BACKGROUND: Soluble beta-amyloid (Aβ) can be cleared from the brain through various mechanisms including enzymatic degradation, glial cell phagocytosis, transport across the blood-brain barrier, and glymphatic clearance. However, the relative contribution of each clearance system and their compensatory effects in delaying the pathological process of Alzheimer's disease (AD) are currently unknown.
METHODS: Fluorescent trace, immunofluorescence, and Western blot analyses were performed to compare glymphatic clearance ability and Aβ accumulation among 3-month-old APP695/PS1-dE9 transgenic (APP/PS1) mice, wild-type mice, aquaporin 4 knock out (AQP4−/−) mice, and AQP4−/−/APP/PS1 mice. The consequence of selectively eliminating microglial cells, or downregulating apolipoprotein E (apoE) expression, on Aβ burden, was also investigated in the frontal cortex of AQP4−/−/APP/PS1 mice and APP/PS1 mice.
RESULTS: AQP4 deletion in APP/PS1 mice significantly exaggerated glymphatic clearance dysfunction, and intraneuronal accumulation of Aβ and apoE, although it did not lead to Aβ plaque deposition. Notably, microglia, but not astrocytes, increased activation and phagocytosis of Aβ in the cerebral cortex of AQP4−/−/APP/PS1 mice, compared with APP/PS1 mice. Selectively eliminating microglia in the frontal cortex via local injection of clodronate liposomes resulted in deposition of Aβ plaques in AQP4−/−/APP/PS1 mice, but not APP/PS1 mice. Moreover, knockdown of apoE reduced intraneuronal Aβ levels in both APP/PS1 mice and AQP4−/−/APP/PS1 mice, indicating an inhibitory effect of apoE on Aβ clearance.
CONCLUSION: The above results suggest that the glymphatic system mediated Aβ and apoE clearance and microglia mediated Aβ degradation synergistically prevent Aβ plague formation in the early stages of the AD mouse model. Protecting one or both of them might be beneficial to delaying the onset of AD.
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This work was supported by grants from the National Natural Science Foundation of China (81671070 and 81772454).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Descriptions of additional files:
Additional file 1
Morris water maze. The Morris water maze test was conducted to measure mouse long-term spatial cognitive function, as described previously. Four Training was performed over 7 consecutive days, with 4 trials per day. During the first two days, mice were trained to find a dark-colored cylindrical platform with a diameter of 10 cm, sitting 0.5 cm above the water surface. Mice did not receive the next hidden platform tests if they had apparent motor and/or visual deficits indicated by low swimming speed (< 75 mm/s) or long escape latency (> 50 s). On the 3rd day, the platform was submerged 1 cm below the surface of the water and moved to the opposite quadrant. Escape latency, swimming distance and swimming speed were calculated. On day 8, the hidden platform was removed, allowing mice to swim freely in the pool for 60 s. The percentage of time spent in the target quadrant and the number of crossing where the platform had been previously located were analyzed. Y-maze test. The Y-maze test was performed to evaluate mouse short-term spatial working memory, as previously described. One arm, termed the novel arm, was blocked by a black baffle, allowing the mice to only move in the other two arms for 5 min. Two hours later, the novel arm was opened, allowing mice to freely move throughout the three arms. The percentage of time traveled in, number of entries into the novel arm, as well as traveling speed during the test, was calculated. Mouse activity in the aforementioned behavioral apparatuses was collected by a digital video camera connected to a computer-controlled system (Beijing Sunny Instruments Co. Ltd., China). All tests were performed by two independent experimenters, who were each blind to the treatment schedule.
Additional file 2
No obvious effects of AQP4 deletion on spatial cognitive function of 3-month-old APP/PS1 mice. a, b The mean escape latency and swimming speed during the training period of the Morris water maze test. c The number of crossing the platform. d The percentage of time in the target quadrant. e The number of entries into the novel arm. f The percentage of time in the novel arm in the Y-maze. Data in S1a, b were analyzed by repeated-measures ANOVA with post hoc Student-Newman-Keuls test. Other Data were analyzed by ANOVA with post hoc Student Newman-Keuls test. Data are means ± SEM. n = 12 per group. Fig. S2. AQP4 deletion did not affect astrocyte activation in 3-month-old APP/PS1 mice. a, b Double immunofluorescence and quantification for GFAP and total-Aβ in the cortex. c, d Immunofluorescence and quantification for GS positive astrocytes in the cortex of APP/PS1 mice and AQP4−/−/APP/PS1 mice. Data are means ± SEM. n = 4 per group, two-way ANOVA with Newman-Keuls post-hoc test. Fig. S3. AQP4 polarization was impaired in the cerebral cortex of 3-month-old APP/PS1 mice. a Double immunofluorescence for AQP4 and GFAP. b Quantitative analyses of the AQP4 polarization. Data are means ± SEM. n = 4 per group, Student’s t-test. **p < 0.01; ***p < 0.001. Fig. S4. Increased astrocyte activation in the cortex in 3-month-old APP/PS1 mice and AQP4−/−/APP/PS1 mice receiving local injection of clodronate liposomes. a, b Double immunofluorescence and quantification for total-Aβ and GFAP in the cortex. Data are means ± SEM. n = 4 per group, two-way ANOVA with Newman-Keuls post-hoc test. Fig. S5. An image shows GFP expression in the cortex one month after injection of AAV encoding apoE siRNAs. a GFP positive area represented where the AAVs was injected. b-d Double immunofluorescence for GFP and GFAP. Note that apoE siRNAs were expressed in GFAP positive astrocytes (arrowheads).
Feng, Weixi; Zhang, Yanli; Wang, Ze; Xu, Hanrong; Wu, Ting; Marshall, Charles; Gao, Junying; and Xiao, Ming, "Microglia Prevent Beta-Amyloid Plaque Formation in the Early Stage of an Alzheimer's Disease Mouse Model with Suppression of Glymphatic Clearance" (2020). Physical Therapy Faculty Publications. 115.