Memory-related deficits following selective hippocampal expression of Swedish mutation amyloid precursor protein in the rat

Experimental Neurology 200 (2006) 371 – 377 www.elsevier.com/locate/yexnr

Memory-related deficits following selective hippocampal expression of Swedish mutation amyloid precursor protein in the rat Yan Gong a , Edwin M. Meyer b , Craig A. Meyers b , Ronald L. Klein d , Michael A. King c,e , Jeffrey A. Hughes a,⁎ b

a Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, FL 32610, USA Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, FL 32610, USA c Department of Neuroscience, College of Medicine, University of Florida, Gainesville, FL 32610, USA d Department of Pharmacology and Therapeutics, LSUHSC-S, Box 33932, Shreveport, LA 71130, USA e Malcom Randall Veterans Affairs Medical Center, Gainesville, FL 32610, USA

Received 2 September 2005; revised 3 February 2006; accepted 22 February 2006 Available online 15 June 2006

Abstract The gene encoding for the Swedish double mutation (K595N/M596L) of amyloid precursor protein (APP695Swe) was expressed bilaterally in adult rat hippocampus to determine its long-term effects on memory-related behavior as well as amyloid deposition. Recombinant adenoassociated viral serotype 2 (rAAV2) vectors were injected that contained either non-expressing DNA or cDNA encoding for APP695Swe under control of a chicken beta actin/cytomegalovirus promoter/enhancer. Immunolabeling human APP with the antibody 6E10 was observed throughout the cytoplasm of aspiny and, to a lesser extent, spine-bearing hippocampal neurons 6 and 12 months post-injection of the APP695Swe but not control vector. Aβ1–42 immunolabeling was identified in unusual immunoreactive objects within the hilus of the dentate gyrus and in the granule cell layer, proximal to the injection site. At 12 months post-transduction, rats that received the APP695Swe gene also demonstrated significant deficits in the acquisition and probe components of the spatial-memory-related Morris water task compared to control animals. These behavioral deficits occurred in the absence of any amyloid plaques, gliosis, or FluoroJade labeling of dying neurons. In conclusion, prolonged and localized APP695Swe expression in hippocampal neurons is sufficient to produce memory deficits without plaque formation or neuronal loss. © 2006 Elsevier Inc. All rights reserved. Keywords: Alzheimer’s disease; Amyloid precursor protein; Adeno-associated virus; Memory-related behavior; Inflammation

Introduction Alzheimer disease (AD) is an age-related neurodegenerative disorder characterized by the progressive loss of cognitive functions. Pathological features of AD include loss of neurons in vulnerable brain regions, the extracellular deposition of amyloid plaques, and intraneuronal accumulation of neurofibrillary tangles and reactive gliosis (Giannakopoulos et al., 1998; Price and Morris, 1999; Shie et al., 2003). Molecular genetic analyses of early onset familial AD indicated that the formation of amyloidogenic and other potentially toxic peptides from amy⁎ Corresponding author. Department of Pharmaceutics, College of Pharmacy, Box 100484, University of Florida, 1600 SW Archer Road, Gainesville, FL 32610, USA. Fax: +1 352 392 4447. E-mail address: [email protected] (J.A. Hughes). 0014-4886/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2006.02.136

loid precursor protein (APP) may be a central event in the pathogenesis of the disease (De Strooper and Annaert, 2000; Selkoe, 2002). A double mutation (K670N/M671L) of the APP gene was described in a Swedish family (Mullan et al., 1993) that resulted not only in early onset of AD, but also in the over-expression of amyloid and subsequent plaque formation when introduced into transgenic mice (Sturchler-Pierrat et al., 1997). Important questions remain, however, about the influence of different APP-derived peptides versus amyloid plaques in the disease process, as well as about the roles of various brain regions underlying the memory-related deficits seen in AD. For example, many studies suggest that C-terminal peptides from APP may be responsible for at least some of the neurotoxicity seen in AD (Oster-Granite et al., 1996; Berger-Sweeney et al., 1999), while others question the causal role of amyloid plaques in this process (Lee et al., 2005). While transgenic mice are important for

