miR-204-3p/Nox4 Mediates Memory Deficits in a Mouse Model of Alzheimer's Disease

Abstract

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia in the elderly, and the mechanisms of AD are not fully defined. MicroRNAs (miRNAs) have been shown to contribute to memory deficits in AD. In this study, we identified that miR-204-3p was downregulated in the hippocampus and plasma of 6-month-old APPswe/PS1dE9 (APP/PS1) mice. miR-204-3p overexpression attenuated memory and synaptic deficits in APP/PS1 mice. The amyloid levels and oxidative stress were decreased in the hippocampus of APP/PS1 mice after miR-204-3p overexpression. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (Nox4) was a target of miR-204-3p, and Nox4 inhibition by GLX351322 protected neuronal cells against Aβ_1-42-induced neurotoxicity. Furthermore, GLX351322 treatment rescued synaptic and memory deficits, and decreased oxidative stress and amyloid levels in the hippocampus of APP/PS1 mice. These results revealed that miR-204-3p attenuated memory deficits and oxidative stress in APP/PS1 mice by targeting Nox4, and miR-204-3p overexpression and/or Nox4 inhibition might be a potential therapeutic strategy for AD treatment.

Keywords: Alzheimer’s disease; Nox4; beta amyloid; memory deficits; miR-204-3p; oxidative stress; synaptic dysfunction.

Graphical abstract

Figure 1

miR-204-3p Was Decreased in the Brain and Plasma of APP/PS1 Mice (A) Heatmap diagram showing different expression patterns of miRNAs in the hippocampus of 6-month-old APP/PS1 and wild-type (WT) mice. n = 3. (B) The levels of miR-204-3p in the hippocampus of 4-, 6-, and 9-month-old APP/PS1 mice were examined by qPCR. n = 4–7. (C) The levels of miR-204-3p in the plasma of 6-month-old WT and APP/PS1 mice were detected by qPCR. n = 5. All data are presented as means ± SEM. ∗p < 0.05, ∗∗p < 0.01. ns, no significance.

Figure 2

Overexpression of Exogenous miR-204-3p Protected against Synaptic and Memory Impairment in APP/PS1 Mice (A) The levels of miR-204-3p in the hippocampus after Lv-miR-204 injection were determined by qPCR. n = 6–7. (B) The levels of miR-204-3p in the plasma after Lv-miR-204 injection were determined by qPCR. n = 8. (C) In NOR tests, the percentage of time to explore new objects was recorded. (D and E) In FC tests, the percentage of freezing time in contextual (D) and cued (E) tests was recorded. n = 17–18. (F) In the acquisition trial, the escape latency was analyzed in the MWM tests. (G–I) The number of target platform crossings (G), time in the target quadrant (H), and latency to the target quadrant (I) were recorded in the probe trial. n = 17–18. (J) The input-output curve (I-O curve) of hippocampal CA1 in Lv-WT, Lv-con, and Lv-miR-204 mice. n = 6–8 slices from three mice in each group. (K and L) LTP induced by high-frequency stimulation was evaluated in hippocampal CA1. n = 6–8 slices from three mice in each group. (M–O) The levels of synaptic proteins in the hippocampus were determined by western blotting, and quantification of the intensities normalized to β-actin was shown. n = 3. (P and Q) Representative images of dendrite morphology and quantitative analysis of dendritic spine density. n = 6 slices from three mice in each group. Scale bars: 10 μm. (R) The percentage of mushroom spines per 10 μm dendrite length in each group. n = 10 slices from three mice in each group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ns, no significance.

Figure 3

Overexpression of miR-204-3p Decreased the Amyloid Levels and Oxidative Stress in the Hippocampus of APP/PS1 Mice (A) Representative images of Aβ staining in the hippocampus of Lv-miR-204-treated APP/PS1 mice and (B) quantification of the area percentage of Aβ plaque load. n = 10 slices from three mice in each group. Scale bars: 250 μm. (C and D) The levels of TBS-soluble, TBS-T-soluble, and FA-soluble Aβ_1–40 (C) and Aβ_1–42 (D) were determined by ELISA in the hippocampus of APP/PS1 mice. n = 5. (E and F) The levels of APP and secretases in the hippocampus were examined by western blotting (E), and quantification of the intensities normalized to GAPDH as a loading control was shown (F). n = 3. (G) The level of ROS in the hippocampus of Lv-miR-204-treated APP/PS1 mice was detected by fluorescence. Scale bars: 250 μm. (H) The level of H2O2 in the hippocampus of Lv-miR-204 treated APP/PS1 mice was examined by spectrophotometry. n = 6. (I–K) The levels of 4-HNE (I), 3-NT (J), and 8-OHdG (K) in the hippocampus of Lv-miR-204-treated APP/PS1 mice were measured by ELISA. n = 4–5. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, no significance.

