Insoluble aggregates of the MAP tau characterize a number of tauopathies. Although there is much evidence linking tau to neurodegeneration, the precise mechanism of tau-mediated neurotoxicity remains to be elucidated. In fact, tau oligomers appear to be the toxic form of tau in neurodegenerative disease. In agreement with this hypothesis, the presence of immunoreactive tau protein in neurons of AD brain tissue, previous to tangle formation, has been shown. Binder and coworkers have produced the novel monoclonal antibody TOC1 that recognizes non-fibrillary tau. This antibody is selective in terms of specifically labeling tau dimers and oligomers, but does not label tau filaments. Timecourse analysis and antibody labeling indicates that oligomers appear as an early event in AD pathogenesis. Aggregated tau, but not monomeric tau, inhibited anterograde fast axonal transport. This inhibition requires a small stretch containing amino acids from the N-terminal region on tau, a phosphatase-activation domain. The molecular chaperone heat-shock protein 70 (Hsp 70) clearly affects tau oligomers formation and stability, as investigated in the squid axoplasm. Hsp 70 preferentially bound to tau oligomers over filaments and prevented anterograde axonal transport inhibition observed with a mixture of both forms of aggregated tau (22).

Tau aggregates comprise abnormally hyperphosphorylated and misfolded tau. Research has traditionally focused on understanding how hyperphosphorylated and aggregated tau mediates dysfunction and toxicity in tauopathies. Recent findings in *Drosophila* and rodent models of tauopathy suggest that large insoluble aggregates such as tau filaments and tangles may not be the key toxic species in these diseases (23). Thus, some investigators have shifted their focus to study pre-filament tau species such as tau oligomers and hyperphosphorylated tau monomers. Interestingly, tau oligomers can exist in a variety of states including hyperphosphorylated and unphosphorylated forms, which can be both

soluble and insoluble. It remains to be determined which of these oligomeric states of tau are causally involved in neurodegeneration and which molecule signal the beginning of the formation of inert/protective filaments. It will be important to better understand this aspect so that tau-based therapeutic interventions can target the really toxic tau species.

Another interesting study showed that oligomers of recombinant full-length human tau protein are neurotoxic *in vivo* after subcortical stereotaxic injection into mice. Data showed that tau oligomers impaired memory consolidation, whereas tau fibrils and monomers did not. In this context it was assumed that tau oligomers can affects synaptic transmission. Thus, synaptic dysfunction seems to result from the action of tau oligomers, potentially reducing the activity of the synaptic vesicle-associated proteins synaptophysin (24). Some studies identify tau oligomers as an acutely toxic tau species *in vivo*, and suggest that tau oligomers induce neurodegeneration by affecting mitochondrial and synaptic function, both of which are early hallmarks in AD and other tauopathies (25). These results open new avenues for neuroprotective intervention strategies for tauopathies by targeting tau oligomers.

# **OLIGOMERS AND THEIR USEFULNESS FOR MONITORING AD**

As mentioned above, tau oligomers are one of the neuropathological hallmarks of AD and other tauopathies. Regarding NFT accumulation in AD, there is evidence showing that progressive neuronal loss and cognitive impairment correlates with the accumulation of soluble species of tau in NFTs in AD mouse models (26). This etiopathological feature has become the target for potential treatments for AD, diagnosis and monitoring evolution of the disease. On this last point, some diagnostic biomarkers for AD based on tau have been developed: (1) biomarkers in cerebrospinal fluid (CSF) and (2) biomarkers in platelet tau (3). Since in the CSF significant exchange of substances with a varied neural environment appears to occur, tau variants are released to the fluid. In this context, one of the most reliable biomarkers reported was based on the ratio between normal tau and hyperphosphorylated tau (Ptau) in the CSF (27). This evaluation about tau and P-tau allows a better correlation with the later stages of synaptic dysfunction and early neurodegeneration (27, 28). Furthermore, the second biomarker is based on the detection of tau in blood platelets (15, 29). Alterations in platelets from AD patients, including modifications in platelets β-amyloid Precursor Protein (APP) have been described previously (30) and APP evaluations have been postulated as a potential biomarker for AD (31). Considering that information we focused our efforts in analyses of platelets tau protein. The innovative method was developed in our laboratory and is based upon the difference between the ratio of molecular species of high molecular weight tau (multimeric) versus low molecular weight (monomeric). In western blot experiments performed with platelet protein extracts obtained from peripheral blood from healthy subjects and patients with AD, it was observed that the latter had tau immunoreactive bands migrating at molecular weights much higher than expected by electrophoresis under denaturing and reducing conditions (SDS-PAGE) (15). These high molecular weight forms of tau (tau HMW) could be attributed to oligomeric

Guzmán-Martinez et al. Tau oligomers for diagnosis and therapeutics

forms of the protein, which are increased in AD patients as compared to healthy elderly subjects (**Figure 1**). Therefore, platelets tau has been postulated as a biomarker for AD (29). Different tau species with variable stages of aggregation are visualized in the electrophoretic patterns. Subsequent studies by Farias et al. (15), have shown that there is a close correlation between the degree of platelet tau modification and level of cognitive impairment, which was measured using neuropsychological tests in patients with AD [for more detailed information about these biomarkers see Ref. (3)]. Moreover, correlations between HMWtau/LMWtau ratio and brain neuroimaging have been observed. This ratio, controlled for age and education, significantly correlated with a clusters of 717 voxels in the right parahippocampal cortex with peak at Talairach coordinates 16, −10, −23 (unpublished observations).

# **TAU AS A THERAPEUTIC TARGET FOR ANTI-ALZHEIMER DRUGS**

Since tau has been recognized as an important actor in neurodegenerative diseases, many molecules that act on tau pathology have been investigated as potential vectors for therapeutic approaches for AD, but also for other tauopathies such as PSP or CBD. One of the molecules that have attracted much attention is the phenothiazine methylthioninium chloride, better known as methylene blue (MB). MB has a very interesting property as an aggregation inhibitor for proteins that adopt beta sheet conformation (32).

Rember™ is the trade name of MB, and a Phase II study randomizing 321 mild to moderate AD patients with placebo or three different doses of Rember™ was presented in 2008 at the International Conference on AD in Chicago. Treatment effects of four points were described on ADAS-Cog on the treatment group as compared to placebo subjects, and also conservation on the cerebral blood flow and brain glucose used in SPECT and FDG-PET scans (33). However, as today, the lack of peer reviewed publications on this compound affect the reliability of the results. In animal models, the effects of MB on cognition appears not to be related to a reduction on NFTs but to a decrease in soluble tau levels (34) (**Figure 2**).

Other groups have focused their efforts on investigating tau kinases inhibitors (16) or tau phosphatase activators (17), as an indirect manner to halt tau hyperphosphorylation, recognized as leading to pathological aggregation of the protein. However, difficulties in finding specific inhibitors/activators with adequate safety profiles have impacted in the lack of new drugs in this area (32, 35). A possible way to decrease adverse effects of tau kinase inhibition might be the use of relatively broad specificity, low power compounds (36).

Davunetide is an eight aminoacids peptide that can be administered intranasally or intravenously and has been described as a tau hyperphosphorylation inhibitor as well as an inhibitor of caspase 3 activation (37). Davunetide has been successfully evaluated in a series of *in vitro* and *in vivo* models (38) for neurodegenerative diseases that include AD, PSP, and schizophrenia (38, 39).

An example of "non-traditional" approaches to tau aggregation regulation includes chaperones modulation. Hsp 70 chaperones assist protein-folding processes and are found up-regulated in several tumors, but also in neurodegenerative diseases such as Pick's

**FIGURE 1 | Representative immunoblots of platelet tau with tau-5 antibody**. High molecular weight tau bands (about 80 kDa) can be appreciated, with greater immunoreactivity in patients with Alzheimer's disease (AD) compared with control subjects (C). Subsequent densitometric analysis allows obtaining the relationship between HMWtau versus LMWtau.

disease,AD, and other tauopathies. Deregulation in Hsp 70 chaperones appears to be implicated in the processes of tau aggregation, so compounds that bind Hsp 70 chaperones are under investigation as possible treatment compounds for neurodegenerative diseases, since there is evidence that Hsp 70 inhibition leads to tau ubiquitination and clearance through ubiquitin-proteasome system (40).

There are some natural compounds that are able to inhibit tau aggregation and possibly, make an impact in neurodegenerative diseases. Shilajit is a natural phytocomplex that has been found in the Himalayan Mountains between India and Nepal and also in the Tibet and Afghanistan and has been used in ayurvedic medicine for centuries as a rejuvenating compound. Our laboratory has worked with the *Andean Compound* (or *Andean shilajit*), a natural compound that can be found in Andean Mountains. *Andean shilajit* is generated by a long-term degradation of certain plants by microorganisms, mostly fungi and is rich in Fulvic Acid (FA) and humic substances among others (**Figure 2**). This natural endemic phytocomplex, resulting from fossilized plants degradation through the years, was discovered in 2008 in the North of Chile and was named *Andean Compound* (41). *In vitro* assays and cell culture data show that Andean Compound and FA strongly interferes with tau aggregation, and interestingly an increase in neurites outgrowth has been observed in neural cell cultures exposed to this natural compound (42). In addition, a placebo-controlled pilot clinical study suggests that consumption of a nutraceutical formulation of *Andean Compound* plus B complex vitamins may produce stabilization of cognitive function in AD patients at a 24-weeks as determined with Global deterioration scale (GDS) and Neuropsychiatric inventory (NPI) measurements (42) (**Figure 2**). On the other hand the same *shilajit* – based compound has a very good safety profile when tested on healthy population (unpublished data).

# **CONCLUDING REMARKS**

Currently, we do not know the exact cause of synaptic dysfunction and neurodegeneration in AD, however, in recent years it has become increasingly clear the importance of tau protein and its post-translational modifications in the pathophysiological processes of AD and other tauopathies.

In this context, determination of different forms of tau protein in brain, CSF (43) and also in blood (44) and peripheral cells (15) has been postulated as a powerful tool for detection and monitoring of the disease in different stages and there is clear evidence of a profile of tau and other biomarkers modifications during AD progression (45, 46). The presence of tau modifications in peripheral cells also points to the inference that AD may be a systemic disease not only confined to nervous tissue. But currently we do not have any information on the functional impact – if any – of oligomeric tau forms in peripheral cells. The latest criteria of AD consider tau-based biomarkers as reliable indicators of neuronal injury processes (47), but as of today there is not a widely available marker of tau modification for clinical use. Hopefully in the near future we will refine techniques for non-invasive assessment of tau. Thereon, are extremely interesting attempts to generate PET markers for tau deposition in brain (48, 49) and we already have promising data regarding measurement of different forms of tau in blood cells (15, 29).

On the other hand, in the absence of positive clinical results in studies with beta amyloid targeted therapies, the need to evaluate therapies that can act on post-translational changes of tau protein has become evident. Particular interest has been paid to therapies that may modulate levels of phosphorylation and oligomerization of tau, whether by direct action on tau or by acting on other related proteins like kinases or heat-shock proteins.

Unfortunately we still need information from large multicenter studies about the usefulness of tau-focused therapies, however,

#### **REFERENCES**

this is a promising field that has attracted the efforts of multiple investigators, so we expect that new and exciting discoveries are right around the corner.

#### **ACKNOWLEDGMENTS**

This research has been financed by grants from Fondecyt (1110373) and by the CORFO project 12IDL4-13071 to Ricardo Benjamin Maccioni.

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for Alzheimer disease: challenges to early intervention. *Neurology* (2007) **69**:1622–34. doi:10.1212/01. wnl.0000295996.54210.69

Alzheimer's disease? Potential therapeutic role of a formulation containing shilajit and complex B vitamins. *Arch Med Res* (2012) **43**:699–704. doi:10.1016/j.arcmed.2012.10.010

NC, et al. Clinical and biomarker changes in dominantly inherited Alzheimer's disease. *N Engl J Med* (2012) **367**(9):795–804. doi:10. 1056/NEJMoa1202753

**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 July 2013; paper pending published: 15 August 2013; accepted: 15 October 2013; published online: 28 October 2013.*

*Citation: Guzmán-Martinez L, Farías GA and Maccioni RB (2013) Tau oligomers as potential targets for Alzheimer's diagnosis and novel drugs. Front. Neurol. 4:167. doi: 10.3389/fneur.2013.00167*

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

*Copyright © 2013 Guzmán-Martinez, Farías and Maccioni. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, providedthe 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.*

# What rendersTAU toxic

#### **Jürgen Götz 1,2\*, Di Xia1 , Gerhard Leinenga1 ,Yee Lian Chew 3 and Hannah R. Nicholas 3**

1 Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia

2 Sydney Medical School, Brain and Mind Research Institute, University of Sydney, Sydney, NSW, Australia

3 School of Molecular Bioscience, University of Sydney, Sydney, NSW, Australia

#### **Edited by:**

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

#### **Reviewed by:**

Wolfgang Härtig, University of Leipzig, Germany Estelle Sontag, University of Newcastle, Australia

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

Jürgen Götz, Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, St Lucia Campus, Brisbane, QLD 4072, Australia e-mail<EMAIL_ADDRESS>
TAU is a microtubule-associated protein that under pathological conditions such as Alzheimer's disease (AD) forms insoluble, filamentous aggregates. When 20 years after TAU's discovery the first TAU transgenic mouse models were established, one declared goal that was achieved was the modeling of authentic TAU aggregate formation in the form of neurofibrillary tangles. However, as we review here, it has become increasingly clear thatTAU causes damage much before these filamentous aggregates develop. In fact, because TAU is a scaffolding protein, increased levels and an altered subcellular localization (due to an increased insolubility and impaired clearance) result in the interaction of TAU with cellular proteins with which it would otherwise either not interact or do so to a lesser degree, thereby impairing their physiological functions. We specifically discuss the non-axonal localization of TAU, the role phosphorylation has in TAU toxicity and how TAU impairs mitochondrial functions. A major emphasis is on what we have learned from the four available TAU knock-out models in mice, and the knock-out of the TAU/MAP2 homolog PTL-1 in worms. It has been proposed that in human pathological conditions such as AD, a rare toxic TAU species exists which needs to be specifically removed to abrogate TAU's toxicity and restore neuronal functions. However, what is toxic in one context may not be in another, and simply reducing, but not fully abolishing TAU levels may be sufficient to abrogate TAU toxicity.

