MicroRNA-338-5p alleviates neuronal apoptosis via directly targeting BCL2L11 in APP/PS1 mice

The expression of miR-338-5p significantly decreased in APP/PS1 mice during the progression of AD

To elucidate the correlation between AD pathology and miR-338-5p, we first detected the expression pattern in the brain of APP/PS1 mice at the age of 2,4,6,8 and 10 months old [22]. The results of qRT-PCR suggested that the expression of miR-338-5p declined dramatically in 8-month-old APP/PS1 mice, which became further lower in 10-month-old APP/PS1 mice, in comparison to the wild-type (WT) mice (Figure 1A). However, among the brains of APP/PS1 mice aged 2, 4 and 6 months old, no significant change was observed in miR-338-5p expression level (Figure 1A). Furthermore, the expression level of Aβ 40 (Figure 1B) and Aβ 42 (Figure 1C) also increased significantly in 8-month-old APP/PS1 mice, which were further shown remarkable elevation in 10-month-old APP/PS1 mice, compared with wild-type mice. All of these synchronous changes between miR-338-5p and Aβ production in APP/PS1 mice indicated the level of miR-338-5p may be negatively correlated with the pathogenesis of AD.

Figure 1. The expression of miR-338-5p decreased in APP/PS1 mice during AD progression. (A) qRT-PCR analysis of miR-338-5p expression in the brains of wild-type (WT) and APP/PS1 mice with increasing age. Data are presented as relative to that of 2-month-old mice. (B, C) ELISA analysis of Aβ 40 (B) and Aβ 42 (C) level in the brains of WT and APP/PS1 mice with increasing age. Data are presented as ng Aβ 40 or Aβ 42 per ml total protein samples. Results are presented as mean ± SD. n = 6 in each group. *P < 0.05; **P < 0.01; ***P<0.001.

MiR-338-5p attenuated cognitive dysfunction in APP/PS1 mice

We reversed the downregulation of miR-338-5p in the brain of APP/PS1 mice aged 8-month-old and 10-month-old through intrahippocampal injection of lentiviral vector overexpressing miR-338-5p to test whether miR-338-5p affects cognitive deficits in APP/PS1 mice [23]. The effectiveness of lentiviral vector overexpressing miR-338-5p in the brain of APP/PS1 mice was validated by qRT-PCR (Figure 2A). By performing the Morris water maze, we then evaluated whether miR-338-5p affects the ability of spatial learning and memory in APP/PS1 mice. In MWM, the relative average escape latency of five training days and the escape latency of each training day were used to show the learning ability. Significant differences were observed in escape latency from day 3 onwards between group APP/PS1+vector and group APP/PS1+miR-338-5p (Figure 2B). Furthermore, in the probe trail, the number of platform-crossing and target quadrant traveling time indicated the memory activities. Moreover, compared to the control group, the time spent in the target quadrant (Figure 2C) and frequency to cross the platform (Figure 2D) were significantly increased in group APP/PS1+miR-338-5p indicated by the probe trials 24 h after the last training session. It is noteworthy that there is no significant difference in the average swimming speedamong the groups (Figure 2E), suggesting that miR-338-5p-ameliorated behavioral performances of APP/PS1 mice resulted from cognitive processes, instead of non-cognitive behavioral activities. Taken together, the results of MWM tests demonstrated that overexpression of miR-338-5p in the brain attenuated impaired spatial learning and memory in APP/PS1 mice.

Figure 2. Lentiviral overexpression of miR-338-5p through intrahippocampal injection improved cognitive dysfunction in APP/PS1 mice. (A) qRT-PCR analysis of miR-338-5p expression in the brain. Data are presented as relative to that of 8-month-old mice infected with lentiviral empty vector. (B) Spatial learning of 8-month-old or 10-month-old APP/PS1 mice was detected as escape latency at different days after training in water maze. (C, D) Spatial memory of 8-month-old or 10-month-old APP/PS1 mice was assessed by probe trials 24 h after the last training session. The number of platform crossing (C), time spent in target quadrant (D) and swimming speed (E) of 8-month-old or 10-month-old APP/PS1 mice were recorded. Results are presented as mean ± SD. n = 6 in each group. *P < 0.05; **P < 0.01; ***P<0.001.

