Neurobiology of Aging 36 (2015) 1316e1332

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Tau immunization: a cautionary tale? Alexandra J. Mably a, Daniel Kanmert a, Jessica M. Mc Donald a, Wen Liu a, Barbara J. Caldarone b, Cynthia A. Lemere a, Brian O’Nuallain a, Kenneth S. Kosik c, Dominic M. Walsh a, * a Laboratory for Neurodegenerative Research, Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Harvard Institutes of Medicine, Boston, MA, USA b Neurobehaviour Laboratory Core, Harvard NeuroDiscovery Center, Boston, MA, USA c Department of Molecular, Cellular and Developmental Biology, Neuroscience Research Institute, University of California, Santa Barbara, CA, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 May 2014 Received in revised form 29 September 2014 Accepted 5 November 2014 Available online 30 December 2014

The amyloid b (Ab)-protein and microtubule-associated protein, tau, are the major components of the amyloid plaques and neurofibrillary tangles that typify Alzheimer’s disease (AD) pathology. As such both Ab and tau have long been proposed as therapeutic targets. Immunotherapy, particularly targeting Ab, is currently the most advanced clinical strategy for treating AD. However, several Ab-directed clinical trials have failed, and there is concern that targeting this protein may not be useful. In contrast, there is a growing optimism that tau immunotherapy may prove more efficacious. Here, for the first time, we studied the effects of chronic administration of an anti-tau monoclonal antibody (5E2) in amyloid precursor protein transgenic mice. For our animal model, we chose the J20 mouse line because prior studies had shown that the cognitive deficits in these mice require expression of tau. Despite the fact that 5E2 was present and active in the brains of immunized mice and that this antibody appeared to engage with extracellular tau, 5E2-treatment did not recover age-dependent spatial reference memory deficits. These results indicate that the memory impairment evident in J20 mice is unlikely to be mediated by a form of extracellular tau recognized by 5E2. In addition to the lack of positive effect of anti-tau immunotherapy, we also documented a significant increase in mortality among J20 mice that received 5E2. Because both the J20 mice used here and tau transgenic mice used in prior tau immunotherapy trials are imperfect models of AD our results recommend extensive preclinical testing of anti-tau antibodyebased therapies using multiple mouse models and a variety of different anti-tau antibodies. Ó 2015 Elsevier Inc. All rights reserved.

Keywords: Tau immunotherapy APP Transgenic mice Alzheimer’s disease Extracellular tau

1. Introduction Persuasive evidence indicates that the amyloid b (Ab)-protein and the microtubule-associated protein, tau, each play important roles in Alzheimer’s disease (AD) (Ballatore et al., 2007; Tanzi, 2012). Indeed, tau and Ab appear to be linked at a molecular level and a large number of studies indicate that Ab can induce changes in tau localization (Hampel et al., 2010; Ittner et al., 2010; Vossel et al., 2010), phosphorylation (Busciglio et al., 1995; Geula et al., 1998; Jin et al., 2011), and aggregation (Gotz et al., 2001). There is also considerable evidence that tau is required for Ab toxicity (Liu

* Corresponding author at: Laboratory for Neurodegenerative Research, Ann Romney Center for Neurologic Diseases, Brigham & Women’s Hospital and Harvard Medical School, Harvard Institutes of Medicine (R 921), 77 Avenue Louis Pasteur, Boston, MA 02115, USA. Tel.: þ1 617 525 5059; fax: þ1 617 525 5252. E-mail address: [email protected] (D.M. Walsh). 0197-4580/$ e see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2014.11.022

et al., 2004; Park and Ferreira, 2005; Rapoport et al., 2002), and Ab-mediated changes in synaptic plasticity (Shipton et al., 2011) and memory (Ittner et al., 2010; Roberson et al., 2007, 2011). Based on these data, it is widely accepted that tau is an essential fulcrum for Ab toxicity and therefore a prime target for therapy (Gotz et al., 2012; Haass and Mandelkow, 2010; Morris et al., 2011). Efforts at therapeutically targeting tau have been centered on modulation of tau aggregation or phosphorylation, however, with the recent realization that tau is normally released from cells (Barten et al., 2011; Chai et al., 2012), and that propagation of tau pathology may involve transfer of tau from one neuron to another (Ahmed et al., 2014; Clavaguera et al., 2009; de Calignon et al., 2012; Dujardin et al., 2014; Kim et al., 2010; Liu et al., 2012), there is growing interest in using antibodies to bind and remove extracellular tau (Clavaguera et al., 2014; Yanamandra et al., 2013). To date, most studies have utilized active immunization to phosphorylated fragments of tau (Asuni et al., 2007; Bi et al., 2011; Boimel et al., 2010; Boutajangout et al., 2010; Rozenstein-Tsalkovich et al., 2013;

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Troquier et al., 2012) or passive immunization with phospho-specific or conformational-specific anti-tau monoclonal antibodies (mAbs) (Boutajangout et al., 2011; Castillo-Carranza et al., 2014; Chai et al., 2011; d’Abramo et al., 2013; Kontsekova et al., 2014b; Walls et al., 2014; Yanamandra et al., 2013). Importantly, all such studies have been conducted in tau transgenic (tg) mice and as such robust analysis of the effects of mAbs on cognition has not been possible. Although amyloid precursor protein (APP) tg mice are also imperfect models of AD (Ashe and Zahs, 2010) certain APP tgs develop readily measureable spatial reference memory deficits that are tau dependent and can be reversed by administration of anti-Ab antibodies (Dodart et al., 2002; Lee et al., 2006; Rasool et al., 2013; Zago et al., 2012). Moreover, in 2 separate APP tg lines (J20 and APP23) genetic ablation of tau has also been shown to recover behavioral and cognitive deficits (Ittner et al., 2010; Roberson et al., 2007, 2011). Consequently, we reasoned that J20 mice would be well suited to study the therapeutic potential of antitau mAbs. Here, we characterized the behavioral phenotype of J20 mice, and then selected an appropriate age and task in which to test the effects of chronic administration of the anti-tau mAb, 5E2. Because we found J20 mice to show mild spatial memory impairments in the radial arm maze (RAM) at 8 months and more pronounced deficits at 12 months, we administered 5E2 starting at 9.5 months and tested animals at 12 months. Although 5E2 was found in the brain of treated mice, antibody administration was unable to attenuate spatial reference memory impairments. Furthermore, 5E2 caused a significant increase in the mortality of J20 but not wild type (Wt) mice. These results recommend the need for: (1) caution when appraising the prior success of anti-tau mAbs administered to tau transgenic mice and (2) extensive testing of tau immunotherapy in preclinical models of AD that do not over-express tau.

2. Methods 2.1. Mice All animal procedures were approved by the Harvard Medical School Institutional Animal Care and Use Committee. Mice were housed under a 12-hour light:dark cycle (lights on 7 AM, lights off 7 PM). Ad libitum food (standard chow, LabDiet, Richmond, IN) was provided unless otherwise indicated. Male hemizygous hAPPSwe/Ind mice (J20) were obtained from Jackson Laboratories (Bar Harbor, ME) and crossed with C57BL/6J female mice to produce hemizygous J20 mice or Wt littermate controls. J20 mice over-express hAPP carrying the Swedish (KM670/671NL) and Indiana (V717F) mutations (Mucke et al., 2000; Palop et al., 2003). Pups were weaned at 20e21 days old, male progeny were tail snipped and genotyped. Tails were digested and DNA extracted using DNeasy blood and tissue kit (Qiagen Sciences, MD). DNA was subjected to polymerase chain reaction using primers for APP (5’: GGT GAG TTT GTA AGT GAT GCC, 3’: TCT TCT TCT TCG ACC TCA GC) and glial fibrillary acidic protein; 5’: GCG CGC TCG TGC ACA CTT ATC ACA C, 3’: CTG CCC CTG ACT TCC TGG AAG CAC). Polymerase chain reaction products were separated on a 0.9% (wt/vol) agarose gel. Male J20 and Wt littermates were group housed with animals of the same genotype and aged to 4 or 8 months before behavioral testing. On retirement from breeding, male J20 mice were individually housed and at 12 months used for behavioral testing. Male C57BL/6J mice were also individually housed and aged alongside the J20 cohort to provide identically treated 12-month Wt controls. For the immunization study, male hemizygous J20 mice or Wt littermate controls were the F1 progeny of 7 of the 12 months group of J20 mice behaviorally characterized, and Wt female mice. Mice were group housed (2e4 animals per cage) until 5 days before behavioral testing, and after this time mice were housed individually.

