Potential Mechanisms and Implications for the Formation of Tau Oligomeric Strains

Abstract

The culmination of many years of increasing research into the toxicity of tau aggregation in neurodegenerative disease has led to the consensus that soluble, oligomeric forms of tau are likely the most toxic entities in disease. While tauopathies overlap in the presence of tau pathology, each disease has a unique combination of symptoms and pathological features, however, most study into tau has grouped tau oligomers and studied them as a homogenous population. Established evidence from the prion field combined with the most recent tau and amyloidogenic protein research suggests that tau is a prion-like protein, capable of seeding the spread of pathology throughout the brain. Thus, it is likely that tau may also form prion-like strains or diverse conformational structures that may differ by disease and underlie some of the differences in symptoms and pathology in neurodegenerative tauopathies. The development of techniques and new technology for the detection of tau oligomeric strains may therefore lead to more efficacious diagnostic and treatment strategies for neurodegenerative disease.

Introduction

In the search for an effective therapeutic strategy against Alzheimer’s disease (AD), the most common cause of dementia worldwide, aggregated amyloid-β (Aβ) emerged as the main target of interest and has remained so for many years. However, as more and more clinical trials against the protein failed, researchers searched for a new and hopefully more effective route to treatment(Godyń et al., 2016). The pathological aggregation of the microtubule-associated protein tau and its subsequent deposition in neurofibrillary tangles (NFTs) are also defining histopathological features of AD that correlate better with symptoms than Aβ(Braak and Braak, 1995). Therefore, the study of tau has become an active field of study with a lot of promise(Šimić et al., 2016). Tau aggregation also plays a role in many other neurodegenerative disorders, collectively known as tauopathies, including Pick’s disease (PiD), progressive supranuclear palsy (PSP), dementia with Lewy bodies (DLB), Parkinson’s disease (PD), frontotemporal dementia (FTD), and corticobasal degeneration (CBD)(Ballatore et al., 2007, Gendron and Petrucelli, 2009). Although this large group of related disorders shares the common pathological hallmark of accumulated tau protein aggregates in the brain, they are diversified with a vast array of pathologies and symptom progressions.

NFTs may play a role in the pathophysiology of tauopathies, but little is known about the mechanisms underlying their formation and the role of tau intermediates soluble in physiological fluids formed between tau monomers and filaments. Hyperphosphorylation contributes to the formation of NFTs, which until recently were believed to trigger processes that lead to neuronal cell death(Andrade-Moraes et al., 2013). However, NFT accumulation alone may be insufficient to cause cell death, as neuropathological studies in AD patients suggest that neuronal loss and cognitive deficits precede NFT formation(Haroutunian et al., 2007). Supporting this, animal models show that tau oligomers appear prior to NFTs and contribute to learning and memory deficits and neuronal cell death, while NFTs are not associated with neuronal death(Lasagna-Reeves et al., 2011b, Cowan et al., 2012, Cook et al., 2015, Kim et al., 2016).

Despite increasing research on this topic, the mechanism by which tau induces toxicity and disease progression remains unknown. One of the largest barriers to conclusive mechanistic investigations of amyloidogenic and tau proteins is inconsistency in the protein species (oligomer, fibril, non-specifically defined aggregate, etc.) studied(Cowan and Mudher, 2013), an important consideration as the aggregation state of tau is critical to its function. Within the same aggregation state, tau exhibits conformational differences that could exert diverse downstream effects(Hyman, 2014, Sanders et al., 2014). Many parallels exist between prions and amyloidogenic proteins present in neurodegenerative disease, suggesting that tau and other aggregate-prone proteins may form prion-like conformers or strains that affect disease progression. Therefore, understanding the characteristics of different tau aggregates may be critical for the investigation of tau’s toxicity and may be responsible for the pathological and symptomatic differences underlying various tauopathies(Hyman, 2014).

