Volume 40, Supplement 1, 2014

Propagation of Tau Pathology (Guest Editors: Miguel Medina and Jesus Avila)

Pages S1-S3
Preface
Miguel Medina, Jesús Avila
Is Tau a Prion-Like Protein?

Page S5
Obituary
William R. Brinkley, George Perry
Lester (Skip) Binder (1949-2013): In the beginning was tau

Pages S7-S15
Review
Jesus Avila, Diana Simon, Miguel Diaz-Hernandez, Jesús Pintor, Felix Hernandez
Sources of Extracellular Tau and its Signaling
Abstract: The pathology associated with tau protein, tauopathy, has been recently analyzed in different disorders, leading to the suggestion that intracellular and extracellular tau may itself be the principal agent in the transmission and spreading of tauopathies. Tau pathology is based on an increase in the amount of tau, an increase in phosphorylated tau, and/or an increase in aggregated tau. Indeed, phosphorylated tau protein is the main component of tau aggregates, such as the neurofibrillary tangles present in the brain of Alzheimer’s disease patients. It has been suggested that intracellular tau could be toxic to neurons in its phosphorylated and/or aggregated form. However, extracellular tau could also damage neurons and since neuronal death is widespread in Alzheimer’s disease, mainly among cholinergic neurons, these cells may represent a possible source of extracellular tau. However, other sources of extracellular tau have been proposed that are independent of cell death. In addition, several ways have been proposed for cells to interact with, transmit, and spread extracellular tau, and to transduce signals mediated by this tau. In this work, we will discuss the role of extracellular tau in the spreading of the tau pathology.

Pages S17-S22
Review
C.L. Sayas, Jesús Ávila
Crosstalk between Axonal Classical Microtubule-Associated Proteins and End Binding Proteins during Axon Extension: Possible Implications in Neurodegeneration
Abstract: During neuronal development, spherical neuroblasts differentiate into mature neurons through the extension of a long axon and several shorter dendrites. Morphological changes that underlie neuronal differentiation are mostly driven by the microtubular cytoskeleton. Regulation of microtubule dynamics and stability during axon and dendrite extension relies on the action of different families of microtubular proteins, such as classical microtubule-associated proteins (MAPs) and microtubule plus-end tracking proteins (+TIPs). This review article addresses recent research on the crosstalk between the main axonal MAPs, tau and MAP1B, and end binding proteins (EBs), the core +TIPs, during axon outgrowth in developing neuronal cells. Furthermore, we discuss the potential implications of a dysregulation of the interplay between tau and EBs in neurodegenerative disorders such as Alzheimer’s disease.

Pages S23-S26
Illana Gozes, Tal Iram, Evgenia Maryanovsky, Carmit Arviv, Liora Rozenberg, Yulie Schirer, Eliezer Giladi, Sharon Furman-Assaf
Novel Tubulin and Tau Neuroprotective Fragments Sharing Structural Similarities with the Drug Candidate NAP (Davuentide)
Abstract: NAP (NAPVSIPQ, davunetide) is a microtubule stabilizing peptide drug candidate. Here, we set out to identify NAP-like peptides that provide neuroprotection and reduce tau pathology. NAP-like peptides were derived using publically available search engines, which identified sequence homologies in the microtubule subunit tubulin and in the microtubule associated protein, tau. NATLSIHQ (NAT) and STPTAIPQ were derived from tubulin, and TAPVPMPD (TAP) was derived from tau. All peptides provided neuroprotection against the Alzheimer’s disease (AD) toxin, the amyloid-β 1-42 peptide, although NAT and TAP were much more potent than STPTAIPQ. NAT also protected astrocytes, while STPTAIPQ was active only at micromolar concentrations. Because NAT and TAP were much more potent than STPTAIPQ in neuroprotection, those peptides were also tested for inhibition of tau-like aggregation (the second protein hallmark pathology of AD). Both NAT and TAP inhibited tau-like aggregation, with NAT being active over a very broad concentration range. NAT also protected in vivo in a frontotemporal dementia transgenic mouse model (Tau-Tg), when tested at the age of ~10 months. Results showed significantly decreased levels of the NAP parent protein, activity-dependent neuroprotective protein in the cerebral cortex of the Tau-Tg which was increased back to normal levels by NAT treatment. This was coupled to protection of Brain-Body weight ratio in the compromised Tau-Tg. With AD being the major tauopathy and with tau taking part in frontotemporal dementia, novel NAP derivatives that reduce tauopathy and provide neuroprotection are of basic and clinical interest.

