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Brain Dicer1 Is Down-Regulated in a Mouse Model of Alzheimer’s Disease Via Aβ42-Induced Repression of Nuclear Factor Erythroid 2-Related Factor 2

Dicer1 is a microRNA-processing enzyme which plays critical roles in neuronal survival and neuritogenesis. Dicer1 deletion induces neurodegeneration or degeneration in retinal pigment epithelium, which is associated with oxidative stress. Oxidative stress is thought to be central in the pathogenesis of Alzheimer’s disease (AD). Therefore, we hypothesize that Dicer1 may play roles in AD. Using immunoblotting and quantitative real-time PCR, Dicer1 protein and mRNA were reduced in the hippocampi of the AD mouse model APPswe/PSEN1dE9 compared with littermate controls. SiRNA-mediated Dicer1 knockdown induced oxidative stress and apoptosis and reduced mitochondrial membrane potential in cultured neurons. Chronic Aβ42 exposure decreased Dicer1 and nuclear factor erythroid 2-related factor 2 (Nrf2) which were reversed by N-acetyl-cystein. Nrf2 overexpression increased Dicer1 mRNA and protein and reverted the Aβ42-induced Dicer1 reduction. We further cloned Dicer1 promoter variants harboring the Nrf2-binding site, the antioxidant response elements (ARE), into a luciferase reporter and found that simultaneous transfection of Nrf2-expressing plasmid increased luciferase expression from these promoter constructs. ChIP assays indicated that Nrf2 directly interacted with the ARE motifs in the Dicer1 promoter. Furthermore, Dicer1 overexpression in cultured neurons reverted Aβ42-induced neurite deficits. Notably, injection of Dicer1-expressing adenovirus into the hippocampus of the mice significantly improved spatial learning. Altogether, we found novel roles of Dicer1 in AD and a novel regulatory pathway for Dicer1. These results suggest that Dicer1 is a target in AD therapy, especially at the early stage of this disorder. In this study, we found that Dicer1 was reduced in the brain of AD mice which is the first report to examine Dicer1 in AD. We further found (i) that Aβ42 exposure decreased Dicer1 via attenuating Nrf2-ARE signaling and (ii) injection of Dicer1-expressing adenovirus into the hippocampus of the AD mice significantly improved spatial learning. Altogether, we found novel roles of Dicer1 in AD and a novel regulatory pathway for Dicer1. This study may open new avenues for investigating potential pathognomonics and pathogenesis in AD. In this study, we found that Dicer1 was reduced in the brain of AD mice which is the first report to examine Dicer1 in AD. We further found (i) that Aβ42 exposure decreased Dicer1 via attenuating Nrf2-ARE signaling and (ii) injection of Dicer1-expressing adenovirus into the hippocampus of the AD mice significantly improved spatial learning. Altogether, we found novel roles of Dicer1 in AD and a novel regulatory pathway for Dicer1. This study may open new avenues for investigating potential pathognomonics and pathogenesis in AD.


Abstract Background
Oxidative stress critically underlies the neurodegenerative pathogenesis of Alzheimer's disease (AD). Depletion of Dicer1, an endoribonuclease central to microRNA maturation, also leads to neurodegeneration. We therefore hypothesized that altered Dicer1 expression may play a role in AD.

