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Behavioral effects of CD40–CD40L pathway disruption in aged PSAPP mice
Brain Research 1015 (2004) 161 – 168 www.elsevier.com/locate/brainres
Research report
Behavioral effects of CD40–CD40L pathway disruption in aged PSAPP mice J. Todd Roach a,b,1, Claude-Henry Volmar a,b,*,1, Samvid Dwivedi a,b, Terrence Town c, Robert Crescentini a,b, Fiona Crawford a,b, Jun Tan d, Michael Mullan a,b a
Roskamp Institute, 2040 Whitfield Avenue, Sarasota, FL 34243, USA b James A. Haley Veteran’s Hospital, Tampa, FL, USA c Yale University School of Medicine, New Haven, CT, USA d University of South Florida, Tampa, FL, USA Accepted 3 May 2004 Available online
Abstract We have shown that, when an amyloid-beta peptide (Ah) overproducing transgenic mouse model (PSAPP) of Alzheimer’s disease (AD) is treated with a depleting antibody against CD40L, it causes marked attenuation of Ah pathology associated with decreased amyloidogenic processing of amyloid precursor protein (APP) and increased cerebral clearance of Ah. Here, we report that, when PSAPP mice receive a regimen of anti-CD40L antibody commencing at an age associated with initial Ah deposition, they demonstrate superior spatial memory on the standard water maze and radial arm water maze tasks, as well as exhibiting superior non-spatial memory in the object recognition test, as compared to control PSAPP mice. Furthermore, PSAPP mice treated with an anti-CD40L antibody regimen commencing at an age associated with extensive Ah deposition demonstrate superior spatial memory on the standard water maze task, as compared to control PSAPP mice. Disruption of CD40L activity has beneficial effects on pathology and cognitive behavior in the PSAPP mouse model, providing support for the therapeutic potential of interrupting the CD40 – CD40L interaction in AD. D 2004 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Degenerative disease: Alzheimer’s—beta amyloid Keywords: Alzheimer’s disease; CD40; Behavior; Transgenic; Mouse; Amyloid precursor protein
1. Introduction Alzheimer’s disease (AD) is the most prevalent form of progressive dementia and is characterized by the accumulation of amyloid beta (Ah) peptide, which is cleaved from the amyloid precursor protein (APP) by h and g secretases [35]. Animal models of AD are available that overproduce and accumulate Ah [18] and such models are key scientific tools in furthering our understanding of AD. Accompanying Ah accumulation, both clinically and in AD mouse models, is an inflammatory cascade in and around Ah aggregates [27,33,35]. This inflammatory reaction may be central to the * Corresponding author. Tel.: +1-941-752-2949; fax: +1-941-752-2948. E-mail address: [email protected] (C.-H. Volmar). 1 The first two authors contributed equally to this work. 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.05.004
development of AD, as clinically, individuals with Ah accumulation but no inflammatory response, do not show clinical expression of AD [33]. We have previously suggested that the costimulatory molecules CD40 and CD40L may play important roles in regulating the microglial response to accumulating Ah [36 –38]. CD40 is a member of the tumor necrosis factor/nerve growth factor receptor superfamily and its natural ligand CD40L is a key immunoregulatory molecule that is a co-stimulator of cells from both the innate and specific arms of the immune system [14,40]. Microglia up-regulate CD40 after Ah challenge and microglial activation induced by nanomolar levels of Ah requires CD40 –CD40L interaction in vitro [36]. It has been reported that CD40 is upregulated in AD patient brains on reactive microglia and in senile plaques, supporting its role in the AD neuroinflammatory processes [6,39]. Work by
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Tan et al. [36] has shown that increased tumor necrosis factor alpha production and induction of neuronal injury occur when Ah-stimulated microglia are treated with CD40L. An Ah overproducing mouse model (APPsw) deficient for CD40L displays reduced microglial activation, suggesting the CD40 –CD40L interaction is necessary for Ah-induced microglial activation [37]. APPsw mice deficient for CD40L also have reduced hyper-phosphorylation of H and show a significant reduction ( f 80%) in Ah burden compared to APPsw mice [37]. Tan et al. conducted further experiments to elucidate possible mechanism(s) of Ah reduction. Subsequent experiments in which doubly transgenic AD mice (PSAPP) were treated with an antiCD40L antibody (anti-CD40L) from 2 to 8 months revealed attenuation ( f 65%) of Ah accumulation associated with increased plasma levels of Ah and increased nonamyloidogenic APP processing [37]. These studies collectively show that reducing CD40L in an Ah overproducing mouse model results in an attenuation of Ah pathology, likely via multiple mechanisms. This attenuation of AD related pathology naturally led to a study assessing the behavioral effects of CD40L disruption in an AD mouse model. We accomplish this by using a battery of validated behavioral tests to compare IgG-treated versus anti CD40L-treated PSAPP mice. Since others have shown, using the Morris water maze and the radial arm water maze spatial memory tests, that PSAPP mice are cognitively impaired when compared to non-transgenic controls [1,13,29], we used those two tests to evaluate PSAPP mice following antibody administration. We also added the object recognition test as a non-spatial component in our memory tests. Sensorimotor tests were also performed using the balance beam and open field tests [1], as well as a grip strength test. The current paper details superior cognitive behavior following administration of anti-CD40L in aging PSAPP mice.
is important to note that the injection regimen was completed at 8 months. Therefore, the mice in the early pathology group did not receive anti-CD40L or IgG during the 8 – 15-month time period. As a passive antibody regimen was utilized and discontinued at 8 months, it is presumed that in the 7 months following the regimen any beneficial effects of the treatment was re-challenged by the transgene driven pathology. The late pathology group received a regimen of antiCD40L or IgG from 10 to 18 months and was then behaviorally tested at 18 months. The late pathology group consisted of 15 mice (anti-CD40L, 3U/4h; IgG, 4U/4h); however, two IgG-treated animals (1U/1h) failed to perform on the water maze task (i.e. constant floating or corkscrew swim path) and were not included in the data analyses, thus reducing the late pathology IgG group to six mice for the water maze. 2.2. Balance beam, grip strength and open field assessment Sensorimotor ability was assessed using a balance beam apparatus, consisting of a 57 cm L 1 cm D wooden (simple) or metal (difficult) dowels suspended above a padded surface. Mice were placed at one end of the beam and the latency to fall was recorded. A 60-s cutoff per trial was used and the mean of three trials was analyzed. Grip strength was assessed by determining the ability of a mouse to hang from an inverted wire cage. A 30-s cutoff was used and the mean of three trials was analyzed. General open field activity was assessed in a circular 1-m arena during a 10-min trial. Activity was assessed using EthoVision tracking software (Noldus Information Technology, Sterling, VA, USA). Distances traveled in the arena and in the 10-cm perimeter of the arena were analyzed. 2.3. Object recognition
2. Materials and methods 2.1. Mice PSAPP mice were bred by crossing TgAPPsw mice (2576 line [16]) crossed with PS1 M146L mice as described [15]. In the PSAPP mice, Ah deposition is detectable at 2– 3 months and is extensive throughout the hippocampus and cortex by 6 months [15,28]. Two groups of mice were utilized in this study, one receiving antiCD40L regimen commencing in the early stage of Ah pathology development (‘‘early pathology’’) and one receiving anti-CD40L regimen commencing in the late stage of Ah pathology development when Ah pathology is quite prominent (‘‘late pathology’’). The early pathology group received either anti-CD40L or isotype-matched control IgG antibody (IgG) from 2 to 8 months and was then behaviorally tested at 15 months. The early pathology group consisted of 12 mice (anti-CD40L, 2U/4h; IgG, 1U/5h). It
The object recognition test utilized a 10-min exposure trial and a 10-min retention trial, separated by a 1-h inter-trial interval (ITI). During the exposure trial, the arena contained two identical objects. During the retention trial, the arena contained a single copy of the previous object and a second novel object. We conducted the test in a circular 1-m field divided into equal hemi-circular arenas. Total time spent investigating each object, as defined as facing the object within 1.5 cm, was recorded during the trials. Normal mice remember a specific object over a delay up to 1 h and spend the majority of their time investigating the novel object during the retention trial [7,9]. A memory index (MI) was calculated using the following formula: [novel object investigation time/(total investigation time of both objects)*100]. 2.4. Morris water maze A mouse version of the Morris water maze [30] was used to assess spatial learning and memory. A 2-m pool, filled
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with opacified water (22j C), located in a cue rich room, was used. The pool contained a submerged 20-cm diameter platform, which remained in a constant location across acquisition trials. Mice were introduced to the pool from four locations and allowed 90 s to locate the hidden platform. Four consecutive acquisition trials (30-s ITI) were given per day for 9 days. At the end of days 3, 6 and 9, the platform was removed and a probe trial was administered to assess memory of the platform location. Swimming behavior was analyzed using EthoVision. The acquisition dependent measures were latency to the platform and path length. The probe dependent measures were path length in each pool quadrant and crosses over the platform location and over corresponding pseudo-platform locations in the nongoal quadrants. A platform preference index was calculated by subtracting the mean of the pseudo-platform crosses from the goal platform crosses.