**Keywords: Alzheimer's disease, C. elegans, frontotemporal dementia, knock-out, PP2A, PTL-1,TAU, transgenic**

# **INTRODUCTION**

TAU belongs to the family of microtubule-associated proteins (MAPs) (Dehmelt and Halpain, 2005). MAPs act in concert with heterodimers of α- and β-tubulin to assemble microtubules. They were named according to the three major size classes of polypeptides: MAP1 (>250 kDa), MAP2 (∼200 kDa), and TAU (50–70 kDa) (Dehmelt and Halpain, 2005; Halpain and Dehmelt, 2006). MAP2 and TAU are expressed together in most neurons, where they localize to separate subcellular compartments. MAP2 is largely found in dendrites, whereas TAU is concentrated in axons (Matus, 1990). TAU has also been found in astrocytes and oligodendrocytes, although, under physiological conditions, its levels are relatively low (Tashiro et al., 1997). In concert with actin and intermediate filaments, microtubules establish and maintain the overall internal architecture of the cytoplasm and thereby comprise a major determinant of overall cell shape (Allen et al., 1985; Vale et al., 1985; Nangaku et al., 1994; Trinczek et al., 1999). Besides interacting with cytoskeletal proteins, MAPs also interact with proteins that have a range of cellular functions, suggesting that TAU is a scaffolding protein (Brandt and Leschik, 2004). Scaffolding proteins are defined as being able to bind at least two signaling proteins, and localizing signaling molecules and transduction pathways to defined cellular locations – for instance the cell body, dendrites or the axon, and to regulate signaling (Shaw and Filbert, 2009). We discuss here that TAU has these properties and that the toxicity of TAU is, at least

in parts, because this scaffolding function is either altered or lost.

When TAU was discovered in 1975 (Weingarten et al., 1975), the subsequent years focused mainly on its tissue distribution and role in microtubule assembly and stabilization. The focus shifted radically when TAU was identified in Alzheimer's disease (AD) brains in a highly phosphorylated form as the filamentous core of the neurofibrillary tangles (NFTs) (Grundke-Iqbal et al., 1986; Goedert et al., 1988). Histopathologically, AD is also characterized by β-amyloid (Aβ)-containing plaques, reduced synaptic density, and neuronal loss in selected brain areas. Interestingly, although NFTs were initially correlated with dementia (Arriagada et al., 1992), it was also found that firstly, CA1 hippocampal neurons can survive with NFTs for about 20 years, and secondly that NFTs may not be obligatory for the death of CA1 hippocampal neurons in AD (Morsch et al., 1999). This does not, however, preclude that NFTcarrying neurons are functionally impaired. For a discussion of the role of NFTs in neurodegeneration, see e.g.,Castillo-Carranza et al. (2013). In addition to AD, neurofibrillary lesions are also abundant in other neurodegenerative diseases such as Pick's disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic grain disease (AGD), and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), where they occur in the absence of overt Aβ deposition. When we, for the first time, expressed human TAU in transgenic mice, we were able to reproduce key aspects of the human TAU pathology (Götz et al., 1995). Specifically, overexpression of human TAU resulted in the accumulation of hyperphosphorylated forms of TAU not only in the axonal compartment, but also in the somatodendritic domain. NFT formation, however, was not achieved by this approach. It was the identification of pathogenic mutations in familial cases of frontotemporal lobar degeneration (FTLD) in the *MAPT* gene encoding TAU (Clark et al., 1998; Hutton et al., 1998; Poorkaj et al., 1998) that enabled us and others to express FTLD mutant forms of TAU in mice. Thus, we not only reproduced NFT formation, but in addition placed TAU downstream of Aβ in a patho-cascade – a central concept in the field (Götz et al., 2001a,b). Despite this, TAU is not simply an innocent bystander in Aβ toxicity, as discussed below. In fact, the dendritic localization of the "axonal" protein TAU not only has a role in physiological postsynaptic scaffolding, but it is also crucial for mediating Aβ's toxicity (Ittner et al., 2010). This and complementary studies in cell culture have put forth the argument that reducing or completely abolishing TAU expression might be beneficial in combating neurodegeneration in AD (Rapoport et al., 2002; Roberson et al., 2007; Ittner and Götz, 2011; Morris et al., 2011b).

In the following, we discuss what we have learned about TAU's toxicity in models that over-express TAU and as a consequence, develop TAU aggregates and NFTs. As this article is part of a series that investigates TAU toxicity from various angles, we will restrict our discussion to selected aspects and will specifically discuss TAU's role in non-axonal compartments, how it affects mitochondrial functions and what we have learned from invertebrate models. We draw the conclusion that TAU is a scaffolding protein and that its mislocalization disrupts cellular processes because it traps proteins with which it would otherwise not interact (or do so to a lesser degree) and thereby prevents them from executing their physiological functions. This does not rule out that the aggregates themselves are toxic, although the current experimental models do not allow for a dissociation of the effects elicited by the aggregates from those elicited by soluble TAU species. In the second part we discuss what we have learned from TAU knock-out models in mice and what the knockout of the worm homolog of TAU, PTL-1, has contributed to an understanding of TAU function. In the final paragraph, we attempt to integrate findings in both TAU over-expressing and knock-out models.

# **WHAT CAUSES TOXICITY WHEN THERE IS TOO MUCH TAU?**

In an attempt to model TAU pathology in mice, the first TAU transgenic mouse model was established 20 years after TAU's discovery by over-expressing the longest of the six major human TAU isoforms, hTau40 (Götz et al., 1995) (For comparison, in the adult mouse brain, there are only three TAU isoforms expressed.). These htau40 transgenic mice reproduce aspects of the human pathology, such as somatodendritic localization and hyperphosphorylation of TAU. Subsequently, stronger promoters were used to drive expression, resulting in more pronounced phenotypes (Ishihara et al., 1999; Spittaels et al., 1999; Probst et al., 2000). Despite the formation of TAU aggregates and an age-dependent decrease in TAU solubility, NFTs did not form until a very old age in these mice (Ishihara et al., 2001). However, signs of Wallerian degeneration including axonal breakdown and segmentation of myelin into ellipsoids were observed. Furthermore, neurogenic muscle atrophy, with groups of small angular muscle fibers, was present in the hind leg musculature of the transgenic mice (Probst et al., 2000). Another characteristic was the presence of large numbers of axonal spheroids in brainstem and spinal cord (Ishihara et al., 1999; Spittaels et al., 1999; Probst et al., 2000). Similar swellings have also been described in transgenic mice with Aβ plaque formation, and in the human AD brain (Stokin et al., 2005).

Following the identification of pathogenic mutations in *MAPT* in FTDP-17, TAU filament formation was achieved in neurons and glia, both by constitutive and doxycycline-regulated expression of mutant human forms of TAU (Götz and Götz, 2009). The first published mouse strain with pronounced NFT formation, JNPL3, expressed P301L mutant human TAU under the control of the murine PrP promoter. This resulted in abnormal TAU filament formation in neurons as well as astrocytes and oligodendrocytes, with NFTs being present in brain and spinal cord (Lewis et al., 2000). Neuronal loss was found in the spinal cord, as evidenced by a twofold reduction in the numbers of motor neurons. Furthermore, the mice developed severe motor disturbances by 10 months of age. We established a second model with NFT formation, pR5 mice, by expressing the same P301L mutation, but using a different human TAU isoform and the neuron-specific mThy1.2 promoter for transgene expression (Götz et al., 2001a). While a motor phenotype was absent, reference memory was impaired and the mice displayed an increased exploratory behavior (Pennanen et al., 2006). Furthermore, owing to a pronounced expression of the transgene in the amygdala, the mice showed an accelerated extinction in the conditioned taste aversion paradigm (Pennanen et al., 2004). When experimental diabetes was induced in pR5 mice, this caused an earlier-onset and increased formation of NFTs, indicating that diabetes can accelerate the onset and increase the severity of disease in individuals with a predisposition to develop a tauopathy (Ke et al., 2009). To determine whether NFTs are integral components of the neurotoxic cascade in AD or whether they represent a protective neuronal response, transgenic mice were generated which allowed for the regulation of P301L TAU expression by adding doxycycline to the drinking water (Santacruz et al., 2005). Because the system resulted in a 15-fold over-expression, this caused a progressive formation of NFTs, a remarkable neuronal loss (70% in the CA1 region), gross atrophy of the brain, and behavioral impairment. Turning the system "off" caused a reduction of TAU levels to 2.5-fold over-expression. Nonetheless, this was sufficient to cause a recovery of memory functions and stabilization of neuron numbers, while NFTs continued to accumulate. These data imply that NFTs *per se* are not sufficient to cause cognitive decline or neuronal death (Santacruz et al., 2005).

While the pR5 mice display memory impairment as a major clinical feature of AD, another feature, Parkinsonism, that characterizes a significant subset of FTLD cases, has been modeled in K369I mutant TAU transgenic K3 mice. We established this strain based on the identification of the K369I mutation of TAU in a single patient with Pick's disease (Neumann et al., 2001), and reproduced the distinct characteristics of Pick's pathology in mice (Ittner et al., 2008). Memory functions were impaired as shown in the novel object recognition test. Owing to a unique expression pattern of the transgene that extends to the Substantia Nigra pars compacta (SNpc), the K3 mice also model early onset

Parkinsonism, i.e., resting tremor, bradykinesia, postural instability, and gait anomalies. They show an increased cataleptic response to haloperidol and an early, but not late, response to l-DOPA, indicating that the dopaminergic system is impaired. We found a selectively impaired axonal transport of distinct cargos including mitochondria and tyrosine hydroxylase (TH)-containing vesicles. An important finding is that at the molecular level, this transport impairment is the result of the accumulation of hyperphosphorylated TAU in the cell body, where it traps the adapter protein and a component of the kinesin motor machinery, JIP1, thereby preventing this molecule from executing its physiological function in the axon (Ittner et al., 2008).  It seems therefore that for TAU to be able to exert toxicity, the subcellular compartments it is aberrantly targeted to are crucial, as this determines with which proteins it pathologically interacts and which cellular functions it impairs. In this process, phosphorylation of TAU has a critical role as reviewed by us recently (Götz et al., 2010). That phosphorylation of TAU is critical in toxicity is also evident from studies in protein phosphatase 2A (PP2A) dominant negative mutant strains. More specifically, to address the role of the TAU phosphatase PP2A in mice with a pre-existing TAU pathology, we crossed the PP2A dominant negative mutant strain Dom5 with pR5 mice that express P301L mutant TAU and found that this exacerbated the TAU pathology of pR5 mice significantly. The double-transgenic mice showed sevenfold increased numbers of hippocampal neurons that specifically phosphorylated the pathological Ser422 epitope of TAU (Deters et al., 2009). The mice showed eightfold increased numbers of NFTs compared with pR5 mice, in agreement with our previous finding that NFT formation is correlated with and preceded by phosphorylation of TAU at the Ser422 epitope (Götz et al., 2001b). We further used the Dom5 mice to show that a small compound, sodium selenate, improves TAU-dependent impairment and neurodegeneration in a PP2A-dependent manner (van Eersel et al., 2010). Together this demonstrates that phosphorylated forms of TAU have a critical role in TAU toxicity.

The tauopathies PSP and CBD are characterized by substantial glial TAU pathology. Aspects of this pathology have been reproduced by expressing G272V mutant TAU under the control of the PrP promoter that resulted in high transgene expression in a subset of both neurons and oligodendrocytes. Electron microscopy established that TAU filament formation was associated with hyperphosphorylation of TAU. Thioflavin S-positive fibrillary inclusions were identified in oligodendrocytes and motor neurons (Götz et al., 2001c), the clinical phenotype of these mice however was subtle. In contrast, when human wild-type TAU was overexpressed in neurons and glial cells under the control of the mouse Tα1 α-tubulin promoter, a glial pathology was obtained that closely resembled the astrocytic plaques of CBD and the coiled bodies of both CBD and PSP (Higuchi et al., 2002). A significant age-related neuronal loss was only found at 18 months of age, whereas oligodendrocytes were lost already at 6 months. The same team employed the 20 ,30 -cyclic nucleotide 30 -phosphodiesterase promoter to express human P301L TAU exclusively in oligodendrocytes (Higuchi et al., 2005). Interestingly, the structural

disruption of myelin and axons preceded the emergence of TAU inclusions in oligodendrocytes; also, impaired axonal transport was found to precede the motor deficits in these mice (Higuchi et al., 2005). Together, these studies highlight a role for glial TAU in disease.