Pages S37-S45
Review
Diane P. Hanger, Dawn H.W. Lau, Emma C. Phillips, Marie K. Bondulich, Tong Guo, Benjamin W. Woodward, Amy M. Pooler, Wendy Noble
Intracellular and Extracellular Roles for Tau in Neurodegenerative Disease
Abstract: Tau has a well-established role as a microtubule-associated protein, in which it stabilizes the neuronal cytoskeleton. This function of tau is influenced by tau phosphorylation state, which is significantly increased in Alzheimer’s disease and related tauopathies. Disruptions to the cytoskeleton in disease-affected neurons include reduced length and numbers of stable microtubules, and their diminished stability is associated with increased tau phosphorylation in disease. Tau is also localized in the nucleus and plasma membrane of neurons, where it could have roles in DNA repair and cell signaling. Most recently, potential roles for extracellular tau have been highlighted. The release of tau from neurons is a physiological process that can be regulated by neuronal activity and extracellular tau may play a role in inter-neuronal signaling. In addition, recent studies have suggested that the misfolding of tau in diseased brain leads to abnormal conformations of tau that can be taken up by neighboring neurons. Such a mechanism may be responsible for the apparent prion-like spreading of tau pathology through the brain, which occurs in parallel with clinical progression in the tauopathies. The relationship between tau localization in neurons, tau release, and tau uptake remains to be established, as does the function of extracellular tau. More research is needed to identify disease mechanisms that drive the release and propagation of pathogenic tau and to determine the impact of extracellular tau on cognitive decline in neurodegenerative disease.

Pages S47-S70
Sudad Saman, Norman C.Y. Lee, Itoro Inoyo, Jun Jin, Zhihan Li, Thomas Doyle, Ann C. McKee, Garth F. Hall
Proteins Recruited to Exosomes by Tau Overexpression Implicate Novel Cellular Mechanisms Linking Tau Secretion with Alzheimer’s Disease
Abstract: Tau misprocessing to form aggregates and other toxic species has emerged as a major feature in our developing understanding of the etiology and pathogenesis of Alzheimer’s disease (AD). The significance of tau misprocessing in AD has been further emphasized by recent studies showing that tau can be secreted from neurons via exosomes and may itself be an important agent in the spreading of neurofibrillary lesions within the brain. Tau secretion occurs most readily under disease-associated conditions in cellular models, suggesting that cellular changes responsible for secretion, possibly including tau oligomerization, could play a key role in the propagation of neurofibrillary lesions in neurodegenerative disease. Here we show that overexpression of 4R0N human tau in neuroblastoma cells recruits mitochondrial and axonogenesis-associated proteins relevant to neurodegeneration into the exosomal secretion pathway via distinct mechanisms. The recruitment of mitochondrial proteins appears to be linked to autophagy disruption (exophagy) in multiple neurodegenerative conditions but has few known direct links to AD and tau. By contrast, the involvement of synaptic plasticity and axonogenesis markers is highly specific to both tau and AD and may be relevant to the reactivation of developmental programs involving tau in AD and the recently demonstrated ability of secreted tau to establish tissue distribution gradients in CNS neuropil. We also found a highly significant correlation between genes that are significantly downregulated in multiple forms of AD and proteins that have been recruited to exosomes by tau, which we interpret as strong evidence for the central involvement of tau secretion in AD cytopathogenesis. Our results suggest that multiple cellular mechanisms may link tau secretion to both toxicity and neurofibrillary lesion spreading in AD and other tauopathies.

Pages S71-S77
Alejandro Martinez-Aguila, Begoña Fonseca, Felix Hernandez, Miguel Díaz-Hernandez, Jesús Avila, Jesus Pintor
Tau Triggers Tear Secretion by Interacting with Muscarinic Acetylcholine Receptors in New Zealand White Rabbits
Abstract: In recent years, in vitro experiments have shown that the spread of Alzheimer’s disease is caused by a non-conventional activation of muscarinic receptors by dephosphorylated extracellular tau protein. However, so far, in vivo data to support this hypothesis has not been obtained. The eye provides a good model where cholinergic (muscarinic) transmission can be analyzed. The role of muscarinic receptors in the stimulation of lacrimal gland secretion has already been described, and it has been suggested that acetylcholine is the main transmitter controlling tear secretion. In this project, we have studied the interaction between tau and muscarinic receptors by analyzing tear secretion in the eyes of white rabbits. Our results show that tau protein increases tear secretion by 47.2% in a similar way to a muscarinic receptor agonist carbachol (84.3%). The use of muscarinic antagonists indicated that tau interacts with M1 and mainly M3 muscarinic receptors. In summary, tau can bind muscarinic receptors in vivo and this may explain the spread of the pathology.