Prion Structural Polymorphisms and Strains

One of the most puzzling phenomena in prion biology is the existence of prion strains, defined as infectious protein isolates that, when transmitted to identical hosts, induce distinct prion-disease phenotypes(Bessen, 1992, Bartz, 2000). The phenotypic traits include incubation times, histopathological lesion profiles, and specific neuronal target areas. These traits typically persist following serial transmissions, thereby stabilizing strain conformation(Bartz, 2000). The strain phenotypes might be encoded by the same aggregate assuming different conformations that have distinct properties, defined as “strains or conformers”. These conformations can also be manipulated(Legname et al., 2005, Tanaka et al., 2006). It has been demonstrated that multiple strains may exist in different brain regions in the same patient, and this is hypothesized to underlie the individual variability in the clinical manifestations of patients with Creutzfeldt-Jakob disease(Schoch, 2006). Prion strains can compete with one another, suggesting that less pathogenic prions could be used as a therapeutic agent against the most toxic strains(Morales et al., 2007). A recent study in the prion field showed that a single mutation leading to a particular prion strain led to complete protection against toxic misfolded prion protein due to an inability to propagate the toxic prion conformation(Asante et al., 2015). The strain phenomenon has recently been hypothesized to extend to many amyloidogenic proteins that form distinct conformations and can be classified as strains, including Aβ(Heilbronner G, 2013, Lu JX, 2013) and α-synuclein(Guo et al., 2013).

Amyloidogenic Protein Oligomeric Strains

Similarly to tau, many groups have reported that fibrils, once thought to be the most toxic species, are actually less toxic than intermediate Aβ aggregates (spherical oligomers and protofibrils)(Glabe and Kayed, 2006). There are a variety of Aβ oligomer conformations(Glabe, 2008) that can be produced by several pathways. Simple manipulation of conditions in which Aβ aggregates can alter conformation, highlighting the complexity of oligomer formation(Kayed et al., 2003, Glabe and Kayed, 2006, Kayed and Glabe, 2006, Kayed et al., 2007, Necula et al., 2007, Glabe, 2008, Mina et al., 2009). Several studies suggest that oligomeric species form via different mechanisms and exhibit different toxicities(Cizas et al., 2010, Zako, 2010, Kayed and Lasagna-Reeves, 2013). A recent study found that two different Aβ oligomers that differ in toxicity can be differentiated using conformation-specific antibodies(Liu et al., 2015). Aβ conformational differences have also been specifically correlated with more rapidly progressive AD(Cohen et al., 2015). Moreover, structurally distinct α-synuclein strains can lead to different behavioral phenotypes and levels of toxicity(Peelaerts et al., 2015) and may be differentiated using similar techniques used in the prion field, such as resistance to proteases(Roberts et al., 2015). Strains of α-synuclein derived from multiple systems atrophy (MSA) were recently shown to be capable of serial transmission in cell culture as well as to induce neuronal dysfunction following successive inoculations in mice(Prusiner et al., 2015, Woerman et al., 2015). Thus a variety of oligomeric species may be common to many proteinopathies and underpin evolution of disease, as well as distinct sub-types of disease.

Classes of Aggregated Tau

Due to the variety of diseases involving aggregated tau with distinct pathological features and symptomology, conformational differences in tau may be a crucial factor distinguishing tauopathies. Different conformers or strains may be present in one or many of the forms of aggregated tau. Soluble aggregation intermediates include protofibrils, annular protofibrils and oligomers, which have subtypes as well that form either en route to insoluble fibril formation or independently to fibrillization.

Oligomers

Tau in its native state exists as an unfolded monomer which has an important function stabilizing microtubules. Tau undergoes alternative splicing to form six different protein isoforms (Fig. 1A) present at varying levels during development, normal adulthood, and disease states. The isoform of tau has important implications for its structure as the number of microtubule binding repeats in the stable core of affects its aggregation stability and the unstructured N- and C-terminal domains may underlie much of the conformational diversity as they provide environmentally-dependent flexibility to the protein(Wegmann et al., 2013, Xu et al., 2016)(Fig. 1B). The unfolded state of monomeric tau, when released from microtubules allows intermolecular contacts and exposure of hydrophobic regions, which combined with a number of environmental factors can lead to the misfolding and aggregation of tau (Fig. 2). Multimeric tau species which form, known as tau oligomers, have repeatedly been found to induce neurotoxicity and cell death in neurodegenerative tauopathies, rather than the large fibrillar structures known as NFTs(Spires et al., 2006, Berger et al., 2007, Maeda et al., 2007, Kopeikina et al., 2011, Cowan and Mudher, 2013, Sahara et al., 2013, Cowan et al., 2015). We have shown that these dynamic tau aggregates are detected in human disease and can propagate through the brain, causing synaptic and mitochondrial dysfunction associated with memory deficits when administered to cognitively normal mice(Lasagna-Reeves et al., 2011a, Lasagna-Reeves et al., 2012b). Importantly, reducing levels of tau oligomers with oligomer-specific antibodies leads to protection against behavioral deficits and tau pathology in multiple mouse models(Castillo-Carranza et al., 2014a, Castillo-Carranza et al., 2014b, Castillo-Carranza et al., 2015).