MiR-338-5p attenuated the amyloid plaque deposition of APP/PS1 mice

We determined whether miR-338-5p decreased amyloid plaque deposition, a hallmark pathologic change scattered in AD brain, to further explore the association between miR-338-5p and AD progression [24]. Compared with control group, the expression levels of Aβ 40 (Figure 3A) and Aβ 42 (Figure 3B) tested by ELISA assay showed remarkable reduction in APP/PS1 mice when overexpressing miR-338-5p in the brain. To further validate these results, Thioflavin-S staining was performed to detect the amyloid plaque in brain slices. Consistent with the results of ELISA assay, both the number (Figure 3C–3E) and area (Figure 3C, 3D, 3F) of amyloid plaque deposition in hippocampus and cortex of APP/PS1 mice significantly decreased when overexpressing miR-338-5p. These results suggested that miR-338-5p decreased amyloid plaque deposition in APP/PS1 mice, which was consistent with its protective effect on cognitive deficits in APP/PS1 mice (Figure 2).

Figure 3. MiR-338-5p attenuated amyloid plaque deposition in APP/PS1 mice. (A, B) ELISA analysis of Aβ 40 (A) and Aβ 42 (B) level in APP/PS1 mice. Data are presented as ng Aβ 40 or Aβ 42 per ml total protein samples. (C, F) Thioflavin-S was used to stain the brain sections to show the number and area of amyloid plaques in hippocampus and cortex. Representative images of plaques in hippocampus and cortex aged 8-month-old (C) or 10-month-old (D). (E, F) Quantification analysis of the number (E) and area (F) of amyloid plaque. The plaques were shown with red fluorescence and cell nuclei were stained with blue fluorescence by DAPI. Scale bar=50 μm. Results are presented as mean ± SD. n = 6 in each group. *P < 0.05; **P<0.01.

MiR-338-5p retarded the apoptotic loss of neurons in APP/PS1 mice

Several miRNAs show the potential to attenuate the apoptotic loss of neurons, such as miR-23a and miR-96 [25, 26]. Furthermore, neuronal loss and the ensuing cognitive dysfunction are inevitable consequences, which are also vital contributing factors to AD progression [27]. As shown by the TUNEL assay, the number of apoptotic neuron in the hippocampus and cortex significantly increased in 8-month-old and 10- month-old APP/PS1 mice, in comparison to wild-type mice (Figure 4A–4C). Interestingly, we showed that the increased number of apoptotic neurons in the brain of APP/PS1 mice was remarkably recovered by overexpressing miR-338-5p (Figure 4A–4C). Hence, these results suggested that miR-338-5p decelerated the apoptotic loss of neurons in APP/PS1 mice.

Figure 4. MiR-338-5p retarded apoptotic loss of neurons in APP/PS1 mice. (A) The representative immunofluorescent images of TUNEL-positive neurons in 8-month-old WT and APP/PS1 mice. Green staining indicated TUNEL-positive cells and red staining indicated neurons. (B, C) Quantification of TUNEL-positive neurons in hippocampus (B) and cortex (C) of 8-month-old or 10-month-old WT and APP/PS1 mice. Scale bar=50 μm. Results are presented as mean ± SD. n = 5 in each group. **P < 0.01.

MiR-338-5p ameliorated neuron apoptosis induced by Aβ accumulation

We isolated the primary hippocampal neurons from APP/PS1 mice brain, culturing them in vitro and then treated them with Aβ40, which can cause cell death of neurons by prolonged accumulation [28] to further probe into the ameliorating role of miR-338-5p in neuron. As shown by the TUNEL assay, the increased number of apoptotic neurons treated by Aβ40 treatment for consecutive 3 and 7 days was remarkably mitigated when overexpressing miR-338-5p in neurons (Figure 5A, 5B). To further validate these results, we then detected the expression level of cleaved caspase-3. Indicated by the results of Western blot, Aβ40-induced increased cleaved caspase-3 expression in neurons was rescued by overexpressing miR-338-5p (Figure 5C. 5D), which was consistent with the results of TUNEL assay. Therefore, miR-338-5p protected against neuron apoptosis caused by Aβ40 accumulation, indicated by these in vitroo experiments.

