# **TAU OLIGOMERS**

# **Topic Editors Naruhiko Sahara and Jesus Avila**

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**ISSN** 1664-8714 **ISBN** 978-2-88919-261-8 **DOI** 10.3389/978-2-88919-261-8

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# **TAU OLIGOMERS**

Topic Editors:

**Naruhiko Sahara,** National Institute of Radiological Sciences, Japan **Jesus Avila,** Centro de Bilogía Molecular Severo Ochoa (CSIC-UAM), Spain

Schematic illustrating the central role of tau oligomers in tauopathies. Image taken from: Gerson J.E. and Kayed R. (2013) Formation and propagation of tau oligomeric seeds. Front. Neurol. 4, 93. doi: 10.3389/fneur.2013.00093.

Neurofibrillary tangles (NFTs) composed of intracellular aggregates of tau protein are a key neuropathological feature of Alzheimer's Disease (AD) and other neurodegenerative diseases, collectively termed tauopathies. The abundance of NFTs has been reported to correlate positively with the severity of cognitive impairment in AD. However, accumulating evidences derived from studies of experimental models have identified that NFTs themselves may not be neurotoxic. Now, many of tau researchers are seeking a "toxic" form of tau protein. Moreover, it was suggested that a "toxic" tau was capable to seed aggregation of native tau protein and to propagate in a prion-like manner. However, the exact neurotoxic tau species remain unclear. Because mature tangles seem to be non-toxic component, "tau oligomers" as the candidate of "toxic" tau have been investigated

for more than one decade. In this topic, we will discuss our consensus of "tau oligomers" because the term of "tau oligomers" [e.g. dimer (disulfide bond-dependent or independent), multimer (more than dimer), granular (definition by EM or AFM) and maybe small filamentous aggregates] has been used by each researchers definition. From a biochemical point of view, tau protein has several unique characteristics such as natively unfolded conformation, thermo-stability, acid-stability, and capability of post-translational modifications. Although tau protein research has been continued for a long time, we are still missing the mechanisms of NFT formation. It is unclear how the conversion is occurred from natively unfolded protein to abnormally mis-folded protein. It remains unknown how tau protein can be formed filaments [e.g. paired helical filament (PHF), straight filament and twisted filament] in cells albeit in vitro studies confirmed tau self-assembly by several inducing factors. Researchers are still debating whether tau oligomerization is primary event rather than tau phosphorylation in the tau pathogenesis. Inhibition of either tau phosphorylation or aggregation has been investigated for the prevention of tauopathies, however, it will make an irrelevant result if we don't know an exact target of neurotoxicity. It is a time to have a consensus of definition, terminology and methodology for the identification of "tau oligomers".

# Table of Contents

# "Tau oligomers," what we know and what we don't know

# **Naruhiko Sahara1\* and Jesus Avila2**

1 Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan

2 Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Madrid, Spain

\*Correspondence<EMAIL_ADDRESS>
#### **Edited by:**

Wendy Noble, King's College London, UK

**Keywords: tau protein, tauopathy, neurodegenerative disease, propagation, tau phosphorylation**

Neurofibrillary tangles, composed of intracellular aggregates of tau protein, are a key neuropathological feature of Alzheimer's disease and other neurodegenerative diseases, collectively termed tauopathies. Tau research has become one of the central players in the investigation of neurodegenerative diseases. Tau protein has several unique characteristics such as natively unfolded conformation, thermo-stability, acid-stability, and capability of posttranslational modifications. We still do not know whether tau itself is toxic. With certain triggers, tau may transit into toxic forms. Researchers are now looking for "tau oligomers" as toxic components. Because "tau oligomers" contain variable species of tau protein [e.g., dimer (disulfide bond-dependent or -independent), multimer (more than dimer), granular (defined as EM or AFM) and perhaps small filamentous aggregates] (**Figure 1**), it is important to have a consensus regarding the definition, terminology, and methodology for the identification of "tau oligomers" (1–6).

Recently, "prion-like" toxicity and propagation mechanisms underlying the progression of disease have been proposed. With this concept, tau may have the ability to translocate between neurons and amplify toxic components (7). Although we do not know the exact forms of toxic tau oligomers, accumulating evidence has shown the probability of tau oligomer propagation (6).

Tau is an intracellular microtubule-associated protein. The mechanism of tau transmission from cell to cell is still unknown. Research focusing on extracellular tau will open potential new avenues for discovering the mechanism of tau propagation (8).

Abnormally hyperphosphorylated tau is a key feature of human tauopathies. Although we are not sure whether phosphorylation rather than oligomerization of tau is an initial molecular event in

tau pathogenesis, investigating the regulatory mechanisms of tau phosphorylation will be essential (9–11).

Here, we provide an overview of the current understandings of "tau oligomers" (1–12). Efforts toward the identification of neurotoxic tau species will ultimately lead to the translational research for developing novel therapeutic strategies for tauopathies.

#### **REFERENCES**

*Received: 19 December 2013; accepted: 02 January 2014; published online: 13 January 2014.*

*Citation: Sahara N and Avila J (2014) "Tau oligomers," what we know and what we don't know. Front. Neurol. 5:1. doi: 10.3389/fneur.2014.00001*

*This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neurology.*

*Copyright © 2014 Sahara and Avila. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Are tau aggregates toxic or protective in tauopathies?

# **Catherine M. Cowan\* and Amrit Mudher \***

Centre for Biological Sciences, University of Southampton, Southampton, UK

#### **Edited by:**

Jesus Avila, Centro de Biología Molecular Severo Ochoa CSICUAM, Spain

#### **Reviewed by:**

Diego Rincon-Limas, University of Florida, USA Efthimios M. C. Skoulakis, Biomedical Sciences Research Centre Alexander Fleming, Greece

#### **\*Correspondence:**

Catherine M. Cowan and Amrit Mudher, Centre for Biological Sciences, University of Southampton, Building 85, Highfield Campus, Southampton SO17 1BJ, UK e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS>
Aggregation of highly phosphorylated tau into aggregated forms such as filaments and neurofibrillary tangles is one of the defining pathological hallmarks of Alzheimer's disease and other tauopathies. Hence therapeutic strategies have focused on inhibition of tau phosphorylation or disruption of aggregation. However, animal models imply that taumediated dysfunction and toxicity do not require aggregation but instead are caused by soluble hyper-phosphorylated tau. Over the years, our findings from a Drosophila model of tauopathy have reinforced this. We have shown that highly phosphorylated wild-type human tau causes behavioral deficits resulting from synaptic dysfunction, axonal transport disruption, and cytoskeletal destabilization in vivo.These deficits are evident in the absence of neuronal death or filament/tangle formation. Unsurprisingly, both pharmacological and genetic inhibition of GSK-3β rescue these tau phenotypes. However, GSK-3β inhibition also unexpectedly increases tau protein levels, and produces insoluble granular tau oligomers. As well as underlining the growing consensus that tau toxicity is mediated by a highly phosphorylated soluble tau species, our findings further show that not all insoluble tau aggregates are toxic. Some tau aggregates, in particular tau oligomers, are non-toxic, and may even be protective against tau toxicity in vivo.This has serious implications for emerging therapeutic strategies to dissolve tau aggregates, which might be ineffective or even counter-productive. In light of this, it is imperative to identify the key toxic tau species and to understand how it mediates dysfunction and degeneration so that the effective disease-modifying therapies can be developed.

**Keywords: Alzheimer's disease, dimer, oligomer, filament, neurofibrillary tangle, insoluble tau**

# **INTRODUCTION**

# **TAU PROTEIN IN ALZHEIMER'S DISEASE AND OTHER TAUOPATHIES**

Deposits of insoluble tau within neurons are defining pathological hallmarks in the group of neurodegenerative diseases known as tauopathies. Tauopathies include Alzheimer's disease (AD), Fronto-temporal Dementia with Parkinsonism on chromosome-17 (FTDP-17), Pick's disease, Corticobasal Degeneration (CBD), Progressive Supranuclear Palsy (PSP), and others (1). In all of these conditions, tau becomes both abnormally hyper-phosphorylated and deposited in insoluble aggregates [reviewed in Ref. (1, 2)]. These diseases differ in their clinical features, differentiallyaffected neuronal populations, and the distinct forms taken by the insoluble tau. Indeed, even within one disease state, the insoluble tau may be found in many distinct morphological forms; some *en route* to the final form of that disease's tau deposits, and others possibly on a different pathway.

In this review we will focus primarily on the forms of insoluble tau observed in AD, since they have been more widely studied. We will describe the different species of insoluble tau that have been identified; briefly review the factors that might promote tau aggregation; and then assess the evidence for and against the toxicity of each type of tau aggregate. Inevitably, this cannot be a comprehensive account of the extensive literature on this subject in the interests of space. Therefore we have selected papers which we believe represent the balance of evidence for and against toxicity, with apologies to those whose work we have not included. In this context we will use the term toxicity rather broadly, meaning either neuronal death, or neuronal dysfunction without death.

# **PHYSIOLOGICAL AND PATHOLOGICAL SPECIES OF TAU**

This section briefly describes the major forms that tau has been shown to take in AD. These different species are treated in approximate order of size, from smallest to largest (**Table 1**). However, there is no intention to imply that each one goes on to form the next in a clear pathway.

#### **MONOMER**

Monomers of tau are highly soluble proteins of 55–74 kDa in size [depending upon splice variant and phosphorylation status – (3)]. There are six splice variants which contain either three or four microtubule-binding repeats, as well as either zero, one, or two N-terminal domains. These isoforms are usually denoted tau0N3R, tau1N3R, tau2N3R, tau0N4R, tau1N4R, and tau2N4R. They usually acquire a predominantly random coil structure under normal physiological conditions (4). Partially folded forms of tau monomers have also been described which are distinct from native tau monomers, and have a reduced level of random coiling but an increased level of β-sheet structure (5). Interestingly, such molecules are immediately positive for Thioflavin (which binds β-sheet). Compact monomers have also been characterized displaying intra-molecular disulfide bonds (6). Only the three isoforms of four-repeat tau can form these compact monomers, since

the second cysteine required for an intra-molecular interaction is in the extra repeat domain.

#### **DIMER/TRIMER**

Dimers are composed of two tau monomers in anti-parallel orientation linked by disulfide bonds. Tau dimers can be observed by electron microscopy (EM) as rod-like particles 22–25 nm long, which is similar in appearance to the monomers (7). Dimers can form from any isoforms of tau. Within that, however, two distinctly different forms of dimers have been described (8). One is cysteine-dependent and reducible; while in contrast the other is cysteine-independent, non-reducible, and has inter-molecular disulfide bridging at the microtubule-binding domain. Both forms have been identified *in vitro*, and in tau transgenic (JNPL3) mice (8). Preparing small oligomers from recombinant tau *in vitro*, dimers have been reported with apparent sizes of 180 kDa (9) and 130 kDa (10), as well as trimers with an apparent size of 120 kDa (11). In human tau transgenic mice, soluble tau species of 140 kDa have been described (8, 12). Small soluble tau species of approximately dimer and trimer size, and probably including tau fragments, have also been isolated from synapses in AD brains (13). It is unclear whether these variously reported dimers and trimers are indeed different tau species or whether they represent subtle variations of the same structure.

#### **SMALL SOLUBLE OLIGOMER**

Small soluble oligomers of tau of very many different sizes have been described *in vitro* and *in vivo*. Often, however (perhaps because of differences in post-translational modifications leading to different apparent sizes on PAGE), it can be difficult to determine if small oligomers described by different groups represent the same species or not. In one study, the soluble dimers described above were shown *in vitro* to develop into small soluble oligomers containing six to eight tau molecules (approximately 300–500 kDa in size) (8). JNPL3 mice, which over-express human tau with the P301L mutation (tau0N4R-P301L) and harbor neurofibrillary tangles (NFTs), additionally have small tau oligomers which run at a wide range of sizes by PAGE [Sahara et al. (8)].

#### **INSOLUBLE GRANULAR TAU OLIGOMER**

Granular tau oligomers (GTOs) are electron-dense granular or globular aggregates of tau. They have been isolated from AD brains, mostly at early and moderate Braak stages (14). GTOs are composed of an average of 40 densely packed tau monomers. This corresponds to a size of 1800 kDa, or 20–50 nm in diameter when observed by EM or by atomic force microscopy (AFM) (15). It is important to note that, on the scale of insoluble protein aggregates generally, this is extremely small. Standard protocols for the sedimentation of insoluble proteins, such as 100,000 × *g* spin for 30–60 min [e.g., Ref. (16)], would fail to sediment GTOs which would remain in suspension in the "soluble" fraction, despite their demonstrable insolubility in SDS (15). Instead, sedimentation of GTOs requires a 200,000 × *g* spin for 2 h (15). The same authors developed a rigorous fractionation/purification protocol for GTOs. They further characterized the GTOs as being positive for MC1 and for Thioflavin, despite clearly being not filamentous in any way. They conclude that GTOs have β-sheet structure, and suggest that they may be composed of the partially folded form of tau monomer (15).

