doi: /brain/awu213 Brain 2014: 137;

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1 doi: /brain/awu213 Brain 2014: 137; BRAIN A JOURNAL OF NEUROLOGY Neuronal uptake of tau/ps422 antibody and reduced progression of tau pathology in a mouse model of Alzheimer s disease Ludovic Collin, 1 Bernd Bohrmann, 1 Ulrich Göpfert, 2 Krisztina Oroszlan-Szovik, 1 Laurence Ozmen 1 and Fiona Grüninger 1 1 Roche Pharmaceutical Research and Early Development, Neuroscience Ophthalmology and Rare Diseases Discovery & Translational Area, Roche Innovation Center Basel, Grenzacherstrasse 124, CH-4070 Basel, Switzerland 2 Roche Pharmaceutical Research and Early Development, Large Molecule Research, Roche Innovation Center Penzberg, Nonnenwald 2, D Penzberg, Germany Correspondence to: F. Grüninger, Roche Pharmaceutical Research and Early Development, NORD DTA, Roche Innovation Center Basel, Grenzacherstrasse 124, CH-4070 Basel, Switzerland fiona.grueninger@roche.com The severity of tau pathology in Alzheimer s disease brain correlates closely with disease progression. Tau immunotherapy has therefore been proposed as a new therapeutic approach to Alzheimer s disease and encouraging results have been obtained by active or passive immunization of tau transgenic mice. This work investigates the mechanism by which immunotherapy can impact tau pathology. We demonstrate the development of Alzheimer s disease-like tau pathology in a triple transgenic mouse model of Alzheimer s disease and show that tau/ps422 is present in membrane microdomains on the neuronal cell surface. Chronic, peripheral administration of anti-tau/ps422 antibody reduces the accumulation of tau pathology. The unequivocal presence of anti-tau/ps422 antibody inside neurons and in lysosomes is demonstrated. We propose that anti-tau/ps422 antibody binds to membrane-associated tau/ps422 and that the antigen-antibody complexes are cleared intracellularly, thereby offering one explanation for how tau immunotherapy can ameliorate neuronal tau pathology. Keywords: tau/ps422 antibody; tau immunotherapy; lipid rafts Introduction The current disease-modifying therapeutic approaches to treating Alzheimer s disease are heavily focused on clearing amyloid-b plaques, one of the two hallmark pathologies of the disease. Clinical trials with amyloid-b therapies, including immunotherapy, have thus far yielded disappointing results and have generated renewed interest in alternative approaches that target other aspects of Alzheimer s disease. Tau-containing neurofibrillary tangles are the other hallmark pathology of this disease. The amyloid cascade hypothesis of Alzheimer s disease places development of tau pathology downstream from amyloid-b plaque deposition, possibly explaining why tau has received less attention as a potential therapeutic target (Hardy, 1992). However, unlike amyloid-b plaques, the number of neurofibrillary tangles in Alzheimer s disease brain measured post-mortem correlates well with antemortem cognitive status, suggesting a more direct link between this pathology and development of disease (Nelson et al., 2012). Tau is an axonal protein that normally associates with and stabilizes microtubules. In Alzheimer s disease, abnormal phosphorylation, misfolding and aggregation of tau leads to neurofibrillary tangle formation and ultimately to neuronal cell death by a mechanism that is not yet fully understood. It is unclear whether neurofibrillary tangles themselves can exert neurotoxic effects or Received April 14, Revised June 27, Accepted June 29, Advance Access publication July 31, 2014 ß The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please journals.permissions@oup.com

2 tau/ps422 antibody ameliorates tau pathology Brain 2014: 137; whether smaller, misfolded, protofibrillar tau species are responsible for neurotoxicity (Congdon and Duff, 2008). Despite uncertainty about the precise aggregation state of the relevant neurotoxic species, clearance of misfolded tau should be of benefit to neurons. Different therapeutic strategies can be envisaged, one of which is immunotherapy with an antibody directed against misfolded tau. Given that tau is an intracellular protein, immunotherapy would seem to have little chance of success: antibodies have to traverse not only the blood brain barrier but also the neuronal plasma membrane. Nevertheless, many groups have shown that immunotherapy in tau transgenic mice leads to reduced pathology (Boutajangout et al., 2011; Chai et al., 2011; d Abramo et al., 2013; Yanamandra et al., 2013; Castillo-Carranza et al., 2014). One explanation for these findings is that antibodies inhibit transcellular spreading of tau aggregates (Clavaguera et al., 2009; Voss et al., 2012). A large body of evidence now supports the hypothesis that tau aggregates can spread in a prion-like manner between interconnected neurons, implying that misfolded tau is