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2.2. Behavioral characterization Initially mice of 3 different ages (4, 8, and 12 months) were tested in open field, spontaneous alternation Y-maze, RAM, and contextual fear conditioning (CFC) paradigms. Thereafter, we chose to test the effect of passive immunization with an anti-tau antibody on the performance of 12 months mice in open field and RAM. Mice were single housed and handled daily for 5 days before beginning behavioral testing. Testing always took place during the light photoperiod, unless otherwise stated. Mice were acclimatized to testing rooms for at least 20 minutes before testing began. 2.2.1. Open field Mice were tested in an open field chamber (27.3 cm  27.3 cm  20.3 cm; Med Associates, St. Albans, VT) for a total of 60 minutes, as described previously (Dillon et al., 2008; Kornecook et al., 2010). Infrared beams across the chamber track the animal’s placement and movement within the chamber. Data were analyzed using Activity Monitor software (Version 5.9; Med Associates) to give distance traveled. Zone analysis gathered using the same system allowed calculation of percentage time spent in the center of the open field chamber. 2.2.2. Spontaneous alternation Y-maze Mice were assessed in the spontaneous alternation Y-maze, as described previously (Dillon et al., 2008; Kornecook et al., 2010). The Y-maze consisted of 3 arms (30 cm  10 cm) with 20 cm high walls, placed at 120o angles to one another and arbitrarily designated from right to left as A, B, and C. Mice were placed in the center of the maze (where all 3 arms intersect) and allowed to freely explore the maze for a 6-minute interval. Movement within the maze and the sequence of arm entries was tracked using TopScan Suite (Version 1.00; CleverSys, Reston, VA). Only when an animal placed all 4 limbs in an arm was it counted as having entered that arm. An alternation was defined as 3 successful consecutive entries to the 3 arms of the maze (i.e., ABC, ACB, BCA, BAC, CBA, or CAB). Percentage alternation was calculated by dividing number of alternations by total number of possible alternations (number of arm entries subtracted by 2) and multiplying by 100. 2.2.3. Radial arm maze Five days before testing in the RAM mice were placed on a food restricted diet. Mice were weighed daily and fed food rations to lower their body weight to approximately 85% of their free-feeding weight. Mice were maintained at approximately 85% free-feeding bodyweight for habituation and testing in the RAM. Testing was carried out on a maze (Lafayette Instruments, Lafayette, IN), consisting of 8 arms (33 cm  5.5 cm), with clear polycarbonate side walls, radiating from a central platform (27 cm diameter), and spaced equally apart from one another (45o), as previously described (Dillon et al., 2008). The maze was surrounded by extramaze cues (cut-out shapes on the walls), which allowed the mice to orientate themselves on the maze. To prevent the animals visualizing food pellets, food cups were situated at the end of each arm 0.5 cm below the surface of the runway. Mice underwent 2 days of habituation during which all the arms of the maze were baited with 20 mg casein pellets (BioServ, Frenchtown, NJ). The mice were allowed 25 minutes to explore the maze and consume the food pellets. On the second day of habituation, once the animal had transversed an arm and collected the food pellet the arm was blocked off by a motorized door, so that the animal could not revisit that arm. This process habituated the animal to the use of the motorized doors. Following habituation mice underwent 8 consecutive days of spatial reference memory (SRM) training. For this only 3 arms were baited with food pellets, with the same 3

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Table 1 Primary antibodies Antibody

Immunogen and/or epitope

Host

Application (dilution)

IHC pretreatment

IHC secondary antibody

Source and reference

AW7

Ab1-40

Rabbit polyclonal

IHC (1:1000)

88% formic acida

21F12

Ab33-42

Mouse monoclonal

IHC (1 mg/mL), ELISA (1 mg/mL)

88% formic acid

Biotinylated goat, anti-rabbit Biotinylated anti-mouse IgG

2G3

Ab33-40

Mouse monoclonal

IHC (1 mg/mL)

88% formic acida

Biotinylated anti-mouse IgG

m266

Ab16-23

Mouse monoclonal

ELISA (3 mg/mL)

N/A

N/A

3D6

Ab1-5, requires free Asp1 of Ab

Mouse monoclonal

ELISA (1 mg/mL)

N/A

N/A

AT8

Phospho-tau Ser202/ Thr205 Specific for prehelical filament-like aggregate

Mouse monoclonal

IHC (5 mg/mL)

Microwaveb

Mouse monoclonal

IHC (5 mg/mL)

Microwaveb

Biotinylated goat, anti-mouse IgG1 Biotinylated goat, anti-mouse IgG1

Tau-1

Tau residues 189e207

Mouse monoclonal

Western blot (1 mg/mL)

N/A

N/A

BT-2

Tau residues 194e198

Mouse monoclonal

ELISA (2.5 mg/mL)

N/A

N/A

Tau-5

Tau residues 210e241

Mouse monoclonal

ELISA (1.7 mg/mL)

N/A

N/A

5E2

Tau residues 214e233

N/A

Tau residues 243e441 HIV glycoprotein 120

N/A N/A

N/A N/A

8E5

APP residues 444e592

Immunization (1 mg/ mL, 250 mL injection) Western blot (1 mg/mL) Immunization (1 mg/ mL, 250 mL injection) IHC (0.1 mg/mL)

N/A

K9JA 46-4

Mouse monoclonal (IgG1) Rabbit polyclonal Mouse monoclonal (IgG1) Mouse monoclonal Rat monoclonal

IHC (0.2 mg/mL)

Walsh Laboratory/Mc Donald et al. (2012) Elan Pharmaceuticals/ Johnson-Wood et al. (1997) Elan Pharmaceuticals/ Johnson-Wood et al. (1997) Elan Pharmaceuticals/ Seubert et al. (1992) Elan Pharmaceuticals/ Johnson-Wood et al. (1997) Pierce/Mercken et al. (1992a) Peter Davies (Albert Einstein College of Medicine, NY)/Jicha et al. (1997) Chemicon/Binder et al. (1985) Pierce/Mercken et al. (1992b) Calbiochem/LoPresti et al. (1995) Kosik Laboratory/Kosik et al. (1986) DAKO ATCC/Reeves et al. (1995) Elan Pharmaceuticals/ Games et al. (1995) AbD Serotec

MC1

CD45

Mouse B-cells

a

Microwaveb 88% formic acid

a

Biotinylated antimouse IgG Biotinylated goat, antirat, mouse adsorbed

Key: ELISA, enzyme-linked immunosorbent assay; IHC, immunohistochemistry; N/A, not applicable. a A total of 8 minutes incubation in 88% formic acid at room temperature. b Boiled in sodium citrate buffer (pH 6.0; BioGenex, San Ramon, CA) and allowed to cool slowly at room temperature.

spatial locations used throughout training. Mice underwent 6 trials a day with an approximately 1 minute inter-trial interval. Once an arm had been traversed, it was blocked by a motorized door, which remained in place for the remainder of that trial. Each trial lasted until the mouse collected all 3 food pellets or for a maximum of 5 minutes. The maze was cleaned with 70% ethanol between each trial and between mice. The extended time taken to run the paradigm prevented testing of the entire age cohort, so interleaved cohorts of 12e14 genotype- and age-balanced mice were tested. Any mice that consistently failed to explore the maze and eat the food pellets during habituation were removed from the task. 2.2.4. Contextual fear conditioning Mice were assessed in the CFC paradigm, conducted as previously described (Chen et al., 1996; Phillips and LeDoux, 1992), with minor modifications. The CFC chamber (30.5 cm  24.1 cm  21.0 cm; Med Associates) had plexiglass side walls, stainless steel end walls, and a floor of steel bars (4.8 mm diameter) with bars spaced 1.6 cm apart. The chamber was housed in a sound attenuation chamber, which also contained a yellow stimulus light and a fan to mask extraneous noise. On day 1, mice were placed in the CFC chambers for a total of 5 minutes; during the first 2 minutes, baseline freezing activity was recorded. Freezing was defined as the absence of ambulatory and stereotypic movement. The threshold for movement was set at 400 pixels per frame. After the initial 2 minutes period, mice received a 0.5 mA foot shock for a 2-second duration. Freezing activity was monitored for a further 2 minutes after which a second identical foot shock was administered.

Freezing activity was monitored for 1 minute, and animals were removed to their home cages. Twenty-four hours later, mice were tested for recall. Animals were placed in the CFC chamber, and freezing activity was recorded for a total of 3 minutes. Movement was monitored, and freezing calculated, using TopScan Suite software (Version 1.00; CleverSys). 2.3. Antibodies A total of 15 different antibodies were used in this study, the details of which are provided in Table 1. 2.4. Antibody administration Two IgG1 antibodies were used for passive immunotherapy in mice: (1) mAb 5E2 which recognizes a mid-region epitope of the tau protein (Kosik et al., 1986) and (2) mAb 46-4 which was raised to HIV coat protein 1 (Reeves et al., 1995). 5E2 was used because it recognizes all murine isoforms of tau irrespective of phosphorylation or aggregation status (Kosik et al., 1986; O’Dowd et al., 2013) and because it should capture many potential fragments of tau (Meredith et al., 2013). 46-4 was used as a control because mice cannot be infected with HIV and because this antibody is not known to recognize any endogenous murine proteins. Antibodies were purified from hybridoma supernatants using protein G and were 95% pure as determined by sodium dodecyl sulphatepolyacrylamide gel electrophoresis and coomassie staining.

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Fig. 1. Schedule of antibody injections and behavioral testing. Mice began receiving antibody injections (250 mg, i.p.) at 9.5 months of age. Animals received a total of 11 injections before behavioral assessment and a further 3 injections during testing. Following the 11th antibody injection, mice were used for open field (OF) testing. Five days later, mice received their 12th i.p. injection of antibody and the following day began habituation to the radial arm maze (RAM). The 13th and 14th i.p. injections of antibody took place on the final day of RAM habituation and on the 2nd day of RAM training, respectively. Antibody was administered between 2 and 5 PM, and on days during behavioral testing antibodies were injected 2 or more hours after testing. Abbreviation: i.p., intraperitoneal. (For interpretation of the references to color in this Figure, the reader is referred to the Web version of this article.)

Antibodies were tested for endotoxin using an LAL chromogenic kit (Pierce, Rockford, IL) and contained 1 U/mg of endotoxin. Beginning at 9.5 months, animals received 11 weekly intraperitoneal injections of antibody (250 mL of 1 mg/mL antibody solution) (Fig. 1). The dose and route of immunization was based on prior studies using tau transgenic mice (Boutajangout et al., 2011). To ensure levels of circulating antibody were kept at a maximum throughout behavioral testing, mice received an additional 3 antibody injections (250 mg) during this period (Fig. 1). Antibody was administered between 2 and 5 PM, and on days during behavioral testing antibody injection was carried out at least 2 hours after training. The investigator carrying out the behavioral testing was blind to the treatment groups.

at 80  C pending analysis. The resulting pellet was rehomogenized in the presence of 5 volumes (wt/vol) TBS containing 1% Triton X100 and protease inhibitors, centrifuged at 90,000g and 4  C for 1 hour, and the supernatant (TBS-TX extract) removed to clean tubes and stored at 80  C. The final pellet was resuspended in 0.5 volume (wt/vol) 88% formic acid and agitated overnight. Samples were centrifuged at 14,000g for 15 minutes, and the entire supernatant (formic acid extract) removed to clean tubes and stored at 80  C pending analysis. Immediately before analysis, formic acid samples were neutralized by diluting 1:27 (vol/vol) with unbuffered 1 M Tris.

