ApoE4 impairs hippocampal plasticity isoform-specifically and blocks the environmental stimulation of synaptogenesis and memory

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Neurobiology of Disease 13 (2003) 273–282

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ApoE4 impairs hippocampal plasticity isoform-specifically and blocks the environmental stimulation of synaptogenesis and memory Ofir Levi,a Ana L. Jongen-Relo,b Joram Feldon,b Allen D. Roses,c and Daniel M. Michaelsona,* a b

The Department of Neurobiochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel The Department of Biology, Laboratory of Behavioral Neurobiology, The Swiss Federal Institute of Technology, Zu¨rich, Switzerland c The Department of Neurology, Duke University, Durham, NC, USA Received 16 October 2002; revised 12 March 2003; accepted 2 April 2003

Abstract Alzheimer’s disease (AD) is associated with genetic risk factors, of which the allele E4 of apolipoprotein E (apoE4) is the most prevalent, and is affected by environmental factors that include education early in life and socioeconomic background. The extent to which environmental factors affect the phenotypic expression of the AD genetic risk factors is not known. Here we show that the neuronal and cognitive stimulations, which are elicited by environmental enrichment at a young age, are markedly affected by the apoE genotype. Accordingly, exposure to an enriched environment of young mice transgenic for human apoE3, which is the benign AD apoE allele, resulted in improved learning and memory, whereas mice transgenic for human apoE4 were unaffected by the enriched environment and their learning and memory were similar to those of the nonenriched apoE3 transgenic mice. These cognitive effects were associated with higher hippocampal levels of the presynaptic protein synaptophysin and of NGF in apoE3 but not apoE4 transgenic mice. In contrast, cortical synaptophysin and NGF levels of the apoE3 and apoE4 transgenic mice were similarly elevated by environmental enrichment. These findings show that apoE4 impairs hippocampal plasticity and isoform-specifically blocks the environmental stimulation of synaptogenesis and memory. This provides a novel mechanism by which environmental factors can modulate the function and phenotypic expression of the apoE genotype. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Alzheimer’s disease; Apolipoprotein E; Enriched environment; Learning; Synaptogenesis; Synaptophysin; Transgenic mice; Working memory

Introduction Alzheimer’s disease (AD) is associated with genetic and environmental risk factors. The genetic factors include the amyloid precursor protein (APP), whose breakdown peptide, ␤-amyloid, is a hallmark of AD; the presenilin proteins; and apolipoprotein E (apoE), which is the major brain lipoprotein (for review see Price et al., 1998; Roses, 1998; Selkoe, 2001). The environmental risk factors include low levels of early childhood education and low socioeconomic background (Katzman, 1993; Kawas et al., 1999; Moceri et al., 2000, 2001; Zhang, 1990). The extent to which the * Corresponding author. Fax: ⫹972-3-6407643. E-mail address: [email protected] (D.M. Michael).

phenotypic expression of the AD genetic risk factors are affected by environmental factors and the mechanisms which mediate such interactions are not known. There are three major apoE isoforms (Weisgraber, 1994) of which apoE3, which contains a cysteine residue at position 112 and an arginine at position 158, is the most common. The two other isoforms, apoE4 and apoE2, contain respectively two arginines and two cysteines at positions 112 and 158. Genetic and epidemiological studies revealed that the APOE ␧4 genotype is an important risk factor for both sporadic and familial AD and that the gene dosage of this allele is inversely related to the age of onset of the disease (Corder et al., 1993; Saunders et al., 1993; Roses, 1996). Animal model studies in which mice expressing human apoE3 and apoE4 were subjected to neuronal stress

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and injury paradigms suggest that apoE plays an important role in neuronal repair, a function which is impaired in the apoE4 form of the protein (Buttini et al., 2000; Hartman et al., 2001; Sabo et al., 2000; White et al., 2001). Such conclusions may also be drawn from human clinical data in a number of diseases (Chapman et al., 2001). The extent to which these isoform-specific effects of apoE are related to actual repair of damaged cellular constituents or to the plastic neogenesis of new cellular components is yet to be determined. The environmental effects on cognitive and neuronal function can be monitored by exposure of rodents to an enriched environment which consists of social interactions and of stimulation of exploratory and motor behavior with objects such as toys and tunnels and a running wheel for exercise. This exposure has pronounced plastic neuronal consequences, which include dendretic aborization, synaptogenesis, elevated NGF levels, and neurogenesis and which are associated with cognitive effects such as marked improvements in learning and memory (Diamond et al., 1966; Rosenzweig, 1966; Van Praag et al., 2000). In the present study we examined the extent to which the phenotypic expression of the different apoE alleles can be affected by environmental conditions. This was pursued by subjecting young mice transgenic to either of the human APOE alleles ␧3 and ␧4 on a null mouse apoE background (Xu et al., 1996; Sabo et al., 2000) and control and apoEdeficient mice to an enriched environment and investigating the extent to which the resulting neuronal and cognitive plastic responses are affected by the human apoE alleles. This revealed that apoE4 impairs hippocampal plasticity isoform-specifically and does not allow the stimulation of synaptogenesis and memory by environmental enrichment.

