Focal expression of adeno-associated viral-mutant tau induces widespread impairment in an APP mouse model

Neurobiology of Aging 34 (2013) 1355e1368

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Focal expression of adeno-associated viral-mutant tau induces widespread impairment in an APP mouse model Elisa Dassie a,1, Melissa R. Andrews a, 2, Jean-Charles Bensadoun b, Matthias Cacquevel b, Bernard L. Schneider b, Patrick Aebischer b, Fred S. Wouters c, Jill C. Richardson d, Ishrut Hussain d, David R. Howlett d, Maria Grazia Spillantini a, James W. Fawcett a, * a

Department of Clinical Neurosciences, Cambridge University Centre for Brain Repair, Cambridge, UK Neurodegenerative Studies Laboratory, Brain Mind Institute, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Laboratory for Molecular and Cellular Systems, Department of Neurophysiology and Sensory Physiology, University of Göttingen, Göttingen, Germany d GlaxoSmithKline, R&D China U.K. Group, Stevenage, UK b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 December 2011 Received in revised form 3 October 2012 Accepted 22 November 2012 Available online 25 December 2012

Adeno-associated virus serotype 6 (AAV6) viral vectors encoding mutant and normal tau were used to produce focal tau pathology. Two mutant forms of tau were used; the P301S tau mutation is associated with neurofibrillary tangle formation in humans, and the 3PO mutation leads to rapid tau aggregation and is associated with pathogenic phosphorylation and cytotoxicity in vitro. We show that adenoassociated viral injection into entorhinal cortex of normal and tau knockout animals leads to local overexpression of tau, and the presence of human tau in axons projecting to and emanating from the entorhinal cortex. Starting at 2 months and increasing by 6 months post-injection neurons expressing mutant tau developed hyperphosphorylated tau pathology, in addition to dystrophic neurites. There was neuronal loss in tau-expressing regions, which was similar in normal and in TASTPM mice injected with mutant tau. There was neuroinflammation around plaques, and in regions expressing mutant tau. We saw no evidence that mutant tau had affected amyloid-beta pathology or vice versa. Morris water maze behavioral tests demonstrated mild memory impairment attributable to amyloid-beta pathology at 2 and 4 months, with severe impairment at 6 months in animals receiving adeno-associated viral-3PO. Therefore, TASTPM mice injected with mutant tau displayed many of the main features characteristic of human Alzheimer’s disease patients and might be used as a model to test new drugs to ameliorate clinical features of Alzheimer’s disease. Ó 2013 Elsevier Inc. All rights reserved.

Keywords: Alzheimer’s disease Tau Tauopathy Viral vectors Adeno-associated virus Neurodegeneration Memory Alzheimer’s precursor protein

1. Introduction Alzheimer’s disease is characterized by b-amyloid-containing plaques, tau-containing neurofibrillary tangles (NFTs), reduced synaptic density, axon and dendrite retraction, neuronal loss in specific brain areas (Gotz et al., 2004), and neuroinflammation around senile plaques (Glass et al., 2010) and at sites of ghost tangles (Janelsins et al., 2008). b-amyloid-containing plaques and neurofibrillary tangles coexist in Alzheimer’s disease (AD), leading to the concept of a neurodegenerative cascade, with amyloid-beta (Ab) triggering tau pathology in familial cases with mutations in the amyloid precursor protein (APP) gene. However, the mechanism by which tau and b-amyloid pathologies might interact is currently unknown. * Corresponding author at: Cambridge University Centre for Brain Repair, Robinson Way, Cambridge CB2 0PY, UK. Tel.: þ44 1223331160; fax: þ44 1223331174. E-mail address: [email protected] (J.W. Fawcett). 1 Current address: Venetian Institute for Molecular Medicine, Padova, Italy. 2 School of Medicine, University of St Andrews, St Andrews, UK. 0197-4580/$ e see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2012.11.011

To study possible tau-b-amyloid interactions, and to generate animals with focal tau pathology, we have generated adenoassociated viral (AAV) vectors expressing mutant forms of tau. NFTs can be induced by overexpressing tau constructs carrying familial mutations linked to frontotemporal dementia (Osinde et al., 2008; Ramirez et al., 2011). One such mutation within the tau gene which is the cause of a human familial tauopathy is the P301S change in the microtubule associated protein tau gene, characterized by 4 repeats (4R) and 0 inserts in the N-terminus (0N). When expressed in transgenic mice, the mutant P301S tau produces widespread functional deficits and neurodegeneration (Allen et al., 2002; Delobel et al., 2008). 3PO tau is not found in spontaneous mutations, but was mutated to enable rapid aggregation and a more rapid pathogenic phosphorylation pattern and cytotoxicity in vitro (Iliev et al., 2006). More specifically, the 3PO (Pattern Optimized) mutation is within the longest human tau isoform (441aa) which presents motifs of alternating polar and non-polar amino acids within the microtubule binding regions (MTBR) created by changing

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one or two residues in the second, third, and fourth MTBR of tau characterized by 4 repeat (4R) and 2 repeats in the N-terminus (2N). These alternating motifs increase the stability of the tau protein resulting from an increased tendency for instant aggregation because of the formation of an alternating “hydrophilic/hydrophobic” pattern associated with cytotoxicity and pathologic phosphorylation patterns (Iliev et al., 2006). Both P301S and 3PO tau mutants have 4 repeats, but differ in length because of the absence of exon 3 in the P301S mutation. As a control, we have used an AAV carrying a wild type (WT) form of tau (AAV-WT tau). Expression of WT tau in in vivo models can confer aberrant phosphorylation with time, however no authentic tau pathology is usually detected (Jaworski et al., 2010). Viral vectors represent useful tools to express proteins of interest in defined anatomical regions because of their stable and safe expression, low rate of integration into the host genome, and preference for transduction of neurons in the central nervous system (Towne et al., 2010). To study interactions between the AAV-induced expression of mutant tau and Ab pathology, we injected the viral vectors into a well-established transgenic animal with two AD-related mutations. The TASTPM transgenic mouse model overexpresses human APPswe and presenilin-1M146V mutant cDNAs under the control of the mouse Thy-1 promoter and has been shown to form Ab deposits from 3e4 months of age (Howlett et al., 2004). Although APP/PS1 transgenic mice show an abnormal murine tau phosphorylation pattern within dystrophic neurites close to amyloid plaques, no typical NFTs are observed. To produce pathology in a region of the brain that is affected and shows aberrant tau pathology in the early stages of AD, we injected AAVs into the entorhinal cortex (ERC) of TASTPM mice. The ERC is one of the most heavily damaged areas in AD patients, playing an important role in memory deficits that herald the onset of the disease and characterize its course (Braak and Braak, 1991; Hyman et al., 1984, 1986). After the AAV-tau injections, evaluation of neuronal loss, aberrant hyperphosphorylated tau, amyloid pathologies, and neuroinflammation within the ERC and CA1 regions of hippocampus were performed along with behavioral assessment using the Morris water maze (MWM) task. 2. Methods 2.1. Tau constructs, green fluorescent protein, and AAV Three tau isoforms were used in this study; the longest human WT tau isoform, the mutant tau isoform identified as 3PO tau (Iliev et al., 2006), both with 441 amino acids, and the shortest human 4 repeat isoform carrying the human P301S mutation (Allen et al., 2002) (GenBank accession number NM_016834.4) normally consisting of 383 amino acids, however, our construct included a 6 amino acid N-terminal linker sequence, resulting in a length of 389 amino acids. These were subcloned into the AAV-phosphoglycerate kinase 1 expression vector and packaged into replication incompetent adeno-associated serotype 6 virus (AAV6). AAV6-green fluorescent protein (GFP) was also used as a control reporter gene for gene expression, anatomic spread, and viral toxicity after AAV6 delivery in the brain. AAV6 expression was driven under phosphoglycerate kinase 1 promoter and was produced as previously described (Grimm et al., 2003; Towne et al., 2009). 2.2. In vitro transduction of HEK-293T cells and embryonic rat cortical neurons HEK-293T cells were maintained in Dulbecco’s Modified Eagle Medium (Invitrogen) containing 10% fetal calf serum (FCS)