Figure 4

Nox4 Was a Target of miR-204-3p (A) Predicted binding sites of miR-204-3p, Nox4 3′ UTR, and Nox4 3′ UTR mutant. (B) N2a cells were cotransfected with miR-204-3p and Nox4 3′-UTR or Nox4 3′-UTR mutant for 48 h, and the relative luciferase activity was assayed. n = 6. (C) The mRNA level of Nox4 was measured in the Lv-con and Lv-miR-204 group. n = 5. (D) The protein level of Nox4 was measured in the hippocampus of Lv-miR-204-treated APP/PS1 mice by western blotting (left panel), and quantification of the intensities normalized to β-actin was shown (right panel). n = 3. (E) Cell viability of neurons treated with Lv-miR-204 or GLX351322 followed by Aβ42 was examined by CCK-8 assays. n = 6. (F) Cell viability of neurons treated with Lv-miR-204 with or without Lv-Nox4 followed by Aβ42 was examined by CCK-8 assays. n = 6. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ns, no significance.

Figure 5

Nox4 Inhibition Protected against Synaptic and Memory Dysfunctions in APP/PS1 Mice (A) The mRNA level of Nox4 subunit p22phox in the hippocampus of APP/PS1 mice treated with GLX351322 was determined by qPCR. n = 4. (B) The protein level of p22phox in the hippocampus of APP/PS1 mice treated with GLX351322 by western blotting. n = 3. (C) In NOR tests, the percentage of time to explore new objects was recorded. n = 16. (D and E) In FC tests, the percentage of freezing time in contextual (D) and cued (E) tests was recorded. n = 15. (F) In the acquisition trial, the escape latency was analyzed in the MWM tests. (G–I) The number of target platform crossings (G), time in the target quadrant (H), and latency to the target quadrant (I) were recorded in the probe trial. n = 14–15. (J) The I-O curve of hippocampal CA1 of WT-vehicle mice, APP-vehicle mice, and APP-GLX35132 mice. n = 8–9 slices from three mice in each group. (K and L) LTP induced by high-frequency stimulation was evaluated in hippocampal CA1. n = 8–9 slices from three mice in each group. (M–O) The levels of synaptic proteins in the hippocampus were determined by western blotting, and quantification of the intensities normalized to β-actin was shown. n = 3. (P and Q) Representative images of dendrite morphology and quantitative analysis of dendritic spine density in each group. n = 6 slices from three mice in each group. Scale bars: 10 μm. (R) The percentage of mushroom spines per 10 μm dendrite length in each group. n = 10 slices from three mice in each group. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ns, no significance.

Figure 6

Nox4 Inhibition Decreased Amyloid Levels and Oxidative Stress in the Hippocampus of APP/PS1 Mice (A and B) Representative image of Aβ staining in the hippocampus of GLX351322-treated APP/PS1 mice and quantification of the area percentage of Aβ plaque load. n = 10 slices from three mice in each group. Scale bars: 250 μm. (C and D) The levels of TBS-soluble, TBS-T-soluble, and FA-soluble Aβ_1–40 (C) and Aβ_1–42 (D) were determined by ELISA in the hippocampus of GLX351322-treated APP/PS1 mice. n = 3. (E and F) The levels of APP and secretases in the hippocampus were examined by western blotting, and quantification of the intensities normalized to GAPDH as a loading control was shown. n = 3. (G) The level of ROS in the hippocampus of GLX351322-treated APP/PS1 mice was detected by fluorescence. Scale bars: 250 μm. (H) The level of H₂O₂ in the hippocampus of GLX351322-treated APP/PS1 mice was examined by spectrophotometry. n = 5. (I–K) The levels of 4-HNE (I), 3-NT (J), and 8-OHdG (K) in the hippocampus of GLX351322-treated APP/PS1 mice were measured by ELISA. n = 5–6. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ns, no significance.