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studying the amyloidogenic component of the AD process, their high levels of mutant APP expression throughout the brain combined with plaque accumulation in multiple brain regions complicate any direct attempt to address these questions (Irizarry et al., 1997; Duff, 2001). These mice also present a concern about potential developmental effects in response to human APP expression, effects that may account for the very modest nature of memory-related deficits seen in them using some established paradigms such as the spatial Morris water task. We therefore used a region-selective approach to evaluate the effects of mutant APP expression in adults, focusing initially in the hippocampus (Klein et al., 2002a, 2004). cDNA encoding the Swedish double mutation (APP695Swe) was packaged into a recombinant adeno-associated virus type 2 (rAAV2) construct under control of a promoter that we found to provide stable expression for up to 2 years. This vector transduces neurons predominantly in the adult brain, bypassing potential developmental effects. Mutant APP transgene expression, astrocytosis, and Morris water task performance were monitored for up to 12 months post-transduction. Materials and methods Vector preparation The two plasmids used in this study for vector preparation contained either APP695Swe cDNA or a control non-expressing cDNA, both flanked by the AAV-2 terminal repeats (TR). The expression cassette included, in 5′ to 3′ order: (1) a 1.7 kb sequence containing the hybrid cytomegalovirus (CMV) immediately-early enhancer/chicken β actin promoter/exon 1/intron (Daly et al., 1999); (2) the human APP695Swe or a non-coding cDNA; and (3) the poly (A) tail from bovine growth hormone. Plasmids were propagated in SURE cells (Stratagene, La Jolla, CA) and CsCl purified as previously described (Zolotukhin et al., 1996). Plasmids were packaged into rAAV2 using the adenovirusfree method developed by Zolotukhin et al. (1999). Human embryonic kidney 293 cells at 70% confluence were transfected by the calcium phosphate method with plasmid containing APPSwe695 or non-coding vector in an equimolar ratio with the plasmid pDG, which contained the AAV2 capsid protein genes and adenovirus 5 genes necessary for helper function in packaging (Grimm et al., 1998). Three days after transfection, cells were harvested and pelleted. The pellet was resuspended in lysis buffer (50 mM Tris pH 8.5, 150 mM NaCl) and freeze–thawed three times. The sample was then incubated with 1500 units of endonuclease (Sigma, St. Louis, MO) for 30 min at 37°C and centrifuged at 2000 × g for 20 min. The resulting supernatant was added to a Beckman Optiseal centrifuge tube. Using a peristaltic pump, a discontinuous gradient of iodixinol (Optiprep; Nycomed, Sheldon, Birmingham, UK) was added to the tube in four layers (60%, 40%, 25% and 15% iodixinol). The tubes were heat sealed, placed in a Beckman 70 Ti rotor, and centrifuged at 100,000 × g for 1 h at 18°C. The interphase containing rAAV2 was then removed and purified through a heparin affinity column (Sigma, St. Louis, MO). After washing