While most studies on TAU use the mouse as a model organism, several wild-type and mutant human TAU transgenic models have been established in the nematode *C. elegans* (Kraemer et al., 2003; Miyasaka et al., 2005; Brandt et al., 2009; Fatouros et al., 2012). Some of these models have been reviewed in detail (Ewald and Li, 2010) and are not discussed here.

In the following, we will discuss two important aspects of TAU toxicity, one is the impairment of mitochondrial functions, and the second the role TAU has in the dendrite. Mitochondrial dysfunction has long been associated with the pathophysiology of AD (Blass and Gibson, 1991). The morphology of a cellular mitochondrial network is maintained by reciprocal rounds of fission and fusion, a process termed mitochondrial dynamics. Elevated fusion produces elongated, interconnected mitochondria, while enhanced fission results in mitochondrial fragmentation. Mitochondrial dynamics cannot be discussed in isolation, as fission (biogenesis), fusion, bioenergetics, motility/transport, and turnover by mitophagy are highly inter-dependent processes (Chen and Chan, 2009). How TAU impairs the fission and fusion of mitochondria, has been reviewed by us recently (Duboff et al., 2013), following the finding of a role of TAU in altering mitochondrial dynamics. Specifically we had found that in human TAU transgenic mice and flies, F-actin is increased, which disrupts the physical association of mitochondria and the fission protein DRP1, leading to mitochondrial elongation (Duboff et al., 2012). The resulting neurotoxicity can be rescued either by reducing mitochondrial fusion, or by enhancing fission, or by reversing actin stabilization.

Earlier, we had analyzed P301L TAU transgenic pR5 mice by MALDI TOF/TOF mass-spectrometric analysis, which revealed a deregulation of mainly metabolism-related proteins including mitochondrial respiratory chain complex components such as the complex V component ATP synthase D chain, antioxidant enzymes, and synaptic proteins (David et al., 2005). A subsequent functional analysis demonstrated a mitochondrial dysfunction in pR5 mice together with a reduced NADH-ubiquinone oxidoreductase activity and, with age, impaired mitochondrial respiration and ATP synthesis. Mitochondrial dysfunction was associated with higher levels of reactive oxygen species (ROS) in aged transgenic mice and an up-regulation of antioxidant enzymes.When complex V levels were analyzed in human FTDP-17 patient brains carrying the P301L TAU mutation, a significant decrease in complexV levels was found in all P301L human brain samples compared with controls, underscoring the validity of the proteomics findings in mice for the human disease (David et al., 2005). A serial analysis of gene expression (SAGE) of the same mouse strain looking specifically at the amygdala also revealed deregulated mitochondrial genes (Ke et al., 2012a).

Mitochondrial functions are also impaired by Aβ and as there are many findings that support the notion of a cross-talk of Aβ and TAU in affecting mitochondrial functions (Eckert et al., 2010), it was reasonable to cross pR5 mice with Aβ plaque-forming

APPswPS2N141I double-transgenic APP152 mice to generate tripletransgenic (tripleAD) mice that combine both pathologies in one model (Grueninger et al., 2010). Quantitative isobaric-tag labeling (iTRAQ) followed by mass spectrometry revealed a massive deregulation of 24 proteins, of which one third were mitochondrial proteins mainly related to complexes I and IV of the oxidative phosphorylation system (OXPHOS). When mitochondrial functions were addressed, this revealed that deregulation of complex I is TAU-dependent, whereas deregulation of complex IV is Aβdependent, both at the protein and activity level (Rhein et al., 2009). In addition, synergistic effects of Aβ and TAU were evident in the tripleAD mice establishing a molecular link between Aβ and TAU protein in AD pathology *in vivo* (Rhein et al., 2009). In the P301L TAU transgenic pR5 mice, a transcriptomic analysis further revealed an up-regulation of glyoxalase I that detoxifies dicarbonyl compounds and thereby reduces the formation of advanced glycation end (AGE) products (Chen et al., 2004). Co-staining with a phospho-TAU antibody suggested glyoxalase I up-regulation as an early defense mechanism to combat elevated levels of aggregated TAU. To understand which processes are disrupted by Aβ in the presence of TAU aggregates, we applied comparative proteomics to Aβ-treated P301L TAU-expressing neuroblastoma cells and the amygdala of P301L TAU transgenic pR5 mice that had been stereotaxically injected with Aβ preparations.We found that a significant fraction of proteins that were altered in both systems belonged to the same functional categories, i.e., proteins involved in the stress-response associated with protein folding (David et al., 2006). Among the deregulated proteins was valosin containing protein (VCP), an essential component of the ER-associated degradation (ERAD) process, whereas members of the peroxiredoxin family were down-regulated. Together this indicates that TAU and Aβ exert both separate and synergistic toxic effects that are mediated by mitochondria and the stress-related unfolded protein response.

The amyloid cascade hypothesis in a patho-cascade places Aβ upstream of TAU (Hardy, 2006). This concept has been proven in P301L mutant TAU transgenic mice that develop an increased number of NFTs, either by crossing them with Aβ plaque-forming transgenic mice (Lewis et al., 2001), or by stereotaxically injecting Aβ into their brains (Götz et al., 2001b). That Aβ toxicity is dependent on TAU has first been shown *in vitro* because primary neuronal cultures derived from TAU knock-out mice were resistant to the toxic effects of Aβ (Rapoport et al., 2002). This finding was subsequently reproduced *in vivo*, by crossing Aβ plaqueforming mice that are characterized by premature mortality, high susceptibility to experimentally induced excitotoxic seizures and memory deficits, onto a TAU knock-out background (Roberson et al., 2007). Mechanistically, this protection appeared to be conferred by a reduced susceptibility to excitotoxicity when TAU was either absent or when its levels were reduced (Roberson et al., 2007). Excitotoxicity is the over-activation of NMDA receptors (NMDARs) that results in neuronal damage and death because of increased calcium influx and nitric oxide (NO) activation (Palop and Mucke, 2009). By using another APP plaque-forming mouse strain and a different TAU knock-out strain, we were able to reproduce the TAU-dependent protection from Aβ-induced premature mortality and memory deficits, and determined that this protection is conferred by a reduced susceptibility to excitotoxicity

(Ittner et al., 2010). Likewise, overexpression of a truncated form of TAU (∆Tau) that lacks the microtubule-binding region MBR also rescued the phenotype of the Aβ plaque-forming mice (Ittner et al., 2010). In a more recent model, tau pathology was shown to develop independent of Aβ (Winton et al., 2011). This was shown by finding no differences in TAU lesion formation when crossing 3xTg-AD mice that co-express mutant forms of APP, presenilin and TAU and hence develop a plaque and TAU pathology, with a mouse strain that lacks the enzyme BACE1 required for Aβ generation. However, it has to be considered that the TAU pathology in the 3xTg-AD mice develops very late and is modest compared with that of other widely used TAU transgenic mouse models (Oddo et al., 2003a,b).

What is the mechanistic explanation for this protection conferred by reduction of TAU? We next showed that TAU is also present in the dendrite (although in low quantities compared with the axon), where it is critically involved in postsynaptic NMDAR downstream signaling by localizing the SRC kinase FYN to the dendrite. FYN phosphorylates the NMDAR that then recruits the postsynaptic scaffolding protein PSD-95 to form a complex. Because levels of postsynaptic FYN are massively reduced in TAU-deficient or ∆Tau-expressing mice, this results in uncoupling of NMDARs from excitotoxic signaling and abrogation of Aβ-mediated toxicity (Ittner et al., 2010; Ittner and Götz, 2011). A subsequent study found that Aβ causes a range of deleterious effects on TAU including its localization to dendrites (Zempel et al., 2010). Aβ, Fyn, and TAU therefore seem to orchestrate neuronal damage (Haass and Mandelkow, 2010; Ittner et al., 2010; Roberson et al., 2011). An essential role for FYN in this process is supported by the fact that the pathology of Aβ plaque-forming mice is enhanced when FYN is overexpressed and ameliorated when FYN is absent (Chin et al., 2004, 2005).

TAU interacts with FYN at least in two ways. Firstly, although TAU is mainly phosphorylated on serine and threonine residues, it also contains three tyrosine residues. Tyr18 is phosphorylated by FYN, and the phosphorylated motif interacts with FYN via FYN's SH2 domain. Secondly, TAU contains seven PXXP motifs located in the amino-terminus (all of which are retained in the ∆Tau construct). Of these, the seventh proline-rich RTPPKSP motif has been shown to be crucial for the interaction with the SH3 domain of FYN and other SRC non-receptor tyrosine kinases (Lee et al., 1998; Bhaskar et al., 2010; Ittner et al., 2010). Interestingly, this motif is also critical in the interaction of TAU with the phosphatase PP2A that exists as a heterotrimeric holoenzyme complex. It has been shown recently that the PP2A regulatory subunit Bα binds to and dephosphorylates TAU, and thereby regulates microtubule stability (Sontag et al., 2012). When FYN is bound to the (seventh) proline-rich RTPPKSP motif that is conserved in both TAU and MAP2, this inhibits the interaction of PP2A/Bα with either TAU or MAP2. The corresponding synthetic RTPPKSP peptide, but not the phosphorylated RpTPPKSP version, competes with TAU and MAP2 for binding to PP2A/Bα. This finding is remarkable because the down-regulation of PP2A/Bα and the deregulation of FYN/TAU interactions have been linked to enhanced TAU phosphorylation inAD (Sontag et al.,2012).MAP2 is mainly a dendritic protein as mentioned above. Why MAP2 cannot compensate for the absence of TAU in TAU knock-out mice and target FYN to the

dendritic spine is not understood, especially as both MAP2 and TAU have been shown to efficiently bind to FYN.

# **WHAT ARE THE EFFECTS OF REDUCED TAU LEVELS?**

As reviewed in detail recently, a series of TAU knock-out mouse strains have been generated to gain insight into the physiological functions of TAU, and while initially reported to be overtly normal, behavioral changes and motor deficits were identified at least for some of these strains (Ke et al., 2012b).

Work in primary neuronal cultures laid the foundation: when TAU was down-regulated using antisense oligonucleotides, this caused a reduced neuronal outgrowth (Caceres and Kosik, 1990). However, the first TAU knock-out mice established by Harada et al. (1994) a few years later were surprisingly normal, with no evidence of either an altered axonal elongation or any macroscopic change. In a compensatory mechanism for the absence of TAU, MAP1A was up-regulated, and the axon caliber was altered to what is typically found in dendrites. Primary neuronal cultures established from these TAU knock-out mice did not display a reduced neuronal outgrowth phenotype, contrasting with earlier observations of neurons in which TAU levels had been reduced using antisense approaches (Harada et al., 1994; Takei et al., 2000). Nonetheless, the absence of TAU is not without consequences as the hypoplasticity of the commissural tract and the disorganization of the neuronal layering found in MAP1B knock-out mice (Takei et al., 1997) is exacerbated by cross-breeding these mice with TAU knock-out mice (Takei et al., 2000).

In 2001, two additional TAU knock-out lines became available. The first was established by Dawson et al. (2001), who used the same strategy as Harada and colleagues by replacing the first coding exon of TAU with a neomycin selection cassette, thereby abrogating TAU expression. The second"knock-in" strain was generated by Tucker et al. (2001),who inserted a GFP cassette in frame, resulting in a fusion protein that contained the first 31 amino acids of TAU. None of these two strains revealed any obvious impairment [at the time, the Tucker strain was only used as a "tool" to study neurotrophins; we have, however, since used this strain for behavioral studies (Ittner et al., 2010)]. Interestingly, in the Dawson strain, MAP1A levels were increased twofold at birth, while they were reduced back to normal levels as the mice became older suggesting that MAP1A may compensate for the loss of TAU during early brain development, but not in the mature brain (Dawson et al., 2001). In keeping with the very first *in vitro* studies (Caceres and Kosik, 1990), primary neurons obtained from this knock-out strain showed a slowed maturation with reduced neurite length throughout all developmental stages and a reduced axon length of stage 3 (onset of polarity) neurons (Dawson et al., 2001). A fourth TAU knockout was established in 2007, again by inserting a selection cassette into exon 1, but the cassette was flanked by FRT (flippase recognition target) recombination motifs to allow for subsequent manipulation of the targeted *MAPT* gene (Fujio et al., 2007). Not surprisingly, the mice looked quite normal, and MAP1A levels were found to be increased as previously reported in two of the three knock-out strains. Taken together, TAU is not essential in mice, although small differences were found between the four strains with regards to compensatory mechanisms and when analyzed *ex vivo*.