transiently exposed in the extracellular space (Clavaguera et al., 2009; Frost and Diamond, 2010; Guo and Lee, 2011; Himmelstein et al., 2012; Voss et al., 2012). Tau in the extracellular space may be targeted and cleared by antibodies. However, additional clearance mechanisms may be involved. We raised an antibody to the S422 phosphorylation site in tau, a disease-specific phospho-epitope that is associated with tau misfolding (Bussiere et al., 1999; Pennanen and Götz, 2005; Guillozet-Bongaarts et al., 2006). Tau/pS422 is prominent in early stages of Alzheimer s disease and persists until late-stage disease, making it an attractive target for antibody therapeutics (Guillozet-Bongaarts et al., 2006; Vana et al., 2011). Due to its location near the C-terminal end of the tau molecule, the tau/ ps422 epitope should be well exposed and available for antibody binding in pathological forms of tau i.e. oligomeric and fibrillar tau aggregates (Patterson et al., 2011; Wegmann et al., 2013). We show that TauPS2APP transgenic mice develop less tau pathology than vehicle-treated controls if treated for 4 months with anti-tau/ ps422 antibody. We further demonstrate the unequivocal presence of antibody inside neurons and propose that antigenmediated uptake of antibody into neurons contributes to efficacy of immunotherapy. These findings provide an additional (spreading-independent) mechanistic rationale for developing tau immunotherapy. Materials and methods Anti-tau/pS422 antibody generation MAb86 Rabbits were immunized with the phosphopeptide tau( )[pS422] coupled to keyhole limpet haemocyanin. Cultured supernatants from single B cells were screened by ELISA for binding to biotinylated tau( )[pS422] and counter-screened against the non-phosphorylated peptide. The variable domains of MAb86 were cloned from a positive B cell by reverse transcriptase PCR. MAb86 was expressed recombinantly in human embryonic kidney cells either with a murine IgG1 or a human IgG1 isotype. MAb86 was used for the in vivo experiments described in this study. The human IgG1 isotype was used for all experiments except for the 16-week chronic efficacy study, for which the murine IgG1 was used. Anti-tau/pS422 clone and Immunohistochemical detection of tau/ps422 on brain sections was performed using one of two fully murine monoclonal anti-tau/ps422 antibodies, referred to as clone and clone The generation and characterization of is described in Grueninger et al. (2010, 2011). Antibody is derived from a sister clone that came out of the same work and has identical specificity. These two murine antibodies were used interchangeably for detection of tau/ps422 on sections. TauPS2APP mice The TauPS2APP mice have been described in detail previously (Grueninger et al., 2010). Briefly, a double transgenic mouse line was created by co-injection of both APP (Swe) and presenilin 2 (PS2, now known as PSEN2) (N141I) transgenes into C57Bl/6 zygotes as already described (Ozmen et al., 2009). The transgenic TauP301L mouse (line pr5) was generated in a mixed C57Bl/6, DBA/2 background (Götz et al., 2001) and backcrossed for seven generations into C57Bl/6. TauPS2APP triple transgenic mice were generated by crossing the APP-PS2 and tau transgenic lines. TauPS2APP mice develop both amyloid-b plaques and neurofibrillary tau pathology with histopathological changes first becoming evident at 6 7 months of age. Mice were housed in single cages and had free access to food and water. All mouse procedures were conducted in strict adherence to the Swiss federal regulations on animal protection and to the rules of the Association for Assessment and Accreditation of Laboratory Animal Care and with the explicit approval of the local veterinary authority. Acute and chronic treatment of TauPS2APP mice with MAb86 There were no gender differences in the development of tau pathology in TauPS2APP mice therefore male and female mice were used as available, except for the chronic study where the gender groups were balanced. For acute studies to investigate antibody distribution in the brain, 16-month-old TauPS2APP mice were treated with two separate doses of MAb86 at 60 mg/kg intraperitoneally with a 3-day interval between doses. Mice were killed 48 h postdosing using a lethal dose of pentobarbital (in accordance with Swiss federal regulations on animal protection). They were then perfused transcardially with 20 ml PBS before brain removal. To investigate efficacy of MAb86 in reducing tau brain pathology, 10-month-old mice were treated weekly intraperitoneally with 30 mg/kg antibody or vehicle (n = 14/group). The mice were treated for a total of 16 weeks then sacrificed 1 week after receiving the final dose. No adverse effects of chronic antibody treatment were observed. The acute studies were done with human IgG1 version of MAb86. The chronic treatment study was performed with MAb86 as murine IgG1.