2.5. Euthanasia and tissue collection

The protein content of TBS extracts was determined using a micro-BCA assay kit (Pierce). Samples were mixed with lithium dodecyl sulphate sample buffer plus beta mercaptoethanol (2.5% vol/vol) and boiled for 6 minutes. Eight micrograms total protein was loaded per well and electrophoresed on a NuPage Novex 4%e 12% polyacrylamide bis tris gel using 4-morpholinepropanesulfonic acid buffer at 180 V for at least 90 minutes. Thereafter, proteins were electrotransferred from the gel onto 0.2 mm nitrocellulose (Optitran, Schleider and Schüll, Germany) at 400 mA for 90 minutes. To improve detection, membranes were microwaved as previously described (Ida et al., 1996). Filters were blocked with 1% (wt/vol) BSA in PBS for 1 hour at room temperature, washed briefly in PBS containing 0.05% tween-20 (PBS-Tw) and incubated overnight at 4  C with the mouse monoclonal antibody Tau-1 (1 mg/mL) and a rabbit polyclonal anti-GAPDH antibody (1:10,000), or polyclonal antibody K9JA (1 mg/mL) and a mouse monoclonal antiGAPDH antibody (1:10,000). The following day, membranes were washed for 15 minutes 4 with PBS-Tw. Flurochrome-coupled antimouse and anti-rabbit secondary antibodies (each at 1:10,000; Rockland, Gilbertsville, PA) were added for 45 minutes, the membranes were washed again (15 minutes, 4) and bands visualized using an LI-COR Odyssey Near Infrared Imaging system (LI-COR Biosciences, Lincoln, NE). To allow comparison between blots, known amounts of recombinant murine tau (4 and 8 ng per well) were loaded onto each gel.

Immediately following completion of the final behavioral test, mice were anesthetized with ketamine/xylazine/acepromazine (100/10/2 mg/kg). Terminal blood was collected by cardiac puncture into ethylenediaminetetraacetic acid charged tubes (10 mL), centrifuged at 1500g for 10 minutes at room temperature and supernatant (plasma) transferred to a clean tube and stored at 80  C. Following blood collection, mice were intracardially perfused with approximately 20 mL ice-cold phosphate-buffered saline (PBS) using a 30-mL syringe. The brain was removed, and the success of the perfusion was assessed by visual inspection; only brains free of blood contamination were processed further. The meninges were carefully removed, and brain cut down the midline. The left hemisphere was immediately frozen on dry ice for biochemical analysis, and the right hemisphere was drop fixed in 10% formalin for 2 hours. Tail snips were collected and genotype retested. 2.6. Tissue preparation The olfactory bulbs and cerebellum were dissected from frozen hemi-brains and the remaining tissue weighed on a top pan balance. Thereafter, the tissue was cut into small pieces using a razor blade and homogenized in 5 volumes (wt/vol) Tris-buffered saline (TBS), pH 7.4, plus protease inhibitors (5 mM ethylenediaminetetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 10 mg/mL leupeptin, 1 mg/mL aprotinin, 1 mg/mL pepstatin A, 1 mM pefabloc and 2 mM 1,10 phenanthroline) using a dounce homogenizer fitted to an overhead stirrer (Wheaton, Millville, NJ). Samples were centrifuged at 90,000g and 4  C for 1 hour. The entire supernatant (TBS extract) was removed to clean tubes and stored

2.7. Western blot assessment of tau

2.8. Tau enzyme-linked immunosorbent assay The antibodies used for capture and detection are described in Table 1. Black half-area high-binding 96-well plates (Greiner BioOne, Monroe, NC) were coated with BT2 (25 mL of 2.5 mg/mL in

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TBS per well) for 1 hour at 37  C. Wells were washed 5 times with TBS containing 0.05% tween-20 and then blocked by incubating with 3% (wt/vol) BSA in TBS for 2 hours. Thereafter, wells were washed 5 times with TBS and 25 mL samples, blanks, or standards (mouse tau 430; 7.8e8000 pg/mL) added and incubated overnight at 4  C. The next day, plates were incubated for 2 hours with shaking at room temperature to allow the assay to return to room temperature. Subsequently, 25 mL Tau-5 (Tau-5 was conjugated to alkaline phosphatase using a Lightning-Link alkaline phosphatase conjugation kit (Innova Biosciences, Cambridge, UK), per manufacturer’s instructions) in TBS containing 0.05% tween-20 containing 1% (wt/vol) BSA was added to each well and incubated for 1 hour with shaking. Wells were washed 5 times, and 50 mL chemiluminescent substrate (Tropix CDP-Star Sapphire II, Applied Biosystems, Grand Island, NY) was added and incubated with shaking at room temperature for 30 minutes. Chemiluminescence was measured using a Synergy H1 plate reader (Biotek, Winooski, VT). Standard curves were fitted to a 5-parameter logistic function with 1/Y 2 weighting, using MasterPlex ReaderFit (MiraiBio Group, San Francisco, CA). The lower limit of quantification (LLOQ) of the assay was 30 pg/mL, and all samples were diluted so that the concentration of tau exceeded the LLOQ but was lower than the 8000 pg/mL standard. To detect tau in the small volumes of cerebrospinal fluid (CSF) available, we adapted the above assay to a 384 well format. All procedures for the 384-well assay were identical to the 96-well assay except for the volumes used. Wells were coated with 10 mL BT2 and 5 mL of samples, standards, blanks, and Tau5-AP conjugate were used. Black high-binding 384-well plates were from Greiner (Bio-One). ˇ

2.9. Ab enzyme-linked immunosorbent assay MULTI-ARRAY 96 well small-spot black microplates (Meso Scale Discovery, Rockville, MD) were coated with 3 mg/mL of monoclonal antibody m266 in PBS and incubated at room temperature for 18 hours. Antibody m266 recognizes an epitope within Ab13-26 (Table 1), thus enabling detection of both N- and C-terminally heterogenous Ab species. Wells were washed for 15 minutes 4 and blocked with 150 mL of 5% Blocker A (Meso Scale Discovery) in PBSTw for 1 hour at room temperature with shaking. Plates were washed 3 times with PBS-Tw before addition of samples diluted to give a final concentration of 1 enzyme-linked immunosorbent assay (ELISA) diluent (1% Blocker A/PBS-Tw). Twenty-five micro liters of samples or synthetic Ab1-40 or Ab1-42 standards (Meso Scale Discovery) were added and incubated for 2 hours at room temperature with shaking. All synthetic peptides were aliquoted and stored at 80  C before use and diluted in TBS and/or ELISA diluent, TBS containing 1% Triton X-100 (TBS-TX)/ELISA diluent or 1M Tris/ ELISA diluent, so as to correspond with the buffer composition of the samples tested. After capture, wells were washed 3 times with PBS-Tw and incubated with biotinylated antibody. To allow the detection of Ab beginning at Asp1 or terminating at Ala42 monoclonal antibodies 3D6 (1 mg/mL) or 21F12 (1 mg/mL) were used (Table 1). Simultaneously, 1 mg/mL of the reporter reagent (SULFOTAG Labeled Streptavadin) (Meso Scale Discovery) was added in ELISA diluent and incubated for 2 hours at room temperature with gentle agitation. Finally, wells were washed 3 times with PBS-Tw and 150 mL of 2 meso scale discovery read buffer was added to allow for electrochemiluminecence detection (Meso Scale Discovery). A SECTOR imager (Meso Scale Discovery) was used to measure the intensity of emitted light, thus allowing quantitative measurement of analytes present in the samples. To control for detection of endogenous murine Ab and reactivity with non-Ab material, we were careful to include 2 important controls, namely: (1) at least 1

or more extracts from an age-matched Wt mouse brain and (2) extracts from 12 months J20 mouse brain immunodepleted of Ab. Extracts were immunodepleted of Ab using the polyclonal anti-Ab antibody, AW7, as previously described (Barry et al., 2011). All samples were diluted so the concentration of the Ab present fell within the linear range of the standard curve. For a given assay (i.e., either x-42 or 1-x), all samples were analyzed on the same day. The LLOQ of the x-42 and 1-x assays was 17 pg/mL and 15 pg/mL, respectively. 2.10. Measurement of anti-tau antibody in plasma and brain The concentration of anti-tau antibodies was measured in both TBS brain extracts and plasma using a solid-phase ELISA. Clear 96 well high-binding plates (Costar, Corning, NY) were coated with recombinant murine tau 430 (50 mL of 5 mg/mL in PBS per well) for 1 hour at 37  C. Wells were washed twice with PBS-Tw and blocked by incubation with 200 mL 1% (wt/vol) BSA in PBS-Tw for 2 hours. Wells were washed again and samples, standards (5E2; 3.9e4000 pg/mL) or blanks (100 mL/well) were incubated for 1 hour at 37  C. Thereafter, wells were washed twice and incubated with biotinylated polyclonal goat anti-mouse IgG (g-heavy chain specific; Sigma Chemical Co, St. Louis, MO) for 45 minutes at 45  C. Wells were washed twice and incubated with Streptavidin-HRP (1:1000; Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) for 45 minutes at 37  C before being washed 3 times with PBSTw and detected with TMB substrate (SureBlue Reserve; KPL, Gaithersburg, MD). Standard curves were fitted to a 5-parameter logistic function with 1/Y 2 weighting, using MasterPlex ReaderFit (MiraiBio Group). The LLOQ of the assay was 70 pg/mL, plasma samples were diluted 1:200,000, and TBS extracts were diluted 1:50 to allow reliable quantification of antibody. ˇ

