Tau: From research to clinical development

Alzheimer’s & Dementia - (2016) 1-7

Tau: From research to clinical development David M. Holtzmana,*, Maria C. Carrillob, James A. Hendrixb, Lisa J. Bainc, Ana M. Catafaud, Laura M. Gaulte, Michel Goedertf, Eckhard Mandelkowg, Eva-Maria Mandelkowg, David S. Millerh, Susanne Ostrowitzkii, Manuela Polydoroj, Sean Smithk, Marion Wittmannl, Michael Huttonm a

Department of Neurology, Hope Center for Neurological Disorders, Knight Alzheimer’s Disease Research Center, Washington University, St. Louis, MO, USA b Medical & Scientific Relations, Alzheimer’s Association, Chicago, IL, USA c Independent Science Writer, Elverson, PA, USA d Piramal Imaging GmbH, Berlin, Germany e AbbVie, North Chicago, IL, USA f Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom g German Center for Neurodegenerative Diseases (DZNE), CAESAR Research Center, Bonn, Germany h Bracket Global, Wayne, PA, USA i F. Hoffmann–La Roche Ltd, Neuroscience, Basel, Switzerland j Novartis Institute of Biomedical Research, Cambridge, MA, USA k Merck, West Point, PA, USA l Biogen, Cambridge, MA, USA m Eli Lilly, Inc, Windlesham, Surrey, UK

Abstract

Alzheimer’s Association Research Roundtable Fall 2015—Tau: From research to clinical development. Tau pathology is recognized as the key driver of disease progression in Alzheimer’s and other neurodegenerative diseases. Although this makes tau an attractive target for the development of novel diagnostic and therapeutic strategies, the mechanisms underlying the onset and progression of taurelated neurotoxicity remain elusive. Recent strides in the development of sophisticated preclinical models and the emergence of tau PET imaging and fluid biomarkers provide new opportunities to increase our understanding of tau biology, overcome translational challenges, and accelerate the advancement of tau therapeutics from bench to bedside. With this in mind, the Alzheimer’s Association convened a Research Roundtable in October 2015, bringing together experts from academia, industry, and regulatory agencies to discuss the latest understanding of tau pathogenic pathways and review the evolution of tau therapeutics and biomarkers currently in development. The meeting provided a forum to share experience and expertise with the common goal of advancing the discovery and development of new treatment strategies and expediting the design and implementation of efficient clinical trials. Ó 2016 Alzheimer’s Association. Published by Elsevier Inc. All rights reserved.

Keywords:

Tau; Alzheimer’s disease; Biomarkers; Tauopathies; Neurofibrillary tangles

D.M.H. co-founded C2N Diagnostics, LLC and has an equity interest along with Washington University. D.M.H. may receive may receive royalty income based on technology developed by D.M.H. and licensed by Washington University to C2N Diagnostics and subsequently licensed to AbbVie. D.M.H. consults for C2N Diagnostics, Genentech, AstraZeneca, Denali, AbbVie, and Neurophage. *Corresponding author. Tel.: 11 314-747-0644; Fax: 11 314 362-1771. E-mail address: [email protected]

1. Introduction Alois Alzheimer described neurofibrillary tangles (NFTs) in Alzheimer’s disease (AD) brain more than a hundred years ago, but it was not until the 1980s that tangles were shown to be comprised of microtubule-associated protein tau in a hyperphosphorylated, insoluble, and filamentous

http://dx.doi.org/10.1016/j.jalz.2016.03.018 1552-5260/Ó 2016 Alzheimer’s Association. Published by Elsevier Inc. All rights reserved.

