polymerization of tau protein in Alzheimer´s disease. (A) The model starts with the appearance of PHF-core tau in cytoplasm of susceptible neurons. (B) The high binding capacity of PHF-tau results in the assembly of dimers of PHF-core and intact tau molecules in the cytoplasm. (C) The phosphorylation of intact tau would be an early event to hide the toxic soluble aggregates of molecules. (D). The high affinity and stability of the proto-filaments that make up the mature intracellular NFT allows tau molecules to form PHFs. (E) With the death of the neuron, the PHF-core subunit becomes exposed again in the extracellular space follow‐ ing proteolysis. Further details are described in the text.

### **Acknowledgements**

Authors express their gratitude to the Mexican families for the donation of brain tissue from their beloved and without which these studies would not be possible. Amparo Viramontes Pintos for the handling of brain tissue.This work was financially supported by CONACyT grants, No. 142293 (to B.F.).