3 2836 Brain 2014: 137; L. Collin et al. Preparation of sarkosyl insoluble tau for western blotting Sarkosyl insoluble tau was prepared according to the method of Götz et al. (2001). Quantitation of tau and tau/ps422 by specific ELISAs Assays were performed according to Grueninger et al. (2010). Lipid raft preparation Detergent-resistant lipid rafts were prepared according to Kumari and Francesconi (2011). Fresh or snap-frozen mouse brain tissue from 16- month-old TauPS2APP transgenic mice was used as a source of material. Sucrose gradients were harvested in 1-ml fractions, beginning at the top of the gradient. Protein content of each fraction was determined by bicinchoninic acid assay (Pierce/Thermo) and 85 mg protein of each fraction was then analysed by western blotting. Blots were developed with MAb86 to detect tau/ps422, anti-flotillin 1 (Thermo Scientific) as a marker of lipid rafts and anti-clathrin (Novus Biologicals) to detect detergent-soluble, non-raft proteins. Quantitative image analysis After PBS perfusion, frozen sagittal sections were prepared and immunostained with a murine monoclonal antibody against tau/ps422 (Roche in-house, clone ) at a concentration of 2 mg/ml. Sections were imaged with a Metafer 4 slide scanning system (MetaSystems). The tau/ps422 immunopositive area was measured within the hippocampus and neocortical region and area of phosphorylated tau-positive immunofluorescence signal determined by an unbiased morphometrical method by means of computer-assisted image analysis. Quantification of phosphorylated tau positive area was done after threshold adjustment with MCID M7 Elite software (InterFocus Ltd.). The ratio of occupied area to region of interest area was calculated and a t-test used with the spreadsheet software Microsoft Excel. As MAb86 and antibody both bind to the region around ps422 in tau, internalized MAb86 may potentially interfere with immunodetection of tau/ps422 by antibody Control staining with AT8 (which binds to a different phospho-epitope upstream from ps422) indicated that this was not the case (data not shown). Immunofluorescence of free-floating brain sections TauPS2APP mouse brain free-floating sections were carried out according to Collin et al. (2004). Briefly, free-floating sections were permeabilized with Triton TM X-100 (0.3 or 0.5%) before incubation with primary antibodies. The following primary antibodies were used: anti-lamp2 (Fitzgerald, 10R-CD107bbMSp), anti-sqstm1/p62 (SantaCruz, sc-25575), anti-tubulin alpha (Serotec, clone Y/L ½), anti-human PHF-TAU AT8 (Pierce, MN1020), anti-flotillin (Thermo Scientific, PA ). With the exception of the images shown in Fig. 1A F, tau/ps422 detection was performed using murine monoclonal anti-tau/ps422, clone or Appropriate donkey and/or goat secondary antibodies conjugated to Alexa Fluor Õ 488, 555, 594 or 647 were used (Molecular Probes). Sections were also stained with 4 0,6 0 -diamidino-2-phenylindole (DAPI). Images were acquired using a Leica TCS SP5 confocal system (Leica Microsystems) using HCX PL APO CS dry UV, HCX PL APO CS oil UV or HCX PL APO LB oil UV objectives. Leica LAS-AF 3D Deconvolution tool and Imaris software (Prism Software) were used to process images, to perform 3D reconstruction, cross-sections and movie. To analyse the co-localization of MAb86 with lipid rafts, brain sections of mice injected acutely with MAb86 were stained with a goat anti-human antibody and an anti-flotillin 1-Cy3 antibody (F8931, Sigma). To visualize the plasma membrane of the dendritic tree, a projection of three images (with a step size of 0.3 mm) was done. The co-localization study was performed in Imaris. Thresholds for MAb86 (Channel A) and flotillin 1 (Channel B) signals were 50 and 80, respectively. The co-localized signal is presented in white. Super resolution microscopy Brain free-floating sections were prepared as described above. First, endogenous biotin-like substances were quenched (Invitrogen kit, # , according to manufacturer s instructions). Primary and secondary antibodies were diluted in PBS containing 5% donkey serum and incubated overnight at 4 C or at room temperature for 1 h, respectively. Biotinylated, in combination with Streptavidin V500 (BD Biosciences, ), or Alexa Fluor Õ 488 secondary antibodies were used. Samples were mounted using ProLong Õ Gold