#### **FILAMENT**

It is well known that tau is capable of polymerization into filamentous forms. In AD, the predominant filaments are Paired Helical Filaments (PHF) and Straight Filaments (SF). In other tauopathies such as FTDP-17, however, there is variability in the morphology of tau filaments depending upon the tau mutations and/or tau isoforms involved. Here, filaments may take on other shapes such as twisted ribbon-like and rope-like filaments (17). A straight filament strand is 10 nm wide, and thus PHFs display alternating widths of 10 and 20 nm, with a half-periodicity of 80 nm (18, 19). Tau filaments exhibit β-sheet structure (20) which forms through the MT-binding repeat region (7, 21). Tau filaments from human AD brain have been shown to contain all six tau isoforms (22), although *in vitro* they can also be formed from single isoforms. They can be considered an amyloid (23, 24).

#### **PRETANGLE**

The pretangle is a slightly confusing concept that historically may have referred to a variety of species of tau, or even the status of a neuron. Generally speaking, a pretangle neuron is one that is positive for abnormal tau epitopes (misfolded and/or hyperphosphorylated), in some insoluble format large enough to be visible by light microscopy, yet free from mature fibrils or tangles by morphology. Bancher et al. (25) helpfully classified tangles into four stages (0–3). In this system1 , stage 0 tangles (later referred to

1For reference, stage 1 in this system is filamentous silver-stained tangles composed primarily of PHF; stage 2 is a classic neurofibrillary tangle and stage 3 is a ghost tangle (See "Neurofibrillary Tangle" and "Ghost Tangle").

by others as pretangles) are identified by cytoplasmic non-fibrillar (granular or diffuse) tau immunoreactivity, visible at the light microscope level. When viewed by EM, the labeled material was found to consist of PHFs, SFs, and smaller granular electron-dense material. Where pretangles were observed as granular via light microscopy, this probably represents non-filamentous clumps of PHFs, SFs, and the ultrastructural granules. Other researchers have described immunoreactivity for certain abnormal AD-associated tau epitopes in neurons containing no fibrils, and have deemed the neurons so labeled to be at a pretangle stage [for example Alz50 (26),the 12E8 epitope S262/S356 (27,28),and T231 (27,29)]. Confusingly, there are a number of conflicting reports in the literature as to whether "pretangles" are silver-staining, thioflavin-positive, and whether or not they contain β-sheet structure. It seems probable that these discrepancies arise from (a) a heterogeneity of what is meant by "pretangle" and (b) a sensitivity issue in regard to the assays for β-sheet. Pretangles should surely be positive for markers of β-sheet, since even the earliest partially-folded monomer (5) and certainly tau filaments (30) demonstrably contain β-sheet structure.

#### **OTHER LARGE NON-FIBRILLAR TAU AGGREGATES**

There are other forms and morphologies of pathological insoluble tau found in human brains which are large enough to be seen with the light microscope, and may be filamentous, yet are non-fibrillar in structure. Such aggregates include Hirano bodies, Pick bodies, and argyrophilic grains.

Hirano bodies have been described in AD, Pick's disease and other tauopathy brains (31, 32). Hirano bodies are large intraneuronal paracrystalline structures of 5–10µm in width by 10–30µm in length, composed of 7 nm filament arrays (32). They contain tau, other microtubule-associated proteins, actin, cofilin, other actin-binding proteins, and a fragment of APP.

Pick bodies are the characteristic morphology assumed by tau filaments in Pick's disease, in which they accumulate in limbic and cortical neurons. They are large structures that vary in size in different neuronal types, but are approximately the size of the nucleus. Pick bodies are formed of disorganized bundles of filaments which comprise only the three 3-repeat isoforms of tau, in contrast to the PHFs and SFs formed in AD which are made of all six isoforms [reviewed in Ref. (33)].

Argyrophilic grains are found in Argyrophilic Grain disease, where they accumulate in both neuronal processes and oligodendrocytes (34). Argyrophilic grains are structures that may be spherical, oval, comma-shaped, or spindle-shaped. As the name suggests, they are readily detectable by conventional silver-staining and light microscopy. The grains are much smaller than Hirano bodies, Pick bodies, and NFTs at approximately 4–9µm in size. Argyrophilic grains are comprised of four-repeat tau in 9–18 nm SF and bundles of 25 nm smooth tubules. They never contain PHFs and ribbon-like filaments (34–36).

#### **NEUROFIBRILLARY TANGLE**

Neurofibrillary tangles are the classic tangles first described by Alzheimer in 1907. Classified by Bancher et al. (25) as stage 2 tangles and often described as "flame-shaped," they are large bundles of fibers consisting of both PHFs and SFs which may fill the entire neuronal cytoplasm. The fibers are silver-staining. Brief mention should be made here also of neuropil threads, which are bundles of SFs and PHFs occupying dendrites and largely displacing the cytoskeleton (37).

### **GHOST TANGLE**

Ghost tangles are the structures that remain when the neuron within which the tangle formed has degenerated. They comprise large extracellular bundles of loosely arranged tau filaments. Compared to NFTs, ghost tangles stain more weakly for tau and more strongly for ubiquitin (25). It is thought that ghost tangles have undergone substantial proteolysis, and that thus the filaments are comprised predominantly of tau fragments, again in contrast to NFTs (38).

# **THE SEQUENCE OF EVENTS IN TAU AGGREGATION**

There is some evidence to suggest that larger tau aggregates like PHFs and NFTs evolve from the successive aggregation of smaller tau species like monomers and soluble oligomers (**Figure 1**). One missing link appears to be whether small oligomers can form directly into GTOs in a linear pathway, or whether they represent two different pathways from monomers to PHFs and NFTs.

# **MONOMER**→**DIMER**

There is evidence from the kinetics of tau polymerization that, once the partially folded conformation of the monomer has formed (however that may be triggered), then the process from monomers to dimers (and thence to oligomers) is energetically favorable and proceeds spontaneously (5). For monomers to be able to form dimers requires the PHF6 hexapeptide in the third microtubule-binding repeat domain (8, 39). However, the compact form of the tau monomer does not participate in this form of aggregation (40).

#### **DIMER**→**SMALL SOLUBLE OLIGOMER**

The tau dimer, in particular the cysteine-independent, nonreducible form (8), is thought to be an important intermediate which is involved in controlling the rate of formation of larger intermediates and fibrillization (6, 7, 41). In addition, more than one group has demonstrated that *in vitro* generated tau dimers aggregate to form larger tau oligomers (8, 9).

#### **SMALL SOLUBLE OLIGOMER**→**GTO**

We are not aware of any direct evidence that small oligomers proceed to form GTOs. There is, however, evidence that tau monomers *in vitro* can form GTOs (15), as well as that both monomers and GTOs can form PHFs. However, whether the sequence always proceeds from monomer→dimer→small oligomer→GTO→filament, or whether GTOs and other types of tau oligomers can be on different pathways, is not clear. In general, it is believed that when tau forms larger structures such as filaments of differing morphology, the interactions between tau molecules remain the same, and subunit packing follows the same plan (40). On these grounds it is plausible that GTOs might be part of the same pathway.

#### **GTO**→**PHF**

Increasing the concentration of GTOs *in vitro* causes them to form filaments, whereas the constituent soluble tau does not. On the basis of this, it is suggested that GTOs are precursors of PHFs (15).

#### **MONOMER/DIMER**→**PHF (POSSIBLY VIA THE OTHER INTERMEDIATES)**

There is a wide variety of evidence showing that monomers can polymerizes into PHF, but that does not address whether this is via the oligomeric intermediates or not. Such evidence includes the early *in vitro* demonstrations that tau at high concentrations will self-assemble into PHFs (42–44), and evidence that dimers are normally rate-limiting intermediates in this process *in vitro* (4, 6). There followed from these studies a large body of work delineating important details such as which motifs within tau are required for fibrillization, in which monomeric tau clearly formed into PHFs (39, 45–47). However, as in the early studies, whether oligomers were formed on the way was not necessarily assessed directly.

The mechanism for PHF formation requires two hexapeptide motifs in the microtubule-binding region of tau. These are PHF6 [(306)VQIVYK(311)] and PHF6∗ [(275)VQIINK(281)]. Formation of PHFs involves these two motifs changing conformation from random coil to β-sheet structure (24, 39). It should be noted that mutant tau containing no cysteines is still able to form PHFs *in vitro*, even though much more slowly than WT tau (40). This means that cysteine-dependent (covalent) dimers are not a requisite stage between monomer and PHF.

#### **PHF**→**NFT**

It is well established that NFTs *in vivo* are composed of PHFs and SFs (25). Furthermore, there is also direct *in vitro* evidence that filaments will spontaneously clump together into NFTs (48). Thus it is highly likely that NFTs are formed by the accumulation of tau filaments.

## **WHAT PROMOTES TAU AGGREGATION?**

Little is known about what first triggers the initiation of tau aggregation. It is known that normal monomers do not spontaneously seed aggregation, and that some sort of trigger is needed (30, 49). However, numerous factors have been identified that can promote or increase tau aggregation, at least *in vitro* [reviewed in Ref. (50)].

Enzymatic cleavage of the tau monomer is one such factor. Truncation of the tau protein at Glu391 (51, 52), truncation by caspases at Asp421 (53), cleavage by thrombin (54), removal of the C terminus of the protein (55, 56), or deamination at asparagine or glutamine residues (57) have all been shown to promote tau aggregation [Reviewed in Ref. (58)].

Local concentration of tau can be key. Tau at high concentrations *in vitro* forms PHFs (42–44). Moreover, the transition of tau from random coil to β-sheet is also known to be concentration dependent (39), further supporting the idea that excessive local accumulation of tau may promote its aggregation (especially if other pro-aggregating factors – such as those discussed below – are also in the near vicinity).

Controversially, tau phosphorylation has been postulated to both stimulate and repress its subsequent aggregation into filaments. Circumstantial evidence for stimulation includes the seminal fact that filamentous tau is highly hyper-phosphorylated (59) at many sites. More direct but *in vitro* evidence shows that tau phosphorylated at AD sites polymerizes more readily into tangles of PHF/SF; dephosphorylation abolishes tau's self-assembly; and hyperphosphorylation of recombinant tau by brain kinases induces its self-assembly into tangles of PHF/SF (60, 61). In a cellular model, it has been shown that all three kinases GSK-3β, MEKK, and JNK3 are required for tau aggregation (62). Phosphorylation of tau specifically at Thr231, Ser396, Ser422, and Ser404 promotes self-aggregation of tau into filaments (55, 63, 64).*In vivo*, overexpression of the kinases GSK-3β or Cdk5 can promote tau aggregation (65, 66). On the other hand, *in vitro* studies have shown that tau phosphorylation is not necessary to drive tau into PHFs (41, 67). On the contrary, phosphorylation of KXGS motifs in the repeat region *inhibits* tau aggregation *in vitro* (54, 68). Furthermore, more recently emerging data showing that tau aggregates made up of recombinant non-phosphorylated tau can "seed" further tau aggregation in cells (discussed below) also supports the idea that phosphorylation is not required to promote aggregation (69, 70). However, it is not yet known whether phosphorylated tau would seed and promote aggregation at a different rate.

Some of the missense and deletion mutations found in tau in cases of fronto-temporal dementia (FTDP-17), when expressed in models, display enhanced aggregation compared to normal tau. *In vitro* PHF formation is faster for recombinant tau harboring one of various such mutations. Human tau with each one of the point mutations G272V, N279K, V337M, or R406W shows significantly faster *in vitro* PHF formation than WT full-length human tau, while the ∆K280 and P301L mutants form PHFs at dramatically greater speeds (46). This phenomenon has been confirmed *in vivo*: mice expressing mutant human tauP301L develop pathology more readily than those expressing WT human tau, both on 0N4R and 2N4R tau backgrounds (71–73).

Many polyanionic cofactors of all kinds can promote PHF assembly. These include glycosaminoglycans (GAGs) such as heparin and neuroparin (40, 74, 75); sulphoglycosaminoglycans (sGAGs) like keratins or chondroitin sulfates (76), RNA (41); polyglutamic acid (30, 41, 74); fatty acids such as arachadonic acid (77, 78) and alkyl sulfate detergents (79).

Other factors which may promote tau aggregation include tissue transglutaminase (80), Congo red (81), ferritin (82), H2O2 in the presence of iron (Fenton's reaction) (83), and quinones (84).

