MiR-191-5p alleviates microglial cell injury by targeting Map3k12 (mitogen-activated protein kinase kinase kinase 12) to inhibit the MAPK (mitogen-activated protein kinase) signaling pathway in Alzheimer’s disease

ABSTRACT Alzheimer’s disease (AD) is a progressive neurodegenerative disease. Multiple reports have elucidated that microRNAs are promising biomarkers for AD diagnosis and treatment. Herein, the effect of miR-191-5p on microglial cell injury and the underlying mechanism were explored. APP/PS1 transgenic mice were utilized to establish mouse model of AD. Amyloid-β protein 1–42 (Aβ1-42)-treated microglia were applied to establish in vitro cell model of AD. MiR-191-5p expression in hippocampus and microglia was measured by reverse transcription quantitative polymerase chain reaction. The viability and apoptosis of microglia were evaluated by Cell Counting Kit-8 assays and flow cytometry analyses, respectively. The binding relationship between miR-191-5p and its downstream target mitogen-activated protein kinase kinase kinase 12 (Map3k12) was determined by luciferase reporter assays. Pathological degeneration of hippocampus was tested using hematoxylin-eosin staining and Nissl staining. Aβ expression in hippocampus was examined via immunohistochemistry. In this study, miR-191-5p was downregulated in Aβ1-42-stimulated microglia and hippocampal tissues of APP/PS1 mice. MiR-191-5p overexpression facilitated cell viability and inhibited apoptosis rate of Aβ1-42-treated microglia. Mechanically, miR-191-5p targeted Map3k12 3ʹ-untranslated region to downregulate Map3k12 expression. MiR-191-5p inhibited Aβ1-42-induced microglial cell injury and inactivated the MAPK signaling by downregulating Map3k12. Overall, miR-191-5p alleviated Aβ1-42-induced microglia cell injury by targeting Map3k12 to inhibit the MAPK signaling pathway in microglia.


Introduction
Alzheimer's disease (AD) is a progressive degenerative disease of the central nervous system (CNS) and is the primary contributor of senile dementia [1]. According to World Alzheimer Report, 46.8 million people are living with dementia in 2015 and people suffered from AD worldwide is predicted to be 131.5 million by 2050 [2]. AD is featured with neuronal death, intraneuronal neurofibrillary tangles, and neuritic senile plaques [3,4]. Clinically, the hallmarks of AD include learning and memory impairments, aphasia, apraxia, agnosia, deficits of visuospatial skills, executive dysfunction and changes in personality and behaviors [5,6]. At present, AD cannot be cured completely and the main drugs for AD clinical treatment are N-methyl-d-aspartate (NMDA) receptors and acetylcholinesterase inhibitors [7]. Moreover, histological examination at autopsy is the only definitive diagnosis for AD and twothirds of patients with AD cannot be diagnosed timely [8]. Therefore, finding effective therapeutical approaches for clinical diagnosis and treatment of AD is of great importance.
Hippocampus is a part of the limbic system between medial temporal lobe and thalamus, which is responsible for transformation, storage and localization of long-term memory [9]. Microglia are immune cells of the CNS that play a key role in injury response, pathogen defense, and maintenance of CNS tissues [10]. Amyloid-β protein (Aβ) is the main component of senile plaque, one of histopathological hallmarks of AD. Aβ is formed by sequential cleavage of amyloid precursor protein by β-secretase (BACE1) and γsecretase, and mainly includes Aβ40 and Aβ42, of which Aβ42 possesses higher cytotoxicity [11,12]. With strong phagocytic capacity, microglia can clear Aβ in the brain [13]. However, Aβ deposition induces inflammatory response of microglia, which subsequently accelerating microglial cell apoptosis [14]. Moreover, Aβ accumulation increases Ca 2+ concentration and activates protein kinases in nerve cells, resulting in abnormal phosphorylation of Tau protein [15]. Aggregation of highly phosphorylated Tau protein contributes to neuronal loss and the enlargement of neurofibrillary tangles [16].
MicroRNAs (miRNAs) are small noncoding RNAs (18-25 nucleotides) found in a variety of organisms that significantly reduce the expression of target genes by binding to the 3ʹ-untranslated region (3ʹ-UTR) of the target messenger RNAs [17]. Accumulating studies have revealed that miRNAs can act as potential biomarkers in AD. For example, miR-338-5p overexpression decelerates neuron loss and mitigates cognitive dysfunction in APP/PS1 mice via directly targeting B-cell lymphoma 2 like 11 (BCL2L11) [18]. MiR-34 c targets synaptotagmin 1 through the ROS-JNK-p53 pathway to mediate synaptic deficits in AD [19]. MiR-124 is significantly elevated in the hippocampal tissues of Tg2576 mice, and suppression of miR-124 reverses memory deficits and synaptic failure by interacting with protein tyrosine phosphatase non-receptor type 1 (PTPN1) in AD development [20]. Particularly, miR-191-5p is an important regulator in attentiondeficit/hyperactivity disorder [21] and multiple sclerosis [22]. Furthermore, miR-191-5p has been reported to be downregulated in AD [23] and is regarded as a serum biomarker for AD [24]. However, the pathological effects of miR-191-5p and its possible molecular mechanisms in AD are still poorly investigated.
Mitogen-activated protein kinases (MAPKs) are serine-threonine kinases that are widely existed in CNS and can mediate intracellular signaling associated with variety various cellular activities, including cell transformation, apoptosis, differentiation and proliferation [25]. MAPK family consists of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 [26]. Neuron apoptosis in AD has been demonstrated to be mediated by the MAPK pathway [27]. p38 can regulate apoptosis of neural and non-neuronal cells through numerous mechanisms, including elevation of TNF-α and c-myc levels, activation of caspase-3, Bax transposition, activation of c-JUN and c-fos, as well as phosphorylation of P53 [28][29][30]. The ERK pathway facilitates the migration and activation of neuros, playing a pivotal role in memory and synaptic plasticity [31]. Therefore, inhibiting neuron apoptosis by regulating the MAPK signaling may offer a potential avenue for AD treatment.
In this study, the role of miR-191-5p in AD progression was preliminarily investigated. We hypothesized that miR-191-5p might affect microglial cell injury by regulating downstream genes. Amyloid precursor protein/presenilin 1 (APP/PS1) mouse model of AD was established to explore miR-191-5p expression in vivo. The biological functions and underlying mechanism of miR-191-5p were investigated by establishing in vitro cell model of AD using amyloid-β protein 1-42 (Aβ1-42)-treated microglia. Our study may provide novel insight into therapeutic targets for AD treatment.