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The time point for behavioral testing (15 or 18 months) was chosen based on studies reporting that memory impairments, specific to the tasks in this study, are present at approximately 14 months in PSAPP mice [1,13,26,29], concurring with our findings of no differences between the IgG and anti-CD40L treatment in the early pathology group when tested at 9 months. 2.7. Statistical analyses Comparisons between the IgG and anti-CD40L groups on single points (e.g. balance beam) were made using t-tests for independent samples. Comparisons across multiple time points (e.g. water maze acquisition) were made using repeated measures analysis of variance, followed by posthoc Tukey comparisons. When analyzing the object recognition task, one-sample t-tests with the test value set at 50 (chance performance) were utilized.
2.5. Radial arm water maze A mouse radial arm water maze [5,8] was used to assess working memory. The water maze was adapted by inserting dividers to create 6 25 75-cm arms radiating out from a 50cm diameter region. A submerged 20-cm diameter platform was located at the end of an arm. On each trial, mice were introduced to the pool from one of five non-goal arms and allowed 90 s to locate the platform. Four consecutive acquisition trials (30-s ITI) were administered each day, followed by a single retention trial (i.e. fifth trial, 30-min ITI). The platform remained in a constant location across all trials in a given day and was relocated at the beginning of each day to one of the arms not utilized the previous day. This testing sequence was conducted for 9 days. Entries into an arm not containing the platform were counted as errors and summed across each trial. Note: to reduce stress, mice were allowed to swim freely and were not returned to the start location following each error, as described by others [1]. 2.6. Behavioral testing sequence The early pathology group was first tested at 9 months of age on a smaller version (1-m pool) of the water maze task. No group differences were found at this 9-month time point (data not shown) and these animals were allowed to age 6 months (antibody regimen suspended) before the behavioral battery at 15 months of age. The sequence of testing at 15 months was (1) sensorimotor tasks, (2) open field, (3) water maze, (4) object recognition and (5) radial arm water maze. Animals received 1 – 2 days rest between sensorimotor, open field and water maze tests, and 1-week rest between water maze, object recognition and radial arm water maze tests. Mice in the late pathology group given antibody regimen from 10 to 18 months of age were naı¨ve to all test procedures at the 18-month test point. The test sequence was (1) sensorimotor ability, (2) open field exploration and (3) water maze.
3. Results 3.1. Early pathology group 3.1.1. Sensorimotor tasks (Table 1) No differences existed in balance beam ability between the anti-CD40L- and IgG-treated mice, t(10) = 0.315, p = 0.76. An easier task using a wooden beam was also administered to these animals and no differences were found between groups, t(10) = 0.770, p = 0.459, with the majority of animals remaining on the beam the entire test duration. Similarly, there was no difference in the grip strength assessment between anti-CD40L- and IgG-treated mice, t(10) = 0.148, p = 0.885. 3.1.2. Open field activity (Table 1) No differences were found in the distance traveled in the open field between mice treated with anti-CD40L or IgG when assessing total distance, t(10) = 0.739, p = 0.477. When the 10-min open field trial was examined across 2min time blocks, there was a significant reduction in activity for both groups as the trial progressed, F(4,40) = 3.734, p = 0.011, suggesting habituation learning across the trial. There were no differences in distance traveled across the time blocks between the anti-CD40L- and IgG-treated mice, F(1,10) = 0.546, p = 0.546, and there was not a significant time block by drug regimen interaction, F(4,40) = 0.481, p = 0.749. Mice treated with anti-CD40L traveled a significantly greater proportion of distance (% distance) in the perimeter of the open field than did IgG-treated mice, t(10) = 2.458, p = 0.034. When the proportion of distance traveled in the perimeter of the arena was analyzed across 2min time blocks, no differences were found across time, F(4,40) =0.486, p = 0.746, and no significant interaction between time block and drug regimen was present, F(4,40) = 0.456, p =0.768; however, the between subjects
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component of this analysis revealed a significant treatment difference, F(1,10) = 6.273, p = 0.031. 3.1.3. Water maze (Fig. 1) Water maze acquisition trial performance across the 9 test days was similar between anti-CD40L- and IgG-treated mice. Path length, F(8,80) = 11.041, p < 0.001 (see Fig. 1A), and latency to the platform, F(8,80) = 11.004, p < 0.001 (data not shown), were significantly reduced across training days, demonstrating the acquisition of spatial learning across the test trials. No differences between the two treatment groups was seen on either total path length, F(1,10) = 0.865, p = 0.374, or latency to the platform, F(1,10) = 1.936, p = 0.194, across the 9 test days. Consistent with sensorimotor testing, swimming ability did not differ between treatment groups, as their swimming speed was nearly identical, t(10) = 0.436, p = 0.672 (see Fig. 1B). On the third probe trial, both groups of mice exhibited a significantly greater than chance (25%) performance, swim-
Table 1 Sensorimotor ability, open field activity and object recognition dependent measures Prophylactic regimen
Balance beam (s) Simple condition Difficult condition Grip strength (s)
Treatment regimen
PSAPPIgG
PSAPP-anti- PSAPPCD40L IgG
55.7 (4.3) 28.8 (8.7) 12.9 (3.9)
59.1 (0.9) 30.5 (7.8) 12.1 (3.0)
11.6 (5.7) 8.8 (3.2)
Open field—distance in arena (cm) Total distance 6730 (389) 7328 (708) 10943 (1838) Distance 0 – 2 m 1329 (195) 1541 (234) 1939 (208) Distance 2 – 4 m 1487 (93) 1629 (141) 2480 (424) Distance 4 – 6 m 1395 (112) 1593 (191) 2240 (338) Distance 6 – 8 m 1213 (123) 1176 (54) 2180 (497) Distance 8 – 10 m 1305 (55) 1386 (157) 2105 (452) Open field—% distance in perimeter Total % distance 50.3 (5.7) 66.4 % distance 0 – 2 m 51.6 (10.1) 67.8 % distance 2 – 4 m 50.6 (5.9) 66.9 % distance 4 – 6 m 50.9 (6.0) 71.1 % distance 6 – 8 m 45.3 (5.0) 65.9 % distance 8 – 10 m 51.4 (3.4) 60.7
(3.2)a (4.2) (6.9) (4.4)a (7.1)a (5.0)
73.4 61.7 76.7 75.7 77.3 73.8
(2.9) (6.8) (3.6) (3.8) (4.2) (3.4)
PSAPP-antiCD40L
16.1 (5.9) 9.0 (3.5)
10624 1718 2428 2319 2175 1983
(2459) (275) (469) (657) (641) (499)
56.9 51.1 61.8 58.5 61.1 55.7
(7.0) (10.0) (7.9) (6.3) (6.9) (7.2)
Object recognition (s) Latency to 41.9 (23.7) 77.9 (35.3) first object Total inspection 21.3 (1.7) 28.3 (7.8) time-exposure trial Data are presented as mean F S.E.M. a Indicates significant increase compared to IgG-treated control.