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2.11. Immunohistochemistry and staining Following formalin fixation hemi-brains were stored in PBS overnight and paraffin embedded using an Excelsior ES tissue processor (Thermo Fisher Scientific Inc, Waltham, MA), 10 mm sagittal sections were cut on a microtome (Leica Microsystems Inc, IL) and mounted onto microscope slides. 2.11.1. Immunohistochemistry Sections were de-paraffinized using Histo-clear (2  3 minutes; National Diagnostics, Somerville, NJ) and then rehydrated through a graded series of ethanol and/or water solutions (100%, 95%, 70%, and 50%) for 3 minutes each, followed by a final incubation in distilled water (3 minutes). Endogenous peroxidase activity was quenched by incubation in 0.3% hydrogen peroxide in methanol, for 10 minutes at room temperature. Sections underwent pretreatment applicable to the antibodies being used (Table 1). Immunohistochemistry with certain mouse monoclonal antibodies (8E5, 21F12, and 2G3) was completed using Vector Mouse-On-Mouse kit (Vector Laboratories Inc, Burlingame, CA) in accordance with the manufacturer’s guidelines. For anti-tau monoclonal antibodies (MC1 and AT8), sections were blocked with 5% non-fat milk in TBS for 1 hour at room temperature and incubated overnight in primary antibodies at the concentrations indicated (Table 1) at 4  C. Sections were washed with TBS and incubated with biotinylated goat antimouse IgG1 secondary antibody diluted in 20% Superblock (Pierce) at room temperature for 2 hours. For polyclonal antibodies (AW7 and CD45), sections were blocked with 10% goat serum in TBS for 20 minutes at room temperature. Sections were incubated overnight in primary antibodies at concentrations indicated (Table 1) and 4  C. Sections were washed with TBS and incubated with biotinylated secondary antibodies (Table 1), diluted in 10%

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Fig. 2. J20 mice are hyperactive from the earliest age tested and over time develop deficits in spatial reference memory. On day 1 of behavioral testing, mice were placed in an open field chamber and the distance traveled (A), and time spent in the center of the chamber (B) were recorded. Results are presented as mean values  SEM for Wt and J20 mice at 4 months (Wt n ¼ 12, J20 n ¼ 12), 8 months (Wt n ¼ 11, J20 n ¼ 9), and 12 months (Wt n ¼ 13, J20 n ¼ 9). (A) J20 mice were significantly more active than their littermate controls (Wt) across the 3 ages studied (2-way ANOVA, p < 0.0001) and within age group at 4 months and 12 months (Bonferroni post tests; 4 months, Wt vs. J20, p < 0.001; 8 months, Wt vs. J20, p > 0.05; 12 months, Wt vs. J20, p < 0.05). (B) J20 mice spent significantly more time in the center of the arena across the 3 age groups (2-way ANOVA, p < 0.0001), but the difference between J20 and Wt mice was only significant at 4 months and 8 months (Bonferroni post tests; 4 months, Wt vs. J20, p < 0.01; 8 months, Wt vs. J20, p < 0.01). (CeH) Mice underwent 8 days of training in the RAM, with 6 trials a day. During each trial, the percentage of turns that were 45 (CeE), and the number of incorrect arm entries (FeH) were recorded. A daily average across all 6 trials was calculated for each mouse, and the average value for each mouse was used to calculate the group mean  SEM. (C) Four months J20 and Wt mice performed similarly well throughout the 8 days of testing. (D) At 8 months, J20 and Wt mice made a comparable percentage of 45 turns, but (G) J20 mice made more incorrect arm entries (2-way ANOVA Wt vs. J20, genotype effect p ¼ 0.019; Bonferroni post tests, day 6 p < 0.01). (E) Twelve months J20 mice made more 45 turns than Wt mice across all time points (2-way ANOVA Wt vs. J20, genotype effect p ¼ 0.0016), and this was significantly elevated versus Wt on day 4 (Bonferroni post tests, Wt vs. J20, p < 0.05). Importantly, after the first day of training J20 mice made more reference errors than Wt mice across all days (2-way ANOVA Wt vs. J20 genotype effect ¼ 0.022), and the number of reference errors were significantly higher for J20 mice than Wt mice on days 4 and 5 (Bonferroni post tests, day 4 Wt vs. J20 p < 0.01, day 5 Wt vs. J20 p < 0.05). Only a subset of mice tested in the open field could also be tested in the RAM due to the extended time taken to run the paradigm. Genotype balanced groups of 12e16 mice were tested at each age group; additionally, mice that consistently failed to explore the maze and eat the food pellets during habituation were removed from the task (4 months, 1 Wt; 8 months, 4 Wt 1 J20; 12 months, 1 J20). Due to the large number of mice removed from the 8 months cohort), a second cohort of 8 months Wt (n ¼ 6) and J20 (n ¼ 5) mice were tested in the RAM. Results for the two 8-month cohorts were pooled. Abbreviations: ANOVA, analysis of variance; SEM, standard error of the mean; Wt, wild type. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. (For interpretation of the references to color in this Figure, the reader is referred to the Web version of this article.)

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goat serum (in TBS) at room temperature. Thereafter, all sections were incubated with horseradish peroxidase (Vector Elite ABC kit, Vector Laboratories Inc) for 45 minutes at room temperature. Diaminobenzidine (DAB; Sigma Chemical Co) was then added and incubated until sufficient color development was obtained on the positive controls sections. Development was stopped by incubation in water. Sections were counterstained with hematoxylin (Fisher Scientific, Pittsburg, PA), dehydrated through graded ethanol and/or water solutions (50%, 70%, 95%, and 100%), cleared by incubation in Histo-clear, and cover-slipped under Permount (Fisher Scientific). 2.11.2. Thioflavin S Sections were deparaffinized using Histo-clear and rehydrated as above and then stained with thioflavin S (ThS) by incubating sections in a 1% (wt/vol) ThS (Sigma Chemical Co) solution for 8 minutes at room temperature, followed by incubations in 80% ethanol (2  3 minutes) and 95% ethanol (1  3 minutes). Sections were rinsed in distilled water before being cover-slipped under Hydromount (National Diagnostics). Staining was visualized and scored using an Olympus BX50 microscope (Olympus, Tokyo, Japan), and images were collected using a QiCam digital camera (QImaging, Surrey, BC, Canada). 2.11.3. Image analysis In immunized mice images from the entire hippocampus were analyzed for AW7 immunoreactivity and ThS staining using Image J software (NIH). Images were converted to an 8-bit format and a threshold manually set. The region of interest was manually assigned, selecting the entire hippocampus bordered dorsally by the corpus callosum. Percentage positive pixels within the selected region of interest were analyzed. AT8 and CD45 immunoreactivity in the hippocampus was quantified using Bioquant Life Science Version 12.5 for Windows (Bioquant Image Analysis Corporation, Nashville, TN). Hemosiderin staining was assessed by counting of microhemorrhages throughout the entire brain. For all analyses, 6 sections from 3 equidistant planes were analyzed by an investigator blind to antibody treatment and an average value determined for each mouse. 2.12. Statistical analysis Statistical analyses were performed with GraphPad Prism 5 for Windows (GraphPad Software, Inc, La Jolla, CA). Differences between pairs of means were assessed by 1-way analysis of variance (ANOVA), where data were normally distributed, or Mann Whitney U test. Differences between multiple means were assessed by Kruskal-Wallis test, 2-way ANOVA or repeated measures ANOVA, as indicated. Differences between survival curves were assessed using a log-rank test (Mantel-Cox). Biostatisticians from the Center for Clinical Investigation, a joint Harvard Catalyst and Brigham and Women’s Hospital entity, were consulted on all statistical matters. Values are presented as mean  standard error of the mean (SEM). Significant differences between groups are indicated by: *p < 0.05; **p < 0.01; and ***p < 0.001. 3. Results Recent studies have demonstrated that lowering neuronal tau expression protects neurons from the adverse effects of soluble Ab oligomers and can lessen behavioral changes in APP transgenic mice (Ittner et al., 2010; Jin et al., 2011; Roberson et al., 2007). Therefore, we examined if chronic immunization with an anti-tau antibody could rescue behavioral deficits in a mouse line that had been previously shown to benefit from genetic ablation of tau (Roberson et al., 2007). Because the behavior of

