Effects of Striatal Amyloidosis on the Dopaminergic System and Behavior: A Comparative Study in Male and Female 5XFAD Mice

Abstract

Background:

Nearly two-thirds of patients diagnosed with Alzheimer’s disease (AD) are female. In addition, female patients with AD have more significant cognitive impairment than males at the same disease stage. This disparity suggests there are sex differences in AD progression. While females appear to be more affected by AD, most published behavioral studies utilize male mice. In humans, there is an association between antecedent attention-deficit/hyperactivity disorder and increased risk of dementia. Functional connectivity studies indicate that dysfunctional cortico-striatal networks contribute to hyperactivity in attention deficit hyperactivity disorder. Higher plaque density in the striatum accurately predicts the presence of clinical AD pathology. In addition, there is a link between AD-related memory dysfunction and dysfunctional dopamine signaling.

Objective:

With the need to consider sex as a biological variable, we investigated the influence of sex on striatal plaque burden, dopaminergic signaling, and behavior in prodromal 5XFAD mice.

Methods:

Six-month-old male and female 5XFAD and C57BL/6J mice were evaluated for striatal amyloid plaque burden, locomotive behavior, and changes in dopaminergic machinery in the striatum.

Results:

5XFAD female mice had a higher striatal amyloid plaque burden than male 5XFAD mice. 5XFAD females, but not males, were hyperactive. Hyperactivity in female 5XFAD mice was associated with increased striatal plaque burden and changes in dopamine signaling in the dorsal striatum.

Conclusion:

Our results indicate that the progression of amyloidosis involves the striatum in females to a greater extent than in males. These studies have significant implications for using male-only cohorts in the study of AD progression.

Keywords

Alzheimer’s disease amyloid-beta dopamine neurotransmission hyperlocomotion 5XFAD

INTRODUCTION

Nearly two-thirds of patients diagnosed with Alzheimer’s disease (AD) are female [1]. Furthermore, neurodegeneration and clinical symptoms of AD occur more rapidly in females compared to males [2 –4]. Female AD patients have greater brain atrophy and amyloid plaque burden than males [5, 6] and clinical Mini Mental-State Examination data of AD patients indicate that female AD patients have greater decline in memory, reasoning, language, and spatial orientation compared to male AD patients at the same stage of disease [4 , 8]. This disparity suggests there are sex differences in AD progression. Still, most published behavioral studies in AD mouse models include only males.

AD patients undergo progressive amyloid-β (Aβ) deposition in the brain, and a higher Aβ plaque density in the striatum accurately predicts the presence of clinical AD pathology [9]. Aβ pathology in the striatum could cause memory impairment by dysregulating dopaminergic activity in the early stages of AD. In fact, progressive loss of striatal dopamine neurons projecting from the ventral tegmental area occurs long before Aβ plaque deposition can even be detected and could have consequences on cognitive function [10 –16].

The striatum includes two nuclei innervated by dopamine neurons. In the ventral striatum, mesolimbic dopamine neurons innervating the nucleus accumbens play a role in motivational control and reward processing [17]. In the dorsal striatum, nigrostriatal dopamine neurons terminating in the caudate nucleus facilitate cognitive control, and dopamine neurons innervating the putamen regulate motor function but also are involved in habit learning and stimulus-response [18, 19]. In addition, mesocortical dopamine neurons originating in the ventral tegmental area innervate the cortex and play a role in executive functions [2, 20].

In humans, antecedent attention-deficit/hyperactivity (ADHD) disorder is associated with an increased risk of dementia [21 –23]. ADHD, in the context of dementia, is an interesting phenomenon because patients with ADHD have deficits that span the functions of each of the dopamine neuronal populations in the brain. These deficits include stimulus-response (nigrostriatal dopamine neurons), executive function (mesocortical dopamine neurons), reward processing (mesolimbic dopamine neurons), and motor function (nigrostriatal dopamine neurons) [24 –27].

Sex-dependent differences in hyperlocomotion are apparent in several transgenic mouse models of dementia, including tTA:APPsi, TDP-43, APPSwe/PS1ΔE9, and J20/TauKO [28 –31]. The widely utilized 5XFAD mouse is an aggressive transgenic mouse model of AD that demonstrates amyloid plaque pathology [32]. With the need to consider sex as a biological variable, we hypothesized that sex would influence striatal plaque burden, behavior, and dopaminergic signaling in prodromal 5XFAD mice. We analyzed the behavior and brains of 5-month-old 5XFAD mice. We found that female 5XFAD mice have higher striatal Aβ plaque density than age-matched WT males. Higher striatal plaque burden in 5XFAD female mice was associated with hyperactivity, reduced dopamine transporter, and increased tyrosine hydroxylase expression in the dorsal striatum. These results highlight the importance of understanding sex as a biological variable in the study of AD progression.