Materials and methods Transgenic mice Human apoE3 and apoE4 transgenic mice were generated on an apoE-deficient C57BL/6J background utilizing human apoE3 and apoE4 transgenic constructs as previously reported (Xu et al., 1996). Accordingly, cosmid libraries were constructed from lymphoblasts of humans known to be homozygous carriers for apoE3 or apoE4, after which fragments containing human regulatory promoter sequences and the coding sequences for human apoE were used to produce the transgenic mice (Xu et al., 1996). The experiments were performed with the apoE3-453 and apoE4-81 lineages which express similar levels of brain apoE. The apoE transgenic mice were backbred with genetically homogenous apoE-deficient mice (Jackson Labs., Cat. No. N10 JAX) for more than 10 generations and were heterozygous for the human apoE transgene and homozygous for mouse apoE deficiency. The apoE genotype of the mice was confirmed by PCR analysis. Accordingly, DNA

was extracted from a small piece of tail tissue that was digested overnight at 55°C with proteinase K (Boehringer, Mannheim, Germany). Four microliters of the digested tissue were then added to 40 ␮l of GeneReleaser (BioVentures Incorporated, TN), which were then treated for 5 min in a microwave (900 W). The presence of mouse and human apoE genes was determined by PCR analysis of the extracted DNA as described by Xu et al. (1996). Differentiation between the human apoE3 and apoE4 transgenes was done by PCR-utilizing primers that amplify the DNA region spanning the apoE polymorphic site (AA 112) (Wenham et al., 1991; Chapman et al., 1996). The PCR products thus obtained (227 bp) were digested with the restriction endonuclease, AFlIII (NEB), after which the resulting products (177 and 50 bp for apoE3 or 227 bp for apoE4) were analyzed on 2% agarose gels as previously described (Chapman et al., 1996). Learning and memory Three-weeks-old apoE3 and apoE4 transgenic mice and control and apoE-deficient mice on the same background (C57BL/6J) were placed in either regular cages or in cages which contained exploratory objects such as toys, tunnels, and a running wheel (n ⫽ 5 males per mouse group in each of the environments) (Van Praag et al., 2000). The learning and memory experiments were initiated 20 weeks later and the animals were kept in their respective cages between tests. Learning was measured by a T-maze paradigm, which is composed of a forced run in which the mouse is directed to a given arm of the maze where it is rewarded, followed by a choice run in which the mouse is scored for its ability to correctly return to the arm in which it was rewarded during the forced run (Anger et al., 1991). The maze was constructed in a water bath from white wooden boards that reached above and below water level and that together formed a T-shaped canal whose arms were 40 cm long and 15 cm wide. The sides of the T-shaped canal were closed. A moveable, visible platform that could be placed at either of its ends served as the reward. The forced run was performed by randomly closing one side of the T-maze and placing the visible platform on the freely accessible side. After being allowed to reach this visible platform, the mouse was subjected to the test run in which both arms of the T-maze were open and the mouse was scored for its ability to return to the platform-containing arm of the T-maze. This was performed 8 times per day for the indicated number of days. Working memory was measured after the mice learned the T-maze paradigm and each reached a criterion of at least seven correct choices per day for 2 consecutive days. This was performed by varying the time interval between the forced and choice runs and by measuring the number of correct choices as a function of the duration of the delay. Working memory was considered impaired when the performance of the mice decreased below seven correct choices of the 8 daily trials. The apoE-deficient mice had swimming-stam-