Despite this wealth of data over many years regarding factors that promote aggregation, questions still remain about what initiates tau aggregation *in vivo* in health and disease. However, once tau aggregation has been initiated, it is believed to promote further"prion"-like"seeding"and propagation of tau aggregation and pathology (85). This was first demonstrated *in vivo* where stereotaxic injections of brain homogenate containing tau aggregates led to induction and propagation of tau aggregation in tau transgenic mice (85). Supportive data also emerged from studies in cell culture showing that incubation of tau-expressing cells with fibrils of recombinant tau leads to induction of tau aggregation in the recipient cells (69, 70).

#### **ARE NEUROFIBRILLARY TANGLES TOXIC?**

#### **NFTs: EVIDENCE FOR TOXICITY**

The evidence that associates NFTs with neuronal dysfunction and neurodegeneration is largely correlative in nature. Studies of human post-mortem brains initially implicated NFTs in toxicity by showing a strong spatial and temporal correlation between NFTs and severity of dementia, and between NFTs and neurodegeneration or neuronal death (86–92). Some tau transgenic mouse models display neuron loss in the same timeframe and/or location as NFT formation. For example, expression of tauP301L under the thy1.2 promoter causes neuronal apoptosis at the same age as filaments and NFTs (93); while tauP301L under the prion promoter causes NFTs in spinal cord, brainstem, and pretangles in cortex, at the same time as loss of motor neurons (71). Furthermore, in tau mouse models, there is a correlation between reduction of NFT and improvement in cognition (94). The limitation of these correlative studies between reduced NFTs and reduced impairments is that, in many cases, other smaller tau species may be reduced also. This leaves open the question of whether it is the reduction of the NFTs or the smaller species which has been beneficial.

More direct evidence in favor of NFT toxicity comes from mice conditionally expressing human tau fragments harboring pro-aggregation or anti-aggregation mutations (95, 96). Proaggregation mice develop PHFs, "pretangles," and NFTs early; followed by synaptic and neuronal loss. In constrast, mice expressing the same tau molecule but with the anti-aggregation mutation never develop aggregates or neuronal pathology. This was verified in a *C. elegans* model of tauopathy which went further to show that treatment with anti-aggregation inhibitors protected against taumediated toxicity (97). This suggests that tau aggregation in the form of PHFs or larger has been the cause of cell death. Moreover, when expression of human tau is suppressed, mice are rescued from toxicity in terms of both cell death and cognition. This represents very strong evidence in favor or PHFs and/or NFTs as the toxic species. These conclusions were difficult to reconcile with a body of evidence detailed below proving that PHFs and NFTs are

neither necessary nor sufficient for toxicity. However, the authors subsequently showed that in the animals expressing pro-aggregant tau, toxicity might in fact be mediated by a species of tau smaller than PHFs and NFTs (98, 99).

#### **NFTs: EVIDENCE AGAINST TOXICITY**

There is now strong evidence from a number of models that NFTs are not required for tau-induced neuronal dysfunction and toxicity. In most *Drosophila* models of tauopathy, neuronal NFTs are usually not formed at all, despite clear neurodegeneration, and functional phenotypes (100–104). In some mouse models where NFTs do form, cognitive/behavioral impairments and cell death can be demonstrated *earlier* in the time course of the disease prior to NFT formation (105). In a different mouse model [PrP44: the shortest human tau isoform (tau0N3R) under the prion promoter] the formation of tau filaments coincides in the time course of the disease with phenotypes such as neurodegeneration and motor deficits, while NFTs form later (106, 107).

There is also compelling evidence that NFTs are not sufficient for toxicity, from mice that conditionally express human tau (tau0N4R-P301L). These mice display age-dependent development of NFTs, neuronal loss, and progressive motor deficits. When tau expression is switched off after the onset of memory impairments and NFT formation, memory improves and cell loss is stabilized, yet NFTs remain (108, 109). Furthermore, when tau is turned off at a timepoint when there are pretangles but no NFTs yet, the pretangles stay stable. This indicates that they also are insufficient for toxicity. This is corroborated in a different study using the same mice, in which a successful treatment reduced motor deficits despite failing to reduce NFTs (110). Furthermore, tanglebearing neurons in this model were shown to be just as active in a functional hippocampal circuit as non-tangle bearing neurons (as evidenced by expression of the immediate early gene *Arc* in response to environmental cues) (111). Further investigations of the mouse conditionally expressing pro- and anti-aggregant form of tau, mentioned in the preceding section, also supports this view. The pro-aggregation mice develop learning and memory deficits from which they recover after tau expression is turned off (98). However, it transpires that after an extended period of tau suppression, NFTs still remain, as in the tau0N4R-P301L mice. This implies that it is a smaller species of tau (soluble or insoluble) which has decreased in correlation with behavioral improvement in these studies (98).

In the light of such evidence, it has been suggested that formation of NFTs is a protective response that ultimately fails (58). This review describes a scenario in which caspases, having become activated because the cell contains toxic tau and is thus under stress, cleave the tau making it more fibrillogenic. Cleavage is unlikely to be the only trigger, since the initial steps of aggregation can involve primarily full-length tau isoforms (5, 112). Either way, the idea is that once tau aggregates seed, they can sequester toxic tau and thus delay cell death. However, the trade-off is that axonal transport is compromised and cellular protein degradation pathways become clogged, and thus the neuron gradually becomes dysfunctional (58). This is supported by evidence that NFT-bearing neurons appear to survive for decades (113) and maintain markers of normal gene expression (111), and may be in fact be longer lived than

those neurons without NFTs in the AD brain that would have died at earlier time-points and hence were not evident at post-mortem.

In conclusion, despite NFTs being a vital historical clue to the involvement of tau in neurodegenerative disease, a wide variety of strong evidence now exists that NFTs themselves are neither necessary nor sufficient to cause tau-induced toxicity and dysfunction.

### **ARE TAU FILAMENTS TOXIC?**

Some of the evidence regarding NFTs is also applicable as indirect evidence about tau filament toxicity. That is, some of the evidence in favor of large aggregates being toxic could really apply to either NFTs or PHFs/SFs or both. Further, the evidence that NFTs are not toxic naturally casts suspicion onto smaller pathological species such as filaments. However, direct evidence for PHFs as the primary toxic species, rather than something smaller, is sparse.

#### **FILAMENTS: EVIDENCE FOR TOXICITY**

As with the evidence in favor of NFT toxicity described above, much of the evidence that implicates filaments as a toxic species of tau is correlative in nature. For example, in some mouse models of tauopathy, filaments coincide in the time course of the disease with phenotypes such as neurodegeneration and motor deficits (106), while NFTs form later (107). Similar results were seen in one *Drosophila* model of tauopathy in which tau filament formation was reported (114). Furthermore, mutations in tau which are responsible for FTDP-17 also promote faster tau filament formation (46), thus circumstantially implicating tau filaments in the disease process.

An immunotherapy study targeting tau in a mouse model provided some more direct evidence in favor of PHF toxicity. In this study, immunization of JNPL3 mice (with a short phosphorylated tau fragment) served to reduce tau aggregation (into PHFs or larger aggregates) and the associated behavioral phenotypes, but failed to reduce smaller species of tau. This suggests that that PHFs or larger tau aggregates are the toxic species in this model (115). In another study, tau filaments but not monomers (at physiological concentrations) were shown to selectively impair anterograde transport in isolated squid axoplasm (116, 117).

#### **FILAMENTS: EVIDENCE AGAINST TOXICITY**

As with the NFT situation, some animal models have impairment but no filaments, or at least not until later in the disease progression. For example, *Drosophila* (100–104) and *C. elegans* (118) expressing human tau display behavioral phenotypes indicative of neuronal dysfunction and toxicity without forming tau filaments or larger aggregates. Even in mice, in the transgenic tau model expressing the longest human tau isoform (tau2N4R), brains contain some form of insoluble tau but nothing as big as PHFs (or NFTs), while the animals display a motor impairment (119). Such evidence indicates that filaments are not necessary for tau-induced toxicity.

There is also evidence that filaments are not sufficient for toxicity, since they continue to form in the conditional tau mice mentioned earlier, in which transgenic tau expression has been turned off and deficits thereby rescued (108). In another mouse model, Andorfer et al. showed that, while there was widespread neurodegeneration, the PHF-containing neurons appeared "healthy" in

terms of nuclear morphology, suggesting that the polymerized protein was probably neuroprotective (105). In an *in vivo* lamprey model (120), administration of a benzothiazole derivative drug (purported to break up tau filaments) successfully improved tau-induced phenotype, but apparently did so without actually breaking up the tau filaments. This provides further evidence that filaments are not sufficient for toxicity. *In vitro*, polymerization of hyper-phosphorylated tau into PHFs abolishes its toxic activity to sequester other MAPs (121). Unlike the soluble form of hyperphosphorylated tau, the filamentous form of tau does not bind MAPs and does not disrupt microtubules *in vitro* (121).

In an inducible cell line, the repeat domain of wild-type tau was non-toxic, whereas a similar construct harboring a point mutation that induced aggregate formation (eventually PHFs) caused cell death (54). Crucially, however, increased cell death was observed *before* PHF formation in the aggregate-prone mutant, demonstrating that PHFs were not necessary for toxicity, and that in fact a smaller form of aggregate was the toxic species.

While the *in vivo* evidence is not quite as extensive as for NFTs, one can also conclude that tau filaments are neither necessary nor sufficient for tau-induced toxicity, and that something smaller than filaments is the most toxic form of tau.

#### **SPECIES OF TAU FOUND IN FILAMENTS**

When the insoluble protein fraction from brains of AD patients or animal models, containing any tau filaments or larger tau aggregates, is solublized using urea or formic acid, the tau species which were building blocks of these large insoluble aggregates can be identified. These species include monomers of 55, 60, 64, and 68 kDa in size (67), and a 170-kDa species. The 64 and 170 kDa species in particular have been implicated in toxicity. The 64-kDa species represents a hyper-phosphorylated monomer. It is found in brains of the tau0N4R-P301L transgenic mouse (122), and increases with age in the insoluble fraction at the expense of the 55-kDa monomer (which is found in both soluble and insoluble fractions).

In the same study that showed NFTs were not necessary for toxicity [because they did not decrease in successfully treated tau mice – (110)], the successfully treated mice displayed a significant reduction in 64 kDa tau from the high-speed insoluble fraction. Confusingly, subsequent commentators have described this species as "soluble aggregated tau" [e.g., Ref. (123)]. However, this 64 kDa species is always a component of an aggregate that sediments at 150,000 × *g*, which is therefore bigger than a GTO. Another study in a conditional mouse model found that three distinct tau species correlated with neuronal dysfunction (12). Two of these species were in a sarkosyl-insoluble fraction (from which NFTs had been previously cleared) which must represent GTOs or filaments: a 64-kDa hyper-phosphorylated monomer and a 170-kDa hyper-phosphorylated oligomer. The third species was a 140-kDa oligomer from the soluble fraction. All of these species arose early in the disease progression, and increased with increasing learning and memory deficits. Conversely, all three decreased upon suppression of transgenic tau expression and recovery from neuronal dysfunction. These results clearly implicate one or more of the three species in toxicity; however, it is not clear whether the culprit is the soluble or insoluble components or both. The same 64 kDa species has also been described in a sarkosyl-insoluble

fraction from transgenic tau0N4R-301L mice that represents SFs and NFTs (71, 108). It is not toxic in this circumstance because it continues to increase after tau expression has been turned off and animals are recovering (108).

In summary, these two species of tau, a hyper-phosphorylated 64 kDa monomer and a 170-kDa oligomer, have frequently been demonstrated as components of an insoluble filament fraction. There is some evidence, although not conclusive, that these species may be associated with toxicity.

# **ARE (INSOLUBLE) GRANULAR TAU OLIGOMERS TOXIC?**

Part of the difficulty in acquiring evidence about GTOs from the existing literature is that, as mentioned earlier, in standard insoluble protein fractionation protocols any GTOs in the sample will be lost. Even though fully insoluble, they are too small to sediment with a standard "high-speed insoluble" fraction (15). They remain in suspension in the "soluble" fraction; and yet as they are around 1800 kDa in size and not readily reducible without 8 M urea, they will not enter a standard PAGE resolving gel and be detected (Cowan, unpublished observation). Therefore when one reads, for example, studies in tau mouse models showing small oligomers and PHFs and drawing conclusions about toxicity, one cannot usually conclude anything about the presence or absence, toxicity or otherwise, of GTOs.

#### **GTOs: EVIDENCE FOR TOXICITY**

In the years following the identification of GTOs, when evidence was beginning to accumulate that PHFs and NFTs might not be toxic, some evidence remained that some insoluble form of tau must be toxic. There was speculation in reviews that insoluble oligomers of tau, perhaps GTOs, might be the most toxic species. However, there is no direct evidence for this. Clues that GTOs might be associated with toxicity come from studies showing that numbers of GTOs increase with progression of AD, that fewer GTOs are observed at Braak stage 0 than at stage 1, and that their peak precedes that of NFTs (14). GTOs are generally believed to consist of toxic phosphorylated species of tau because phosphorylated tau levels are high in the AD brain at the time-points when GTOs are abundant. Such clues have led to the suggestion that reducing GTOs may prove to be a promising therapeutic strategy; however, the authors of these publications acknowledge that the effect of GTOs on neuronal vulnerability is unknown.

