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- Materials and methods
The ε4 allele of apolipoprotein E (apoE4) is the predominant genetic risk factor for late-onset Alzheimer's disease (AD) and is also implicated in cognitive deficits associated with normal aging. The biological mechanisms by which APOE genotype affects cognitive processes or AD pathogenesis remain unclear, but interactions of apoE with amyloid β peptide (Aβ) are thought to play an important role in mediating apoE's isoform-specific effects on brain function. Here, we investigated the potential isoform-dependent effects of apoE on behavioral and cognitive performance in human apoE3 and apoE4 targeted-replacement (TR) mice that also overexpress the human amyloid precursor protein (APP). Beginning at 6–7 months of age, female APP-Yac/apoE3-TR (‘poE3’) and APP-Yac/apoE4-TR (‘poE4’) mice were tested on a battery of tests to evaluate basic sensorimotor functioning, spatial working memory, spatial recognition, episodic-like memory and attentional processing. Compared with apoE3 mice, a generalized reduction in locomotor activity was observed in apoE4 mice. Moderate, but significant, cognitive impairments were also detected in apoE4 mice in the novel object-location preference task, the contextual fear conditioning test, and a two-choice visual discrimination/detection test, however spontaneous alternation performance in the Y-maze was spared. These results offer additional support for the negative impact of apoE4 on both memory and attention and further suggest that APP-Yac/apoE-TR mice provide a novel and useful model for investigating the role of apoE in mediating susceptibility to cognitive decline.
Initial investigations of transgenic mouse models in which expression of human apoE isoforms were under control of either the neuron-specific-enolase (NSE) or glial fibrillary acidic protein (GFAP) promoters have emphasized the differential effects of human apoE isoforms on brain function. Both NSE-apoE4 and GFAP-apoE4 mice were impaired in learning a water maze task, with deficits being more pronounced in female animals and exacerbated with increasing age (Hartman et al. 2001; Raber et al. 1998; van Meer et al. 2007). These results suggest that human apoE4 has negative effects on spatial memory processing in mice independent of its cellular source in the brain. More recent studies with human apoE targeted-replacement (TR) mice, which express apoE in a temporal and spatial pattern similar to wild-type mice (Sullivan et al. 2004), have supported these earlier findings as age- and gender-dependent impairments have also been reported in apoE4-TR mice on a range of spatial learning and memory tasks (Bour et al. 2008; Grootendorst et al. 2005).
Mounting evidence points to interactions of apoE and amyloid β peptide (Aβ) as important modulators of the pathological events associated with the amyloid cascade (Brendza et al. 2002; Manelli et al. 2004; Sadowski et al. 2006). As such, earlier groups have examined the effects of human apoE in transgenic mice expressing a mutant form of the human amyloid precursor protein (APP) (e.g. Fagan et al. 2002; Holtzman et al. 1999), but to date there has been no study of the effects of apoE status on cognitive functions in mouse models expressing the nonmutated form of h-APP characteristic of most cases of late-onset AD. While mice that slightly overexpress wild-type human APP exhibit normal neuromotor and cognitive function up to 15 months of age (Murai et al. 1998), we investigated whether the isoform-specific effects of h-apoE on behavioral performance would be exacerbated in these genomic h-APP mice. Thus, neurological functions of adult h-APP mice with TR of apoE3 or apoE4 were assessed in the grip strength, rotarod, open-field and home-cage activity tests. In the same mice, learning and memory abilities were evaluated in the Y-maze spontaneous alternation, contextual fear conditioning, and novel object-location preference tasks; sustained attention was assessed in a two-choice visual discrimination task.
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- Materials and methods
This is the first report of isoform-specific effects of human apoE on both cognitive and noncognitive behavioral measures in an animal model that also expresses nonmutated, genomic human APP. We report that ‘poE4’ female mice show impairments in contextual fear memory and spatial recognition memory, reduced attentional capacity and slower reaction times, as well as a generalized decrease in activity levels as compared with ‘poE3’ mice.