Figure 5. MiR-338-5p rescued neuron apoptosis induced by Aβ accumulation. (A–C) Primary hippocampal neurons were isolated infected with lentiviral miR-338-5p expressing vector or lentiviral empty vector. Two days later, neurons were cultured for consecutive 3 or 7 days with or without 5 mM Aβ40. (A, B) The representative immunofluorescent images (A) and quantification (B) of TUNEL-positive hippocampal neurons in vitro. (C, D) The representative western blot images (C) and quantification analysis of cleaved caspase-3 expression (D). Scale bar=50 μm. Results are presented as mean ± SD. n = 3 in each group. *P < 0.05; **P<0.01.

MiR-338-5p attenuated neuron apoptosis by directly targeting BCL2L11

To further clarify the underlying mechanism of miR-338-5p decelerating neuron apoptosis under Aβ40 treatment, Targetscan was used to conduct targeting prediction analysis based on bioinformatics [29]. We focused on the BCL2-like 11 (BCL2L11) (Figure 6A), since it has been previously reported to contribute to neuron apoptosis [30], The 3ʹ-UTR of BCL2L11 with wild-type or mutant seed sequence recognition sites was cloned into a luciferase reporter to determine whether BCL2L11 was a direct target of miR-338-5p. The results showed that the overexpressing miR-338-5p resulted in a significant reduction in the luciferase activity of the plasmid carrying BCL2L11 3ʹUTR-WT, while luciferase activity in cells transfected with the BCL2L11 3ʹUTR-Mut plasmid did not change significantly (Figure 6B). Conversely, silencing miR-338-5p showed remarkable increase in luciferase activity of wild-type 3’UTR of BCL2L11 with the mutant construct unaffected (Figure 6C). These results suggest that BCL2L11 can be targeted by miR-338-5p directly. Furthermore, we next determined whether miR-338-5p inhibits BCL2L11 expression in neurons. The results showed that overexpressing miR-338-5p reduced BCL2L11 expression, and conversely, silencing miR-338-5p increased BCL2L11 expression in neurons, further supporting its negative role in modulating BCL2L11 expression (Figure 6D–6F). Consistent with the in vitroo experiments, overexpressing miR-338-5p also significantly decreased the expression level of BCL2L11 in the brain of APP/PS1 mice (Supplementary Figure 1). We then hypothesized the suppressed BCL2L11 expression contributed to the ameliorating role of miR-338-5p against Aβ40-induced neuron apoptosis. To verify this hypothesis, BCL2L11 expression in neurons was restored through overexpression mediated by transient transfection. Astonishingly, we found that the protecting effect of retarding apoptotic loss of neuron (Figure 6G, 6H) and ameliorating neuron apoptosis elicited by miR-338-5p (Figure 6I, 6J) were completed reversed along with BCL2L11 restoration. Therefore, all of these results suggested that the anti-apoptotic effect of miR-338-5p on neurons treated by Aβ40 depended on the expression of its target, BCL2L11, unveiling the important role of miR-338-5p/BCL2L11 axis in attenuating neuron apoptosis treated by Aβ40 in vitro.