Results
Using immunoblotting and quantitative real-time PCR, we found that Dicer1 protein and mRNA levels were reduced in the hippocampi of animals of the AD mouse model APPswe/PSEN1dE9 compared with littermate controls. SiRNA-meditated Dicer1 knockdown induced oxidative stress, reduced mitochondrial intermembrane potential, and increased apoptosis in cultured neurons. Aβ42 exposure decreased Dicer1 and also down-regulated the oxidative stress-induced transcriptional regulator nuclear factor erythroid 2-related factor 2 (Nrf2). Conversely, Nrf2 overexpression increased Dicer1 mRNA and protein levels and reverted the Aβ42-induced Dicer1 reduction. To further investigate Dicer1 regulation, we cloned Dicer1 promoter variants harboring the Nrf2binding site, the antioxidant response elements (ARE), into a luciferase reporter and found that simultaneous transfection of Nrf2-expressing plasmid increased luciferase expression from these promoter constructs. ChIP assays indicated that Nrf2 directly interacted with the ARE motifs in the Dicer1 promoter. Furthermore, Dicer1 4 Background Alzheimer's disease (AD) is an inexorable neurodegenerative disorder, characterized with extracellular amyloid plaque deposition, intracellular neurofibrillary tangle formation, extensive neuronal loss, and cognitive dysfunction [1,2]. The pathogenesis of AD involves the key component of oxidative stress which is dependent on the balance between reactive species and anti-oxidation systems [3]. Accumulation of reactive oxygen/nitrogen species generates oxidative stress and produces ensued cytotoxicity [4] as well as does ferroptosis due to iron accumulation in AD brain [5,6].
Amyloid peptide inactivates membrane channel and transporter including sodium/calcium exchanger, calcium ATPase, glutamate and glucose transporter--these hazardous effects involve the roles of reactive oxygen species and lipid peroxidation [7][8][9]. Anti-oxidation systems are used to cope with oxidative injury, in which the regulation of the key enzymes or proteins, used for synthesizing glutathione or recycling peroxiredoxins, revolve around a transcriptional factor, nuclear factor erythroid 2related factor 2 (Nrf2) [10,11]. Nrf2 is a basic leucine zipper protein that regulates expression of a multitude of antioxidant proteins in response to oxidative injuries. Under quiescent situation, Nrf2 is retained in cytoplasm by Keap1, but upon oxidative stimulation, it is released from the binding and translocated into nucleus, triggering the transcription of antioxidant genes [12].
Human Dicer1 is an enzyme with multidomains consisting of an amino-terminal domain with helicase activity, a DUF383 domain with unknown function, a PAF (Piwi-Argonaute-Zwille) domain, two RNase III domains, and a carboxyl-terminal domain for double-stranded RNA binding [13]. In addition to traditional roles such as in generating small regulatory RNAs including miRNAs or siRNAs [14], Dicer1 also participates in maintaining heterochromatin structure [15,16], involving in inflammation [17,18], and breaking chromosomal DNA in Caenorhabditis elegans undergoing apoptosis [19].
Degeneration of retinal pigment epithelial cell (RPE), a characteristic of advanced dry age-related macular degeneration (AMD), is associated with Dicer1 depletion through which AluRNA, a specific substrate of Dicer1 degradation, accumulates and results in inflammasome activation and MyD88 signaling which lead to ensued cell death [20,21].
In the central nervous system, Dicer1 is widely expressed in neurons and glia.
Conditional loss of Dicer1 in neuron or glia produces different phenotypes and differentially impacts the brain function--the inducible impairments depend on developmental stages [22][23][24][25]. RPE degeneration induced by Dicer1 depletion involves inflammasome activation which likely accompanies with oxidative stress which is an etiological factor in neurodegeneration occurring in AD. As such, we exploited the roles of Dicer1 in AD and further explored whether Dicer1 expression was regulated by Nrf2, a transcription factor balancing redox level. We further investigated potential therapy by overexpressing Dicer1 in the hippocampus of APPswe/PSEN1dE9 (APP/PS1) mice.