How do TAU knock-out mice fare in behavioral and motor studies?  At 10–12 months, the Dawson mice performed like wildtype in the radial arm and the Morris water maze (Dawson et al., 2010). Subtle motor deficits were detected at 3–3.5 months when the knock-out mice showed an increased latency to cross a beam and also made more slipped steps, but otherwise showed normal motor functions (Morris et al., 2011a). However, at 12 months of age, the Harada mice displayed signs of muscle weakness in the wire-hanging test, reduced balance in the rod-walking test, hyperactivity in a novel environment and impaired contextual fear conditioning (Ikegami et al., 2000). Interestingly, muscle weakness was also evident in heterozygous knock-out mice. Spatial learning, however, of this TAU knock-out strain was normal, when assessed in the eight-arm radial and the Morris water maze (Ikegami et al., 2000). The most pronounced phenotype was recently reported in the Dawson strain kept on a C57BL/6/SV129 background, with Parkinsonism evident at 12 months of age due to a massive loss of dopaminergic neurons in the SNpc, associated with motor impairments such as a reduced performance on the Rotarod and decreased locomotion in the open field (Lei et al., 2012). Because these findings contradict the earlier studies, Morris et al. (2013) performed a detailed behavioral and motor analysis of the Dawson strain that was kept on a C57BL/6 background. They found in their mice that the chronic lack of TAU did not impair learning and memory functions in the Morris water maze and the novel object recognition test, neither at 11–17 nor at 21–22 months of age (Morris et al., 2013). Interestingly, at 12–15 months the knockout mice weighed more (with 21–22 month-old mice showing a trend) (Morris et al., 2013),while K369I TAU over-expressing mice weigh less (Ittner et al., 2008). The knockout did not alter rearing or activity in the open field, however, both aging and TAU ablation reduced the latency to fall off the Rotarod and this was correlated with body weight. At 12–15 months, TAU knock-out mice took longer to descend from the pole than heterozygous knockout or wild-type mice, whereas for the older age group latencies did not differ. Together this suggests that complete TAU ablation causes subtle motor deficits that are related to an increased body weight. Finally, the newer study did find up to 16% reductions in dopamine levels but this was not sufficient to cause Parkinsonism nor did the administration of l-DOPA improve the performance of the mice in the pole test (Morris et al., 2013). In contrast to the work by Lei et al. (2012), no iron accumulation was found in brain areas such as the hippocampus, striatum or SN. A reason for these discrepancies may be the difference in the genetic background (C57BL/6/SV129 versus C57BL/6) as this has been reported to affect brain metal levels in both transgenic and normal mice (Maynard et al., 2006).

It is apparent that differences in genetic background and design of the targeting construct have an impact on the phenotype of transgenic animals. Another confounding factor for the study of TAU is the presence of other MAPs, which appear to share several biological roles (Dehmelt and Halpain, 2005; Sontag et al., 2012). Potential compensatory functions attributable to closely related MAPs can be excluded in the nematode *C. elegans*, where

protein with TAU-like repeats (PTL-1) is the sole homolog of TAU/MAP2/MAP4 in the worm (McDermott et al., 1996; Gordon et al., 2008), meaning that shared physiological functions of the different family members can be addressed.

PTL-1 contains a high level of sequence homology to TAU/MAP2/MAP4 within the microtubule-binding repeat (MBR) domain in the carboxy-terminus.Analysis of a *ptl-1* transcriptional reporter line demonstrates that PTL-1 has a neuronal expression pattern in adult worms, and in addition PTL-1 has been shown to regulate microtubule assembly *in vitro* (Goedert et al., 1996; McDermott et al., 1996). PTL-1 has been implicated in the regulation of microtubule-based motility in several neurons (Tien et al., 2011) and in the response to gentle touch (Gordon et al., 2008). Aging-related damage in *C. elegans* neurons is evident by neurons displaying abnormal structures such as branching or blebbing from the cell body or axon (**Figure 1**), and these changes progressively accumulate as the worms age (Pan et al., 2011; Tank et al., 2011; Toth et al., 2012). Using two *ptl-1* mutant strains, one a null knockout (Gordon et al., 2008) and the other putatively generating a protein product containing only the Nterminal region [i.e., similar to our ∆Tau truncation construct (Ittner et al., 2010)], it was observed that *ptl-1* mutant strains showed an increased incidence of abnormal structures compared with wild-type animals (Chew et al., 2013). This suggests that the neurons of *ptl-1* mutant animals develop signs of aging at a faster rate than in wild-type animals. In addition, *ptl-1* mutant animals are also short-lived compared with wild-type controls (Chew et al., 2013). These phenotypes of accelerated neuronal aging and shortened organismal lifespan were rescued by re-expressing PTL-1 in a *ptl-1* null mutant. Interestingly, increasing the number of gene copies of *ptl-1* by incorporating the *ptl-1* transgene in

a wild-type background was observed to mirror both the neuronal and lifespan phenotypes observed in the *ptl-1* mutant strains, indicating that gene dosage of PTL-1 is vital. This demonstrates a key role of PTL-1 in maintaining neuronal health with age and in regulating whole organism lifespan (Chew et al., 2013). These findings in *C. elegans* show that levels of PTL-1 need to be tightly regulated, suggesting that therapeutic strategies involving the reduction of TAU levels should not lead to a complete reduction of TAU.

# **CONCLUDING REMARKS**

What causes sporadic forms of AD is not known, but it is tempting to speculate that Aβ and TAU act through a combination of excitotoxicity, inhibition of axonal transport and aberrant localization as well as combined effects on mitochondria. It is at the synapse where Aβ induces damage and impairs memory-related electrophysiological properties (Masliah, 1995; Arendt, 2009; Wu et al., 2010). Under basal conditions, mild activation of the NMDAR results in physiological ROS production, while under neurodegenerative conditions, triggered by Aβ, over-activation of NMDARs causes excessive calcium influx and generates NO (Nakamura and Lipton, 2011). These changes in calcium can affect mitochondria (Stanika et al., 2012). Furthermore, axonal transport of cargoes including mitochondria is impaired when TAU detaches from the microtubules and localizes to the somatodendritic domain (Ishihara et al., 1999; Ittner et al., 2008). It has been proposed that in disease conditions such as AD, rare species of toxic TAU exist that need to be removed in order to restore neuronal functions. Other possibilities include a role for non-coding RNAs including mi-RNAs and truncated forms of TAU in toxicity, as discussed elsewhere (Schonrock et al., 2010; Hebert et al., 2012; Zilka et al.,

**FIGURE 1 | Neuronal aging in C. elegans is demonstrated by the accumulation of abnormal neuronal structures**. Reporter lines expressing GFP in particular neuronal subsets are used to track these phenotypes as animals age. In touch receptor neurons (top), branching from the cell body or axon, as well as beading along the axon, can be observed. In GABAergic

motor neurons (bottom), branching from commissures extending dorsally can be visualized. These phenotypes usually accumulate in late adulthood in wild-type animals, but in ptl-1 mutant strains these structures can be seen starting in early to mid-adulthood. Arrows indicate branching, asterisks indicate beading. Scale bar = 50µ. Ventral is down.

2012). As discussed here, simply reducing TAU levels may be therapeutically beneficial. Work in TAU knock-out mice and PTL-1 knock-out worms suggests that especially in an aging brain, one would not aim for a full ablation of TAU expression. This is also because the TAU/FYN interaction is required for oligodendrocyte functions including myelination. Expression of the N-terminal domain of TAU (a construct similar to ∆Tau) alone causes abnormal sorting of FYN, poor myelination and seizures (Klein et al., 2002). However, if one were to succeed in reducing TAU levels by any form of therapeutic intervention, in practical terms it is unlikely that this reduction will be complete. For a positive therapeutic outcome, it might even be sufficient to reduce TAU expression after a pathology has developed as suggested by studies in mice with inducible TAU expression, including those that express shorter variants of TAU (Van der Jeugd et al., 2012; Hochgrafe et al., 2013).

From a therapeutic point of view, besides manipulating TAU levels, its localization and the interaction with other proteins, a range of alternative strategies can be pursued. One is restoring neuronal functions by transplanting stem cells to provide neurotropic support (Yamasaki et al., 2007). As far as TAU is concerned, evidence is accumulating that its phosphorylation is critical for TAU to be toxic, and that reducing TAU phosphorylation is a promising strategy (Gong and Iqbal, 2008). What is not entirely clear is whether this requires distinct phosphorylation events or whether a generally elevated level of phosphorylation is

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Other suitable therapeutic strategies are the restoration of mitochondrial function, blocking the interaction of TAU with FYN or JIP1, or disrupting the excitotoxic complex of NMDAR and PSD-95. Other strategies are the activation of chaperones (Ward et al., 2012) and the use of aggregation blockers (Akoury et al., 2013). While it is clear that Aβ plays a key role in the pathogenesis of AD, it is becoming even clearer that there is a future of therapeutics for AD beyond amyloid (Lane et al., 2012; Yoshiyama et al., 2012). Whether in practical terms it will be necessary for the efficacy of emerging therapeutic strategies to exactly determine which TAU species is the most toxic form, remains to be determined.

# **ACKNOWLEDGMENTS**

This study was supported by the Estate of Dr Clem Jones AO, and grants from the Australian Research Council and the National Health and Medical Research Council of Australia to Jürgen Götz.

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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: 18 April 2013; paper pending published: 06 May 2013; accepted: 28 May 2013; published online: 10 June 2013.*

*Citation: Götz J, Xia D, Leinenga G, Chew YL and Nicholas HR (2013) What renders TAU toxic. Front. Neurol. 4:72. doi: 10.3389/fneur.2013.00072*

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

*Copyright © 2013 Götz, Xia, Leinenga, Chew and Nicholas. This is an openaccess 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.*

# Tangles, toxicity, and tau secretion in AD – new approaches to a vexing problem

# **Kerry L. Gendreau and Garth F. Hall \***

Department of Biological Sciences, University of Massachusetts Lowell, Lowell, MA, USA

#### **Edited by:**

Naruhiko Sahara, National Institute of Radiological Sciences, Japan

#### **Reviewed by:**

Naruhiko Sahara, National Institute of Radiological Sciences, Japan Tsuneya Ikezu, Boston University, USA

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

Garth F. Hall, Department of Biological Sciences, University of Massachusetts Lowell, 198 Riverside Street, Lowell, MA 01854, USA e-mail<EMAIL_ADDRESS>
When the microtubule (MT)-associated protein tau is not bound to axonal MTs, it becomes hyperphosphorylated and vulnerable to proteolytic cleavage and other changes typically seen in the hallmark tau deposits (neurofibrillary tangles) of tau-associated neurodegenerative diseases (tauopathies). Neurofibrillary tangle formation is preceded by tau oligomerization and accompanied by covalent crosslinking and cytotoxicity, making tangle cytopathogenesis a natural central focus of studies directed at understanding the role of tau in neurodegenerative disease. Recent studies suggest that the formation of tau oligomers may be more closely related to tau neurotoxicity than the presence of the tangles themselves. It has also become increasingly clear that tau pathobiology involves a wide variety of other cellular abnormalities including a disruption of autophagy, vesicle trafficking mechanisms, axoplasmic transport, neuronal polarity, and even the secretion of tau, which is normally a cytosolic protein, to the extracellular space. In this review, we discuss tau misprocessing, toxicity and secretion in the context of normal tau functions in developing and mature neurons. We also compare tau cytopathology to that of other aggregation-prone proteins involved in neurodegeneration (alpha synuclein, prion protein, and APP). Finally, we consider potential mechanisms of intra- and interneuronal tau lesion spreading, an area of particular recent interest.

**Keywords: tau oligomerization, tau toxicity, tau secretion, interneuronal lesion spread, exosome**

# **OVERVIEW**

Cytotoxicity associated with the accumulation of abnormal protein aggregates has emerged as a central common mechanism underlying human neurodegenerative disease. Neurons are unique among differentiated cell types in that they do not re-enter the cell cycle and thus cannot use mitosis as a method for clearing abnormally aggregated proteins. As a result, they are inherently vulnerable to disruption of protein turnover mechanisms such as the ubiquitin/proteasome pathway and autophagy, especially in aged individuals. The abnormal turnover of aggregation-prone proteins such as alpha synuclein (SNCA), prion protein (PrP), amyloid beta (Ab) peptide, and tau are thus key factors in most (95%) of the neurodegenerative diseases that affect humans. Protein aggregation is typically accompanied and potentiated by abnormal phosphorylation, ubiquitination, covalent crosslinking, and the abnormal activation of autolytic proteases (1–6). A common feature of such proteins is an "intrinsically disordered" structure (4), in which the normal conformation can be readily changed into a beta-sheet rich structure with high aggregation propensity (6). Moreover, conditions in which SNCA (Parkinson's disease, Lewy Body Dementia), tau (Corticobasal Degeneration, Pick's disease, Frontotemporal Dementia), and PrP [Creutzfeldt–Jakob disease (CJD), Gerstmann–Straussler–Scheinker disease] form abnormal aggregates typically show overlapping neuropathology, suggesting that synergistic interactions may occur between these proteins in each of these conditions (7). The aggregation of the microtubule (MT)-associated protein tau, which is heat stable and normally exhibits a random coil conformation in aqueous solution, plays a central role in the neurodegeneration seen in Alzheimer's disease and non-Alzheimer's tauopathies that form the core of the paired helical filament (PHF) (8, 9). PHFs and related filamentous aggregates such as straight and ribbon-like filaments (10) in turn make up the hallmark tau lesions [neurodegenerative diseases (NFTs), Pick bodies, etc.] seen in Alzheimer's disease and other tauopathies. However it is likely that at least some of the characteristic features that distinguish tauopathies from other neurodegenerative syndromes have roots in specific normal cellular functions of tau.