Fig. 1. Alternative splicing leads to the formation of six different tau protein isoforms.

(A) Alternative splicing of tau and tau isoform makeup may alter protein folding and aggregate conformation. Tau forms six different isoforms, three of which have three repeats in the microtubule binding domain (3R0N, 3R1N, 3R2N) and three of which have four repeats (4R0N, 4R1N, 4R2N). (B) Tau isoforms may lead to differential stability and structure in tau oligomers depending upon the rigidity of the structure. Modifications and environmental conditions can alter the unstructured C- and N-termini surrounding the stable, protease-resistant core.

Fig. 2. Tau oligomers are the most toxic species in disease.

A number of complex and inter-related factors, including alterations in post-translational modifications, truncation alternative splicing, mutations, co-factors, chaperones, oxidative stress and seeds (Aβ, α-synuclein) can lead to the formation of soluble, intermediate tau aggregates—tau oligomers. Tau oligomers may go on to form stable filaments, however, these highly dynamic structures appear to be far more toxic than tau fibrils and may lead to the spread of pathology and a number of downstream toxic effects. Thus, tau oligomers represent a bottleneck in the process of tau toxicity in disease and a likely important therapeutic target.

Protofibrils

Protofibrils are intermediate, metastable species that form en route to fibrillization. Protofibrils share some structural similarities with mature fibrils, including a rope-like fibrillar structure detectable by EM and AFM and stable hydrogen bonding. However, protofibrils are inherently more dynamic and unstable and do not show high response to thioflavin T(Williams et al., 2005a), which recognizes the β-sheet structure characteristic of amyloid fibrils. Protofibrils can be detected with oligomer-specific conformational antibodies, indicating their similar structure (Kayed et al., 2003). Similarly to oligomers, these soluble intermediates have been shown to be more toxic and relevant to disease pathogenesis than the larger, more stable fibrillar structures and NFTs (Harper et al., 1997, Walsh et al., 1997, Habicht et al., 2007, Habicht et al., 2008, Rijal Upadhaya et al., 2012)

Annular Protofibrils

The formation of pore-like structures, known as annular protofibrils, has been known for amyloid proteins, including amyloid-β and α-synuclein for years(Hafner et al., 2001, Kril et al., 2002, Lasagna-Reeves et al., 2011c). Recently, the formation of tau annular protofibrils was shown in vitro and in human disease tissue(Lasagna-Reeves et al., 2014). Results suggest that tau oligomers can go on to form annular protofibrils by a specific off-pathway mechanism from fibril formation. These pore-like structures are believed to disrupt permeability of the cell membrane, leading to non-specific ion leakage.

Tau Fibrils

Paired helical filaments (PHF) and straight filaments (SF) that make up NFTs are comprised of hyperphosphorylated tau(Grundke-Iqbal et al., 1986, Lee et al., 1991) in a β-sheet conformation(von Bergen et al., 2005). Differences in NFT location and structure exist among various tauopathies. In a study by Clavaguera et al., brain homogenate derived from various tauopathies was injected into the brains of ALZ17 mice, which express non-mutated human tau, and this induced the deposition and spread of characteristic tau aggregates from each disease(Clavaguera et al., 2013). Recently, Boluda et al. extended these results, showing that at much earlier time points than the original study, CBD and AD-specific pathology propagates in ALZ17 mice following brain homogenate injection, with distinct cell specificity(Boluda et al., 2015).

While this is strong evidence for the existence of tau strains, it is critically important to accurately identify the species in total brain homogenate responsible for seeding the spread of unique tau pathologies. In addition to isoform composition, research suggests that disulfide linkages impact fibril structure(Furukawa et al., 2011). Sanders et al. (2014) reported that conformationally distinct tau fibrils generated from the tau repeat domain propagate in cells over multiple passages. Additionally, when injected bilaterally in the hippocampi of P301S mice, different strains are propagated over multiple generations and can be differentiated by their biochemical characteristics(Sanders et al., 2014). However, fibrillar strains were derived from tau fragments, which may not accurately reflect natural conditions in human disease and as no behavior was completed, toxicity in vivo is difficult to assess from this study(Sanders et al., 2014). Nonetheless this study provided good evidence for the idea that the conformational diversity of tau strains dictates its toxic potential and transmissibility. In addition, expressing specific point mutations in truncated tau also leads to diversification of fibrillar structure(Meyer et al., 2014).