Figure 6. miR-338-5p ameliorated neuronal apoptosis by directly targeting BCL2L11. (A) TargetScan was used to predict the binding sites of miR-338-5p within the 3ʹ-UTR of BCL2L11. (B) Overexpressing miR-338-5p resulted in a remarkable decrease in luciferase activity of BCL2L11-WT and exerted no effect on luciferase activity of BCL2L11-Mut in primary hippocampal neurons. (C) Silencing miR-338-5p caused a significant increase in luciferase activity of BCL2L11-WT and exerted no effect on luciferase activity of BCL2L11-Mut in primary hippocampal neurons. (D–F) Relative BCL2L11 mRNA level (D) and protein expression (E, F) of primary hippocampal neurons transfected with NC mimic or miR-338-5p mimic, or NC antagomir or miR-338-5p antagomir determined by qRT-PCR and Western blot respectively. (G–I) Primary hippocampal neurons were transfected with pcDNA3.1-vector or pcDNA3.1-BCL2L11. Two days later, neurons were cultured for consecutive 7 days with or without 5 mM Aβ40. (G, H) The representative immunofluorescent images (G) and quantification (H) of TUNEL-positive hippocampal neurons in vitro. (I, J) The quantification of TUNEL-positive hippocampal neuron in vitro. The representative western blot images (I) and quantification analysis (J) of cleaved caspase-3 expression. Scale bar=50 μm. Results are presented as mean ± SD. n = 3 in each group. *P < 0.05; **P<0.01.

Animals

In this study, all experimental protocols and procedures were approved by the Medical School of Sun Yat-Sen University, and were conducted in strict accordance with National Institutes of Health Guidelines for the use of experimental animals. Wild-type mice (male, C57BL/6) and APP/PS1 transgenic mice (male, APP/PS1) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animals were housed in polypropylene cages and the relative humidity was maintained at 50 ± 10% and the room temperature was maintained at 22°C, with a 12 h light-dark cycle. All animals were sacrificed by exposure to carbon dioxide at the end of the experiment.

Western blot

Samples of tissues and cells were lysed in RIPA lysis buffer with phosphatase and protease inhibitors. Protein samples were subjected to 10% SDS-PAGE and transferred to PVDF membranes. 5% non-fat milk in 0.1% TBST buffer was used to blocked membranes at 4 °C overnight. The membranes were subsequently incubated with antibodies cleaved caspase-3 (Cell Signaling Technology #9661, 1:1000), BCL2L11 (Cell Signaling Technology #2933S, 1:1000), β-actin (Cell Signaling Technology #3700S, 1:2000). The protein–antibody complex was detected with HRP-conjugated secondary antibodies and enhanced chemiluminescence. Image J software (GE Healthcare, USA) was used to perform the analysis of western blot.

Quantitative RT-PCR

Total RNA was extracted from cultured cells in accordance with the manufacturer’s protocol (Invitrogen). By using Superscript First-Strand cDNA Synthesis Kit (18080-051, Invitrogen, Carlsbad, CA), Total RNA (1 μg) was reverse transcribed into cDNAs. SYBR Premix Ex Taq II kit (DRR081A, TAKARA, Japan) on LightCycler 480 System (Roche, Switzerland) was used to performed quantitative RT-PCR.

Intrahippocampal injection of lentivirus

APP/PS1 mice aged 7-month-old or 9-month-old were anesthetized with chloral hydrate and placed in a stereotactic frame (stereotaxic apparatus 51600, Stoelting, USA), positioned in a stereotaxic instrument. Then, 2 μL lentivirus miR-338-5p, or lenti-vector was injected into the hippocampus bilaterally using the following coordinates: −2.7 mm dorsal/ventral, −2.7 mm anterior/posterior, ±3.2 mm medial/lateral from the bregma [23]. By using a syringe (Syringe pumps 51600z, Stoelting, USA) and a 27-gauge needle, the preparation was injected at a speed of 0.5 μL/min over a period of 4 min. One month after intrahippocampal injection of lentivirus, mice were used for subsequent biochemical and behavior analyses.

Measurement of Aβ40 and Aβ42

An enzyme-linked immunosorbent assay (ELISA) was performed to detect the concentration of Aβ40 and Aβ42 in the hippocampus of APP/PS1 mice and wild-type mice. Briefly, mice were first anesthetized by chloral hydrate and we removed the brain tissues quickly. Next, brain tissues were homogenized with RIPA buffer on ice, and then centrifuged at 12,000 rpm for 10 min to collect the supernatants. Aβ40 Mouse ELISA Kit (Invitrogen) and Aβ42 Mouse ELISA Kit (Invitrogen) were used to measure the concentration of Aβ40 and Aβ42 in strict accordance with the manufacturer's protocols.