Fig. 1. Water maze performance in 15-month PSAPP mice that were treated with anti-CD40L (n = 6) or control IgG (n = 6) from 2 to 8 months of age. Data are presented as mean F S.E.M. (A) Swim path length across nine blocks (4 trials/day/block) of acquisition training. There is no difference in acquisition performance between the two groups. (B) Swim speed across nine blocks of acquisition trials. Inset represents the mean across all trials. (C) Slope across the proportion of distance swam in the goal quadrant for the three probe trials. Positive slope indicates memory improvement for the platform location across probe trials. The anti-CD40L group had a greater slope across this memory measure than did the IgG group, t(10) = 3.835, p = 0.003.
ming further than 60% of their path length in the goal quadrant. However, there is a trend for the anti-CD40Ltreated mice to focus a greater proportion of their swim path in the quadrant previously containing the escape platform, t(10) = 1.876, p = 0.09 (data not shown). When examining the change in performance level across the three probe trials, an interesting effect emerged. To best assess the change in probe trial performance (memory retention), the slopes of the dependent measures across the three probe trials for each animal were calculated. Data from anti-CD40L-treated mice presented a positive slope value, indicating an increase in the proportion of distance swam in the goal quadrant across the three probe trials (see Fig. 1C). The slope of the proportion of distance swam in the goal quadrant was significantly higher in anti-CD40L-treated mice as compared to the control IgG-treated mice, t(10) = 3.835, p = 0.003. Similar significant effects were found for the other probe trial dependent measures (data not shown). 3.1.4. Object recognition (Fig. 2) No behavioral differences were evident between mice treated with anti-CD40L or IgG during the exposure trial of the object recognition task. The total time spent investigat-
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ing the two identical objects during the exposure trial did not differ, t(10) = 0.881, p = 0.399, nor did the latency to the first object investigation, t(10) = 0.846, p = 0.417 (see Table 1). On the retention trial of the object recognition task, mice treated with anti-CD40L showed strong memory retention for the previously presented object, spending a significantly greater than chance (50%) proportion of time investigating the novel object, t(5) = 4.233, p = 0.008, as compared to the IgG-treated mice, which investigated the novel and previously presented objects equally (chance performance), t(5) = 0.027, p = 0.979 (see Fig. 2). This test was only given to the early pathology group due to time constraints. 3.1.5. Radial arm water maze (Fig. 3) A difference between the two treatment groups was apparent on the final day (9th) of radial arm water maze (RAWM) testing. Mice treated with anti-CD40L show a significant reduction in the number of errors committed across the five trials of day 9, F(4,20) = 5.723, p = 0.003 (see Fig. 3A), whereas the error reduction, across the five trials, is marginal, F(4,20) = 2.463, p = 0.078 in the IgG-treated animals. When RAWM trials 2 –5 of day 9 are combined, the anti-CD40L-treated mice commit significantly fewer errors than did IgG-treated mice, t(10) = 2.583, p = 0.027 (see Fig. 3B). 3.2. Late pathology group 3.2.1. Sensorimotor tasks (Table 1) As in the early pathology regimen, no differences existed in balance beam walking ability, t(11) = 1.12, p = 0.286, or grip strength assessment, t(11) = 0.277, p = 0.714, between the anti-CD40L- and IgG-treated mice. 3.2.2. Open field activity (Table 1) No differences were found in the total distance traveled in the open field between mice treated with anti-CD40L or IgG
Fig. 2. Object recognition performance presented as mean ( F S.E.M.) memory index in 15-month-old PSAPP mice treated with anti-CD40L (n = 6) or control IgG (n = 6) from 2 to 8 months. The anti-CD40L group showed memory retention greater than chance performance (horizontal line), t(5) = 4.233, p = 0.008, whereas the IgG group showed no memory retention over the 1-h delay.
Fig. 3. Radial arm water maze performance in 15-month PSAPP mice treated with anti-CD40L (n = 6) or control IgG (n = 6) from 2 to 8 months of age. Data are presented as mean F S.E.M. (A) Errors committed across the 5 trials on the final day of radial arm water maze testing. The anti-CD40L group showed improvement across the five trials, F(4,20) = 5.723, p = 0.003, whereas the IgG-treated group shows no improvement, F(4,20) = 2.463, p = ns. (C) Errors committed across trials 2 – 5 on the final day of radial arm water maze testing. The anti-CD40L group committed fewer errors than the IgG group, t(10) = 2.583, p = 0.027.
antibody, t(11) = 0.893, p = 0.720. When the 10-min open field trial is examined across 2-min time blocks, no change in distance traveled was found across the five time blocks, F(4,44) = 1.688, p = 0.170. There were no differences in distance traveled across time blocks between the antiCD40L- and IgG-treated mice, F(1,11) = 0.135, p = 0.720, and a time block by drug regimen interaction did not exist, F(4,44) = 0.378, p = 0.823. Mice treated with anti-CD40L or IgG were similar in the total proportion of distance they traveled along the perimeter of the open field arena, t(11) = 1.870, p = 0.088, and in the proportion of distance traveled along the perimeter across 2-min time blocks, F(1,11) = 3.484, p = 0.089. 3.2.3. Water maze (Fig. 4) Acquisition trial performance was similar between antiCD40L- and IgG-treated mice. Path length, F(8,88) = 18.187, p < 0.001 (see Fig. 4A), and latency to the platform, F(8,88) = 20.844, p < 0.001 (data not shown), were significantly reduced across training days, demonstrating the acquisition of spatial learning across the test trials. No significant differences between the two treatment groups were seen on either total path length, F(1,11) = 0.521, p = 0.486, or latency to the platform, F(1,11) = 3.665, p = 0.082 across the 9 days of training; however, a separation between the two groups begins to appear on the final 2 days of
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memory retention probe trials, as compared to the control IgG regimen. Mice treated with anti-CD40L swam a greater proportion of their path length in the goal quadrant than did IgG-treated mice, when analyzed across the three probe trials, F(1,11) = 7.024, p = 0.023. Mice treated with antiCD40L also showed a significant increase in performance over the three probe trials, F(2,12) = 6.914, p = 0.01, whereas the performance of the IgG-treated animals did not significantly change over the three probe trials, F(2,10) = 1.807, p = 0.214 (see Fig. 4C). A similar pattern of effects was found using the platform preference index, as the antiCD40L group had a marginally higher platform preference index across the three probe trials, F(1,11) = 4.455, p = 0.059, and displayed significant improvement across the three probe trials, F(2,12) = 16.651, p < 0.001, while the IgG-treated group did not, F(2,10) = 3.241, p = 0.082 (see Fig. 4D). Taken together, our results show an improvement of cognitive abilities in PSAPP mice treated with antiCD40L versus IgG for both the early and the late pathology regimen.