and analysed using a Leica gated-sted microscope with a HCX PL APO oil objective. Quantification of tau/ps422 neurons in the CA1 region of the hippocampus Sagittal brain cryosections (20 mm) from TauPS2APP mice at baseline (10 months) or mice treated for 16 weeks with MAb86 or vehicle were acetone-fixed and stained with an anti-tau/ps422 antibody coupled to Alexa Fluor Õ 488 and DAPI. Five animals per condition, with three sections per animal, were analysed. Z-stack confocal images were acquired using a HCX PL APO CS dry UV objective. Tau/ ps422-positive neurons contained within the first 600-mm of CA1 (starting from the tip) were counted. Results are expressed as the mean number of tau/ps422 cells per 600-mm or per 200-mm intervals. One-way ANOVA with Bonferroni s comparison test was used for statistical analysis (Prism Software). Quantification of lysosomes in tau/ps422 neurons Z-stack confocal images of brain sections co-stained with tau/ps422 and LAMP2 antibodies were acquired in the CA1 region of the hippocampus using a HCX PL APO LB oil UV (step size of 0.21 mm). 3D reconstruction of individual tau/ps422 neuronal soma was done after deconvolution (Imaris software, Bitplane) to quantify tau/ps422 amount (volume of tau-ps422/mm 3 ) and lysosome number (spots/100 mm 3 ). A total of 80 neurons were analysed. Statistical analysis was compiled using Imaris measurement pro-module. Correlation analysis was done using the Spearman rank correlation method (Prism Software).

4 tau/ps422 antibody ameliorates tau pathology Brain 2014: 137; Figure 1 MAb86 is a tau/ps422-specific antibody that binds neurofibrillary tau. (A) Tau western blots using MAb86 (top) and human tau-specific antibody HT7 (bottom) for detection. MAb86 binds specifically to ERK-phosphorylated tau (lane 1) but not to unphosphorylated tau (lane 2), ERK-phosphorylated mutant tau S422A (lane 3) or unphosphorylated tau S422A (lane 4). (B) Western blots of brain extracts from TauPS2APP mouse brains using MAb86 (top) and human tau-specific antibody HT7 (bottom) for detection. Extracts were prepared from 16-month-old wild-type mice (lane 1) or triple transgenic mice at 8, 12, 16 and 20 months of age (lanes 2 5). M = molecular weight markers. (C) Immunofluorescent detection of tau/ps422-containing neurons in the somatodendritic compartment of CA1 pyramidal layer using MAb86 (in red). Nuclei are counterstained with DAPI (blue). (D F) Higher magnification reveals diverse neurofibrillary tau structures (in green). (G and H) Peripherally administered MAb86 targets tau/ps422 in neurons. Unspecific signal from the vasculature is detected in the vehicle-injected mice (G). MAb86, detected using an anti-human IgG-AF555, is present in a subpopulation of CA1 pyramidal neurons (H arrows). Nuclei are counterstained with DAPI (G and H).

5 2838 Brain 2014: 137; L. Collin et al. Figure 2 Tau/pS422 locates in lipid raft microdomains on neuronal membranes in untreated TauPS2APP mice. (A D) Pathological tau/ ps422 is detected in lipid raft-like microdomains on the periphery of a dendritic shaft (A and B). (C and D) High magnification of the boxed area in B. Microtubule cytoskeleton is in green (B and D), nuclei are counterstained with DAPI in B. (E G) Tau/pS422 partially localizes with flotillin 1 as indicated by arrows. (H) Sucrose density gradient fractionation of mouse brain proteins shows that some tau/ps422 is found in flotillin-positive fractions. Bottom and top refer to the bottom and topmost fractions of the gradient. P is the detergent insoluble material that pellets at the bottom of the gradient. Molecular weight markers are denoted by M and are 64 kda (top), 51 kda (middle) and 28 kda (bottom). Results MAb86 binds specifically to tau phosphorylated at S422 and detects hyperphosphorylated tau aggregates in TauPS2APP mouse brain We generated a monoclonal rabbit antibody, MAb86, that is specific for tau phosphorylated at Ser422, as determined in vitro by western blotting of tau, ERK2-phosphorylated tau and ERK2-phosphorylated taus422a (Fig. 1A). In mouse brain extracts, MAb86 detects a 64 kd sarkosyl-insoluble tau species that accumulates with age in TauPS2APP transgenic mice but not in wild-type mice (Fig. 1B). The 64 kd tau band corresponds to hyperphosphorylated human tau (Götz et al., 2001). There is no cross-reactivity with any protein in wild-type mouse brain extracts, consistent with MAb86 being specific for phosphorylated human tau. MAb86 binds to AT8-positive, tangle-like aggregates on brain sections prepared from TauPS2APP mice, demonstrating that the antibody is specific for misfolded, hyperphosphorylated tau (Fig. 1C and Supplementary Fig. 2). MAb86 does not