(Invitrogen) and 1% penicillin streptomycin fungizone (PSF) (Invitrogen). Embryonic day 18 rats were sacrificed and the brains were removed and collected in Hank’s Balanced Salt Solution (without calcium and magnesium). The cerebellum, meninges, and blood vessels were removed and the cortical tissue was minced before enzymatic dissociation with Accutase (300 mL, PAA Laboratories) at 37  C for 15 minutes. The tissue was washed with Dulbecco’s Modified Eagle Medium containing 10% FCS and 1% PSF and further dissociated mechanically into a single-cell suspension with a Pasteur pipette. Cells were plated on glass coverslips coated with 10 mg/mL poly-D-lysine (Sigma-Aldrich) (100,000 HEK-293T cells per coverslip or 150,000 cortical neurons per coverslip) and allowed to attach. The viral titers are indicated in Table 1. Viruses were diluted in phosphate-buffered saline (PBS) to obtain 200 transducing units (TU)/cell. The following day for HEK-293T cells or 8 days after plating the cortical neurons, cells were transduced with the AAVs at 200 TU per cell. The appropriate amount of virus was added to the culture medium, containing camptothecin (Sigma Aldrich) (1 mM for HEK293T cells or 0.5 mM for cortical neurons). Three days after AAV transduction, cultures were fixed with 4% paraformaldehyde (PFA), washed with PBS, and blocked with 10% normal goat serum (Dako) in 0.1% Tween-20 (Sigma Aldrich) in PBS. Cells were incubated with primary antibody (listed in Table 2) overnight at 4  C. The next day, cells were incubated with fluorescent-conjugated secondary antibody (AlexaFluor goat anti-rabbit or AlexaFluor goat anti-mouse). The total number of cells was visualized with the fluorescent nuclear dye Hoechst (Sigma-Aldrich). Coverslips were mounted with Fluorosave (Calbiochem) and examined with a Leica CTR 6000 fluorescent microscope. 2.3. Animals Experiments were conducted in accordance with the United Kingdom Animals (Scientific Procedure) Act of 1986 and UK Home Office regulations. TASTPM mice (provided by GlaxoSmithKline), Jae Tau KO (bred in house), and C57BL/6J (Charles River) were used for in vivo experiments. Animals were kept in standard housing conditions with a 12-hour light/dark cycle, and given water and a standard mouse chow pellet diet ad libitum. Transgenic mice overexpressing the hAPP695swe mutant cDNA (TAS10) and transgenic mice overexpressing the presenilin-1 M146V mutant cDNA (TPM) were generated as previously described (Howlett et al., 2004; Richardson et al., 2003). Jae Tau KO mice (gift from M. Goedert and bred in house) are a tau knockout (/) line expressing enhanced GFP in postmitotic neurons in the developing nervous system and were generated as previously described (Tucker et al., 2001). Male C57BL/6J (Charles River) mice were used as control animals for immunohistologic and quantification analysis, and for behavioral studies. 2.4. Surgical procedures TASTPM transgenic mice received a unilateral injection in the ERC at 2 months of age and were sacrificed at 4, 6, or 8 months of Table 1 Viral titers Virus

Titer (TUs/mL)

AAV-GFP AAV-WT tau AAV-P301S tau AAV-3PO tau

8.69 6.26 7.8 8.5

   

1010 1010 109 1010

Key: AAV, adeno-associated viral; GFP, green fluorescent protein; TUs, transducing units; WT, wild type; 3PO, pattern-optimized.

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Table 2 List of antibodies used for this study Type

Specificity/epitope

Host

Concentration or dilution

Primary antibody HT7 Polyclonal anti-human tau Tau (clone tau-5) AT8 AT100 20G10 11C5 GFAP

Monoclonal Polyclonal Monoclonal Monoclonal Monoclonal Monoclonal Monoclonal Polyclonal

Mouse Rabbit Mouse Mouse Mouse Mouse Mouse Rabbit

IF: (1:500) IF: (1:1000) WB: (1:1000) IF: (1:500) IF: (1:500) IF: (1:1000) IF: (1:1000) IF: (1:1000)

Iba1

Polyclonal

Rabbit

IF: (1:1000)

Wako

NeuN GFP bIII tubulin

Monoclonal Polyclonal Polyclonal

Human tau Human tau Total tau Ser202/Thr205 Thr212/Ser214 Ab x-42 Ab x-40 Astrocytes and some CNS ependymal cells Cytoplasmic of microglia and macrophages Neuronal nuclei Green fluorescent protein Neuron specific class III b-tubulin (bIII)

Mouse Rabbit Rabbit

IF: (1:500) IF: (1:1000) IF: (1:1000)

Chemicon Invitrogen Covance

Goat

IF: (1:500)

AdB, Serotec

Goat Goat Goat

IF: IF: IF: IF: IF:

Molecular Molecular Molecular Molecular Molecular

Secondary antibody Biotinylated goat anti-mouse Goat anti-rabbit 568 Goat anti-rabbit 488 Goat anti-rabbit 660 Streptavidin 568 Streptavidin 647

(1:1000) (1:1000) (1:500) (1:500) (1:500)

Antigen retrieval

Formic acid 70% Formic acid 70%

Supplier Innogenetics DAKO Biosource Innogenetics Innogenetics Glaxo Smith Kline Glaxo Smith Kline DAKO

Probes, Probes, Probes, Probes, Probes,

Invitrogen Invitrogen Invitrogen Invitrogen Invitrogen

Key: Ab, amyloid beta; CNS, central nervous system; IF, immunofluorescence; WB, western blot.

age (see Table 3 for numbers of animals injected and time of sacrifice). During surgery, mice were anesthetized with 3% isoflurane in 2 L/min oxygen (carrier gas) and placed in a stereotaxic frame (David Kopf Instrument, Tujunga, CA, USA). Anesthesia was maintained during surgery at 2.5%e2% isoflurane (in 0.6 L/min oxygen and 0.4 L/min nitrous oxide). The coordinates used for the ERC were anterior/posterior: 3, medial/lateral: 3.7, dorsal/ ventral: 4 according to K. Franklin and G. Paxinos (1997). ERC lesion consisted of a unilateral injection lesion using a 34-gauge inox tube inserted into a 26-gauge inox tube (Cooper’s Needle Works, Ltd) attached to a 26-gauge 10 mL Hamilton syringe (Hamilton Company) driven by an infusion syringe pump (World Precision Instruments, Stevenage, UK) at 0.1 mL/min. One microliter of AAV-GFP plus sterile PBS or AAV-WT tau, AAV-P301S tau, AAV-3PO tau (6  1010 TU/mL) was injected into the ERC over a 10-minute period, followed by 4 minutes before capillary removal. Validation of tau expression by histology was performed at the end of the experiment. 2.5. MWM task After receiving a unilateral viral injection at 2 months of age, mice were assessed behaviorally at 8 months of age in the MWM task. A water tank (black polypropylene, diameter, 200 cm; height, 40 cm) was filled to a depth of 40 cm with water (23  C) and rendered opaque by the addition of a nontoxic white paint powder. Four positions around the edge of the tank were Table 3 Numbers of animals used