with 1× TD buffer (1× PBS, 1 mM MgCl2, 2.5 mM KCl) and eluting with 1× TD/1 M NaCl, the sample was concentrated in Millipore (Bedford, MA) Biomax 100 Ultrafree-15 units precoated overnight with 2% rat serum/lactated Ringer’s solution. The viral stock was then diluted twice with lactated Ringer’s solution to decrease the salt concentration by 100 times. Recombinant AAV2 vectors were titered for total genomic particles by a previously described quantitative DNA slot blot analysis (Kube and Srivastava, 1997). Briefly, a 4-μl aliquot of the virus stock was treated with Dnase I (Roche, Mannheim, Germany) at 37°C for 1 h followed by incubation with proteinase K (Boehinger Mannheim, Germany) for 1 h at 37°C to obtain the encapsulated DNA. Dnase I buffer (10×) was 50 mM Tris–HCl (pH 7.5) and 10 mM MgCl2. The 10× proteinase K buffer contained 10 mM Tris–HCl (pH 8.0), 10 mM EDTA, and 10% sodium dodecyl sulfate. The sample was then phenol-chloroform extracted twice and chloroform extracted once and precipitated with sodium acetate and ethanol. The pellet was dissolved in 40 μl of water (a 1:10 dilution of the original aliquot). The sample was quantified by a DNA slot blot assay using 1.7 kb EcoRI segment of pTR-UF12 (Klein et al., 2002b) as probe and a series of dilutions of pTR-UF12 as standard curve. Titers for the rAAV2 used were: rAAV2-APP, 6 × 1012 total genomic particles/ ml; non-expressing vector, 5 × 1012 total genomic particles/ml. Stereotaxic surgery Male Sprague–Dawley rats were obtained from the Harlan Sprague–Dawley farm (Indiana) and housed and bred in animal facility in the University of Florida Health Science Center. Rats (∼250 g) were anesthetized with 4% of isoflurane/oxygen. Analgesic flunixin meglumine (2 mg/kg) was administered intramuscularly before surgery to minimize pain. Vectors were injected bilaterally through a 27-gauge cannula connected via 26gauge polyethylene tubing to a 10 μl Hamilton syringe mounted to a CMA/100 microinjection pump. The injection coordinates for the hippocampus were −3.5 mm bregma, 2.0 mm medial/ lateral, and 3.1 mm dorsal/ventral (Paxinos and Watson, 1986). The pump delivered up to 2 μl virus (1010 genomic particles) at a rate of 0.15 μl/min. The cannula was removed slowly 2 min after the injection. The skin was sutured, and the animals were placed on a heating pad until they began to recover from the surgery, before being returned to their individual cages. All animal care and procedures were in accordance with institutional IACUC and NIH guidelines. Morris water task The Morris water task was conducted 12 months post-transduction as described (Schenk and Morris, 1985). Each rat was tested for three trials per day for 7 days, with one of four starting locations varied between trials, and allowed a maximum of 60 s to locate the platform. If the platform was not located within the maximum time, the rat was guided to the location. The rat was allowed 30 s on the platform. For each trial, latency to find the platform (maximum 60 s), path length (cm) to the platform, and swim speed were recorded by a video-tracking/computer-digitizing

