Inhibition of Drp1 Ameliorates Synaptic Depression, Aβ Deposition, and Cognitive Impairment in an Alzheimer's Disease Model

Abstract

Excessive mitochondrial fission is a prominent early event and contributes to mitochondrial dysfunction, synaptic failure, and neuronal cell death in the progression of Alzheimer's disease (AD). However, it remains to be determined whether inhibition of excessive mitochondrial fission is beneficial in mammal models of AD. To determine whether dynamin-related protein 1 (Drp1), a key regulator of mitochondrial fragmentation, can be a disease-modifying therapeutic target for AD, we examined the effects of Drp1 inhibitor on mitochondrial and synaptic dysfunctions induced by oligomeric amyloid-β (Aβ) in neurons and neuropathology and cognitive functions in Aβ precursor protein/presenilin 1 double-transgenic AD mice. Inhibition of Drp1 alleviates mitochondrial fragmentation, loss of mitochondrial membrane potential, reactive oxygen species production, ATP reduction, and synaptic depression in Aβ-treated neurons. Furthermore, Drp1 inhibition significantly improves learning and memory and prevents mitochondrial fragmentation, lipid peroxidation, BACE1 expression, and Aβ deposition in the brain in the AD model. These results provide evidence that Drp1 plays an important role in Aβ-mediated and AD-related neuropathology and in cognitive decline in an AD animal model. Therefore, inhibiting excessive Drp1-mediated mitochondrial fission may be an efficient therapeutic avenue for AD.SIGNIFICANCE STATEMENT Mitochondrial fission relies on the evolutionary conserved dynamin-related protein 1 (Drp1). Drp1 activity and mitochondria fragmentation are significantly elevated in the brains of sporadic Alzheimer's disease (AD) cases. In the present study, we first demonstrated that the inhibition of Drp1 restored amyloid-β (Aβ)-mediated mitochondrial dysfunctions and synaptic depression in neurons and significantly reduced lipid peroxidation, BACE1 expression, and Aβ deposition in the brain of AD mice. As a result, memory deficits in AD mice were rescued by Drp1 inhibition. These results suggest that neuropathology and combined cognitive decline can be attributed to hyperactivation of Drp1 in the pathogenesis of AD. Therefore, inhibitors of excessive mitochondrial fission, such as Drp1 inhibitors, may be a new strategy for AD.

Keywords: Alzheimer's; Drp1; amyloid; mitochondria; synaptic depression.

Figure 1.

mdivi-1 inhibits Aβ-mediated mitochondrial fragmentation and dysfunction in neuroblastoma cells. A–C, SK-N-MC cells stably expressing mito-YFP (SK/mito-YFP) were treated with Aβ (1–42) oligomer (10 μm) in the presence or absence of mdivi-1 (25 μm) for 4 h. Then, the cells were imaged using a fluorescence microscope (A), cells with fragmented mitochondria were counted (B), and mitochondrial length was measured (C). D–F, SK-N-MC cells were treated with Aβ (10 μm) in the presence or absence of mdivi-1 (25 μm) for 8 h. D, Then, the alteration of mitochondrial membrane potential was monitored by the Mito-Probe JC-1 assay. E, Intracellular ROS level was measured by a H₂DCF-DA fluorescence ROS detection assay. F, Next, the cellular total ATP level was examined by an ATP bioluminescence detection assay. Scale bar, 10 μm. Data are presented as the means ± SEM (n = 4). One-way ANOVA followed by Turkey post hoc comparisons tests were performed in all statistical analyses (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 2.

mdivi-1 inhibits mitochondrial fragmentation and ROS production in Aβ-treated primary hippocampal neurons. A–D, Primary hippocampal neurons treated with Aβ (1–42) oligomer (5 μm) in the presence or absence of mdivi-1 (25 μm) for 6 h and then the mitochondria were stained with a selective mitochondrial probe (100 nm) (MitoTracker). Mitochondria were imaged by a confocal microscopy (A) and the length (B) and intensity (C) of the mitochondria were measured in confocal 3D images. Intracellular ROS accumulation of Aβ-treated hippocampal neurons was assayed using H₂DCF-DA fluorescent dye (D). E, Mitochondrial ROS induction by Aβ was detected by MitoSox Red staining in hippocampal neurons. Scale bar, 20 μm. Data are presented as the means ± SEM (n = 4). One-way ANOVA followed by Turkey post hoc comparisons tests were performed in all statistical analyses (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3.

mdivi-1 suppresses synaptic depression in Aβ-treated primary cultured hippocampal neurons. A, Representative images of primary hippocampal neurons transfected with vG-pH in each condition. Top images are the resting condition, middle images are ΔF images of the 100 action potential. Bottom is the visualization of all synaptic boutons by applying NH₄Cl. B, Representative traces of synaptic vesicle exocytosis stimulated by 100 action potential from control, mdivi-1-treated, Aβ-treated, and mdivi-1+Aβ-treated neurons. Neurons transfected with vG-pH were stimulated at 10 Hz 10 s with or without mdivi-1 and Aβ. Intensities were normalized to the peak NH₄Cl response. C, Mean values of amplitudes of 100 action potential response in control (n = 9 cells), mdivi-1-treated (n = 8), Aβ-treated (n = 15), and mdivi-1+Aβ-treated (n = 12) neurons. Scale bar, 5 μm. Data represent means ± SEM. One-way ANOVA followed by Turkey post hoc comparisons tests were performed in all statistical analyses (**p < 0.001).