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ina problems and their T-maze experiment was therefore terminated after 4 days of acquisition. Motor ability Motor performance and coordination were monitored by the rotarod test (Jones and Roberts, 1968). Accordingly, the mice were placed on a rod rotating at 10 revolutions per minute and the latency to fall from the rod was recovered during 60-s trials. The test was performed in three sessions, each consisting of three consecutive trials with a 1 h resting period between them. Preparation of brain samples ApoE3 and apoE4 transgenics and control mice were sacrificed after exposure to the indicated environment for 20 weeks and the subsequent completion of the T-maze learning and memory experiments. The apoE-deficient mice were treated similarly, except that, due to lack of stamina, they were subjected to the T-maze for only 4 days. The mice were anesthetized with ketamine and sacrificed by transcardial perfusion with phosphate-buffered saline (PBS), after which their brains were removed and halved. The hippocampus and cortex of the left hemisphere were then homogenized in lysis buffer [20 mM Tris–HCl, (pH 8.0) which contained 140 mM NaCl, 10% glycerol, 1% NP40 and protease inhibitors; Calbiochem], after which they were aliquoted and stored frozen at ⫺70°C. The right brain hemispheres, which were used for immunohistochemistry, were fixed overnight in 4% formaldehyde in PBS, after which they were cryoprotected by immersion in 30% sucrose for 24 h at 4°C and frozen in a tissue-freezing medium (Jung, Leica Instruments). Immunoblot analysis Equal amounts of the hippocampal and cortical homogenates [1 ␮g protein/lane for synaptophysin and, respectively, 10 and 5 ␮g protein/lane for apoE and the glial fibrillary acidic protein (GFAP) immunoblots] were loaded onto 12% polyacrylamide SDS gels that contained 26 lanes (Criterion System from BioRad) and were then electrophoresed and blotted. Synaptophysin and GFAP were detected by antisynaptophysin (Sigma) and anti-GFAP (Pharmagen) mAbs, whereas human apoE was visualized with a polyclonal goat antihuman apoE that reacts similarly with apoE3 and apoE4 (CalBiochem). The synaptophysin, GFAP, and apoE levels were determined by quantitation of the intensities of the corresponding immunoblot bands utilizing the BIS-202 BioImaging system. The use of wide 26-lane gels allowed the electrophoresis of up to 24 different samples and two standards on the same gel. This enabled quantitative comparison of up to four individual mice in each of six groups on the same gel (e.g., apoE3, apoE4 and control mice exposed to either enriched or regular environments).

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Measurements of NGF NGF levels in hippocampal and cortical homogenates of the different mice groups were measured by ELISA using a Promega kit. Immunohistochemistry The brains were cut at the level of the anterior hippocampus (2 mm posterior to bregma) on a freezing microtome into six adjacent series of 30-␮m-thick coronal sections. A one-in-six series was mounted immediately onto gelatinecoated slides and stained with cresyl violet. The other five series were collected into a cryoprotectant solution [30% ethylene glycol (Fluka) and 30% glycerol (Fluka) in 50 mM PB, pH 7.4] and stored at ⫺20°C until use. After the cresyl violet staining, the sections were dehydrated through an alcohol series, cleared with xylene, and coverslipped with DPX (BDH, Poole, England). Prior to the immunohistochemical processing, sections were rinsed in 0.1 M Tris– HCl (pH 7.4) and mounted onto gelatine-coated slides. Immunohistochemistry was performed in slice mounted sections using a synaptophysin monoclonal antibody (S-5768 clone SVP-38, dilution 1:100; Sigma, St. Louis, MO). The sections were incubated with the primary antibody overnight at room temperature diluted in 5% milk powder/0.2% Tween-20 in Tris–HCl. After rinsing in 0.1 M Tris–HCl, sections were incubated for 2 h at room temperature in biotinylated goat antimouse serum (1:500, Vector Laboratories, Burlingame, CA) in secondary diluent consisting of 5% milk powder/0.2% Tween-20 in Tris–HCl. After more rinses in 0.1 M Tris–HCl, sections were incubated for 2 h in avidin-biotin-horseradish peroxidase complex (1:200, ABC-Elite, Vector Laboratories) in 0.1 M Tris–HCl/0.2% Tween-20. Following rinses in Tris–HCl, sections were placed for 30 min in chromagen solution consisting of 0.05% diaminobenzidine (Sigma) and 0.01% H2O2 (Fluka). The reaction was visually monitored and stopped in rinses of 0.1 M Tris–HCl. In order to minimize variability, sections from all animals were stained simultaneously. The immunostained sections were viewed at a magnification of ⫻50 and analyzed utilizing the Image-Pro plus system for image analysis (version 4.0, Media Cybernetics, Silver Spring, MD, USA) attached to a Zeiss light microscope (Axioskop, Oberkochen, Germany) interfaced with a CCD video camera (Kodak Megaplus, Rochester, NY). The levels of synaptophysin immunoreactivity in the hippocampal CA1, CA3, and dentate gyrus subfields were determined by measurements of the optical density of synaptophysin immunohistochemical labeling, which, as was recently shown, correlates linearly with the number of synaptophysin-immunoreactive sites (Li et al., 2002). The optical densities were measured in rectangular cursors placed on the hippocampal CA1, CA3, and dentate gyrus areas (250 ⫻ 750 ␮m for CA1 and CA3 and 250 ⫻ 250 ␮m for the dentate gyrus). For background staining, optical densi-

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ties were measured within open rectangular cursors of 100 ␮M ⫻ 100 ␮M placed along the corpus callosum. To control for variation in background staining across sections, the density reading from the corpus callosum was subtracted from that of the indicated hippocampal areas of each section. Three randomly selected and coded coronal sections at the level of the anterior hippocampus (2 mm posterior to bregma) were analyzed for each animal, and the results of the effects of Group ⫻ Treatment on the synaptophysin optical density of each of the areas were analyzed by ANOVA as described below. Statistical analysis Values of the behavioral experiments are expressed as mean ⫾ SEM, whereas the immunoblot, ELISA, and optical density results are expressed as mean ⫾ SD. Differences among means of the experimental groups were analyzed by two-way ANOVA with treatment and genotype as two independent factors. Post hoc comparisons of the results were performed when ANOVA showed a significant difference, using t tests with Bonferroni corrections for multiple comparisons.