TASTPM-WT tau TASTPM-P301S tau TASTPM-3PO tau TASTPM-GFP TASTPM con C57BL/6J

Injected at 2 mo of age

Sacrificed at 4 mo of age

Sacrificed at 6 mo of age

Sacrificed at 8 mo of age

40 38 40 27 30 45

13 13 14 9 10 15

12 11 13 8 8 15

12 11 11 8 8 15

arbitrarily designated north (N), south (S), east (E), and west (W) to provide 4 alternative start positions and to divide the tank into 4 quadrants: NE, SE, SW, and NW. A video camera was fixed 1.6 m above the center of the tank, and connected to an HVS tracking system (HVS Image 2020, Hampton, UK). The N and E sides of the pool were in proximity with the walls of the room (1 meter from each cardinal point) and a poster board was placed behind the SW quadrant to hide the experimenter and to provide a richer environment for the side of the maze. Extramaze visual cues were provided by printouts of black shapes on a white background placed on the walls and the board. Four halogen lamps (300 W) placed on the outer wall of the pool at floor level provided a condition of low luminosity. Total numbers of animals tested in the MWM using an established protocol (Morris, 1984) and an alternative protocol (Gulinello et al., 2009) are shown in Table 3. The established protocol was performed in 12 days. Learning time was performed during 5 days (Days 1e5), followed by a probe trial (Day 6). Reverse training was performed again during 5 days (Days 7e11), followed by reverse probe trial (Day 12). Mice that failed to locate the platform within the time limit ascribed an escape latency of 60 seconds and were placed onto the platform by hand. Then, in the alternative protocol (Gulinello et al., 2009), animals were trained in a series of visible platform trials on Day 1 (D1). The diameter of the platform was the same for visible and hidden trials. The test required 4 visible platform trials (D1V1eD1V4) at Day 1. The platform was outlined by a dark ring. The last visible platform trial was considered to be its posthabituation baseline (D1V4). Mice that failed to locate the platform within the time limit were ascribed an escape latency of 180 seconds and were placed on the platform by hand. Animals remained on the platform for 15 seconds, before being returned to the home cage during the intertrial interval. Twenty-four hours after the last visible platform trial, the animals were tested in a series of 3 hidden platform trials (D2T1eD2T3). The platform remained in the same position (N) for all trials and animals were delivered to either the W or E quadrant because they were equidistant from the target. All trials were videotaped and subsequently analyzed using purposedesigned image analysis software (HVS, Hampton, UK).

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2.6. Immunohistochemistry Animals were terminally anesthetized with sodium pentobarbitol (2 mL/kg intraperitoneal; Rhone Merieux) at 4, 6, 8, or 10 months postoperatively and transcardially perfused with 50 mL of PBS, pH 7.4, followed by 50 mL of cold 4% PFA, pH 7.4. Brains were postfixed for 24 hours in 4% PFA and cryoprotected in 30% sucrose in PBS. After cryoprotection, brains were sectioned using a sledge microtome in the coronal plane at 35 mm thickness and stored at 4  C in Trizma (Sigma)-buffered saline (TBS) with 0.05% sodium azide until processed for histology. Histological analysis was performed on a minimum of 3 animals per group from the group of animals used for behavioral testing (MWM). Sections were washed with TBS (pH 7.4) and incubated in 5% normal goat serum with TBS containing 0.2% Triton X-100 (Sigma; TXTBS) for 1 hour at room temperature to block nonspecific binding. Sections were incubated overnight with primary antibody in TXTBS at 4  C. After washing in TBS, sections were incubated in secondary antibodies in TXTBS. Antibodies that required enhancement of signal were incubated with biotinylated goat anti-mouse antibody (AbD; Serotec), followed by washes of TBS and incubation with fluorescent streptavidin. Total number of cells was visualized with the fluorescent nuclear dye Hoechst in TXTBS (1:10,000). Finally, sections were washed in Tris non-saline, mounted onto 1% gelatin-coated slides and coverslipped with Fluorosave (Calbiochem). Stained sections were examined with Leica CTR 6000 fluorescent microscope. Ab antibodies required antigen retrieval treatment (70% formic acid for 20 seconds) before the blocking step to expose antigens which had been masked by the fixation process. In some cases, Congo red derivative (trans,trans)1-fluoro-2,5-bis(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (FSB) was used at 5 mM concentration in 3% bovine serum albumin, 0.1% Triton-X 100 PBS to visualize b-sheet protein aggregates (Gasparini et al., 2009; Velasco et al., 2008).

a 20 objective, converted to 8-bit black and white images, and a fixed intensity threshold was applied to define the region of staining. The percent area occupied by Ab immunoreactive pixels was calculated with ImageJ (NIH). Sections were analyzed at approximately 170-mm intervals between 2.70 and 4 mm relative to bregma. Results were statistically analyzed using analysis of variance (ANOVA). This interval was chosen to prevent remeasuring and/or counting of individual plaques. Five sections from each animal were quantified, and 3 animals per group were used for analysis. 2.10. Quantification of GFAP and Iba1 Immunofluorescent images of the ERC stained for glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule 1 (Iba1) were used for quantification. Images were captured with a 20 objective and the mean immunofluorescence value was calculated with LAS AS Leica software. Sections were analyzed at approximately 170-mm intervals between 2.70 and 4 mm relative to bregma. Results were then statistically analyzed using ANOVA. Five sections from each animal were quantified, and 3 animals from each group were used for analysis. 2.11. Statistical analysis

Using descriptions from previously published work (Shi et al., 2009), we identified dystrophic neurites as aberrant neuritic sproutings, swollen dendrites, and/or swollen axons.

All quantification data are graphically presented and were analyzed with Student t test (ipsilateral compared with the contralateral side). The mean values and standard error of the means are represented in all figures, with significance levels set at p < 0.05. Graphs were generated using Excel, and for MWM analysis, 2-way ANOVA with repeated measures was used to analyze data from the training sessions. To evaluate the performance of mice during training, we considered 2 dependent variables related to learning and memory skills (escape latency and path length) and 3 dependent variables related to behaviors that might affect learning and memory performance without being directly linked to these cognitive processes (swim speed, time spent floating, and thigmotaxis). Time spent floating during each trial was analyzed as an approximate measure of depression, and thigmotaxis (time spent in the outer area of the maze) was analyzed as a measure of anxiety (Malleret et al., 1999).

2.8. Neuronal counting in ERC and CA1

3. Results

TASTPM mouse brains were stained for NeuN at approximately 170-mm intervals throughout the brain. Immunofluorescent images in selected areas (same anatomical location used for each animal) of the ERC or CA1 were recorded. For the ERC, quantification was performed at the injection site, where the area of interest was defined from 2.70 to 4 mm posterior to bregma. Magnification of photographs for analysis was 20 optical zoom and NeuN-positive cells were counted. Each area subjected to neuronal counting was set as 200  650 mm. Five sections from each animal were quantified, and 3 animals per group were used for analysis. For the CA1, magnification of photographs for analysis was 63 optical zoom and NeuN-positive cells were counted. Each area subjected to neuronal counting was set as 100  150 mm and 3 boxes were counted for each side for each section. Four sections per animal were quantified, and 3 animals per group were used for analysis.