4. Discussion
Fig. 4. Water maze performance in 18-month-old PSAPP mice treated with anti-CD40L (n = 6) or control IgG (n = 6) from 10 to 18 months of age. Data are presented as mean F S.E.M. (A) Swim path length across nine blocks (4 trials/day/block) of acquisition training. There was no difference in acquisition performance between the two groups. (B) Swim speed across nine blocks of acquisition trials. Inset represents mean swim speed across all trials. (C) Proportion of distance swam in goal quadrant for three probe trials. The anti-CD40L group swam further in the goal quadrant than did the IgG group, F(1,11) = 7.024, p = 0.023, and also showed improvement across the probe trials, F(2,12) = 6.914, p = 0.01, that was not apparent in the IgG group, F(2,10) = 1.807, p = ns. (D) Platform preference index for the three probe trials. The anti-CD40L group swam further in the goal quadrant, t(11) 2.53, p = 0.028, than did the IgG group, and also showed improvement across the probe trials, F(2,12) = 16.651, p < 0.001, that was not present in the IgG group, F(2,10) = 3.241, p = ns.
training, t(11) = 1.774, p = 0.104. Consistent with sensorimotor testing, swimming ability did not differ between treatment groups, as swim speed was nearly identical between the two treatment groups, t(11) = 0.563, p = 0.585 (see Fig. 4B). As in the early pathology regimen, the anti-CD40L late pathology regimen was associated with improvement on the
Paralleling the mitigation of AD pathology demonstrated by Tan et al. [37], mice treated with anti-CD40L from 2 to 8 months (early pathology regimen), and then behaviorally tested at 15 months of age, showed enhanced spatial and nonspatial reference and spatial working memory as compared to control IgG antibody treated mice. Similarly, mice treated with anti-CD40L from 10 to 18 months (late pathology regimen) showed superior spatial reference memory as compared to IgG-treated mice. During the open field test, although there was no significant difference in exploratory behavior when data were analyzed in 2-s time blocks, mice treated with anti-CD40L spent significantly more time in the perimeter than the IgG-treated ones, a behavior that is usually associated with anxiety [4]. Presumably, anxiety would cause the anti-CD40L-treated animals to perform poorly in the behavioral tasks used [3,32]; however, we saw the opposite. It is of note that the general sensorimotor ability and activity levels between all anti-CD40L and the IgG antibody treatment groups were similar, thus allowing cognitive behavioral interpretation of the differences on the physically dependent water maze and object recognition tasks. Water maze acquisition trial performance following the early pathology regimen of anti-CD40L was not different from that of the control IgG-treated mice. It is crucial to note that comparisons are made between two groups of treated (IgG or anti-CD40L) PSAPP mice and thus similar water maze acquisition performance does not suggest a lack of behavioral impairment at the ages tested. The purpose of this study was to determine the cognitive behavioral benefits of anti-CD40L in the PSAPP mouse, as it has previously been demonstrated that PSAPP mice show water maze and
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radial arm water maze impairments at 14 –15 months when compared to nontransgenic controls [1,13,29]. Although differences in the learning phase of the hidden platform water maze task were not present, differences in the memory retention phase (probe trials) were evident. This pattern of effects on the water maze is not unique; others have found strong differences on probe trial performance conducted at time points where acquisition trial performance was similar [21]. Differences were detected between the treatment groups by calculating the slope of probe trial performance across the three probe trials, thus quantifying the rate of change in probe trial performance across the test period. It was found that mice treated with anti-CD40L had substantial improvement in their reference memory over time, whereas the control IgG-treated showed no change in reference memory across the test period. The use of slope (rate of change) analyses is useful in elucidating fundamental changes in learning and memory that are not revealed by single time point analyses [20], such as across multiple water maze acquisition or probe trials. Superior reference memory in the anti-CD40L-treated mice was not limited to spatial reference memory. The early pathology group treated with anti-CD40L also showed superior non-spatial reference memory on a difficult (60-min ITI) object recognition task, as compared to the control IgG-treated mouse group. To assess working memory performance, the RAWM was included in the behavioral test battery. Previous studies have shown RAWM deficits in aged PSAPP mice compared to nontransgenic controls and have shown attenuation of this deficit following Ah antibody injection [1,29]. If the RAWM task is considered in terms of a match to sample task, it is crucial to evaluate the level of performance following the sample trial (trial 1). When the RAWM match trials of the final day (day 9) are averaged, it was found that the mean error rate of the anti-CD40L-treated mice was half that of the IgG-treated mice. This is a reflection of superior reference and working memory following anti-CD40L administration. Although the current protocol was slightly different, it is noteworthy that the errors committed on the 4th and fifth trials of day 9 were comparable to that of 15 – 17-month-old PSAPP mice reported elsewhere [1]. We have previously shown that a regimen of anti-CD40L will dramatically reduce accumulation of Ah in PSAPP mice brain mechanisms of increased clearance to the periphery [37]. The enhanced cognitive ability found in 15month-old mice treated with anti-CD40L from 2 to 8 months is impressive, given that at some point during the period from 8 to 15 months the accumulation of Ah returned to normal PSAPP levels due to the suspension of the antibody regimen. To achieve the maximal cognitive benefit, the early pathology anti-CD40L regimen should perhaps have continued until behavioral testing was completed. Future studies will determine the full extent of anti-CD40L’s beneficial effects upon cognitive behavior in an AD mouse model by continually administering anti-CD40L until an old age. The late pathology regimen experiment utilized the strategy of
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continually administering anti-CD40L until the beginning of behavioral testing; however, this regimen commenced at an age associated with the presence and increased rate of h amyloid deposition [15]. Thus, the late pathology regimen was an assessment of the efficacy of anti-CD40L to suspend or even reverse high-level Ah accumulation. The cognitive behavioral benefits of anti-CD40L were also evident following the late pathology regimen. Water maze probe trial performance data showed that the reference memory of the mice treated with anti-CD40L was superior to those treated with control IgG antibody. The anti-CD40L group outperformed the IgG group across all three probe trials. Also, as in the early pathology regimen, anti-CD40Ltreated mice showed significant development of reference memory, where IgG-treated mice showed no reference memory improvement. A growing body of research indicates that several types of treatment reduce amyloid-associated pathology and improve behavioral impairments in an in vivo AD mouse model : anti-CD40L treatment [36,37] and the current study, Ah immunization [2,10,12,19,22,29,34], non-steroidal antiinflammatory drugs [11,17,23,25,31] and antioxidants such as the curry spice curcumin [24]. Given the current limitation of therapeutic strategies for AD, it is of strong interest that CD40L interruption has such beneficial effects in an AD mouse model. The current study shows that this novel therapeutic approach, which results in the reduction of Ah burden, microglial activation, amyloidogenic APP processing and the increased clearance of Ah [37], is associated with beneficial cognitive effects. Acknowledgements We thank Omar Hammad and Maykel Jimenez for their technical assistance and we are grateful for the generous support of Diane and Robert Roskamp, which made this work possible. References [1] G.W. Arendash, D.L. King, M.N. Gordon, D. Morgan, J.M. Hatcher, C.E. Hope, D.M. Diamond, Progressive, age-related behavioral impairments in transgenic mice carrying both mutant amyloid precursor protein and presenilin-1 transgenes, Brain Res. 891 (2001) 42 – 53. [2] F. Bard, C. Cannon, R. Barbour, R.L. Burke, D. Games, H. Grajeda, T. Guido, K. Hu, J. Huang, K. Johnson-Wood, K. Khan, D. Kholodenko, M. Lee, I. Lieberburg, R. Motter, M. Nguyen, F. Soriano, N. Vasquez, K. Weiss, B. Welch, P. Seubert, D. Schenk, T. Yednock, Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease, Nat. Med. 6 (2000) 916 – 919. [3] G. Bongers, R. Leurs, J. Robertson, J. Raber, Role of H3-receptormediated signaling in anxiety and cognition in wild-type and Apoe / mice, Neuropsychopharmacology 29 (3) (2004 (Mar)) 441 – 449. [4] J. Buccafusco, Methods of behavior analysis in neuroscience, Methods and New Frontiers in Neuroscience, 1st edition, CRC Press, Boca Raton, FL, USA, 2001.
