Background/Aims: MicroRNA-9 (miR-9) plays important roles in nervous system diseases such as glioblastoma and neurodegenerative disorders. However, how miR-9 contributes to dementia requires further study. In this study, we evaluated the role of miR-9 in dementia and the molecular mechanisms underlying its effects. Methods: A rat model of dementia was created by occlusion of the bilateral common carotid artery (2VO) for 8 weeks. Learning and memory were assessed using the Morris Water Maze (MWM). MicroRNA expression profiling was performed according to a protocol provided by LC Sciences, and quantitative real-time PCR (qRT-PCR) was used to detect the level of miR-9. Transmission electron microscopy (TEM) and hematoxylin-eosin (HE) staining were used to assess pathological changes in brain tissue. Western blot and immunofluorescence were employed to detect the expression of β-site APP cleaving enzyme 1 (BACE1) and c-AMP response element-binding protein (CREB). Results: Learning and memory were significantly impaired in 2VO rats, and these changes were accompanied by neuronal loss and glial activation in brain tissues. miR-9 was greatly upregulated in both the hippocampus and cortex of rats following 2VO. Knockdown of endogenous miR-9 via lentiviral vector-mediated delivery of its antisense molecule (lenti-pre-AMO-miR-9) reduced the vulnerability to dementia, reversed the increase in BACE1 expression, and ameliorated the reduction in CREB expression triggered by 2VO. BACE1 protein levels were significantly increased, but CREB protein levels were significantly decreased in the presence of miR-9 in cultured neonatal rat neurons (NRNs). AMO-miR-9 rescued the upregulation of BACE1 and downregulation of CREB elicited by miR-9 in rats. Dual luciferase assay experiments showed that overexpression of miR-9 inhibited the expression of CREB by targeting its 3’UTR domain. CREB protein was downregulated by miR-9 overexpression which was reversed by miR-9 inhibition in cultured NRNs. TEM imaging showed that miR-9 caused damage to NRNs, which was reversed by addition of AMO-miR-9. Conclusion: We conclude that miR-9 plays an important role in regulating the process of dementia induced by 2VO in rats by increasing BACE1 expression via downregulation of CREB.

Chronic brain hypoperfusion (CBH) is a common clinical feature of Alzheimer’s disease (AD) and vascular dementia (VD), which can cause deficiencies in learning and memory [1, 2]. CBH has been demonstrated to induce autophagic-lysosomal neuropathology [3], reduced dendritic spine density [4], glial activation [5], inflammatory reactions and white matter damage [6].

Various factors, such as hypertension, diabetes, high cholesterol, cardiovascular diseases, and smoking, increase the risk for CBH, which eventually results in decreased blood supply to the brain [7]. It has recently been demonstrated that CBH can cause obvious cognitive impairment. The mechanisms underlying this impairment include β amyloid (Aβ) overproduction, tau hyperphosphorylation [7], oxidative stress impairment [8], inflammation [9], synaptic dysfunction [10] and neuronal loss [11]. Previous studies have identified factors that regulate CBH, which include hypoxia inducible factor-1 (HIF-1) [12], β amyloid precursor protein (β-APP), cyclin dependent kinase 5 (CDK5), glycogen synthase kinase-3β (GSK-3β) [13], mitogen-activated protein kinase (MAPK) [14] and non-protein molecules such as microRNAs [1, 3, 15, 16]. However, the precise molecular mechanisms of CBH are not fully understood.

MicroRNAs are a family of small, well-conserved non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. Previous studies have shown that miR-195, miR-27a, and miR-9 play a role in the process of CBH. In particular, miR-9 was reported to induce a deficit in Nav1.1/Nav1.2 trafficking through post-transcriptional regulation of the SCN2B gene during CBH. However, whether miR-9 induced impairments in learning and memory and its precise mechanisms require further study. miR-9 has been reported to play a role in many different diseases, including colorectal cancer [17], breast cancer [18], cardiac fibrosis [19], stroke [20], AD [21], and Huntington’s disease (HD) [22] and so on. It has also been found that miR-9 is upregulated in the serum of AD patients [23]. miR-9 also regulates inflammatory processes in atherosclerosis by inhibiting activation of the NLRP3 inflammasome [24]. However, how miR-9 regulates the development of dementia is not fully understood. In this study, we performed microRNA expression profiling in a rat model of CBH induced by bilateral common carotid artery occlusion (2VO) for 8 weeks. The aim of this study was to identify the role of miR-9 in a rat model of CBH induced by 2VO and the molecular mechanisms underlying its effects.

Animals

Male Sprague-Dawley rats (6 months old, 220-260 g) were obtained from the Animal Centre of the Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China. The rats were housed at 23 ± 1°C and 55 ± 5% humidity and maintained on a 12-h light-dark cycle (lights on at 07:00 A.M.) with food and water available ad libitum. Rats among the different study groups were age- and sex-matched. Rats were anesthetized with chloral hydrate (300 mg/kg, intraperitoneal (i.p.) for the surgical induction of permanent, bilateral common carotid artery occlusion (2VO). Forty-eight male Sprague-Dawley rats were randomly divided into four equal groups (n = 12 per group). Thirty-six rats were used to establish animal models of bilateral common carotid artery occlusion (2VO). Our experiments were divided into four groups: the sham group (with surgery to isolate the bilateral common carotid artery but without ligation); the 2VO group (with bilateral common carotid artery occlusion surgery); the 2VO+AMO-miR-9 group (with bilateral common carotid artery occlusion surgery and injection of a lentiviral vector-mediated antisense molecule (lenti-AMO-miR-9); and the 2VO+NC group (with bilateral common carotid artery occlusion surgery and injection of lentiviral vector-mediated negative control molecule). Each group included 12 rats. The experimental protocol was approved by the Animal Ethics Committee of the Harbin University of Commerce and was carried out in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals issued by the Ministry of Science and Technology of China.