Materials
The following primary antibodies were used in this study: Dicer1 (

Animal
All mice were fed water and food ad libitum in a temperature-and humidity-

Intrahippocampal injection of adenovirus expressing Dicer1
The adenovirus (Ad) incorporating sequences of either Ad-pCMV-EGFP or Ad-pCMV-Dicer1:T2A:EGFP in which the transcription of Dicer1 and EGFP is driven by independent promoter were packaged and generated by Cyagen Biosciences Inc.
(Guangzhou, China). Before stereotactic injection, animals were anaesthetized by intraperitoneal injection of ketamine/xylazine (0.1/0.05 mg/g body weight) and mounted on a stereotactic frame (KOPF, KD Scientific). The virus were injected into bilateral hippocampus with 2µl of viral titre (1.2 × 10 9 vg/mL) in each hemisphere using a 10-µl Hamilton syringe (Hamilton Medical, Reno, NV, USA) connected to a 30-gauge micropipette, at an injection rate of 0.2 µl/min. The injection coordinates were anteriorposterior, −2 mm, mediolateral, ± 2mm, dorsoventral, −2mm from bregma. The person in charge of injection wore protective equipment and the injections were conducted in a biological safety cabinet in a biological safety level II lab setting.
Autoclave of disposals was used to prevent contamination. The injections were conducted in 3.5-/4-month-old mice and behavior test was performed during 17-23 days after injection in which learning curve was acquired during 17-22 days and probe trial conducted at the 23rd day after injection. Halves of mice were used for immunohistochemistry and halves of them used for western blot.

Morris Water Maze
Water maze tests were performed using three groups of mice, WT mice (with intrahippocampal injection of Ad-EGFP) and APP/PS1 mice (intrahippocampal injection of Ad-EGFP or Ad-Dicer1-T2A:EGFP). The water maze (1.2-m diameter) was filled with water (24°C) and made opaque by the addition of nontoxic white paint. The water maze was surrounded by a black curtain (placed 80 cm away) that held three salient visual cues. Initially, mice were randomly trained in four quadrants of the water maze, and were allowed a maximum of 60 s to find a hidden platform (10-cm diameter, 1 cm under the water surface). Mice were trained for 6 days (4 trials per day, 10-min interval between trial). On day 7, mice were given a 60-s probe test (scanning the platform) to test their spatial memory. SLY-WMS Morris Water Maze System (Beijing Sunny Instruments Co. Ltd) consisting of a frame grabber, a video camera, a water pool, and an analysis software was used to monitor the animal's swimming pattern, distance, speed, and the amount of time spent in each of the four quadrants.

Cell culture
Newly born mice were used for culturing dissociated hippocampal and cortical neurons (CNs and HNs, respectively). Hippocampal or cortical tissue was mechanically dissociated after digestion with 0.25% trypsin (Beyotime, Haimen, China) and 0.1% DNaseI (Takara, Dalian, China) at 37°C for 15 min. DMEM/F12 containing 10% fetal bovine serum (Gibco, Billings, MA,USA) was then added, and the cells were centrifuged at 120 g for 3 min and the supernatants were then plated on poly-D-lysine-coated 8 cmculture dishes in neurobasal medium supplemented with 2% B27 (Invitrogen, Carlsbad, CA,USA) and 10 μM cytosine arabinoside (Sigma-Aldrich, Saint Louis, MO, USA). After 24 hours, the culture medium was replaced with fresh neurobasal medium supplemented with 2% B27 and the neuronal cultures were used for experiments after three days in vitro culture (DIV 3). Before transfection, the medium was replaced with DMEM/F12 containing 0.1% bovine serum albumin. After 24 h in culture, neurons were used for transfection, Aβ42 oligomer treatment or adenovirus infection. N2A, SK-N-BE (2) or HEK 293T cells was cultured in Dulbecco's modified Eagle's Medium(DMEM) containing 10% Fetal bovine serum(FBS).
The bold letters indicated the predicted AREs sites with consensus sequence TGA(C/G)NNNGC. A sequence not containing AREs site was also amplified by changing a forward primer CGACGCGTGGGCAGTCAGAGAGAGAGGAAAGGAAGG spanning -581 to +3 (promoter 0). The region spanning individual ARE sequence was also amplified by using forward primer CGACGCGTAGGAGATCAAGACCATCCTGGG and reverse primer CCCAAGCTTTAGTGGCGCAATCTCAGCT for ARE3 (124 bp).