Experimental animals
A total of 24 male APP/PS1 mice (25 ± 2 g, 4 months old) and 8 wild-type (WT) C57BL/6 J female mice (25 ± 2 g, 4 months old) were purchased from Beijing HFK Bioscience Co., Ltd (Beijing, China). All mice were given free access to water and food and housed under a 12-hour light/dark cycle. All animal experiments were performed following the ethical requirements approved by the Ethics Committee of Hubei Provincial Hospital of Traditional Chinese Medicine (Hubei, China).

Animal grouping
APP/PS1 mice were utilized as the model group and C57BL/6 J mice were utilized as control group. All mice were anaesthetized using 3% pentobarbital sodium (30 mg/kg) (Sigma-Aldrich, St. Louis, MO, USA) via intraperitoneal injection [32]. After anesthetization, mice were decapitated and the hippocampi were stripped. A part of hippocampal tissues was fixed with 4% paraformaldehyde, dehydrated, paraffin-embedded and sliced up for histological observation.

Hematoxylin-eosin (H&E) staining
Paraffin-embedded hippocampal sections were stained with hematoxylin-eosin solution (Sigma-Aldrich) according to the manufacturer's instructions. To assess mean microvascular density, a light microscope (Olympus, Tokyo, Japan) was applied to determine the number of microvessels per unit area (/mm 2 ) [33].

Nissl staining
After dewaxing and rehydration, paraffin-embedded hippocampal sections were stained with Nissl staining solution (Linmei Biotechnology, Hefei, China) for 30 min at 60°C. Sections were then subjected to dehydration with anhydrous ethanol, made transparent with xylene, and sealed with neutral gum [33]. The morphology of Nissl bodies in hippocampus was observed under a light microscope (Olympus).