Results Control apoE3- and apoE4-transgenic mice and apoEdeficient mice were placed after weaning for 20 weeks in either a regular or an enriched environment. The consequences of this environmental exposure were first assessed behaviorally by T-maze tests that monitor learning and memory (Anger et al., 1991). The results thus obtained with control mice are depicted in Fig. 1. As can be seen, both groups improved their performance over days (Fig. 1A). However, the environmentally stimulated controls learned more effectively and reached the learning criterion (at least seven correct choices of eight daily trials on 2 consecutive days) after 6 days of acquisition, whereas control mice that were maintained in a regular environment reached the learning criterion after 9 days of acquisition (Fig. 1A). Analysis of these results by ANOVA in terms of the day of acquisition on which the mice achieved the learning criterion revealed a significant effect of treatment (P ⬍ 0.001). The effects of environmental stimulation on the working memory of the control mice were measured by a delayed match to sample test, in which the effects of varying delays between the choice and forced runs on the performance of the mice were monitored. As can be seen in Fig. 1B, environmental stimulation had a marked effect on working memory and prolonged it from about 20 s in control mice maintained in a regular environment to over 45 s in those exposed to environmental stimulation. Analysis of these results by ANOVA, in terms of the delay at which the performance of the mice deteriorated to fewer than seven correct choices, revealed a significant effect of treatment (P ⬍ 0.001).

Fig. 1. The effects of environmental enrichment on learning and working memory of control mice. Three-weeks-old C57BL/6J control mice were placed for 20 weeks in either regular cages or in an enriched environment (n ⫽ 5 per treatment). Learning (number of correct choices of eight daily trials) and working memory (delayed match to sample) were measured as described under Materials and Methods. The T-maze learning curves of control mice maintained in the different environments are depicted in A, whereas their corresponding performances in the working memory test are shown in B. Results presented at each time point are the average ⫾ SEM of five control mice exposed to either enriched (●) or regular (䡩) environments; P ⬍ 0.001 for the effects of the enriched environment on acquisition and on working memory.

The effects of environmental stimulation on the performance of the apoE3- and apoE4-transgenic mice in the learning and working memory tests are depicted in Fig. 2. As can be seen, the performance of the apoE3- and apoE4transgenic mice in the T-maze acquisition test improved over days for animals that were kept in either environment (Fig. 2A and 2B). Environmental stimulation of the apoE3transgenic mice decreased the time required to reach the learning criteria, from 17 days for mice maintained in a regular environment to 12 days. In contrast, this treatment had no effect on the learning of the apoE4-transgenic mice, and both the regular and environmentally stimulated apoE4 mice reached the learning criteria after 16 days of acquisition. Analysis of these results by ANOVA, in terms of the day at which each of the mice achieved the learning criteria, revealed a significant effect of treatment (P ⬍ 0.001) and group (P ⬍ 0.006). This effect was due to improvement in the learning curve of the enriched apoE3-transgenic mice relative to those maintained in regular cages (P ⬍ 0.002), whereas the learning of the apoE4-transgenic mice was not significantly affected by environmental enrichment and was similar to that of apoE3-transgenic mice that were maintained in the regular environment. The effects of environmental stimulation on the working memory of the apoE3- and apoE4-transgenic mice, as measured in the T-maze by a delayed match to sample test, are depicted in Fig. 1C and 1D. As can be seen, environmental stimulation markedly improved the working memory of the apoE3-transgenic mice from ⬃20 s to ⬃45 s (Fig. 2C). In contrast, environmental stimulation had no effect on the working memory of the apoE4-transgenic mice, which was similar to that of the regularly treated apoE3 and apoE4 mice (⬃20 s). ANOVA revealed significant effects of

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Fig. 2. Comparison of the effects of environmental enrichment on learning and working memory of apoE3- and apoE4-transgenic mice. Three-weeksold apoE3- and apoE4-transgenic mice were placed for 20 weeks in either regular cages or in an enriched environment (n ⫽ 5 per Group ⫻ Treatment). Learning (number of correct choices of eight daily trials) and working memory (delayed match to sample) were measured as described under Materials and Methods. The T-maze learning curves of the two mice groups maintained in the different environments are shown in A and B, whereas their corresponding performances in the working memory test are shown in C and D. Results presented at each time point are the average ⫾ SEM of five mice of each genotype which were exposed to either enriched (●) or regular (䡩) environments; P ⬍ 0.006 for the acquisition and P ⬍ 0.001 for the working memory results of apoE3-transgenic mice maintained in the enriched environment relative to apoE3 mice kept in regular cages.