Chromatin immunoprecipitation (CHIP) assay
The CHIP assay kit (Beyotime Biotechnology, cat# P2078) was used to examine the interaction between Nrf2 and Dicer1 promoter. All the procedures were performed according to the manufacturer's instructions. SK-N-BE cells were plated on 10-cm culture plate until ~70% confluency and added with 37% formaldehyde diluted to a 1% final concentration. To crosslink the protein-DNA complexes, the cells were incubated with formaldehyde on a shaker for 10 min at 37 o C. 1X glycine solution at a final dilution was added to quench the formaldehyde, which was rocked at room temperature for 5 min, and then the media were removed. The cells were immediately washed twice and harvested with ice-cold phosphate buffered saline (PBS) containing 1mM phenylmethylsulfonyl fluoride (PMSF), with 2 X 10 8 cells in each microcentrifuge tube.
The harvested cells were subject to centrifuge with 1000 x g at 4 o C, resuspended in SDS (sodium dodecyl sulfate) lysis buffer containing 1 mM PMSF, and incubated on ice for 10 min. Following the lysis, the samples were sonicated to shear chromatin, centrifuged for 5 min at 13,000 x g at 4 o C, and the supernatants were harvested and diluted 10-fold in ChIP dilution buffer containing 1 mM PMSF. After dilution, 15% of each sample was collected to use as a positive input in PCR analysis. The rest of each sample (~ 2 mL) were pre-absorped with addition of 70 µL protein A+G agarose/salmon sperm DNA, and the mixture were rocked for 30 min at 4 o C, centrifuged for 1 min at 1,000 x g, and the supernatants were collected. After this pre-absorption, rabbit anti-Nrf2 antibody (Proteintech) was used to precipitate DNA-protein complexes; rabbit IgG (Beyotime Biotechnology, cat#A7016) was used as a negative control. To capture the antibody, 60 µL protein A+G agarose/salmon sperm DNA was added, rocked for 60 min at 4 o C, and centrifuged for 1 min at 1,000 x g. The supernatants were discarded and the pellets were washed sequentially with Low, High Salt, and LiCl Immune Complex Wash buffer, and TE buffer. The pellets were used for PCR amplification and the following specific primers were used in PCR analyses for amplifying fragment containing

Aβ42 oligomer preparation
The oligomer form of Aβ42 was prepared as described [26]. Briefly, the peptide was dissolved in 1,1,1,3,3,3-Hexafluoro-2-propanol (Sigma-Aldrich) to remove any aggregates, stored in 4°C for 30 min, dried under room temperature,and dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) to 1 mM. The dissolved peptide was diluted in phenol-free F12 medium to 500 nM and incubated at 4°C for 24 h. The solution was subjected to centrifugation at 14,000 x g for 30 min at 4°C. The supernatant was collected as the oligomer preparation which was used at a final concentration of 100 nM.

Cell viability assay
Neurons were cultured in 96-well plates at a density of 5 X 10 3 per well in DMEM/F-

Measurement of mitochondrial membrane potential and reactive oxygen species (ROS) activity
Primary cultured cortical neurons at 9×10 5 cells/well and hippocampal neurons at After incubation at 37°C for 30 min, the neurons were added into 96-well plate at the density of 4×10 4 per well and subjected to measurement with plate fluorescence reader at 485nm (SpectraMax M5, Molecular devices).

Neurite outgrowth assay
The neuronal cultures were treated by oligomer Aβ42 at 100 nM for 24 h and then infected with Ad-EGFP or Ad-Dicer1-T2A:EGFP virus (5 X10 7 vg/mL) for 48 h. For determination of neurite, the neurites at least two times longer than the soma diameter were included in the measurement (Image pro plus, Olympus Optical Co., Ltd, Tokyo, Japan). The measurement of neurite length was conducted in three independent culture preparation and the values were averaged from 150 infected neurons in either group.