Pages S79-S89
Javier de Cristóbal, Luis García-García, Mercedes Delgado, Mar Pérez, Miguel A. Pozo, Miguel Medina
Longitudinal Assessment of a Transgenic Animal Model of Tauopathy by FDG-PET Imaging
Abstract: Abnormal levels and hyperphosphorylation of tau protein have been proposed as the underlying cause of a group of neurodegenerative disorders collectively known as ‘tauopathies’. The detrimental consequence is the loss of affinity between this protein and the microtubules, increased production of fibrillary aggregates, and the accumulation of insoluble intracellular neurofibrillary tangles. A similar phenotype can be observed in various preclinical models, which have been generated to study the role of tau protein in neurodegenerative disorders. In this study, we have analyzed the brain metabolic activity in an animal model of tauopathy (tauVLW transgenic mice), which has been previously reported to mimic some of the phenotypic features of these disorders. By using a non-invasive technique, positron emission tomography (PET), a longitudinal non-clinical follow up study was carried out during most of the lifespan of these transgenic mice, from the youth to the senescence stages. The results obtained point out to an aging-dependent decrease in 18F-fluoro-deoxyglucose (FDG) uptake in the cerebral areas analyzed, which was already significant at the adult age, i.e., 11 months, and became much more prominent in the oldest animals (19 months old). This observation correlates well with the histopathological observation of neurodegeneration in brain areas where there is overexpression of tau protein.

Pages S91-S96
Short Communication
Naruhiko Sahara, Yan Ren, Sarah Ward, Lester I. Binder, Tetsuya Suhara, Makoto Higuchi
Tau Oligomers as Potential Targets for Early Diagnosis of Tauopathy
Abstract: The discovery of tau mutations in frontotemporal dementia has been a key event in neurodegenerative disease research. The rTg4510 mouse line expressing human tau with P301L FTDP-17-tau mutation has been established to understand the role of tau in neurodegeneration. Our histological analyses with tau antibodies and fluorescent tau ligands on rTg4510 mice revealed that tau oligomer formation was distinct from tangle formation. While in vivo imaging of mature tangles is now available, imaging biomarkers for tau oligomers would be useful for clarifying their roles in neurotoxicity and for diagnosing early-stage tau pathology.

Pages S97-S111
Diana L. Castillo-Carranza*, Julia E. Gerson*, Urmi Sengupta, Marcos J. Guerrero-Muñoz, Cristian A. Lasagna-Reeves, Rakez Kayed *These authors contributed equally to this work.
Specific Targeting of Tau Oligomers in Htau Mice Prevents Cognitive Impairment and Tau Toxicity Following Injection with Brain-Derived Tau Oligomeric Seeds
Abstract: Neurodegenerative disease is one of the greatest health crises in the world and as life expectancy rises, the number of people affected will continue to increase. The most common neurodegenerative disease, Alzheimer’s disease, is a tauopathy, characterized by the presence of aggregated tau, namely in the form of neurofibrillary tangles. Historically, neurofibrillary tangles have been considered the main tau species of interest in Alzheimer’s disease; however, we and others have shown that tau oligomers may be the most toxic form and the species responsible for the spread of pathology. We developed a novel anti-tau oligomer-specific mouse monoclonal antibody (TOMA) and investigated the potential of anti-tau oligomer passive immunization in preventing the toxicity of tau pathology in Htau mice. We injected pure brain-derived tau oligomers intracerebrally in 3-month-old wild-type and Htau mice and investigated the protective effects of a single 60 µg TOMA injection when compared to the same dose of non-specific IgG and found that TOMA conferred protection against the accumulation of tau oligomers and cognitive deficits for up to 1 month after treatment. Additionally, we injected pure brain-derived tau oligomers intracerebrally in 3-month-old wild-type and Htau mice and treated animals with biweekly injections of 60 µg TOMA or non-specific IgG. We found that long-term administration of TOMA was effective as a preventative therapy, inhibiting oligomeric tau and preserving memory function. These results support the critical role of oligomeric tau in disease progression and validate tau oligomers as a potential drug target.