Prior to characterization on the cognitive tasks, ‘poE3’ and ‘poE4’ mice were first assessed for their gross neurological functions, as sensorimotor anomalies can confound behavioral analysis (Crawley 1999). Whereas ‘apoE4’ mice were slightly lighter than ‘apoE3’ mice, there were no differences between the two lines in terms of grip strength or latency to fall from the rotarod, nor were any ataxic or otherwise abnormal neurologic phenotypes observed throughout the entire test period. As mentioned above, though, a general pattern emerged in which ‘apoE4’ mice were found to show significant reductions in overall activity levels. This difference manifested itself not only as less distance traveled in activity tests, but also as fewer arm entries committed during Y-maze spontaneous alternation testing. Nonetheless, ‘apoE3’ and ‘apoE4’ mice showed comparable vertical rearing events, rates of habituation to the open field, as well as comparable patterns of diurnal activity. Interestingly, reduced locomotor activity has also been reported for female apoE4 TR mice (Bour et al. 2008; Grootendorst et al. 2005). In contrast, Raber and colleagues (1998) failed to observe a difference in total distance traveled in the open-field test between mice expressing human apoE3 or apoE4 in neurons (NSE-apoE mice), while instead detecting a reduction in rearing events in NSE-apoE4 transgenics. This suggests that the physiological distribution of human apoE expression in genetically modified mouse models may have significant effects on the manner in which APOE genotype can influence locomotor and exploratory activity. Most importantly, however, the results of our sensorimotor testing make it highly unlikely that impairments in balance, strength, coordination or other noncognitive factors can account for the current differences observed between ‘poE3’ and ‘poE4’ mice on tests of learning, memory and attention.
The ability to remember and process spatial information is impaired in humans with AD (Lawrence and Sahakian 1995). In the present study, we found evidence for impaired spatial memory in female ‘poE4’ mice compared with ‘apoE3’ mice when performance was assessed in the novel object-location preference task, which is based on an animal's tendency to spend more time exploring an object encountered in a novel location vs. an identical object encountered in a familiar location (Ennaceur et al. 1997; Thinus-Blanc et al. 1996). Whereas ‘poE3’ mice displayed a significant preference for exploring the displaced object more so than the nondisplaced object during the test session, ‘apoE4’ mice did not differentiate between the two objects. Moreover, the greater recognition index in the apoE3 group compared with the apoE4 group is reflective of more robust long-term spatial recognition memory in ‘apoE3’ mice. Although ‘apoE4’ mice displayed generalized decreases in locomotor activity across several tasks, both groups showed comparable numbers of contacts and total time spent exploring the objects during the acquisition phase. While these data argue against potential disparities in object-directed exploratory behaviors, and are instead indicative of a spatial recognition memory impairment in female ‘poE4’ mice, the possible contribution of differences in novelty preference or neophobia to this observed effect cannot be entirely ruled out. Nonetheless, the observation that both groups also showed equal exploration of the stimulus objects upon first presentation during the training session, together with the fact that these results are in agreement with previous findings in female h-apoE TR mouse lines showing intact spatial recognition performance in mice expressing h-apoE3 but not in mice expressing h-apoE4 (Grootendorst et al. 2005), suggest a true difference in spatial memory abilities.
The contextual fear conditioning test has been used extensively in mice and rats to assess the integrity of hippocampal-dependent memory systems (Anagnostaras et al. 2001), dysfunction of which may underlie the episodic memory deficits that characterize AD and related dementias (Gold and Budson 2008). In this task, the strength of the pairing between the conditioned stimulus (i.e. chamber context) and unconditioned stimulus (i.e. electric footshock) is inferred by the animal's conditioned fear response (i.e. freezing behavior). Thus, the greater the freezing to the conditioning context, in the absence of aversive shock stimuli, the greater the strength of the contextual fear memory. We have shown here that ‘poE4’ mice exhibit less freezing to the conditioning context following a 24-h retention interval than do ‘poE3’ mice. The comparable levels of freezing observed at the end of the acquisition session imply that the difference observed on the test session does not reflect a generalized loss of fear, or the inability to acquire a conditioned fear response. The fact that, at the time of retention testing, ‘apoE4’ mice freeze at substantially higher levels than those seen at pre-shock baseline also indicates that significant memory of the conditioning procedure has been consolidated and recalled. Nonetheless, these results do suggest that the strength of the contextual fear association is subtly, but significantly, reduced in ‘apoE4’ mice. Whether the extent of this impairment might be exacerbated by decreasing the salience of the conditioning context, decreasing the number of pairings between conditioned and unconditioned stimuli or increasing the length of the retention delay, all of which have been shown to affect the nature or appearance of contextual fear memory deficits in transgenic mouse models of AD (Corcoran et al. 2002; Dineley et al. 2002; Kimura and Ohno 2009), remains a topic for future investigation.