3.1. AAV-tau in vitro

2.7. Identification of dystrophic neurites and aberrant tau morphology

2.9. Quantification of Ab plaques TASTPM mouse brains were stained for 11C5 and 20G10 antibodies at approximately 170-mm intervals throughout the brain after performing antigen retrieval. Images were captured with

AAV-GFP, AAV-WT tau, AAV-P301S tau, and AAV-3PO tau were first tested in vitro to confirm tau GFP and expression. We tested the AAV6 vectors for GFP and tau gene transfer in HEK-293T cells and primary cortical neurons. Seventy-two hours post-transduction, immunostaining with HT7 antibody in HEK-293T cells showed robust expression in cells transduced by tau viruses (data not shown). Immunocytochemical analysis of rat primary cortical neurons indicated that cells transduced by AAV-GFP displayed normal morphology with GFP immunoreactivity observed in cell bodies and along the processes (Supplementary Fig. 1a). Cells transduced by AAV-WT tau, as observed after AAV-GFP transduction, mainly displayed normal morphology but with human tau immunoreactivity observed in cell bodies and along the processes (Supplementary Fig. 1b). After AAV-P301S tau and 3PO tau transduction, striking differences in cell morphologies were observed when compared with controls including dystrophic morphology with dystrophic neurites and nuclei appearing fragmented (Supplementary Fig. 1c and d). Cell clustering was often observed in AAV-tau mutant transduced cells.

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3.2. AAV-tau selectively transduces neurons GFP and WT, P301S, 3PO tau viruses delivered via AAV6 showed neuroselectivity which was confirmed with NeuN counterstaining. Brains injected with GFP showed colocalization of GFP expression with NeuN, indicating that the AAV6 viral vectors specifically transduce neurons. Additionally, double staining with GFAP or Iba1 did not reveal colocalization of glial markers with virallytransduced cells (data not shown). Moreover, brains of TASTPM mice injected with WT tau double-stained with polyclonal rabbit anti-human tau and NeuN demonstrated that NeuN antibodies colocalized with human tau expression in neurons (Fig. 1). Results were similar in C57BL/6J and Jae Tau KO mice injected with AAV-tau viruses. 3.3. Anatomical characterization of tau expression in vivo Expression of AAV-WT tau, -P301S tau, -3PO tau, and -GFP in the ERC was first investigated by immunohistochemistry in two control mouse models, WT C57BL/6J and Jae Tau KO mice. AAV-GFP was used in this study as a control for gene expression, anatomical spread, and viral toxicity after AAV delivery in the brain. The expression of GFP and WT, P301S, and 3PO tau viruses was confirmed 4 weeks and 4 months post-injection in WT C57BL/6J mice and at 2, 4, and 6 months post-operatively in Jae Tau KO mice by 2 phosphorylation independent anti-human tau specific antibodies (polyclonal rabbit anti-human tau and HT7). Anatomically, robust GFP immunoreactivity in the injected brains was mainly observed in 3 areas: ERC (injection site) (Supplementary Fig. 2a and d), CA1 and CA3 hippocampal regions (Supplementary Fig. 2b), and usually in the neuropil of the dentate gyrus (Supplementary Fig. 2c), and in the neuropil of the contralateral hippocampus. Injection of AAV-WT (Fig. 2), -P301S, and -3PO tau led to expression of human tau (polyclonal rabbit anti-human tau and HT7 antibodies co-labeled) in the mouse brain with the same anatomical pattern of distribution as AAV-GFP. Exactly the same distribution pattern of tau expression was seen when we injected the viruses into TASTPM mice. We observed robust expression of human (immunolabeled

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with anti-human tau) or phosphorylated tau (immunolabeled with phosphorylation-dependent anti-AT8) over the course of our experimental time points (2, 4, 6, and 8 months post-injection) in TASTPM-injected animals (Supplementary Fig. 3). Further confirmation of tau expression was performed with Western blot analysis from AAV-injected ERC tissue in which a strong increase in tau levels (including human tau) was found in the WT, P301S, and 3PO groups (data not shown). Human tau or GFP positive axons and cells were also observed in the regions of the ipsilateral and contralateral brain that send or receive connections to the ERC including the neuropil of the hippocampus, but not the contralateral ERC. We did not observe any human tau immunoreactivity (HT7) in non-tau injected groups (Supplementary Fig. 3), including in the AAV-GFP injected group (data not shown). These results demonstrate that both normal and mutant tau proteins are transported down axons, and that the AAV6 vector is transported retrogradely back to the cell body of neurons whose axons project to the ERC. The expression induced by the viruses remained robust up to 8 months postinjection, the last time point analyzed in this study. 3.4. AAV-3PO and P301S tau cause neuronal loss in ERC and CA1 of TASTPM tau-injected mice We quantified neuronal loss at two sites in injected mice; the ERC injection site, and in the CA1 region of the hippocampus in which there were many neurons expressing tau after retrograde transport of virus from the injection site. The ERC of TASTPM-tau was analyzed in animals injected at 2 months of age and sacrificed at 4, 6, 8, and 10 months of age. There was no neuronal loss in the contralateral ERC relative to TASTPM controls at any stage, confirming the contralateral side as a reliable negative control. We saw no neuronal loss in the ipsilateral injected ERC 2 months after injection in animals sacrificed at 4 months of age. Four months after injection (at 6 months of age) significant neuronal loss was found in TASTPM-3PO tau (p < 0.05) mice relative to the contralateral side, but not in other groups (TASTPM-WT, TASTPM-P301S tau mice, and TASTPM non-injected mice). At 6 months post-injection (8 months of age), significant differences between ipsi- and contralateral sides

Fig. 1. Adeno-associated virus serotype 6 (AAV6) viral vector is neuronal-specific. Images show polyclonal rabbit anti-human tau (anti h-tau; arrow in A) and NeuN (arrow in B) co-staining (C, merged image) in the entorhinal cortex with higher magnification (in D, from boxed insert in C) of a neuron immunoreactive for both antibodies. Sections represent a TASTPM-WT tau injected mouse in which AAV-tau virus was delivered at 2 months of age and the animal was sacrificed at 4 months of age. Scale bar ¼ 50 mm.

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Fig. 2. Tau-injected mice showed HT7 immunoreactivity (A) in CA1 and CA3 hippocampal regions (B) and in the neuropil of the dentate gyrus (C), and the entorhinal cortex injection site (D). Sections from a Jae Tau KO mouse at 2 months post-AAV injection, and sacrificed at 4 months. Similar HT7 immunoreactivity pattern was observed in C57BL/6J and TASTPM injected mice. Scale bar ¼ 50 mm.

were found in both TASTPM-3PO (p < 0.05) and TASTPM-P301S (p < 0.05) mice, but not in TASTPM-WT tau and control mice. At the last time point examined, 8 months after injection (10 months of age), significant differences were apparent between ipsi- and contralateral sides in all tau groups, the TASTPM-WT tau (p < 0.05), TASTPM-P301S (p < 0.01), and TASTPM-3PO (p < 0.01) tau mice (Fig. 3A). At no point were any significant differences found in neuronal cell counts between ipsi- and contralateral sides in TASTPM-GFP and control non-injected mice (Fig. 3A). These findings together suggest that neuronal loss is caused by tau protein expression and not by AAV toxicity. Moreover, toxicity of 3PO tau is displayed at an earlier stage than P301S tau. Additionally, AAVs injected into C57BL/6J mice resulted in a similar trend of neuronal loss at the injection site. We counted neurons at 8 months postinjection, and compared with the non-injected control side demonstrating with C57 non-injected (0% loss), C57-GFP (1.5% loss),