#### **MT AND NON-MT ASSOCIATED FUNCTIONS OF TAU**

Tau is expressed from a single gene on chromosome 17 and is alternatively spliced to yield six different isoforms in the adult central nervous system (CNS). Each of these contains a C-terminal microtubule binding domain (MTBR) consisting of three or four tandem repeat motifs. The best understood "normal" function of tau in mature neurons involves its binding to and stabilizing axonal

**Abbreviations:** AD, Alzheimer's disease; APP, amyloid precursor protein; CJD Creutzfeldt–Jacob disease; CNS, central nervous system; CSF, cerebrospinal fluid; ECF, extracellular fluid; ER, endoplasmic reticulum; FAD, familial AD; GA, Golgi apparatus; GO, gene ontology; HD, Huntington's disease; HSPG, heparan sulfate proteoglycan; LOAD, late onset AD; MAP, microtubule associated protein; MT, microtubule; MTBR, microtubule binding repeat domain; NDD, neurodegenerative disease; NFT, neurofibrillary tangle; NT, neuropil thread; PD, Parkinson's disease; PrP, prion protein; PrPSc, misfolded, pathogenic prion protein; SNCA, alpha synuclein; TGN, trans-Golgi network.

MTs via the MTBR. Tau belongs to a family of microtubule associated proteins (MAPs) that includes other neuronal proteins such as MAP1A, MAP1B, and MAP2, and also non-neuronally expressed members (MAP4). Each of these proteins contains a conserved region in and around the MTBR that suggests a common origin via gene duplication. The poor conservation of areas outside of the MTBR in both mammalian tau family molecules and in taulike proteins in various vertebrate and invertebrate species suggests that they may play species-specific functions.

The regulation of tau-MT binding has been heavily studied and is now well established. Tau-MT binding associated with MT stabilization is mediated by the phosphorylation of serine and threonine residues at sites immediately adjacent to and within the MTBR by a wide variety of kinases [Ref. (11) for a good review]. Phosphorylation of the regions flanking the MTBR produce a stoichiometrically graded reduction in the affinity of tau for MTs, whereas phosphorylation at specific sites within the MTBR (Ser262 and Ser356) abolish virtually all tau-MT interactions (12). Subtleties of tau-MT binding appear to be particularly dependent on the phosphorylation pattern of proline-associated serine and threonine resides on the N-terminal side of the MTBR by proline-directed kinases (e.g., CDK5, MAP kinase 1, GSK3b).

It has become clear that tau has functions in addition to axonal MT stabilization in both mature and developing neurons that involve alternative binding partners for the MTBR (e.g., actin- and actin-associated proteins, heparin sulfate proteoglycan) and/or other parts of the tau molecule, such as the amino terminal projection domain (13). These functions include the integration of cellular cytoskeletal functions with interneuronal signaling pathways. Important developmental functions of tau include various aspects of axonogenesis, such as the establishment of axonal identity (i.e., neuronal polarization) (14) and the subsequent outgrowth (15) and myelination of developing axons. Each of these developmental functions involves MTBR interactions with the subcortical actin network and plasma membrane (16, 17), via either the MTBR itself [actin – Ref. (18)] or via the N-terminal projection domain, which interacts with Src family non-receptor tyrosine kinases such as fyn (13, 19, 20).

#### **DISEASE-ASSOCIATED TAU MODIFICATIONS ARE CORRELATED WITH THEIR DISSOCIATION FROM MTs**

#### **Tau hyperphosphorylation**

A key alteration that is associated with NFT formation is the phosphorylation of multiple serine and threonine residues in and around the MTBR that normally regulate tau binding to MTs (hyperphosphorylation). Hyperphosphorylation reversibly decreases the affinity of tau for MTs (21, 22) and is consistently seen in PHFs isolated from AD and non-AD tauopathy brains even at early stages in the development of disease (23–27). Hyperphosphorylated tau defined as containing 10 or more moles of phosphate per tau molecule (28) isolated from AD brains has been shown to be capable of self-assembly *in vitro* (29), suggesting that tau hyperphosphorylation may directly induce aggregate formation as well as altering the normal functions of tau. Tau mutations associated with frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) increase the rate and amount of tau phosphorylation and decrease the number of phosphate groups required for aggregation (11, 28). Although clearly associated with PHF formation, hyperphosphorylation may not be a prerequisite for tau aggregation, since tau constructs containing only the MTBRs are able to form filaments in the unphosphorylated state (29, 30). However, dephosphorylation prevents the self-assembly of full-length isoforms, suggesting that the N- and extreme C-terminal regions of tau inhibit this (31, 32). Curiously, phosphorylation of tau at certain residues (Ser262 and Ser 214) that decrease tau-MT affinity act to prevent formation of PHFs (33). This, together with multiple reports of phosphorylation state-contingent kinase specificity for individual sites in the flanking domains, suggests that regulation of both tau:tubulin and tau:tau affinity by phosphorylation is highly subtle and remains incompletely understood.

#### **Tau truncation**

The evolution of tangle-intrinsic tau from full-length isoforms to MTBR-only fragments (34) suggests that proteolysis at both the C and N termini of tau may play a significant role in NFT formation. This is directly supported by a number of studies of tau filament formation. Temporal analysis of filament formation in AD brains shows that tau misfolding [recognized by the Alz50 antibody (35)] precedes initial truncation at D421, with subsequent truncation at E391 appearing at later stages of the disease (34). Truncated C-terminal tau fragments can act as nucleation seeds *in vitro*, sequestering full-length tau in both mutant and wild-type forms (11). Such nucleation may be related to conformation specific tau:tau interactions that have recently been proposed to mediate the intercellular propagation of neurofibrillary lesions (36, 37). Truncation of tau at E391 has been identified within the core of PHFs (38) and at D421 in the brains of AD patients (39). The presence of the C terminal has an inhibitory effect on polymerization of full-length tau (31) and C-terminal truncation accelerates tau filament formation *in vitro* (40–42). A study using cells expressing the MTBR of tau containing an FTDP-17 mutation demonstrated that proteolysis of the N and C regions flanking the MTBR produced aggregation-prone fragments capable of seeding further aggregation, while blocking N-terminal truncation of this fragment prevented C-terminal proteolysis, suggesting that this region (near K257) may act to shield downstream residues (11). It should be noted that although C-terminal truncation is sufficient to cause tau aggregation in cellular models, additional mechanisms might drive filament formation in diseased neurons. Tangle-bearing neurons in a transgenic mouse model contained little D421-cleaved tau (43), suggesting that multiple pathways to aggregate formation are likely active in neurons affected by tauopathy. This is consistent with the presence of multiple types of lesions in the various forms of non-AD tauopathy and in AD brain, where neuropil threads (NTs) occur with or perhaps even before the onset of NFT formation (44) and with the characteristic differences in neurofilament content and tau conformation seen in NTs (45) and Pick bodies (10, 46) versus NFTs.

#### **Other variables affecting tau aggregation**

In addition to phosphorylation and truncation, there are a number of factors that appear to modulate tau aggregate formation, some of which are associated with tauopathy cytopathogenesis. Foremost among these are the intronic and exonic point and deletion mutations in and around the MTBR that cause familial non-AD tauopathies [reviewed in Ref. (47)]. The restriction of such mutations to the MTBR and their ability to drive the neurofibrillary pathogenesis of presenile tauopathies constitutes some of the strongest evidence for the importance of tau aggregation in human disease. Polyanions such as heparan sulfate proteoglycan (HSPG) are abnormally distributed in pre-tangle and tangle-bearing neurons and can catalyze the formation of straight filaments and PHFs *in vitro* (48, 49) regardless of the phosphorylation state of tau (30, 50, 51). Specific isoforms of tau also differ in their ability to form filaments *in vitro*. Different ratios of three and four repeat (3R and 4R) tau isoforms are characteristic of tau aggregates found in specific tauopathies (26, 52) and the presence of intronic mutations interfering with the splicing of exon 10 in patients with FTDP-17 (30, 53) confirms that the resulting change in the ratio of 3R:4R tau is sufficient to drive tau filamentation and neurodegeneration (54). Three and four repeat isoforms differ in characteristics that may affect their participation in NFT formation, including the ability to form disulfide bridges (55) and their relative affinity for MTs and fyn kinase (56). The presence of 3R tau may reduce the tendency of 4R tau to form filaments (57), possibly by interfering with disulfide bond formation. Finally, the larger cellular and genetic context is likely to have a significant bearing on the tendency of tau to aggregate into NFTs. Early cellular changes involving lysosomal and autophagy pathway abnormalities may either result from or modulate tau aggregate formation (58, 59). Genetic factors such as the H1 haplotype, which affects the overall expression levels of tau and the splicing of exon 10 and possibly exons 2 and 3 (60), and the presence of ApoE4 allele have been reported to be associated with high disease incidence (61). Additional genetic factors that affect tau aggregation and thus disease propensity include interactions with elements on chromosome 21 that affect tau splicing and the function of non-APP proteins such as DYRK1A (53, 62) as well as APP itself (63). Overall, tau interactions with non-MT elements via its MTBR and in particular, tau:tau interactions associated with aggregation and their involvement in tau-mediated neurodegeneration have been a subject of intensive investigation. However, while a great deal is now known about both the mechanisms of tau aggregation and the circumstances of tau modifications associated with aggregate formation, it remains unclear exactly how each of these elements contributes to tau cytopathogenesis. This uncertainty has been exacerbated by the identification of tau toxicity mechanisms that are not associated with the MTBR but which may also play important roles in neurodegeneration.

#### **REACTIVATION OF DEVELOPMENTAL TAU FUNCTIONS IN NEURODEGENERATION**

While tau aggregation is clearly a central factor in tauopathy pathogenesis, the role tau plays in the development of axonal identity and other aspects of axonogenesis has increasingly been linked to neurodegenerative disease mechanisms. These include the appearance of cell cycle markers (64) and ectopically sprouting axonlike processes (NTs) emerging from the dendrites (45, 65–67) during the development of neurofibrillary lesions in neurodegenerative tauopathies. Dendritic NTs in particular suggest that tau functions in the development of axonal identity may play a role in the early stages of AD where they may reflect damage to mechanisms that maintain the terminally differentiated neuronal state (68, 69). The "fetal" (3R0N) tau isoform is the shortest three repeat tau isoform with the lowest binding affinity for MTs (30), possibly reflecting the far greater importance of tau N-terminal interactions and functions during development relative to the role tau plays in the mature CNS. A key tau interactor during development is the non-receptor tyrosine kinase fyn, which plays a critical role in axonal outgrowth and myelination (15, 70). Interactions between the tau N terminus and signaling molecules such as fyn typically occur in the actin-rich cortical cytoskeleton largely in the absence of MTs and are necessary for functions such as growth cone motility (16). Fyn, like tau, localizes to NFTs (20, 71) and is essential to the development and possibly the propagation of Abeta-mediated toxicity in mouse models of AD (72). Fyn-mediated interactions with tau play a role in the localization of a small amount of tau to the plasma membrane (73), particularly at dendritic loci (74) where it is involved in synaptic functions associated with learning and memory (75–77). Abnormal interactions of tau with fyn kinase increasingly appear to play a critical role in membrane-associate tau dysfunction especially via the generation of synaptotoxic species of Abeta from APP. This occurs in the context of fyn-mediated phosphorylation of APP in early endosomes (78) and in turn exacerbates both tau localization to rafts and tau phosphorylation by fyn (79). Such correlations suggest that tau mislocalization to dendrites and the generation of abnormal amounts of Abeta may interact synergistically to produce both cytotoxicity and abnormal developmental events such as NT growth and cell cycle re-entry (80) in AD neuropathogenesis.

#### **INTERNEURONAL ASPECTS OF TAUOPATHY**

Tau interactions with lipid raft proteins such as fyn and (possibly) APP may be centrally important in two recently emerging interneuronal aspects of tau pathobiology: (a) tau secretion and interneuronal transfer via unconventional mechanisms and (b) tau:tau interactions involved in templated misfolding and "prionlike" lesion spreading mechanisms. Membrane localization favors tau oligomerization, which increasingly appears to be a key event in at least some forms of tau toxicity. Membrane-associated tau functions mediated by its N terminus appear to be linked to the diversion of tau from the cytosol to membrane-bound vesicles, in particular those associated with the trans-Golgi network and the autophagosome-lysosome pathway (81). As a consequence, it is likely that both cellular and molecular aspects of interneuronal lesion spreading have their roots in membrane-associated tau misprocessing mechanisms.

#### **Selective neuronal vulnerability to tau toxicity**

Ever since it became apparent in the mid-1980s that Alzheimer's disease is a common neurodegenerative condition of the elderly as opposed to a relatively rare familial syndrome, it has been clear that the number and distribution of NFTs is strongly correlated with progressive cognitive loss (82, 83). There are two possible mechanisms that might account for disease-associated stereotyped patterns of lesion development with increasing disease severity. One possibility is that loci that are affected early in the disease sequence are more vulnerable to the mechanisms underlying tau-induced degeneration at the cellular level. A hierarchy of vulnerability to tau toxicity might then result in a stereotyped sequence of lesion development over time. One characteristic shared by brain regions that develop early tau lesions in AD is synaptic plasticity; highly plastic cortical pyramidal neurons such as those of the hippocampus are inherently vulnerable to excitotoxic insults by virtue of their glutamatergic pharmacology and the prevalence of LTP-associated plasticity mechanisms (84). Selective vulnerability may also be acquired via injury, as with the greatly increased risk posed by antecedent head trauma (85–87). It seems increasingly likely that both intrinsic and extrinsic selective vulnerability factors interact with lesion spreading mechanisms associated with synaptic connectivity patterns (discussed at length below) to produce the variety of clinical syndromes associated with neurofibrillary degeneration.