Figure 4.

Treatment of mdivi-1 attenuates hippocampal memory impairment in APP/PS1 mice. APP/PS1 mice and age-matched WT littermates were administered with vehicle or mdivi-1 (10 mg/kg, 40 mg/kg) daily for 4 weeks. The effect of mdivi-1 on learning and memory in APP/PS1 mice versus vehicle-treated control mice by MWM test and passive avoidance test was determined. The average latency to escape to the hidden platform during each day of training sessions (A), the time spent in the target quadrant where the hidden platform was previously placed during probe trial (B), the mean number of mice crossing the target quadrant during probe trial (C), typical swimming traces during probe test (D), and visual swimming speed test (E) of MWM test were performed. F, Latency time in passive avoidance task was measured. Data represent means ± SEM (n = 7 per group). One-way ANOVA followed by Bonferroni's post hoc comparisons tests were performed in statistical analyses (*p < 0.05, **p < 0.01, ***p < 0.001 compared with the vehicle-treated control group).

Figure 5.

Administration of mdivi-1 reduces mitochondrial fragmentation and ROS production in the brain of APP/PS1 mice. A, APP/PS1 mice were administered with mdivi-1 (10 and 40 mg/kg) daily for 38 d and control mice were give vehicle. After brain dissection, the tissues were stained with DAPI and MitoTracker for 20 min. Then, the fluorescence images (63× and zoom in 10×) were captured by a confocal microscopy and length of mitochondrial length was measured. B, C, APP/PS mice were administered with mdivi-1 (10 mg/kg, 40 mg/kg) daily for 38 d. After brain dissection, the tissues were stained with DAPI and anti-4-HNE antibody. Then, the fluorescence images (40×) were captured by confocal microscopy (B) and the fluorescence intensity of 4-HNE was measured (C). D, E, Brain sections of the indicated groups were stained with anti-Iba-1 antibody, anti-NeuN antibody, anti-GFAP antibody, and anti-MAP2 antibody and immunohistochemical staining in hippocampus (CA1) was imaged with a confocal microscope. Scale bar, 20 μm. Data represent means ± SEM (n = 5 per group). One-way ANOVA followed by Turkey post hoc comparisons tests were performed in all statistical analyses (*p < 0.05, **p < 0.01, ***p < 0.001 vs the vehicle-treated control group).

Figure 6.

mdivi-1 reduces the expression of BACE1 and APP CTF-β in the brain of APP/PS1 mice. APP/PS1 mice were administered with mdivi-1 (10 mg/kg, 40 mg/kg) daily for 38 d. A, Expression of BACE1 and sAPPβ was examined by Western blot analysis with whole-brain protein extracts. B, C, Optical density of BACE1 (B) and sAPPβ (C) protein by Western blotting was quantified with Image software. D, Expression level of BACE1 mRNA was determined by quantitative RT-PCR analysis with mRNA extracted from whole-brain tissues. Values are means ± SEM (n = 6 per group). One-way ANOVA followed by Turkey post hoc comparisons tests were performed in all statistical analyses (*p < 0.05, **p < 0.01, ***p < 0.001 vs the vehicle-treated control group).

Figure 7.

mdivi-1 reduces cerebral Aβ load in the brain of APP/PS1 mice. APP/PS1 mice were administered with mdivi-1 (10 mg/kg, 40 mg/kg) daily for 38 d. Brain sections from vehicle-treated or mdivi-1-treated APP/PS1 mice were incubated with an antibody against Aβ (6E10) and visualized by DAB staining. The amyloid plaques in hippocampus and cortex were identified with microscopy (A) and the neurite plaques in hippocampus (B) and cortex (C) were quantified. Scale bar, 800 μm. Values are means ± SEM (n = 5 per group). One-way ANOVA followed by Turkey post hoc comparisons tests were performed in all statistical analyses (**p < 0.01).

Figure 8.

There was no adverse effect of chronic mdivi-1 treatment in APP/PS1 mice. To assess the toxicity of mdivi-1, APP/PS1 mice were given 0, 10, or 40 mg/kg/d mdivi-1 orally for 40 d. mdivi-1-administered or vehicle-treated male mice from 7 months of age were assessed for final body weights and organs weights. A, Body weights measured at the end of the dosing period were indistinguishable from vehicle-administered APP/PS1 mice or mdivi-1-treated APP/PS1 mice, which verified the toxicity of mdivi-1 in APP/PS1 mice. B, In addition, there were no differences in major organ weights among the study cohort, including brain weight. There was no statistically significant change among any of the group within the measures as determined by one-way ANOVA with Tukey's post hoc analysis.