C57-WT tau (4% loss), C57-P301S (10.2% loss), and C57-3PO (11.3% loss). In the CA1 region of the hippocampus there was no difference in neuronal numbers between groups contralateral to the injections. Comparison of neuronal numbers between ipsilateral and contralateral sides, showed significant neuronal loss ipsilaterally in TASTPM-WT (p < 0.05), -P301S (p < 0.05), and 3PO (p < 0.01) in mice injected at 2 months of age and sacrificed at 8 months of age (Fig. 3B). This indicates that mutant tau expression after retrograde transport of the AAVs is toxic to neurons. 3.5. Phosphorylated tau and dystrophic neurons in the brain after AAV-tau injection The pattern of expression of human tau protein in the TASTPM mouse brain after AAV injections was the same as in the normal

Fig. 3. NeuN quantification of entorhinal cortex and CA1 hippocampal regions. (A) Neuronal loss in the entorhinal cortex region of each mouse group: in TASTPM mice AAV-injected at 2 months of age and sacrificed at 4 months (2), 6 months (4), 8 months (6), and 10 months of age (8). Neuronal loss occurs earlier in 3PO tau-injected mice, beginning at 4 months post-injection. Neuronal loss is observed later in the 3PO1S tau-injected and wild type tau-injected mice (starting at 6 or 8 months postinjection, respectively). (B) Data obtained from NeuN quantification of hippocampal region CA1 of AAV-injected TASTPM mice. NeuN quantification of the CA1 region of the hippocampus of the ipsilateral (J) side was statistically compared with the contralateral (C) side in TASTPM mice injected at 2 months and sacrificed at 8 months of age. Neuronal loss is more evident in mutant tau-injected mice than in wild type tau-injected mice. Additionally, there was no difference in neuronal loss in the injected side compared with the non-injected side in the GFP-injected C57BL/ 6J group. * p < 0.05, ** p < 0.01. Numbers in parentheses indicate the number of months post-injection.

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Fig. 4. Human and hyperphosphorylated tau expression was robust in entorhinal cortex region. Human tau immunoreactivity was very robust in AAV-tau injected mice (high magnification of TASTPM mice AAV-tau injected and sacrificed at 2 months post-injection). AT8 immunoreactivity displayed dystrophic neurites in TASTPM-P301S and -3PO tau mice at 2 (E, F) and 8 (H, I) months post-injection. In TASTPM-WT tau mice, AT8 immunoreactivity was observed as punctate bodies, dystrophic neurites, and skeletons of dead neurons (D, G). TASTPM-mutant tau injected mice showed dystrophic morphology as early as 2 months post-injection (B, C), whereas TASTPM-WT tau mice showed normal morphology at 2 months post-injection (A). Scale bar ¼ 10 mm.

and tau knockout animals described above. Neurons expressing P301S or 3PO tau displayed either normal morphology or atrophic morphology, especially at the later time points examined (Fig. 4). The number of neurons characterized by dystrophic morphology qualitatively increased with age. At 8 months post-injection, only a few of the transduced neurons expressing human tau displayed a normal morphology compared with the abundant dystrophic transduced neurons. Tau-transduced neurons showed variable immunoreactivity to human tau. Some were intensely and uniformly labeled (mainly the transduced neurons showing normal morphology), some displayed a brighter immunoreactivity, and others displayed a weak granular immunoreactivity (mainly neurons characterized by atrophic morphology) (Fig. 4B, C, E, F, H, I). Neurons transduced by

AAV-WT tau showed normal morphology (Fig. 4A and D), except at later time points analyzed (8e10 months of age), in which TASTPMWT tau-injected mice in a few instances displayed dystrophic neurons (Fig. 4G). Tau is physiologically phosphorylated on several serine and threonine sites (Avila, 2006). We investigated the presence of abnormal phosphorylation of tau in the two control mouse lines with no APP-related mutations (C57BL/6J, Jae Tau KO) and in TASTPM mice. Mice were injected with the various AAV vectors, and examined at 2, 4, 6, and 8 months after virus injection. The presence of abnormally phosphorylated tau was detected by immunohistochemical analysis using phosphorylation-dependent anti-tau antibodies, AT8 and AT100 (which has also been shown to recognize the

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sarkosyl-insoluble fraction from P301S transgenic mice, and show positive staining for NFTs) (Allen et al., 2002; Augustinack et al., 2002; Yoshida and Goedert, 2006), and with the fluorescent Congo red derivative FSB, which stains tau aggregates. In animals with no AAV injections or AAV-GFP, no phosphorylated tau was seen in C57BL/6J (or Jae Tau KO) non-injected or AAVGFP injected mice. In TASTPM controls, as described previously, there was weak AT8 staining in close association with Ab deposits, further increased at 6 and 8 months of age (Howlett et al., 2008). In addition, there were occasional instances of AT100 immunoreactivity in association with plaques or occasionally as highly immunoreactive punctate spots. Animals that received AAV-P301S and 3PO tau were analyzed by staining for AT8 and AT100. Results in all the animals, including the Jae Tau KO, were similar. In the ERC at the injection site, we saw high levels of AT8 hyperphosphorylated tau beginning from 4 weeks post-injection with increasing numbers of heavily stained neurons up to 8 months, the latest time point analyzed. Though at 4 weeks after injection only a small proportion of neurons positive for human tau were also positive for AT8, at 8 months almost all cells expressing human tau were also AT8-positive. Additionally, by 8 months we also found that a small proportion of human tau-expressing neurons were AT8-positive in animals injected with WT tau, although the staining was very weak and was mainly visible along neuronal processes. At 4 months post tau virus injection, a higher percentage of human tau-positive neurons were AT8 positive also in 3PO tauinjected mice compared with P301S tau-injected mice (Fig. 5). AT8 immunoreactivity accumulated particularly in the somatodendritic compartment, and we saw little staining along axon tracts. At 4 weeks, the AT8 staining was diffuse and cytoplasmic. By 6 months, most AT8 positive neurons showed aggregated structures within the cytoplasm, and phosphotau in the proximal dendrites and nuclei was sometimes displaced by inclusions. Mutant tau-injected brains often showed collapsed or misaligned dendrites, with neurons often appearing atrophic. Densely stained neuropil threads were often detected near atrophic AT8-positive neurons, representing the destruction of dendritic and axonal structures (Fig. 4). Similar staining was observed in the CA1 region in hippocampal neurons expressing human tau. We also saw occasional AT8