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Research report
Behavioral effects of CD40–CD40L pathway disruption in aged PSAPP mice J. Todd Roach a,b,1, Claude-Henry Volmar a,b,*,1, Samvid Dwivedi a,b, Terrence Town c, Robert Crescentini a,b, Fiona Crawford a,b, Jun Tan d, Michael Mullan a,b a
Roskamp Institute, 2040 Whitfield Avenue, Sarasota, FL 34243, USA b James A. Haley Veteran’s Hospital, Tampa, FL, USA c Yale University School of Medicine, New Haven, CT, USA d University of South Florida, Tampa, FL, USA Accepted 3 May 2004 Available online
Abstract We have shown that, when an amyloid-beta peptide (Ah) overproducing transgenic mouse model (PSAPP) of Alzheimer’s disease (AD) is treated with a depleting antibody against CD40L, it causes marked attenuation of Ah pathology associated with decreased amyloidogenic processing of amyloid precursor protein (APP) and increased cerebral clearance of Ah. Here, we report that, when PSAPP mice receive a regimen of anti-CD40L antibody commencing at an age associated with initial Ah deposition, they demonstrate superior spatial memory on the standard water maze and radial arm water maze tasks, as well as exhibiting superior non-spatial memory in the object recognition test, as compared to control PSAPP mice. Furthermore, PSAPP mice treated with an anti-CD40L antibody regimen commencing at an age associated with extensive Ah deposition demonstrate superior spatial memory on the standard water maze task, as compared to control PSAPP mice. Disruption of CD40L activity has beneficial effects on pathology and cognitive behavior in the PSAPP mouse model, providing support for the therapeutic potential of interrupting the CD40 – CD40L interaction in AD. D 2004 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Degenerative disease: Alzheimer’s—beta amyloid Keywords: Alzheimer’s disease; CD40; Behavior; Transgenic; Mouse; Amyloid precursor protein
1. Introduction Alzheimer’s disease (AD) is the most prevalent form of progressive dementia and is characterized by the accumulation of amyloid beta (Ah) peptide, which is cleaved from the amyloid precursor protein (APP) by h and g secretases [35]. Animal models of AD are available that overproduce and accumulate Ah [18] and such models are key scientific tools in furthering our understanding of AD. Accompanying Ah accumulation, both clinically and in AD mouse models, is an inflammatory cascade in and around Ah aggregates [27,33,35]. This inflammatory reaction may be central to the * Corresponding author. Tel.: +1-941-752-2949; fax: +1-941-752-2948. E-mail address: [email protected] (C.-H. Volmar). 1 The first two authors contributed equally to this work. 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.05.004
development of AD, as clinically, individuals with Ah accumulation but no inflammatory response, do not show clinical expression of AD [33]. We have previously suggested that the costimulatory molecules CD40 and CD40L may play important roles in regulating the microglial response to accumulating Ah [36 –38]. CD40 is a member of the tumor necrosis factor/nerve growth factor receptor superfamily and its natural ligand CD40L is a key immunoregulatory molecule that is a co-stimulator of cells from both the innate and specific arms of the immune system [14,40]. Microglia up-regulate CD40 after Ah challenge and microglial activation induced by nanomolar levels of Ah requires CD40 –CD40L interaction in vitro [36]. It has been reported that CD40 is upregulated in AD patient brains on reactive microglia and in senile plaques, supporting its role in the AD neuroinflammatory processes [6,39]. Work by
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Tan et al. [36] has shown that increased tumor necrosis factor alpha production and induction of neuronal injury occur when Ah-stimulated microglia are treated with CD40L. An Ah overproducing mouse model (APPsw) deficient for CD40L displays reduced microglial activation, suggesting the CD40 –CD40L interaction is necessary for Ah-induced microglial activation [37]. APPsw mice deficient for CD40L also have reduced hyper-phosphorylation of H and show a significant reduction ( f 80%) in Ah burden compared to APPsw mice [37]. Tan et al. conducted further experiments to elucidate possible mechanism(s) of Ah reduction. Subsequent experiments in which doubly transgenic AD mice (PSAPP) were treated with an antiCD40L antibody (anti-CD40L) from 2 to 8 months revealed attenuation ( f 65%) of Ah accumulation associated with increased plasma levels of Ah and increased nonamyloidogenic APP processing [37]. These studies collectively show that reducing CD40L in an Ah overproducing mouse model results in an attenuation of Ah pathology, likely via multiple mechanisms. This attenuation of AD related pathology naturally led to a study assessing the behavioral effects of CD40L disruption in an AD mouse model. We accomplish this by using a battery of validated behavioral tests to compare IgG-treated versus anti CD40L-treated PSAPP mice. Since others have shown, using the Morris water maze and the radial arm water maze spatial memory tests, that PSAPP mice are cognitively impaired when compared to non-transgenic controls [1,13,29], we used those two tests to evaluate PSAPP mice following antibody administration. We also added the object recognition test as a non-spatial component in our memory tests. Sensorimotor tests were also performed using the balance beam and open field tests [1], as well as a grip strength test. The current paper details superior cognitive behavior following administration of anti-CD40L in aging PSAPP mice.
is important to note that the injection regimen was completed at 8 months. Therefore, the mice in the early pathology group did not receive anti-CD40L or IgG during the 8 – 15-month time period. As a passive antibody regimen was utilized and discontinued at 8 months, it is presumed that in the 7 months following the regimen any beneficial effects of the treatment was re-challenged by the transgene driven pathology. The late pathology group received a regimen of antiCD40L or IgG from 10 to 18 months and was then behaviorally tested at 18 months. The late pathology group consisted of 15 mice (anti-CD40L, 3U/4h; IgG, 4U/4h); however, two IgG-treated animals (1U/1h) failed to perform on the water maze task (i.e. constant floating or corkscrew swim path) and were not included in the data analyses, thus reducing the late pathology IgG group to six mice for the water maze. 2.2. Balance beam, grip strength and open field assessment Sensorimotor ability was assessed using a balance beam apparatus, consisting of a 57 cm L 1 cm D wooden (simple) or metal (difficult) dowels suspended above a padded surface. Mice were placed at one end of the beam and the latency to fall was recorded. A 60-s cutoff per trial was used and the mean of three trials was analyzed. Grip strength was assessed by determining the ability of a mouse to hang from an inverted wire cage. A 30-s cutoff was used and the mean of three trials was analyzed. General open field activity was assessed in a circular 1-m arena during a 10-min trial. Activity was assessed using EthoVision tracking software (Noldus Information Technology, Sterling, VA, USA). Distances traveled in the arena and in the 10-cm perimeter of the arena were analyzed. 2.3. Object recognition
2. Materials and methods 2.1. Mice PSAPP mice were bred by crossing TgAPPsw mice (2576 line [16]) crossed with PS1 M146L mice as described [15]. In the PSAPP mice, Ah deposition is detectable at 2– 3 months and is extensive throughout the hippocampus and cortex by 6 months [15,28]. Two groups of mice were utilized in this study, one receiving antiCD40L regimen commencing in the early stage of Ah pathology development (‘‘early pathology’’) and one receiving anti-CD40L regimen commencing in the late stage of Ah pathology development when Ah pathology is quite prominent (‘‘late pathology’’). The early pathology group received either anti-CD40L or isotype-matched control IgG antibody (IgG) from 2 to 8 months and was then behaviorally tested at 15 months. The early pathology group consisted of 12 mice (anti-CD40L, 2U/4h; IgG, 1U/5h). It
The object recognition test utilized a 10-min exposure trial and a 10-min retention trial, separated by a 1-h inter-trial interval (ITI). During the exposure trial, the arena contained two identical objects. During the retention trial, the arena contained a single copy of the previous object and a second novel object. We conducted the test in a circular 1-m field divided into equal hemi-circular arenas. Total time spent investigating each object, as defined as facing the object within 1.5 cm, was recorded during the trials. Normal mice remember a specific object over a delay up to 1 h and spend the majority of their time investigating the novel object during the retention trial [7,9]. A memory index (MI) was calculated using the following formula: [novel object investigation time/(total investigation time of both objects)*100]. 2.4. Morris water maze A mouse version of the Morris water maze [30] was used to assess spatial learning and memory. A 2-m pool, filled
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with opacified water (22j C), located in a cue rich room, was used. The pool contained a submerged 20-cm diameter platform, which remained in a constant location across acquisition trials. Mice were introduced to the pool from four locations and allowed 90 s to locate the hidden platform. Four consecutive acquisition trials (30-s ITI) were given per day for 9 days. At the end of days 3, 6 and 9, the platform was removed and a probe trial was administered to assess memory of the platform location. Swimming behavior was analyzed using EthoVision. The acquisition dependent measures were latency to the platform and path length. The probe dependent measures were path length in each pool quadrant and crosses over the platform location and over corresponding pseudo-platform locations in the nongoal quadrants. A platform preference index was calculated by subtracting the mean of the pseudo-platform crosses from the goal platform crosses.