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system (HVS Image, Hampton, UK). On day 8, animals were given a probe test in which the platform was removed and the rats had 60 s to search for the platform. The swim distance and percentage of time spent in each quadrant were recorded. This behavioral study was powered to detect effect size of 1.5 times of standard deviation at P = 0.05 (two-sided) and 80% power. The latency to find the platform during the 7-day acquisition training in the Morris water task was analyzed with repeated measure ANOVA using SAS GLM procedure. rAAV2APP695Swe and non-expressing vector were designated treatments, and days 1 to 7 treated as repeated measurement variables. For each animal, the latency times for each of the 3 trials were averaged and used as the response for that day. For the probe trials, the percentages of total time spent in each quadrant of the pool were determined and analyzed using a two-sample t test. All statistical analyses were performed using SAS Software version 9.13 (SAS Institute Inc., Cary, NC). Power analysis was performed in G*Power software. Graphics was done in Graphpad Prism version 4 (GraphPad software, Inc.) A P value b 0.05 was considered statistically significant. Immunohistochemistry Anesthetized animals were perfused with 100 ml of cold PBS followed by 400 ml cold 4% paraformaldehyde/PBS. The brains were removed and equilibrated in a cryoprotectant solution of 30% sucrose/PBS and stored at 4°C. Coronal sections (50 μm) were cut on a sliding microtome with freezing stage. Antigen detection was conducted on free-floating sections by incubation in a blocking solution (3% goat serum/0.3% Triton X-100/PBS) for 1 h at room temperature followed by primary antibody incubation overnight at 4°C. Endogenous peroxidase was quenched by incubation in 0.5% H2O2/PBS for 10 min before blocking. Primary antibodies used were: 6E10 (1:1000, Signet, Dedham MA), glial fibrillary acidic protein (GFAP) (1:2000, Chemicon, Temecula, CA), NeuN (1:1000) (Chemicon), anti-Aβ1–42 (1:1000, Serotec), anti-synaptophysin (1:500, Sigma, St. Louis, MO). The sections were washed in PBS and incubated with biotinylated anti-mouse IgG (1:1000, Dako, Carpinteria, CA) for 1 h at room temperature. The sections were then washed with PBS and labeled with ExtrAvidin peroxidase (HRP) conjugate (1:1000, Sigma) for 30 min at room temperature. Development of color was conducted with a solution of 0.67 mg/ml diaminobenzidine (DAB, Sigma)/0.1 M sodium acetate/8 mM imidazole/2% nickel sulfate/0.003% H2O2. The sections were then mounted on Superfrost plus microscopic slides (Fisher, Hampton, NH), air-dried for 30 min and dehydrated by passing through H2O, 70%, 95%, 100%, 100% ethanol, xylene twice and coverslipped with Eukitt (Calibrated Instruments, Hawthorne, NY). Thioflavin S staining To stain for amyloid plaques, sections were treated with the following solutions for 5 min each: deionized H2O, Mayer’s hematoxylin solution (filtered; Zymed, San Francisco, CA), running tap water, deionized H2O, 1% thioflavin S (in dH2O,

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filtered, fresh, Sigma), 70% alcohol, deionized H2O and PBS (Rudelli et al., 1984). The sections were then mounted on slides, air-dried and coverslipped with glycerol gelatin and viewed under fluorescent microscope. FluoroJade staining To stain for degenerating neurons, a FluoroJade staining procedure modified from the published method by Schmued et al. (1997) was used. The free-floating sections were treated with 100% ethanol for 3 min followed by 70% ethanol and dH2O for 1 min each, 0.06% potassium permanganate for 15 min and washed with dH2O for 1 min. Then, the sections were treated in the dark with 0.001% FluoroJade staining solution for 30 min followed by three washes of dH2O. The sections were then mounted, air-dried and coverslipped with glycerol gelatin for epifluorescence microscopy. Stereology measurements The numbers of cells expressing APP immunoreactivity were estimated by an unbiased stereological optical fractionator method (West et al., 1991). Tissue sections spaced regularly throughout the hippocampus were analyzed using a microscope with a motorized stage. Optical dissectors were 50 × 50 × 15 μm cubes spaced in a systematically random manner 150 μm apart and offset 3 μm from the section surface. The fractionator sampling was optimized to yield approximately 100–200 counted cells per animal, for error coefficients less than 0.01. Using the Image Pro Plus software (Media Cybernetics, Silver Spring, MD), the perikarya of 6E10 antibody-labeled cells were outlined on calibrated digital micrographs for area measurement. This process was conducted for three sections regularly spaced through the hippocampus. The area values were then totaled for each injection and analyzed using previously described methods (King et al., 2002). Results APP was detected with the human-selective 6E10 antibody 6 months and 12 months post-transduction with the APP695Swe vector. Strong labeling was observed throughout the cytoplasm (perikarya, dendrites, axons) of neurons with an aspiny morphology (inhibitory, GABAergic). Less intense immunoreactivity characterized spine-bearing (excitatory, glutamatergic) neurons (Fig. 1). No immunolabeling of human APP was found in either non-neuronal cells following this procedure or in neurons after transduction with the non-expressing vector. A quantitative stereological evaluation of APP695Swe-expressing neurons demonstrated APP expressing neurons at both 6 months (7408 ± 2000, mean ± SD, n = 3) and 12 months post-injection (6249 ± 1356, n = 3). This apparent lack of APP695Swe-induced neurotoxicity over an extended interval was corroborated by the lack of detectable FluoroJade labeling of degenerating neurons at either posttransduction interval. Furthermore, both treatment groups had similar levels of hippocampal synaptophysin and GFAP staining

