Disruption of metabolic, sleep, and sensorimotor functional outcomes in a female transgenic mouse model of Alzheimer's disease

Abstract

Alzheimer's Disease (AD) is the most prevalent form of dementia globally, and the number of individuals with AD diagnosis is expected to double by 2050. Numerous preclinical AD studies have shown that AD neuropathology accompanies alteration in learning and memory. However, less attention has been given to alterations in metabolism, sleep, and sensorimotor functional outcomes during AD pathogenesis. The objective of this study was to elucidate the extent to which metabolic activity, sleep-wake cycle, and sensorimotor function is impaired in APPSwDI/Nos2^-/- (CVN-AD) transgenic mice. Female mice were used in this study because AD is more prevalent in women compared to men. We hypothesized that the presence of AD neuropathology in CVN-AD mice would accompany alterations in metabolic activity, sleep, and sensorimotor function. Our results showed that CVN-AD mice had significantly decreased energy expenditure compared to wild-type (WT) mice. An examination of associated functional outcome parameters showed that sleep activity was elevated during the awake (dark) cycle and as well as an overall decrease in spontaneous locomotor activity. An additional functional parameter, the nociceptive response to thermal stimuli, was also impaired in CVN-AD mice. Collectively, our results demonstrate CVN-AD mice exhibit alterations in functional parameters that resemble human-AD clinical progression.

Keywords: Alzheimer’s disease; Metabolism; Neuroinflammation; Sensorimotor; Sleep.

Fig. 1.. Experimental design for the assessment of metabolic, sleep, and sensorimotor function.

Three cohorts of CVN-AD and WT mice (8-9 months old) were used in this study. The first cohort of mice was used to examine indirect calorimetry (i.e. metabolism), sleep, and activity. The second cohort of mice was used to examine sensorimotor function. Animals in the second cohort underwent the open field, rotarod, and hot plate tests on Days 1, 2, and 3 respectively. Furthermore, the open field and rotarod tests were used to assess passive and evoked locomotion respectively while the hot plate test was used to assess sensorimotor function and response to thermal stimuli. A third cohort of mice was used for immunohistochemistry. Brain regions of interest used in the quantification of Iba-1 and GFAP immunohistochemistry were the somatosensory cortex, hippocampal CA1, and hippocampal CA3 (outlined in black). Image Credit: Allen Institute and Biorender.

Fig. 2.. Beta-amyloid burden is coupled to neuroinflammation in 8-9 months old CVN-AD mice.

(A) Representative images showing cell nuclei (blue) and beta-amyloid deposition (green) in the somatosensory cortex and hippocampus (CA1) of CVN-AD mice compared to age-matched WT mice. Higher magnification images (right, 20X) were derived from 5X magnified images (left, white boxes). (B) Microgliosis (p = 0.02, Student’s unpaired t-test) and astrogliosis (p = 0.03, Student’s unpaired t-test) were significantly increased in the cortex of CVN-AD mice compared to WT mice. (C) Microgliosis is significantly increased (p = 0.043, Mann Whitney test) in the hippocampus (CA1 and CA3) of CVN-AD mice compared to WT mice; however, there was no significant difference in astrogliosis between CVN-AD and WT mice in the hippocampus (CA1 and CA3). (D) CVN-AD mice microglia displayed an amoeboid morphology with shorter processes (i.e. activated microglia) compared to WT mice microglia which displayed longer ramified processes (i.e. surveillance microglia) in the cortex and hippocampus (CA1 shown). (E) Cortical and hippocampal (CA1 shown) astrocytes of CVN-AD mice displayed intense GFAP staining, cell body hypertrophy (red arrow), and increased fiber-like processes (yellow arrows) compared to WT mice. Data represented as mean ± SEM; n = 3/group; *p < 0.05, **p < 0.01. Scale bar in (A) = 75 μm, (B, C) = 25 μm, and (D, E) = 75 μm. Images were taken at (A) 5X and 20X magnification and (B, C) 40X magnification. Magnified images in (D, E) were derived from images taken at 40X magnification in (B, C).

Fig. 3.. Energy utilization and expenditure is altered in CVN-AD mice.

(A, B) VO₂ (light cycle (white horizontal bars): p = 0.0064; dark cycle (black horizontal bars): p = 0.001) and VCO₂ (light cycle: p = 0.0001; dark cycle: p = 0.001) are significantly decreased in CVN-AD mice compared to WT mice. (C) Respiratory exchange ratio (RER) was decreased in the light cycle (p = 0.0001) of CVN-AD mice compared to WT mice. However, no significant differences in RER were seen between CVN-AD and WT mice in the dark cycle. (D) CVN-AD mice revealed a significant decrease in energy expenditure in the light (p = 0.01) and dark (p = 0.0003) cycles compared to WT mice. Data was analyzed using the FDA analysis of variance and p-values were based on the Fmax test statistic; n = 5 (WT) and n = 14 (CVN-AD). 1 interval = a 26 min period.

Fig. 4.. Sleep is disrupted in the CVN-AD mice.

(A) There was no significant difference in percent time spent sleeping between CVN-AD and WT mice in the light cycle (sleep phase). However, in the dark cycle (awake phase) CVN-AD mice spent an increased percent time sleeping compared to WT mice, though, this was not statically significant (p = 0.087, Student’s unpaired t-test). (B) Number of sleep bouts in the light cycle was not significantly different between CVN-AD and WT mice. However, in the dark cycle CVN-AD mice had a significantly increased (p = 0.01, Student’s unpaired t-test) number of sleep bouts compared to WT mice. (C) No significant differences in average bout duration were observed in the light and dark cycles between CVN-AD and WT mice. (D) Analysis for activity during the sleep test revealed that CVN-AD mice had a significantly decreased activity (p = 0.02) in the dark cycle (white horizontal bars) compared to the WT mice. No significant differences in locomotor activity were seen in the light cycle (black horizontal bars) between CVN-AD and WT mice. Data in (A-C) represented as mean ± SEM. Data in (D) was analyzed using the FDA analysis of variance and p-values were based on the Fmax test statistic; n = 5 (WT) and n = 13-14 (CVN-AD); *p < 0.05. 1 interval = a 26 min period.

Fig. 5.. CVN-AD mice exhibit sensorimotor deficits.

(A, B) Open field testing revealed a significant decrease in spontaneous horizontal (p = 0.0009, repeated measures two-way analysis of variance (ANOVA)) and vertical (p = 0.006, repeated measures two-way analysis of variance (ANOVA)) movements in the CVN-AD mice compared to WT mice. (C) Evoked locomotion was not significantly different between CVN-AD and WT mice on the rotarod test over 4 trials. (D) CVN-AD mice displayed an increased latency to respond to nociceptive stimuli compared to WT mice, however, this difference was not statistically significant (p = 0.06, Student’s unpaired t-test). Furthermore, the total number of nociceptive behavior was significantly decreased (p = 0.006, Student’s unpaired t-test) in the CVN-AD mice compared to WT mice. Data represented as means ± SEM; n = 6 (WT), n = 8 (CVN-AD); *p < 0.05, **p < 0.01, ***p < 0.001.