Pages S113-S121
Erin E. Congdon, Senthilkumar Krishnaswamy, Einar M. Sigurdsson
Harnessing the Immune System for Treatment and Detection of Tau Pathology
Abstract: The tau protein is an attractive target for therapy and diagnosis. We started a tau immunotherapy program about 13 years ago and have since demonstrated that active and passive immunotherapies diminish tau pathology and improve function, including cognition, in different mouse models. These findings have been confirmed and extended by several groups. We routinely detect neuronal, and to a lesser extent microglial, antibody uptake correlating with tau pathology. Antibodies bind tau aggregates in the endosomal/lysosomal system, enhancing clearance presumably by promoting their disassembly. Extracellular clearance has recently been shown by others, using antibodies that apparently are not internalized. As most pathological tau is neuronal, intracellular targeting may be more efficacious. However, extracellular tau may be more accessible to antibodies, with tau-antibody complexes a target for microglial phagocytosis. The extent of involvement of each pathway may depend on numerous factors including antibody properties, degree of pathology, and experimental model. On the imaging front, numerous tau ligands derived from β-sheet dyes have been developed by several groups, some with promising results in clinical PET tests. Postmortem analysis should clarify their tau specificity, as in theory and based on histological staining, those are likely to have some affinity for various amyloids. We are developing antibody-derived tau probes that should be more specific, and have in mouse models shown in vivo detection and binding to pathological tau after peripheral injection. These are exciting times for research on tau therapies and diagnostic agents that hopefully can be applied to humans in the near future.

Pages S123-S133
Alberto Rábano, Izaskun Rodal, Raquel Cuadros, Miguel Calero, Félix Hernández, Jesús Ávila
Argyrophylic Grain Pathology as a Natural Model of Tau Propagation
Abstract: Argyrophylic grain disease (AGD) is a sporadic 4R tauopathy that usually presents in combination with other sporadic tauopathies or with Alzheimer’s disease (AD) pathology, and may contribute to dementia in older age patients. In previous studies, a detailed analysis of AGD pathology in the medial temporal lobe has been hampered by the common presence of concurrent AD changes. With the objective to assess the potentiality of AGD in research on tau propagation, here we present a study of a series of AGD postmortem cases (n = 53). The total series was divided in a subgroup of cases with Braak-stage ≤ II (n = 23) and a subgroup with Braak-stage > II or indeterminate (n = 30) in order to minimize interference with AD pathology. A detailed neuropathological evaluation of the medial temporal lobe was performed at three coronal levels with Gallyas stain, and immunostains with p62, AT8, and AT100 antibodies. Western blot analysis of the entorhinal and hippocampal cortex was performed in 8 cases with a panel of anti-tau antibodies. Cases were genotyped for APOE polymorphism and for H1/H2 alleles of the MAPT gene. All cases, and particularly lower-Braak stage cases, displayed a highly homogeneous pattern of involvement by argyrophylic grains and pretangles between connected regions (primarily basolateral nuclei of the amygdala, entorhinal/transentorhinal cortex, and hippocampal cortex). Staging of cases reveals progression of pathology along well-established neuroanatomical pathways. Western blot studies yielded a specific pattern of isoforms with a characteristic predominant band at 64 kDa. Genetic analysis showed a strong association with the H1 allele of the MAPT gene. AGD may thus be an optimal natural disease model for testing hypotheses related to tau propagation in human tissue.

Pages S135-S145
Kunie Ando*, Anna Kabova*, Virginie Stygelbout, Karelle Leroy, Céline Heraud, Christelle Frédérick, Valérie Suain, Zehra Yilmaz, Michèle Authelet, Robert Dedecker, Marie-Claude Potier, Charles Duyckaerts, Jean-Pierre Brion *These authors equally contributed to this work.
Vaccination with Sarkosyl Insoluble PHF-Tau Decrease Neurofibrillary Tangles Formation in Aged Tau Transgenic Mouse Model: A Pilot Study
Abstract: Active immunization using tau phospho-peptides in tauopathy mouse models has been observed to reduce tau pathology, especially when given prior to the onset of pathology. Since tau aggregates in these models and in human tauopathies are composed of full-length tau with many post-translational modifications, and are composed of several tau isoforms in many of them, pathological tau proteins bearing all these post-translational modifications might prove to be optimal tau conformers to use as immunogens, especially in models with advanced tau pathology. To this aim, we immunized aged wild-type and mutant tau mice with preparations containing human paired helical filaments (PHF) emulsified in Alum-adjuvant. This immunization protocol with fibrillar PHF-tau was well tolerated and did not induce an inflammatory reaction in the brain or adverse effect in these aged mice. Mice immunized with four repeated injections developed anti-PHF-tau antibodies with rising titers that labeled human neurofibrillary tangles in situ. Immunized mutant tau mice had a lower density of hippocampal Gallyas-positive neurons. Brain levels of Sarkosyl-insoluble tau were also reduced in immunized mice. These results indicate that an immunization protocol using fibrillar PHF-tau proteins is an efficient and tolerated approach to reduce tau pathology in an aged tauopathy animal model.