### **Interneuronal movement of misprocessed tau**

For many years, the interconnectedness of affected regions and the highly stereotyped, disease-specific pattern of neurofibrillary lesion spread within the brain in AD and non-AD tauopathies has suggested that the actual interneuronal transfer of a toxic factor must be involved in the progression of neurodegenerative tauopathies (88–91). The actual movement of toxic tau species between neurons is now thought by many to be the primary mechanism mediating the progressive appearance of tau pathology and clinical dysfunction in tauopathy, including those associated with repeated head injury (87). In particular, the templated misfolding mechanism that mediates the infectivity of prion diseases such as CJD is increasingly invoked as a model for the interneuronal transfer of tau-mediated toxicity (36, 92–97), especially in murine transgenic models of tauopathy (98–100). Evidence that different abnormal conformations of PrP may in fact define different prion-mediated diseases (101, 102) raises the possibility that very different clinical presentations and neuronal vulnerabilities could be attributable to molecular level differences in a single protein. However, the study of tau as a "tauon" [a term for a tau species that spreads toxicity via templated misfolding coined by Novak et al. (93)] has been conducted largely in the absence of a cellular context for the actual transfer of tau between neurons, making it difficult to connect the molecular mechanisms involved in protein templating to specific cellular events and mechanisms associated with tau cytopathology. In particular, while the secretion (103–105) and uptake (92) of tau itself in cellular models has been demonstrated and linked to the elevated tau levels in early AD (106), it is unclear how secretion is related to the spreading of neurofibrillary lesions and whether that occurs via an oligomer-associated mechanism, such as templated misfolding or ionophore formation (107, 108), or via some mechanism that does not require the tau MTBR at all (109–112). The subtle but important questions that remain about the relationships between the development of NFTs, toxic oligomer formation, templated misfolding, N-terminal tau toxicity, and the actual cellular mechanisms responsible for secretion and uptake of misprocessed tau species will be explored at greater length below.

# **CURRENT FOCI OF TAUOPATHY RESEARCH**

#### **THE BIOGENESIS OF NEUROFIBRILLARY TANGLES**

The development of NFTs, the characteristic tau lesions that develop in cortical pyramidal neurons during the course of AD, has been relatively well characterized but is still incompletely understood. NFT development begins with the abnormal phosphorylation of tau at multiple sites in and around the MTBR, accompanied by abnormal somatodendritic tau accumulation resulting in the appearance of pre-fibrillary phosphorylated punctate deposits in the cell body and dendrites of affected neurons (24, 113). These eventually form condensed fibrillar deposits near the nucleus and near dendritic branch points, displacing normal cytoskeletal elements such as MTs (113, 114). Over time, these fill most of the cell and take on a characteristic flame-shaped appearance. Eventually, the neuron containing the NFT dies and the NFT remains as a "tombstone" lesion or "ghost" tangle.

While all tau isomers and cleavage fragments that contain the MTBR appear to be capable of forming filaments, their precise morphology may vary considerably; they can appear as straight, ribbon-like, or any of a variety of PHF-like structures, all of which are reproducible under *in vitro* conditions (10, 46, 115). Moreover, the wide variety of tau modifications that affect aggregation suggest that considerable subtlety exists in the mechanisms responsible for tau assembly. Such factors include the presence of tauopathy-inducing point mutations (116, 117) the presence/absence of the C-terminal (31) or N-terminal (10, 32) domains, and variations in the experimental conditions used (118). The fact that the range of filaments and aggregate types seen with human tauopathies can be reproduced in sporadic tauopathy syndromes without benefit of tau mutations indicate that additional relevant variables to tau aggregate form come from cellular factors, such as the inclusion of NFs (Pick bodies) or internal membrane elements (granulovacuolar degeneration) or even the shape of the host cell. Understanding how molecular and cellular factors interact to produce a specific type of aggregate can thus be seen as an index of our overall grasp of tauopathy pathogenesis.

#### **Direct formation of NFTs from cytosolic tau**

A key unresolved issue in NFT formation is the cellular context in which NFTs form during the course of neurodegenerative disease. In particular, it is unclear whether NFTs form as a result of cytosolic or even MT-templated interactions between tau species or whether NFT formation requires additional cellular elements, such as membrane-bound vesicles, to occur. The most straightforward hypothesis of NFT generation and growth is direct assembly from free tau monomers and/or oligomers in the cytosol. The ability of tau filaments to form *in vitro* by MT-templated mechanisms (119), and the ability of tauopathy mutations to actively disrupt MT networks (116) as well as favor filament formation (117, 120) are all consistent with the idea that tau aggregate formation, eventually leading to NFTs, begins immediately upon dissociation from MTs or even by conformational changes that occur in MT-bound tau. For instance, tau oligomerization has been observed to occur on the surface of MTs (119), which might then generate cytosolic tau oligomers capable of directly seeding NFT growth. This seems to account directly for the granular pre-fibrillary deposits that constitute the earliest visible stage of NFT formation (121, 122) and

is consistent with *in vitro* experiments demonstrating that tau filament assembly resulting in NFT formation is potentiated by tau interactions with fatty acids and/or polyanionic molecules such as heparin sulfate proteoglycans and RNA (30, 50, 123, 124). Moreover, electron and atomic force microscopy studies have revealed numerous intermediates in tau fibril formation, including abnormally folded monomers capable of aggregation (125) and granular tau aggregates that may be formed from these monomers (121, 122). All of these observations are consistent with the direct formation of NFTs from cytosolic tau oligomers and this mechanism is therefore widely assumed to account for the biogenesis of most, if not all, NFTs.

#### **Does vesicle-associated tau contribute to NFT formation?**

Studies of tau biology raise the possibility of an alternative (or perhaps additional) mechanism to direct oligomerization in the cytosol. The catalysis of tau oligomerization by fatty acids is as consistent with a membrane-mediated assembly mechanism as it would be with a cytosol-only mechanism. More direct support for membrane-mediated NFT formation includes electron microscopic observations of membrane-associated PHFs in AD brain (126) and the experimental generation of tau filaments *in vitro* on the surfaces of anionic micelles (127). This occurs via tau intermediates containing β structures whose conformations are dependent on the presence of the anion and which are capable of seeding tau fibril formation. Tau that is not associated with MTs interacts with actin (18) and/or actin-associated proteins (128, 129) causing misprocessed tau to accumulate under the plasma membrane and in membrane-bound vesicles in cellular tauopathy models (19, 81, 105, 130, 131). This could plausibly result in tau endocytosis leading to vesicle-associated tau being returned to the perinuclear region via the retromer pathway, followed by NFT formation via membrane-templated oligomerization (125, 127). The Golgi apparatus and the autophagy-lysosome pathway are additional possible sources of tau-bearing vesicles that could contribute to NFTs. Abnormalities in Golgi structure have been identified both in association with tau overexpression (132) and with NFTs in AD brain (133). A recent study of PHF tau uptake in neuronal cultures suggested that endocytosed tau aggregates eventually form perinuclear aggresome-like deposits, which also suggests involvement of trans-Golgi "retromer" pathways in NFT formation (134). Similarly, the disruption of proteasome/macroautophagy (autophagy) mediated tau turnover is a prominent early element in pre-fibrillar changes in AD cytopathology (59), as evidenced by the polyubiquitination of tau in NFTs (135). Experimental disruption of lysosomal function via chloroquine administration induced lysosomal accumulation of tau aggregates (136), suggesting that diseaseassociated changes might contribute to NFT formation in AD via a similar mechanism. Similar patterns of somatodendritic tau distribution and associated signs of toxicity are seen in cortical pyramidal neurons with pre-fibrillar tau deposits or nascent NFTs (24, 113, 114, 137). These patterns suggest a role for vesicle-associated tau in both local tau cytotoxicity and in the generation of NFTs (81, 138). Examples are shown in **Figure 2**. In both cases, tau accumulates first in distal, membrane-rich dendritic structures (113, 139) and then at branch points and along dendritic shafts, where it is correlated with local MT loss, causing varicosities to appear

in dendrites (113, 131). In the lamprey model, which is the only *in situ* tauopathy model from which high resolution localization information is available, these are associated with the accumulation of tau-bearing vesicles (and membrane-bound organelles such as mitochondria) at either end of the varicosity, suggesting that they are due to the failure of bidirectional MT mediated transport (**Figure 1B**). The accumulation of tau-containing vesicles at dendritic branch points is likely due to the shift in MT polarity patterns (140) typically found there, which induces the accumulation of vesicular organelles such as mitochondria. This vesicular build-up may be either the cause or consequence of MT-mediated transport failure (141–143).

#### **TAU TOXICITY MECHANISMS – ARE NFTs TOXIC?**

The widely observed correlation between NFT distribution and neurodegeneration in nearly all tauopathies including AD (82, 83, 113, 114) has led to the widespread assumption that NFTs are an integral feature of tau neurotoxicity. Although toxic tau aggregates are notoriously difficult to generate in cell culture from wild-type tau isoforms (144), studies with hyperaggregating tau mutants have demonstrated that cleavage products are toxic when expressed in culture, with aggregate formation and apoptotic cell death occurring within 24–48 h of tau expression (145, 146). That said, it is becoming increasingly evident that NFTs may not be the agent driving neurotoxicity in whole animal tauopathy models. In *Drosophila*, expression of both mutant and wild-type human tau leads to AD-like pathology (late onset neurodegeneration, selective toxicity of cholinergic neurons) in the absence of NFTs (147). In addition, the long time courses (up to 20 years) proposed for NFT formation based on imaging data from AD brain (148–150) are not consistent with direct causality between NFTs and tau toxicity. While NFT growth is largely irreversible in inducible mouse tauopathy models, more dynamic aspects of tau toxicity are clearly reversible (151–154),suggesting that the mature NFT itself is much less toxic than the events associated with building it. Dendritic and axonal changes associated with tau accumulation and NFT formation in both transgenic mice (155) and AD brains (114, 156) appear to be correlated with abnormal mitochondrial distribution, which in turn recruits low ATP and Ca++ mediated toxicity mechanisms (144). Ca++ mediated tau toxicity is also suggested by the effects of specific tauopathy mutations on Ca++ channel properties (55) and high resolution correlations between localized secretion, MT loss, and accumulations of vesicular tau (81, 131) as illustrated in **Figure 1**. These findings and others have complicated our understanding of the relationship between NFT distribution and abundance and the actual toxicity mechanisms driving human neurodegenerative disease. Recent studies have also tended to dissociate degenerative cellular changes from NFT formation. It has even been suggested that large tau aggregates such as NFTs may serve a neuroprotective role (157, 158), preventing hyperphosphorylated tau from sequestering normal, MT-bound tau. Studies in other tauopathy models have also called into question the role played by hyperphosphorylation in the chain of events leading to degeneration; recent fly model studies (159) have suggested that hyperphosphorylation can be neuroprotective by blocking other aspects of tau toxicity, such as apoptotic changes associated with cell cycle re-entry (80, 160).

**FIGURE 1 | Accumulation of vesicular tau in ABC dendrites and at dendritic branch points causes local transport failure, MT loss, and localized secretion from dendrites**. High resolution confocal imaging of somatodendritic tau accumulation in the lamprey tauopathy model suggests a cellular mechanism to account for the relationship between localized tau toxicity, somatodendritic MT loss, and the pattern of NFT evolution in pyramidal neurons as described by Braak et al. (113), Braak and Braak (137), and Blazquez-Llorca et al. (139). **(A)** Cell body (left) and dendritic branch point (center) of a lamprey ABC expressing full-length 4R0N human tau bearing the P301L tauopathy mutation before the onset of tau-induced degeneration. Tau is triple labeled: MT associated (Tau5, green channel), MT dissociated (9G3, blue channel), and total tau (GFP epitope tag red channel). Tau phosphorylated at Y18 (pY18 tau or 9G3 positive MT dissociated tau) is accumulating at the base of large dendrites and at branch points, a pattern typical of MT-transported vesicles (asterisks). The rightmost panel shows pY18 tau accumulating at either end of dendritic varicosities (arrows). **(B)** Left panels show pY18 distribution in non-degenerating (Stage 1) and degenerating (Stage 2) ABC dendrites. Distally transported tau is distributed throughout distal (but not proximal) dendrites in non-degenerating cells, but becomes localized to dendrite branch points and varicosities with the onset of degeneration [arrows – see Refs (81, 165)]. Center and right: Dendritic beading is caused by the localized failure of MT mediated transport, resulting

in the accumulation of pY18 (fyn phosphorylated) tau associated with vesicles and membrane-bound organelles. The accumulation of mitochondria (COX2 label) is particularly well marked. With the onset of dendritic degeneration, total and pY18+ tau accumulates at each end and eventually in the center of dendritic varicosities in what appear to be MT-transported vesicles. The localized secretion occurring in the vicinity of such deposits suggests that tau-bearing vesicles first destabilize the MTs responsible for their transport, accumulate in the resulting varicosities and are then secreted. While the mechanism responsible for this has not been demonstrated directly, the concomitant loss of MTs and localized secretion suggests a Ca++ flux mediated mechanism. **(C)** A model for vesicle-associated tau in NFT formation and cytodegeneration. Failure of tau to become axonally localized and bind axonal MTs results in actin association and endocytosis (1). Tau-bearing vesicles are transported both distally and proximally on dendritic MTs, accumulating at dendritic branch points (where MT polarity patterns favor localized cargo accumulation Aronov 01) and near synaptic terminals (2), where it may become locally toxic possibly via interacting with Abeta in synapse-associated endosomes, resulting in structural failure of dendrites (top right) and uptake by afferents resulting in retrograde trans-synaptic movement. Synaptic activity may also result in the centripetal transport of tau-bearing vesicles to the Golgi apparatus (3) where it may modulate NFT formation. Scale bars: **(A)**: 20µ, **(B)** 100µ (left), 5µ (right).