Behavioral test

Morris water maze (MWM) was used to assess the spatial memory performance of mice according to the previous report [50]. In brief, animals were first placed in the water maze pool (temperature 22 ± 1°C, depth 50 cm, diameter 150 cm) for 2 days to adapt to the environment. In training trials, mice were released from three different quadrants and trained to find the hidden platform for five consecutive days. The platform was removed on the sixth day, and platform-crossing times and target quadrant traveling time were recorded, which indicated the memory ability of the animals. In probe trials, the platform was removed 24 hours after the last training. Mice were set free to swim at the starting point for 60 s, and duration in the target quadrant and the number of platform crossing were recorded. ANY-maze software (Stoelting Co.) was used to track the animal behavior automatically.

Tissue preparation and immunofluorescence

Mice were anesthetized with chloral hydrate and perfused with 4% paraformaldehyde transcardially. The brain tissues were paraffin embedded and sectioned at the thickness of 5 μm. For thioflavin-S staining, we used 0.2% thioflavin-S (T1892, Sigma-Aldrich) to stain brain sections for 10 min. After the brain sections were washed with PBS for three times, the brain sections were photoed by an IX53 fluorescence microscope (Olympus). Image J software (GE Healthcare, USA) was employed to analyze the quantification of images.

TUNEL assay

For TUNEL staining of brain sections, the anti-NeuN antibody (Cell Signaling Technology #24307S, USA, 1:200) diluted in PBS was incubated with the sections overnight at 4°C. Goat anti-rabbit antibody 546 (red, Santa Cruz Biotechnology, USA, 1:200) were then biotinylated the tissue sections for 60 min. TUNEL staining was performed in accordance with the manufacture’s instructions of TUNEL system kit (Promega, USA). Image-pro Plus software (GE Healthcare, USA) was employed to analyze the number of neurons stained positively for TUNEL and NeuN in hippocampus and cortex by two persons blinded to the treatments.

For TUNEL staining of neuron, the cultured primary neurons were washed once with PBS, fixed with 4% paraformaldehyde at room temperature for 1 h and permeabilized using 0.1% Triton X-100. The anti-NeuN antibody (Cell Signaling Technology #24307S, USA, 1:200) was incubated with the fixed neuron overnight at 4°C. Then TUNEL reaction mixture were incubated with the fixed neurons at 37 °C for 1 h. DAPI was added to the wells for 5 min to stain nuclear after rinsing the cells with PBS. We counted the TUNEL-positive cells manually, and calculated the percentage of positive cells for each sample.

Culture and treatment of primary hippocampal neurons

The hippocampi were removed from the brains under a light microscope. Hippocampal neurons were dissociated with DNase and 0.125% trypsin. Then the neurons were seeded in 6-well culture plates for Western blotting at a density of 1 × 10⁵ cells/cm² or in poly-d-lysine-coated glass coverslips for immunocytochemistry staining at a density of 1 × 10⁴ cells/cm². Cultures were maintained in neurobasal A medium (Invitrogen, Carlsbad, CA) containing 0.5 mM glutamine and 2% B27 supplement in an incubator (95% air, 5% CO2) at 37 °C. For the treatment of Aβ40 (FC3-018-01, Phoenix Pharmaceuticals), the neurons were cultured for consecutive 3 or 7 days with and without 5 mM Aβ40. Fresh medium was replaced every 2 days in the presence and absence of 5 mM Aβ40 until the end of the experiment.

Statistical analysis

The Shapiro–Wilk test was used to test whether the data were normally distributed and Levene's test was used to confirm that the data had no significant heterogeneity of variance. Data were presented as means ± SD and analyzed by one-way ANOVA followed by Tukey’s post-hoc test. Sample size was calculated by using the SPSS 11 software to achieve an 80% power at a significance level of 0.05. The GraphPad Prism software (version 7.0, CA, USA) was used to conduct the statistical analyses. In all cases, statistical significance was accepted at P < 0.05.