There is also evidence that an insoluble oligomer(s) of some sort is probably the culprit, without clear evidence that it is GTOs. For example, in the study mentioned as evidence against PHF toxicity in an inducible cell line (54), the soluble tau construct was not toxic, whereas the aggregate-prone mutant tau species caused cell death *prior* to PHF formation. This therefore represents evidence against both soluble tau toxicity and PHF toxicity, but rather implicates some intermediate species. Similarly, in another study, very small insoluble tau oligomers (of up to a few hundred kDa) were isolated from synaptosomes derived from AD brains and were associated with impaired ubiquitin proteasome function (124). If any GTOs had been present in this preparation, they might not have been observed with the protocols used. Clearly small insoluble tau oligomers exist and are associated with toxicity but whether

they can be classified as GTOs or indeed their precursors is not clear.

#### **GTOs: EVIDENCE AGAINST TOXICITY**

We have recently observed, in a *Drosophila* model of tauopathy, the formation of GTOs which are non-toxic (125) When flies express human tau0N3R in neurons they exhibit a clear behavioral phenotype, but no insoluble tau. However, upon pharmacological or genetic manipulations which inhibit GSK-3β, the phenotype is rescued and GTOs are produced. We demonstrate that these GTOs produced in flies are the same size as those isolated from human brain and comprise of non-phosphorylated full-length tau monomers. Like us, another group has also demonstrated the formation of large insoluble tau oligomers in *Drosophila,* in conditions associated with rescue of tau-mediated toxicity (126). They showed that rescue of human tau0N4R or tau0N4R-R406W induced neurodegeneration and behavioral deficits by co-expression of Nicotinamide mononucleotide adenylyltransferase (NMNAT) also led to increased formation of insoluble tau oligomers. However whether these insoluble tau oligomers were the same as the GTOs that we described in our model is not clear. Nonetheless, both our studies collectively imply that tau aggregation can correlate with rescue from tau-induced phenotype. However, whether this is because of sequestration of smaller toxic tau species, or the presence of non-phosphorylated tau in the conditions in which the GTOs form, or something else about their structure is not clear.

Overall, the pathological significance of GTOs has yet to be fully understood. It is possible that different GTOs form in different circumstances and the phosphorylated status of their constituent tau proteins and/or the extent of β-sheet structure may play a role in determining their toxicity.

# **ARE SOLUBLE (MONOMERIC OR OLIGOMERIC) TAU SPECIES TOXIC?**

As has already been alluded to, there are numerous species of tau that are soluble, and it seems probable that they possess very different properties. Just considering monomers, there is clearly a multiplicity of species: the three major conformations described (regular, compact, and partially folded); the six splice variants; and of course the array of phosphorylation combinations, both demonstrated and possible, which have been barely touched upon here. Then there are at least two conformations of dimer (cysteine-dependent and independent), as well as trimers, and small oligomers of various sizes and phosphorylation states, as described earlier. We would speculate that there are probably many variations of soluble oligomeric tau species occurring in nature in the brain that have not yet been specifically described. Further, some of the evidence regarding the toxicity or otherwise of soluble tau cannot (or did not) differentiate between these species.

#### **SOLUBLE TAU: EVIDENCE FOR TOXICITY**

There are many examples of studies conducted *in vivo* and *in vitro* showing that soluble tau is sufficient to cause dysfunction and toxicity. To give a few examples, pseudophosphorylated tau causes cell death when virally expressed in hippocampal slices, without becoming SDS-insoluble (127). Soluble tau monomer applied extracellularly to cells in culture causes intracellular calcium increase and cell death (128, 129). Additionally, some tauopathy models in *Drosophila* (100–102, 130) exhibit significant neuronal dysfunction and degeneration and yet contain no insoluble tau providing strong evidence that soluble tau is sufficient to cause dysfunction and toxicity. Similar evidence exists in mouse models: for example, mice expressing WT tau2N4R under the CamK-II promoter (131) as well as those expressing mutant tauN279K (132) both display learning defects but no NFTs or insoluble tau and no cell death. However, these mouse studies are subject to the caveat discussed in Section "Are (Insoluble) Granular Tau Oligomers (GTOs) Toxic?" that if there were any GTOs present they would not have been detected by the protocols used. Therefore, strictly speaking, we feel that the conclusion from such studies is: either soluble tau or small insoluble tau oligomers are sufficient for toxicity.

Further evidence supporting soluble tau toxicity comes from many studies showing rescue of tau-mediated phenotypes after suppression of tau expression which leads to reductions in soluble tau but persistence of tangle pathology (58, 108, 111, 133). In one such study, transgenic tauP301L mice were treated with methylene blue at a dose sufficient to rescue their memory deficit, and reduce total and soluble tau without affecting insoluble tau aggregates (134). This study went one step further in showing that the reduction of soluble tau is required for the rescue of phenotype.

Whether the toxic soluble species of tau is monomeric or oligomeric (or both) in these studies is not always clear. There is a convincing body of evidence showing that certain specific dimers, trimers, and other very small soluble oligomers are sufficient to cause toxicity both *in vitro* and *in vivo*. Patterson et al. (135) show that the 180-kDa dimers that they produced *in vitro* can suppress fast axonal transport in a squid axoplasm model. The 120-kDa trimers produced by Lasagna-Reeves et al. have been shown to be toxic *in vitro* (11) and *in vivo* (136). These trimers cause significantly more cell death than tau monomers or filaments when applied to SH-SY5Y cells in culture (11). Intra-hippocampal injections of trimers cause significant loss of synapses and neurons resulting in memory deficits, whereas injections of tau monomers or fibrils do not (136). Overall, these findings show that dimers and trimers of tau can be toxic.

Tau species of this size range have also been demonstrated *in vivo,* indicating that they are physiologically relevant. Both the Kayed and the Binder laboratories have used the oligomers that they made *in vitro* to raise oligomer-specific anti-tau antibodies, TOC-1 (9) and T22 (137), which they demonstrate recognize tau *in situ* in the post-mortem AD brain. Both oligomerspecific antibodies react with "pretangles" in early Braak stages, and co-localized with some disease-associated phospho-tau epitopes.

Small soluble oligomers also arise in many transgenic tau animal models in a context that implicates them in toxicity. For example, transgenic *Drosophila* expressing either human tau0N4R or tau0N4R-S406W in brain display soluble tau oligomers of 150– 250 kDa in size (114, 126) and in both studies oligomer formation was associated with degeneration. Berger et al. (12) and Sahara et al. (8) independently identified small soluble tau oligomers of approximately 140 kDa (believed to be dimers) in brain homogenates of tau0N4R-P301L transgenic mice. The oligomers detected by Berger et al. (12) (in the inducible tau0N4R-P301L mice) appeared at very early stages of disease when memory deficits were evident in the absence of tangle formation or neuronal loss. Either this 140 kDa soluble species and/or the two small insoluble species of tau discussed in Section "Species of Tau Found in Filaments" are implicated in causing toxicity in this model.

Like the oligomers identified by the Binder and Kayed laboratories described above, the 140-kDa oligomers identified by Berger et al. (12) in tau transgenic mice are also detectable in the brains of AD and FTDP-17 patients. It is not clear whether all of these oligomers are one and the same tau multimer or whether they represent tau oligomers at different stages of maturation during the disease process. In addition, it has not been determined whether aggregation into larger oligomers alters the toxicity of the small tau oligomers.

#### **SOLUBLE TAU: EVIDENCE AGAINST TOXICITY**

Evidently, not all soluble phosphorylated tau can be toxic. Soluble tau of many species is obviously found physiologically in healthy individuals. One specific example of a soluble species known to be non-toxic is the compact monomer with intra-molecular disulfide bonds, which appears to be a species that is relatively protective and, notably, does not go on to form larger aggregates (40).

In the tau immunization study mentioned earlier as evidence in favor of PHF toxicity (115), it was found that immunization causes reduced aggregation of tau into PHFs or larger aggregates, and was associated with a reduced behavioral phenotype. However, this also causes an *increase* of PHF-1 immunoreactivity in the "high-speed soluble" fraction, which in this case would represent any species of GTO size or smaller. This suggests not only that PHFs might be toxic, but also that the soluble species present were not sufficient for toxicity (115).

The inducible pro-aggregation and anti-aggregation mutants of the tau repeat regions created by the Mandelkow laboratory argue against toxicity of soluble tau. In both the cell lines [the study mentioned as evidence against PHF toxicity: (54)], and in the mice (95, 98), the common theme was that the anti-aggregation mutant, which would always be a soluble form of tau, was never toxic. The mouse lines provide some additional correlative evidence against toxicity of any form of tau smaller than PHFs, in that the levels of "soluble" tau were constant between the pro-aggregation mouse, which experienced neurotoxicity, and the anti-aggregation mouse, which did not.

Clearly, physiological tau is soluble and non-toxic. However, under pathological conditions tau may undergo changes that render it toxic, even though it may remain soluble.

#### **DISTINGUISHING BETWEEN SMALL (SOLUBLE) TAU OLIGOMERS AND TAU MONOMERS**

In the above paragraphs, a general trend is that the evidence for soluble tau toxicity centers on some reasonably well-defined dimers and trimers which are demonstrably sufficient for toxicity; whereas the evidence against either has not distinguished between the myriad species, or has only said that one particular type of soluble tau is not toxic. In that sense, the evidence against is not conclusive. While the specific data cited in favor of dimer and trimer toxicity is compelling, we still know very little about the properties (or indeed existence) of all the other soluble forms.

# **CONCLUSION**

In conclusion, there is a body of evidence demonstrating that small soluble tau oligomers are the most toxic form of tau. Filamentous and fibrillar tau is neither necessary nor sufficient for tau-induced toxicity, and may very well represent a neuroprotective strategy. Such ideas are not new, and a number of reviews over the past few years have drawn the same conclusions (138–140). Nevertheless, this conclusion is still not broadly accepted. Even if it were to be accepted, many questions remain. We still have little idea which of the multiplicity of soluble tau species is the culprit or culprits: is it monomers, dimers or trimers or all three, and in which conformation(s) and phosphorylation state(s)? Also, we have incomplete information about the sequence of events on the pathway(s) of tau aggregation (**Figure 2**). Especially, where do GTOs fit in? Are they made gradually from increasing sizes of smaller oligomers? Or do they have a different conformation that makes them so compact and insoluble? Can they really go on to form PHFs *in vivo*? Such questions are important for informing the strategies to be implemented when developing treatments for AD and tauopathies. We do not yet know which species of tau would represent the best target for tau-based therapies. If a certain specific set of small soluble tau oligomers are toxic, while insoluble GTOs and larger insoluble tau species are not, then perhaps strategies aimed at breaking up large insoluble tau aggregates might prove ineffective. Especially if it transpires that GTOs are not only non-toxic but on an alternative pathway to PHFs, then perhaps *encouraging* GTO formation might even turn out to be a valid approach. Alternatively, it might not be size and solubility alone of the tau species that are the

#### **REFERENCES**

key toxicity-determining factors: levels of β-sheet structure and phosphorylation at certain sites may also be influential.

There is a precedent from other proteinopathies for a small soluble species being the most toxic, while the smallest insoluble form is relatively protective. This has been demonstrated for huntingtin protein in Hungtington's disease (141, 142), alpha-synuclein in Parkinson's disease (143), and amyloid beta in AD (144): however, evidence for this phenomenon in the case of tau aggregation is only beginning to emerge now.

# **ACKNOWLEDGMENTS**

We would like to thank Dr. Ayodeji A. Asuni (University of Southampton) for reading the manuscript. CMC is funded by a Neurodegenerative Disease Specialist Grant from the Alzheimer's Society and Bupa.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 13 June 2013; accepted: 29 July 2013; published online: 13 August 2013. Citation: Cowan CM and Mudher A (2013) Are tau aggregates toxic or protective in tauopathies? Front. Neurol. 4:114. doi: 10.3389/fneur.2013.00114*

*This article was submitted to Frontiers in Neurodegeneration, a specialty of Frontiers in Neurology.*

*Copyright © 2013 Cowan and Mudher. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Characteristics of tau oligomers

# **Yan Ren and Naruhiko Sahara\***

Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, College of Medicine, University of Florida, Gainesville, FL, USA

#### **Edited by:**

Jesus Avila, Centro de Biología Molecular Severo Ochoa CSIC-UAM, Spain

#### **Reviewed by:**

Jesus Avila, Centro de Biología Molecular Severo Ochoa CSIC-UAM, Spain Emmanuel Planel, Centre Hospitalier

de l'université Laval, Canada

#### **\*Correspondence:**

Naruhiko Sahara, Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, College of Medicine, University of Florida, 1275 Center Drive, Gainesville, FL, 32610, USA e-mail<EMAIL_ADDRESS>
In Alzheimer disease (AD) and other tauopathies, microtubule-associated protein tau becomes hyperphosphorylated, undergoes conformational changes, aggregates, eventually becoming neurofibrillary tangles (NFTs). As accumulating evidence suggests that NFTs themselves may not be toxic, attention is now turning toward the role of intermediate tau oligomers in AD pathophysiology. Sarkosyl extraction is a standard protocol for investigating insoluble tau aggregates in brains.There is a growing consensus that sarkosyl-insoluble tau correlates with the pathological features of tauopathy.While sarkosyl-insoluble tau from tauopathy brains has been well characterized as a pool of filamentous tau, other dimers, multimers, and granules of tau are much less well understood. There are protocols for identifying these tau oligomers. In this mini review, we discuss the characteristics of tau oligomers isolated via different methods and materials.