Group ⫻ Treatment (P ⬍ 0.001), which were due to improvements in the working memory of the enriched apoE3transgenic mice relative to the other groups (P ⬍ 0.001). Interestingly, control mice whose apoE is endogenous seem to perform better in the learning and memory tests than do the corresponding apoE3-transgenic mice, which express human apoE3 on a null mouse apoE background (compare Figs. 1 and 2). T-maze experiments with the apoE-deficient mice revealed that they experienced swimming stamina problems. The testing was therefore terminated prematurely after 4 days before the mice reached the learning criterion. Measurements of the performance of the mice in the rotarod test revealed that the latencies in the first trial of apoE4- and apoE-deficient mice that were kept in a regular environment (respectively 18 ⫾ 4 and 38 ⫾ 20 s) were lower than those of the corresponding control and apoE3transgenic mice (respectively 60 and 55 ⫾ 5 s). However, the performance of these apoE4- and apoE-deficient mice improved in subsequent trials and by the third trial was maximal (⬃60 s) and similar to those of the control and apoE3-transgenic mice. In contrast, following exposure to

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the enriched environment, the four mice groups performed equally and optimally in all the rotarod trials. This suggests that the learning and memory deficits of the environmentally stimulated apoE4-transgenic mice are not associated with impairments in motor performance and coordination. The extent to which the cognitive deficits of the apoE4transgenic mice are related to impairments in synaptic plasticity was investigated by immunoblot assays in which synaptic density was assessed by measurements of the level of the presynaptic protein synaptophysin (Saito et al., 1994). Since exposure to learning paradigms can by itself stimulate brain synaptogenesis and neuronal plasticity (Gould et al., 1999; Ramirez-Amaya et al., 2001), we first focused on the three mice groups (e.g., apoE3- and apoE4-transgenic mice and normal control mice) that were also subjected to the learning and memory T-maze tests. As shown in Fig. 3, environmental stimulation induced a pronounced increase in the intensity of the hippocampal synaptophysin immunoblot bands (MW ⫽ 38 kDa) of the apoE3-transgenic and control mice, whereas those of the apoE4-transgenic mice were unaffected by this treatment. This effect was specific to neurons as the levels of the astrocytic protein GFAP (MW ⫽ 50 kDa) were not affected either by the apoE genotype or by environmental stimulation (Fig. 3). Quantitation of the synaptophysin results followed by ANOVA (n ⫽ 4 per Group ⫻ Treatment) revealed a significant effect of group (P ⬍ 0.003) and treatment (P ⬍ 0.001) and of Group ⫻ Treatment (P ⬍ 0.02). Further examination of the results revealed that the synaptophysin levels of the apoE3-transgenic and control mice were similarly increased following environmental stimulation. In contrast, those of the apoE4 transgenic mice were unaffected by the environmental stimulation and were similar to those of apoE3-transgenic and control mice that were maintained in the regular environment (Fig. 4). The levels of cortical synaptophysin in the apoE transgenic and control groups are depicted in Fig. 5. As can be seen, the cortical synaptophysin levels of the apoE4- and apoE3-transgenic mice and of the control mice, unlike those of the hippocampus, were all similarly elevated following environmental stimulation. Analysis of these results by ANOVA (n ⫽ 4 per Group ⫻ Treatment) revealed a significant effect of Treatment (P ⬍ 0.001), but not of

Fig. 3. Synaptophysin and GFAP levels in the hippocampus of apoE transgenic and control mice maintained in regular and enriched environments. ApoE3 (ApoE3-tg.) and apoE4 (ApoE4-tg.) transgenic mice and control (Control) mice were maintained in either a regular or an enriched environment, after which their hippocampi were excised, homogenized, and immunoblotted with antisynaptophysin (upper panel) and anti-GFAP (lower panel) mAbs as described under Materials and Methods. Results shown correspond to three individual mice in each of the six groups.

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Fig. 4. Quantitation of the effects of the enriched environment on hippocampal synaptophysin levels of control (Control), apoE3-transgenic (ApoE3-tg.), and apoE4-transgenic (ApoE4-tg.) mice following exposure to either a regular or an enriched environment. Hippocampal homogenates were immunoreacted on Western blots and the results obtained were quantitated by computerized densitometry as described under Materials and Methods. Results are the mean ⫾ SD of four mice in each of the six groups, which were maintained either in a regular environment (light symbols) or in an enriched environment (dark symbols); *P Ⰶ 0.005 for enriched vs regular environments and **P ⬍ 0.005 for enriched apoE3 vs enriched apoE4 transgenic mice.