Western blot analysis
The 4-or 11-month-old mice were fasted overnight , and at the next morning, mice were deeply anesthetized by isoflurane and transcardially perfused with ice-cold phosphate buffer (PBS). The hippocampi were isolated and freshly frozen in liquid nitrogen. For analyzing the neurons treated by amyloid peptide, the cytoplasmic and nuclear proteins were extracted by plasma and nuclear protein isolation kit (Cat# P0027, Beyotime Biotechnology), respectively. For analyzing the cells for overexpressing Nrf2 plasmid, SK-N-BE (2) or N2A cells were seeded in 6-well plates at the density of 1×10 6 or 5×10 5 cells/well, respectively. Tissue or cell samples were homogenized in Super RIPA buffer composed of components in mM: 460 Tris-HCl, pH 7.4, 138 NaCl, 1 EDTA, 2.5 NaF, 2.5 Na3VO4, 1 phenylmethanesulfonylfluoride, 1 dithiothreitol, supplemented with 0.1% Nonidet P-40, and 1 X protease/ 1 X phosphatase inhibitor cocktail (Sigma, St.Louis, MO). Proteins were resolved on 12% SDS-PAGE gels (Bio-Rad) and transferred to nitrocellulose membranes (ThermoFisher). Membranes were blocked in blocking buffer (Beyotime Biotechnology) and then incubated with primary antibody.
Following wash, the membranes were then incubated with secondary antibody, and developed with chemiluminescence system kit (ThermoFisher). The blots were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).

Quantitative real-time polymerase chain reaction (qRT-PCR)
The hippocampi were isolated and freshly frozen in liquid nitrogen. SK-N-BE cells or Reaction specificity, indicated by the presence of a single amplification peak for each PCR reaction, was examined by use of dissociative curves. 2 -△△ Ct method was used to quantify the reaction, which was normalized to sham treatment or transfection of pCMV3 empty vector. Primary cultured cortical neurons and hippocampal neurons were plated on 6-well plate, and treated with Aβ42 oligomer (100 nM) or sham treatment for 48 h in neurobasal medium with 2% B27, respectively. Dicer1 mRNA levels or B1 RNA levels were assessed by qRT-PCR as above.

Immunofluorescence
The 4-or 11-month-old WT, APP/PS1 non-fasted mice were deeply anesthetized by isoflurane, killed, and then transcardially perfused with ice-cold PBS followed by 4% paraformaldehyde (PFA) in PBS. The isolated brains were postfixed in 4% PFA overnight, then stored in PBS containing 0.01% sodium azide. Brains were mounted in agarose and 10 µm coronal sections were obtained using a Leica VT1000S Vibratome.
Antigen demasking was performed in 0.01M sodium citrate solution (pH 6.0) at 99°C for 40 min. After washing the sections in PBS three times, 5 min each, at room temperature (RT), the sections were blocked using 5% normal donkey serum in PBS

Experimental design and statistical analysis
During Water Maze test, mice were evenly allocated to each experimental group according to the gender and ages, and the tests were conducted in a blinded manner, that is, the investigator was blinded to the group allocation of the genotype or treatment during the experiment and when assessing all the results. Animal experiment sample size calculator: http://www.lasec.cuhk.edu.hk/sample-size-calculation.html was used to determine the sample sizes, assuming 40% difference in the mean and standard deviation of 20% at power 95% and type I error 0.01 [27]. Similarly, determination of neurite outgrowth was conducted in a blinded manner. All the data acquired during experiments were included for analysis and there were no sample size differences between the beginning and end of experiments. All data were presented as means ± SD unless specified. Single data points were shown as overlaying dot-plot on bar-graph when sample sizes were smaller than 15, otherwise, shown as bar-graph.
For parametric data, Student's t-test was used for comparing differences between two groups. In experiments with more than two groups, one-way ANOVA was performed followed by Tukey's post hoc test for comparisons among groups. For analysis of two groups of non-parametric data, the Mann-Whitney U-test was used for determining the differences. For Morris water maze experiment, two-way ANOVA with repeat measures followed by Tukey's post hoc test was used for analyzing the time spent in four quadrants, frequency to crossing platform, swimming speed and distance. Statistical analyses were performed with Graphpad Prism 7.04 software (Graphpad software, Inc. La Jolla, CA, USA). Difference was regarded as significance when p<0.05.