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The time point for behavioral testing (15 or 18 months) was chosen based on studies reporting that memory impairments, specific to the tasks in this study, are present at approximately 14 months in PSAPP mice [1,13,26,29], concurring with our findings of no differences between the IgG and anti-CD40L treatment in the early pathology group when tested at 9 months. 2.7. Statistical analyses Comparisons between the IgG and anti-CD40L groups on single points (e.g. balance beam) were made using t-tests for independent samples. Comparisons across multiple time points (e.g. water maze acquisition) were made using repeated measures analysis of variance, followed by posthoc Tukey comparisons. When analyzing the object recognition task, one-sample t-tests with the test value set at 50 (chance performance) were utilized.
2.5. Radial arm water maze A mouse radial arm water maze [5,8] was used to assess working memory. The water maze was adapted by inserting dividers to create 6 25 75-cm arms radiating out from a 50cm diameter region. A submerged 20-cm diameter platform was located at the end of an arm. On each trial, mice were introduced to the pool from one of five non-goal arms and allowed 90 s to locate the platform. Four consecutive acquisition trials (30-s ITI) were administered each day, followed by a single retention trial (i.e. fifth trial, 30-min ITI). The platform remained in a constant location across all trials in a given day and was relocated at the beginning of each day to one of the arms not utilized the previous day. This testing sequence was conducted for 9 days. Entries into an arm not containing the platform were counted as errors and summed across each trial. Note: to reduce stress, mice were allowed to swim freely and were not returned to the start location following each error, as described by others [1]. 2.6. Behavioral testing sequence The early pathology group was first tested at 9 months of age on a smaller version (1-m pool) of the water maze task. No group differences were found at this 9-month time point (data not shown) and these animals were allowed to age 6 months (antibody regimen suspended) before the behavioral battery at 15 months of age. The sequence of testing at 15 months was (1) sensorimotor tasks, (2) open field, (3) water maze, (4) object recognition and (5) radial arm water maze. Animals received 1 – 2 days rest between sensorimotor, open field and water maze tests, and 1-week rest between water maze, object recognition and radial arm water maze tests. Mice in the late pathology group given antibody regimen from 10 to 18 months of age were naı¨ve to all test procedures at the 18-month test point. The test sequence was (1) sensorimotor ability, (2) open field exploration and (3) water maze.
3. Results 3.1. Early pathology group 3.1.1. Sensorimotor tasks (Table 1) No differences existed in balance beam ability between the anti-CD40L- and IgG-treated mice, t(10) = 0.315, p = 0.76. An easier task using a wooden beam was also administered to these animals and no differences were found between groups, t(10) = 0.770, p = 0.459, with the majority of animals remaining on the beam the entire test duration. Similarly, there was no difference in the grip strength assessment between anti-CD40L- and IgG-treated mice, t(10) = 0.148, p = 0.885. 3.1.2. Open field activity (Table 1) No differences were found in the distance traveled in the open field between mice treated with anti-CD40L or IgG when assessing total distance, t(10) = 0.739, p = 0.477. When the 10-min open field trial was examined across 2min time blocks, there was a significant reduction in activity for both groups as the trial progressed, F(4,40) = 3.734, p = 0.011, suggesting habituation learning across the trial. There were no differences in distance traveled across the time blocks between the anti-CD40L- and IgG-treated mice, F(1,10) = 0.546, p = 0.546, and there was not a significant time block by drug regimen interaction, F(4,40) = 0.481, p = 0.749. Mice treated with anti-CD40L traveled a significantly greater proportion of distance (% distance) in the perimeter of the open field than did IgG-treated mice, t(10) = 2.458, p = 0.034. When the proportion of distance traveled in the perimeter of the arena was analyzed across 2min time blocks, no differences were found across time, F(4,40) =0.486, p = 0.746, and no significant interaction between time block and drug regimen was present, F(4,40) = 0.456, p =0.768; however, the between subjects
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component of this analysis revealed a significant treatment difference, F(1,10) = 6.273, p = 0.031. 3.1.3. Water maze (Fig. 1) Water maze acquisition trial performance across the 9 test days was similar between anti-CD40L- and IgG-treated mice. Path length, F(8,80) = 11.041, p < 0.001 (see Fig. 1A), and latency to the platform, F(8,80) = 11.004, p < 0.001 (data not shown), were significantly reduced across training days, demonstrating the acquisition of spatial learning across the test trials. No differences between the two treatment groups was seen on either total path length, F(1,10) = 0.865, p = 0.374, or latency to the platform, F(1,10) = 1.936, p = 0.194, across the 9 test days. Consistent with sensorimotor testing, swimming ability did not differ between treatment groups, as their swimming speed was nearly identical, t(10) = 0.436, p = 0.672 (see Fig. 1B). On the third probe trial, both groups of mice exhibited a significantly greater than chance (25%) performance, swim-
Table 1 Sensorimotor ability, open field activity and object recognition dependent measures Prophylactic regimen
Balance beam (s) Simple condition Difficult condition Grip strength (s)
Treatment regimen
PSAPPIgG
PSAPP-anti- PSAPPCD40L IgG
55.7 (4.3) 28.8 (8.7) 12.9 (3.9)
59.1 (0.9) 30.5 (7.8) 12.1 (3.0)
11.6 (5.7) 8.8 (3.2)
Open field—distance in arena (cm) Total distance 6730 (389) 7328 (708) 10943 (1838) Distance 0 – 2 m 1329 (195) 1541 (234) 1939 (208) Distance 2 – 4 m 1487 (93) 1629 (141) 2480 (424) Distance 4 – 6 m 1395 (112) 1593 (191) 2240 (338) Distance 6 – 8 m 1213 (123) 1176 (54) 2180 (497) Distance 8 – 10 m 1305 (55) 1386 (157) 2105 (452) Open field—% distance in perimeter Total % distance 50.3 (5.7) 66.4 % distance 0 – 2 m 51.6 (10.1) 67.8 % distance 2 – 4 m 50.6 (5.9) 66.9 % distance 4 – 6 m 50.9 (6.0) 71.1 % distance 6 – 8 m 45.3 (5.0) 65.9 % distance 8 – 10 m 51.4 (3.4) 60.7
(3.2)a (4.2) (6.9) (4.4)a (7.1)a (5.0)
73.4 61.7 76.7 75.7 77.3 73.8
(2.9) (6.8) (3.6) (3.8) (4.2) (3.4)
PSAPP-antiCD40L
16.1 (5.9) 9.0 (3.5)
10624 1718 2428 2319 2175 1983
(2459) (275) (469) (657) (641) (499)
56.9 51.1 61.8 58.5 61.1 55.7
(7.0) (10.0) (7.9) (6.3) (6.9) (7.2)
Object recognition (s) Latency to 41.9 (23.7) 77.9 (35.3) first object Total inspection 21.3 (1.7) 28.3 (7.8) time-exposure trial Data are presented as mean F S.E.M. a Indicates significant increase compared to IgG-treated control.