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Fig. 1. Long-term APP expression probed with 6E10 antibody after rAAV2-APPSwe or non-expressing vector injection. (A) Six months post-injection of rAAV2APPSwe (12.5×); (B) hilar neurons in dentate gyrus (32.5×); (C) 12 months post-injection of rAAV2-APPSwe (12.5×); (D) CA3 neurons (32.5×); (E) higher magnification in granule cell layer (130×); (F) dentate gyrus (130×); (G) dendrites of granule neurons (130×); (H) 6 months post-injection of non-expressing vector.

at each time point, suggesting no long-term loss of synaptic arborization or astrocytosis. Selective labeling of Aβ1–42 immunoreactivity was seen the hilus of the dentate gyrus and in the granule cell layer, proximal to the injection site 10–15 months after APP695Swe but not control vector injections (Fig. 2A). 6E10 immunostaining highlighted human APP-expressing

cells (Fig. 2B). The Aβ1–42-labeled objects were generally smaller than the 6E10-positive cells and were neither plaque- nor neuron-like in size and shape. The absence of amyloid plaques was confirmed by thioflavin S staining, which was not seen in any treatment group. Despite the absence of amyloid plaques or neurotoxicity, APP695Swe-vector-treated animals performed more poorly

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Fig. 2. Aβ1–42 (left) and APP (right) staining at 12 months post-rAAV2-APPSwe injection. (A) β amyloid 1–42 (20×); (B) APP (20×).

than controls in the Morris water task both in the acquisition (P = 0.0397, F(1,4) = 5.14) and probe phases at 12 months post-transduction (Fig. 3). For the probe phase, the control and APP695Swe vector values for percent of time in the target quadrant were 55.0% ± 4.2% and 38.2% ± 3.5%

Fig. 3. Morris water maze performance 12 months post-injection of either the control or APPSwe expression vector into hippocampus. Differences between these treatment groups were seen in the acquisition phase (A, P = 0.0397, F(1,14) = 5.14) and probe trial (B, P value b 0.01) (n = 12/group).

(mean ± SD, respectively; P b 0.01). No difference in swim distance was observed between the treatment groups. Discussion A significant discovery from this study is that long-term APP695Swe expression in rat hippocampal neurons is sufficient to interfere with spatial-memory-related behavior, without neurotoxicity or amyloid plaque accumulation. These deficits in both acquisition and retention/recall phases of the Morris water task were more substantial than seen in transgenic mice carrying this double mutation, even when laden with amyloid deposits throughout the hippocampus and neocortex (Pompl et al., 1999; Westerman et al., 2002). Whether this is due to the high local levels of human APP found in the cytoplasm of these transduced adults, to the lack of developmental effects that may occur in the transgenic mouse, or other factors is not clear. However, it is apparent that expression of this gene in a very localized region of the brain is sufficient to mimic a cardinal behavioral condition associated with Alzheimer’s disease. It was unexpected to find no amyloid plaques, neurotoxicity, or loss of synaptophysin staining in the hippocampus despite robust expression of APP695Swe and the accumulation of Aβ1– 42 immunoreactivity in the region. We continued to look for plaques for up to 15 months without success. Several C-terminal fragments of this peptide as well as Aβ1–42 have been found to be highly toxic when administered directly to neurons (Pike et al., 1991; Davenport Jones and Fox, 1998; Kim et al., 2003). Aβ1– 42 is of course only one component of amyloid plaques, and additional factor(s) may be necessary for their accumulation that are not expressed in these animals (Yankner et al., 1989; Yankner, 1996). Alternatively, Aβ1–42 efflux from the hippocampus may be sufficient to obviate plaque formation (DeMattos et al., 2002). Intraneuronal Aβ has also been implicated in Alzheimer’s disease (Hartmann, 1999; Wilson et al., 1999; Gouras et al., 2005). Several studies have reported that Aβ42 processing occurs intracellularly, including in the ER (Hartmann et al., 1997), Golgi apparatus (Xu et al., 1997), and endosomal–lysosomal system (Cataldo et al., 2000; Pasternak et al., 2004). Aβ42 normally resides in the outer limiting membrane of multivesicular bodies