Dicer1 or Nrf2 expression was reduced in the hippocampus of APP/PS1 mice
Dicer1 is reduced in the RPE cells in advanced dry AMD [20] and knockout of Dicer1 induces neurodegeneration [22,23]. In light of these evidence, we explored Dicer1 expression in the brain of the AD mice, APP/PS1. The staining of Dicer1 was reduced in the CA1, CA3, and dentate gyrus (DG) of APP/PS1 mice compared to WT littermate, at the age of 4 months or 11 months (Figure.1A). The protein was also significantly reduced in the homogenates from the hippocampus of 4-month APP/PS1 mice compared to WT littermate (p=0.0092, n=6 for each genotype, 3 female and 3 male) and this reduction became severe at the age of 11 months (p=0.0002, n=6 for each genotype, 3 female and 3 male) ( Figures.1B and 1C). Consistent with reduced protein, Dicer1 mRNA was also reduced in the hippocampus compared to WT at the age of 4 months (p=0.0335), and at the age of 11 months as well (p=0.0304) ( Figure.1F). Similarly, Dicer1 was significantly reduced in the homogenates from the parietal lobe of APP/PS1 mice at the age of 6 months when compared to WT littermate (p=0.022, n=6 for each genotype, 3 female and 3 male, )(Supplemental Figure.1).
Nrf2 is a master regulator of anti-oxidation genes regulating redox homeostasis [28]; Dicer1 depletion induces cytotoxicity via oxidative stress [20]. Thus, we simultaneously examined the protein levels of Nrf2 and activated caspase3 in the homogenates from APP/PS1 hippocampi. Similarly, Nrf2 protein levels in AD mice were significantly reduced compared to WT littermate at the age of 4 months (p=0.0001) and 11 months (p=0.0038), respectively ( Figures. 1B and 1D). In accordance with these findings, Nrf2 mRNA in hippocampus from AD mice was reduced compared to WT littermate at 4 months (p=0.0167) and at 11 months as well (p=0.0195), respectively ( Figure. 1G) Activated caspase3 in AD mice was significantly increased compared to WT littermate at 4 months (p=0.0098) and 11 months (p=0.0008), respectively ( Figures. 1B and 1E).

Dicer1 knockdown induced neurotoxicity
Dicer1 depletion induced cytotoxicity involving the roles of ROS and inflammation in the RPE cells [21]. Thus, we explored the relevant effects by knocking down Dicer1with specific siRNA in the primary murine hippocampal neuronal cultures (HNs). The Dicer1 siRNA used was selected from three siRNAs basing on efficacy (Supplemental Figure 2E and 2G).
Compared to transfection with NC siRNA, the Dicer1 siRNA significantly decreased Dicer1 protein in the HNs (p=0.044) ( Figures. 2E and 2F). The effects of Dicer1 knockdown on ROS production, mitochondrial integrity and neuronal survival were also replicated in the primary murine cortical neuronal cultures (Supplemental Figures. 2B-2H).
Aβ42 oligomer decreased Dicer1 and Nrf2 in neurons upon chronic exposure As above, we found that Dicer1 was reduced in APP/PS1 mice; the mice contain robust level of soluble Aβ and the deletion mutation (dE9) significantly accelerates the shift of the Aβ40/ Aβ42 ratios and elevates the levels of Aβ42 [29,30]. Thus, we  Figure 3B). We further tested whether reduction of Dicer1 by Aβ42 could be rescued by N-acetyl-cysteine (NAC), an antioxidant. As expected, Aβ42 treatment for 48 h reduced Dicer1 in CNs compared to sham treatment (p=0.00001), which was significantly rescued by NAC (p=0.032)( Figures.3D and 3E).
Simultaneously, we examined total Nrf2, which was also reduced by Aβ42 (p=0.0005), the effect rescued by addition of NAC (p=0.0009) ( Figures. 3D and 3F (Figures. 3Hand 3J), which was rescued by addition of NAC (p=0.00001 for nucleic)or (p=0.0419 for cytosolic), respectively ( Figures. 3G and 3H).  4F-4H). We scanned the promoter of Dicer1 with software (promoter 2.0 prediction server) and found three AREs motif upstream of transcription start site (tss), that is, Dicer1 promoter region. We cloned the ARE1-ARE3-containing promoter fragments into PGL6-basic plasmid containing firefly luciferase-coding sequence (-Luc), respectively, and also cloned the promoters containing different ARE motif into PGL6-basic plasmid, respectively ( p=0.00001) ( Figures. 4I and 4J). Transfection of Nrf2-expressing plasmid (pCMV Nrf2) increased the ratios between firefly and renilla in constructs containing ARE1(p=0.000219) or ARE3 motif (p=0.043) when compared to the cells transfected with vehicle plasmid, pCMV3 ( Figure. 4J). When compared the constructs containing different AREs under transfection with pCMV Nrf2, we found that the ratios of firefly/renilla from ARE1 constructs were significantly from the construct ARE2