Fig. 1. Water maze performance in 15-month PSAPP mice that were treated with anti-CD40L (n = 6) or control IgG (n = 6) from 2 to 8 months of age. Data are presented as mean F S.E.M. (A) Swim path length across nine blocks (4 trials/day/block) of acquisition training. There is no difference in acquisition performance between the two groups. (B) Swim speed across nine blocks of acquisition trials. Inset represents the mean across all trials. (C) Slope across the proportion of distance swam in the goal quadrant for the three probe trials. Positive slope indicates memory improvement for the platform location across probe trials. The anti-CD40L group had a greater slope across this memory measure than did the IgG group, t(10) = 3.835, p = 0.003.
ming further than 60% of their path length in the goal quadrant. However, there is a trend for the anti-CD40Ltreated mice to focus a greater proportion of their swim path in the quadrant previously containing the escape platform, t(10) = 1.876, p = 0.09 (data not shown). When examining the change in performance level across the three probe trials, an interesting effect emerged. To best assess the change in probe trial performance (memory retention), the slopes of the dependent measures across the three probe trials for each animal were calculated. Data from anti-CD40L-treated mice presented a positive slope value, indicating an increase in the proportion of distance swam in the goal quadrant across the three probe trials (see Fig. 1C). The slope of the proportion of distance swam in the goal quadrant was significantly higher in anti-CD40L-treated mice as compared to the control IgG-treated mice, t(10) = 3.835, p = 0.003. Similar significant effects were found for the other probe trial dependent measures (data not shown). 3.1.4. Object recognition (Fig. 2) No behavioral differences were evident between mice treated with anti-CD40L or IgG during the exposure trial of the object recognition task. The total time spent investigat-
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ing the two identical objects during the exposure trial did not differ, t(10) = 0.881, p = 0.399, nor did the latency to the first object investigation, t(10) = 0.846, p = 0.417 (see Table 1). On the retention trial of the object recognition task, mice treated with anti-CD40L showed strong memory retention for the previously presented object, spending a significantly greater than chance (50%) proportion of time investigating the novel object, t(5) = 4.233, p = 0.008, as compared to the IgG-treated mice, which investigated the novel and previously presented objects equally (chance performance), t(5) = 0.027, p = 0.979 (see Fig. 2). This test was only given to the early pathology group due to time constraints. 3.1.5. Radial arm water maze (Fig. 3) A difference between the two treatment groups was apparent on the final day (9th) of radial arm water maze (RAWM) testing. Mice treated with anti-CD40L show a significant reduction in the number of errors committed across the five trials of day 9, F(4,20) = 5.723, p = 0.003 (see Fig. 3A), whereas the error reduction, across the five trials, is marginal, F(4,20) = 2.463, p = 0.078 in the IgG-treated animals. When RAWM trials 2 –5 of day 9 are combined, the anti-CD40L-treated mice commit significantly fewer errors than did IgG-treated mice, t(10) = 2.583, p = 0.027 (see Fig. 3B). 3.2. Late pathology group 3.2.1. Sensorimotor tasks (Table 1) As in the early pathology regimen, no differences existed in balance beam walking ability, t(11) = 1.12, p = 0.286, or grip strength assessment, t(11) = 0.277, p = 0.714, between the anti-CD40L- and IgG-treated mice. 3.2.2. Open field activity (Table 1) No differences were found in the total distance traveled in the open field between mice treated with anti-CD40L or IgG
Fig. 2. Object recognition performance presented as mean ( F S.E.M.) memory index in 15-month-old PSAPP mice treated with anti-CD40L (n = 6) or control IgG (n = 6) from 2 to 8 months. The anti-CD40L group showed memory retention greater than chance performance (horizontal line), t(5) = 4.233, p = 0.008, whereas the IgG group showed no memory retention over the 1-h delay.
Fig. 3. Radial arm water maze performance in 15-month PSAPP mice treated with anti-CD40L (n = 6) or control IgG (n = 6) from 2 to 8 months of age. Data are presented as mean F S.E.M. (A) Errors committed across the 5 trials on the final day of radial arm water maze testing. The anti-CD40L group showed improvement across the five trials, F(4,20) = 5.723, p = 0.003, whereas the IgG-treated group shows no improvement, F(4,20) = 2.463, p = ns. (C) Errors committed across trials 2 – 5 on the final day of radial arm water maze testing. The anti-CD40L group committed fewer errors than the IgG group, t(10) = 2.583, p = 0.027.
antibody, t(11) = 0.893, p = 0.720. When the 10-min open field trial is examined across 2-min time blocks, no change in distance traveled was found across the five time blocks, F(4,44) = 1.688, p = 0.170. There were no differences in distance traveled across time blocks between the antiCD40L- and IgG-treated mice, F(1,11) = 0.135, p = 0.720, and a time block by drug regimen interaction did not exist, F(4,44) = 0.378, p = 0.823. Mice treated with anti-CD40L or IgG were similar in the total proportion of distance they traveled along the perimeter of the open field arena, t(11) = 1.870, p = 0.088, and in the proportion of distance traveled along the perimeter across 2-min time blocks, F(1,11) = 3.484, p = 0.089. 3.2.3. Water maze (Fig. 4) Acquisition trial performance was similar between antiCD40L- and IgG-treated mice. Path length, F(8,88) = 18.187, p < 0.001 (see Fig. 4A), and latency to the platform, F(8,88) = 20.844, p < 0.001 (data not shown), were significantly reduced across training days, demonstrating the acquisition of spatial learning across the test trials. No significant differences between the two treatment groups were seen on either total path length, F(1,11) = 0.521, p = 0.486, or latency to the platform, F(1,11) = 3.665, p = 0.082 across the 9 days of training; however, a separation between the two groups begins to appear on the final 2 days of
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memory retention probe trials, as compared to the control IgG regimen. Mice treated with anti-CD40L swam a greater proportion of their path length in the goal quadrant than did IgG-treated mice, when analyzed across the three probe trials, F(1,11) = 7.024, p = 0.023. Mice treated with antiCD40L also showed a significant increase in performance over the three probe trials, F(2,12) = 6.914, p = 0.01, whereas the performance of the IgG-treated animals did not significantly change over the three probe trials, F(2,10) = 1.807, p = 0.214 (see Fig. 4C). A similar pattern of effects was found using the platform preference index, as the antiCD40L group had a marginally higher platform preference index across the three probe trials, F(1,11) = 4.455, p = 0.059, and displayed significant improvement across the three probe trials, F(2,12) = 16.651, p < 0.001, while the IgG-treated group did not, F(2,10) = 3.241, p = 0.082 (see Fig. 4D). Taken together, our results show an improvement of cognitive abilities in PSAPP mice treated with antiCD40L versus IgG for both the early and the late pathology regimen.