Permanent, bilateral common carotid artery occlusion (2VO) in the rat

The 2VO rat model was generated according to the methods of a previous study [1, 16]. Briefly, after induction of anesthesia with chloral hydrate (300 mg/kg, intraperitoneal (i.p.)), both common carotid arteries were exposed through a midline ventral incision. The vagus nerves were carefully separated, and both common carotid arteries were permanently ligated with 5-0 silk suture. The surgical wound was then sutured, and the animals were allowed to awaken before being returned to their cages. The sham-operation group underwent an identical procedure without ligation of the bilateral common carotid arteries. After surgery, rats were monitored daily for 8 weeks.

Transmission Electron Microscopy (TEM)

The method for preparation was similar to the previous study with minor modifications [3]. The hippocampus samples from each rat were fixed in 3% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) and post-fixed with 1% osmium tetroxide in Sorensen’s phosphate buffer. Following dehydration in ethyl alcohol, the hippocampal tissue was embedded in Epon-Araldite resin (Canemco Inc.). Ultrathin sections were cut serially with a Leica ultramicrotome (EM UC6, Leica Microsystems, Wetzlar, Germany), stained with uranyl acetate and lead citrate, and examined using a JEOL TEM (JEM-1011; JEOL Ltd., Tokyo, Japan). Three mice per group were used for TEM.

Hematoxylin-Eosin (HE) Staining

Rats were anesthetized with 10% chloral hydrate (500 mg/kg, intraperitoneal (i.p.) and perfused transcardially with 4% buffered paraformaldehyde (PFA), pH 7.4. The brains were removed, fixed with 4% PFA, dehydrated and embedded in paraffin. Two-micrometer frozen sections were prepared, and hematoxylin prime-eosin (HE) staining was performed to assess pathological changes in the CA1 and DG regions of hippocampal tissue by light microscopy. Three mice per group were used for HE staining.

Morris Water Maze (MWM)

To assess spatial learning and memory, the Morris water maze was used in accordance with the classic Morris protocol with modifications [1]. Briefly, the maze consisted of a circular tank (2.0 m in diameter) filled with water (opaque water at 25 ± 1°C) and conceptually divided into four quadrants. The pool was surrounded by black curtains to occlude extra-maze cues. A small white platform (20 cm diameter, 20 cm high) was submerged 2 cm below the water level, which was marked by black food pigment within the pool. Rats were trained to find an escape platform in the center of first quadrant from a distal start location. For cued training (three trials per day over 5 days), the rats were released into the water facing the side walls. The rats were allowed to rest on the platform for 20 s. The escape latency was defined as the time that the rats spent finding the platform. After the last trial on day 5, the platform was removed. The rats were tested in one 120-s swim probe trial on day 6. The escape latency (s), swimming distance (m), swimming speed (m/s), swimming route, and number of platform crosses were monitored using an on-line DigBehav-Morris water maze Video Analysis System (Mobile datum Software Technology Co., Ltd., Shanghai, China). Eight mice per group were used for the water maze experiments.

RNA extraction

For microarray analysis, 3 hippocampal tissues were collected 8 weeks after 2VO. Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions. Briefly, the hippocampal tissue was ground and crushed in liquid nitrogen, and then 0.2 ml chloroform per 1 ml of RNA in Trizol was added. The sample was centrifuged at 13,500 rpm at 4°C for 15 min. The aqueous phase was removed, isopropanol was added to the tube, and the sample was incubated at room temperature for 10 min. The sample was again centrifuged at 13,500 at 4°C for 10 min. Finally, the pellet was washed with 1 ml of 75% ethanol. The RNA was dissolved in DEPC water. Six mice per group were used for RNA extraction.

MicroRNA microarray hybridization

Total RNA obtained from the hippocampus of 3 sham and 3 2VO rats was labeled and hybridized on microRNA microarrays. MicroRNA expression profiling was performed using LC Sciences technology (LC Sciences, Houston, Texas, USA) as described previously [25, 26]. The arrays were designed to detect miRNA transcripts corresponding to rat miRNA listed in the Sanger miRBase release v. 14.0. The analysis of microarray data was also performed by LC Sciences (Houston, Texas, USA). T-tests were performed to identify significant differences in microRNA expression between sham-operated rats and 2VO rats. After RNA hybridization, tag-conjugation Cy5 dyes were circulated through the microfluidic chip for dye staining. Fluorescence images were collected and digitized. The data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (locally weighted regression). For two-color experiments, the ratio of the two sets of detected signals was log2 transformed, and differentially expressed m-RNAs were identified through fold change filtering and normalization.