MATERIALS AND METHODS

Animals

All experimental procedures comply with the ARRIVE guidelines and were approved by MSU-IACUC. Protocols were performed following the NIH Guide for the Care and Use of Laboratory Animals. Five-month-old male and female wild-type (WT; C57BL/6J) mice and 5XFAD (Tg(APPSwFlLon,PSEN1*M146L *L286V)6799Vas, MMRRC Stock No: 34848-JAX) were purchased from Jackson Labs at eight weeks old and were fed standard rodent chow (Research Diets, Inc) ad libitum for up to 24 weeks. Five-month-old prodromal 5XFAD [33, 34] and age-matched C57BL/6 mice were divided into four groups (n = 12 per group): WT male, 5XFAD male, WT female, and 5XFAD female. Unless otherwise noted, twelve animals per group were used for behavior analysis and five per group were used for immunohistochemistry.

Nesting test

We used the nesting test as a measure of daily living activities. The primary experimentor could not be blinded to mouse strain due to differences in coat color. To eliminate bias, a primary exerimentor placed each mouse alone in a clean cage with two round paper nestlets. After 24 h, the primary experimentor assigned a random letter to each cage, photographed the nest and then placed the mouse back in its home cage. A second experimentor inspected the nests and rated each on a 1–5 scale according to published methods [35].

Y-maze, Spontaneous alternation test

The spontaneous alternation behavior test was used to measure spatial working memory. This test takes advantage of a rodent’s preference to investigate a new arm of the maze rather than returning to a previously visited arm. A Y-shaped maze (arms 5 cm wide, 35 cm long, and 20 cm high, at 120 angles from one another) was used for this test, which was conducted according to published methods [36]. To provide unbiased analysis, each mouse was recorded using an overhead camara over three minutes using Ethovision software (Noldus, Wageningen, Netherlands). Maximum possible alternations were defined as the total number of arms entered –2. Animals were removed from the study if they did not enter at least six arms during the trial. The percent correct alternations were calculated as the number of entries into three separate arms/maximum possible alternations×100.

Y-maze, novel arm test

The novel arm test was used to measure hippocampal-dependent spatial memory. The test was conducted in a Y-shaped maze (arms 5 cm wide, 35 cm long, and 20 cm high, at 120 angles from one another) as previously published [37]. Ethovision software recorded each trial using an overhead camara. The percent time in the novel arm was defined as the time spent with all four paws in the novel arm divided by the time spent in all arms.

Open field (OF) testing

The locomotive activity of each mouse was evaluated using VersaMax OF beam break apparatus and Fusion Software. A transparent acrylic box measuring 40×40×30 cm was placed inside the infrared beam-break device. Mice were placed in the arena’s center and allowed to move freely for 30 min. Fusion software recorded the activity of each mouse and parsed the activity into 5 min bins for analysis. Distance traveled, average velocity, and stereotypic behaviors were analyzed. Distance traveled was calculated as the number of centimeters traveled over the allotted time. The average velocity was calculated as cm/sec over the allotted time. Stereotyped behaviors are repetitive, rhythmic, bilateral movements with fixed patterns and regular frequency. Stereotyped behaviors were counted when the mouse broke the same beam in succession without breaking an adjacent beam and was expressed as the number of counts over the allotted time [38].

Immunofluorescent imaging

Free-floating brain hemi sections containing the striatum (20μm) were taken approximately 1.40 from bregma, treated with 1% sodium-borohydride in PBS for 15 min, and washed in PBS containing 10% methanol and 3% hydrogen peroxide for 15 min. Following three washes with PBS, samples were blocked in 3% bovine serum albumin and 10% normal donkey serum with 0.5% Triton X-100 in PBS. Brain sections were incubated overnight with primary antibodies against tyrosine hydroxylase (TH; 1:2000; Chemicon AB152), dopamine transporter (DAT; 1:1000, Chemicon AB369), amyloid beta (Aβ; 1:500, Cell Signaling, 8243), or neuronal nuclei antigen (NeuN; 1:500, Millipore MAB377). Goat anti-rabbit Cy3 (for TH), goat anti-rat FITC (for DAT), donkey anti-mouse Alexa-488 (for NeuN), or donkey anti-rabbit Alexa-594 (for Aβ) were used to identify immunoreactivity. Brain sections without primary antibodies were used as negative controls. Brain sections were mounted onto slides using ProLong Gold antifade reagent (Invitrogen). A Nikon TE2000-U microscope with a 4X and 10X objective was used for imaging. We evaluated striatal fiber immunoreactivity (ir) in three adjacent sections per mouse by measuring TH-ir or DAT-ir integrated density within the dorsal striatum using FIJI software. An integrated density of the overlying cortex was subtracted from the value generated from the striatum. We used rainbow RGB-LUT to highlight the difference between striatal fibers in WT and 5XFAD mice.