either Group or Group ⫻ Treatment. In contrast, the cortical levels of GFAP were not affected either by the apoE genotype or by environmental stimulation (not shown). Exposure to an enriched environment increases the expression of nerve growth factor (NGF) (Pham et al., 1999). Measurements of hippocampal NGF levels revealed that they were the same in apoE3, apoE4, and control mice that were maintained in regular cages and that the enriched environment increased the hippocampal NGF levels of the control and apoE3-transgenic mice but had no effect on those of the apoE4-transgenic mice (Fig. 6A). ANOVA of these results (n ⫽ 5 per Group ⫻ Treatment) revealed a significant effect of Treatment (P ⬍ 0.003) and of Group ⫻ Treatment (P ⬍ 0.01). In contrast, environmental enrichment stimulated similar increases in the cortical NGF levels of the apoE3 as well as of the apoE4-transgenic and control mice (Fig. 6B). Accordingly, ANOVA of the cortical NGF results (n ⫽ 5 per Group ⫻ Treatment) revealed a significant effect of Treatment (P ⬍ 0.001) and no effect of either Group or Group ⫻ Treatment. The observed parallelism between the effects of the apoE genotype and of environmental enrichment on hippocampal and cortical synaptophysin and NGF levels suggest that similar mechanisms may play a role in the brain area and isoform-specific effects of apoE4 on these neuronal parameters. The effects of environmental stimulation on the cortical and hippocampal synaptophysin levels of apoE-deficient mice are depicted in Fig. 7. As can be seen, environmental stimulation of the apoE-deficient mice resulted, as in control and apoE3-transgenic mice and unlike in the apoE4 transgenics, in elevated synaptophysin levels in both the hippocampus and cortex. Thus, the apoE-deficient mice, which unlike the other groups were not subjected to the learning and memory tests, did respond plastically to the enriched environment. This suggests that the inhibitory effects of

apoE4 on hippocampal synaptogenesis following environmental stimulation are not due to a loss of function but rather to the gain of an inhibitory function. The effects of the apoE genotype and environmental stimulation on hippocampal synaptophysin levels were also investigated immunohistochemically. Representative micrographs thus obtained from apoE3- and apoE4-transgenic mice that were exposed to either a regular or an enriched environment are presented in Fig. 8. As can be seen, exposure of the apoE3-transgenic mice resulted in a pronounced increase in synaptophysin immunoreactivity in the dentate gyrus as well as the CA1 and CA3 areas. In contrast, the corresponding hippocampal synaptophysin levels of the apoE4-transgenic mice were not affected by environmental stimulation and were similar to those of the apoE3-transgenic mice that were maintained in the regular environment (Fig. 8). Computerized densitometric measurements of the synaptophysin levels in the dentate gyrus of the apoE3- and apoE4-transgenic mice that were maintained in the different environments and of similarly treated apoE-deficient and control mice (n ⫽ 4 per Group ⫻ Treatment) are shown in Fig. 9. An ANOVA of these results revealed a significant effect of Group (P ⬍ 0.01), Treatment (P ⬍ 0.001), and of Group ⫻ Treatment (P ⬍ 0.01). Further examination of the results revealed that the synaptophysin levels of the apoE3transgenic, apoE-deficient and control mice were similarly and significantly increased following environmental stimulation to, respectively, 195 ⫾ 39% (P ⬍ 0.01), 197 ⫾ 50% (P ⬍ 0.020), and 170 ⫾ 18% (P ⬍ 0.001) of the corresponding mice that were maintained in the regular environment. In contrast, the synaptophysin levels of the dentate gyrus of the apoE4-transgenic mice were unaffected by environmental stimulation and were similar to those of the control, apoE3-transgenic and apoE-deficient mice that were maintained in the regular environment (Fig. 9). Similar results were obtained in the CA3 and CA1 hippocampal subfields, which were, however, significant only for CA3. There was a small increase in the hippocampal apoE levels of the two mice groups following exposure to the enriched environment. However, the hippocampal apoE lev-

Fig. 5. Cortical synaptophysin levels in the hippocampus of apoE-transgenic mice and control mice maintained in regular and enriched environments. ApoE3- (ApoE3-tg.) and apoE4- (ApoE4-tg.) transgenic and control (Control) mice were maintained in either a regular or an enriched environment, after which their cortices were excised and immunoblotted with an antisynaptophysin mAb as described under Materials and Methods. Results shown correspond to three individual mice in each of the six groups.

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Fig. 6. NGF levels in the hippocampus (A) and cortex (B) of apoE transgenic and control mice maintained in regular and enriched environments. ApoE3 (ApoE3-tg.) and apoE4 (ApoE4-tg.) transgenic mice and control (Control) mice were maintained in either a regular or an enriched environment, after which their hippocampi and cortices were excised and homogenized as described under Materials and Methods. NGF levels were then determined by ELISA using a Promega kit. Results presented (in nanograms per milligram of protein) are the mean ⫾ SD of five mice per each of the mice groups which were maintained either in a regular environment (light symbols) or in an enriched environment (dark symbols) 100% ⫽ 4.7 ⫾ 0.4 and 1.2 ⫾ 0.1 pg/mg tissue for respectively the hippocampus and cortex; *P ⬍ 0.001 in the cortex and P ⬍ 0.02 in the hippocampus for enriched vs regular environments.

els were the same for apoE3- and apoE4-transgenic mice maintained in either the regular or the enriched environments (Fig. 10). Similar results were obtained in the cortex (not shown). This suggests that neuronal and cognitive deficits of apoE4-transgenic mice following environmental stimulation are not due to differences in their brain apoE levels.