Thus it is of great interest to determine whether toxic tau oligomers form distinct strains that differ by disease. Similarities between tau and other amyloidogenic proteins with unique conformational strains suggest that this is likely the case (Fig. 3).

Fig. 3. Hypothetical models for the formation of tau oligomeric conformers.

(A) Risk factors cause tau misfolding and lead to the formation of conformationally distinct misfolded monomers that aggregate into different tau oligomeric strains. (B) Misfolded tau induces the formation of a flexible, dynamic, master oligomeric conformer that transforms into different oligomeric strains depending on less-defined risk factors, such as changes to cell autonomous and non-autonomous factors. (C) Conformational differences in tau oligomeric strains could lead to differential seeding of the spread of strains in different brain regions and cell populations.

Other Potentially Toxic Species

Misfolded tau monomer is another possibly toxic species in tauopathies. While most studies show that tau monomer is non-toxic and incapable of propagating tau pathology(Lasagna-Reeves et al., 2012a, Wu et al., 2013), an abnormal, higher molecular weight form of tau monomer known as PHF-tau or A68 has been shown to exhibit some potentially toxic characteristics(Liu, 1993). The 68 kD tau is found in higher proportions in tau aggregates in disease brains and is more highly phosphorylated than native tau monomer(Bramblett et al., 1993, Shin et al., 1993, Miller, 1999). Truncated tau may also be a toxic species of interest. Fragmentation of tau has also been shown to be affected in disease states, led to increased aggregation in vitro, and increased toxicity(Zilka et al., 2006, De Strooper, 2010, Reifert et al., 2011).

Tau Oligomeric Strains

Collectively the evidence suggests that dynamic tau intermediate soluble aggregates (tau oligomers) are the toxic tau entities and initiators of tau pathology and propagation. The formation of other amyloidogenic protein oligomeric strains and evidence for tau fibrillar strains suggests that tau oligomers may exist in different conformationally distinct conformers (strains) that play a critical role in disease phenotypes. One pre-fibrillar tau oligomer species that has been characterized and appears to have differential structure and toxicity is the granular tau oligomer(Maeda et al., 2006, Maeda et al., 2007), reviewed by Cowan and Mudher(Cowan and Mudher, 2013). This is a 40mer structure identified in AD brains and believed to be a precursor of tangles. However, it does not appear to be as clearly toxic as other oligomeric tau species since it can be found in animal models in the absence of toxicity(Cowan et al., 2015), similarly to the fibrillar Aβ oligomer strain that was found to be less toxic than other conformational states(Liu et al., 2015). The reason for the lowered toxicity could be due to a lack of beta pleated sheet conformation in granular tau oligomers. This illustrates the fact that tau aggregates, whether small or intermediate in size, vary in constituent tau composition and conformation and this may play a role in determining their toxicity. Thus, the elucidation of the different tau oligomeric conformers may reveal new targets for novel therapeutic strategies tailored for different neurodegenerative tauopathies and perhaps could be used to screen the best candidates and experimental drugs.