Statistical analysis

Statistical analysis was performed with GraphPad Prism (v9.0). Twelve animals per group were initially tested in all behavior tests. For the spontaneous alternation test, one male WT was removed from the study because they did not complete the minimum of six arm entries. A female WT was removed from the study because it froze during the trial due to noise in the room. In all cases where continuous data was collected and all statistical assumptions were met, a two-way ANOVA followed by Sidak’s post-hoc test was used. In cases where data was nonparametric, a Kruskal-Wallis test was used. For y-maze analysis, a one sample Wilcoxian t-test was also used to determine if animals performed differently than would be expected by chance (hypothetical value set at 33.33%). We considered p-values of ≤0.05 statistically significant. In all bar graphs the bar represents the mean, error bars represent the standard error of the mean, and individual points represent each sample measured.

RESULTS

Amyloid plaque burden in the striatum is higher in female 5XFAD mice

The widely utilized 5XFAD mouse is an aggressive transgenic mouse model of amyloidosis that rapidly develops amyloid plaque pathology [32]. Amyloid deposition begins at approximately two months of age. By six months, amyloid plaques develop in the hippocampus, cortex, and striatum [32]. In humans, a higher Aβ plaque density in the striatum is predictive of clinical AD pathology [9]. With the need to consider sex as a biological variable, we measured Aβ plaque burden in the cortex, dorsal striatum, and ventral striatum of male and female WT and 5XFAD mice (Fig. 1). In both male and female 5XFAD mice, Aβ plaque burden in the cortex was significantly increased compared to WT mice (Fig. 1B, % immunoreactive area; male 5XFAD versus male WT, 4.057±0.4897 versus 0.1234±0.1234, ANOVA, p = 0.0.0143; female 5XFAD versus WT, 6.350±1.9610 versus 0.0110±0.0050, ANOVA, p = 0.0004). Additionally, Aβ plaque burden in the cortex was not significantly different between male and female 5XFAD mice (Fig. 1B, % immunoreactive area; male 5XFAD, 4.057±0.4897 versus 6.350±1.961, ANOVA, p = 0.1289). In the dorsal striatum, Aβ plaque burden in male 5XFAD mice did not significantly increase compared to WT males, while female 5XFAD mice had significantly higher Aβ plaque burden than WT females (Fig. 1C, % immunoreactive area; female 5XFAD versus female WT, 5.5.3820±1.1900 versus 0.4480±0.2985, ANOVA, p < 0.0001). In the ventral striatum, Aβ plaque burden in male 5XFAD mice was not significantly increased, while female 5XFAD mice had significantly higher Aβ plaque burden than WT females (Fig. 1D, % immunoreactive area, female 5XFAD versus female WT, 3.108±0.8217 versus 0.0844±0.0630, ANOVA, p = 0.0002). These results indicate that while cortical Aβ plaque burden in the cortex at the level of the striatum did not significantly differ between male and female 5XFAD mice, dopaminergic systems in the dorsal and ventral striatum of 5XFAD mice are more affected in females than in males. Staining of brain sections containing the striatum with the neuronal marker NeuN indicated that amyloid plaques in the striatum were neuritic in nature (Fig. 1E).

Fig. 1

Aβ plaque burden is higher in the striatum of 5XFAD females. A) Representative images of amyloid immunoreactivity in the striatum of 5XFAD (right panels) and WT mice (left panels). Amyloid was detected by immunolabeling with Aβ antibodies (D54D2; red), which detects Aβ₃₇, Aβ₃₈, Aβ₃₉, Aβ₄₀, and Aβ₄₂. Neurons are labeled with NeuN (green). Scale bars, 1000μm. B) Aβ plaque burden in the cortex at the level of the striatum was increased in male and female 5XFAD mice compared to WT mice ((% immunoreactive area; male 5XFAD versus male WT, 4.057±0.4897 versus 0.1234±0.1234, ANOVA, p = 0.0143; female 5XFAD versus female WT, 6.350±1.9610 versus 0.0110±0.0050, ANOVA, p = 0.0004). There was no difference between male and female 5XFAD mice (% immunoreactive area; male 5XFAD versus female 5XFAD, 4.057±0.4897 versus 6.350±1.961, ANOVA, p = 0.1289). C). Aβ plaque burden was increased in the dorsal striatum of female 5XFAD mice compared to female WT mice (% immunoreactive area; female 5XFAD versus female WT, 5.5.3820±1.1900 versus 0.4480±0.2985, ANOVA, p < 0.0001). Male 5XFAD mice had significantly lower Aβ plaque burden than female 5XFAD mice (% immunoactive area; male 5XFAD versus female 5XFAD, 0.8422±0.5139 versus 5.382±1.190, ANOVA, p = 0.0002). D. In the ventral striatum, female 5XFAD mice had significantly higher Aβ plaque burden than WT females (% immunoreactive area, female 5XFAD versus female WT, 3.108±0.8217 versus 0.0844±0.0630, ANOVA, p = 0.0002). male 5XFAD mice had significantly lower Aβ plaque burden than female 5XFAD mice (% immunoactive area; male 5XFAD versus female 5XFAD, 0.4429±0.3178 versus 3.108±0.8217, ANOVA, p = 0.0006). E) Representative images of NeuN (green) and Aβ (red) co-localization (orange) in the striatum of female 5XFAD mice. Scale bars, 50μm.