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D.M. Holtzman et al. / Alzheimer’s & Dementia - (2016) 1-7

state [1–3]. Tau pathology is now recognized as a key driver of a broad spectrum of neurodegenerative diseases, collectively known as tauopathies. They include diseases where tau plays a primary role, as well as tauopathies such as AD, where tau aggregation and spreading appear to be enabled by amyloid-b aggregation. Primary tauopathies include frontotemporal lobar degeneration with tau inclusions (FTLD-tau), Pick’s disease, progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD), argyrophilic grain disease (AGD), and chronic traumatic encephalopathy. These diseases differ by affected brain regions and cell types, as well as by biochemical features of aggregated tau [4]. The recent emergence of in vivo tau imaging sparked further interest in tau as a treatment target and prompted the Alzheimer’s Association’s Research Roundtable to choose tau as the topic of its Fall 2015 meeting. The meeting brought together experts from academia, industry, and regulatory agencies to review what is known about the structure, function, and biology of tau and the clinical manifestations of tau pathology, and to explore pathways toward expedited development of tau-targeted therapeutics.

in MAPT were shown to cause cases of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17T) [9–11], a dementing disease characterized by aggregates of tau but not beta-amyloid. More than 50 MAPT mutations have now been identified [5]. These mutations appear to enhance aggregation, either by disrupting tau protein structure or by interfering with mRNA splicing of exon 10, resulting in increased relative levels of 4R tau. Genetic variants in genes other than MAPT have also been linked to increased risk of tauopathy [12]. Two haplotypes of MAPT gene—H1 and H2—produced by an unusual inversion event some 3 million years ago have also been identified [13]. The H1 haplotype shows a strong association with an increased risk of PSP, CBD, and AGD, likely caused by the presence of certain singlenucleotide polymorphisms that may cause subtle changes in splicing or gene expression [14]. The H1 haplotype is also associated with an increased risk of Parkinson’s disease [15] and multiple system atrophy [16], as well as a significant but small increased likelihood of late-onset AD (LOAD), whereas the H2 haplotype leads to a reduced risk of LOAD [17].

2. Tau biology and structure

3. Tau pathogenesis

Tau is a multifunctional protein that is believed to play a role in microtubule binding, axonal transport, modulation of signaling pathways, and adult neurogenesis. Six major isoforms are expressed in the human brain from a single MAPT, the tau gene; they vary as a result of the alternative mRNA splicing of exons 2, 3, and 10. These isoforms are described in terms of the number of C-terminal repeat sequences (3R or 4R) and the number of N-terminal repeats (0N, 1N, or 2N). In healthy human brain, about half of the tau forms are 3R and the rest are 4R. Tau also undergoes a variety of post-translational modifications, including phosphorylation, acetylation, methylation, O-GlcNAcylation, ubiquitination, sumoylation, nitration, prolylisomerization, and truncation [5]. In healthy neurons, tau binds to microtubules as well as to a variety of other binding partners [1]. Knocking out tau protein in mouse models yields a relatively benign phenotype, indicating that it is not critical for normal function, although some knockout lines have shown changes in synaptic function and neuronal hyperexcitability [6]. Tau may become neurotoxic as a result of post-translational modifications that lead to its aggregation [1]. Alternatively, conformational changes in tau may precede post-translational modifications. Filamentous deposits of hyperphosphorylated tau characterize all tauopathies. However, these deposits occur in different brain regions and cell types and are composed of somewhat different filamentous structures. Some studies suggest that the patterns of pathology are consistent with the trans-synaptic, network-based spread of tau [7,8]. Genetic studies have further elucidated the link between structural changes in tau and pathology. In 1998, mutations

In AD, neurodegeneration correlates with the presence of NFTs. However, at autopsy, some NFTs in hippocampal and parahippocampal regions are seen in almost all individuals over the age of 50 years, regardless of their cognitive status [18]. This finding has led to the proposal that a new category of tauopathy, called primary age-related tauopathy (PART) [19], exists when NFTs containing tau accumulate predominantly in the hippocampal and parahippocampal regions in the absence of Ab pathology. People with PART may have symptoms ranging from normal to amnestic cognitive decline. Rarely, there can be more significant cognitive impairment. It is notable, however, that in most individuals who go on to develop NFT pathology in the neocortex in regions beyond the parahippocampal and hippocampal areas, significant Ab deposition is also present [20]. This suggests, along with other data, that while NFT development in PART may in some cases lead to cognitive decline, substantial decline along with dementia is usually precipitated by spread of tau into the neocortex which is in some way facilitated by Ab [21]. It will be important in future studies to sort out the relationship between Ab and tau. What is known is that in AD, NFTs accumulate after a hierarchical pattern, appearing first in the entorhinal cortex and later in limbic and association cortices [22,23]. Although this pattern of tau spreading has been relatively well defined and linked to anatomical connectivity, the mechanisms by which tau aggregation subsequently leads to propagation and spreading remain unclear [24]. Also unresolved is how, in the various tauopathies, particular neural networks are affected by different tau isoforms; and which tau species are critical for driving neuronal dysfunction.

