Chronic cerebral hypoperfusion causes decrease of O-GlcNAcylation, hyperphosphorylation of tau and behavioral deficits in mice

Abstract

Chronic cerebral hypoperfusion (CCH) is one of the causes of vascular dementia (VaD) and is also an etiological factor for Alzheimer's disease (AD). However, how CCH causes cognitive impairment and contributes to Alzheimer's pathology is poorly understood. Here we produced a mouse model of CCH by unilateral common carotid artery occlusion (UCCAO) and studied the behavioral changes and brain abnormalities in mice 2.5 months after UCCAO. We found that CCH caused significant short-term memory deficits and mild long-term spatial memory impairment, as well as decreased level of protein O-GlcNAcylation, increased level of tau phosphorylation, dysregulated synaptic proteins and insulin signaling, and selective neurodegeneration in the brain. These findings provide mechanistic insight into the effects of CCH on memory and cognition and the likely link between AD and VaD.

Keywords: Alzheimer’s disease; O-GlcNAcylation; brain insulin signaling; chronic cerebral hypoperfusion; cognitive impairment; neurodegeneration; synaptic plasticity markers; tau phosphorylation.

FIGURE 1

Animal study design (A) and verification of the UCCAO model (B,C). Homogenates of the cortices and hippocampi of mice 2.5 months after sham or UCCAO surgery were analyzed by Western blots developed with the indicated antibodies (B). Each lane represents an individual mouse sample. The blots were then quantified densitometrically, and the data after normalization with the GAPDH level are presented as mean ± SEM (n = 6/group), where the values of the sham group are set as 1.0 (C). *p < 0.05 vs. sham group; #p < 0.05 vs. contralateral group.

FIGURE 2

General behavior and short-term memory of mice with CCH. The body weight and food intake were monitored once a week after sham or UCCAO surgery (A,B). Spontaneous locomotor and exploratory activity was assessed in an open field (C). Anxiety-like behaviors were evaluated in an elevated plus maze, and the CAs entries are shown (D). Gait was evaluated using footprint test, and different limb strides are shown (E). Motor coordination and balance were evaluated using accelerating Rotarod, and the fall latency of each trial is shown (F). Depression was evaluated using forced swim test, and last 4 min immobility out of total 6 min is shown (G). One-trial object recognition task was carried out in an open field. Time spent exploring two identical objects during sample phase are shown as percentage of object exploring time (H). Object discrimination during test phase is presented by the recognition index (time exploring the novel object/total time for exploring) (I). Data are reported as mean ± SEM.*p < 0.05 vs. sham mice.

FIGURE 3

Morris water maze tests of mice with CCH. Spatial memory of the mice was tested in the Morris water maze test (A–D) and the consecutive reversal test (E–L). The average swim speed in the training phase (A,E), the distance traveled to the hidden platform during training (B,F), the number of the platform site crossings during the probe trial (C,G), the percentage of distance traveled in the target, adjacent left (Adj.L), adjacent right (Adj.R), and opposite (Opp) quadrants during probe trial (D,H) and the gradual reduction in percent time/distance in the quadrant of previous platform and the gradual shift to the new quadrant (I–L) are shown. Data are reported as mean ± SEM.*p < 0.05 vs. sham mice.

FIGURE 4

Effects of CCH on O-GlcNAcylation and tau phosphorylation. Homogenates of the cortices and hippocampi of mice 2.5 months after sham or UCCAO surgery were analyzed by immuno-dot blots developed with a mixture of monoclonal antibodies RL2 and CTD110.6 against O-GlcNAcylated proteins and Western blots developed with the indicated antibodies (A,C). The blots were then quantified densitometrically, and the data after normalization with the GAPDH (B) or R134d for total tau level (D) are presented as mean ± SEM (n = 6/group), where the values of the sham group are set as 1.0.*p < 0.05 vs. sham group.

FIGURE 5

Effects of CCH on synaptic proteins. Homogenates of the cortices and hippocampi of mice 2.5 months after sham or UCCAO surgery were analyzed by Western blots developed with the indicated antibodies (A). The blots were then quantified densitometrically, and the data after normalization with the GAPDH level are presented as mean ± SEM (n = 6/group), where the values of the sham group are set as 1.0 (B).*p < 0.05 vs. sham group. #p < 0.05 vs. contralateral group. The coronal brain sections were immunostained with antibody against synaptophysin (C). CA, Cornu Ammonis; DG, dentate gyrus; Scale bar: 100 μm.

FIGURE 6

Effects of CCH on the GFAT2 and the insulin signaling pathway. Homogenates of the cortices and hippocampi of mice 2.5 months after sham or UCCAO surgery were analyzed by Western blots developed with the indicated antibodies (A). The blots were then quantified densitometrically, and the data after normalization with the GAPDH (B) or the corresponding protein level (C) are presented as mean ± SEM (n = 6/group), where the values of the sham group are set as 1.0; *p < 0.05 vs. sham group. #p < 0.05 vs. contralateral group.

FIGURE 7

Effects of CCH on neuronal degeneration. Representative microphotographs of Fluoro-Jade stainining of cerebral cortex and dentate gyrus sections of mice 2.5 months after sham or UCCAO surgery (A). The Fluoro-Jade positive neurons were then quantified by using the Image J software, and the data are presented as mean ± SEM (n = 6/group) (B). Coexistence of hyperphosphorylated tau (detected using antibody against pS262) and Fluoro-Jade positive neurons was seen in adjacent sections (C).*p < 0.05 vs. sham group.