both transgenic and wild-type mice can be influenced by multiple factors, including genetic drift, housing conditions, and diet (Crabbe et al., 1999; Wahlsten et al., 2003); we first systematically characterized J20 and Wt mice before testing the effects of anti-tau immunotherapy. 3.1. J20 mice exhibit aberrant behavior and develop impairments in spatial reference memory Performance in the open field is a simple and reliable means to identify aberrant behavior and/or locomotor disturbances. J20 mice traveled significantly further than Wt controls (Fig. 2A; 2-way ANOVA, p < 0.0001; Bonferroni post tests; 4 months old, Wt vs. J20, p < 0.001; 8 months, Wt vs. J20, p > 0.05; 12 months, Wt vs. J20, p < 0.05), and spent more time in the center of the open field chamber, than Wt mice (Fig. 2B; 2way ANOVA, p < 0.0001; Bonferroni post tests; 4 months, Wt vs. J20, p < 0.01; 8 months, Wt vs. J20, p < 0.01). These data indicate that young J20 mice are more active and less anxious than their Wt littermates, but that this phenotype dissipates somewhat with age. Impairment of SRM is an early and frequent symptom of AD (Henderson et al., 1989; Monacelli et al., 2003) and of certain mouse models of AD (Ashe, 2001; Ashe and Zahs, 2010). To test spatial reference learning and memory, we used a modified version of the 8-arm radial maze, first reported by Olton and Samuelson (1976). Three measures of behavior were recorded over the training days: (1) latency to collect all 3 pellets from the maze; (2) the percentage of 45 turns made; and (3) the number of incorrect arm entries made (SRM errors). The radial arm maze requires that animals both learn and recall the task, such that the first trials require that animals learn the task, and after learning the task the animals can recall that experience and thus exhibit improved performance on subsequent trials. In this regard, the most important read out is the number of reference errors (Wenk, 2004). J20 and Wt mice displayed similar latencies at all 3 ages tested (data not shown). Four-month-old J20 mice showed similar levels of 45 turns and SRM errors to Wt controls (Fig. 2C and F). Eight-month-old J20 mice displayed similar percentages of 45 turns to Wt mice (Fig. 2D), but made more SRM errors than Wt controls (Fig. 2G; 2-way ANOVA, J20 vs. Wt, genotype effect p ¼ 0.019), and post hoc testing revealed this impairment to be significant (within day) on day 6 (Fig. 2G; Bonferroni post tests, Day 6, p < 0.01). At 12 months, J20 made significantly more 45 turns than Wt controls (Fig. 2E; 2-way ANOVA Wt vs. J20, genotype effect p ¼ 0.0016), and post hoc analyses revealed that this effect became significant (within day) on day 4 (Fig. 2E; Bonferroni post tests, day 4, p < 0.05). An increase in 45 turns has been suggested as an egocentric method to strategically search the maze, whereas the Wt mice are likely using an allocentric method utilizing the extra-maze cues to orientate themselves and locate the food pellets. J20 mice have previously been shown to be more likely to use egocentric strategies compared with Wt controls (Deipolyi et al., 2008), and hippocampal lesioned mice make more 45 turns on the 8-arm radial maze (Dillon et al., 2008). Importantly, 12 months J20 mice made significantly more reference errors on every day of training except the first day, and this effect was significant (within day) on days 4 and 5 (Fig. 2H; Bonferroni post tests, day 4 Wt vs. J20 p < 0.01, day 5 Wt vs. J20 p < 0.05). These results indicate that SRM impairments in J20 mice are present at approximately 8 months and become more pronounced at 12 months. In addition to the SRM and open field tests, we also investigated performance of J20 and Wt mice using spontaneous alternation Ymaze and CFC tests. Wt and J20 mice exhibited expected alternation

A.J. Mably et al. / Neurobiology of Aging 36 (2015) 1316e1332 Table 2 J20 mice accumulate amyloid with increasing age and exhibit signs of aberrant tau metabolism and activation of microglia Age

Genotype

AW7

21F12

2G3

ThS

MC1

AT8

8E5a

CD45

4 mo 4 mo 4 mo 4 mo 4 mo 4 mo 4 mo 4 mo 4 mo 4 mo 4 mo 4 mo 8 mo 8 mo 8 mo 8 mo 8 mo 8 mo 8 mo 8 mo 8 mo 12 mo 12 mo 12 mo 12 mo 12 mo 12 mo 12 mo 12 mo 12 mo

J20 J20 J20 J20 J20 J20 J20 J20 J20 Wt Wt Wt J20 J20 J20 J20 J20 J20 J20 Wt Wt J20 J20 J20 J20 J20 J20 J20 Wt Wt

0.5 0 0 0.5 0 0.5 0.25 0 0 0 0 0 3 3 3.25 3 2.75 2.5 3.5 0 0 5 5 5 5 5 5 4.5 0.5 0.75

0.5 0.5 0 0.5 0.5 0.5 1 1 0.5 0 0 0 4 3 4 3.5 3 3.5 3 0 0 5 5 5 5 5 5 5 0 0

0.5 0 0 0.5 0 0.5 0 0 0 0 0 0 3 2 2.5 2 1.5 2 2 0 0 3.5 4 4 4 3 3.5 4 0 0

0 0 0 0 0 0 0 0 0 0 0 0 1 1.5 1.25 2 2.25 1.25 1 0 0 3.75 4 3.75 3.5 2.75 3 3 0 0

0.5 0 0 0.5 0 0 0 0.5 0 0 0 0 1 0.5 1 0.5 0.5 1.5 1 0 0 1 1.5 1.5 2 2 1.5 2 0 0

0.5 0 0 0.5 0 0 0 0 0 0 0 0 1.5 1 2 1 1.5 1.5 2 0 0 2 2 2 2 2 2 2 0 0

0 0 0 0 0 0 0 0.5 0 0 0 0 1.5 1.5 1.5 2 2 2.5 1 0 0 2.5 3 2.5 3 3 2.5 3 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0.5 0 1.5 1 0.5 2 0 0 3 3 4 3.5 2.5 3 3 0 0

The amount of staining was scored qualitative with 0.5 indicating minimal staining and 5 extensive staining throughout the cerebrum. Key: ThS, thioflavin S; Wt, wild type. a Focal 8E5 staining associated with amyloid deposits, distinct from the diffuse 8E5 staining of transgenically expressed APP seen in J20 mice.

patterns in the Y-maze (Dillon et al., 2008) and learning and recall of CFC (Supplementary Fig. 1), but there was no difference between the J20 and Wt groups in either assay. Thus the major behavioral deficits evident in our J20 mice were altered activity, which waned with increasing age; and decreased SRM, which became more prominent with increasing age. 3.2. Brains of J20 mice exhibit an age-dependent increase in ADrelevant pathology We examined Ab and tau pathology and biochemistry in the same J20 mice used for behavioral analysis. Hemi-brains from J20 and Wt brains were analyzed by immunohistochemistry to assess amyloid load and the presence of amyloid-associated pathology. Staining was assigned a semiquantitative score by an investigator blind to age and genotype. Total amyloid deposition was assessed using the polyclonal anti-Ab antibody, AW7, and fibrillar amyloid was quantified by binding of ThS. Abnormally, phosphorylated and/or aggregated tau was assessed using the monoclonal antibodies AT8 and MC1, respectively. ThS positive amyloid was neither detected in the brains of 4 months J20 mice nor in Wt mice at any age. AW7 immunoreactive Ab was not detected in the hippocampus or cortex of 4 months or 8 months Wt mice (Table 2). The majority (5/9) of 4 months J20 mice lacked Ab immunoreactive deposits, but a significant number of mice (4/9) did have small quantities of AW7 reactive deposits (Table 2). At 8 months, all J20 mice had at least some ThS positive and AW7 reactive deposits in both the hippocampus and cortex (Table 2; Fig. 3). At 12 months, large numbers of AW7

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reactive deposits were evident throughout the hippocampus and cortex, a portion of which bound ThS (Table 2; Fig. 3, arrows). Abnormally, phosphorylated tau was detected with the monoclonal antibody, AT8, which is specific for tau phosphorylated at Ser202 and Thr205 (Mercken et al., 1992a). AT8 immunoreactivity was found associated with Ab deposits in brains from 4 months (2/9), 8 months (7/7), and 12 months (7/7) J20s (Table 2; Fig. 3). MC1 recognizes aggregated, but not monomeric or microtubule-associated tau (Jicha et al., 1997). MC1 staining was evident in a third of 4 months J20’s and in all 8 and 12 months J20’s (Table 2; Fig. 3). As with AT8, MC1 was greatest in 12 months J20s and was not detected in Wt mice at any age (Table 2). Thus, although a recent study (Petry et al., 2014) raised important questions about the specificity of AT8 and MC1 when used for Western blotting, the fact that neither mAb produced appreciable staining in littermate controls and that MC1 and AT8 immunoreactivity increased in J20 mice with age strongly argues that the staining seen with these 2 very different antibodies is attributable to a modest accumulation of nonphysiological forms of tau in dystrophic neurites found associated with plaques. 3.3. J20 mice evince age-dependent increases in water-soluble, detergent-soluble, and formic acid-soluble Ab, but levels of tau were comparable with Wt controls Hemi-brains were serially extracted in TBS, TBS-TX, and formic acid, to give water-soluble, detergent-soluble, and formic acidsoluble extracts, respectively. Extracts were analyzed for their Ab and tau content by ELISA. Across all 3 extracts Abx-42 and Ab1-x levels followed a similar pattern with the levels of Ab1-x higher than Abx-42. Both Abx-42 and Ab1-x in the TBS extract increased with age in J20 mice (Fig. 4A; Abx-42, 1-way ANOVA, p < 0.0001; Ab1-x, 1-way ANOVA, p < 0.0001). Twelve-month-old J20 mice had significantly higher TBS Ab than both 4 months and 8 months J20 mice (Fig. 4A; Bonferroni post tests; Abx-42, 4 months vs. 12 months, p < 0.001; 8 months vs. 12 months, p < 0.001; Ab1-x, 4 months vs. 12 months, p < 0.001; 8 months vs. 12 months, p < 0.001). Detergent-soluble Ab detected in J20 mice also increased with age (Fig. 4C; Abx-42, 1-way ANOVA, p ¼ 0.0032; Ab1-x, 1-way ANOVA, p ¼ 0.026). Twelve-month-old J20 mice had significantly higher levels of TBS-TX Ab than both 4 months and 8 months mice (Fig. 4C; Bonferroni post tests; Abx-42, 4 months vs. 12 months, p < 0.01; 8 months vs. 12 months, p < 0.05; Ab1-x, 4 months vs. 12 months, p < 0.01; 8 months vs. 12 months, p < 0.01). Formic acidsoluble Ab was readily detected in hemi-brains from 4 months J20 mice, and steadily increased with age, with the levels in 12 months J20 mice some 100-fold higher than those in 4 months animals (Fig. 4E; Abx-42, 1-way ANOVA, p < 0.0001, Bonferroni post tests, 4 months vs. 8 months, p < 0.01, 4 months vs. 12 months, p < 0.001; 8 months vs. 12 months, p < 0.001; Ab1-x, 1-way ANOVA, p < 0.0001, Bonferroni post tests, 4 months vs. 12 months, p < 0.001; 8 months vs. 12 months, p < 0.001). Although 4 months J20 mice have relatively low levels of detectable formic acid Ab compared with 8 months and 12 months J20 mice, this was still greater than levels of Ab detected in the TBS or TBS-TX extracts of the oldest J20 mice tested. Comparison of Ab levels and reference errors made by 8 and 12 months J20 mice during days 4, 5, and 6 of RAM training (when significant differences between J20 and Wt mice were observed), revealed that all 6 measured forms of Ab tended to be increased in mice that made more reference errors (data not shown). Unlike Ab, transgenic expression of APP had no discernible effect on the levels or distribution of tau (Fig. 4B, D, and F). Levels of TBS soluble tau fluctuated with age, with the highest values found in 4 months mice and the lowest in 8 months mice (Fig. 4B; 2-way