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Recent work using a variety of animal and cellular models [25] has begun to tease out the complexities of taumediated stress and injury but has also revealed both the promise and perils of relying on such mice for clarifying the role of tau in human pathogenesis. Most mouse models are based on the P301 L and P301S mutations of MAPT. Two commonly used P301 L lines are the Tg4510 Tet-off mouse and the 3X transgenic mouse. Both lines originally developed aggressive phenotypes, but the 3X transgenic mice available from Jackson Laboratories have been noted to develop a less aggressive disease. Changes like these complicate the interpretation of results. In the rTg4510 mice, even when tau expression was reduced, neurofibrillary tangle formation continued. Although this might suggest a prion-like mechanism, there is an alternative interpretation. It has been proposed that when the toxic soluble tau entity is turned off in some neurons, there are enough remaining neurons that can function at an adequate level to continue functioning despite the presence of NFTs. In 2010, Alix De Calignon, working in Brad Hyman’s laboratory, proposed that in these mice, caspase activation preceded tangle formation but the neurons survived even after the formation of NFT’s. It may be that soluble tau species, rather than fibrillar tau, is the toxic moiety in neurodegeneration [26]. However, subsequent work failed to show significant caspase 3 activation in rTg4510 mice [27]. Newer data from Karen Ashe’s laboratory suggest that caspase-2 cleaves tau, resulting in truncated forms that infiltrate dendritic spines. Non-animal models have also proved useful for studying the pathways by which tau may become toxic. For example, hippocampal organotypic slice models mimic events in live mice on a shorter time scale, showing a loss of dendritic spines, as well as a loss of nerve cells [28]. There is also a variety of nonmouse models enabling the study of interactions between Ab and tau. For example, Rudy Tanzi et al. generated a three-dimensional human neural cell culture model that recapitulated both amyloid and tau pathologies in a dish; Ab aggregates formed first, followed by the appearance of tau hyperphosphorylation and NFTs. There is no question that tau pathology is linked to neurodegeneration in AD and other tauopathies; there is also overwhelming evidence that aggregation and accumulation of Ab drive further development of tauopathy [29]. Although NFTs are common in limited brain regions in people over the age of 50 years, symptoms usually develop only in those who have also Ab deposition and spread of tau from the medial temporal lobe to neocortical regions. In a recent study, Tau PET patterns are shown to correspond well with Braak staging of NFT pathology [30]. In addition, increasing Braak stage was associated with increasing amyloid burden as determined by amyloid PET [20]. In addition, cerebrospinal fluid (CSF) studies have provided useful insights. It is known that Ab42 levels in CSF decrease due to the presence of Ab deposition, whereas total Tau and phosphorylated (p)Tau levels increase with disease progression. It is likely that