6 tau/ps422 antibody ameliorates tau pathology Brain 2014: 137; detect anything on brain sections prepared from control (WT B6) mice (Supplementary Fig. 1), consistent with its specificity. Tau/pS422 tangles are detected in the soma and the main apical and basal dendrites of hippocampal neurons illustrating the pathological nature of tau/ps422-containing structures (Fig. 1C arrows). High magnification of CA1 neurons shows different types of tau/ps422-positive neurons, recapitulating bona fide Alzheimer s disease tau pathology (Fig. 1D F) (Augustinack et al., 2002). MAb86 also detects Alzheimer s disease-tau and typical neurofibrillary pathology on human Alzheimer s disease brain sections (Supplementary Fig. 3). After intraperoneal administration to TauPS2APP mice, MAb86 is detected in the soma and dendrites of CA1 neurons that contain tau pathology (Fig. 1H and Supplementary Fig. 4). No signal is detectable in vehicle-injected TauPS2APP mice (Fig. 1G) or in MAb86-injected wild-type mice (data not shown). The selective internalization of MAb86 by neurons that contain tau pathology suggests target-dependent uptake mechanism. Tau/pS422 is present in lipid rafts We hypothesized that entry of MAb86 in neurons may occur through direct or indirect interaction with tau/ps422 at the neuronal plasma membrane. In CA1 neurons, tau/ps422 is largely excluded from and mainly located around the dendritic microtubule cytoskeleton. It localizes in membrane-like microdomains along the dendritic shaft that resembles lipid rafts (Fig. 2A D, arrows). We demonstrated the presence of tau/ps422 in lipid rafts by double immunostaining with MAb86 and flotillin 1 antibodies (the latter being a marker of lipid rafts) (Staubach and Hanisch, 2011). Cross-sections of confocal images show the peripheral distribution of flotillin 1 in a neuronal process and some regions of co-localization with tau/ps422 (Fig 2E G). Furthermore, tau/ps422 co-fractionated with detergent-resistant flotillin-containing membrane compartments (Fig. 2H). Together these data show that some tau/ps422 localizes at the plasma membrane and to some extent within lipid rafts. Peripherally administered MAb86 is detectable in lipid rafts MAb86 present in the parenchyma may be able to bind to tau/ps422 located in lipid rafts at the neuronal membrane surface. We therefore analysed the distribution of MAb86 in CA1 neurons of antibody-treated mice. MAb86 distributes in puncta/granular structures along and within the dendrites of targeted neurons, with a patchy distribution similar to that reported for phosphorylated tau above, as well as within the neuronal soma (Fig. 3A F and Supplementary Video 1). This suggests that MAb86 indeed binds to lipid raft-associated tau/ps422 at the plasma membrane. We next analysed whether MAb86 could bind to flotillin 1-positive lipid rafts on the plasma membrane of CA1 neurons. MAb86 is detected in granular structure both in the soma and the dendrite of the neuron (Fig. 4A). Flotillin 1 rafts are present on the Figure 3 Peripherally administered MAb86 is detected at the membrane and in granular structures in CA1/3 neurons. (A D) Peripherally administered MAb86 localizes to the periphery and inside soma, apical dendrites and basal processes of hippocampal neurons (A). A tubulin antibody was used to highlight the neuronal morphology (B). Nuclei are counterstained with DAPI in C and D. (E and F) Higher magnification of the boxed areas in D shows MAb86-containing granules along and inside the basal process (E) and dendritic shaft (F) (cross-sections, arrows). membrane of the dendrite and the soma where co-localized signal was found (Fig. 4C and D). Cross-sections of the dendritic tree (boxed area in Fig. 4C) confirm the presence of injected MAb86 on lipid rafts (Fig. 4E H). Internalized MAb86 is detectable in lysosomes The intraneuronal localization of MAb86 further suggests that binding of MAb86 to raft-associated tau/ps422 may promote internalization of the protein complex.