Cultured primary hippocampal neonatal rat neurons (NRNs)

The hippocampal regions were removed from rat pups on postnatal day 1, placed in phosphate buffered saline (PBS) solution on ice, and minced. The hippocampal tissue was cut in pieces and incubated for 15 min at 37°C with 0.125% trypsin (Gibco, USA). The cells were seeded in poly-D-lysine-coated 6-wells plates (10 µg/ml, Sigma, USA). The neurons were maintained in neurobasal medium A (Gibco, USA) containing 2% B27 supplement (Invitrogen, USA) and 10% fetal bovine serum (FBS, HyClone, Logan, UT). The cells were incubated in a 5% CO2 humidified chamber at 37°C for growth. After 3 days, 5 µmol/L cytosine arabinoside (Sigma, USA) was added into the neurobasal medium A to inhibit astrocyte proliferation. For all experiments, the neurons were used 10-14 days after plating.

Synthesis of miR-9, AMO-MIR-9 and other oligonucleotides

miR-9 mimics (MIMAT0000781) and AMO-miR-9 (miR-9 antisense) were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). AMO-miR-9 contains 2’-O-methyl modifications at every base and a 3’ C3-containing amino linker. A scrambled RNA was used as the negative control (sense: 5’-UUCUCCGAACGUGUCACGUAA-3, and antisense: 5’-ACGUGACACGUUCGGAGAAUU-3’).

Transfection Procedures

After the neurons were cultured for 12 days, 75 pmol/mL miR-9 and/or AMO-miR-9, or negative control RNA (NC) were transfected into neonatal hippocampal neurons. Small interfering RNA (siRNA) targeting CREB were transfected into neonatal hippocampal neurons with X-treme GENE siRNA transfection reagent (Cat. #04476093001, Roche, USA) according to the manufacturer’s instructions. 48 h after transfection, cells were collected for total RNA isolation, protein purification, or immunofluorescence staining. The procedure was repeated for 6 batches of cultured neonatal rat neurons. A pool of CREB siRNAs (containing 4 different sequences to ensure knockdown of the target mRNA) was designed and synthesized by Thermo Fisher scientific (Thermo Scientific, Bremen, Germany). The pooled CREB siRNA were designed to target the following sequences in CREB: GCTGTAACAGAAGCTGAAA, GCCACAGATTGCCACATTA, GCTCCCACTGTAACCTTAG and TCAGTCTCCACAAGTCCAA.

Construction of Lentiviral Vectors

Using the BLOCK-iT polII miR-RNAi expression vector with the EmGFP kit from Invitrogen (Carlsbad, CA, USA), the pre-miR-9 overexpression and pre-AMO-miR-9 vectors were identified after analyzing the plasmid sequence (Invitrogen, Shanghai, China). The lentiviral titers used for the experiments were 9.25 × 108 transducing U/ml. Lentiviral suspensions were stored at -80°C until use and were briefly centrifuged and kept on ice immediately prior to injection.

Stereotaxic injection of the lentiviral vectors

After anesthetic induction, rats were placed onto a stereotaxic frame (RWB Life Science Co., Ltd., China) as previously described [1]. Injection coordinates relative to bregma were as follows: AP (anteroposterior), -4.52 mm; ML (mediolateral), ± 3.2 mm; DV (dorsoventral), -3.16 mm below the surface of the dura. The coordinates were derived from the atlas of Paxinos and Watson. Two microliters (10,000TU/µl) of lenti-pre-miR-9 and/or lenti-pre-AMO-miR-9 were injected into the CA1 of the hippocampus. The vectors were injected at both hippocampal sites before animals were subjected to the 2VO procedure. Lentivirus-mediated negative control molecules were used in our study. The negative control (“top strand” oligo: TGC TGA AAT GTA CTG CGC GTG GAG ACG TTT TGG CCA CTG ACT GAC GTC TCC ACG CAG TAC ATTT) and its complementary sequence (“bottom strand” oligo: CCT GAA ATG TAC TGC GTG GAG ACG TCA GTC AGT GGC CAA AAC GTC TCC ACG CGC AGT ACA TTTC) were cloned, and the double-stranded oligonucleotides were generated by annealing the top and bottom strand. The oligos were cloned into the pcDNA6.2-GW/EmGFP-miR vector, and the ligated constructs were transformed into competent Escherichia coli. After the colony was purified and identified as the correct expression clone, the NC expression cassette was transferred to the Gateway-adapted destination vectors using PolII promoters to form a new expression clone containing attR substrates. The vector was identified after analyzing the plasmid sequence.

Quantitative real-time PCR (qRT-PCR)

Quantitative real-time PCR (qRT-PCR) was performed to confirm the results of microarray analysis. Total RNA was extracted from the hippocampal tissue of 2VO rats and from the sham-operation rats using Trizol reagent (Invitrogen). One microgram of total RNA was converted to cDNA, which was used as template in a 20 µl reaction containing 7 µl of ddH2O, 10 µl of 2x SYBR Green mixture (Roche), 1 µl of 10x forward primer and 1 µl of 10 x reverse primer (Invitrogen). The forward primers of miR-9 in the quantitative real-time PCR assay were “GGGGTCTTTGGTTATCTAG” and the reverse primers of miR-9 were “ATCCAGTGCGTGTCGTGGA,” Quantitative RT-PCR was performed using a 7500 Real-Time PCR system (Applied Biosystems, California, USA) at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s.