Overexpression of Dicer1 improved spatial learning in APP/PS1 mice
We further explored the effect of overexpression of Dicer1 in APP/PS1mice. Figure 6). Consistent with previous observation in Figure.

Discussion
In our study, reduction of Dicer1 was detected as early as around 4 months in APP/PS1 mice in which Aβ deposition begins in the brain at the age of 6 months [29].
Furthermore, knockdown of Dicer1 in cultured neurons induced neurite deficit before soma degeneration, and intrahippocampal injection of Dicer1-expressing virus improved spatial learning. These evidence suggest that reduction of Dicer1 is an early event in AD, possibly before overt Aβ deposition and targeting Dicer1 may provide a new strategy for AD therapy, with target at the early stage of this disorder.
The expression or activity of Dicer1 is delicately maintained with the regulation at transcriptional, post-transcriptional, and post-translational levels. For instance, SOX4, a transcriptional factor involved in embryonic development and cell fate determination, positively regulates Dicer1 transcription [34]. Similarly, a transcription factor linked with microphthalmia, melanocyte inducing transcription factor, increases its transcription during melanocyte differentiation [35]. At post-transcriptional level, microRNA such as let-7 miRNA or miR-292 regulates Dicer1 via targeting its coding region or 3'-UTR, respectively, thereby inhibiting its expression [36,37]. At post-translation level, phosphorylation of Dicer1 at its RNase IIIb and double-stranded RNA-binding domains promotes its nuclear translocation and inhibits its function [38]--particularly, a phosphomimetic of phospho-serine by replacing serine 1836 with aspartate impairs Dicer1 function and causes postnatal lethality [39]. Distinct with these studies, we delineate a novel regulation of Dicer1, that is, by Keap1-ARE-Nrf2 pathway, which is unreported before. Aβ42 reduced Dicer1 protein in neurons at 48 h treatment which was possibly due to reduced nuclear Nrf2 from chronic Aβ treatment. By contrast, at 6 h treatment, Aβ42 reduced Dicer1 to a significant difference but Nrf2 was unchanged which suggested that Dicer1 diminution by acute Aβ treatment occurred at posttranslational level which was independent on Nrf2. In other words, dicer1 depletion induces oxidative stress causing neurotoxicity; chronic Aβ treatment reduced Dicer1 which involved the roles of reduced Nrf2 whereas acute Aβ treatment--6 h treatment-reduced Dicer1 which was independent on Nrf2 and possibly took place at posttranslational level.
Oxidative stress is thought to be central in the pathogenesis of AD, for example, oxidative stress exacerbates insulin resistance in AD brain [40] and disrupts mitochondria [41], resulting in failure to produce sufficient ATP for Na + -K + -ATPase and Na + -Ca ++ exchanger in order to maintain ion homeostasis in neurons [42][43][44]. Dicer1 depletion may be an internal sources contributing to oxidative stress by leading to accumulation of Alu RNA [21]. Intrahippocampal injection of Nrf2-expressing lentivirus improves spatial learning in APP/PS1 mice [45] but Nrf2 promotes cancer cell proliferation [46]. As such, manipulation of Nrf2 expression may lead to undesired side effects although it indicates potential benefit in AD brain [45]. By contrast, appropriate expression of Dicer1 may be an alternative strategy to oppose oxidative stress or other pathogenic factors in AD brain.
Dicer1 is ubiquitously expressed in the brain including in neurons, astroglia, and oligodendrocytes [23,24,47]. With immunofluorescence, we at least demonstrated that neuronal Dicer1 was decreased in AD hippocampus compared to that in littermate control mice. The glial Dicer1 in astrocytes or in oligodendrocyes was not investigated in this study. Thus, Dicer1 reduction detected from APP/PS1 brain homogenates and secondary neurodegeneration are the combinatory effects from neurons and glia. Therefore, it is important to determine the percentage of Dicer1 reduction in different cell type in AD brain for therapy purpose.
Dicer1 is central to the processing from pre-microRNAs to mature microRNAs, which regulate neuronal survival and neuritogenesis [48,49]. Deletion of functional Dicer1 has been found to induce accumulation of Alu RNA and thereby to activate NLRP3 inflammasome and to induce oxidative damages in advanced dry AMD [20,21].
Conditional ablation of Dicer1 in oligodendrocytes induces neuronal impairment via demyelination, oxidative damage, inflammatory gliosis in mice [23]. There is also evidence showing that conditional knockout of Dicer1 in dopaminergic neurons of ventral midbrain leads to neuronal loss through inhibiting micoRNAs biosynthesis [50].
Similarly, blocking microRNAs biosynthesis by Dicer1 deletion disrupts morphogenesis in the cortex and in the hippocampus [22]. Apart from Alu RNA accumulation, microRNAs deficiency due to Dicer1 depletion may play unexpected roles in AD neurodegeneration due to mature miRNA deficiency [22,47].

