Antenatal Hypoxia Accelerates the Onset of Alzheimer's Disease Pathology in 5xFAD Mouse Model

Abstract

Alzheimer's disease (AD) is a chronic neurodegenerative disorder associated with cognitive impairment and later dementia among the elderly. Mounting evidence shows that adverse maternal environments during the fetal development increase the risk of diseases later in life including neurological disorders, and suggests an early origin in the development of AD-related dementia (ADRD) in utero. In the present study, we investigated the impact of antenatal hypoxia and fetal stress on the initiation of AD-related pathology in offspring of 5xFAD mice. We showed that fetal hypoxia significantly reduced brain and body weight in the fetal and the early postnatal period, which recovered in young adult mice. Using spontaneous Y-maze, novel object recognition (NOR), and open field (OF) tasks, we found that antenatal hypoxia exacerbated cognitive decline in offspring of 5xFAD compared with normoxia control. Of interest, fetal hypoxia did not alter intraneuronal soluble amyloid-β (Aβ) oligomer accumulation in the cortex and hippocampus in 5xFAD mouse offspring, indicating that antenatal hypoxia increased the vulnerability of the brain to synaptotoxic Aβ in the disease onset later in life. Consistent with the early occurrence of cognitive decline, we found synapse loss but not neuronal death in the cerebral cortex in 5xFAD but not wild-type (WT) offspring exposed to antenatal hypoxia. Furthermore, we also demonstrated that antenatal hypoxia significantly increased microglial number and activation, and reactive astrogliosis in the cerebral cortex in WT offspring. Moreover, antenatal hypoxia resulted in an exacerbated increase of microgliosis and astrogliosis in the early stage of AD in 5xFAD offspring. Together, our study reveals a causative link between fetal stress and the accelerated onset of AD-related pathology, and provides mechanistic insights into the developmental origin of aging-related neurodegenerative disorders.

Keywords: 5xFAD mouse; Alzheimer’s disease; antenatal hypoxia; cognitive impairment; gliosis; synapse loss.

Figure 1

Antenatal hypoxia caused growth restriction in 5xFAD offspring. Pregnant mice were treated with normoxia or hypoxia from day 14.5 to day 17.5 of gestation. Brain weight (A), body weight (B), and the ratio of brain to body weight (C) of fetuses at embryonic day 18.5 (E18.5), and offspring at postnatal day 3 (P3) and 21 (P21) were evaluated. Data are mean ± SEM. Student’s t-test was applied to each data set. ***p < 0.0001; **p < 0.001; *p < 0.05; NS, not significant. E18.5, n = 17–18; P3, n = 11–14; P21, n = 6.

Figure 2

Antenatal hypoxia exacerbated cognitive impairment in 5xFAD offspring. Spontaneous Y-maze test (sp-Y, A,B) and novel object recognition (NOR, C,D) were conducted at 2 and 4 months. (A) Schematic of the sp-Y test. (B) Spontaneous alternation of 5xFAD offspring exposed to antenatal hypoxia or normoxia. (C) Schematic of the NOR test. (D) Discrimination index (DI) of 5xFAD offspring exposed to antenatal hypoxia or normoxia. Open field test (OF, E–H) was performed at 4 months. (E) Schematic of the OF test and movement tracks from normoxia- or hypoxia-treated 5xFAD offspring. The red square separates inner zone (center) and outer zone. (F) Distance in center, (G) time in center, and (H) entries in center of offspring exposed to antenatal hypoxia or normoxia. Data are mean ± SEM. Student’s t-test was applied to each data set. *p < 0.05. Sp-Y test at 2 months: Norm, n = 15; Hy, n = 12. Sp-Y test at 4 months: Norm, n = 15; Hy, n = 7. NOR at 2 months: n = 11. NOR at 4 months: Norm, n = 12; Hy, n = 8. OF: Norm, n = 12, Hy, n = 8.