Western blot

The total protein samples were extracted from primary cultured neurons or from 6 hippocampus and cortex samples from rats. Frozen tissue was homogenized with 1000 µl of solution containing 40% SDS, 60% RIPA and 1% protease inhibitor. The homogenate was then centrifuged at 13,500 rpm for 30 min and the supernatants were collected. Protein concentrations were measured using a BCA kit (Universal Microplate Spectrophotometer; Bio-Tek Instruments, Winooski, VT, USA). Protein samples were fractionated by SDS-PAGE and then transferred to PVDF membrane. The samples were incubated for 2 h in primary antibodies against BACE1 (Cat# ab2077,1:200, Abcam, Cambridge, MA) and cAMP response element-binding protein (CREB) (Cat# ab32515,1:200, Abcam, Cambridge, MA), and then incubated in the secondary goat anti-rabbit (926-32210, LI-COR, 1:10000) or goat anti-mouse (926-32211, LI-COR,1:10000) antibody at room temperature for 1 h. The bands were captured using an Odyssey Infrared Imaging System (LI-COR) and quantified in each group using Odyssey v. 3.0 software (area × OD) with normalization to the internal control.

Immunofluorescence staining in cultured neurons

The culture medium was discarded, and the neurons were washed 3 times with PBS. The cultured neurons were perfused with 4% buffered paraformaldehyde (PFA) for 30 min, washed 3 times with PBS, and then incubated in blocking solution (0.1% Triton X-100 + 10% goat block serum + PBS) for 1 h. Following this step, the cultured neurons were incubated with the primary antibodies anti-β-Tubulin III (neuronal) antibody (Cat no. T8578; 1:5000; Sigma, Saint Louis, USA) or anti-BACE1 antibody (catalog #ab2077; 1:1000; Abcam) overnight at 4°C. The neurons were then washed and incubated with the secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594 (Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. The immunofluorescence intensity of BACE1 was measured by image Plus 5.0 software.

Immunofluorescence staining of rat brain sections

Rats were anesthetized with 10% chloral hydrate (500 mg/kg, i.p.) and perfused transcardially with 4% buffered PFA, pH 7.4. The brains were removed, dehydrated and frozen in OCT, and 20 µm sections were mounted on glass slides. After blocking, the sections were incubated with the anti-NeuN (catalog# ab104225; 1:200; Abcam) and anti-GFAP (catalog #ab7260; 1:500) primary antibodies overnight at 4°C. The sections were then washed and incubated with the secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 594 (Molecular Probes, Eugene, OR, USA) for 1 h at room temperature.

Luciferase assay

Using the Lipofectamine 2000 (Invitrogen, USA) transfection reagent according to the manufacturer’s instructions, HEK293T cells were transfected with 20 µmol/L miR-9, AMO-miR-9, or negative control siRNAs (NC); 0.5 µg psi-CHECKTM-2-target DNA (firefly luciferase vector); and 1 μL blank plasmid. After 48 h of transfection, the luciferase activity was measured with a dual luciferase reporter assay kit and luminometer (GloMaxTM 20/20, Promega, USA). Mutagenesis nucleotide-substitution mutations were carried out using direct oligomer synthesis for the 3’UTRs of the CREB-binding site. All constructs were sequence verified.

Statistics

The data are expressed as the mean ± SEM. Statistical analysis was performed using a Student’s t-test. A two-tailed P < 0.05 was considered to indicate a statistically significant difference.

Chronic brain hypoperfusion induced by bilateral common carotid artery occlusion (2VO)

To study the potential biological functions of miRNAs in chronic brain hypoperfusion (CBH), we established a model of CBH induced by bilateral common carotid artery occlusion (2VO) for 8 weeks in rats. Transmission electron microscopy of hippocampal sections was employed to examine pathological changes after CBH. As shown in Fig. 1A, in the 2VO group, the ultrastructure of the hippocampus underwent pathological changes, including shrinkage of the hippocampal cell nuclei, irregular and unclear nuclear membranes, swelling of the mitochondria, and synaptic damage. (Fig. 1A). Our immunofluorescence staining showed a reduction in NeuN (a neuronal specific nuclear protein, red) and an increase in glial fibrillary acidic protein (GFAP, green) in the hippocampus of 2VO rats compared to sham rats (Fig. 1B). Hematoxylin and eosin (HE) staining showed obvious damage to the hippocampus and cortex in 2VO rats compared to the sham group (Fig. 1C). In the cued learning trial, the latency to find the hidden platform was longer in 2VO rats than in sham rats (P < 0.05, Fig. 1D). In addition, in the probe trial, 2VO rats showed a reduction in the number of platform crosses compared to the sham control rats (P < 0.05, Fig. 1E).

Fig. 1.

Pathological injury and learning and memory dysfunction after chronic brain hypoperfusion (CBH) induced by bilateral common carotid artery occlusion (2VO) in rats. (A) Electromicrographs showing the ultrastructural features of the cell nuclei and synapses in the hippocampus in Sham and 2VO rats 8 weeks after surgery to induce CBH. The black arrows indicate the described pathological features in the brain tissue on TEM. (B) Assessment of cells in the hippocampus of 2VO and Sham rats using immunofluorescence staining. The neuronal marker NeuN was stained red, and the astrocyte marker GFAP was stained green. (C) Hematoxylin and Eosin (HE) staining of representative brains 8 weeks after 2VO. (D) Comparison of the average latency to find the platform for the Sham group and 2VO group. (E) The number of times the target platform location was crossed during the probe trial. Data are expressed as the mean ± SEM; *P < 0.05 vs. Sham.

Fig. 1.