#### **Oligomer-associated toxicity mechanisms**

Tau oligomers have been the most widely proposed candidate for the toxic intermediate species in NFT biogenesis responsible for the correlation between neurofibrillary lesions and neurodegeneration in AD and non AD tauopathies (83). The toxicity of oligomeric tau is suggested by numerous correlative studies (161) and in particular with respect to the dynamic effects of tau aggregation; for instance, the concentration of tau multimers (162) but not large aggregates or monomers (95) in the brains of tauopathy mice are correlated with memory and cognitive deficits.

*Oligomer-mediated membrane permeability changes.* The structural similarity between amyloid proteins associated with neurodegeneration supports the existence of a common toxicity mechanism based on common properties of such proteins such as their propensity for oligomerization and close association with membrane. The toxicity of amyloid oligomers unrelated to neurodegenerative diseases suggests that a specific, shared conformation may be responsible, with toxicity being mediated by mitochondrial dysfunction associated with an increase in reactive oxygen species (141, 163). Oligomers of several different amyloids cause an increase in ion conductance across lipid bilayers (164) raising the possibility that they might alter or the permeability of the plasma membrane, resulting in increased internal [Ca++] and associated toxic changes. Both Ab and SNCA can form oligomeric structures that increase ion permeability of synthetic vesicular membranes and which may allow them to create pores within cell membranes (107). Leakage of cellular contents across the plasma membrane in SH-SY5Y cells was observed after the external application of tau oligomers (108), suggesting a similar toxicity mechanism for tau. These results collectively suggest that tau oligomers may mediate any of several toxicity mechanisms associated with Ca++ dysregulation or abnormal generation of reactive oxygen species. Such changes are typically seen with excitotoxicity and mitochondrial dysfunction, both of which appear to be important elements of AD-associated neurotoxicity (141). Studies

# **FIGURE 2 | The context of exosomal tau secretion and transneuronal**

**neurodegeneration**. **(A)** Connectivity diagrams summarizing proteomic analysis of the exosomal proteome associated with tau overexpression from neuroblastoma cell cultures (106) using the String online dataset (104). GO term analysis shows that exosomal tau secretion involves factors with known links to tau misprocessing (APP, oligomerization, Wnt pathway) and also suggests the involvement of mechanisms with less established and no apparent links to tau, AD, and exosomes. Of the ∼660 proteins identified, 50 were both present on the AlzGene list (http://www.alzgene.org/) of 616 AD related proteins and had clear links (0.4 confidence or more (104)) to tau and/or exosomal markers (CD9 and CD81 were not present in our set, but were part of the probe query) – see ExoCarta (239) via the String 9.05 connectivity algorithm. Note the strong linkage to tau (MAPT – shown as a black circle for reference). **(B)** Another group (right diagram) consisted of internally connected proteins that did not have clear functional links to tau on String (confidence <0.4 or not detected). These nonetheless had significant signals for AD, PD, and HD that were tightly linked to mitochondrial markers, suggesting the association of abnormal autophagy with tau secretion, and with synaptic plasticity (LTP, LDP). All terms shown reflect significant enrichment (p < 0.001) by the String algorithm. **(C)** Connectivity diagram from GeneMANIA (http://www.genemania.org) showing physical interactions between proteins isolated from exosomes using the same set as shown in **(A)**. In order to identify proteins typically localized to exosomes, we scored each protein for the number of times it appeared in the nearly 70 different

mammalian exosomal datasets at ExoCarta. Links between tau and highly exosome associate proteins (dark green icons), mildly exosome associated (light green icons), and aggregation-prone proteins such as SNCA and APP (Abeta) are shown as well to illustrate the plausibility of tau diversion to exosomes in the context of these co-purified proteins. **(D)** Expression of 4R0N tau in lamprey ABCs produces dendritic localization of tau (red channel), dendritic degeneration, and localized focal secretion (left box, orange label) as shown in a confocal micrograph after 10 days of expression (81). Immunolabel for LC3/MAP1b (green channel) shows cytoskeletal localization in non-transfected giant axons (seen in cross section – ax), and in circumferential axons (arrow axon). One of the latter has taken up secreted tau shown at higher magnification (right) and is exhibiting toxicity in the form of varicosities (arrows) similar to those shown in ABC dendrites (dendrite, center). Highly co-localized tau (blue channel) shows cleaved, autophagosome-associated LC3II associated with tau (arrows) in a pattern similar to that seen for dendritic mitochondria in **Figure 1B**. Scale bar: 50µ (left), 10µ (right). **(E)** Consensus sets of 1575 downregulated and 1383 upregulated proteins in both LOAD (238) and early onset FAD (237). **(F)** Schematic outlining a hypothesis of AD cytopathogenesis that accounts for tau associated exosomal secretion and downregulation of synaptic and axonal proteins in AD (red) in the context of current knowledge. Likely triggers for tau secretion and trans-synaptic lesion spreading (blue) may occur either directly from the axon due to loss of axonal MT integrity and secretion regulation, or indirectly from dendrites as the results of toxicity caused by somatodendritic tau accumulation.

in cellular tauopathy models showing a correlation between localized cytotoxicity, tau membrane association, and localized MT loss (81, 130, 165) are consistent with this (see examples in **Figure 2**), as is the presence at the plasma membrane of polyanionic molecules known to catalyze tau oligomer/filament formation such as HSPGs (30, 166) and RNA (123).

*Oligomers as prions.* The "prionlike" toxicity and propagation mechanisms recently proposed for tau may be considered as a special category of oligomer-mediated toxicity, if one makes the assumption that a prionlike misfolded tau conformation would be toxic and would propagate in a manner similar to that of the PrP itself. However, despite the considerable amount of research effort devoted to understanding the relationship between propagation and toxicity mechanisms of the PrP, this relationship remains quite unclear. Mutant forms of PrP that do not create large amounts of misfolded, pathogenic prion protein (PrPSc) with high beta-sheet content in plaques have still been shown to generate CJD-like syndromes in mice (167). Moreover, interference with the normal GPI linkage that anchors PrP to the membrane can affect the ability of the mutant form to propagate interneuronally and generate a clinical syndrome but without affecting local cytotoxicity (168). Tau misprocessing appears to have a number of parallels to PrP toxicity in this regard; like PrP, tau oligomerization, and post-translational modifications that favor tau oligomer formation (e.g., hyperphosphorylation, truncation) are closely associated with toxicity. In the case of tau, it remains unclear whether higher-level oligomers and/or polymers propagate interneuronally in human tauopathies. Recent studies using murine tauopathy models have suggested that they are capable of this (98, 99, 169).

*Disruption of protein turnover pathways.* The dysfunction of proteasomal, autophagosomal, and lysosomal pathways with protein aggregate formation in tauopathy is a candidate for mediating tau toxicity as well as NFT formation, and may play an important role in the development of other tau containing cellular lesions, such as granulovacuolar degeneration [Ref. (170), reviewed in Ref. (171)] in which tau accumulations appear to be membrane associated. The polyubiquitinated state of NFT-tau suggests involvement of the ubiquitin-proteasome pathway (135). Additionally, the colocalization of tau aggregates with high concentrations of acid hydrolases in granulovascular degeneration (GVD) bodies in the hippocampal neurons of AD patients suggests that they may be the result of incomplete autophagy (172, 173). Lysosomal activation is also observed in cultured cells transfected with mutant tau and in mice expressing mutant tau transgenes (174). Inhibition of lysosomal proteases by tau misprocessing causes the accumulation of amphisome-resembling vacuoles in cultured neurons that are morphologically similar to those seen in AD brains. Inhibition of vesicular transport by tau, especially N-terminal tau fragments (175) may also prevent the fusion of autophagosomes and lysosomes (176), causing the retention of tau-bearing autosomes in axons and dendrites, where their accumulation may result in localized degeneration and even tau secretion [**Figure 2**, also see Ref. (81)]. One way that dysfunction of the autophagy-lysosomal machinery caused by tau aggregates may induce toxicity is enhancing tau oligomerization by the generation of hyperaggregating

tau cleavage fragments due to the incomplete activity of lysosomal proteases. Cathepsin L has been shown to generate aggregation-prone fragments mutant but not wild-type tau via association with Lamp2A and Hsc70 on the cytosolic face of the lysosomal membrane (177). Tau-induced disruption of autophagy may also recruit synergistic effects related to the production of Abeta from APP, since PS1 is also necessary for autophagosomelysosome fusion and lysosomal proteolysis (178). It should also be noted that the vesicle trafficking pathways associated with aggresome formation may also become abnormally involved in tauopathy as demonstrated by a recent study in which tau aggregates were endocytosed and then localized to perinuclear deposits (134).

#### **Non-oligomer-mediated tau toxicity mechanisms**

*Receptor-mediated toxicity.* An alternative to oligomer/aggregate associated mechanisms of tau toxicity to explain the selective vulnerability of neurons and spread of NFTs through synaptically connected regions in Alzheimer's disease is toxicity in response to tau binding to extracellular receptors, especially those for synaptic transmitters such as glutamate and acetylcholine. Tau is toxic to neurons in culture when applied extracellularly (179), apparently via the generation of Ca++ fluxes via the activation of M1 and M3 muscarinic acetylcholine receptors (180), which bind tau with a greater affinity than acetylcholine. This is consistent with the preferential distribution of muscarinic acetylcholine receptors on entorhinal and hippocampal pyramidal neurons, and accounts for their vulnerability in early stages of Alzheimer's disease (181). Interestingly, the dephosphorylation of secreted tau by tissue-non-specific alkaline phosphatase could potentiate its high-affinity binding to muscarinic receptors of nearby neurons (182), thus accounting for the "clustering" lesion spread patterns characteristically seen in tauopathies (90, 91, 183).

The generation of Ca++ fluxes via receptor-mediated toxicity need not directly involve aggregate or oligomer formation as a variety of MTBR− tau species appear to form multiple toxic fragments via Ca++ mediated activation of calpains and caspases in neuroblastoma cultures (109, 184) in at least some cases by NMDA receptor activation (109). A well studied fragment generated by calpain activity is a 17-kDa N-terminal fragment consisting of tau residues 45–230; this is toxic when applied to aged primary hippocampus cells and when expressed in the brains of transgenic *Drosophila* and in ABCs in the lamprey tauopathy model (142, 185, 186). The 17-kDa fragment is elevated in cortical neurons of AD and tauopathy patients that exhibit increased calpain activity (112) and is unusual among toxic tau species in its ability to produce neuritic pathology in neuroblastoma lines (185). However, it remains unclear if the 17-kDa fragment produced by calpain cleavage is a cause of neuronal death in tauopathy (187). Another N-terminal tau fragment with a molar mass of 20–22 kDa was found enriched specifically in the synaptosomes of AD brains in comparison to control brains (184). When overexpressed in primary rat neurons, an N-terminal tau fragment containing residues 26–44 impaired mitochondrial function and caused neuronal death (188). Downregulation of calpains in a tauopathy model of *Drosophila* was associated with decreased neurodegeneration (186). Both the tau N-terminal domain and fyn-tau interactions are associated with the mechanism of Ab toxicity that is mediated by the presence of

tau (63, 74, 111), as are many fragments generated by similarly activated caspases (189, 190).

*Can "prionic" and receptor-mediated N-terminal tau toxicity coexist?* The ability of tau to mediate Ab toxicity in the absence of the MTBR (111, 190) and the potential for NFTs to act as neuroprotective agents (157, 158, 191) raise questions about how large a role tau aggregate toxicity plays as a direct agent of neurodegeneration, at least in AD (112, 192). The links between toxic N-terminal tau fragments and excitotoxicity are consistent with the known vulnerability of cortical neurons with glutamatergic inputs and high levels of synaptic plasticity to AD (84) and the strong association between Ab mediated toxicity in early stage disease with synaptic dysfunction (193), suggesting that oligomer toxicity is not necessarily required for neurodegenerative disease pathogenesis. We recently pointed out that receptor-mediated toxicity mechanisms could account for neurofibrillary lesion propagation via Ca++ mediated activation of calpains and caspases, which could (at least in theory) generate both NFTs and secretable toxic fragments in downstream neurons, which could then repeat the cycle. Ironically, such a mechanism would technically fulfill the requirements of the original Prion Hypothesis, which makes no mention of templated misfolding (194). It thus seems safe to say that coexistence is not only possible but necessary given what we know (and don't know) about how tau toxicity actually operates in neurodegenerative disease.