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(MVBs) and smaller tubulo-vesicular organelles in neurons of mouse, rat, and human brain (Takahashi et al., 2002). Our results indicate that neuronal APP695Swe expression and resulting Aβ42 accumulation may be early key events in Alzheimer’s disease memory-related deficits, occurring prior to amyloid deposits and explicit neuronal loss. The vector in this study was selected for its tropism for brain neurons, which accounts for the lack of microglial or astrocytic APP or Aβ42 expression. While there are several potential processes underlying these behavioral deficits, based on recently characterized effects of APP-derived peptides on neuronal function, one possibility involves their highly potent inhibition of hippocampal alpha7 nicotinic receptors (Liu et al., 2001). Selective antagonism of these receptors in hippocampus is sufficient to interfere with memory-related behavior (Bettany and Levin, 2001), while their activation improves behavioral performance in several models including the Morris water task (Meyer et al., 1997). Expression of high levels of APP would therefore be expected to interfere with alpha7 receptor function locally in the hippocampus. Along this line, hippocampal alpha7 nicotinic receptor binding is increased in APP695Swe-expressing transgenic mice (Bednar et al., 2002), suggesting up-regulation of the receptor typical of long-term receptor antagonism (Almeida et al., 2004). We also found increased hippocampal alpha7 receptor binding following hippocampal transduction with the APP695Swe AAV expression vector (unpublished observation), consistent with a role for these receptors in the memory deficits. Inflammation is increasingly recognized as a potential component of Alzheimer’s disease, but its relationship with intraneuronal APP processing and accumulation remains incompletely understood. No vector-induced astrocytosis was apparent at 6 or 12 months, suggesting that this inflammatory response was not involved. A preliminary analysis of microglial activation of the MHC II complex also found no difference between treatment groups at 6 or 12 months, though additional study will be required to rule out a role for microglia in these transgenic APP-induced behavioral changes. Acknowledgment This study was funded by NIH grant AG10485. References Almeida, L.E., Pereira, E.F., Camara, A.L., Maelicke, A., Albuquerque, E.X., 2004. Sensitivity of neuronal nicotinic acetylcholine receptors to the opiate antagonists naltrexone and naloxone: receptor blockade and up-regulation. Bioorg. Med. Chem. Lett. 14, 1879–1887. Bednar, I., Paterson, D., Marutle, A., Pham, T.M., Svedberg, M., Hellstrom-Lindahl, E., Mousavi, M., Court, J., Morris, C., Perry, E., Mohammed, A., Zhang, X., Nordberg, A., 2002. Selective nicotinic receptor consequences in APP(SWE) transgenic mice. Mol. Cell. Neurosci. 20, 354–365. Berger-Sweeney, J., McPhie, D.L., Arters, J.A., Greenan, J., Oster-Granite, M.L., Neve, R.L., 1999. Impairments in learning and memory accompanied by neurodegeneration in mice transgenic for the carboxyl-terminus of the amyloid precursor protein. Brain Res. Mol. Brain Res. 66, 150–162. Bettany, J.H., Levin, E.D., 2001. Ventral hippocampal alpha 7 nicotinic receptor blockade and chronic nicotine effects on memory performance in the radialarm maze. Pharmacol. Biochem. Behav. 70, 467–474.

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