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Fig. 3. J20 exhibit age-dependent accumulation of Ab throughout the cerebrum together with modest levels of misfolded and aberrantly phosphorylated tau. The presence of Ab deposits was investigated using the polyclonal anti-Ab antibody, AW7, and amyloid-binding dye thioflavin S (ThS). Abnormally, phosphorylated or aggregated tau was detected with the monoclonal antibodies AT8 and MC1, respectively. Ab deposits were seldom detected in the hippocampus of either 4 months J20 or Wt mice. Although Ab deposits were always detected in the hippocampus and cortex of 8 months J20 mice. Large quantities of both core (arrows) and diffuse Ab deposits were evident in all 12 months J20 mice. In both 8 and 12 months J20 mice, small quantities of ThS positive deposits were detected. Four-month-old J20 mice seldom exhibited MC1 (3/9 mice) or AT8 (2/9 mice) reactivity, whereas MC1 and AT8 positive deposits were detected in all 8 and 12 months J20 mice. Figures are at 4 magnification, scale bar 200 mm; insets 20 magnification, scale bar 50 mm. Abbreviation: Ab, amyloid-b. (For interpretation of the references to color in this Figure, the reader is referred to the Web version of this article.)

ANOVA, age effect p < 0.0001, genotype effect p ¼ 0.77). TBSTX-soluble tau levels remained constant across all 3 ages tested (Fig. 4D), whereas formic acid extractable tau was present at only approximately 1/10th the level of TBS tau and was the highest in 4 months mice and lowest in 8 months mice (Fig. 4F, 2-way ANOVA formic acid, 2-way ANOVA, age effect p < 0.0001, genotype effect p ¼ 0.53). The fact that AT8 and MC1 staining increased with age in the hippocampus of J20s (Table 2) suggests that the modest changes detected by immunostaining are too small to be detected when whole brain homogenates are examined. That is, small differences in specific pools of tau and/or specific brain regions are masked by the much higher concentrations of physiologic tau present throughout the brain.

3.4. Behavioral and cognitive disturbances in J20 mice are refractory to anti-tau immunotherapy Because it had been previously demonstrated that genetic ablation of tau recovered behavioral and cognitive deficits in J20 mice (Roberson et al., 2007), we investigated if chronic treatment with an anti-tau antibody could have a similar effect. To control for nonspecific effects on learning and memory, Wt littermate mice also received injections of 5E2 and 46-4. Approximately 20 hours following the 11th antibody injection (Fig. 1), mice were placed in an open field arena for 60 minutes, and the distance traveled along with the time spent in the center of the arena was recorded. Consistent with our earlier

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Fig. 4. J20 mice show age-dependent increases in water-soluble, detergent-soluble, and formic acid-soluble Ab, but not total tau. Hemi-brains from J20 and Wt littermate controls were sequentially extracted to yield water-soluble, detergent-soluble, and formic acid-soluble fractions which were analyzed by ELISA for Abx-42, Ab1-x, and total tau. (A) Water-soluble Ab increases with age in J20 mice (Abx-42, 1-way ANOVA, p < 0.0001; Ab1-x, 1-way ANOVA, p < 0.0001). Twelve months mice have significantly higher water-soluble Abx-42 and Ab1-x than both 4 months and 8 months mice (Bonferroni post tests; Abx-42, 4 months vs. 12 months, p < 0.001; 8 months vs. 12 months, p < 0.001; Ab1-x, 4 months vs. 12 months, p < 0.001; 8 months vs. 12 months, p < 0.001). (C) The levels of Ab detected in the TBS-TX fraction also increased with age (Abx-42, 1-way ANOVA, p ¼ 0.0032; Ab1-x, 1-way ANOVA, p ¼ 0.026), and 12 months mice had significantly higher detergent-soluble Ab than both 4 months and 8 months mice (Bonferroni post tests, Abx-42, 4 months vs. 12 months, p < 0.01; 8 months vs. 12 months, p < 0.05; Ab1-x, 4 months vs. 12 months, p < 0.01; 8 months vs. 12 months, p < 0.01). (E) The levels of Ab in the formic acid fraction were on average approximately 130 times higher than the other 2 fractions and increased with age (Abx-42, 1-way ANOVA, p < 0.0001; Bonferroni post tests, 4 months vs. 8 months, p < 0.01; 4 months vs. 12 months, p < 0.001; 8 months vs. 12 months, p < 0.001. Ab1-x, 1-way ANOVA, p < 0.0001; Bonferroni post tests; 4 months vs. 12 months, p < 0.001; 8 months vs. 12 months, p < 0.01). In all fractions and at all ages, Ab1-x was detected at higher levels than Abx-42 (B, D, and F). The levels of tau in Wt and J20 mice were highly comparable at all ages and extracts studied. The levels of tau in the TBS and formic acid fractions fluctuated significantly with age (TBS, 2-way ANOVA, age effect p < 0.0001, genotype effect p ¼ 0.26; formic acid, 2-way ANOVA, age effect p < 0.0001, genotype effect p ¼ 0.19). Values are presented as means  SEM for 4 months (Wt n ¼ 11, J20 n ¼ 9), 8 months (Wt n ¼ 11, J20 n ¼ 7), and 12 months (Wt n ¼ 13, J20 n ¼ 7) mice. Abbreviations: ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; SEM, standard error of the mean; TBS, Tris-buffered saline; TX, Triton X; Wt, wild type. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. (For interpretation of the references to color in this Figure, the reader is referred to the Web version of this article.)

behavioral characterization (Fig. 2A), J20 mice, irrespective of the antibody they received were more active than immunized Wt mice (Fig. 5A; 2-way ANOVA, genotype effect, p < 0.0001; Bonferroni post tests, Wt þ 46-4 vs. J20 þ 46-4 p < 0.05; Wt þ

5E2 vs. J20 þ 5E2 p < 0.001). J20 mice treated with 5E2 or 46-4 exhibited similar activity levels (Fig. 5A; 2-way ANOVA, genotype effect, p ¼ 0.51), indicating that 5E2 treatment was not able to modify hyperactivity in J20 mice. Although 5E2-treated J20

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Fig. 5. The hyperactive phenotype and deficits in spatial reference memory evident in J20 mice are refractory to chronic passive immunization with the anti-tau antibody 5E2. Twenty-four hours following the 11th antibody administration, mice were placed in the open field arena, and (A) distance traveled and (B) time spent in the center of the arena were measured over 60 minutes. (A) J20 mice are consistently more active in the open field than Wt mice (2-way ANOVA, genotype effect, p < 0.0001; Bonferroni post tests, Wt þ 46-4 vs. J20 þ 46-4 p < 0.05; Wt þ 5E2 vs. J20 þ 5E2 p < 0.001), and this phenotype was not attenuated by immunization with 5E2. (B) J20 mice spend more time in the center of the open field arena than Wt mice (2-way ANOVA, genotype effect, p < 0.0001; Bonferroni post tests, Wt þ 46-4 vs. J20 þ 46-4 p < 0.001; Wt þ 5E2 vs. J20 þ 5E2 p < 0.05), but again this behavior was unaltered by immunization with 5E2. Following open field testing, mice were assessed for SRM in the radial arm maze. An average across all 6 daily trials was calculated for each mouse, and the average value for each mouse was used to calculate the group mean  SEM. (C) Both J20 treatment groups show an increase in the percentage of 45o turns made over the first 3 days, followed by a decrease over days 4e8. Wt mice treated with 46-4 or 5E2 show a similar pattern of 45o turns that is distinct from their J20 counterparts (2-way ANOVA, Wt þ 46-4 vs. J20 þ 46-4, interaction effect p < 0.0001, Wt þ 5E2 vs. J20 þ 5E2, interaction effect p < 0.0001). There were no differences between J20 mice treated with 5E2 versus 46-4 (2-way ANOVA, J20 þ 5E2 vs. J20 þ 46-4, treatment effect p ¼ 0.75). (D) Twelve-month-old J20 mice show impaired SRM compared with Wt mice (2-way ANOVA, Wt þ 46-4 vs. J20 þ 46-4, genotype effect p ¼ 0.0004, interaction effect p < 0.0001). J20 mice treated with 5E2 were not significantly different to 46-4 treated J20 mice (2-way ANOVA, J20 þ 5E2 vs. J20 þ 46-4, treatment effect p ¼ 0.66). Asterisk (*) denotes Wt þ 46e4 versus J20 þ 46-4 statistics and y denotes Wt þ 5E2 versus J20 þ 5E2 statistics. Results are presented as mean values  SEM for Wt and J20 mice treated with 5E2 or 46e4. The levels of anti-tau antibody in plasma samples and water-soluble extracts of PBS perfused brains from 5E2- and 46-4-treated J20 and Wt mice were measured by ELISA. (E) TBS extracts from Wt and J20 mice that received 5E2 contained approximately 69 ng/g and 42 ng/g 5E2 antibody, respectively. Significantly less antibody was available for capture in J20 mice than Wt mice (t test, p ¼ 0.022). (F) Plasma samples from 5E2-treated Wt and J20 mice contained approximately 155 mg/mL and approximately 145 mg/mL 5E2 antibody, respectivelydthese observed values are remarkably close to the expected value of 170 mg/mL. Levels of anti-tau antibody detected in the TBS extract and plasma of 46-4-treated mice were below the lower limit of detection for the ELISA. Error bars are SEM; (AeB) Wt (5E2, n ¼ 13; 46-4, n ¼ 12) and J20 (5E2, n ¼ 9; 46-4, n ¼ 12); (EeF) Wt (5E2, n ¼ 10; 46-4, n ¼ 3) and J20 (5E2, n ¼ 9; 46-4, n ¼ 3). Abbreviations: ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; SEM, standard error of the mean; Wt, wild type. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. y indicates p < 0.05, yy indicates p < 0.01. (For interpretation of the references to color in this Figure, the reader is referred to the Web version of this article.)