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increases in tau and p-tau in CSF is due to ongoing synaptic and neuronal damage and not specifically due to the presence of NFTs. Interestingly, CSF tau and p-tau are not increased in the presence of NFTs in non-AD tauopathies such as PSP. Reflecting the fact that increases in CSF tau somehow reflect the process of neurodegeneration in AD, it has been shown that cognitively normal people with high levels of tau combined with low Ab42 almost all progressed to mild dementia or mild cognitive impairment (MCI) [31]; and those with MCI progressed to AD [32]. To study the interactions of Ab and tau in mice, a variety of models have been developed, including one generated by G€otz et al, where mice from a line transgenic for human P301L tau were injected with Ab42 fibrils, producing a synergistic response with a five-fold increase in NFTs [33]. Other models with mutant genes for both MAPT and APP have also been produced, supporting the idea that tau and Ab interact to produce AD pathology [34]. Finally, when tau was knocked out in an Ab-overproducing transgenic mouse, Ab continued to aggregate, but every aspect of the clinical phenotype was rescued, leading to the suggestion that tau enhanced the toxicity of Ab [35]. There is also evidence that in a model of AD, cortical Ab plaques accelerates the spread of neurofibrillary degeneration throughout the cortex, amplifying neuronal loss [36], although the mechanisms by which Ab mediated tau spreading remain unclear. There may also be a role for the innate immune system. Indeed, in a mouse model of tauopathy, it has been shown that microglial cells were involved in tau spread via exosome secretion and that inhibiting exosome synthesis blocked tau propagation [37]. Animal models, as well as human studies, have led to the suggestion that normal tau may mediate Ab toxicity [35,38–42]. However, as mentioned earlier, a consensus seems to be emerging that in AD and other tauopathies, toxicity is mediated by a gain of toxic function caused by one or more conformationally altered species of tau, which form oligomers and aggregates. Tau pathology seems to be associated with is its ability to selfassemble, into either oligomers or full-blown filaments. In a regulatable transgenic mouse model, it is possible to initiate pathology by inducing “pro-aggregant” mutations which increase the propensity for beta structure, but perhaps more importantly, as a proof-of-concept, it is also possible to completely eliminate tau pathology by “anti-aggregant” mutations, which do not allow beta structure [43]. These data suggest that preventing or disrupting tau aggregation and subsequent spreading could be an important therapeutic target in AD and other tauopathies. 4. Biomarkers and other tools Developing programs for tau therapeutics will require not only new and improved animal models, including those mentioned above but also new imaging technologies and biomarkers. For example, development of ligands for

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in vivo imaging of tau aggregates has recently moved from the research laboratory into the clinic, showing good correlations with neuropathologic studies from patients with AD and suggesting that these ligands may make it possible to follow the progression of tau accumulation longitudinally [44]. Development of blood and CSF biomarkers has also progressed, focusing not only on improved measurements of tau and phosphorylated tau but also other markers of neurodegeneration. For example, CSF levels of neurofilament light chain correlated better than tau as a measure of disease severity in several primary tauopathies [45]. Novel methods for measuring various species of tau in CSF have also advanced, recognizing the fact that tau is an active, kinetically dynamic protein. Stable isotope-labeling kinetics quantitation, for example, enables investigators to assess the kinetics of tau metabolism under physiological conditions, as tau is generated, post-translationally modified, spreads, and is degraded over the course of the disease [46]. 5. Therapeutics Diverse therapeutic approaches have been proposed, as illustrated in Fig. 1. They include reducing tau expression, inhibiting the assembly of tau, disrupting tau aggregates, boosting cellular mechanisms to break down and eliminate toxic forms of tau, stabilizing microtubules, and inhibiting the spread of misfolded tau. A number of small molecule inhibitors are in development to target tau along multiple points in the pathogenic pathway. For example, a small molecule inhibitor of the deubiquitinating enzyme Usp14 has been shown to enhance proteasome activity [48], which may help to clear toxic forms of tau in the brain [49]. Another strategy uses an orally available small molecule inhibitor of O-GlcNAase (OGA) to increase O-GlcNAcylation in the brain, which has been shown by several groups to protect against tau and Ab toxicity in preclinical AD mouse models [50]. Studies in bigenic MAPT/APP mutant mice showed that inhibition of OGA

prevented cognitive decline and amyloid plaque formation [51] and in JNPL3, tauopathy mice acted to block the formation of hyperphosphorylated tau, the development of tau aggregates, and neurodegeneration [52]. Various protective mechanisms are possible; however, it is interesting to note that O-GlcNAcylation of tau was recently shown to attenuate tau aggregation in vitro [53]. Inhibiting acetylation of tau has also been proposed as a therapeutic strategy. Tau acetylation at K174 has been identified as an early change in AD brains as well as a mediator of neurodegeneration [54]. Inhibition of acetyltransferase p300 with salsalate has been shown to lower levels of soluble tau, reduce hippocampal atrophy, and improve performance in behavioral studies [55]. Small molecules may also be useful as inhibitors of fibril formation and aggregation [56]. Biologics are also in development as tau therapeutics. For example, tau anti-sense oligonucleotides and siRNAs may work through a variety of mechanisms, including a reduction in the levels of phosphorylated tau, moderation of RNA splicing, tau silencing, and blockade of tau seeding [57,58]. Immunotherapy also offers several possible therapeutic pathways, including blocking neuronal uptake of tau aggregates and promoting clearance of aggregates by microglia [59–62]. 5.1. Strategies for developing therapeutics Developing treatments for tauopathies as a strategy for AD drug development offers potential advantages in terms of more precise targeting, increased efficiency of trials because of more rapid progression of disease, and opportunities to access specific regulatory pathways. For example, the Orphan Drug Act, passed by the U.S. Congress in 1983, provides incentives for sponsors to develop drugs for diseases that affect fewer than 200,000 Americans. PSP is one such disease; thus, targeting PSP in a therapeutic trial may offer a way to reduce risk to sponsors and accelerate tau-based AD drug development because the Food and Drug Administration has stated that “studies in etiologically

