“An Evaluation of the Cognitive Effects of a Short-Term and a Long-Term Ovarian Hormone Deprivation in a Transgenic Mouse Model of Alzheimer’s Disease: Addressing the Critical Window”
Introduction
Alzheimer’s disease (AD) is a progressive, age-related neurodegenerative disease that impairs many aspects of cognition including memory. AD affects 10% of the population 65 years and older, and 50% of the population 85 years and older (Alzheimer’s Association, 2017). This highlights a desperate need to understand the neurobiological underpinnings of the disease in order to discover appropriate targets for effective treatments. Initially, clinical symptoms are manifested as impairments in working memory and consolidation, and then eventually will progress to impact other cognitive domains such as planning, language, and judgment (Selkoe, 2001). Diagnoses of AD is contingent on two pathological hallmarks in the brain known as plaques, which are aggregations of the Aβ peptide, and tangles, which are aggregations of hyperphosphorylated tau. Importantly, research has shown that the build up of these toxic species occur years before the onset of clinical symptoms (Sperling et al., 2011). Women are at a greater relative risk for developing AD than age-matched men, and one hypothesis is that the loss of ovarian hormones (i.e. estrogens and progesterone) after menopause might contribute to this differential risk between men and women (Rocca et al., 2007, 2012). Additionally, women can enter menopause via surgical removal of the ovaries (known as an oophorectomy). Research has shown that the younger age of oophorectomy is correlated with increased risk of AD-related dementias (Rocca, 2012). Further, hormone therapy following oophorectomy has been shown to potentially reduce this risk (Rocca et al., 2012; Paganini-Hill and Henderson, 1994, Tang et al., 1996). However, there are mixed results in the literature about the detrimental impacts of ovarian hormone loss and subsequent benefits of hormone therapy (HT) (Shumaker et al., 2004). Likely, there is a wide range of variables that are at play. Specifically, this study evaluates the length of ovarian-hormone deprivation because studies have shown that women that underwent an oophorectomy before the age of 49, but not given HT until the age of 50 had an increased risk for dementia. Conversely, women with an oophorectomy at the same age and given HT until the age of 50 had no increased risk for AD (Phung et al., 2010). This suggests a potential critical window where the brain is susceptible to the detriments of ovarian-hormone loss, but receptive to the beneficial effects of HT. Therefore, the overarching research question of this project is to evaluate how a short term (ST) and a long-term (LT) ovarian hormone deprivation impacts spatial memory and AD-like neuropathology. To study the question of interest, transgenic mice were used. Importantly, rodents do not naturally develop AD neuropathology. Therefore, transgenic mice that have genes that are associated with familial onset of AD have been utilized to evaluate AD-like pathological changes in-vivo. Additionally, ovariectomy (Ovx) is the surgical removal of the ovaries in rodents. This paradigm is used to give a blank slate of ovarian hormones. Taken together, using transgenic mice in an Ovx model can allow us to systematically investigate behavior and AD-like neuropathological changes following ovarian hormone loss.
Methods
Female transgenic (Tg) mice expressing both APP and PS1 mutations on the background of C57B/6 mice were bred. Six breeding cages were set up housing two females and one male per cage. The housing conditions were controlled with a 12-hour light cycle (7:00am-7:00pm) and food and water were kept freely available. Both female Tg and wildtype (Wt) control mice were used for this study. At 21 days old, mice were weaned and pair housed. On occasions when an odd number of females were weaned, mice were triple-housed to avoid single-housing stress. All procedures were approved by the Institutional Animal Care and Use Committee and adhered to the National Institutes of Health standards. Mice were randomly assigned to one of eight treatment groups to evaluate the effects of ST or LT ovarian-hormone deprivation: Wt Sham ST (n=14), Wt Sham LT (n=14), Wt Ovx ST (n=14), Wt Ovx LT (n=13), Tg Sham ST (n=13), Tg Sham LT (n=12), Tg Ovx ST (n=14) and Tg Ovx LT (n=12). At approximately three months of age, all mice underwent either sham or Ovx surgery. Approximately three weeks following Ovx, mice underwent vaginal smears for eight consecutive days to evaluate estrous cyclicity in the sham mice and to confirm hormone deprivation in Ovx mice (Goldman et al., 2007). Behavior testing for the ST group began one month following surgery, and behavior testing for the LT group began three months following surgery. All mice were tested on the same battery of behavior tasks including the Morris water maze, delayed-matched-to sample water maze (DMS), and visible platform task. All the water mazes utilize positive and negative reinforcement to motivate the mouse to find a hidden platform beneath the surface of the water. Removing the mouse from the cool temperature (18-20C) of the water once it finds the platform acts as the negative reinforcement, and placing the mouse in a heated testing cage between trials acts as a positive reinforcement. Additionally, the MM and DMS mazes utilized spatial cues around the room for the mouse to navigate through the maze. MM was used to assess spatial reference memory by having a consistent platform location beneath the water across five days of testing. DMS was used to assess spatial short-term and working memory by changing the platform location across 13 days of testing. The visible platform task was then used to assess motor and visual competence, the procedural components of a water maze. All mice were euthanized one day after visible platform testing. Mice were deeply anesthetized under isoflurane prior to decapitation.