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Fig. 6. 5E2 treatment did not alter levels of cerebral tau. Tau content in CSF (A) and water-soluble (B), detergent-soluble (C), and formic acid-soluble (D) fractions from J20 and Wt mouse brain were analyzed by ELISA. (AeD) No difference was detected in the levels of tau present in 5E2-treated mice compared with 46-4-treated mice (2-way ANOVA, treatment effect, CSF p ¼ 0.45; water-soluble p ¼ 0.75; detergent-soluble p ¼ 0.17; formic acid-soluble p ¼ 0.53). Results are presented as mean values  SEM for Wt and J20 mice; Wt (5E2, n ¼ 10; 46-4, n ¼ 12) and J20 (5E2, n ¼ 9; 46-4, n ¼ 12). Abbreviations: ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; SEM, standard error of the mean; Wt, wild type. (For interpretation of the references to color in this Figure, the reader is referred to the Web version of this article.)

mice spent slightly less time in the center of the open field arena than 46-4-treated J20 mice, this was not significant (Fig. 5B; 2way ANOVA, genotype effect, p ¼ 0.18). J20 mice that received the control antibody 46-4 made more SRM errors and 45 turns than Wt controls receiving 46-4 (Fig. 5C, 2-way ANOVA, Wt þ 46-4 vs. J20 þ 46-4, interaction effect p < 0.0001; Fig. 5D, 2-way ANOVA, Wt þ 46-4 vs. J20 þ 46-4, genotype effect p ¼ 0.0004, interaction effect p < 0.0001). 5E2-treated and 46-4-treated J20 mice made similar numbers of 45 turns (Fig. 5C; 2-way ANOVA, J20 þ 5E2 vs. J20 þ 46-4, treatment effect p ¼ 0.75, interaction effect p ¼ 0.07). Moreover, J20 mice receiving 5E2 made a similar number of SRM errors as J20 mice treated with 46-4 (Fig. 5D; 2-way ANOVA, J20 þ 5E2 vs. J20 þ 46-4, treatment effect p ¼ 0.66, interaction effect p ¼ 0.42). Thus, chronic anti-tau immunotherapy was unable to attenuate any of the behavioral impairments found in 12 months J20 mice. 3.5. Anti-tau antibody is present and active in the brain and periphery of 5E2-immunized mice, but did not affect the levels of cerebral tau In light of the failure of 5E2 to improve cognition in J20 mice, it was important to confirm that active antibody reached the brain.

Accordingly, we measured the concentration of anti-tau antibodies in brain (that had been thoroughly perfused with PBS and was free of detectable blood contamination) and plasma of animals receiving 5E2 and 46-4. As expected, anti-tau antibody was detected in the plasma and brain of animals that received 5E2, but not in animals that received 46-4 (Fig. 5E and F). On average approximately 70 ng/ g of anti-tau antibody was detected in TBS fractions of 5E2-treated Wt mice, whereas significantly less antibody (approximately 40 ng/ g) was found in TBS fractions of 5E2-treated J20 mice (Fig. 5E; t-test, J20 þ 5E2 vs. J20 þ 46-4 p ¼ 0.022). The ELISA used to measure antitau antibodies uses plate-immobilized recombinant tau, such that free antibody can readily bind to immobilized tau and be detected, whereas antibody already bound to tau will not be detected. The finding that similar levels of antibody are found in plasma of 5E2treated Wt and J20 mice (Fig. 5F; J20 þ 5E2 approximately 155 mg/mL; Wt þ 5E2 approximately 145 mg/mL), whereas approximately 40% less 5E2 was detected in the brains of 5E2treated J20 mice versus 5E2-treated Wt mice suggests that a significant portion of the 5E2 in J20 brain bound to endogenous tau, but did not nonspecifically interact with the large amount of intracellular tau released during homogenization (not shown). Given that the amount of 5E2 in brain was relatively low versus the high concentration of intracellular tau, one would not expect to

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with 5E2, does not overcome behavioral deficits associated with APP/ Ab over-expression. Like genetic knock-out of tau, antibody treatment did not alter the number or distribution of amyloid deposits, AT8/MC1 immunoreactivity or activated microglia (Supplementary Fig 2), but worryingly, administration of 5E2 specifically led to sudden and unexplained death in a significant number of J20, but not Wt, mice. 4. Discussion

Fig. 7. Administration of the anti-tau antibody, 5E2, to J20 but not Wt mice was associated with increased mortality. Beginning at 9.5 months mice received 10 weekly injections and 4 injections over 2 weeks during behavioral testing. During immunization deaths were recorded. All Wt animals survived to the end of the study irrespective of whether they received 5E2 (14/14 mice) or 46-4 (13/13 mice). Similarly, all J20 mice that received 46-4 (12/12 mice) survived until the end of the study, whereas 4 of 13 J20 mice that received 5E2 died within 80 days of receiving their first injection. These animals died at, 54, 66, 75, and 80 days into the study. Mortality rates for 5E2 treated J20 mice were significantly increased compared with 46-4-treated J20 mice (J20 þ 5E2 vs. J20 þ 46-4, p ¼ 0.041). Antibody injections (arrows). Abbreviation: Wt, wild type. (For interpretation of the references to color in this Figure, the reader is referred to the Web version of this article.)

be able to detect a change in a small pool of extracellular tau because this would be masked by the huge excess of intracellular tau released during homogenization. As expected, we detected no differences in the levels of tau in CSF, and in TBS, TBS-TX, and formic acid extracts of J20 and Wt mice that received 5E2 and 46-4 (Fig. 6AeD; 2-way ANOVA, treatment effect, CSF p ¼ 0.45; watersoluble p ¼ 0.75; detergent-soluble p ¼ 0.17; formic acid-soluble p ¼ 0.53). Using Western blotting with 2 different anti-tau antibodies (K9JA and tau-1, Table 1), we also investigated if 5E2 treatment altered the forms or fragments of tau. The levels of full-length tau (migrating between approximately 48 and 58 kDa) were highly similar in Wt and J20 mice, and antibody treatment did not alter the levels of full length tau (Supplementary Fig. 3).

3.6. Chronic anti-tau immunization leads to increased mortality in J20, but not Wt mice Of the 13 J20 mice that received 5E2 treatment, 4 died, within 80 days of beginning antibody treatment (Fig. 7; J20 þ 5E2 vs. J20 þ 46-4, p ¼ 0.041), whereas there were no deaths among J20 mice that received 46-4 or Wt mice that received 5E2 or 46-4 (Fig. 7). These findings indicate that 5E2 is exerting specific adverse effects on mice that over-express APP. The fact that there was no loss of viability among J20 mice receiving 46-4 suggests that the adverse effects of 5E2 involve the APP/Ab-tau axis. There were no visible abnormalities in any major organ of the 4 mice that died, but tissue was not collected for detailed analysis. We reasoned that the surviving 5E2-treated mice also were negatively (but less severely) affected by 5E2 and searched for defects in these animals. When stained with hematoxylin and eosin, no gross anatomic abnormalities were detected (Supplementary Fig. 4D and E). Microhemorrhage following anti-Ab immunization is a common reported negative side-effect in hAPP transgenic mice (Burbach et al., 2007; DeMattos et al., 2004; Pfeifer et al., 2002; Racke et al., 2005; Wilcock et al., 2004); however, administration of 5E2 did not increase microhemorrhage in J20 mice compared with 464-treated J20 mice (Supplementary Fig. 4AeC). In sum, our experiments demonstrate that the anti-tau mAb, 5E2, can readily enter the brain but that unlike ablation of tau, treatment