7 2840 Brain 2014: 137; L. Collin et al. Figure 4 Peripherally administered MAb86 co-localizes with flotillin 1 in the dendritic shaft of CA1 neurons. (A C) Represents a projection of three confocal images showing peripherally administered MAb86 (A) in granular structures both in the soma and the dendrite of a CA1 neuron. Flotillin 1 rafts (B) are present on the membrane of the dendrite and at the periphery of the soma. The white signal in (D) shows the co-localization between MAb86 and flotillin-1 from a single plan confocal image. (E H) Cross-sections of the boxed area in C showing co-localization of MAb86 (E) with flotillin 1-positive rafts (F). Lipid rafts can mediate endocytosis of cargos and their delivery to lysosomes for degradation (Pooler et al., 2013). We therefore investigated whether MAb86-containing granules could be delivered to lysosomes. Mice treated with MAb86 show MAb86- containing granules in and along the dendritic process of neurons as well as in the neuronal soma, where they accumulate with LAMP2 positive lysosomes (Fig. 5A C). Interestingly, lysosomes are also detected in the dendritic shaft, in close proximity to the neuronal membrane, where they co-localize with MAb86 granules (Fig. 5D F). These data suggest that endocytosed MAb86 granules are targeted for lysosomal degradation. As MAb86 selectively targets phosphorylated tau-positive neurons, this may provide a mechanism for delivery of the MAb86/phosphorylated tau complex to lysosomes for degradation, ultimately leading to a reduction in tau pathology. Chronic treatment of TauPS2APP mice with MAb86 reduces tau/ps422 pathology To investigate the effect of MAb86 on the development of tau pathology, TauPS2APP mice were injected weekly with MAb86 (30 mg/kg) or vehicle for 16 weeks. At the end of treatment, immunoassay of whole brain extracts showed a significant reduction in tau/ps422 levels in MAb86-treated mice compared to vehicle-treated mice (Fig. 6A C). This reduction becomes highly significant when adjusted for the small variability in total tau expression i.e. by calculating the ratio tau/ps422:total tau (**P ). These ELISA data are supported by quantitative immunohistochemistry analysis of individual brain regions (Supplementary Fig. 5). Quantification of tau/ps422-positive cells found in the first 600 mm of the CA1 pyramidal layer confirm that chronic MAb86 treatment significantly prevents the development of pathology with 50% fewer tau/ps422-positive cells found in MAb86-treated mice compared to vehicle (Fig. 6D E, ***P ). These data demonstrate that chronically administered MAb86 delays the progression of pathology in TauPS2APP mice. Tau/pS422 accumulation leads to lysosomal deficiency To understand how MAb86 can be of therapeutic benefit, it is important to know how tau/ps422 disrupts neuronal function. Accumulation of tau/ps422 aggregates could result from the decline of protein clearance and/or lysosomal dysfunction. Lysosomes are clearly discernible in the perinuclear and dendritic region of CA1 pyramidal neurons (Fig. 7B and C). However, in neurons with well-established tau/ps422 pathology there is a significant inverse correlation between the number of lysosomes and the pathological tau/ps422 load (Fig. 7D). These data suggest that progressive accumulation of tau/ps422 can affect lysosomal integrity. We provided additional evidence of defective lysosomal clearance in tau/ps422-positive neurons by demonstrating the accumulation of p62/sqstm1 in these cells (Fig. 7E G).

8 tau/ps422 antibody ameliorates tau pathology Brain 2014: 137; Figure 5 Peripherally administered MAb86 is targeted to the lysosomes. (A C) MAb86-containing vesicles are delivered to the lysosomes. Immunodetection of peripherally administered MAb86 (A) and LAMP2-positive lysosomes (B) in neurons from the CA1 region. MAb86 is detected in vesicular structures in the soma (arrow) and the dendrites where lysosomes also accumulate. Nuclei are counterstained with DAPI in the merged image (C). (D F) Higher magnification of the boxed area in C showing MAb86 vesicles contained within LAMP2- positive lysosomes (F, arrowheads). Accumulation of aggregate-prone protein has been shown to negatively impact protein clearance machinery (Yang et al., 2009; Freeman et al., 2013). Using gated stimulation emission depletion microscopy we investigated whether tau/ps422 accumulation could impact lysosomal integrity. Granular pre-tangle tau/ps422 structures depicted by confocal microscopy are revealed to be fibrillar structures by super resolution (Fig. 7H I). Lysosomes, the lumens of which are indiscernible by confocal microscopy, are distinguished as doughnut-like shapes with LAMP2 signal delimitating the lysosomal membrane (Fig. 7J and K). By combining both stainings, tau/ps422 fibrils are seen to partially overlap with the lysosomal membrane suggesting that phosphorylated tau fibrils can bind to and/or get inserted into lysosomes (Fig. 7L N). Together these data suggest that the insertion of tau/ps422 fibrils may compromise the integrity of lysosomal membranes and ultimately lead to lysosomal depletion. Discussion Several passive immunization studies in mice provide a strong rationale for developing tau immunotherapy for Alzheimer s disease (Boutajangout et al., 2011; Chai et al., 2011; d Abramo et al., 2013; Castillo-Carranza et al., 2014). In this work we investigated how MAb86, an antibody directed against tau/ps422, reduces tau/ps422 levels in the brains of TauPS2APP mice. This particular phosphorylated epitope in tau was selected as a target for antibody generation because data suggest a close link between this epitope and the development of tau pathology and because it had already been the target of a successful active immunization study (Vana et al., 2011; Troquier et al., 2012). We demonstrate that MAb86 is internalized specifically by neurons that contain tau/ps422 and conclude that antibody uptake is mediated by antigen that is associated with the plasma membrane. Lysosomal degradation of antigen-antibody complexes may contribute to the efficacy of MAb86 in slowing the development of tau pathology.