Tau Aggregation as a Secondary Amyloidosis

The critical role of amyloidogenic oligomers has been revealed for multiple neurodegenerative diseases(Walsh and Selkoe, 2004). Recently published works support the idea that prefibrillar soluble oligomers of amyloidogenic proteins, including α-synuclein and amyloid-beta (Aβ) are more toxic than insoluble Lewy bodies and senile plaques(Klein et al., 2001, Walsh et al., 2002, Gispert et al., 2003, Kayed et al., 2003, Lesne et al., 2006, Walsh and Selkoe, 2007, Danzer et al., 2009, Tsika et al., 2010). Pathogenic proteins involved in different neurological disorders often interact with one another and modulate downstream activities that disrupt their normal function. Although Alzheimer’s disease and Parkinson’s disease have unique pathological features, there is considerable overlap between these two disorders. The effects of Aβ aggregation on tau misfolding and aggregation are well-established(Hardy, 2002, Oddo et al., 2006, Clinton et al., 2010, Bloom, 2014). It is also well-established that aggregated Aβ is an important contributor to tau phosphorylation and aggregation in animal models and cell cultures. In primary neuronal culture, Aβ is capable of inducing tau phosphorylation(Busciglio et al., 1995). Aβ42 fibrils induced formation of NFTs in P301L tau transgenic mice(Gotz et al., 2001), and pre-aggregated Aβ42 induced tau paired helical filament (PHF) formation in cells over-expressing human tau(Ferrari et al., 2003, Pennanen and Gotz, 2005). These experiments were performed with aggregated Aβ, which implies that they contain different prefibrillar and fibrillar Aβ aggregates. Recently, it has been shown that soluble Aβ oligomers trigger tau aggregation in vitro and in vivo(Chabrier et al., 2012). We previously demonstrated that Aβ and α-synuclein oligomers prepared in vitro are capable of inducing toxic tau oligomer formation(Lasagna-Reeves et al., 2010), targeting tau oligomers in an Aβ precursor protein (APP) overexpression mouse model also leads to a decrease in toxic Aβ aggregates and a genome-wide association study reported genetic interactions between tau and α-synuclein in Parkinson’s disease pathogenesis(Simon-Sanchez et al., 2009). Moreover, fibrillar α-synuclein has been shown to induce tau tangle formation in vitro(Waxman and Giasson, 2011) and accelerate tau phosphorylation, further enhancing its likelihood to form aggregates(Jensen et al., 1999). Recently two different α-synuclein fibrillar seeds have been identified with varying capacities of causing tau aggregation both in neuronal cultures and in vivo(Guo et al., 2013). A study also provided evidence that α-synuclein induces fibrillar tau formation and found that both tau and α-synuclein exert synergistic effects on each other leading to the formation of fibrillar amyloid structure(Giasson et al., 2003). Although insoluble forms of α-synuclein and tau proteins have been observed to co-exist in DLB patients(Iseki et al., 2002), our recent report was the first published evidence that these proteins occur together within oligomeric organizations that further perpetuate their aggregation(Sengupta et al., 2015).

Collectively this illustrates that the complexity of neurodegenerative tauopathies is likely to be partially dependent upon interactions between other proteins, particularly other amyloids. Cross-seeding between aggregant proteins may induce the formation of diverse tau conformers differing between diseases with mixed protein pathologies. However, in addition to the common mixed pathology disorders—AD, PD, and DLB—phenotypes and pathologies also differ between tauopathies lacking aggregation of other amyloidogenic proteins. Therefore, other mechanisms must be at play that are independent of other aggregant proteins.

Tau Modifications

Modifications to tau take place in the form of alternative splicing, mutations, post-translational modifications, and truncation. These changes could alter the folding of the protein and lead to the formation of different conformations. In support of this idea is the fact that tauopathies are associated with different tau isoform compositions. For example, PSP and CBD specifically display primarily 4R tau pathology and 3R tau aggregation, respectively, while PiD and AD display both 3R and 4R aggregates. Studies have found that 3R tau forms mainly twisted PHFs, while 4R tau fibrils are mainly SFs in vitro(Goedert, 1996, Barghorn and Mandelkow, 2002). However, the in vivo fibrillar conformations of isoform-specific aggregates in PiD and CBD vary(Munoz-Garcia, 1984, Ksiezak-Reding, 1994). Another likely cause of conformational differences in disease proteins is the presence of genetic mutations. While tau mutations are not associated with AD or PD, they do induce other tauopathies. The identification of around 40 tau mutations that cause familial dementia, such as FTD and Parkinsonism linked to chromosome 17 (FTDP-17) demonstrated that tau alone could cause neurodegeneration(Goedert and Jakes, 2005). However, the mechanisms by which tau mutations cause neurological disorders are still unclear, though the effect of mutations on tau aggregation and fibril formation has been studied extensively. While tau protein will not spontaneously aggregate in vitro without the addition of polyanionic cofactors, tau fragments expressing point mutations, P301L and ΔK280, rapidly aggregate due to an increase in the shift towards β-sheet structure(von Bergen et al., 2001). Multiple mutations have been identified in the microtubule binding repeat region (P301S, P301L, and P301T), which highlights its importance in aggregation. Additionally, in a study on the effect of mutations on heparin-induced fibrillar assembly, P301L and P301S mutations led to the largest increase in filament formation. G272V also increased filament formation in both 3R and 4R tau, while V337M had a small stimulatory effect only in 3R tau isoforms(Goedert et al., 1999). Studies carried out both in vitro and in vivo suggest that different mutations may have varied phenotypic and pathogenic effects. The P301S mutation is associated with early onset of clinical symptoms of FTDP-17, reduced microtubule assembly, and increased filament assembly. P301S mice show motor symptoms, including severe partial paralysis of the hindlimbs as well as hyperphosphorylated tau aggregates in the brain and spinal cord, but no signs of apoptosis (Allen et al., 2002). On the other hand, P301L mice have increased levels of apoptosis(Götz et al., 2001, Ho et al., 2001). These mice also display motor deficits and NFTs in the brain and spinal cord, as well as deposits in glial cells(Lewis J, 2000, Götz et al., 2001). Meanwhile, the R406W mutation associated with FTDP-17 induces similar clinical symptoms and neuropathology seen in Alzheimer’s disease. Mice expressing the mutation develop NFTs of similar morphology to AD inclusions in the forebrain and cognitive deficits, but no sensorimotor symptoms(Tatebayashi et al., 2002). Moreover, a recent study revealed that mutations give rise to varied tau fibrillar strains(Meyer et al., 2014). Phosphorylated tau is well-known to be altered in disease and to affect aggregation. However, the relationship between phosphorylation state and toxicity is complex, with different sites leading to widely varying effects, as was illustrated recently in a Drosophila model of tauopathy(Papanikolopoulou and Skoulakis, 2015). In fact, elevated levels of tau phosphatase has been shown to increase toxicity of tau in the extracellular space(Díaz-Hernández et al., 2010). Moreover in a Drosophila model of tauopathy, reduced tau phosphorylation was associated with rescue of phenotype but increased oligomerisation of tau(Cowan et al., 2015). Tau has also been shown to be altered by a number of other post-translational modifications including acetylation, glycation, and others(Gerson et al., 2014), suggesting that this wide variability could have an effect on tau oligomer conformation. Tau has also been shown to form disease-specific fragments that enhance its ability to seed the formation of oligomers and spread through the extracellular space(Wang et al., 2009, Kanmert et al., 2015, Matsumoto et al., 2015). These results suggest that tau modifications could be involved in the formation of unique oligomeric tau conformations that result in different disease phenotypes.