Pathological injury and learning and memory dysfunction after chronic brain hypoperfusion (CBH) induced by bilateral common carotid artery occlusion (2VO) in rats. (A) Electromicrographs showing the ultrastructural features of the cell nuclei and synapses in the hippocampus in Sham and 2VO rats 8 weeks after surgery to induce CBH. The black arrows indicate the described pathological features in the brain tissue on TEM. (B) Assessment of cells in the hippocampus of 2VO and Sham rats using immunofluorescence staining. The neuronal marker NeuN was stained red, and the astrocyte marker GFAP was stained green. (C) Hematoxylin and Eosin (HE) staining of representative brains 8 weeks after 2VO. (D) Comparison of the average latency to find the platform for the Sham group and 2VO group. (E) The number of times the target platform location was crossed during the probe trial. Data are expressed as the mean ± SEM; *P < 0.05 vs. Sham.

Close modal

Abnormal expression of miRNAs in the hippocampus and cortex after CBH

MicroRNAs control gene expression at the post-transcriptional level and are essential for neuronal function. Therefore, we first analyzed the expression profile of miRNAs in the hippocampus of 2VO rats and sham rats using microarrays. After normalizing the microarray values using a LOWESS filter with significant differences (P < 0.05), the results were displayed as a heat map (Fig. 2A). Seven microRNAs (let-7b, let-7f, miR-652, let-7a, miR-223, miR-125a-5p, miR-9) were upregulated in the 2VO group compared to the sham group, as indicated by the red color in the heat map. Downregulated microRNAs included miR-l32, miR-23a, miR-151, miR-206, miR-23b and are shown as shades of green in the heatmap (Fig. 2A). miR-9 is enriched in the CNS and involved in many physiological and pathological neuronal processes. Further experiments using qRT-PCR showed that miR-9 expression was significantly increased in both the hippocampus (P < 0.05, Fig. 2B) and cortex (P < 0.05, Fig. 2C) of 2VO rats.

Fig. 2.

MicroRNA expression profiling in the hippocampus and cortex after chronic brain hypoperfusion using microarray analysis and validated by real-time PCR. (A) MicroRNA expression profiling by microarray analysis in Sham and 2VO rats. The color scale in the heatmap reflects the expression levels of the various miRNAs in each sample (red: high expression, green: low expression, black: no change). The bar code at the top represents the color scale of the log2 values. Each column represents the data from the Sham and 2VO rats and includes three replicates at the top of the heatmap. (B, C) miR-9 expression detected by real-time PCR in the hippocampus and cortex of Sham and 2VO rats after normalization to U6 levels. *P < 0.05 vs. Sham, mean ± s.e.m, n = 6.

Fig. 2.

MicroRNA expression profiling in the hippocampus and cortex after chronic brain hypoperfusion using microarray analysis and validated by real-time PCR. (A) MicroRNA expression profiling by microarray analysis in Sham and 2VO rats. The color scale in the heatmap reflects the expression levels of the various miRNAs in each sample (red: high expression, green: low expression, black: no change). The bar code at the top represents the color scale of the log2 values. Each column represents the data from the Sham and 2VO rats and includes three replicates at the top of the heatmap. (B, C) miR-9 expression detected by real-time PCR in the hippocampus and cortex of Sham and 2VO rats after normalization to U6 levels. *P < 0.05 vs. Sham, mean ± s.e.m, n = 6.

Close modal

Knockdown of miR-9 reversed the dementia phenotype induced by 2VO

Since 2VO increased miR-9 expression in rats, we predicted that the downregulation of miR-9 could prevent the dementia phenotype induced by 2VO. To test this hypothesis, lenti-pre-AMO-miR-9 was injected into hippocampus of 2VO rats. The level of miR-9 was significantly increased in both the hippocampus (Fig. 3A) and cortex (Fig. 3B) of 2VO rats after 8 weeks, and this effect was reversed by lenti-pre-AMO-miR-9 treatment. The lenti-pre-AMO-miR-9 rescue prolonged the latency of 2VO rats to reach the platform and significantly improved their memory, as indicated by a progressive reduction in the latency to reach the platform each day. These effects were not observed in age-matched 2VO rats transfected with NC RNA (Fig. 3C). In addition, lenti-pre-AMO-miR-9 transfection in 2VO rats increased the number of platform crosses in the probe trial (Fig. 3D). We also assessed histological changes after AMO-miR-9 treatment in the 2VO rats; the HE staining results are shown in Fig. 3E. As Fig. 3E shows, AMO-miR-9 treatment clearly alleviated neuronal damage in 2VO rats.

Fig. 3.

The effect of miR-9 knockdown on the learning and memory deficits in the 2VO model. (A, B) Quantitative real-time PCR detection of miR-9 in the hippocampus and cortex 8 weeks after stereotaxic injection. 2VO rats with or without injection of lenti-pre-AMO-miR-9, or NC. **P < 0.01 vs. Sham; ##P < 0.01 vs. 2VO, n = 6. (C) The mean escape latency (seconds) to locate the hidden platform after 2VO and lenti-AMO-pre-miR-9 treatment for 8 weeks. *P < 0.05 vs. Sham; #P < 0.05 vs. 2VO, n =6. (D) The number of times the target platform location was crossed during the probe trial. 2VO rats with or without injection of lenti-pre-AMO-miR-9, or NC. * P < 0.05 vs. Sham; #P < 0.05 vs. 2VO, n = 6. The data represent the mean ± s.e.m. (E) Hematoxylin and Eosin (HE) staining of representative brains 8 weeks after 2VO with or without AMO-miR-195 treatment.