**Keywords: tau, oligomers, dimer, sarkosyl-insoluble, antibody**

# **INTRODUCTION**

Tau is a phospho-protein that belongs to the family of microtubule (MT)-associated proteins. The primary function of tau protein is to modulate MT dynamics for maintaining neuronal processes and regulating axonal transport. During pathogenesis, tau protein abnormally aggregates into intracellular, filamentous inclusions, or neurofibrillary tangles (NFTs) in the brains of individuals with neurodegenerative disorders. These are termed tauopathies [reviewed in Ref. (1)].

In human tauopathies, intracellular aggregates of abnormally hyperphosphorylated tau protein and neuronal cell loss typically coincide within the same brain regions (2). Several transgenic mouse models that overexpress human tau protein have demonstrated how tau pathology and neuronal loss progresses [mouse models are reviewed in Ref. (3)]. However, recent data suggest that tau is involved in neuronal dysfunction before NFTs are formed (4, 5). *In vitro* tau polymerization studies indicated that NFT formation consists of several steps: dimerization, multimerization, oligomerization, and protofibril formation (6–11).

About two decades ago, tau aggregation intermediates (also referred to as AD P-tau) were isolated from the buffer-soluble fraction derived from brains of AD patients (12). More than 10 years later, attention has focused on oligomeric tau species in human (13) and transgenic mouse (14) brains in order to identify the exact neurotoxic components of tau protein. However, the potential role of tau oligomers is poorly understood because they exist in various states (e.g., dimers, multimers, and granules). Here, we review various protocols used to isolate tau oligomers and propose a general outline for the identification of tau oligomers.

#### **SOLUBLE PRE-FIBRILLAR TAU IN HUMAN AD BRAINS**

Greenberg and Davies first reported to isolate sarkosyl-insoluble tau from paired helical filament (PHF)-enriched fraction from human AD brain homogenates (15). Cortical gray matter was homogenized in buffer containing 10 mM Tris-HCl (pH 7.4), 1 mM EGTA, 0.8 M NaCl, and 10% sucrose, and then centrifuged at 27,200 × *g*. PHF-associated tau was enriched from the supernatant by taking advantage of their insolubility in the presence of a detergent, sarkosyl. PHF-associated tau migrated at around 57–68 kDa on one-dimensional PAGE gels. This enriched supernatant was more acidic on two-dimensional PAGE gels compared to extracts from normal brains. Although this PHF-associated tau was not extracted from highly insoluble fraction containing NFTs, the sarkosyl-insoluble tau displayed the same structural and antigenic properties as PHFs isolated from NFTs (16–18) and was distinguishable from normal, soluble tau proteins.

Kopke et al. isolated non-PHF hyperphosphorylated tau from AD brains (12). In their protocol, cortical gray matter was homogenized in buffer containing 20 mM Tris-HCl (pH 8.0), 0.32 M sucrose, 10 mM β-mercaptoethanol, 5 mM EGTA, 1 mM EDTA, 5 mM MgSO4, and proteinase inhibitors. The homogenate was then subjected to differential centrifugation, and the fraction resulting from centrifugation between 27,000 and 200,000 × *g* was collected. This fraction was further extracted with 8 M urea to separate out the PHF-enriched pool. The supernatant contained abnormally phosphorylated non-PHF tau. These tau species were named AD P-tau and had a molecular weight of 67–70 kDa. They were three to fourfold more phosphorylated than tau extracted from control brains and could be detected by Tau1 antibody after alkaline phosphatase treatment. These highly phosphorylated AD P-tau proteins lost their normal MT assembly-promoting activity, which could be recovered upon dephosphorylation with alkaline phosphatase (19). Moreover, AD P-tau could sequester normal tau into filamentous tau aggregates, resulting in MT de-polymerization (20).

These studies suggest that a pool of intermediate pathological tau species exists and that this pool can be recovered in buffer-soluble fractions. The physiological activity and function of these tau species is reduced compared to normal tau species due to hyperphosphorylation. Since the intracellular mobility dynamics of these intermediate tau species is much greater than that of condensed tau aggregates in NFTs, it is possible that intermediate tau species induce neuronal death and/or synaptic dysfunction. Therefore, the isolation and characterization of these tau species is paramount for understanding the pathogenesis of AD and for searching therapeutic methods.

### **TAU OLIGOMERS IN MOUSE MODELS OF TAUOPATHY**

The first tau transgenic mouse model of frontotemporal dementia with Parkinsonism linked to tau on chromosome 17 (FTDP-17- Tau) was the JNPL3 line, which overexpresses P301L mutant 4R0N tau (21). The biochemical characterization of insoluble tau in these mice was done by a modified Greenberg and Davies method (21, 22). In this protocol, mouse brains were homogenized in buffer containing 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, phosphatase inhibitors, and protease inhibitors. A pellet collected from 150,000 × *g* centrifugation was re-homogenized in high-salt/sucrose buffer [10 mM Tris-HCl (pH 7.4), 0.8 M NaCl, 10% sucrose, 1 mM EGTA, 1 mM PMSF] and centrifuged again at 150, 000 × *g*. The resulting supernatant was incubated with 1% sarkosyl, and then centrifuged at 150, 000 × *g*. The pellet was resuspended in TBS as the sarkosyl-insoluble fraction. A 64-kDa tau predominantly existed in the sarkosyl-insoluble fraction; this tau was phosphorylated at multiple sites. Most notably, the amount of 64 kDa tau increased in an age-dependent manner, correlating well with the pathogenesis in JNPL3 mouse brain.

Noble et al. proposed a slightly different protocol for tissue homogenization using RIPA buffer without SDS (23). This modification allows for the study of both cytosolic and membraneassociated proteins involved in AD pathogenesis, such as amyloid precursor protein (APP), in the same extracts (24).

Another broadly used tauopathy mouse model is rTg4510 mice, which express repressible P301L mutant 4R0N tau and develop progressive age-related NFTs, neuronal loss, and behavioral impairment (5). Using a protocol similar to the one for JNPL3 mice with an additional 13,000 × *g* centrifugation as the first step, Berger et al. identified 140 and 170 kDa multimeric tau species in rTg4510 mouse brains (14). The 140 kDa tau was recovered in the supernatant fraction resulting from 150,000 × *g* centrifugation, while the 170 kDa tau was mostly in the sarkosyl-insoluble fraction. Both multimers were not affected by the presence or absence of reducing agent, indicating that the multimers are disulfidebond independent. Importantly, the accumulation of 140 kDa tau coincided with the behavioral impairments of rTg4510 mice (14). Although this finding has had a huge impact on our understanding the neurotoxic mechanisms of tau oligomers, it is still unclear whether 140 and 170 kDa tau multimers can induce neuronal dysfunction.  It should be noted that tau multimers with apparent molecular weights of ∼140 and

∼170 kDa are in fact tau dimers of ∼120 and ∼130 kDa, based on Bis-Tris or Tris-acetate SDS-PAGE migration (11, 25). This was further supported by mass spectrometry analysis of cross-linked tau dimers (26).

Most recently, we demonstrated that TBS-extractable 64 kDa tau species represents better the species involved in the progression of brain atrophy than does the sarkosyl-insoluble tau species (25). These 64 kDa tau species can be recovered in the supernatant following centrifugation of brain homogenates at 27,000 × *g* and further separation from normal tau by 150,000 × *g* centrifugation. TBS-extractable 64 kDa tau and normal tau are similar in thermo-stability but differ in other properties. Under non-reducing gel electrophoresis conditions, nearly all 64 kDa tau species are detected as dimers (∼130 kDa, according to size of molecular markers), whereas most normal tau proteins are detected as monomers. Immuno-electron microscopy revealed that the TBS-extractable 64 kDa tau enriched fraction contains tau-positive granules and filaments (25). This morphological finding was supported by MC1 immunoreactivity and Ab39 insensitivity (25). The MC1 antibody recognizes an early pathogenic conformation of tau (27), while the Ab39 antibody only detects mature tangles (28, 29). Overall, the characteristics of TBS-extractable 64 kDa tau are similar to AD P-tau from human brains.

# **IN VITRO TAU OLIGOMERIZATION**

With tau assembly modeled *in vitro*, unphosphorylated recombinant tau can be polymerized by inducers such as heparin, heparan sulfate, polyunsaturated fatty acids, RNA, or quinones (30–34). Using the heparin-induced tau self-assembly method, we produced and isolated granular-shaped tau oligomers from soluble tau and filamentous tau by sucrose density gradient ultracentrifugation (10). These granular tau oligomers were morphologically defined by atomic force microscopy (AFM) to be 15–25 nm granules, and their molecular mass corresponded to about 40 tau molecules (10). Once formed, granular tau can continue to form filaments without any inducers in a concentration-dependent manner (10).

More recently, Lasagna-Reeves et al. proposed a method to prepare tau oligomers by using amyloid seeds (35). In their protocol, tau oligomerization can be induced in a relatively short period (1 h incubation at room temperature) after adding Aβ42 oligomers. After a total of three rounds of seeding procedures, Aβ42 seeds could be diluted to below the detection limit (35). Examination of these tau oligomers by transmission electron microscopy or AFM revealed a spherical morphology (35). This unique method of producing tau oligomers is a reasonable representative model supporting the amyloid hypothesis (36, 37), in which Aβ oligomers trigger NFT formation. Interestingly, tau oligomers, but not tau monomers or tau fibrils, can cause memory impairment in wild-type mice (13) and can decrease long-term potentiation in hippocampal brain slices (38).

The production of granular tau oligomers must be initiated by dimerization of tau monomers. Heparin-induced tau polymerization allows us to detect initial dimers because of the relatively slower kinetics compared to arachidonic acid-induced tau polymerization (6, 39). It begins by increasing the formation of cysteine-dependent dimers, which occur prior to the detection of thioflavin T (ThT) binding (11). The kinetics of tau polymerization is dependent on oxidative/reducing state. Higherorder oligomers and aggregates assemble more rapidly in the absence of the reducing agent dithiothreitol (DTT) (11). However, cysless-tau (4R tau with both C291A and C322A mutations) forms dimers, which eventually aggregate into fibrils after 24 h incubation with heparin, suggesting that tau aggregation occurs without disulfide-bond formation (11).

Two distinct tau dimers (i.e., cysteine-dependent and cysteineindependent dimers) have been identified in tauopathy mouse models, including JNPL3 mice (11) and rTg4510 mice (14, 25). These dimers have also been shown in cell cultures (11, 40). Dimer formation is an essential step for their further assembly into higher-order oligomers. Although these dimers themselves may not exist in a steady state, it is important to detect the initial step of tau dimerization.

# **GENERATION OF TAU OLIGOMER-SPECIFIC ANTIBODIES**

A monoclonal antibody that selectively recognizes tau dimers and higher-order oligomers has been generated by Binder's group (26). This antibody, named tau oligomeric complex 1 (TOC1), was made against benzophenone-4-maleimide cross-linked recombinant tau dimers (26). Immunogold labeling and dot-blot analysis of aggregated recombinant tau revealed that TOC1 selectively labels tau dimers or oligomers but not filaments (26). Based on their preliminary mapping of the TOC1 epitope, the proline-rich region (Gly155-Gln244) and the Cterminal portion (Leu376-Ser421) were identified as potential binding segments for forming cross-linked tau dimer (26). Since these two regions are on the opposite sides of the MT-binding domains, Patterson et al. advanced the idea of the formation of an antiparallel dimer (26).

TOC1 immunoreactivity is selectively detectable in the early stage of AD pathogenesis; however, TOC1 antibody fails to label mature tangles in AD brains (26). In rTg4510 mice, TOC1 immunoreactivity was observed in the TBS-extractable 64 kDa tau enriched fraction and linked to early pathological changes (Sahara, in preparation). Notably, the immunohistochemical staining pattern of TOC1 antibody was clearly different from those of MC1 and Ab39 antibodies (**Figure 1**). Since NFTs themselves might be protective [reviewed in Ref. (41)], other harmful tau species such as tau oligomers are currently of particular interest. If TOC1 antibody selectively interacts with tau dimers and higher-order oligomers but not tau filaments, and if those species cause neurotoxicity, this antibody can be a useful tool to track the pathway of tau neurotoxicity.