immunoreactivity in neurons in the dentate gyrus, which have no axonal connection with the ERC and therefore might have acquired human tau from the neuropil formed by ERC axons. AT100 antibody recognizes a phosphorylation motif on tau that has been shown to be associated with the presence of tau filaments in P301S mice (Allen et al., 2002; Yoshida and Goedert, 2006). AT100 immunoreactivity was found in TASTPM-P301S and TASTPM-3PO tau animals as early as 2 months after tau injection within the ERC (Fig. 6), although the number of AT100-positive cells was considerably smaller than that of AT8-positive cells. AT100 mainly labeled cell bodies, axons, and dendrites of neurons characterized by dystrophic morphology and additionally, its immunoreactivity increased with time. The AT100 staining appeared earlier (2 months after injection) in animals that received 3PO tau than in animals that received P301S tau, suggesting that phosphorylation at Thr212 and Ser214 (recognized by AT100 antibody) occurs earlier in 3PO tau-injected brains compared with P301S tau-injected brains. However, by 8 months the AT100 staining was comparable in the 2 groups. We also saw a delayed appearance in AT100 staining in the Jae Tau KO animals relative to WT and TASTPM mice, with no staining at 2 months and less staining at 4 months. TASTPM tau-injected brains were stained with AT8 antibody and co-labeled with the fluorescent Congo red derivative FSB, which stains FSB labeled b-sheet protein aggregates (Gasparini et al., 2009; Sato et al., 2004; Velasco et al., 2008). In uninjected TASTPM mice, there was very robust FSB staining of amyloid plaques, which was confirmed by co-labeling with anti-Ab40 antibody (data not shown). In animals injected with P301S and 3PO tau, in addition to plaque staining, we saw neurons that stained for both AT8 and FSB at the injection site (Fig. 7AeH). The number of neurons stained with AT8 and FSB increased in TASTPM-P301S and TASTPM-3PO tau mice in later time points. 3.6. Interactions between tau expression and Ab plaques One of the objectives of this experiment was to investigate interactions between Ab pathology and tau pathology. We therefore asked whether the tau pathology differed around plaques, and whether the plaque load was increased in regions of tau pathology.

Fig. 5. AT8 expression in CA1 hippocampal region. AT8 staining is present in the hippocampus in TASTPM mice injected with AAV-tau mutants. Many human tau positive neurons are also AT8 positive in the 3PO tau injected mice (C, D) and in the P301S (A, B). Scale bar ¼ 50 mm.

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Fig. 6. TASTPM-P301S and 3PO tau-injected mice displayed AT100 immunoreactivity. AT100 immunoreactivity was present at 2 months post-injection. High magnification of double labeling (arrows indicate double-labeled cells) of anti-human tau (B, D) and AT100 (A, C) positive neurons in TASTPM mice injected with AAV-tau mutants, sacrificed at 6 months post-injection. Scale bar ¼ 50 mm. Abbreviations: inj, injection; sac, sacrificed.

TASTPM animals were injected with AAV at 2 months and sacrificed at 4, 6, 8, and 10 months of age. Amyloid pathology in the form of plaques was visible in TASTPM mice starting at 3 months of age and increased thereafter (Howlett et al., 2004). Extracellular Ab deposits were observed in the cerebral cortex and hippocampus by 4 months of age, whereas between 6 and 10 months of age, TASTPM mice displayed extensive cerebral plaque pathology. In TASTPM mice, the Ab42 load was greater than Ab40, and Ab content indicated the presence of multiple forms of the peptide, including full length and N-terminally truncated material (Howlett et al., 2004, 2008). Assessment of Ab deposition was evaluated using antibodies specific for Ab40 (11C5), Ab42 (20G10), and fluorescent FSB Congo red derivative. Plaques were present mainly in cortical and hippocampal areas. Ab load quantification was first evaluated based on the amount of 11C5 immunoreactivity on the ipsilateral side compared with the contralateral side. No difference was found in the amount of Ab40 load in the ipsilateral (AAV-injected) ERC compared with the contralateral side after AAV injection at any of the time points (data not shown). Expression of mutated tau, normal tau, or GFP did not therefore influence the Ab40 load (we did not analyze Ab42 load). We also examined the appearance of staining for hyperphosphorylated tau with AT8 and AT100 staining. As previously reported, there is a light enhancement of AT8 staining and occasional AT100 staining around plaques in the TASTPM brain (Howlett et al., 2008). In animals transduced with mutant tau, there was an increase in tau protein staining in neurons positive for AT8 in the injection area, with differences depending on the type of tau transduced as described above. We saw no evidence for a change in this pattern of staining in the vicinity of plaques (Fig. 7IeK). There was also no qualitative change in the neurons stained with AT100 or FSB in the TASTPM compared with control mice at the same period after injection. 3.7. Activation of astrocytes and microglia in TASTPM tau-injected mice Numerous studies have demonstrated a neuroinflammatory response, especially microglial activation and astrocyte reactivity in

AD (McGeer et al., 1988; Rogers et al., 1988). In addition, inflammatory changes have been also described in several different APP transgenic mouse models (Benzing et al., 1999; Wirths et al., 2008). Focal and diffuse gliosis has also been described at sites of ghost tangles and angiopathic capillaries in the late stage of AD (Janelsins et al., 2008) and in some lines of transgenic mice expressing mutant tau, such as P301S (Bellucci et al., 2004). Howlett et al. have also previously demonstrated that inflammatory cells were observed in close proximity to the Ab in TASTPM transgenic mice and additionally GFAP- and Iba1-positive cells were found to be closely associated with amyloid deposits (Howlett et al., 2008). Thus, we examined glial activation in brain sections of TASTPM mice injected at various ages with WT, P301S, 3PO tau, and GFP and stained with antibodies against GFAP and Iba1. More robust microglial staining was observed on the ipsilateral side along the needle track compared with the contralateral side (Fig. 8), as expected, not only in tau-injected but also in GFP-injected animals, indicating that the lesion induced by the needle caused an inflammatory response. We confirmed, as previously described by Howlett and colleagues, that TASTPM mice display robust GFAP and Iba1 immunoreactivity in close association with amyloid plaques and this immunoreactivity increases with increasing amyloid plaque load with age (Howlett et al., 2008). In addition, we observed an increase in GFAP and Iba1 immunoreactivity in the injection site in association with phosphorylated tau deposition in TASTPM-P301S tau and TASTPM3PO tau mice (Fig. 8 and Supplementary Fig. 4). No apparent increase in GFAP and Iba1 immunoreactivity in the CA1 region of hippocampus was observed between ipsilateral and contralateral sides of TASTPM-WT tau-, TASTPM-P301S tau-, and TASTPM-3PO tau-injected mice. 3.8. Focal expression of AAV-3PO tau leads to impairment in spatial and learning memory at 4 and 6 months post-lesion Previous studies performed in TASTPM animals have shown that TASTPM mice start to show cognitive impairment in an object recognition test by 6 months of age (Howlett et al., 2004; Pugh et al., 2007). We tested animals after tau injection into the ERC in the

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Fig. 7. FSB co-labeled with some AT8 positive neurons in TASPM mice injected with AAV-tau mutants. Low magnification of the entorhinal cortex region near injection site of TASTPM-WT tau (AeC), TASTPM-P301S tau (DeF), and TASTPM-3PO tau (GeI) injected mice and labeled with AT8 (A, D, and G) and FSB (B, D, and H) shows the overall distribution of tau expression and FSB-labeled plaques. High magnification from an AAV-P301S tau-injected animal indicating dense amyloid plaques co-labeled with phospho-tau AT8 (I) and Congo-red derivative FSB (J) with some indication of FSB-positive dystrophic neurites (merged image in K). Sections taken from a TASTPM-tau injected mouse sacrificed at 6 months post-injection. Scale bar ¼ 200 mm (H, I), 10 mm (K).