Despite the impairments observed in ‘poE4’ mice on both the contextual fear conditioning and novel object-location preference tasks, no difference was detected between ‘apoE4’ and ‘apoE3’ mice in terms of spontaneous alternation behavior in the Y-maze. Although all three tasks are known to be affected by hippocampal dysfunction (Anagnostaras et al. 2001; Lalonde 2002; Thinus-Blanc et al. 1996), spontaneous alternation is thought to reflect, in part, the operation of intact spatial working or short-term memory, whereas the contextual fear and novel object-location paradigms require the successful consolidation and recall of more long-lasting memory traces. These results raise the possibility that the brain circuitry mediating spontaneous alternation performance in mice is somehow less vulnerable to the negative consequences of human apoE4 expression than are the respective neural systems underlying longer-term spatial recognition and episodic-like memory. Another possibility that must be considered, is that the difference in the total number of arm entries committed by ‘apoE3’ and ‘apoE4’ mice during the test session could impact the final rates of alternation observed, perhaps by affecting the overall level of proactive interference resulting from previous arm visits.
It has become increasingly clear in recent years that the deficits which characterize AD are often accompanied by dysfunction in certain aspects of attentional processing (Lawrence and Sahakian 1995), yet in most animal models of the disease this cognitive domain has gone unexamined. This may be explained, in part, by the fact that assessment of attentional functioning in rodents has typically involved the use of behavioral tests requiring several weeks to months of training (De Bruin et al. 2006; Patel et al. 2006). We report here that, compared with ‘apoE3’ mice, ‘poE4’ mice display impairments on a two-choice discrimination task that measures aspects of sustained visual attention. Training and testing on this task can be completed within only 2–3 weeks, and performance has been shown to be sensitive to both prefrontal cortex damage and cholinergic blockade (Dillon et al. 2009). Both ‘apoE4’ and ‘apoE3’ mice readily acquired the visual discrimination rule, but ‘poE4’ mice showed a significant impairment as the attentional demands of the task increased. Although deficits in both choice accuracy and reaction time were apparent in ‘apoE4’ mice only at the 2- and 10-second stimulus-duration trials, ‘loor’ effects are likely to have prevented potential differences at the shorter stimulus durations, at which both groups are performing at chance levels. Nonetheless, these data provide the first evidence of an isoform-specific effect of apoE on attentional functioning in mice. In this respect, it is worth noting that the observed effects in ‘poE4’ mice recapitulate some of the changes in speed-of-processing (O’Hara et al. 2008) and visuospatial attention (Greenwood et al. 2005) that have been reported in non-demented human subjects carrying the ε4 allele.
Although previous reports have shown deleterious effects of human apoE4 expression on learning and memory in different genetically modified mouse models, the current study is the first to examine of the effects of apoE status on cognitive performance in a mouse line expressing the nonmutated form of human APP characteristic of most cases of late-onset AD. In vitro studies have shown that apoE binds to Aβ in an isoform-specific manner (Strittmater et al. 1993) and that it influences Aβ fibrillization (Wisniewski et al. 1994), while in vivo studies have shown that apoE colocalizes with Aβ deposits in the brains of demented patients (Namba et al. 1991) and that an increased gene dose of the ε4 allele correlates with increased number of Aβ-containing senile plaques (Rebeck et al. 1993). Additionally, numerous animal studies have shown that apoE is an important cofactor in amyloid-related neuropathology and support the hypothesis that apoE's ability to affect the structure and clearance of Aβ underlies its role as the main genetic risk factor for late-onset AD (Brendza et al. 2002). Given the many complex interactions between apoE and Aβ, the identification of a mouse model in which isoform-specific effects of h-apoE on cognitive function occur against a backdrop of genomic h-APP expression should prove valuable toward efforts in clarifying how apoE status mediates susceptibility to cognitive decline.
In summary, we report here on the phenotypic characterization of novel transgenic mouse lines and provide evidence that the apoE4 genotype is associated with a generalized reduction in locomotor activity, and subtle impairments in spatial recognition, contextual fear memory, and attentional processing when compared with animals expressing human apoE3. Interestingly, these effects appear comparable in many ways to the moderate deficits previously reported in human apoE4 carriers on neuropsychological test measures. Whereas previous studies using other transgenic mice have pointed to the importance of both age and gender in influencing the apoE4-related cognitive decline (Bour et al. 2008; Grootendorst et al. 2005; Raber et al. 1998; van Meer et al. 2007), it is important to note that our present study was not designed to investigate age- and gender-dependent effects of apoE on behavior. Therefore, further study is necessary to clarify the contributions of these factors to the behavioral phenotype observed in our current mouse model. Preliminary findings from our laboratory do, however, suggest that cognitive impairments in ‘poE4’ vs. ‘apoE3’ mice are more pronounced in females than in males (Kornecook et al. 2008). While future experiments should also address the neuropathological mechanisms underlying the behavioral changes, our data support the notion that APP-Yac/apoE4-TR mice represent an excellent model for facilitating our understanding of apoE's effects on both normal and diseased brain functioning.