 Formalin-fixed paraffin sections were stained with TOC1 (1:2500), MC1 (1:1000), and Ab39 (1:250) antibodies by the Dako Universal Autostainer (Dako, Carpinteria, CA, USA). The sections were then counterstained with

hematoxylin. TOC1 diffusely stained cytoplasmic regions of neurons (top panels, rTg4510 and AD brains), while MC1 and Ab39 densely stained these neurons (middle and bottom panels, respectively). This difference in staining pattern is due to the specific binding of TOC1 antibody to premature tau aggregates. Scale Bar, 100µm.

WB, Western blot; EM, electronic microscopy; IHC, immunohistochemistry; SEC, size-exclusion chromatography; AFM, atomic force microscopy.

Another tau oligomer-specific antibody, T22, was generated by Kayed's group (42) against antigenic tau prepared from Aβ seeding of tau oligomers (35). The specificity of this antibody was confirmed by ELISA and dot blotting. It detects only tau oligomers but not tau monomers or PHF fibrils prepared by the heparin-induced tau polymerization method (42). Immunohistochemically, this antibody selectively stains pretangles, neuritic plaques, and neuropil threads but not ghost tangles in AD brain sections (42). Western blotting showed that T22 antibody recognizes higher-molecular-weight tau species (e.g., dimers, trimers, and tetramers) but not monomers (42). It should be noted that the SDS-PAGE samples were not denatured by boiling before running on gels and that T22 immunoreactivity was diminished by denaturing agents such as 8 M urea (42).

These novel tau oligomer-specific antibodies provide a new method to diagnose the early pathological changes that occur in tauopathy. It would be extremely useful to develop methods employing cerebrospinal fluid biomarkers combined with total tau, phosphorylated tau, tau oligomers, and other biomarker measurements to differentially diagnose dementias, such as AD, frontotemporal lobar degeneration, progressive supranuclear palsy, corticobasal degeneration, dementia with Lewy bodies, vascular dementia, and prion disease.

### **CONCLUSION**

Abnormal tau aggregation is considered to be a critical pathological feature of tauopathy. However, the initial molecular event of tau pathogenesis is yet unclear. The hyperphosphorylation of tau is strongly suggested to be directly correlated with the severity of AD pathology (43). Iqbal and colleagues demonstrated that hyperphosphorylated tau extracted from AD brain reduces MT stabilization, sequesters normal tau from MT, and aggregates themselves in the absence of inducer molecules (20, 44). Many studies attempting to identify tau oligomers have demonstrated the existence of hyperphosphorylated tau oligomers in human and transgenic mouse brains (e.g., AD P-tau, 140 and 170 kDa tau multimers, TBS-extractable 64 kDa tau, and T22 antibodypositive tau oligomers) (**Table 1**). Thus, hyperphosphorylation of tau could be the initial event of NFT formation. However, the amount of 140 kDa tau multimer (normal tau dimer) correlates well with behavioral deficits (14), suggesting that hyperphosphorylated tau oligomers may not be essential for neurotoxicity. If antibodies can be generated to recognize non-phosphorylated and hyperphosphorylated tau dimers independently, we will be able to better identify toxic tau species and optimize potential oligomerization inhibitors as possible novel therapies. Standardized isolation methods of tau oligomers are in need to improve consistency between researchers.

In summary, accumulating evidence from biochemistry, immunology, and molecular imaging reveal the existence of tau oligomers as mainly buffer-soluble, non-filamentous, granularshaped conformers. The neurotoxicity of these oligomers has been confirmed in both *in vitro* and *in vivo* experiments (4, 5, 14, 42, 45). The next step of tau oligomer research should investigate whether tau dimers and/or non-granular oligomers exist and functionally correspond to neuronal dysfunction.

# **ACKNOWLEDGMENTS**

Work in the authors' laboratory is supported in part by an R21 grant (NS067127) from the National Institutes of Health/National Institute of Neurological Diseases and Stroke, and a grant from the Thomas H. Maren Junior Investigator Fund of the University of Florida.

# **REFERENCES**

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expressing mutant (P301L) tau protein. *Nat Genet* (2000) **25**:402–5. doi:10.1038/78078

fluorescence in solution. *Biochemistry* (1998) **37**:10223–30. doi:10. 1021/bi980537d

et al. Identification of oligomers at early stages of tau aggregation in Alzheimer's disease. *FASEB J* (2012) **6**:1946–59. doi: 10.1096/fj.11-199851

*U S A* (2001) **98**:6923–8. doi: 10.1073/pnas.121119298

45. Khlistunova I, Biernat J, Wang Y, Pickhardt M,Von Bergen M, Gazova Z, et al. Inducible expression of Tau repeat domain in cell models of tauopathy: aggregation is toxic to cells but can be reversed by inhibitor drugs. *J Biol Chem* (2006) **281**:1205–14. doi:10.1074/ jbc.M507753200

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 21 May 2013; paper pending published: 03 July 2013; accepted: 09 July 2013; published online: 19 July 2013.*

*Citation: Ren Y and Sahara N (2013) Characteristics of tau oligomers. Front. Neurol. 4:102. doi: 10.3389/fneur.2013.00102*

*This article was submitted to Frontiers in Neurodegeneration, a specialty of Frontiers in Neurology.*

*Copyright © 2013 Ren and Sahara. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

# Formation and propagation of tau oligomeric seeds

# **Julia E. Gerson1,2,3 and Rakez Kayed1,2,3\***

1 George P. and Cynthia Woods Mitchell Center for Neurodegenerative Diseases, University of Texas Medical Branch, Galveston, TX, USA

2 Department of Neurology, University of Texas Medical Branch, Galveston, TX, USA

3 Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, TX, USA

#### **Edited by:**

Naruhiko Sahara, University of Florida, USA

#### **Reviewed by:**

Naruhiko Sahara, University of Florida, USA

Irving E. Vega, University of Puerto Rico-Rio Piedras Campus, Puerto Rico

#### **\*Correspondence:**

Rakez Kayed, George P. and Cynthia Woods Mitchell Center for Neurodegenerative Diseases, 301 University Blvd, Medical Research Building, Room 10.138C, Galveston, TX 77555-1045, USA e-mail<EMAIL_ADDRESS>
Tau misfolding and aggregation leads to the formation of neurofibrillary tangles (NFTs), which have long been considered one of the main pathological hallmarks for numerous neurodegenerative diseases known as tauopathies, including Alzheimer's Disease (AD) and Parkinson's Disease (PD). However, recent studies completed both in vitro and in vivo suggest that intermediate forms of tau, known as tau oligomers, between the monomeric form and NFTs are the true toxic species in disease and the best targets for anti-tau therapies. However, the exact mechanism by which the spread of pathology occurs is unknown. Evidence suggests that tau oligomers may act as templates for the misfolding of native tau, thereby seeding the spread of the toxic forms of the protein. Recently, researchers have reported the ability of tau oligomers to enter and exit cells, propagating from diseaseaffected regions to unaffected areas. While the mechanism by which the spreading of misfolded tau occurs has yet to be elucidated, there are a few different models which have been proposed, including cell membrane stress and pore-formation, endocytosis and exocytosis, and non-traditional secretion of protein not enclosed by a membrane. Coming to an understanding of how toxic tau species seed and spread through the brain will be crucial to finding effective treatments for neurodegenerative tauopathies.

**Keywords: tauopathies, tau oligomers, oligomeric seeding, propagation of tau pathology, Alzheimer's**

# **TAU OLIGOMERS ARE THE TOXIC TAU SPECIES IN NEURODEGENERATIVE TAUOPATHIES**

The formation of tau aggregates and neurofibrillary tangles (NFTs) is one of the main pathological hallmarks of numerous diseases termed tauopathies, including the two most common neurodegenerative diseases, Alzheimer's Disease (AD) and Parkinson's Disease (PD) (1, 2). However, it is evident that cell death occurs initially prior to NFT formation in AD (3–6) suggesting that NFTs are not the pathogenic species responsible for the spread of the disease. Recent evidence points to the presence of multimeric tau species which are intermediates between tau monomers and NFT – known as tau oligomers – as the toxic species inducing synaptic dysfunction and cell death in neurodegenerative tauopathies (7–12).

Numerous researchers have investigated tau pathology using animal models, yielding a better understanding of the toxicity of different tau structures. A study in aged mice expressing native human tau (htau mice) found that while NFT formation occurred as animals aged, there was no correlation between the presence of tau filaments and cell death (13). Additionally, a study examining the P301S mouse model, which expresses mutant human tau, found that hippocampal synaptic dysfunction occurred prior to NFT formation (14). Studies using the rTg4510 mouse model, which conditionally expresses P301L mutant tau, found that cell death occurred prior to NFT formation and that cell loss and behavioral impairments could be suppressed by inhibiting tau expression without removing NFTs or preventing their continued accumulation (7, 15). In accordance with this finding, it has been shown that NFTs are protective in the same mouse model

(16), and only pro-aggregate human tau mice (TauRD) show behavioral deficits (17). Another study in the same mouse model characterized tau oligomers biochemically that appeared early and correlated with cognitive deficits (8, 12). Similar results have also been seen in *Drosophila* AD models, where expression of mutant tau causes neurodegeneration, synaptic dysfunction, and axonal transport deficiencies in the absence of NFTs (18, 19). Usage of the protein nicotinamide mononucleotide (NAD) adenylyl transferase (NMNAT) was shown to decrease behavioral and morphological deficiencies in a frontotemporal dementia *Drosophila* model by decreasing levels of tau oligomers (20).

Biochemical analysis of human AD brain tissue has also yielded results suggesting that tau oligomers may initiate toxicity, rather than NFTs. When compared to control brains, levels of tau oligomers were found to be significantly increased in AD brains early in the disease, prior to when NFTs appear and clinical symptoms are evident (9, 21–23). In addition to correlative evidence for the importance of tau oligomers to toxicity, treatment with tau oligomers has also been shown to cause adverse effects in animals. Isolated tau oligomers, but not monomers or NFTs, induced memory impairments, synaptic dysfunction, and mitochondrial dysfunction when given intracerebrally to wild-type mice (24). Therefore, it is possible that NFTs are actually neuroprotective, sequestering toxic forms of tau into large aggregates with less flexibility and surface area to interact with cells. All of these studies form the framework for the model of the progression of neurodegenerative tauopathies beginning with the seeding and propagation of toxic tau oligomers (**Figure 1**).

(72, 138).

# **TAU OLIGOMERS ARE SEEDS FOR THE PROPAGATION OF PATHOLOGICAL TAU**

tauopathies, rather than monomeric tau or hyperphosphorylated NFTs

Recently,researchers have begun to make comparisons between the spread of neurodegenerative disease and prion disease, as studies suggest that misfolded protein templating, known as seeding, may underlie the progression of disease (25). Understanding how tau seeds pathological forms of the protein which propagate to different brain regions is critical to devising a solution to stop the spread of disease. There are two main models for the formation and seeding of pathological tau, oligomer-nucleated conformational induction – based upon the mechanism of action of prion protein, Sup35 (26) and amyloid β (Aβ) (27) – and template-assisted growth. Template-assisted growth proposes that tau fibrils act as template molecules for unfolded monomers. When monomers come in contact with filaments, they are integrated into the filament in organized, parallel stacked β sheets, optimizing hydrogen bonding for stabilization (28). It has been difficult, however, to find spontaneous tau aggregation which occurs experimentally. When fibrils are cleaved, leaving only three microtubule binding repeats, the fragments aggregate spontaneously *in vitro* (29). However, on its own, tau will not polymerize *in vitro* without the addition of certain reagents, post-translational modifications, such as phosphorylation, or induction of mutations.