MWM task using the traditional protocol (Morris 1984) and a modified protocol (Gulinello et al., 2009) more suitable for delicate mice. Deficits in spatial learning and memory have been seen after unilateral ERC and hippocampal lesions using this method (Kopniczky et al., 2006; van Praag et al., 1998). We tested and

compared data from TASTPM tau injected mice, control GFPinjected, TASTPM non-injected, and WT C57BL/6J mice. MWM performance studied at 2 months post-injection using the traditional protocol (5 days of training, probe trial, 5 days of reverse training, and reverse probe trial), showed that TASTPM-tau injected

Fig. 8. Neuroinflammatory response is increased in TASTPM-tau injected mice. TASTPM injected mice showed an increase in GFAP and Iba1 immunoreactivity in the injection site after AAV-tau injection, mainly after P301S (B, F) and 3PO (C, G) tau injection. (AeD) GFAP and Iba1 immunoreactivity observed in sections of mice sacrificed 2 months postinjection. (EeH) GFAP and Iba1 immunoreactivity observed in sections of mice sacrificed 8 months post-injection. Neuroinflammation was observed along the needle track. Arrows indicate inflammation in close association with amyloid plaque deposition. Scale bar ¼ 100 mm.

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and TASTPM control groups were significantly different from C57BL/6J mice during training but not during the 2 probe trials (at Day 6 and Day 12), indicating that the APP mice are impaired compared with the control mouse model. However, no significant difference within the TASTPM groups with or without tau virus was observed in any of the variables analyzed at 2 months postinjection. At 4 months post-injection, the pattern was similar, with all the TASTPM groups showing deficits relative to WT mice. At 6 months post injection, 8 months of age, TASTPM mouse pathology showed extensive cerebral Ab distribution and expression of the virus was still robust in the brain, however the animals are not suitable for a traditional MWM protocol (Supplementary Fig. 5). We therefore used an alternative 2-day protocol to test short-term memory (Gulinello et al., 2009). The mice were trained on the first day in the presence of a visible platform, and the next day we tested their memory in the presence of a hidden platform. Most of the mice showed decreased latency to escape and path length over the first day trials with a visible platform, with many finding the platform in less than 40 seconds. Twenty-four hours later, the platform was in the same position but invisible. At the first trial (D2T1), all TASTPM groups showed an initial deficit in

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finding the platform relative to the previous day. During the successive trials, all the animals generally improved their performance except the TASTPM-3PO tau mice which tended to get worse at finding the platform. Bonferroni post hoc test revealed that TASTPM-3PO tau mice were significantly different from TASTPM control non-injected mice in the second trial on the second day (D2T2). In the last trial of the second day (D2T3), TASTPM-3PO tau mice were significantly different from TASTPMWT tau, TASTPM-P301S tau, TASTPM-GFP, and C57BL/6 in path length and escape latency (Fig. 9A, B, D, and E). In addition, correlating the behavioral data with the amount of neuronal loss, it is apparent that 3PO tau-injected mice demonstrated the highest amount of neuronal loss and the largest behavioral deficit. P301S tau-injected mice also showed high amounts of neuronal loss, however the behavioral deficit was not as severe as in the 3PO tauinjected group (Fig. 9C and F). The other groups (non-injected, GFP-injected, and WT tau-injected) had a strong correlation between a low amount of neuronal loss and a low amount of behavioral deficit (Fig. 9C and F). C57BL/6J performed significantly faster than all the other TASTPM groups particularly on the second day (Fig. 9G). In thigmotaxis, there was no significant difference

Fig. 9. Results from the modified Morris water maze task with a hidden platform in position 2, 4 months post-AAV injection. On Day 1 (D1), mice were tested in the presence of a visible platform, and on Day 2 (D2) the platform was in the same location but hidden. Path length (A, B): 2-way analysis of variance (ANOVA) with repeated measures revealed significant effects of time (F(6,360) ¼ 27.77, p < 0.001) but not of groups or of the interaction between these 2 variables. Escape latency (D, E): 2-way ANOVA with repeated measures revealed significant effects of time (F(6,360) ¼ 25.93, p < 0.001) but not of groups or of the interaction between these 2 variables. Graphical representation (C, F) of the relationship between neuronal loss and cognitive impairment demonstrate a correlation between the percentage of neuronal loss and behavioral performance (n ¼ 3 animals for each group) TASTPM-P301S and TASTPM-3PO mice displayed higher neuronal loss corresponding to a higher behavioral deficit compared with TASTPM-injected (WT tau and GFP), non-injected, and C57BL/6J mice. Interestingly, TASTPM-3PO tau mice (top right quadrant) were also more impaired in the Morris water maze task (path length and escape latency) compared with TASTPM-P301S tau mice (top left quadrant). Speed (G): 2-way ANOVA with repeated measures revealed significant effects of groups (F(5,60) ¼ 5.179, p < 0.001) but not of time and of the interaction between these 2 variables. Floating (H): 2-way ANOVA with repeated measures revealed significant effects of groups (F(5,60) ¼ 5.858, p < 0.001) but not of time and of the interaction between these 2 variables.  p < 0.05, ** p < 0.01, ### p < 0.001.

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between all TASTPM groups (data not shown). In time spent floating (depression), these tests reveal both TASTPM-3PO tau and TASTPM-P301S mice showed a significant increase (4%) compared with the other groups, however this increase is too small to have affected the overall conclusion (Fig. 9H). 4. Discussion Our study had two main aims, to establish a novel model of neurodegeneration through injection of AAV vectors encoding mutant forms of tau and to study the interaction of tau and Ab pathology. We have documented the evolution of expression of the transduced tau, amyloid deposition, tau phosphorylation, neuronal death, astrogliosis, and microglial activation after injection of AAV serotype 6 encoding three forms of tau (WT, P301S, and 3PO tau) and GFP as a control for viral toxicity after AAV delivery. We injected the AAV vectors into the ERC for several reasons. It shows early neurofibrillary tangle pathology in AD, has widespread connections to the hippocampus and elsewhere, and lesions of the ERC in experimental animals produce measurable memory deficits. The use of viral vector gene transfer to model neurodegenerative diseases offers several advantages. When the transgene is delivered unilaterally, the contralateral side can be used as an internal control. By inducing equal levels of tau expression at different ages and in different in vivo models, it is possible to evaluate potential differences in susceptibility to neurodegeneration. Because lesions are focal and can be delivered at any age, it is possible to produce pathology in specific brain regions known to produce behavioral deficits when lesioned, making it possible to test methods for alleviating the deficits. Before studying their effect in an in vivo model, characterization of viral expression was conducted in HEK-293T cells and rat primary cortical neurons. Rat cortical neurons transduced with P301S or 3PO and immunostained with human tau antibody showed a change in cell morphology, with dystrophic neurites and cell clustering, and cells transduced with WT tau and control GFP displayed a normal morphology. These data suggest that P301S and especially 3PO tau are toxic compared with WT tau and control GFP. The next step was to evaluate the pattern of expression of normal and mutant tau after AAV injections into the ERC of WT C57BL/6J mice and Jae Tau KO mice. Using anti-human tau antibodies to trace expression, we found that the AAV6 vectors specifically transduced neurons. The expressed tau was transported down the axons of ERC neurons, most prominently to the hippocampal neuropil, and the virus was also transported retrogradely to label neurons in the hippocampus and elsewhere. The pattern of tau distribution was identical when we injected AAV vectors into TASTPM brains. The next question was whether the expression of the transgenes would cause pathologic changes in transduced neurons. Our findings were very similar in the three types of animals that we injected; C57BL/6J, Jae Tau KO and TASTPM. P301S and 3PO tau expression caused hyperphosphorylation pathology at 3e4 weeks visible using the AT8 antibody, and the proportion of human tau-expressing neurons that stained for AT8 increased with time until 6 months after injection at which time almost all transduced neurons were AT8-positive. Staining with the AT100 antibody showed a similar pattern in fewer neurons, but the staining started to appear later, not apparent until 4 months postinjection. Changes in cell morphology were also observed with an increasing number of dystrophic neurons, most of which stained for AT8 and AT100. Dystrophic neurites were often found in neurons which have abnormal tau deposits (accumulations of hyperphosphorylated tau). It is likely that these abnormal tau deposits eventually led to the death of the neuron, in some cases leaving the appearance of extracellular ghost tangles after the neuron