Impaired behavior in 5XFAD mice

Cognitive impairment in AD is characterized most notably by memory deficits and impairments of activities of daily living [39]. We used a nest-building test to assess activities of daily living at five months of age. There were no significant differences in the quality of nests in male and female WT mice (Fig. 2A–D; mean nest scores; male WT, 4.8±0.1 versus female WT 4.5±0.2, Kruskal-Wallis test, p = 0.3412) Likewise, there was no significant difference between male and female 5XFAD mice (mean nest scores; male 5XFAD, 3.2±0.3 versus female 5XFAD, 2.7±0.2, Kruskal-Wallis test, p = 0.3412). However, both male and female 5XFAD mice built poor nests compared to age and sex-matched WT mice (Fig. 2A–D; mean nest scores; male 5XFAD versus male WT, 3.2±0.3 versus 4.8±0.1, Kruskal-Wallis test, p = 0.0002; female 5XFAD versus female WT, 2.7±0.2 versus 4.5±0.2, Kruskal-Wallis test, p = 0.0001). These data indicate that daily living activities are disrupted in both male and female 5XFAD mice.

Fig. 2

Cognitive impairment in 5-month-old male and female 5XFAD mice. A–D) The nest building test (E, F), the spontaneous alternation test, and the novel arm test (G, H) were used to assess cognition. Bars on each graph represent the mean±SEM individual points represent each mouse. A) Representative images of nests built by WT and 5XFAD male mice. B) Representative images of nests built by WT and 5XFAD female mice. C, D) There were no significant differences in the quality of nests in male and female WT mice (mean nest scores; male WT, 4.8±0.1 versus female WT 4.5±0.2, Kruskal-Wallis test, p = 0.3412). There was no significant difference between male and female 5XFAD mice (mean nest scores; male 5XFAD, 3.2±0.3 versus female 5XFAD, 2.7±0.2, Kruskal-Wallis test, p = 0.3412). Both male and female 5XFAD mice built poor nests compared to age and sex-matched WT mice (mean nest scores; male 5XFAD versus male WT, 3.2±0.3 versus 4.8±0.1, Kruskal-Wallis test, p = 0.0002; female 5XFAD versus female WT, 2.7±0.2 versus 4.5±0.2, Kruskal-Wallis test, p = 0.0001). E, F) Spontaneous alternation behavior in 5XFAD male and female mice was not impaired in any of the groups (One-sample t-test, theoretical value 33.33%, p < 0.0001 for each group). G, H) Novel arm exploration. There was no significant difference between male and female WT mice (% time in novel arm; male WT, 45±3 versus female WT 41±2, Kruskal-Wallis test, p = 0.2591). There was no significant difference between male and female 5XFAD mice (% time in novel arm; male 5XFAD, 38±2 versus female 5XFAD, 34±2, Kruskal-Wallis test, p = 0.1766). Female 5XFAD explored the novel arm significantly less than female WT mice (% time in novel arm; male 5XFAD versus male WT, 38±2 versus 45±3, two-way ANOVA, p = 0.0641; 5XFAD females versus WT females, 34±2 versus 41±2, two-way ANOVA, p = 0.0374). Female 5XFAD mice did not spend more time in the novel arm than would be expected by chance (Wilcoxon t-test, hypothetical value 33.33%, 5XFAD male, p = 0.07, WT male, p = 0.0009, 5XFAD female, p = 0.9, WT female, p = 0.008).

Spatial working memory was measured using the y-maze spontaneous alternation test (Fig. 2E, F). This test of spatial working memory demands intact executive function as the test requires retention and manipulation of visuospatial information. All groups performed more correct alternations than would be expected by chance (Wilcoxon t-test, hypothetical value 33%, p < 0.0001 for all groups). There were no statistical differences between strain or sex, indicating that amyloidosis, at this stage, did not impair spatial working memory (mean % alternations; male 5XFAD, 57±4, male WT, 60±4, female 5XFAD, 60±4, female WT, 64±3, Kruskal-Wallis test).