Cell and organelle specificity

Regional and cellular differences in accumulation of tau aggregates between neurodegenerative diseases have been long known and used to differentiate disorders, even prior to the proposal of the prion-like attributes of tau and other aggregant proteins. Alzheimer’s disease is characterized by the presence of flame-shaped NFTs in neurons and rarely glial inclusions. In Pick’s disease, tau aggregates are primarily found in spherical Pick bodies in neurons, but can also be detected in lower levels in astrocytes and oligodendrocytes, while in both PSP and CBD there is widespread deposition of astrocytic and oligodendroglial tau aggregates(Yoshida, 2006). Moreover, we found that while multiple tauopathies contain tau annular protofibrils, cellular specificity of their formation differed by disease, suggesting the presence of disease and cell-specific conformations(Lasagna-Reeves et al., 2014). Additionally, alterations to the intracellular localization of tau may correlate with disease. While tau in its native state is commonly located in the axonal cytoskeleton, recent studies suggest that it also has a nuclear role, binding and protecting DNA and RNA under oxidative stress conditions that also lead to the formation of toxic tau oligomers(Sultan et al., 2011, Violet et al., 2014). In this environment, functional tau translocates from the cytosol to the nucleus, however tau oligomer formation appears to both inhibit the movement of protective tau to the nucleus and lead to the buildup of nuclear tau aggregates(Violet et al., 2015). Tau may also differentially interact with RNA in disease states in the endoplasmic reticulum (ER) as tau has been shown to be associated with ribosomal and other ER proteins(Meier, 2015). The redistribution of tau protein may also underlie synaptic dysfunction seen in tauopathies as tau aggregates have been found in higher proportions in the somatodendritic compartment and can be specifically detected at the pre- and post-synaptic densities in disease states(Meyer et al., 2014).

Spread of tau oligomeric strains

The ability of amyloid and tau fibrils to seed soluble monomers and convert them to aggregates capable of propagating has been known for more than 20 years and is well standardized and documented(Jarrett et al., 1993, Kelly, 2000, Margittai and Langen, 2004, O’Nuallain et al., 2004, Siddiqua and Margittai, 2010, Dinkel et al., 2011). Recently, our lab and others have shown that amyloid oligomers also can seed and propagate in vitro(Kayed et al., Lee et al., 2011). Moreover, we discovered that tau oligomers prepared from recombinant tau can seed the aggregation of tau in vitro(Lasagna-Reeves et al., 2010). It is hypothesized that brain-derived tau oligomers seed and propagate by a mechanism known as oligomer-nucleated conformational induction. Unlike the seeding mechanism proposed for tau fibrils prepared in vitro—template-assisted growth(Margittai and Langen, 2004, Congdon et al., 2008)—this mechanism excludes the addition of tau monomer to the nucleus/template, stating instead that all monomers will assemble into oligomers before fibril formation. In this mechanism, different conformational strains of tau oligomers would propagate rapidly, seeding endogenous monomer to oligomerize prior to forming stable fibrillar structures.