underwent cell death. Overexpression of WT tau caused low levels of AT8-positive hyperphosphorylation after 4 months. The expression of mutant tau also caused neuronal loss in the ERC and CA1 region of the hippocampus. Neuronal loss was observed at 2 months after injection of 3PO tau, and was not observed until 4 months postinjection in the P301S group. We next investigated an interaction between tau and Ab pathology. Tau pathology is seen in familial cases of AD resulting from mutations in APP or presenilin-1 genes, and also in nonfamilial AD, so it is reasonable to suppose that the Ab pathology in some way triggers tau pathology (reviewed by LaFerla, 2010) although the pathway for this link is unknown. Though mutations in the tau gene have been reported in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), in which they cause tau aggregation and neurodegeneration, no tau mutations have been identified in AD (Shahani and Brandt, 2002). However, tau pathology is one of the main aspects of AD and tau is involved in Ab-induced cell death (Rapoport et al., 2002). The amyloid cascade hypothesis recognizes changes in tau downstream of the Ab pathology (Hardy and Selkoe, 2002), and Ab can induce tau phophorylation at disease-relevant sites (Ferreira et al., 1997; Leschik et al., 2007; Zheng et al., 2002). Additionally, though the amount of NFTs correlate with the degree of dementia (Braak and Braak, 1991), little is known about how tau and Ab directly interact. Some experimental approaches show that FTDP17 tau and WT tau react differently to Ab. Tackenberg and Brandt showed that Ab alone is not neurotoxic but can induce toxicity through phosphorylation of WT tau in an N-methyl-D-aspartate receptor- dependent pathway. They show that tau is essential for Ab-induced neurodegeneration, and indicate that the mechanism by which Ab confers toxicity via tau is different for WT tau and mutant FTDP-17 tau (R406W and P301L) (Tackenberg and Brandt, 2009). Previous studies in triple transgenic mice have shown that though blocking Ab42 or Ab oligomeric accumulation delayed the onset and progression of tau pathology (Oddo et al., 2008), augmenting tau levels did not modulate the onset or progression of Ab pathology (Lewis et al., 2001; Oddo et al., 2007). As observed previously, in TASTPM mice there is abnormal tau phosphorylation in dystrophic neurites in close association with amyloid plaques, demonstrated with AT8 staining. However, expression of the two forms of mutant tau did not augment the amyloid pathology. There was no change in the time of onset or the progression of amyloid plaque pathology, and overall area of the plaques was the same on the tau-transduced and control sides. We also investigated additional changes in tau phosphorylation near to plaques in animals expressing mutant tau, but we could see no evidence that the presence of plaques had locally changed AT8 or AT100 staining. The enhancement of tau pathology reported in P301L mice on injection of fibrillar Ab42 was therefore not reproduced in our model (Gotz et al., 2001). In addition, we did not see a significant increase in neuronal loss from tau pathology in TASTPM compared with WT mice. As observed in AD patients, an increase in the inflammatory response was observed in close association with plaque deposits (as observed in various APP transgenic models), and there was also an increase in GFAP and staining with the Iba1 microglial marker in the ERC region transduced with mutant tau, which was not seen in animals transduced with GFP or normal tau. Early stages of AD pathology are characterized by neurodegeneration in the temporal cortex, affecting the perforant pathway, entorhinal cortex, and hippocampus. Cognitive impairments, deficits in short-term memory, and attention represent some of the earliest symptoms of AD with degeneration of the perforant pathway probably contributing to memory deficits (Kirkby and Higgins, 1998). The ERC plays an important role in memory processing, receiving multimodal sensory information from areas of association, and sensory cortex, and relaying this information to the hippocampal dentate gyrus via the perforant pathway. It also

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receives outputs from the hippocampus via the subiculum and sends afferents back to the association cortex. AD patients show considerable atrophy of the perforant pathway, which consequently affects cognitive behavior (Kirkby and Higgins, 1998). Thus, integrity of this pathway is crucial for normal cognitive function. Previous studies have shown that lesions in the ERC, both unilaterally and bilaterally, might induce alterations of behavioral, and spatial learning and working memory, including AAV-P301L injections that caused a spatial memory deficit (Hardman et al., 1997; Kopniczky et al., 2006; Ramirez et al., 2011; van Praag et al., 1998). The MWM is the standard method for assessing these deficits in rodents. We performed an extensive assessment of normal and AAV-injected animals at 2, 4, and 6 months. The behavioral assays at 2 and 4 months showed that the TASTPM animals were impaired relative to WT, but there was little or no additional deficit from the expression of mutant tau. At 6 months we performed a simpler analysis of shortterm memory, because of the fragility of the animals. This showed that animals expressing the highly toxic 3PO mutated tau had a marked deficit in short-term memory, correlating with neuronal loss and dystrophic neurites in the ERC of these animals. TASTPM transgenic mice injected with mutant tau displayed many of the main features characteristic of human AD patients: neuronal cell loss, aberrant tau pathology, and tau aggregation, amyloid pathology, increased inflammatory response, and memory impairment. The use of viral vectors for expression of mutant tau therefore provides a useful alternative to traditional models of AD. Disclosure statement Jill C. Richardson, Ishrut Hussain, and David Howlett are employees of GlaxoSmithKline. James W. Fawcett is a paid consultant for Acorda Therapeutics, Novartis and Covidien. Experiments were conducted in accordance with the United Kingdom Animals (Scientific Procedure) Act of 1986 and UK Home Office regulations. Acknowledgements This work was supported by Medical Research Council, GlaxoSmithKline, the John and Lucille van Geest Foundation, the NIHR Cambridge Biomedical Research Centre, and the European Union Framework 7 project Plasticise. The authors thank Marc Smith and David Story for technical support, Dr Alessandro Ciamei for behavioral advice, and Dr Manuela Mellone, Dr David A. Tumbarello, Dr Aviva Tolkovsky, Dr Jessica Kwok, and Dr Elske Franssen for constructive advice. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neurobiolaging.2012. 11.011. References Allen, B., Ingram, E., Takao, M., Smith, M.J., Jakes, R., Virdee, K., Yoshida, H., Holzer, M., Craxton, M., Emson, P.C., Atzori, C., Migheli, A., Crowther, R.A., Ghetti, B., Spillantini, M.G., Goedert, M., 2002. Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein. J. Neurosci. 22, 9340e9351. Augustinack, J.C., Schneider, A., Mandelkow, E.M., Hyman, B.T., 2002. Specific tau phosphorylation sites correlate with severity of neuronal cytopathology in Alzheimer’s disease. Acta Neuropathol. 103, 26e35. Avila, J., 2006. Tau phosphorylation and aggregation in Alzheimer’s disease pathology. FEBS Lett. 580, 2922e2927. Bellucci, A., Westwood, A.J., Ingram, E., Casamenti, F., Goedert, M., Spillantini, M.G., 2004. Induction of inflammatory mediators and microglial activation in mice transgenic for mutant human P301S tau protein. Am. J. Pathol. 165, 1643e1652.

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