9 2842 Brain 2014: 137; L. Collin et al. Figure 6 Chronic administration of MAb86 to TauPS2APP mice slows progression of tau pathology. (A C) Ratio of tau/ps422 to total tau in brain extracts measured by ELISA. There is a significant increase in ratio between the start and end of experiment (***P ) and there is a significant reduction of the ratio in antibody-treated mice compared to the vehicle-treated group (**P ). (D) Pathology starts in the subiculum as shown at baseline and progress along the CA1 with time in vehicle injected mice. (E) MAb86 treatment significantly reduces the number of neurons developing tau/ps422 pathology in the first 600 mm of CA1 (***P ).

10 tau/ps422 antibody ameliorates tau pathology Brain 2014: 137; Figure 7 Progressive accumulation of phosphorylated tau impairs lysosomal function in untreated TauPS2APP mice. (A C) Hippocampal region of 24-month-old TauPS2APP mice. Co-staining of CA1 neurons for tau/ps422 (A) and LAMP2, a marker of lysosomes (B, arrow). Lysosomal depletion is apparent in neurons containing substantial tau/ps422 pathology (dotted area in B). (D) Quantification of the number of lysosomes (spots) contained per neuronal soma relative to the amount tau/ps422. The number of lysosomes per neuron inversely correlates with the amount of pathological tau/ps422 (Spearmann correlation: r = , P ). (E G) tau/ps422- containing neurons (E) accumulate the adaptor protein SQSTM1/p62 (F) confirming lysosomal dysfunction in these cells. (H K) Superresolution images of tau/ps422 aggregates (H and I) and LAMP2-positive lysosomes (J and K). Whereas confocal image shows tau/ps422 aggregates (H and insert), gated STED microscopy reveal tau/ps422 fibrils that can be straight and curved (I and insert). LAMP2 lysosomes are resolved as granular structures by confocal microscopy (J and insert) whereas super resolution specifically resolved the LAMP2 positive membrane of the lysosomes with their lumen devoted of any signal (K and insert). (L N) Super resolution images of a double staining between tau/ps422 antibody (L) and a LAMP2 antibody (M) showing co-localization of tau/ps422 fibril with the lysosomal membrane at the exception of an hairpin-like structure (L and N arrow).