Hippocampal-dependent spatial recognition memory was measured using the Y-maze novel arm test (Fig. 2G, H). There was no significant difference between male and female WT mice (% time in novel arm; male WT, 45±3 versus female WT 41±2, two-way ANOVA, p = 0.2591) Likewise, there was no significant difference between male and female 5XFAD mice (% time in novel arm; male 5XFAD, 38±2 versus female 5XFAD, 34±2, two-way ANOVA, p = 0.1766). Female 5XFAD groups performed significantly worse than female WT mice (% time in novel arm; male 5XFAD versus male WT, 38±2 versus 45±3, two-way ANOVA, p = 0.0641; female 5XFAD versus female WT, 34±2 versus 41±2, two-way ANOVA, p = 0.0374). Only the female 5XFAD mice did not spend more time in the novel arm than would be expected by chance (Wilcoxon t-test, hypothetical value 33.33%, 5XFAD male, p = 0.07; WT male, p = 0.0009; 5XFAD female, p = 0.9; WT female, p = 0.008). These data indicate that hippocampal-dependent spatial memory is more severely affected in female 5XFAD mice than in males.

Female 5XFAD mice are hyperlocomotive

Dopamine neurons innervating the dorsal striatum regulate motor function, and abnormalities within the fronto-striatal-cerebellar circuits may cause hyperactivity [40, 41]. We used open field testing to test if 5XFAD female mice are hyperactive. Locomotion was not significantly different between male and female WT mice (total distance traveled; male WT, 5249±260 cm versus female WT, 4511±411 cm, two-way ANOVA by sex p = 0.2198; average velocity; male WT, 13±1 versus female WT, 14±1, two-way ANOVA by sex, p = 0.1631). However, 5XFAD female mice traveled further than female WT mice (Fig. 3A; total distance traveled; female 5XFAD versus female WT, 8147±538 cm versus 4511±411 cm, two-way ANOVA by strain, p < 0.0001). 5XFAD female mice also traveled further than male 5XFAD mice (total distance traveled; male 5XFAD, 5837±456 cm versus female 5XFAD, 8147±538 cm, two-way ANOVA by sex, p = 0.0004). Female 5XFAD mice traveled at higher velocity than WT females (Fig. 3B, mean velocity; female 5XFAD versus female WT, 17±1 cm/s versus 14±1 cm/s, two-way ANOVA by strain, p < 0.0001). 5XFAD females also traveled at higher velocity than 5XFAD males (average velocity; male 5XFAD, 13±1 cm/s versus female 5XFAD, 17±1 cm/sec, two-way ANOVA by sex, p < 0.0001). Taken together, these results indicate that female but not male 5XFAD mice, are hyperlocomotive.

Fig. 3

Hyperlocomotion and stereotyped behavior in 5-month-old female 5XFAD mice. A) Distance traveled across 5 min time bins in the open field. There was no difference in the mean distance traveled between WT male and WT female mice (total distance traveled; male WT, 5249±260 cm versus female WT, 4511±411 cm, two-way ANOVA by sex p = 0.2198) while there was a significant difference in distance traveled between male and female 5XFAD mice (total distance traveled; male 5XFAD, 5837±456 cm versus female 5XFAD, 8147±538 cm, two-way ANOVA by sex, p = 0.0004). Female 5XFAD mice traveled a greater distance than WT females (total distance traveled; female 5XFAD, 8147±538 versus female WT, 4511±411 cm, two-way ANOVA by strain, p < 0.0001). B) Average velocity across 5 min time bins in the open field. There was no difference in the average velocity between WT male and WT female mice (average velocity; male WT, 13±1 cm/s versus female WT, 14±1 cm/s, two-way ANOVA by sex p = 0.1631) while there was a significant difference in the average velocity of male and female 5XFAD mice (average velocity; male 5XFAD, 13±1 cm versus female 5XFAD, 17±1 cm, two-way ANOVA by sex, p < 0.0001). Female 5XFAD mice traveled faster than WT females (average velocity; female 5XFAD 17±1 cm/s versus female WT 14±1 cm/s, two-way ANOVA by strain, p < 0.0001). C) Stereotypic activity counts across 5 min time bins. Stereotypic behavior was significantly different between male and female WT mice (mean stereotypic movements; male WT, 51±1 versus female WT, 45±1, two-way ANOVA by sex, p = 0.0193). In both sexes, 5XFAD mice had less stereotypic behavior compared to WT mice (mean stereotypic movements, 5XFAD males 43±2 versus WT males 51±1, two-way ANOVA by strain, p = 0.0037; 5XFAD females 37±3 versus WT females 45±1, two-way ANOVA by strain, p = 0.0029, male versus female 5XFAD mice, two-way ANOVA by sex, p = 0.0151). For all graphs, data represent the mean±SEM. Black squares represent male WT, blue squares represent male 5XFAD, black circles represent female, and purple circles represent female 5XFAD.