Discussion

astrocyte marker GFAP were not significantly affected by either the apoE genotype or by environmental stimulation. The effects of environmental stimulation on the brain are related to an arousal response of animals confronted with a new and complex environment and to activation of the cellular and molecular learning processes associated with learning and memory (Rosenzweig and Bennett, 1996). Although the neuronal and cognitive effects of the enriched environment depend on and are associated with an increase in exercise, the enriched environment improves T-maze performance markedly more than exposure to a running

The present study revealed that exposure of apoE3- and apoE4-transgenic mice to an enriched environment elicited improvements in learning and memory of apoE3-transgenic mice, whereas apoE4-transgenic mice were unaffected by environmental stimulation and their learning and memory were similar to those of the nonenriched apoE3-transgenic mice. These cognitive effects are associated with elevation of the levels of the presynaptic protein synaptophysin and of NGF in the hippocamus of the apoE3- but not of the apoE4transgenic mice. In contrast, the cortical synaptophysin and NGF levels of the apoE3- and apoE4-transgenic mice were stimulated to the same extent by environmental enrichment. Furthermore, the cortical and hippocampal levels of the

Fig. 7. The effects of apoE deficiency on hippocampal and cortical synaptophysin levels of mice maintained in regular and enriched environments. ApoE-deficient mice were maintained in either a regular or an enriched environment, after which their hippocampal (Hipp.) and cortical (Cortex) synaptophysin levels were determined by immunoblot assays as described under Materials and Methods. Results shown correspond to three individual mice in each of the groups.

Fig. 8. The effects of environmental stimulation on hippocampal synaptophysin immunoreactivity of apoE3- and apoE4-transgenic mice. Synaptophysin in the hippocampus of apoE3-transgenic mice (A and B) and apoE4-transgenic mice (C and D) that were maintained in either regular (A and C) or enriched environments (B and D) was visualized immunohistochemically using antisynaptophysin mAb as described under Materials and Methods. Bar ⫽ 500 ␮.

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Fig. 9. Quantitation of the effects of enriched environment on synaptophysin levels in the dentate gyrus of control (Control), apoE3-transgenic (ApoE3-tg), apoE4-transgenic (ApoE4-tg), and apoE-deficient (ApoE-def.) mice. Coronal brain sections were immunoreacted with an antisynaptophysin mAb, after which the levels of synaptophysin immunoreactivity were quantitated by computerized densitometry as described under Materials and Methods. Results are the mean ⫾ SD of four mice per group that were maintained either in a regular (light symbols) or in an enriched environment (dark symbols); *P ⬍ 0.001.

wheel (Van Praag et al., 2000). Accordingly, and in view of the central role of the hippocampus in learning and memory, it is likely that the deficient cognitive response of the apoE4-transgenic mice to environmental stimulation is mediated by impairments in distinct hippocampal learning and memory processes. The parallel phenotypic effects of apoE4 on cognition and on hippocampal synaptophysin suggest that the lack of improvement in learning and memory of the apoE4-transgenic mice following environmental stimulation may be related to impairments in synaptogenesis and synaptic plasticity. The findings that hippocampal synaptophysin levels of apoE-deficient mice were elevated following environmental stimulation (Fig. 7) and that the apoE3- and apoE4-transgenic mice have similar apoE levels both prior to and following environmental stimulation (Fig. 10) suggest that the inhibitory effect of apoE4 on synaptogenesis is due to gain of a pathological function and not to loss of an apoE3related beneficial property. This is probably also true for the effects of apoE4 on learning and memory. However, as the apoE-deficient mice did not complete the T-maze tests due to stamina problems, this point needs to be further examined. It has previously been shown that apoE deficiency during aging and following brain injury is associated with neuronal and cognitive impairment (Chen et al., 1997; Masliah et al., 1997; Veinbergs and Masliah, 1999). Together with the present findings, this suggests that apoE deficiency affects synaptic and cognitive functions under harsh, stressful conditions, but not following mild treatments such as exposure to an enriched environment. Importantly, the pathological effects of apoE4 are apparent even under conditions in which those of apoE deficiency are not (e.g., environmental stimulation). The finding that the isoform-specific effects of apoE3 and apoE4 on hippocampal and cortical synaptophsysin and NGF levels are the same suggests that both effects are