Fig. 3.

The effect of miR-9 knockdown on the learning and memory deficits in the 2VO model. (A, B) Quantitative real-time PCR detection of miR-9 in the hippocampus and cortex 8 weeks after stereotaxic injection. 2VO rats with or without injection of lenti-pre-AMO-miR-9, or NC. **P < 0.01 vs. Sham; ##P < 0.01 vs. 2VO, n = 6. (C) The mean escape latency (seconds) to locate the hidden platform after 2VO and lenti-AMO-pre-miR-9 treatment for 8 weeks. *P < 0.05 vs. Sham; #P < 0.05 vs. 2VO, n =6. (D) The number of times the target platform location was crossed during the probe trial. 2VO rats with or without injection of lenti-pre-AMO-miR-9, or NC. * P < 0.05 vs. Sham; #P < 0.05 vs. 2VO, n = 6. The data represent the mean ± s.e.m. (E) Hematoxylin and Eosin (HE) staining of representative brains 8 weeks after 2VO with or without AMO-miR-195 treatment.

Close modal

Knockdown of miR-9 reversed the increase in expression of BACE1 induced by 2VO

The expression of BACE1 was significantly increased in the hippocampus and cortex of 2VO rats compared to the sham control group. Interestingly, lenti-pre-AMO-miR-9 inhibited the increased BACE1 expression induced by 2VO in the hippocampus (Fig. 4A) and cortex (Fig. 4B).

Fig. 4.

The effect of miR-9 knockdown on the increased expression of BACE1 induced by 2VO. BACE1 protein expression in the hippocampus (A) and cortex (B) in 2VO rats with or without lenti-AMO-pre-miR-9 treatment for 8 weeks *P < 0.05, **P < 0.01 vs. Sham, #P < 0.05 vs. 2VO, mean ± SEM, n = 3.

Fig. 4.

The effect of miR-9 knockdown on the increased expression of BACE1 induced by 2VO. BACE1 protein expression in the hippocampus (A) and cortex (B) in 2VO rats with or without lenti-AMO-pre-miR-9 treatment for 8 weeks *P < 0.05, **P < 0.01 vs. Sham, #P < 0.05 vs. 2VO, mean ± SEM, n = 3.

Close modal

miR-9 mediates the expression of BACE1 and induces morphological changes in cultured NRNs

To assess whether miR-9 could influence the expression of BACE1 protein, we measured BACE1 protein levels in cultured neonatal rat neurons (NRNs) co-transfected with 75 pmol/mL miR-9 and/or AMO-miR-9 or left untreated (negative control, NC). The successful transfection of miR-9 was determined by qRT-PCR (Fig. 5A). miR-9 and AMO-miR-9 lentivirus were labeled with GFP to permit observation of the transfection efficiency. Morphological changes were observed by transmission electron microscopy, as shown in Fig. 5B. The cultured NRNs showed irregular nuclei, unclear nuclear membranes, swelling of the mitochondria, and damaged synapse morphology, all of which were reversed by AMO-MIR-9 treatment. BACE1 protein levels were significantly increased in the presence of miR-9 (Fig. 5C). AMO-MIR-9 rescued the upregulation of BACE1 elicited by miR-9, and transfection with scrambled miRNA (negative control) failed to affect the protein levels (Fig. 5C). These data suggested that BACE1 is the target of miR-9. The miR-9 and AMO-miR-9 transfection efficiency was assessed using green fluorescent protein (GFP) labeling, as shown in Fig. 5A. Confocal immunofluorescence microscopy revealed that BACE1 was increased after miR-9 treatment, and this effect was reversed by AMO-miR-9 treatment (Fig. 5D).

Fig. 5.

miR-9 regulates the expression of BACE1 and induces morphological changes in cultured NRNs. (A) Left, Verification of uptake of miR-9 by NRNs after transfection. *P < 0.05 vs. NC, mean ± SEM, n = 3 independent RNA samples for each group. Right, the miR-9 and AMO-miR-9 transfection efficiency was also assessed using green fluorescent protein (GFP) labeling. (B) Electromicrographs showing the ultrastructural features of the nuclei and synapses in cultured neonatal rat neurons (NRNs). The black arrows indicate the described pathology in the TEM for NRNs. Cells were transfected with miR-9, AMO-MIR-9, miR-9+AMO-MIR-9, or NC. (C) The effects of miR-9 on the BACE1 protein levels in primary cultured neonatal rat neurons (NRNs) using western blot analysis. Cells were transfected with miR-9, AMO-MIR-9, miR-9+AMO-MIR-9, or NC. n = 3. *P < 0.05 vs. NC; #P < 0.05 vs. miR-9. (D) Representative confocal microscopy images showing primary culture hippocampal neurons stained for tubulin (green, upper), BACE1 (red, middle). A merged image depicting double positivity (yellow) is shown on the bottom after transfection with miR-9 mimics or/and AMO-miR-9, negative control. Measures of optical density for BACE1 are shown on the right. Data represent the mean ± SEM (n = 6 different batch of NRNs). *P < 0.05 vs. NC; #P < 0.05 vs. miR-9.