Conclusions
In this study, we indicated that Dicer1 was reduced in the hippocampus and in the cortex of APP/PS1 mice in vivo, and further showed that Dicer1 was reduced by Aβ42 oligomer in vitro. Dicer1 depletion led to neurite deficit and apoptosis by stress responses including increased ROS production, activated caspase3, and disrupted mitochondrial membrane potential. We further indicated that Nrf2 regulated Dicer1 expression which was linked with Keap1-Nrf2-ARE signaling pathway. Overexpression of Dicer1 increased neuronal survival and recovered neurite deficit by Aβ42 oligomer in vitro, and in vivo, Dicer1 overexpression in CA3 hippocampus improved spatial learning in APP/PS1 mice. Altogether, we reveal a novel role of Dicer1 in AD and a novel regulation of Dicer1. Our findings suggest Dicer1 may be a target in AD therapy.

Ethical approval and consent to participate
All experiments and data reports followed the SfN Policy on Ethics and the study was approved by the Animal Care and Use Committee of Wenzhou Medical University (Approval number# wydw2019-0141). This study did not use human samples or tissues.

Availability of data and materials
The data and materials are available from corresponding author on reasonable request.

Consent for publication
All the authors and funding agencies agree with publication of this study.

Conflict of Interests
All the authors have found no conflict of interests to disclose.

Funding
This study is supported by Natural Science Foundation of Zhejiang Province  relative to βIII-tubulin from F were normalized to NC siRNA group, and averaged from three independent culture preparation. Student's t-test was used to compare the differences, *p=0.02 for comparison.  The protein levels of Dicer1/ GAPDH in C were normalized to pCMV3 group and averaged from three independent culture preparation.