#### **TAU SECRETION**

#### **Cellular mechanisms of tau secretion**

Tau secretion by neurons via multiple biologically distinct pathways has been demonstrated both in culture and *in situ* (105, 106, 195), despite its lack of a signal peptide and of lipidation or GPI anchor sites that would permit its secretion via the conventional ER/Golgi route. Tau resembles other aggregation-prone proteins with key roles in neurodegenerative disease (i.e., SNCA, PrP, and Ab) in that it is secreted at least in part via the exosome pathway (106, 196–198); however the full range of secretion routes and their significance to tauopathy pathogenesis remain unclear. Human tau phosphorylated at Thr181 (epitope AT270) is secreted in exosomes in culture and is also found in exosomes in the cerebrospinal fluid (CSF) of early stage AD patients (106). The early appearance of exosomal CSF tau in AD argues strongly in favor of CSF-tau biogenesis by active secretion rather than passive postmortem release, since it occurs at a stage (Braak Stage 3) when neurofibrillary degeneration is restricted to a small proportion of the brain (199). The N terminal of tau, which interacts with the plasma membrane and membrane-associated proteins, is required for secretion in culture and in an *in situ* lamprey model (195), where tau secretion occurred in two distinct patterns depending on the presence of the MTBR,a pattern consistent with the CSF-tau species observed in AD (200). In the lamprey model, N-terminal tau species lacking the MTBR become distributed in a diffuse, gradient-like pattern with secretion occurring from the soma, while full-length tau was secreted from the dendrites in discrete foci (195), where it set up much steeper tissue gradients, suggesting a role for interactions between the tau MTBR and extracellular matrix elements in the distribution of extracellular tau that could

have relevance to interneuronal tau toxicity patterns and lesion spreading mechanisms (81). Interestingly, the secreted tau in the lamprey model is largely dephosphorylated, which is consistent with the phosphatase activity described by Diaz-Hernandez and co-workers (182), although no overt toxicity to non-expressing cells was observed. Secreted tau cleaved at the C terminal has been observed in cell culture (105), and in transgenic mouse models expressing human tau as well (201, 202). One of these studies suggested that cleavage at D421 as well as phosphorylation increased the rate of tau secretion (203), which is consistent with the low level oligomerization observed in exosomal tau purified from CSF samples (106).

The method(s) by which tau undergoes secretion remain elusive, although most evidence supports an unconventional secretion pathway resulting in the release of tau in membrane-bound vesicles (81, 106, 195, 203), where it is favored by the absence of the MTBR and/or the E2/E3 inserts in the secreted tau species (195). The failure of 4R2N tau to localize to and be secreted via exosomes is consistent with this as well (134), as are recent demonstrations that minute amounts of full-length, non-vesicle-associated tau can be released by induced forebrain iPS cells in culture (204). Such findings are consistent with a specific, non-universal release mechanism for tau that is associated with exosomes, as is the failure of an earlier study to detect exosomal tau secretion from healthy cortical neurons (205). Vesicle-free tau secretion has also been reported from neuroblastomas (106, 204) and may also occur in the lamprey tau secretion model (195). It thus remains possible that the presence of non-vesicular tau in the extracellular space in any model is due to post-secretion release of tau from exosomes or other microvesicles (105, 106, 195, 201, 202). Conversely, it could be that the presence of tau in exosomes may be due to the passive adherence of extracellular tau to exosomes that have already been released. This seems unlikely, given: (a) the restriction to E2− tau seen for exosomal tau and (b) the degree of interconnectivity of exosomal proteins associated with E2− overexpression via both functionally (**Figure 2C**) and via observed physical interactions (**Figure 2C**). Overexpression of tau in culture causes cells to secrete exosomes containing tau that has been phosphorylated at several proline-directed sites (106), possibly as a protective response to high concentrations of membrane-associated tau (206). Overall, it appears that tau may be secreted via multiple mechanisms from neurons in tauopathy, including microvesicle shedding, exosomal secretion via endocytosis and fusion with multivesicular bodies, and exophagy, a pathway involving the diversion of autophagosomes to exosomes (69, 81, 134, 207). Uptake mechanisms into trans-synaptic and adjacent cells have been characterized in even less detail that have tau secretion pathways. In the lamprey system, uptake has largely mirrored the "focal" and "diffuse" secretion mechanisms, with specificities for MTBR+ and tauopathy mutations and MTBR−, E2− tau respectively. A recent study of PHF tau uptake found evidence for perinuclear localization of tau in aggresome-like bodies (134), which is consistent with the scheme suggested in **Figure 1**. While the uptake of tau aggregates has been addressed by a number of recent studies directed at "prionlike" interneuronal transmission mechanisms via oligomeric tau species, the general mechanisms that might mediate tau uptake have not yet been characterized in detail in cell culture.

#### **Interneuronal propagation of tau lesions and prions**

Injection of brain extract from mutant human tau P301S transgenic mice into the brains of mice expressing wild-type human tau caused aggregation of wild-type tau in anatomically connected regions of the brain at a distance from the inoculation site (169). Sequential progression of tau aggregation (but not the transfer of individual tau molecules) between synaptically connected regions of the brain was later confirmed in mouse models in which human tau expression was spatially limited to synaptically connected areas of known vulnerability to neurofibrillary degeneration such as the entorhinal (98) and cingulate cortices (99). Further studies using antibodies specific to oligomerized tau have demonstrated that pre-fibrillar tau in AD brain is in fact oligomeric (208) and that tau oligomers derived from the brains of AD patients can recruit endogenous tau to oligomers both *in vitro* (97) and *in vivo* (96) and that intracerebral injection of oligomeric but not fibrillar wild-type tau caused aggregate formation from endogenous tau in synaptically connected but distant areas (96). Interestingly, regions receiving propagated tau in this study do not exhibit signs of tau toxicity, unlike SNCA, which did mediate toxicity as well as protein propagation via oligomer inoculation (100), a finding that illustrates the current ambiguous status of the"tauon"both as a toxicity agent and as a lesion spreading mechanism. In cell culture models, intercellular movement of misprocessed tau between cells and the apparent transfer of conformational alterations to endogenous tau has been more directly demonstrated. Endocytic tau uptake (verified using dextran co-localization) followed by fibril formation was observed in the proximity of labeled aggregates, suggesting recruitment of endogenous tau (92). Double immunolabeling (36) and fluorescence resonance energy transfer (37) has confirmed association of internalized tau with newly formed aggregates composed of endogenous tau. The uptake and axonal transport (retrograde and anterograde) of exogenous full-length tau aggregates has since been reported in differentiated primary neurons grown in Campenot chambers (209). Uptake was described as occurring via a process resembling bulk endocytosis, with the internalized tau associated with general lysosomal and endosomal markers (209). Common features of both cellular and mouse model observations of prionlike tau transfer include a specificity for oligomeric tau species and a requirement for the tau MTBR. The successful use of the 4R2N tau isoform for oligomer propagation (36) and the uptake of MTBR-only tau species (92) are obviously very different from the N-terminal specific E2− favored pattern observed for tau secretion in the lamprey model and in NB2A and M1C neuroblastoma cells (195). It should be noted that these differences reflect the tightly focused nature of the question being posed in the "prion" studies, i.e., whether oligomeric tau could be shown to propagate its oligomeric conformation between cells by recruiting endogenous tau in the recipient cell in the manner now established for misfolded PrP. This is very likely relevant to the one point of agreement between these approaches – that tauopathy mutations favor trans-synaptic tau transfer – since this type of movement may well be associated with oligomerization.

Recent investigations into interneuronal aspects of tauopathy pathogenesis have suffered from a bifurcated focus on either: (1) "prionlike" mechanisms of lesion spreading in terms of templated misfolding mechanisms derived from our understanding

of prion diseases at either the whole animal or molecular level or (2) the morphological changes associated with tau secretion in cell autonomous models. These disparate approaches have (inevitably) been limited by: (a) their strict focus on molecular mechanism at the expense of cellular context and (b) exclusion of biochemical methodology in favor of spatial co-localization (respectively). These differences have dictated the choice of experimental models suited to the experimental approach taken (e.g., transgenic mice or lampreys), but have resulted in largely non-overlapping datasets devoid of a common context with which to consider them. This problem is exacerbated by a notable peculiarity of tau biology – its sensitivity to the terminally differentiated neuronal state. The inability of experimenters to induce tau-specific toxicity responses in cell lines (144) is likely due to the greater dependence of tau toxicity on axonal and synaptic functions relative to other "disease" proteins that are readily toxic in culture (210). While this has until now been a formidable obstruction to finding a broadbased approach to the characterization of mechanisms underlying tau toxicity and lesion spreading, it offers important hints for the direction of future studies on the topic – i.e., to focus on synaptic functions and developmental axon identity and guidance mechanisms to identify tauopathy-specific mechanisms, and to focus on aggregation-mediated mechanisms for identifying common themes of neurodegenerative disease pathogenesis.

#### **IS SYNAPTIC DYSFUNCTION THE POINT AT WHICH TAU OLIGOMERIZATION, AUTOPHAGY DISRUPTION, TAU SECRETION, AND TAU TOXICITY MEET?**

While the means by which tau becomes membrane associated and then diverted into unconventional secretion pathways is not yet clear, the interaction between tau and fyn kinase and the effects of that interaction on synaptic function is emerging as a key feature that might provide clarity. Fyn is rapidly localized to membrane raft domains by virtue of its double lipidation anchor sites (211). Fyn is capable of inducing exocytosis and/or endocytosis of membrane localized proteins (212, 213) and binds tau strongly via its proline rich region (19). Interactions between tau, fyn, and actin potentiate fyn-driven endocytosis of lipid raft markers flotillin-1 and flotillin-2 (129, 214). As shown in **Figure 1**, the Y18 residue of tau, a fyn substrate, is phosphorylated in distal dendritic vesicle accumulations localized to sites of focal tau secretion via microvesicles that contain both pY18 tau and endogenous fyn (81). This offers direct support for a mechanism by which fyn-activated endocytosis of raft domains containing tau results in localized tau secretion via unconventional pathways, one of which is exosomes. The phosphorylation of membrane and vesicle-associated tau by tauopathy-associated kinases (e.g., GSK3b, MARK kinases, and fyn) and their association with the exosome pathway is consistent with both receptor-mediated and oligomer-associated spreading and toxicity mechanisms, especially in the context of synaptic dysfunction.

#### **Mislocalized tau and synaptotoxicity at glutamatergic synapses**

Toxicity resulting from overexpression or extracellular application of N-terminal tau fragments in primary neurons appears to be caused at least in part by NMDA receptor-mediated Ca++ dysregulation (109), suggesting a potential link between Ca++ mediated transmitter release, tau secretion and plasticity-associated excitotoxicity and the dendritic accumulation of misprocessed tau at postsynaptic densities. A central role in this interaction may be mediated by fyn, particularly at highly plastic, glutamatergic synapses. NMDA receptor activity at glutamatergic synapses may mediate the tau-induced dendritic degeneration seen in lamprey ABCs, where fyn associates with and phosphorylates dendritically localized tau, resulting in both localized MT loss and tau secretion (81). Fyn-mediated activation of NMDA receptors normally appears to prevent excitotoxicity at glutamatergic synapses that exhibit LTP and LTD (77), but this mechanism may be vulnerable to disruption by abnormal perisynaptic tau accumulations (74, 215) and may account for the increased resistance to Ab-induced excitotoxicity observed in tau-reduced mice (216). Both the P301S (217) and P301L tauopathy mutations (75) in mice cause changes in hippocampal synaptic transmission and plasticity well before the onset of neuronal loss. This is consistent with the dependence of Ab toxicity on tau at synapses that exhibit plasticity (218) and suggests a modulatory role for both tau and Ab in synaptic plasticity that could increase sensitivity to excitotoxicity with the onset of neurodegenerative disease (216), accounting for the selective vulnerability of the hippocampus in AD. Similarly, mislocalized tau may also disrupt the AMPA receptor recycling associated with NMDA receptor activation via an increase in fyn-tau association and as a resulting increase in the rate of internalization of AMPA receptors on dendritic spines (77).

Chemical induction of LTD under conditions that prevent autophagosome/lysosome fusion causes LC3II-positive puncta to build up in dendritic spines, suggesting a link between AMPA recycling and autophagy (219). This could provide another point of access for misprocessed tau to toxicity and secretion mechanisms in AD, since inhibition of lysosomal acidification and/or the increased generation of Ab associated with *PSEN1* mutations associated with AD can increase Wnt signaling (220) as well as induce the collection of tau aggregates in lysosomes (221). Tau overexpression in both cell culture (136) and *in situ* tauopathy models (81) causes this as well, leading to the recruitment of both GSK3b, MARK and fyn kinases, which are all key mediators of Wnt pathway activity and tauopathy-associated tau kinases (222). This is consistent with the aberrant Wnt activity seen in AD (223) and the tau-dependent nature of Ab-induced toxicity via LTP in hippocampal synapses (218). Moreover, inhibition of GSK3b decreases the number of GR1 AMPA receptor subunits on the cell surface and increases their intracellular concentration (224). This pathway requires endocytosis of GSK3b into multivesicular bodies (225, 226). This provides potential links between the NMDA/fyn-mediated endocytosis of AMPA receptors seen with LTD (227–229) and the recruitment of tau into unconventional secretion pathways.

#### **A unifying hypothesis**