Fig. 5.

miR-9 regulates the expression of BACE1 and induces morphological changes in cultured NRNs. (A) Left, Verification of uptake of miR-9 by NRNs after transfection. *P < 0.05 vs. NC, mean ± SEM, n = 3 independent RNA samples for each group. Right, the miR-9 and AMO-miR-9 transfection efficiency was also assessed using green fluorescent protein (GFP) labeling. (B) Electromicrographs showing the ultrastructural features of the nuclei and synapses in cultured neonatal rat neurons (NRNs). The black arrows indicate the described pathology in the TEM for NRNs. Cells were transfected with miR-9, AMO-MIR-9, miR-9+AMO-MIR-9, or NC. (C) The effects of miR-9 on the BACE1 protein levels in primary cultured neonatal rat neurons (NRNs) using western blot analysis. Cells were transfected with miR-9, AMO-MIR-9, miR-9+AMO-MIR-9, or NC. n = 3. *P < 0.05 vs. NC; #P < 0.05 vs. miR-9. (D) Representative confocal microscopy images showing primary culture hippocampal neurons stained for tubulin (green, upper), BACE1 (red, middle). A merged image depicting double positivity (yellow) is shown on the bottom after transfection with miR-9 mimics or/and AMO-miR-9, negative control. Measures of optical density for BACE1 are shown on the right. Data represent the mean ± SEM (n = 6 different batch of NRNs). *P < 0.05 vs. NC; #P < 0.05 vs. miR-9.

Close modal

miR-9 alters BACE1 levels by directly regulating CREB protein expression

We performed experiments to determine the mechanism by which miR-9 increases the BACE1 level. The expression of the transcriptional repressor CREB was significantly downregulated in 2VO rats (Fig. 6A). We conducted a computational analysis using the miRNA database TargetScan to identify whether miR-9 has the potential to regulate the expression of CREB. Using immunoblotting in cultured NRNs (Fig. 6B), we observed that AMO-9 rescued the downregulation of CREB elicited by miR-9. The scrambled negative control miRNA failed to affect the protein levels, indicating that the observed changes were attributable specifically to the action of miR-9. miR-9 was shown to have conserved binding sites for the 3’UTR of CREB (Fig. 6C). To experimentally verify CREB as a target of miR-9, we first cloned the miR-9 binding sites in the 3’UTR of CREB genes into the luciferase-expressing reporter plasmid to determine the effects of miR-9 on reporter activities in HEK293T cells. As shown in Fig. 6C, co-transfection of miR-9 with the plasmid consistently produced less luciferase activity than transfection of the plasmid alone, whereas mutation of the binding sites abolished the effect of miR-9. To detect whether CREB can regulate BACE1, siRNA of CREB and rolipram (an inhibitor of CREB) were applied, and these treatments significantly increased the expression of BACE1 in primary cultured hippocampal neurons (Fig. 6D). The results imply that CREB regulates the expression of BACE1.

Fig. 6.

The influence of miR-9 on the expression of CREB protein and the effect of CREB on BACE1 level. (A) western blot analysis of CREB in sham and 2VO rats. Upper panel: representative immunoblots of CREB; lower panel: the quantitative analysis of the immunoblots. *P < 0.05 vs. Sham, mean ± s.e.m, n = 6. (B) The effects of miR-9 on the levels of CREB protein in primary cultured neonatal rat neurons (NRNs) assessed by western blot analysis. Cells were transfected with miR-9, miR-9+AMO-9, or NC. n = 3. *P < 0.05 vs. NC; #P < 0.05 vs. miR-9. (C) Complementarity between the miR-9 seed-matched sequence and 3’UTR for CREB predicted by bioinformatic software. The luciferase reporter gene assay for interactions between miR-9 and the CREB binding site in the 3’UTR in HEK293T cells, **P < 0.01 vs. NC, mean ± s.e.m, n = 3. (D) Effect of CREB on the level of BACE1 following CREB siRNA treatment or CREB inhibitor (Rolipram, 10 µmol/L) treatment. **P < 0.01 vs. Ctl, mean ± s.e.m, n = 3.

Fig. 6.

The influence of miR-9 on the expression of CREB protein and the effect of CREB on BACE1 level. (A) western blot analysis of CREB in sham and 2VO rats. Upper panel: representative immunoblots of CREB; lower panel: the quantitative analysis of the immunoblots. *P < 0.05 vs. Sham, mean ± s.e.m, n = 6. (B) The effects of miR-9 on the levels of CREB protein in primary cultured neonatal rat neurons (NRNs) assessed by western blot analysis. Cells were transfected with miR-9, miR-9+AMO-9, or NC. n = 3. *P < 0.05 vs. NC; #P < 0.05 vs. miR-9. (C) Complementarity between the miR-9 seed-matched sequence and 3’UTR for CREB predicted by bioinformatic software. The luciferase reporter gene assay for interactions between miR-9 and the CREB binding site in the 3’UTR in HEK293T cells, **P < 0.01 vs. NC, mean ± s.e.m, n = 3. (D) Effect of CREB on the level of BACE1 following CREB siRNA treatment or CREB inhibitor (Rolipram, 10 µmol/L) treatment. **P < 0.01 vs. Ctl, mean ± s.e.m, n = 3.

Close modal

Chronic brain hypoperfusion plays a major role in the pathology of dementia. The present study demonstrated that upregulation of miR-9 mediates the pathophysiological effects of chronic brain hypoperfusion in dementia by increasing the expression of BACE1, which directly regulates the expression of CREB. Our results included the following observations: (1) chronic brain hypoperfusion leads to obvious learning and memory impairment, which results from severe morphological damage, neuronal loss, and glial activation; (2) the downregulation of miR-9 reverses the deterioration in learning and memory induced by CBH; (3) downregulation of miR-9 reverses the decrease in BACE1 induced by CBH; (4) miR-9 disrupts the normal morphology of cultured NRNs and directly regulates the expression of BACE1; and (5) miR-9 directly regulates the expression of CREB, which inhibits BACE1 expression.