There is increasing interest in understanding the spread of disease from the early stages of tauopathies. In the specific case of AD, NFTs progressively spread throughout the brain in an anatomically stereotypical manner(Braak and Braak, 1991, Delacourte et al., 2002). Based on these and several other studies, it has been postulated that tau proteins spread via a prion-like mechanism(Clavaguera et al., 2009, Brundin et al., 2010, Frost and Diamond, 2010). Clavaguera and coworkers support this concept by demonstrating that intracerebral injections of brain extracts from mice expressing human P301S tau induce the formation and spread of tau aggregates. The pathology spreads from the injection site to neighboring brain regions 15 months post-injection in mice transgenic for human wild type tau (ALZ17 mouse model)(Clavaguera et al., 2009) and 12 months post-injection in wild-type mice. The observation that some areas of the human brain show evidence of neurodegeneration prior to the formation of NFTs and clinical diagnosis(Kril et al., 2002) suggests that tau oligomers could be responsible for neuronal death and the spread of tau pathology. Up to this point, all studies investigating the propagation of tau in cells and in vivo have used heterogeneous populations of aggregated tau; therefore, whether tau oligomers—as opposed to other species—play a leading role in tau aggregation and propagation has been difficult to ascertain. A recent publication by Mathias Jucker and Larry Walker demonstrated that soluble Aβ assemblies (oligomers) are the most potent amyloid-inducing factors, 50-fold stronger than large fibrillar aggregates(Langer et al., 2011).

These results combined with evidence for the existence of different tau oligomeric strains suggest that varying regional distribution and time to acquire disease may be due to differences in the ability of tau conformers to seed and spread through the brain. Within the same disease, some strains may correlate with certain disease stages. There may be diversity by brain region as well as by cell type as different environmental conditions including pH(Sneideris et al., 2015, Verasdonck et al., 2015) and temperature(Tanaka et al., 2006) could lead to conformational changes. The impact of interactions with chaperones, as well as post-translational modifications are also important areas of exploration as the formation of amyloid strains has been shown to depend on variability of chaperone interaction(Sporn and Hines, 2015) and tau is known to interact with a number of different chaperones that affect its aggregation(Petrucelli et al., 2004, Dickey et al., 2006). Additionally, differences in conformers or strains may characterize different tauopathies, explaining pathological differences in tau diseases.

Tau strains outside the brain

A simple, non-invasive, and inexpensive test for the early detection of AD is urgently needed. It is well-established that tau levels are increased in the cerebrospinal fluid (CSF) of patients with AD(Olsson et al., 2016, Takeda S, 2016). As the central nervous system (CNS) is in direct contact with the CSF, an increase in tau oligomers in the CNS would theoretically be evident in the CSF. Furthermore, CSF is accessible via lumbar puncture in living patients. Evidence that amyloidogenic proteins may exhibit different properties in the CSF when compared to the brain was recently provided by a study that investigated seeding potential of Aβ oligomers from brain tissue and CSF. Despite a greater than 10-fold increase in levels of Aβ oligomers in the CSF compared to the brain, when injected in the brains of transgenic mice, these CSF Aβ oligomers were not capable of seeding, while those collected from the brain did seed amyloid aggregation in vivo(Fritschi et al., 2014). Therefore, differences in the conformation of oligomers in the brain versus the CSF, as well as potential heterogeneity of seeding potential of oligomer populations in the CSF may be of great importance for the development of biomarkers and diagnostic tools. Though CSF would provide a useful diagnostic tool, detecting biomarkers with a simple blood test might be preferable. Studies show that Aβ can be detected in the platelets, however high molecular weight tau that may be comprised of oligomers and other aggregates correlates better with disease symptoms(Neumann, 2011, Farías, 2012).