In this study, we undertook the first ever assessment of anti-tau immunotherapy in a well-characterized colony of hAPP tg mice. We selected the mAb, 5E2, because it is known to recognize all full length and mid-region fragments of human and murine isoforms of tau irrespective of phosphorylation or aggregation status (Kosik et al., 1986; O’Dowd et al., 2013) and because its specificity is equal to or higher than other mid-region anti-tau antibodies (Alexandra J. Mably and Dominic M. Walsh, unpublished observations). For our animal model, we choose the J20 mouse line because prior studies had shown that these mice exhibit spatial reference memory deficits that are responsive to both anti-Ab antibodies (Maier et al., 2006) and genetic ablation of tau (Roberson et al., 2007). That is, J20 mice tolerate immunotherapy and their cognitive deficits require expression of tau. To determine an appropriate age at which J20 mice could be used for antibody experiments, we tested behavior and measured levels of various forms of Ab, tau, and indices of amyloid-associated pathology at 3 discrete ages. Consistent with prior reports, our J20 colony evinced increased mortality between 0 and 7 months of age (not shown), and at the earliest age studied showed signs of hyperactivity (Cheng et al., 2004; Roberson et al., 2007). These mice performed normally on Y-maze and CFC, but demonstrated a robust age-dependent impairment of SRM. In agreement with earlier studies, the temporal deposition and relative solubilities in TBS, TBS-TX, and FA indicate that Ab begins to deposit in very young animals and that multiple assembly forms of Ab are likely to be present throughout the life of J20 mice (Cheng et al., 2007; Hong et al., 2011; Shankar et al., 2009). To the best of our knowledge, this is the first study to investigate the levels and biochemical distribution of tau in brains of both Wt and APP tg mice. As one would expect for a highly soluble protein that can associate with the cytoskeleton, the overwhelming majority of tau is found in the water- and detergent-soluble fractions (Ksiezak-Reding et al., 1988; Yamada et al., 2011). In the TBS and TBS-TX extracts examined, tau accounts for approximately 0.16% of total protein. Indeed, it is interesting to note that even in aqueous brain extracts from APP tg mice the molar concentration of tau is approximately 100-fold higher than that of Ab. Of course the compartmentalization of tau and Ab are likely to be very different, such that most Ab is extracellular and readily available to antibodies (Schenk, 2002; Schenk et al., 2012), whereas the vast majority of tau is intracellular and less accessible to antibodies (Binder et al., 1985; Brion et al., 1988). Thus one might expect that the usefulness of anti-tau antibodies will depend on both the nature of extracellular tau and the contribution extracellular tau makes to memory impairment. Despite previous evidence that knock-out of tau can improve spatial reference memory in J20 mice (Roberson et al., 2007), we report that administration of 5E2 has no beneficial effect on memory. A trivial explanation for this outcome could have been that active 5E2 did not reach the brain. However, our analysis shows that appreciable levels of anti-tau antibodies were present in brains of both J20 and Wt mice (approximately 0.01% of blood borne 5E2), and at levels comparable with that documented in other passive immunization studies (Levites et al., 2006; Reiber and Felgenhauer, 1987). Given that animals were transcardially perfused with physiological saline and that only brains free of blood contamination

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were used, it is reasonable to assume that the 5E2 detected was not an artifact of blood contamination, but 5E2 present in the interstitial fluid or cells of the brain (Levites et al., 2006; Reiber and Felgenhauer, 1987). An alternate explanation for the lack of effect of 5E2 could have been that the dose was too low. That is, the amount of 5E2 present in brain was insufficient relative to the amount of soluble tau. In this regard, it is important to note that the ELISA used to measure 5E2 depends on the antibody being unoccupied (free) and available to bind to plate immobilized tau, thus in Wt and J20 mice 5E2 was not limiting. These results suggest that in the brains of Wt animals only very tiny concentrations of tau exist outside neurons, an observation consistent with the femtomolar levels of tau found in interstitial fluid (Maia et al., 2013; Yamada et al., 2011) and CSF of Wt mice (Maia et al., 2013; Yamada et al., 2011) (Fig. 6A). Intriguingly, although free 5E2 was readily detected in J20 mouse brain, the levels were significantly lower (approximately 40% lower) than those detected in Wt littermatesdthis despite the fact that levels of circulating 5E2 were highly similar in both Wt and J20 mice. We take these results to indicate that a higher fraction of 5E2 is bound to antigen in J20 mouse brain than in Wt brain. Nonetheless, our detection of an appreciable amount of free 5E2 in J20 brain demonstrates that the amount of antibody reaching the brain was not limiting. Thus, because appreciable levels of 5E2 reach the brain yet memory impairment persists, it is reasonable to conclude that the memory impairment evident in J20 mice is not mediated by extracellular tau or at least not by a form of tau that is recognized by 5E2. Of 16 tau immunotherapy studies, the vast majority utilized antibodies or vaccinations that target conformational or phospho epitopes and all were tested in tau transgenic mice (Asuni et al., 2007; Bi et al., 2011; Boimel et al., 2010; Boutajangout et al., 2010, 2011; Chai et al., 2011; Kontsekova et al., 2014a, 2014b; Rozenstein-Tsalkovich et al., 2013; Troquier et al., 2012; Walls et al., 2014; Yanamandra et al., 2013). All studies that used mAbs to conformational- or phospho-specific epitopes were successful at attenuating tangle pathology, and several showed improvements in functional outcomes such as locomotion and survival (Asuni et al., 2007; Boutajangout et al., 2010, 2011; Chai et al., 2011; Kontsekova et al., 2014a; Yanamandra et al., 2013). In contrast, the only 2 studies that tested sequence-specific anti-tau mAbs failed to influence tau pathology (Oddo et al., 2004; d’Abramo et al., 2013). In the preeminent tau immunotherapy study by Oddo et al. (2004), a single injection of the mid-region human-specific, anti-tau mAb, HT7, was unable to clear tau pathology when injected into the hippocampus of 12 months 3xtg mice, whereas injection of certain anti-Ab mAbs were partially effective. Another mid-region antibody, DA31, failed to attenuate tangle pathology in P301L mice under conditions where the conformation-specific MC1 mAb significantly reduced the rate of tau accumulation (d’Abramo et al., 2013). Taken together, these studies strongly suggest that mid-region “pan-tau” mAbs are not effective at clearing tau pathology. However, the relationship between tau pathology and cognitive impairment is not well understood, and there is burgeoning evidence that frank tau pathology per se is not a cause of dysfunction (Spires-Jones and Hyman, 2014). For instance, memory deficits in rTg4510 mice and pro-aggregant TauRD mice are reversible when tau transgene expression is suppressed even in the continued presence of neurofibrillary tangle pathology (Santacruz et al., 2005; Sydow et al., 2011). Moreover, the nature of extracellular tau remains enigmatic (Meredith et al., 2013; Wagshal et al., 2014; Yamada et al., 2011). Consequently, the outcome of the present study recommends that prior tau immunotherapy results in tau tgs should be interpreted with caution. This is particularly important because many of the tau tg models used in earlier immunizations studies over-express very high levels of tau and develop

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motor deficits which compromise their use for studying learning and memory (Boutajangout et al., 2012). In addition to the lack of positive effect of anti-tau immunotherapy, we also documented a significant increase in mortality among J20 mice that received 5E2 (4 of 13 treated animals died). This occurred at an age when J20 mice exhibit a morality rate indistinguishable from their littermate controls. In contrast, in Wt mice, 5E2 had no negative effects on viability, memory and learning, or brain pathology. Thus, because 5E2 was not associated with adverse effects in the 11 Wt mice that received 5E2, nor in the 12 J20 mice given an isotype-matched control IgG, it is evident that the increased mortality in 5E2-immunized J20 mice was highly specific and required expression of human APP/Ab. However, given that J20 mice have no real neurofibrillary pathology, it is not clear if 5E2 would prove toxic under conditions when tangles are abundant. We found no evidence for the sort of chronic neuroinflammatory disease that is known to cause death in Wt mice immunized with various fragments of tau (Rosenmann et al., 2006; RozensteinTsalkovich et al., 2013) and as yet the cause of the sudden death of 4 of 13 5E2-immunized J20s remains unexplained. New studies will be required to shed light on the molecular mechanisms that led to these sudden deaths, but we speculate that 5E2 may be acting on the tau/APP/Ab axis to enhance aberrant excitatory neuronal activity (Lalonde et al., 2012; Palop et al., 2007; Roberson et al., 2011; Sanchez et al., 2012) and that this leads to fatal convulsive seizures. Similarly, it will be important to determine whether the adverse effects seen with 5E2 are replicated in other models of AD and with antibodies that recognize other domains of tau. With regard to the latter, one would anticipate that antibodies directed against different epitopes may have different effects. Such future studies may also provide new insights about the normal physiological role of extracellular tau and forms of tau specifically altered by APP/Abdependent mechanisms. Moreover, given that epileptiform activity is driven by APP over-expression, such pathways may not be active in tau transgenics and consequently 5E2 (Born et al., 2014), and perhaps other mid-region anti-tau mAbs, may not exert toxicity in the absence of APP over-expression. It is important to emphasize that we tested only a single anti-tau mAb and used but 1 APP tg mouse line; nonetheless, the effects of 5E2 are both disappointing and worrying and serve to underscore the complexity of studying and targeting tau. Clearly, future studies should look not only at a selection of anti-tau mAbs that target distinct epitopes and conformations but also use a variety of preclinical models. To avoid drawbacks associated with overexpression models of AD, it will be important to develop knock-in models that allow the expression of the full complement of human tau and APP in an appropriate spatial and temporal pattern. Such animals should not only better represent AD but also provide more accurate preclinical models in which to test trial therapies such as anti-tau mAbs. Disclosure statement The authors have no conflicts of interest to disclose. Acknowledgements The authors thank Jeffrey Frost, Patrick McKinney, Gregory Dillon, and Dr Roderick Bronson for technical assistance. They also thank Drs Donna Barten and Sethu Sankaranarayanan (BristolMyers Squibb) for advice on the tau ELISA and Dr Lennart Mucke for useful discussions. Monoclonal antibodies 8E5, m266, 21F12, and 2G3 were a kind gift from Drs Peter Seubert and Dale Schenk,

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Tau immunization: a cautionary tale?

The amyloid β (Aβ)-protein and microtubule-associated protein, tau, are the major components of the amyloid plaques and neurofibrillary tangles that t...
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