In order for aggregation to occur, tau must be released from microtubules to reach a high concentration of free cytosolic tau, conformational changes must occur to allow for aggregation, possibly by increasing β sheet content, and dimerization must occur (30, 31). The addition of polyanions, such as heparin or RNA can induce fibrillization of tau (32), causing a conformational change from random coil structure to β sheet structure (33). Free fatty acids, such as arachidonic acid can also increase aggregation (34, 35) due to the presence of an alkyl chain, which induces micellization, and a negatively charged head group on the fatty acid to create a negatively charged surface on the micelle. In the presence of tau, the critical concentration for micelle formation is greatly decreased, allowing anionic micelles to attract tau to the negatively charged surface and thereby compensate for positive charges in tau and enable tau aggregation (36,37). Phosphorylation may also play a role in fibrillization. Paired helical filaments (PHF) and straight filaments (SF), which make up the NFTs found in the brains of patients with AD, are comprised of hyperphosphorylated tau (38, 39). Phosphorylated tau has a higher tendency toward aggregation

than unphosphorylated tau and kinases involved in the phosphorylation of these sites in tau have been shown to be altered in AD (40). Hyperphosphorylated tau has been shown to aggregate *in vitro,* possibly due to the addition of negative charge which would increase aggregation, similarly to the addition of polyanions and free fatty acids. Furthermore, this process can be inhibited by dephosphorylation (41, 42). Phosphorylation may also induce aggregation by reducing the interaction of tau with microtubules and allowing it to interact instead with unphosphorylated tau and form aggregates (43–45). Mutations, such as those that lead to frontotemporal lobar degeneration (FTLD), can increase tau aggregation through different mechanisms. Many mutations lead to a decrease in microtubule assembly kinetics, which could lead to more free cytosolic tau and increase aggregation (46–49). Some mutations lead to a decrease in the dissociation constants (*K*d) for dimer and filament formation, while others increase the rate of nucleation without affecting *K*d (30). Mutations that cause increased formation of β-sheets lead to heightened aggregation due to an increase in hydrophobic interactions, deviating from the highly hydrophilic native tau protein (50, 51). Fibrillar tau can thereby be recognized by dyes which interact with β-sheets, such as Congo Red and thioflavin S (52). However, at high concentrations, these dyes can induce fibrillization due to an attraction between positive charges formed in the core of PHFs and negative charges of the anionic dyes (53–55).

In amyloid proteins in which seeding has been well-established, such as prion proteins and Aβ, oligomers have been shown to be the most potent seeds (56, 57), working by way of oligomernucleated conformational induction (26, 27). Due to the increased interest in the toxicity of tau oligomers, evidence has emerged in support of the oligomer-nucleated conformational induction model as more studies have begun to explore the importance of tau oligomers in the initialization of tauopathies. Oligomer-nucleated conformational induction entails oligomers or conformational changes irreversibly stabilizing the highest energy protein states, known as the nucleus, allowing stable monomers to aggregate into oligomeric structures. Oligomers are driven to further elongation to form lower energy, stable filaments (58). As opposed to template-assisted growth, monomers are not incorporated directly into fibrils, but are instead entirely aggregated into oligomers prior to filament formation (59). Tau dimerization increases the tendency to aggregate and can be induced by oxidation (60), which suggests that tau oligomerization may be an important step in the fibrillization pathway. The appearance of oligomeric species of other amyloid proteins has been observed on the path to fibril formation (61–63).

While the addition of reagents and mutations used to induce fibrillization has been integral to understanding how tau aggregation occurs, it does not explain how fibril formation may occur spontaneously in sporadic disease. The mechanism by which tau aggregation occurs physiologically has not yet been elucidated, however there have been some advances in the understanding of how certain steps in the process may occur. Release of tau from microtubules may occur following post-translational modifications, such as phosphorylation (43, 44). Localization to anionic surfaces, alternative splicing, and post-translational modifications stabilizing aggregated conformations may all act as enhancers to

increase speed of nucleation (64). Under physiological conditions, nucleating cofactors can induce tau aggregation in a similar fashion to agents used *in vitro*. There is evidence that polyanionic species, such as tubulin, RNA, and α-synuclein can increase the tendency of tau to aggregate (65–67) The formation of disulfide bridges is critical for the initial creation of dimers from monomers, as well as intermolecular crosslinking of the microtubule binding domain independent of cysteine to continue oligomerization of three-repeat tau (68). Prior to monomer aggregation into oligomers, the free energy of solvation decreases, causing a shift in preference for peptide-solvent interactions toward peptide– peptide interactions, as water is evacuated due to poor interaction with the peptide backbone and sidechains. Water release increases entropy of the solvent, thereby balancing the loss in conformational entropy caused by aggregation. The interaction of side chains with the backbone in the form of hydrogen bonding leads to the creation of β-sheet structure and aggregate stabilization. While oligomers form a similar structure to fibrils, they are not as ordered, which likely increases their toxicity (69). Proteolytic processing by endogenous proteases has also been shown to create self-aggregating fragments, which nucleate and co-aggregate with full-length protein effectively enough for a small amount of fragment to seed PHFs (70). Direct interactions between misfolded tau and native protein may be the underlying mechanism of seeding as experiments have shown tau protein–protein interactions occur when tau aggregates enter cells containing native tau (71).

Tau oligomers – identified with the tau oligomer-specific antibody, T22, which does not recognize monomers or fibrils (59) – which have been seeded with oligomers derived from brain tissue have been shown to be highly toxic (23, 72). When tested with Bis-ANS, which recognizes exposed hydrophobic patches, oligomers had higher affinity for Bis-ANS than PHFs, which may underlie toxicity. The toxic effects of tau oligomers formed by seeding recombinant tau with oligomeric seeds, however, can be prevented when pre-treated with T22 (23, 72).

Some tauopathies, such as progressive supranuclear palsy (PSP), only have one pathogenic species involved in disease progression (1). However, most tauopathies contain other amyloid proteins in addition to tau, such as Aβ in AD and α-synuclein in PD. In such diseases, cross-seeding of heterologous protein species is an additional mechanism which is important for tau seeding (59, 67, 73–77).

# **TAU OLIGOMERS PROPAGATE FROM AFFECTED BRAIN REGIONS TO UNAFFECTED REGIONS**

Aβ has been shown to propagate from affected brain areas to unaffected areas in mice over-expressing Aβ precursor protein that have been injected with Aβ isolated from the brains of AD patients and AD transgenic mice (78, 79), suggesting that perhaps tau could spread in a similar fashion. A few years later, a similar mechanism was demonstrated for the propagation of tau. When tau extracted from P301S mice was injected into the brains of mice over-expressing wild-type human tau (ALZ17 mice), which do not form tau aggregates, tau pathology was observed to have spread from the injection site to neighboring brain regions (80). Additionally,in transgenic mice which differentially express pathological tau in the entorhinal cortex, where tau pathology is first observed in AD, human tau has been shown to spread to both neighboring and synaptically connected neurons which do not express human tau mRNA. Translocated human tau was able to seed mouse tau misfolding (81, 82). However, these studies did not specifically investigate which tau species specifically induced seeding and propagation of tau pathology and the usage of transgenic mouse models is not analogous to sporadic forms of AD. When wild-type mice were injected with both tau oligomers and PHFs isolated from AD brains, tau oligomers induced the spread of tau pathology from the injection site to neighboring brain regions and impaired memory, as measured by object recognition. Conversely, mice injected with PHFs only exhibited tau pathology near the injection site and did not exhibit any memory impairments on the behavioral task, suggesting that tau oligomers, but not fibrillar tau, is capable of seeding and propagating pathology (72). Furthermore, similar results have been found using primary neurons. Neurons were exposed to low molecular weight aggregates – recognized by the tau oligomer-specific antibody, T22, and examined via electron microscopy for oligomeric characteristics – as well as to fibrils formed *in vitro*, filaments formed *in vivo*, and monomers. Low molecular weight aggregates and short fibrils exhibited uptake into the cell, but monomers and filaments were not internalized (83). Other studies have shown tau aggregate uptake using cell culture, but did not specifically identify the type of aggregates being internalized. Neural stem cells treated with tau monomers and aggregates formed using the tau microtubule binding repeat region induced to fibrillize with arachidonic acid, exhibited significantly more tau aggregate uptake than monomer uptake. Additionally, aggregates, but not monomers, induced seeding of endogenous tau misfolding (84).

On the other hand, Guo and Lee hypothesized that seeding of pathological tau in cultured cells would be able to occur more quickly by seeding with pre-formed tau fibrils, thereby omitting the step where monomer must be converted to oligomer prior to fibril formation. Fibrillization of recombinant tau was induced with the addition of heparin and was verified using thioflavin T. Fibril-treated cells exhibited seeding and propagation of aggregates via endocytosis. However, following fibril confirmation with thioflavin T and prior to cell treatment, fibrils used in this study were sonicated (85). Previous research investigating Aβ seeding found that sonication increased seeding ability by fragmenting fibrils into smaller, soluble species (57) and sonicated prions have also been shown to have more potent seeding potentials than unsonicated fibrils (56). Since it has been shown that both prion and Aβ oligomers, rather than fibrils, are the seeds for pathological protein templating (26, 27), it is likely that sonication partially converts insoluble fibrils into soluble oligomeric forms. Sonication of tau fibrils has also been shown to cause shearing of filaments, particularly those in PHF form (86). Therefore, it is likely that sonicated tau fibrils used to treat cells in the previous study (85) also contained tau in oligomeric form, which may explain why seeding and propagation was successful.

Recently, Wu et al. studied propagation of tau in primary neurons using microfluidic chambers which allow somatodendritic compartments to be isolated from axonal compartments, enabling not only the analysis of tau uptake from the extracellular space into the cell, but also propagation within the neuron. They found that low molecular weight tau aggregates specifically recognized by tau oligomer-specific antibody, T22, propagate between isolated neuronal compartments both anterogradely and retrogradely (83). Importantly, tau is primarily found in the axons of healthy neurons (87), though tau may also be found in the dendrite where it colocalizes with the src kinase, fyn (88, 89). In AD, however, misfolded and hyperphosphorylated tau accumulates in the axon, dendrites, and the cell body (90), suggesting that intracellular transport may also be important for the spread of disease. In lamprey neurons expressing low levels of tau, tau was primarily localized to the axon and proximal dendrites, both regions consistent with tau functioning as a microtubule-associated protein. However, in neurons expressing high levels of tau, tau was found in distal dendrites and near the soma membrane, both areas lacking microtubules. High-expressing tau cells showed more degeneration and secretion of tau. Moreover, as tau can modulate activity of microtubule-associated motor proteins involved in dendritic transport, tau localized to the dendrite may have implications for its propagation (91). Phosphorylated tau localized at the synapse in AD brain samples appears to correlate with ubiquitin-proteasome system (UPS) dysfunction, suggesting that tau oligomer accumulation at the synapse impairs the UPS, which is a crucial player in the breakdown of tau. Accumulation of tau at the synapse may also suggest a mechanism for trans-synaptic tau propagation (92).

Phosphorylation clearly plays a role in the toxicity and localization of tau, however, its exact role in neurodegenerative disease is unknown and appears to be quite complicated. While hyperphosphorylated tau has been shown to have toxic results in the cell, increasing aggregation and abnormal tau localization (40, 90), dephosphorylated tau can also have harmful effects. Phosphorylated tau released into the medium of cultured neuroblastoma cells through muscarinic receptor activation that is dephosphorylated by tissue-non-specific alkaline phosphatase (TNAP) led to excitotoxicity, increasing calcium levels in nearby cells. Additionally, levels of TNAP are heightened in AD brains compared to control brains (93). Another study of primary cortical neurons also found that extracellular tau is largely dephosphorylated (94). Conversely, one study found that phosphorylation of tau increased its secretion from HeLa cells (95). Inflammation and activation of microglia has been shown to increase tau phosphorylation as well as aggregation, but is complicated by the fact that the opposite effect is seen in Aβ (96–99). The localization of tau in the cytosol, cell membrane, and the nucleus also appears to be important for tau toxicity, and is mediated by phosphorylation. Oxidative stress and heat shock induce the dephosphorylation of cytosolic tau and its transport into the nucleus. Once relocated to the nucleus, tau appears to protect neuronal DNA from damage under cell stress (100), which may be important in AD where DNA damage has been shown to occur (101). One possibility is that abnormal phosphorylation of tau in AD may prevent tau from being dephosphorylated and translocated to the nucleus to protect against DNA damage. The localization of tau to the cell membrane may also depend upon its dephosphorylation, as tau with lower levels of phosphorylation in its proline region was shown to be associated with the cell membrane, while phosphorylated tau was found in the cytoplasm (102, 103). However, interaction with membrane bound proteins, such

as aforementioned fyn may stabilize association of phosphorylated tau with the membrane.

# **MECHANISM OF TAU PROPAGATION**

The entry of prion proteins, Aβ, and other amyloid proteins into the cell via different mechanisms has been well-established (104– 107). One hypothesis for amyloid oligomer toxicity and entrance into cells is through protein interaction with the cell membrane. One model suggests that oligomers embed themselves into the cell membrane and form pores. However, it appears as though the formation of pore-like annular protofibrils occurs through a separate pathway from fibril formation (108). An alternative model suggests that oligomers interact with lipid rafts in the bilayer, causing membrane thinning and increased membrane permeabilization, which may play a role in oligomer toxicity, allowing non-specific ion entrance, as well as leakage of cellular compartments. Several types of amyloid oligomers have been shown to increase membrane permeability, including Aβ, α-synuclein, and prion protein (107, 109–111). Tau has been reported to interact with the lipid rafts in the cell membrane and undergo conformational changes leading to membrane stress (112–115). Additionally,permeabilization of the membrane could mediate internalization of oligomers into the cell.