It is well-known that in addition to the accumulation of toxic amyloidogenic proteins in the parenchyma of the AD brain, there is also a vascular component to the disease. High levels of comorbidity between AD and vascular conditions suggest a possible common mechanism underlying vascular dysfunction and amyloidogenic protein dysregulation in AD(Yarchoan et al., 2012, Sudduth et al., 2014). Notably, animal models of AD also manifest Aβ-dependent vascular pathology(Lasagna-Reeves et al., 2014, Lin, 2014, Park et al., 2014). Vascular changes can induce hypoxia, which has been shown to facilitate the formation and deposition of Aβ and tau aggregates(Villarreal et al., 2014). The strongest correlation between vascular pathology and neurodegenerative disease is for AD, but dementia in PD and LBD has also been associated with vascular alterations(Toledo et al., 2013). Deformation of capillaries in the hippocampus and cortex is a common change in AD(Baloyannis, 2012), which could result from the over-secretion of vascular endothelial growth factor stimulated by Aβ(Dal Prà et al., 2014, Lin, 2014). Importantly, alterations in blood vessel walls may include perforations that could impair blood brain barrier (BBB) function(Scheibel, 1989). Notably, BBB impairments have been described in both neurodegenerative disease patients and mouse models(Tanifum et al., 2014).

Additionally, oligomers from amyloidogenic proteins, including Aβ, α-synuclein, and TDP43, are found in the cerebrovasculature of AD patients(Guerrero-Muñoz et al., 2014). The relationship between tau and the cerebrovasculature in tauopathies has not been thoroughly investigated, although tau has been shown to accumulate in perivascular areas affected by Aβ deposits in the AD brain(Williams et al., 2005b). Additionally, the tuft-shaped astrocytes commonly found in PSP brain frequently have vascular contact, and tau deposits are located in close proximity to blood vessels(Shibuya et al., 2011). Decreases in blood pressure in elderly patients with hypertension, which are associated with negative effects on the vasculature, and chronic cerebral hypoperfusion, which is the leading cause of vascular dementia, lead to increases in phosphorylated tau and memory deficits(Glodzik et al., 2014, Zhao et al., 2014). Moreover, tau pathology in a mouse model of tauopathy (P301L mice) is associated with brain capillary constriction prior to cell death(Jaworski et al., 2011). Collectively, the evidence suggests that vascular dysfunction is a critical component in neurodegenerative disease and that tau aggregation may play a role in this process. However, the extent to which vascular pathology varies among diseases is unknown. More research will be needed to determine whether tau aggregates may present in the cerebrovasculature and if they may differ between disorders or individuals based on conformation.

Conclusion

Evidence from the prion field and similarities between prions, other amyloidogenic proteins and tau suggest that tau likely forms different conformational strains. As is the case for prions, it is possible that the toxic entities in tauopathies include a range of strains responsible for templating and that these populations may overlap(Collinge and Clarke, 2007, Li et al., 2010). Complexity may also arise from different amyloid oligomeric seeds interacting with tau, as well as interaction with chaperones and other proteins. The characterization of tau strains may impact a variety of fields as the concept of prion-like induction and spreading of pathogenic proteins is linked to additional neurodegenerative diseases(Polymenidou and Cleveland, 2011, Munch and Bertolotti, 2012). It is important to keep in mind that the term “prion-like” refers to the self-propagation of tau and other amyloidogenic proteins across cells and tissues. It does not imply that these aggregates are infectious(Hardy and Revesz, 2012). Further multi-disciplinary study and structural characterization is needed to fully understand the most toxic tau species in disease and the impact of aggregation state and conformation on disease phenotype. The evaluation and unique detection of these structures could lead to the determination of dominant toxic strains as well as potentially neuroprotective conformers. Improved detection of tau oligomeric strains in living patients through imaging technologies such as positron emission tomography (PET) could open doors to new and more accurate diagnostic tools for neurodegenerative disease(Okamura et al.). These results could have massive impact on diagnostics as well as therapeutic approaches, thus research into this field should continue.

Biographies

Dr. Julia E. Gerson is a post-doctoral fellow in Rakez Kayed’s laboratory at the University of Texas Medical Branch where she studies the toxicity of tau oligomers as they relate to neurodegenerative tauopathies.

Dr. Amrit Mudher is an Associate Professor in Neurosciences within the Centre for Biological Sciences at the University of Southampton. Her laboratory investigates the mechanisms that underpin tau-mediated dysfunction and degeneration in tauopathies such as Alzheimer’s disease and fronto-temporal dementia

Dr. Rakez Kayed is an expert on amyloid oligomers and neurodegeneration. He is an associate professor in the University of Texas Medical Branch, Department of Neurology. His laboratory studies the mechanisms of protein misfolding and aggregation using biophysical and biochemical analyses, cell and animal models and human tissue.