As previous studies have reported, the permanent bilateral occlusion of the common carotid arteries (2VO) is a classic animal model to study chronic cerebral hypoperfusion [4, 27, 28]. To our knowledge, chronic cerebral hypoperfusion induced by 2VO is a continuous process, and extending the time of ligation and reduced brain blood flow induces corresponding pathological changes. The 2- to 3-day period after 2VO constitutes acute ischemia; 8 weeks to 3 months after 2VO constitutes chronic ischemia; and after three months of ligation, cerebral blood flow gradually returns to normal levels. The most obvious features of neurodegenerative diseases are a loss of neurons and reduced synaptic connections. Previous studies have shown that after 1 week of 2VO, the quantity of hippocampal neurons in rats was not significantly reduced [29]. After 2 weeks of 2VO, 6 to 29% of the animals showed hippocampal CA1 neuronal damage [30, 31]. After 4 weeks of 2VO, 55% of the animals showed neuronal damage, and after 8 to 13 weeks, 67% of the animals showed damage in all regions of the hippocampus [32, 33]. These results suggest that 2VO causes a progressive increase in neurodegeneration in the hippocampus in rats. Our results also demonstrated that the neurons and synaptic connections were damaged after 8 weeks of 2VO (Fig. 1A). The learning and memory impairment resulting from CBH is caused by glial activation and neuronal loss. The timing of glial activation corresponds closely to that of the behavioral deficits in the setting of CBH [5]. Our data also showed a reduction in cognitive function with glial activation and a loss of hippocampal neurons after CBH (Fig. 1B-E).

Previous studies have shown that miRNAs can regulate proteins involved in neurodegenerative diseases and thus may play an important role in disease development [34]. MicroRNAs play important functions in the regulation of gene expression by targeting mRNA to affect its stability and translation [35]. Evidence has shown that the levels of APP and BACE1 protein are increased in the cortex and hippocampus after CBH in rats. This study also detected a large quantity of soluble Aβ, which was accompanied by the downregulation of miR-195. Further experiments showed that miR-195 can regulate the expression of APP and BACE1, affecting the generation of Aβ in dementia [1]. Other studies have shown that miR-29 expression is decreased in patients with AD, while the content of the potential target protein BACE1 is significantly increased [36, 37]. In both AD patients and AD model mice, miR-107, miR-328 and miR-298 levels are gradually reduced [38, 39]. miR-124, miR-186, miR-339 were reported to be the key factors that regulate BACE1 expression in dementia [40-42]. Our microarray data showed that CBH induces extensive miRNA changes in rat following CBH. We detected seven upregulated microRNAs: let-7b, let-7f, miR-652, let-7a, miR-223, miR-125a-5p, and miR-9 (Fig. 2A, Fig. 2B). Previous studies have shown that the level of miR-9 is increased in the hippocampus and cortex of AD patients [43, 44]. However, some reports have shown that miR-9 attenuates Aβ-induced synaptotoxicity [45]. This discrepancy implies that the changes in miR-9 levels in AD may be dependent on different inducers. Our studies showed that miR-9 expression was significantly increased under CBH conditions, which is consistent with previously reported trends.

The inhibition of miR-9, which is upregulated in CBH-induced dementia, markedly improved the cognitive impairment caused by 2VO (Fig. 3B, Fig. 3C), suggesting that inhibiting miR-9 expression could be a valid therapeutic approach for AD. The majority of miRNAs targeting BACE1 have been reported to be downregulated in AD brains. However, in our study, the miR-9 level was increased, and miR-9 promoted the expression of BACE1 (Fig. 4, Fig. 5). These findings provide a molecular basis for the deregulation of BACE1 in CBH and offer new perspectives on the pathology of dementia. Ultimately, the upregulation of BACE1 in dementia was counteracted by the delivery of anti-miR-9 to the brain with the aim of reducing neuronal damage (Fig. 5B-5D).

Although our results suggest that inhibiting miR-9 in CBH represents a viable therapeutic approach to treat dementia, there were also some limitations to our study. In this study, we showed that miR-9 might increase the levels of BACE1 by inhibiting the transcriptional repressor CREB (Fig. 6); however, the precise pathways should be further studied. A previous study demonstrated that nimodipine protected the brain from CBH by activating the Akt/CREB signaling pathway, suggesting that CREB plays an important role in CBH-related conditions [46]. Studies have also shown that liraglutide exerts its neuroprotective effects via activation of the cAMP/PKA/CREB pathway [47]. CREB was previously reported to contribute to the regulation of BACE1 [48]. Our data indicate that CREB plays a key role in CBH by regulating BACE1 expression (Fig. 6). Overall, this study provides evidence to suggest that the miR-9 is of functional importance in the development of dementia.

This work was supported in part by grants from the Funds for the Natural Science Foundation of Heilongjiang Province (H201382), The Opening Foundation of Key Laboratory of Chinese Materia Medica (Heilongjiang University of Chinese Medicine), Ministry of Education of the People‘s Republic of China(2016051544).

The authors declare no competing financial interests.

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