4. Discussion
Fig. 4. Water maze performance in 18-month-old PSAPP mice treated with anti-CD40L (n = 6) or control IgG (n = 6) from 10 to 18 months of age. Data are presented as mean F S.E.M. (A) Swim path length across nine blocks (4 trials/day/block) of acquisition training. There was no difference in acquisition performance between the two groups. (B) Swim speed across nine blocks of acquisition trials. Inset represents mean swim speed across all trials. (C) Proportion of distance swam in goal quadrant for three probe trials. The anti-CD40L group swam further in the goal quadrant than did the IgG group, F(1,11) = 7.024, p = 0.023, and also showed improvement across the probe trials, F(2,12) = 6.914, p = 0.01, that was not apparent in the IgG group, F(2,10) = 1.807, p = ns. (D) Platform preference index for the three probe trials. The anti-CD40L group swam further in the goal quadrant, t(11) 2.53, p = 0.028, than did the IgG group, and also showed improvement across the probe trials, F(2,12) = 16.651, p < 0.001, that was not present in the IgG group, F(2,10) = 3.241, p = ns.
training, t(11) = 1.774, p = 0.104. Consistent with sensorimotor testing, swimming ability did not differ between treatment groups, as swim speed was nearly identical between the two treatment groups, t(11) = 0.563, p = 0.585 (see Fig. 4B). As in the early pathology regimen, the anti-CD40L late pathology regimen was associated with improvement on the
Paralleling the mitigation of AD pathology demonstrated by Tan et al. [37], mice treated with anti-CD40L from 2 to 8 months (early pathology regimen), and then behaviorally tested at 15 months of age, showed enhanced spatial and nonspatial reference and spatial working memory as compared to control IgG antibody treated mice. Similarly, mice treated with anti-CD40L from 10 to 18 months (late pathology regimen) showed superior spatial reference memory as compared to IgG-treated mice. During the open field test, although there was no significant difference in exploratory behavior when data were analyzed in 2-s time blocks, mice treated with anti-CD40L spent significantly more time in the perimeter than the IgG-treated ones, a behavior that is usually associated with anxiety [4]. Presumably, anxiety would cause the anti-CD40L-treated animals to perform poorly in the behavioral tasks used [3,32]; however, we saw the opposite. It is of note that the general sensorimotor ability and activity levels between all anti-CD40L and the IgG antibody treatment groups were similar, thus allowing cognitive behavioral interpretation of the differences on the physically dependent water maze and object recognition tasks. Water maze acquisition trial performance following the early pathology regimen of anti-CD40L was not different from that of the control IgG-treated mice. It is crucial to note that comparisons are made between two groups of treated (IgG or anti-CD40L) PSAPP mice and thus similar water maze acquisition performance does not suggest a lack of behavioral impairment at the ages tested. The purpose of this study was to determine the cognitive behavioral benefits of anti-CD40L in the PSAPP mouse, as it has previously been demonstrated that PSAPP mice show water maze and
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radial arm water maze impairments at 14 –15 months when compared to nontransgenic controls [1,13,29]. Although differences in the learning phase of the hidden platform water maze task were not present, differences in the memory retention phase (probe trials) were evident. This pattern of effects on the water maze is not unique; others have found strong differences on probe trial performance conducted at time points where acquisition trial performance was similar [21]. Differences were detected between the treatment groups by calculating the slope of probe trial performance across the three probe trials, thus quantifying the rate of change in probe trial performance across the test period. It was found that mice treated with anti-CD40L had substantial improvement in their reference memory over time, whereas the control IgG-treated showed no change in reference memory across the test period. The use of slope (rate of change) analyses is useful in elucidating fundamental changes in learning and memory that are not revealed by single time point analyses [20], such as across multiple water maze acquisition or probe trials. Superior reference memory in the anti-CD40L-treated mice was not limited to spatial reference memory. The early pathology group treated with anti-CD40L also showed superior non-spatial reference memory on a difficult (60-min ITI) object recognition task, as compared to the control IgG-treated mouse group. To assess working memory performance, the RAWM was included in the behavioral test battery. Previous studies have shown RAWM deficits in aged PSAPP mice compared to nontransgenic controls and have shown attenuation of this deficit following Ah antibody injection [1,29]. If the RAWM task is considered in terms of a match to sample task, it is crucial to evaluate the level of performance following the sample trial (trial 1). When the RAWM match trials of the final day (day 9) are averaged, it was found that the mean error rate of the anti-CD40L-treated mice was half that of the IgG-treated mice. This is a reflection of superior reference and working memory following anti-CD40L administration. Although the current protocol was slightly different, it is noteworthy that the errors committed on the 4th and fifth trials of day 9 were comparable to that of 15 – 17-month-old PSAPP mice reported elsewhere [1]. We have previously shown that a regimen of anti-CD40L will dramatically reduce accumulation of Ah in PSAPP mice brain mechanisms of increased clearance to the periphery [37]. The enhanced cognitive ability found in 15month-old mice treated with anti-CD40L from 2 to 8 months is impressive, given that at some point during the period from 8 to 15 months the accumulation of Ah returned to normal PSAPP levels due to the suspension of the antibody regimen. To achieve the maximal cognitive benefit, the early pathology anti-CD40L regimen should perhaps have continued until behavioral testing was completed. Future studies will determine the full extent of anti-CD40L’s beneficial effects upon cognitive behavior in an AD mouse model by continually administering anti-CD40L until an old age. The late pathology regimen experiment utilized the strategy of
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continually administering anti-CD40L until the beginning of behavioral testing; however, this regimen commenced at an age associated with the presence and increased rate of h amyloid deposition [15]. Thus, the late pathology regimen was an assessment of the efficacy of anti-CD40L to suspend or even reverse high-level Ah accumulation. The cognitive behavioral benefits of anti-CD40L were also evident following the late pathology regimen. Water maze probe trial performance data showed that the reference memory of the mice treated with anti-CD40L was superior to those treated with control IgG antibody. The anti-CD40L group outperformed the IgG group across all three probe trials. Also, as in the early pathology regimen, anti-CD40Ltreated mice showed significant development of reference memory, where IgG-treated mice showed no reference memory improvement. A growing body of research indicates that several types of treatment reduce amyloid-associated pathology and improve behavioral impairments in an in vivo AD mouse model : anti-CD40L treatment [36,37] and the current study, Ah immunization [2,10,12,19,22,29,34], non-steroidal antiinflammatory drugs [11,17,23,25,31] and antioxidants such as the curry spice curcumin [24]. Given the current limitation of therapeutic strategies for AD, it is of strong interest that CD40L interruption has such beneficial effects in an AD mouse model. The current study shows that this novel therapeutic approach, which results in the reduction of Ah burden, microglial activation, amyloidogenic APP processing and the increased clearance of Ah [37], is associated with beneficial cognitive effects. Acknowledgements We thank Omar Hammad and Maykel Jimenez for their technical assistance and we are grateful for the generous support of Diane and Robert Roskamp, which made this work possible. References [1] G.W. Arendash, D.L. King, M.N. Gordon, D. Morgan, J.M. Hatcher, C.E. Hope, D.M. Diamond, Progressive, age-related behavioral impairments in transgenic mice carrying both mutant amyloid precursor protein and presenilin-1 transgenes, Brain Res. 891 (2001) 42 – 53. [2] F. Bard, C. Cannon, R. Barbour, R.L. Burke, D. Games, H. Grajeda, T. Guido, K. Hu, J. Huang, K. Johnson-Wood, K. Khan, D. Kholodenko, M. Lee, I. Lieberburg, R. Motter, M. Nguyen, F. Soriano, N. Vasquez, K. Weiss, B. Welch, P. Seubert, D. Schenk, T. Yednock, Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease, Nat. Med. 6 (2000) 916 – 919. [3] G. Bongers, R. Leurs, J. Robertson, J. Raber, Role of H3-receptormediated signaling in anxiety and cognition in wild-type and Apoe / mice, Neuropsychopharmacology 29 (3) (2004 (Mar)) 441 – 449. [4] J. Buccafusco, Methods of behavior analysis in neuroscience, Methods and New Frontiers in Neuroscience, 1st edition, CRC Press, Boca Raton, FL, USA, 2001.
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