Motor stereotypies are repetitive, rhythmic, bilateral movements with a fixed pattern and regular frequency. These types of behaviors are prevalent in laboratory mice [42]. Decreased D2R autoreceptor activity is known to suppress stereotypic behavior [43], and expression of the 5XFAD transgenes reduces D2-like receptor (D2R) density in the striatum [44]. Therefore, we examined stereotypic behavior in the open field test. Stereotypic behavior was significantly different between male and female WT mice (Fig. 3C, mean stereotypic movements; male WT, 51±1 versus female WT, 45±1, two-way ANOVA by sex, p = 0.0193). In both sexes, 5XFAD mice had less stereotypic behavior compared to WT mice (Fig. 3C, mean stereotypic movements, 5XFAD males versus WT males, 43±2 versus 51±1, two-way ANOVA by strain, p = 0.0037; 5XFAD females versus WT females, 37±3 versus 45±1, two-way ANOVA by strain, p = 0.0029). Further male 5XFAD mice exhibited more stereotypic behavior than female 5XFAD mice (mean stereotypic movements, 5XFAD males versus 5XFAD females, 43±2 versus 37±3, two-way ANOVA by sex, p = 0.0151). These results suggest dopaminergic signaling changes in both male and female 5XFAD mice.

Dopaminergic changes in the striatum

Female 5XFAD mice were hyperlocomotive with reduced stereotypic behavior. Therefore, we examined the dorsal and ventral striatum for DAT and TH immunoreactivity (ir). There were no significant differences in either DAT-ir or TH-ir between male and female WT mice. There were also no significant differences in either DAT-ir or TH-ir in the male 5XFAD mice compared to WT males. While DAT-ir in the dorsal striatum of male 5XFAD mice was not different from WT males, DAT-ir in the dorsal striatum of female 5XFAD mice was 20% less than in the dorsal striatum of WT female mice (Fig. 4A, B; % integrated DAT-ir density of 5XFAD females versus WT females; 87.4±2.0% versus 109.4±8.6%, ANOVA, p = 0.0188). DAT-ir in the ventral striatum was not significantly different between any of the groups (Fig. 4C). While TH-ir in the dorsal striatum of male 5XFAD mice was not significantly different than male WT mice, TH-ir in the dorsal striatum of female 5XFAD mice was increased 24% compared to the WT females (Fig. 4D; % integrated density; 5XFAD females versus WT females, 121.1±4.7 versus 97.6±4.3, ANOVA, p = 0.0029). TH-ir was also increased in the ventral striatum of 5XFAD females (Fig. 4E; % integrated density; 5XFAD females versus WT females, 121.2±4.4 versus 97.5±6.1, ANOVA, p = 0.0262). The increase in TH and decrease in DAT suggest that dopamine signaling in the dorsal striatum of female 5XFAD mice is perturbed.

Fig. 4

Effects of 5XFAD transgenes on DAT-ir and TH-ir in male and female mice. A) Representative rainbow RGB LUT colored immunofluorescent images of the DAT-ir in the striatum (scale bars, 500μm). B) DAT-ir in the dorsal striatum was compared in male and female WT and 5XFAD mice. Integrated density of DAT-ir is expressed as % male WT (DAT-ir % male WT; male 5XFAD, 101.2±1.9, ANOVA, p = 0.8896; female WT, 109.4±8.6, p = 0.2825; female 5XFAD, 87.4±2.0, ANOVA, p = 0.0188). C) Percent integrated density of DAT-ir in the ventral striatum (DAT-ir % male WT; male 5XFAD, 111.7±2.8, ANOVA, p = 0.1555; female WT, 101.3±6.3, p = 0.8709; female 5XFAD, 93.7±4.1, ANOVA, p = 0.3502). D) Representative rainbow RGB LUT colored immunofluorescent images of TH-ir in the striatum (scale bars, 500μm). E) TH-ir in the dorsal striatum was compared in male and female WT and 5XFAD mice. Integrated density of TH-ir is expressed as % male WT (TH-ir % male WT; male 5XFAD, 101.6±6.0, ANOVA, p = 0.8152; female WT, 97.6±4.3, ANOVA, p = 0.7282; female 5XFAD, 121.1±4.7, ANOVA, p = 0.0029). F) TH-ir in the ventral striatum was compared in male and female WT and 5XFAD mice. Integrated density of TH-ir is expressed as % male WT (TH-ir % male WT; male 5XFAD, 104.9±8.9, ANOVA, p = 0.6180; female WT, 97.5±6.1, ANOVA, p = 0.2565; female 5XFAD, 121.2±4.4, ANOVA, p = 0.0262).

