Early postnatal defects in neurogenesis in the 3xTg mouse model of Alzheimer's disease

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to dementia. The hippocampus, which is one of the sites where neural stem cells reside and new neurons are born, exhibits the most significant neuronal loss in AD. A decline in adult neurogenesis has been described in several animal models of AD. However, the age at which this defect first appears remains unknown. To determine at which stage, from birth to adulthood, the neurogenic deficits are found in AD, we used the triple transgenic mouse model of AD (3xTg). We show that defects in neurogenesis are present as early as postnatal stages, well before the onset of any neuropathology or behavioral deficits. We also show that 3xTg mice have significantly fewer neural stem/progenitor cells, with reduced proliferation and decreased numbers of newborn neurons at postnatal stages, consistent with reduced volumes of hippocampal structures. To determine whether there are early changes in the molecular signatures of neural stem/progenitor cells, we perform bulk RNA-seq on cells sorted directly from the hippocampus. We show significant changes in the gene expression profiles at one month of age, including genes of the Notch and Wnt pathways. These findings reveal impairments in neurogenesis very early in the 3xTg AD model, which provides new opportunities for early diagnosis and therapeutic interventions to prevent neurodegeneration in AD.

Fig. 1. Reduced volume of hippocampal structures in 3xTg mice at early ages.

a Representative Cresyl violet staining of a coronal section of NTG mice brain. GCL, DG, hippocampus and cortical thickness are labelled. Scale Bar 1 mm. b Absolute volume measurements of GCL, c DG, d hippocampus, and e whole brain for NTG and 3xTg at postnatal day 7 (P7) and 3 months of age (3Mo). f Relative volume measurements of GCL, g DG, h hippocampus, to the whole brain for NTG and 3xTg at postnatal day 7 (P7) and 3 months of age (3Mo). i Average cortical thickness for NTG and 3xTg at 1 month of age. Data represented as mean ± SD. A two-tailed, unpaired Student’s t-test was used for statistical analysis (*p < 0.05), n = 3–6 for each group.

Fig. 2. Decline of neural stem/progenitor cells and immature neurons in the SGZ of 3xTg mice at various time points.

a Representative immunostaining of Sox2 (in red) and DCX (in green) at 1 month of age in coronal sections of NTG (Top) and 3xTg (Bottom). GCL is visualized by DAPI. Scale bar 100 µm. b Quantification of the total number of Sox2-positive cells in the SGZ at postnatal day 5 (P5); postnatal day 7 (P7); 1 month of age (1Mo); 3 months of age (3Mo); 6 months of age (6Mo); and 9 months of age (9Mo). c Quantification of the total number of DCX-positive cells in the SGZ at 1 month of age (1Mo); 3 months of age (3Mo); 6 months of age (6Mo); and 9 months of age (9Mo). d Representative immunostaining of Sox2 (in red) and S100b (in green) at 1 month of age in coronal sections of NTG (Top) and 3xTg (Bottom). GCL is visualized by DAPI. Scale bar 100 µm. e Quantification of the total number of Sox2-positive/S100b-negative cells and f Sox2-positive/S100b-positive cells in the SGZ at 1 month of age (1Mo); 3 months of age (3Mo); and 9 months of age (9Mo). g Graphs representing the percentage of S100b-positive cells out of the total Sox2-positive cells in the SGZ at 1 month of age (1Mo); 3 months of age (3Mo); and 9 months of age (9Mo). Data represented as mean ± SD. A two-tailed, unpaired Student’s t-test was used for statistical analysis (p < 0.05*, p < 0.01**), b and c: n = 3 for P5 and 6Mo; n = 4 for P7, 1Mo and 9Mo; n = 6 for 3Mo. e, f and g: n = 3 for 1Mo and 9Mo; n = 4 for 3Mo.

Fig. 3. Decline in the Hopx-expressing population in the SGZ of 3xTg mice at as early as postnatal days.

a Representative fluorescent images of Hopx (in green) and Sox2 (in red) at 1 month of age in coronal section of NTG (Top) and 3xTg (Bottom). GCL is visualized by DAPI. Scale bar 100 µm. b Quantification of the total number of Hopx-positive/Sox2-positive cells in the SGZ at postnatal day 5 (P5); postnatal day 7 (P7); 1 month of age (1Mo); 3 months of age (3Mo); 6 months of age (6Mo) and 9 months of age (9Mo). c Representative fluorescent images of Hopx (in yellow), Sox2 (in green) and DAPI (Blue) at postnatal day 0 in coronal section of NTG (Top) and 3xTg (Bottom). Scale bar 100 µm. Magnified view of fluorescent images were shown for merged, Sox2 and Hopx on the left. d Quantification of the total number of Hopx-positive/Sox2-positive cells in the primitive structure of the dentate gyrus at postnatal day 0. Data represented as mean ± SD. A two-tailed, unpaired Student’s t-test was used for statistical analysis (p < 0.05*, p < 0.01**), n = 4 for P5 and P7; n = 3 for P0, 1Mo, 3Mo, 6Mo and 9Mo.

Fig. 4. Reduced proliferation of neural stem/progenitor cells in the SGZ of 3xTg mice at as early as postnatal days.

a Representative fluorescent images of EdU staining in the SGZ after 2 hours of incorporation. Scale bar 100 µm. b Quantification of EdU-positive cells in the SGZ of NTG and 3xTg mice at multiple time points. postnatal day 5 (P5); postnatal day 7 (P7); 1 month of age (1Mo); 3 months of age (3Mo) and 6 months of age (6Mo). c Representative fluorescent images of pHH3 staining in the SGZ of NTG and 3xTg mice at multiple time points. Scale bar: 100 µm. d Quantification of pHH3-positive cells in the SGZ of NTG and 3xTg mice at multiple time points. postnatal day 5 (P5); 1 month of age (1Mo); 3 months of age (3Mo); 6 months of age (6Mo) and 9 months of age (9Mo). Data represented as mean ± SD. Statistical analysis was performed with an unpaired two-tailed Student’s t-test (p < 0.05*, p < 0.01**). b n = 6 for P5; n = 4 for P7; n = 3 - 4 for 1Mo and 6Mo; and n = 4 - 5 for 3Mo. d n = 3.

Fig. 5. Early transcriptional changes in the SGZ of 3xTg at 1 month of age.

a Methodology. b Cell sorting strategy of neural stem/progenitor cells using Nestin-YFP. c MA plot of RNA-seq. Significant genes are labelled in Blue with 1391 significant genes, 720 genes as down-regulated and 671 as up-regulated in the 3xTg cells. n = 3. d Gene ontology (GO) analysis of genes shown in c, up- or down- regulated respectively. e Heatmap showing the expression pattern of significantly altered genes grouped under different GO terms: Oxidative phosphorylation, mitochondrion, cell cycle, Alzheimer disease, calcium signaling, and extracellular matrix. Color density indicates the z-score computed from the normalized read counts. f RT-PCR validation of selected genes. Data represented as mean ± SEM. Statistical analysis was performed with an unpaired two-tailed Student’s t-test, p < 0.05*. n = 7.