11 2844 Brain 2014: 137; L. Collin et al. Tau/pS422 association with neuronal membranes We detected tau/ps422 in neuronal membrane microdomains using high resolution confocal microscopy. In further experiments these membrane microdomains were shown to be flotillin-positive lipid rafts. This is consistent with the findings of Kawarabayashi et al. (2004) who showed that phosphorylated tau is predominantly found in the lipid raft fraction from Alzheimer s disease brains, complementing data showing that APP, amyloid-b and the secretases are also present in this compartment (Vetrivel and Thinakaran, 2010). Rafts therefore constitute a lipidic microenvironment where the key pathological entities of Alzheimer s disease converge. Despite being a very soluble protein, tau has a high propensity to interact with lipid membranes (Maas et al., 2000; Eidenmuller et al., 2001; Pooler and Hanger, 2010). It can undergo structural compaction and insert into the bilayer (Ferrari et al., 2003; Jones et al., 2012). Tau/pS422 in lipid rafts may therefore be exposed to the extracellular environment and provide a neuronal entry point for MAb86. In experiments using acute brain slices from tau transgenic mice, Sigurdsson (2008) and Congdon et al. (2013) showed that IgG can enter neurons by clathrin-mediated endocytosis following binding to low-affinity FcgII/III receptors. This mechanism would be expected to operate in all neurons and cannot therefore explain the selective uptake of MAb86 into phosphorylated tau-containing neurons. Although we cannot exclude uptake by a receptor binding modality, our data support the uptake of MAb86 by direct, target-mediated binding to tau/ps422 at the neuronal membrane. A similar mechanism has been demonstrated for uptake of antibodies directed against cell-surface expressed rabies virus (St Pierre et al., 2011). Phosphorylated tau may have a more general propensity to associate with membranes and, interestingly, flotillin 1 positive raftlike lipid microdomains have also been described to be present in the lysosomal membrane (Kokubo et al., 2003; Kaushik et al., 2006). We show in Fig. 7 that small tau/ps422 fibrils seem to integrate into the lysosomal membrane and that there is an inverse correlation between fibrillar tau/ps422 levels and lysosome number in pyramidal neurons in TauPS2APP mice. Binding of phosphorylated tau fibrils to lysosomal membranes and resultant lysosomal damage and depletion may ultimately cause accumulation of autophagic vacuoles, a phenotype that has been reported in some models of neurodegeneration (Lin et al., 2003). This would be consistent with our observation that neurons with late stage tau/ps422 pathology accumulate p62/sqstm1 protein suggesting impairment of autophagosomal-lysosomal flux (Götz et al., 2000; Kins et al., 2003). Kuusisto et al. (2002) showed that p62/ SQSTM1 accumulates in the soma of neurofibrillary tangle-positive neurons in Alzheimer s disease, thereby providing a link between our findings in transgenic mice and human disease. MAb86 uptake into neurons and mechanism of action Peripherally administered MAb86 can be visualized in the cytoplasm of neurons that contain tau/ps422 aggregates, consistent with data from Sigurdsson and coworkers (2008) who showed cellular uptake of labelled tau antibodies both in vitro and in vivo (Asuni et al., 2007; Krishnamurthy et al., 2011). We further showed that MAb86 binds to phosphorylated tau present at the neuronal membrane in raft-like domains, is detectable inside neurons in granular structures and is also found in lysosomes. MAb86 bound to lipid raft-associated tau/ps422 may induce endocytosis and enable clearance of tau/ps422 through lysosomal degradation. Although this hypothesis still needs to be validated experimentally, it is supported by recent data showing the presence of tau antibody in lysosomes following addition of the antibody to brain slices prepared from tau transgenic mouse (Gu et al., 2013). Lipid raft-associated proteins are known to be trafficked through the endocytic pathway and degraded in lysosomes (Ferrari et al., 2000; Pooler et al., 2013). However, most of the MAb86-positive granular structures do not co-localize with LAMP2. They may correspond instead to the small fibrillar tau structures discernable by gated stimulation emission depletion. These aggregates are similar to neurofibrillary tangles and accumulate in the cytoplasmic space. Some internalized MAb86 must therefore escape lysosomal degradation and reach the cytoplasm. MAb86 that enters the cytoplasm may protect lysosomes from fibrillar damage by binding tau/ps422 and reducing fibrillization or by inhibiting fibril insertion into the lysosomal membrane. In summary, internalized MAb86 may slow progression of tau pathology by two principal routes, namely, by promoting lysosomal clearance of membrane-associated tau/ps422 and by protecting the clearance system itself from damage by tau/ps422. Implications for tau immunotherapy Recent papers suggest that misfolded tau can move between synaptically connected neurons, thereby contributing to the spreading of tau pathology (Clavaguera et al., 2009; Goedert et al., 2010; de Calignon et al., 2012; Liu et al., 2012; Ahmed et al., 2014). It is not clear to what extent pathological tau spreading contributes to the development of pathology in TauPS2APP mice. In Fig. 6 we show progression of pathology along the CA1 axis as mice age (see also Grueninger et al., 2010). However, it is difficult to distinguish true spreading from cell autonomous effects that arise due to intracellular differences in tau overexpression in this transgenic system. Promotion of intracellular clearance of antigenantibody complexes is an alternative or additional route by which tau immunotherapeutics could reduce pathological tau accumulation in the brain. Acknowledgements We thank A. Girardeau, A. Kronenberger, M. Prummer, F. Gerber, M. Amman and J. Messer for excellent technical assistance and A. Ghosh for constructive criticisms on the manuscript. U. Schwarz, N. Garin and L. Kammermeier from Leica Microsystems kindly helped to generate the gated stimulation emission depletion images.

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