There has also been evidence for endocytosis as a route of amyloid entry into the cell. Propagation of α-synuclein, prion protein, Sup35, and Aβ has been shown to be associated with the endosomal pathway (116–118). One study found that tau aggregates colocalize with dextran in neural stem cells, implying that entry into the cell occurs via macropinocytosis. However, the aggregate type was not specifically tested (84). Aggregates identified specifically as tau oligomers colocalized with fluid-phase endocytosis marker, dextran, as well as with early endosomal marker, Rab5, and late endosomal/lysosomal marker, Lamp1. When endocytosis was inhibited with dynamin inhibitor, Dynasore, tau uptake was blocked, while inhibition of clathrin-mediated endocytosis with Pitstop2B did not impact internalization (83). These studies together suggest a mechanism for tau propagation in which tau is internalized via pinocytosis and enters the endosomal pathway. Tau can move through the endosomal pathway to the lysosome where toxic species may be degraded or recycled back to the cell membrane, where they may be released to be internalized by adjacent neurons. More research is needed to determine how membrane-enclosed tau oligomers are released inside of the cell, though it appears likely that the majority are degraded in the lysosome, while those that avoid degradation may cause the endosomal membrane to burst and be released in the cytoplasm, where they can seed aggregation of healthy tau (83). While clathrin-mediated endocytosis did not appear to be involved in tau propagation, endocytosis inhibitors are often found to be non-specific (119), and therefore, the possibility of other types of endocytosis in tau spread bears more study.

Receptor-mediated endocytosis could be another route of entry into the cell as amyloids have been reported to bind to cell surface receptors. Internalization of α-synuclein has been shown to be dependent upon receptor-mediated endocytosis, potentially through caveolin-mediated endocytosis (117). Additionally, Aβ binds to NMDA, α7 nicotinic acetylcholine, and APOE receptors, inducing receptor endocytosis (120–124). Aβ oligomers also bind cellular prion protein, PrPc , which is complexed with the Src tyrosine kinase, Fyn. This interaction has been shown to increase tau dysfunction and prevents native tau from binding to fyn (125). Under normal conditions, tau binds to fyn in oligodendrocytes (126) and in neurons, activating the Ras/MAPK pathway (103). Mutations to the microtubule binding region in tau lead to decreases in oligodendrocyte process number and length and disease-related missense mutations increase tau association with Fyn (127). Results indicate that the interaction between tau and fyn may be important for neurodegeneration, both through a loss in native tau interaction and through a gain in toxic tau function. Interaction with PrPc complexed to fyn could also mediate tau entry into the cell as the PrPc complex is associated with and endocytosed with caveolin (128). Tau may also enter the cell through a direct interaction with fyn. Lee et al. used a lamprey ABC tauopathy model in which tau is expressed in specifically identified ABC neurons to investigate the spread of tau. They report that tau phosphorylated at Y18, the site most commonly phosphorylated by fyn kinase, is associated with vesicular organelles. Additionally, when tau is overexpressed and localizes to the dendrite, dendritic vesicle accumulation is observed. Phosphorylated tau is colocalized with vesicles which bear resemblance to endosomes, as well as to fyn. Fyn has also been shown to colocalize with exosomes, suggesting a possible mechanism for fyn-tau transport in which fyn-associated tau is endocytosed, transported from early endosomes to late endosomal compartments, and then transported out of the cell via exosomes (91).

While one mechanism for tau oligomer release is through oligomer toxicity leading to cell death, causing the cell to lyse and release its contents (129), studies show that this likely does not account for the majority of tau release. In primary neurons treated with tau oligomers, extracellular tau only increases once levels as high as 40% cell death are reached, which does not correspond to physiological levels of cell death during the initial spread of neurodegenerative disease (130). Additionally, treatment with tau oligomers in primary neurons does not lead to significant levels of apoptosis (83). There has however been some evidence for nonapoptotic membrane blebbing as a possible secondary mechanism for tau release (91, 131).

Exocytosis has been implicated as a mechanism of amyloid spread as prion proteins and α-synuclein have been shown to be associated with exosomes in cell culture (132, 133). However, investigations of a similar mechanism for tau release have been unclear. Simón et al. found that when tau was overexpressed in kidney-derived cell lines, tau was secreted contained within membrane vesicles (129, 134). While tau secreted by neuroblastoma cells and tau in human CSF was found to be associated with exosomes in one study (135), another reported that tau was not detected in isolated exosomes from neuroblastoma cells (130). However, these studies used cell models where tau was overexpressed. In an attempt to approach more similar conditions to those seen physiologically, researchers cultured primary cortical neurons containing endogenous tau and found that tau was released by a mechanism unrelated to cell death and was regulated by AMPA receptor activation. Inhibition of synaptic vesicle release decreased extracellular tau, while tau was not found to be associated with exosomes, indicating that release of tau through traditional synaptic exocytosis following AMPA receptor activation may be one mechanism of tau release (94). Another study found that cells constitutively release tau which is not contained within a membrane under conditions inhibiting cell death (136). Therefore, more research is warranted to investigate the conditions under which tau is associated with exosomes and the specific tau conformations found in exosomes. Tunneling nanotubes – long, temporary channels that allow for long-distance transport between cells – have recently been discovered as a transport mechanism for prion protein (137). While they have not yet been studied directly in the context of tau, similarities between the spread of prions and tau suggest that tunneling nanotubes may be another potential mode of tau propagation meriting study.

# **CONCLUSION**

Determining how neurodegenerative tauopathies initiate and propagate toxic species will be crucial to finding a treatment for these diseases. Recent evidence suggests that tau oligomers, not NFTs, are the toxic tau species mediating the initiation, seeding, and propagation of neurodegenerative tauopathies and are the best target for anti-tau therapeutics. The mechanism by which tau seeding occurs remains to be elucidated, but oligomer-nucleated conformational induction, whereby native tau monomers are

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# **ACKNOWLEDGMENTS**

We thank Drs. Diana L. Castillo-Carranza, Marcos J. Guerrero-Munoz, and George R. Jackson for useful suggestions. This work was supported by: The Cullen Family Trust for Health Care, the Alzheimer's Drug Discovery Foundation (ADDF), the Michael J. Fox Foundation, and the Mitchell Center for Neurodegenerative Diseases.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 12 June 2013; paper pending published: 19 June 2013; accepted: 01 July 2013; published online: 17 July 2013.*

*Citation: Gerson JE and Kayed R (2013) Formation and propagation of tau oligomeric seeds. Front. Neurol. 4:93. doi: 10.3389/fneur.2013.00093*

*This article was submitted to Frontiers in Neurodegeneration, a specialty of Frontiers in Neurology.*

*Copyright © 2013 Gerson and Kayed. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

# Tau oligomers as potential targets for Alzheimer's diagnosis and novel drugs

# **Leonardo Guzmán-Martinez 1,2, Gonzalo A. Farías 2,3 and Ricardo Benjamin Maccioni 1,2,4\***

1 Laboratory of Cellular and Molecular Neurosciences, Faculty of Sciences, University of Chile, Santiago, Chile

2 International Center for Biomedicine (ICC), Santiago, Chile

3 Department of Neurology and Neurosurgery North, University of Chile, Santiago, Chile

4 Department of Neurological Sciences East, Faculty of Medicine, University of Chile, Santiago, Chile

#### **Edited by:**

Jesus Avila, Centro de Biología Molecular Severo Ochoa CSIC-UAM, Spain

#### **Reviewed by:**

Cheng-Xin Gong, The City University of New York, USA Efthimios M. C. Skoulakis, Biomedical Sciences Research Centre Alexander Fleming, Greece George Perry, University of Texas at San Antonio, USA

#### **\*Correspondence:**

Ricardo Benjamin Maccioni, International Center for Biomedicine, Avda Vitacura 3568, OF. 513, Vitacura, Santiago, Chile e-mail<EMAIL_ADDRESS>
A cumulative number of approaches have been carried out to elucidate the pathogenesis of Alzheimer's disease (AD). Tangles formation has been identified as a major event involved in the neurodegenerative process, due to the conversion of either soluble peptides or oligomers into insoluble filaments. Most of recent studies share in common the observation that formation of tau oligomers and the subsequent pathological filaments arrays is a critical step in AD etiopathogenesis. Oligomeric tau species appear to be toxic for neuronal cells, and therefore appear as an appropriate target for the design of molecules that may control morphological and functional alterations leading to cognitive impairment.Thus, current therapeutic strategies are aimed at three major types of molecules: (1) inhibitors of protein kinases and phosphatases that modify tau and that may control neuronal degeneration, (2) methylene blue, and (3) natural phytocomplexes and polyphenolics compounds able to either inhibit the formation of tau filaments or disaggregate them. Only a few polyphenolic molecules have emerged to prevent tau aggregation. In this context, fulvic acid (FA), a humic substance, has potential protective activity cognitive impairment. In fact, formation of paired helical filaments in vitro, is inhibited by FA affecting the length of fibrils and their morphology.

**Keywords: tau oligomers, PHFs, Alzheimer's disease, tauopathies, diagnosis and treatment**

# **INTRODUCTION**

Tau protein, a member of the microtubule-associated protein (MAPs) family, plays a fundamental role in the assembly and stabilization of microtubules, as well as on axonal transport and neurite outgrowth (1–3). In this context, tau protein plays an important role in the maintenance of neuronal polarity and in the stabilization of the morphology of differentiated neurons. In developing neurons, tau activity is crucial for the morphogenesis of the growth cones and, it has been suggested to play a key role in promoting axonal growth (4). Tau is encoded by a single gene located on chromosome 17 (17q21), possessing 16 exons in its primary transcript. Six different isoforms are expressed by post transcriptional modifications (alternative splicing) from the primary transcript. Mature protein length is about 352 up to 441 amino acid residues, and a molecular weights of 45–65 kDa depending on the tau isoforms (5, 6). The C-terminal region has a domain containing the microtubulebinding repeats, which is critical for microtubule assembly (2), whereas the affinity of tau for microtubules is finely regulated by an orchestrated set of phosphorylations. The motif KXGS is one of several different motifs located within these repeats susceptible to be phosphorylated (7). In turn, tau is characterized as an hydrophilic cationic protein, unfolded under native conditions, and with a low ordered secondary structure (8). In addition to roles in stabilization of microtubules, tau plays a

major role in bridging the different cytoskeletal structures. Thus, besides microtubules, tau interacts with actin and intermediate filaments (2, 9).

Tau is located primarily in neurons, however, traces of this protein have been found in certain non-neuronal cells. Under pathological conditions, tau can be also expressed in glial cells (10). It is also possible to find tau or it's mRNA in several peripheral tissues such as heart, liver, lung, skeletal muscle, among others (11– 13). Interestingly, tau variants have been also observed in human platelets (3, 14, 15).

Tau phosphorylation plays an important role in regulating its binding to microtubules and thereby regulating their stability within neuronal cells. However, under pathological situations, tau protein is abnormally phosphorylated or dephosphorylated in specific residues, perhaps due to the activities of various protein kinases and phosphatases (such as GSK-3β and Cdk-5, and PP1A and PP2, respectively) (16, 17). This change in the phosphorylation state of tau can lead to irreversible changes in the dynamics of microtubules, cellular dysfunction, ultimately triggering cell death of the neuron (6, 18). Moreover, tau protein may have other post-translational modifications, including: (i) glycosylations, (ii) ubiquitinations, (iii) truncations, and (iv) nitrations (6).

Hyperphosphorylated tau protein is the main component of abnormal protein aggregates found in the neurons of patients with neurodegenerative brain disorders known as tauopathies. Those neurodegenerative diseases have in common the presence of intraneuronal aggregates of tau. These aggregates are known as Neurofibrillary Tangles (NFTs) and are made up of paired helical filaments (PHFs) and straight filaments mainly composed of hyperphosphorylated tau. The formation of PHFs from tau molecules may follow different steps and could involve tau phosphorylations, followed by limited proteolysis and conformational changes in tau protein (19), and finally its polymerization into aberrant polymers in neuronal cells. These neuronal disorders include: Pick's disease, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), frontotemporal dementia with Parkinsonism linked to chromosome 17 (DFTP-17) and Alzheimer's disease (AD). AD is considered the most prevalent tauopathy worldwide (20). In AD, abnormal phosphorylations occur and specifically tag certain amino acids of tau protein: Ser202, Thr205, Ser235, and Ser404 (21). These post-translational modifications are catalyzed by two main protein kinases: the Cdk-5/p35 system and GSK-3β (6). In this paper, we focus on how tau oligomers became the focus for the search for new drugs, and also potential targets for accurate diagnosis of AD.

# **ARE TAU OLIGOMERS RESPONSIBLE FOR NEURONAL DEGENERATION? TECHNICAL APPROACHES**