Results
Here, we present results from the DMS and visible platform task, as the MM data are still being quantified. Our dependent measures for DMS were quantifying the first time a mouse enters an arm without a platform (First Errors), and any subsequent times a mouse enters an arm without a platform (Repeat Errors). Our dependent measure for the visible platform task was latency to the platform (s). The repeated measures ANOVA for Repeat Errors for the testing trials within the acquisition phase (Days 2-6, Trials 2-4) revealed a main effect of Trial [F(2,94)=11.91; p<0.0001] with errors decreasing across trials, indicating mice learned within a day. For the acquisition phase, there was no main effect of Genotype or Treatment, there was a Genotype x Treatment interaction [F(1,47)=4.63; p<0.05], and there was a Trial x Genotype x Treatment interaction [F(2,94)=6.46; p<0.005]. Post hoc analysis of Trial 2 alone, the working memory trial, for the acquisition phase showed a significant Genotype x Treatment interaction [F(1,47)=12.84; p<0.001]. The Genotype x Treatment interaction was null for Days 2-6, Trial 3 alone [F(1,47)=0.03; p=0.86] and Trial 4 alone [F(1,47)=1.33; p=0.25]. There was no significant Genotype effect for the two-group comparison between Wt Sham and Tg Sham mice for Repeat Errors in the acquisition phase, Trials 2-4 [F(1,23)=1.39; p=.25]. There was a main effect of Genotype for the two-group comparison for Wt Ovx and Tg Ovx mice for the acquisition phase, Trials 2-4 [F(1,24)=4.35; p<.05], whereby the Tg Ovx mice made more Repeat Errors than the Wt Ovx mice, indicating that Ovx induced a Genotype effect. For this analysis of Ovx mice only, there was also a significant Genotype x Trial interaction [F(2,48)=5.76; p<0.01], whereby the Tg Ovx mice made more Repeat Errors than Wt Ovx mice on Trial 2 alone, the working memory trial. Indeed, in a separate analysis for Trial 2 alone, there was a main effect of Genotype [F(1,24)=7.72; p<.025]; whereby the Tg Ovx mice made more Repeat Errors on this working memory trial than the Wt Ovx mice in the acquisition phase of testing. There were no main effects or interactions for Genotype or Treatment for the asymptotic phase (Days 7-10). For the LT cohort, the repeated measures ANOVA for Repeat Errors in the acquisition phase, Trials 2-4, revealed a Genotype x Treatment interaction [F(1,47)=4.11; p<0.05]. The two- group comparison between the Tg Ovx and Tg Sham mice during the acquisition phase revealed a marginal effect of Treatment [F(1,22)=3.35; p=0.08], whereby the Tg Ovx group made marginally more errors than the Tg Sham group. There were no main effects or interactions for Genotype or Treatment for the asymptotic phase (Days 7-10). There were no significant Genotype or Treatment main effects or interactions for neither ST nor LT cohorts in the visible platform task, suggesting all groups comparably performed the procedural components of water maze testing. For the ST cohort, there was a main effect of Trial [F(5,235)=24.95; p<0.0001] For the LT cohort, there was a main effect of Trial [F(5,235)=9.794; p<0.0001].
Discussion
The goal of this study was to evaluate the impacts of ST and LT ovarian-hormone deprivation on cognition in a transgenic model of AD. A Genotype x Treatment x Trial interaction was shown for the ST cohort in the acquisition phase (Days 2-6), indicating that depending on trial and genotype, Sham and Ovx mice performed differently. Further analyses revealed effects specific to Trial 2 because this trial represents the working memory trial, as mice are required to update the location of the platform from the previous day with the new location learned from the previous information trial (Trial 1). As expected, mice made the largest number of errors within a day on this trial, making it the hardest trial. A two-group comparison of Ovx mice alone for the working memory trial revealed a Genotype main effect such that the Tg Ovx mice were impaired compared to the Wt Ovx group; the Genotype main effect was not seen when the Sham mice were analyzed alone. This indicates that spatial memory ability in the Tg mice may be more sensitive to the loss of ovarian hormones than the Wt mice. The neurobiological mechanisms underlying these differences are currently unknown. One possibility is that the differences seen between the Tg Ovx mice and the Wt Ovx mice could be mediated by the cholinergic system, which is known to be critical for learning and memory processes. The cholinergic system is altered by AD in humans, and disruption in this neurotransmitter system contributes to the memory impairments typically seen in AD (Gibbs, 2010). Estrogens can modulate and protect cholinergic neurons by increasing the enzyme responsible for synthesizing acetylcholine, choline acetyltransferase (ChAT), in the basal forebrain (Luine, 1985). Further, evidence from in-vitro cell culture indicates that estrogen treatment has been shown to reduce Aβ formation (Xu et al., 1998). This cumulative evidence suggests that Ovx-induced estrogen loss could make the brain more susceptible to disruptions in the cholinergic circuitry and subsequent cognitive impairments, which may be amplified or compounded by the presence of AD-like neuropathology. In the acquisition phase for the LT cohort, there was a similar significant Genotype x Treatment interaction. However, these effects were not specific to a particular trial. Further, comparisons between the Tg Ovx mice and the Tg Sham mice were marginal. Interactions for both the ST and LT cohorts were not apparent in the asymptotic phase of testing (Days 7-10). This suggests that differences between treatment groups are the most pronounced when mice were still learning the task. Brain tissue from these mice will be analyzed for AD-like pathology to correlate it with maze performance. Overall this will help reveal how the removal of a mouse’s ovarian hormones via Ovx impacts the build up of AD-like pathology in both the ST and the LT. Future studies can expand on a potential critical window for intervention in order to target specific proteins to reduce AD-like pathology, with the ultimate goal of preventing or postponing behavioral impairments associated with AD.
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