Figure 3

Antenatal hypoxia did not affect soluble Aβ accumulation in the brain cortex and hippocampus of 2-month-old 5xFAD offspring. The brain cortex was separated from the brain of 2-month-old 5xFAD offspring exposed to normoxia or hypoxia. (A) Western blotting was performed to detect the soluble Aβ levels with primary antibody against soluble Aβ (oAβ). Actin was used as internal control. Data are mean ± SEM. Student’s t test was applied to each data set. NS, not significant, n = 4. (B) Confocal imaging of soluble Aβ (green) distribution in the cortex (upper panel) and hippocampus (lower panel) in the brain of 2-month-old 5xFAD offspring exposed to normoxia or hypoxia. DAPI stains nuclei (blue). Scale bar: 200 μm.

Figure 4

Antenatal hypoxia resulted in synaptic loss in the brain cortex of 2-month-old 5xFAD offspring. (A–C) Western blotting analysis of the presynaptic marker Syn1 and postsynaptic marker PSD95 in the cortex of 2-month-old wild-type (WT) and (D–F) 5xFAD mice exposed to normoxia or hypoxia. Actin was used as internal control. Data are mean ± SEM. Student’s t-test was applied to each data set. n = 3–4. (G) Confocal images of brain slices from 2-month-old 5xFAD offspring exposed to normoxia or hypoxia stained with antibody against Synapsin 1 (green). DAPI stains nuclei (blue). Scale bar: 10 μm. NS, not significant.

Figure 5

Antenatal hypoxia increased microglial activation in the brain cortex of 2-month-old 5xFAD offspring. (A,B) qRT-PCR of mRNA levels of microglial markers Iba1, TMEM119 in the cortex of WT and 5xFAD offspring with antenatal hypoxia or normoxia treatment. Data are mean ± SEM. Student’s t-test was applied to each data set. n = 4. (C) Confocal images of coronal brain slices of 2-month-old 5xFAD offspring with antenatal hypoxia or normoxia treatment stained with antibody against Iba1 (green). DAPI strains nuclei (blue). Insert (b1) shows the resting microglia and (b2) shows the activated microglia. Scale bar: (B), 100 μm; (b1, b2), 20 μm. (D) Quantification of Iba1-positive cell numbers in the cerebral cortex with ×20 objective. Student’s t-test was applied to each data set. A total of nine sections from three animals were used for analysis. (E) qRT-PCR of mRNA levels of the activated microglia marker CD68 in the cortex of the WT and 5xFAD mice exposed to antenatal hypoxia and normoxia. Data are mean ± SEM. Student’s t-test was applied to each data set. n = 4. (F) Confocal images of coronal brain slices of 2-month-old 5xFAD mouse offspring with antenatal hypoxia or normoxia treatment stained with antibody against CD68 (red) an Iba1 (green). DAPI stains nuclei (blue). (G) Quantification of CD68 fluorescence intensity in the cerebral cortex with ×20 objective. Data are mean ± SEM. Student’s t-test was applied to each data set. A total of nine sections from three animals were used for analysis. Scale bar: 20 μm. (H) Regression analysis of the correlation between spontaneous Y-maze performance with relative mRNA level of Iba1, TMEM119, and CD68, respectively. Individual data points are from 5xFAD normoxia (red) and hypoxia treated mice (blue).

Figure 6

Antenatal hypoxia increased reactive astrogliosis in the brain cortex of 2-month-old 5xFAD offspring. (A) qRT-PCR of GFAP mRNA levels in the cortex of offspring exposed to normoxia or hypoxia. Student’s t-test was applied to each data set. n = 4. (B) Confocal images of brain slices from 2-month-old 5xFAD offspring exposed to normoxia or hypoxia stained with antibody against GFAP (red). DAPI stains nuclei (blue). Insert (b1) shows normal astrocyte and (b2) shows the reactive astrocyte. Scale bar: (B), 100 μm; (b1,b2), 20 μm. (C) Quantification of GFAP fluorescence intensity in the cerebral cortex with ×20 objective. Data are mean ± SEM. Student’s t-test was applied to each data set. In total, nine sections from three animals were used for analysis. (D) Regression analysis of the correlation between spontaneous Y-maze performance with GFAP relative mRNA level. Single data points are from 5xFAD normoxia (red) and hypoxia treated mice (blue).