Fig. 1. Potential tau therapeutic strategies [47].

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or pathophysiologically related conditions. can support each other, allowing initial approval of several uses or allowing additional claims based on a single adequate and wellcontrolled study” [63]. Another approach would be to test tau-targeted therapies in patients with FTLD. To this end, an international group of clinical investigators, with support from the NIH, has established an FTLD registry that includes hundreds of FTLD mutation carriers who may be candidates for clinical trials; as well as a research network including clinical sites and cores for imaging, genetics, and biospecimens to support such trials. It should be noted, however, that more research is needed to confirm that tau’s role in FTLD, PSP, AD, and other tauopathies is similar enough such that Tau therapeutics will be effective across a range of tauopathies. 6. Conclusions In recent years, pharmaceutical and biotech companies have invested substantial resources into the development of tau-targeted therapeutics, particularly immunotherapeutics. However, many questions remain to be answered regarding the species of tau (soluble, aggregated, posttranslationally modified, and so forth) that drives neuronal dysfunction, as well as the treatment approach most likely to arrest or reverse tau-mediated pathogenesis. Trying to draw inferences about biology from structural changes, we see at autopsy is limited; thus, it will be essential to continue improving in vivo imaging tools as well as other biomarkers that enable quantitation of the biochemical changes associated with tauopathy. There is strong evidence that in AD, Ab exacerbates pathologic changes in tau and pathogenesis, albeit through unknown mechanism(s). Understanding these mechanisms is a key goal of current AD research, as it may uncover, not only a better understanding of disease but also novel treatment strategies. More efficient and successful drug development also requires the identification of biomarkers of disease progression and treatment effectiveness, as well as trial designs and infrastructure, to maximize the resources available, including the most precious of all, the patients themselves. Acknowledgments Funding: DMH: Tau Consortium, The JPB Foundation, NIH grants NS090934, AG04867801. The authors thank Heidi Jurgens for editorial input and Meredith McNeil for logistical planning of the roundtable as well as the contributing speakers: Karen Ashe, MD, PhD, Randy Bateman, MD, Kaj Blennow, MD, PhD, Adam Boxer, MD, PhD, Joel Braunstein, MD, Kurt Brunden, PhD, Peter Davies, PhD, David Eisenberg, PhD, Li Gan, PhD, Alfred Goldberg, PhD, Mansuo Hayashi, PhD, Brad Hyman, MD, PhD, Michael Irizarry, MD, Tim Miller, MD, PhD, Mark Mintun, MD, Tom Montine, MD, PhD, Gil Rabinovici, MD, Andrew Stephens, MD, and David Vocadlo, PhD.

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RESEARCH IN CONTEXT

1. Systematic review: We reviewed the literature of recent work exploring the role of Tau pathology in Alzheimer’s and other neurodegenerative diseases. The development of sophisticated preclinical models and the emergence of tau PET imaging and fluid biomarkers have improved our understanding of tau biology and may affect the diagnosis and treatment of clinical Alzheimer’s disease (AD) and other tauopathies. 2. Interpretation: The present paper posits that Tau is a key driver of disease progression making it both an attractive therapeutic target but also a valuable biomarker. 3. Future directions: A better understanding of the mechanism of how Ab exacerbates pathological changes in tau and pathogenesis is needed and could lead to new therapeutic strategies. In addition, many questions remain regarding the species of tau (soluble, aggregated, post-translationally modified, etc.) that drives neuronal dysfunction, as well as the treatment approach most likely to arrest or reverse taumediated pathogenesis.

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