DISCUSSION

Females are more often diagnosed with AD than males, and females have more significant cognitive impairments than males at the same stage of the disease [1 , 6]. This difference in disease presentation may occur because amyloid pathology is more severe in females than males. Buckley et al. found in individuals on the AD trajectory, early tau and amyloid deposition were elevated in females compared to males [45]. This is also true in mouse models of AD, where females have increased amyloid plaque burden compared to age-matched males [46]. With the need to consider sex as a biological variable, we investigated the hypothesis that sex would influence striatal plaque burden, behavior, and dopaminergic signaling in prodromal 5XFAD mice. Our results suggest that deposition of Aβ in the striatum is associated with hyperactivity in female 5XFAD mice. For the first time, we demonstrate that prodromal 5XFAD female mice are hyperlocomotive with significant changes in amyloid deposition, DAT, and TH expression in the striatum.

Amyloid pathology is thought to lead to dysfunctional dopamine neurotransmission, which may cause deficits in daily living, executive function, and memory. 5XFAD transgenic mice begin to deposit Aβ as early as two months of age and exhibit memory deficits at 5–6 months of age [32 , 47–51]. In these mice, Aβ plaques are pronounced in the cortex, hippocampus, and striatum [32, 52]. The presence of amyloid plaques in the striatum of these mice is remarkable because, in humans, plaque burden in the striatum is predictive of clinicopathological AD [9]. These data suggest that sex differences in behavior are associated with the higher plaque burdens within the striatum.

Recent work in mouse models of amyloidosis implicates Aβ deposition in the development of early neuronal hyperexcitability that precedes the loss of dopamine neurons [11 , 53–56]. We found that diffuse NeuN immunoreactivity was strongly co-localized with Aβ in the striatum. The Aβ plaques in the striatum were both neuritic and dopaminergic, with strong diffuse co-localization in the 5XFAD mice. This suggests that there is significant loss of dopamine neurons in the nigrostratial pathway. Indeed, Vorobyov et al. demonstrated in 5XFAD mice, there is a significant decrease in the number of TH immunoreactive neurons in both the ventral tegmental area and the substantia nigra by 6 months [56]. We found an increase in TH and decrease in DAT in the striatum of female 5XFAD mice. This perturbation in the expression of dopaminergic machinery in females could be explained as a compensatory response to loss of dopaminergic neurons in the midbrain. As dopamine neurons are lost, females might compensate by increasing dopamine synthesis and decreasing synaptic dopamine reuptake.

It is largely accepted that accumulation of Aβ is implicated in the loss of dopamine neurons in AD [57]. The amyloid hypothesis states that toxic Aβ accumulation triggers tau hyperphosphorylation and deposition that contributes to neuronal dysfunction, cell death, and cognitive impairment [58, 59]. This is especially true for dopamine neurons, which are particularly sensitive to cell death. The susceptibility of dopamine neurons has been extensively described in Parkinson’s disease [60]. Dopamine neurons have long and highly arborized projections that are mostly unmyelinated [61]. The length of these dopamine neurons necessitate efficient transport systems and proteostatic mechanisms. In addition, dopamine neurons have autonomous pacemaker activity [62]. These structural and functional qualities of dopamine neurons require a large amount of energy that must be supplied by healthy mitochondria. This energy requirement leaves dopamine neurons exposed to injury from toxic dopamine metabolites, reactive oxygen species, and impaired proteostasis.

Amyloid pathology in the striatum can impair daily living activities, executive function, memory, and locomotion. We used a nest-building test to assess activities of daily living at five months of age. Nest building was poor in both the male and female 5XFAD mice, and there was no significant difference in the quality of nests built by male or female 5XFAD mice. Both males and females have plaque burden in the striatum, but females have a more severe plaque burden. The fact that female nest building was not more severely affected than males might suggest that plaque burden in the striatum is not associated with nest building skills. It is also possible that the lower plaque burden in the male striatum of 5XFAD mice was sufficient to produce poor nest-building skills. The timeline of Aβ deposition in the striatum and behavior deficits in male and female 5XFAD mice is being addressed in separate studies.

Executive function is impaired in patients with AD and could also be associated with amyloid plaque burden in the striatum. We used the spontaneous alternation test to assess working spatial memory. The spontaneous alternation test takes advantage of a healthy rodent’s preference to explore novel environments. In the spontaneous alternation test, a mouse with intact spatial working memory will prefer to visit the arm of the maze that is the newest environment, resulting in the mouse subsequently exploring a different arm of the maze. This test requires retention and manipulation of visuospatial information, which places demands on executive functions. At five months of age, 5XFAD male and female mice had no apparent deficits in this test compared to age and sex-matched WT mice. These data indicate that amyloid pathology in the striatum, at this stage, was not associated with impaired executive function. Interestingly, patients with Parkinson’s disease can complete the Tower of London task (a task commonly used to diagnose executive impairment in patients) just as well as healthy control patients. Positron emission tomography during this test demonstrates that healthy patients utilize the right caudate nucleus during this task. In contrast, Parkinson’s disease patients compensate for the loss of dopaminergic innervation to the caudate nucleus by activating the hippocampus instead [63].