mediated by a similar mechanism. Such a mechanism may act directly on the nerve terminal or via an upstream effect on neuronal functions which precede synapse formation. Previous in vitro studies revealed that apoE4 inhibits, whereas apoE3 stimulates, neurite outgrowth by isolated neurons in culture (Nathan et al., 1994; Pitas et al., 1998). Future studies of the time course of the effects of the apoE genotype and of environmental stimulation on synaptic, dendritic, axonal, and other neuronal parameters are needed in order to identify the neuronal parameters that are directly affected by apoE4 and that mediate its inhibitory effects on synaptogenesis. The cortical and hippocampal apoE levels of the apoE3- and apoE4-transgenic mice are similar and are marginally elevated only following environmental stimulation (Fig. 10). Accordingly, the presently observed hippocampal specificity of the effects of apoE4 on the synaptophysin and NGF levels may be due either to differences in the response of hippocampal and cortical apoE receptors to apoE4 or to differential susceptibility of these brain areas to the metabolic changes which are induced by apoE4. ApoE is the major lipoprotein in the brain and as such its effects on neuronal function have been linked to lipids, in particular cholesterol transport and homeostasis (Mahley and Rall, 2001). This perception has now been enlarged by recent studies which have linked apoE and its receptors to brain development via novel signaling pathways (Herz and Beffert, 2000; Herz, 2001). It remains to be determined which apoE receptor and which of these mechanisms mediate the isoform- and brain-area-specific neuronal and cognitive effects of apoE4. We have recently shown that activation of brain astrocytes by icv injection of LPS is impaired in apoE-deficient mice and that this effect is reversed in apoE3 but not in apoE4 transgenic mice (Ophir et al., 2003). These findings, together with results which were obtained with other lines of apoE transgenic mice (Buttini et al., 2000; Fagan and Holtzman, 2000; Sabo et al., 2000; White et al., 2001), suggest that the pathological effects of apoE4 fall into two main categories: gain of a negative function, as presently reported, and loss of a positive apoE3-related function. It is thus possible that the pathological effects of apoE4 may be mediated by several mechanisms that differ in their cellular and molecular properties and whose pheno-

Fig. 10. ApoE levels in the hippocampus of apoE-transgenic mice maintained in regular and enriched environments. ApoE3- and apoE4-transgenic mice were maintained in either regular or enriched environments, after which their hippocampi were excised and immunoblotted with antihuman apoE antiserum as described under Materials and Methods. Results shown correspond to three individual mice in each of the groups.

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typic expression is affected by the experimental paradigm. Enriched environment can also stimulate neuronal and cognitive function when applied at an adult age (Van Praag et al., 2000) and it remains to be determined whether the age-dependent pathological effects of apoE4 can be offset by such a treatment. The present animal model findings are in accordance with neuropathological studies of AD brains that revealed that apoE4 is associated with impairments in plastic neuronal remodeling (Arendt et al., 1997). They are also in accordance with the epidemiological observations that the risk for AD of apoE4 carriers is modified by early-life socioeconomical environment (Moceri et al., 2001) and that education in early life lowers the risk of AD in apoE3 but not in apoE4 subjects (Kalmijn et al., 1997). This suggests that, with the expected worldwide improvement in the quality of early life, the importance of apoE4 as an AD risk factor will increase in the future. In summary, the present findings reveal that the pathological effects of apoE4 are elicited by environmental enrichment and that they are mediated by specific impairments in neuronal plasticity and synaptogenesis in the hippocampus. Acknowledgments We thank Duke University, NC, USA, and Glaxo-Wellcome for kindly providing the transgenic mice; Dr. Zipora Speiser for her help in the rotarod experiments; Drs. Ina Weiner and Lee Zuckerman for their help and advice in the behavioral studies; Mrs. Angela Cohen for her editorial assistance; and Ms. Liz Weber for the histological preparation. This work was supported partly by grants to D.M.M. from the European Community (Grant 2001/00972), from the Fund for Basic Research sponsored by the Israel Academy of Sciences and Humanities (Grant 43/00-1), from the Harry Stern National Center for Alzheimer’s Disease and Related Disorders, from the Eichenbaum Foundation, and by a grant to J.F. from the Swiss Federal Institute of Technology, Zurich. D.M.M. is the incumbent of the Myriam Lebach Chair in Molecular Neurodegeneration. References Anger, W.K., 1991. Animal test systems to study behavioral dysfunctions of neurodegenerative disorders. Neurotoxicology 12, 403– 413. Arendt, T., Schindler, C., Bruckner, M.K., Eschrich, K., Bigl, V., Zedlick, D., Marcova, L., 1997. Plastic neuronal remodeling is impaired in patients with Alzheimer’s disease carrying apolipoprotein E4 allele. J. Neurosci. 17, 516 –529. Buttini, M., Akeefe, H., Lin, C., Mahley, R.W., Pitas, R.E., Wyss-Coray, T., Mucke, L., 2000. Dominant negative effects of apolipoprotein E4 revealed in transgenic models of neurodegenerative disease. Neuroscience 97, 207–210. Chapman, J., Estupinan, J., Asheron, A., Goldfarb, L.G., 1996. A simple and efficient method for apolipoprotein E genotype determination. Neurology 46, 1484 –1485.

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