The striatum and hippocampus are linked via the prefrontal cortex [64]. It is thought nondopaminergic inputs from the striatum via the basal ganglia-thalamocortical loops [65] are involved in episodic memory tasks [66]. Landau et al. demonstrated the importance of striatal dopamine in memory function. In healthy elderly patients, there is a positive correlation between striatal dopamine and performance in memory tasks [67].

We used the y-maze novel arm test to assess episodic spatial memory. In this test, both male and female 5XFAD groups spent less time in the novel arm than WT males or females. However, the female 5XFAD mice did not spend more time in the novel arm than would be expected by chance. This data indicates that episodic spatial memory is more severely affected in female 5XFAD mice than in males.

In humans, there is an association between antecedent ADHD and increased dementia risk [21 –23]; female mice from several mouse models of dementia are hyperlocomotive [28 –31]. We showed that female 5XFAD mice are hyperlocomotive. We also found a reduction in stereotypic behavior in male and female 5XFAD mice compared to WT mice. Aged female 5XFAD mice have reduced D2 autoreceptors and reduced TH in the striatum [44]. Decreased D2R autoreceptor activity suppresses stereotypic mouse behavior [43]. D2 autoreceptors regulate dopamine signaling by providing feedback inhibition that controls dopamine synthesis, release, and reuptake [68].

Prodromal female, but not male, 5XFAD mice were hyperlocomotive with reduced stereotypic behavior. One explanation for sex differences in locomotion might be altered expression of dopaminergic machinery in the striatum. TH is the rate-limiting enzyme for dopamine synthesis, and DAT is necessary for postsynaptic dopamine reuptake. While both sexes of 5XFAD mice had reduced stereotyped behavior, only the female 5XFAD mice had reduced DAT-ir and increased TH-ir in the striatum. These changes suggest prodromal 5XFAD females likely have increased dopamine synthesis and release with limited reuptake, thus increasing extraneuronal dopamine and motor activity. Female rodents have more nigrostriatal dopamine neurons than males [69]. In addition, extracellular dopamine in the striatum varies with the estrous cycle. When estrogen is at its highest, D2 autoreceptor activity is reduced, and extracellular dopamine is increased. Estrogen treatment of ovariectomized female rodents results in similar increases in dopamine release [70, 71]. Finally, estrogen also reduces GABA release, which indirectly increases dopamine release via disinhibition of dopamine terminals [72, 73].

Extravesicular dopamine is converted to dopamine quinones or other toxic dopamine metabolites that damage dopamine neurons and lead to subsequent motor dysfunction. Cataldi et al. found that the toxic monoamine oxidase product of cytosolic dopamine, 3,4-dihydroxyphenylacetaldehyde (DOPAL) can stabilize toxic Aβ oligomers and exacerbate Aβ deposition [74]. We found that female 5XFAD mice had increased deposition of Aβ in the striatum compared to males. If Aβ deposition is important for dopamine neurotransmission in the striatum, females would have decreased dopamine synthesis in the striatum with more significant motor impairment than males as they age. O’Leary et al. assessed age-related changes in motor function in male and female 5XFAD mice from 3–16 months old. As mentioned previously, Son et al. described reduced TH immunoreactivity in 9-month-old female 5XFAD mice [31]; in a rotarod test, both male and female 5XFAD mice had impaired motor coordination by ten months, and by thirteen months, 5XFAD female mice appeared to have worsened compared to males [75, 76].

In conclusion, amyloid pathology in 5XFAD mice is associated with sex differences in dopaminergic function in the striatum, which likely results in hyperactivity in female mice. Hyperactive behavior should be considered when assessing learning and memory in mouse models of amyloidosis. Since antecedent ADHD is associated with an increased risk for dementia, more studies are needed to determine the role of striatal dopamine and estrogen in memory function. These differences may play a role in worsened cognitive impairment in females compared to males.

The current study has certain limitations. Striatal plaque burden is predictive of clinicopathological AD. Therefore, we chose to focus on the characterization of amyloid plaque burden and dopamine signaling solely in the striatum and did not determine the effects of amyloid pathology on terminals of the dopamine neuronal populations. Future studies will be directed toward other dopaminergic nuclei in the brain.

Footnotes

ACKNOWLEDGMENTS

The authors have no acknowledgments to report.

FUNDING

This research was partly supported by NIH R01DK121272-01A1 and R01DK121272-3S1.

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

DATA AVAILABILITY

The data supporting the findings of this study are available within the article and/or its supplementary material.

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