Immunohistochemistry
Hippocampi sections were dewaxed in xylene and rehydrated by gradient ethanol. After washing, the sections were sealed with 3% (v/v) H 2 O 2 and treated with sodium citrate buffer (10.2 mM) at 95°C for 20 min. After sealing with 10% (w/v) BSA in phosphate buffered saline for 10 min, the sections were incubated with anti-Aβ (Abcam, Cambridge, MA, USA) at 4°C overnight according to the manufacturer's instructions. At last, hippocampus sections were incubated with HRP-conjugated secondary antibody and counterstained with hematoxylin. Images of hippocampus were obtained by DP2-TWAN image acquisition system (Olympus) and analyzed by Image-Pro Plus software (Media Cybernetics, USA) for Aβ quantification [33].

Cell culture
Primary microglia were isolated by mild trypsinization from the cortex of newborn (postnatal day 0-2) C57BL/6 J mice (Beijing HFK Bioscience) as previously described [34]. In brief, the cortex was shredded and digested into a single cell suspension which was incubated in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and 100 U/ml 1% streptomycin/penicillin (Gibco). After incubation for 15 d, mixed glial cells were shaken at 37°C for 2 h. Separated microglial cells were collected for the subsequent experiments.

Cell treatment
The Aβ used in this study was human Aβ1-42 (China Peptides, Shanghai, China). The Aβ powder was dissolved, incubated with dimethyl sulfoxide (Gibco) and then diluted to a stock solution with phosphate buffered saline (Boster Biological Technology, Wuhan, China) [35]. After cell culture, microglial cells were treated with 10 μM Aβ1-42 for 24 h [35]. Aβ1-42-stimulated microglial cells served as the Aβ1-42 group. Microglial cells without Aβ1-42 treatment acted as the control (Con) group.

Cell transfection
MiR-191-5p mimics were used to overexpress miR-191-5p with negative control (NC) mimics as the control. Full sequence of Map3k12 was subcloned into the pcDNA3.1 vector to elevate Map3k12 expression with empty pcDNA3.1 vector as a negative control. All plasmids and vectors were purchased from GenePharma (Shanghai, China) and transfected into microglia using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) [36]. The transfection efficiency was examined using RT-qPCR after 48 h.

Cell counting Kit-8 (CCK-8) assay
The viability of microglial cells was detected by CCK-8 assay [37]. Microglial cells with or without Aβ1-42 treatment and miR-191-5p mimics/NC mimics transfection were seeded into 96-well plate at a density of 5 × 10 3 cells/well. After that, Cell Counting Kit-8 (CCK-8) solution (10 μL; Yeasen, Shanghai, China) was supplemented to the culture plate and co-incubated with the cells for 4 h at 37°C. The optical density of each well at 450 nm wavelength was examined using a Microplate Reader (Multiscan EX; Labsystems, Helsinki, Finland). All experiments were conducted in triplicate.

Flow cytometry analysis
The apoptosis of microglia was evaluated using an Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, CA, USA) via flow cytometry analysis [38]. After washed with phosphate buffered saline, microglial cells with indicate treatments were resuspended in binding buffer and then stained with Annexin V-FITC (5 µL; 10 min) and propidium iodide (PI) (5 µL; 5 min) at room temperature in the darkness according to the manufacturer's instructions. The apoptosis rate of stained microglia was analyzed by a flow cytometry (FACScan, BD Biosciences) using Cell Quest Pro software (Beckman Coulter, CA, USA). All experiments were repeated three times.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA was isolated from microglial cells and hippocampus tissues of mice using TRIzol reagent (Invitrogen) [39]. Then, extracted RNA was reverse transcribed to complementary DNA using First Strand cDNA synthesis kit (Roche, Switzerland). PCR was performed using the PrimeScript RT reagent Kit (Invitrogen) on IQ5 real-time PCR system (Bio-Rad, USA) following the manufacturer's protocols. The expression levels of miR-191-5p and Map3k12 were analyzed using the 2 −ΔΔCt method and normalized to U6 and GAPDH respectively. Sequences of primers were listed in Table 1.
As Figure 1d demonstrated, miR-191-5p was downregulated in the hippocampal tissues of APP/PS1 mice compared with that in tissues of WT mice, suggesting that miR-191-5p may participate in AD progression.

MiR-191-5p overexpression relieves Aβ1-42induced microglial cell injury
To establish in vitro cell model of AD, Aβ1-42 was utilized to treat microglia. MiR-191-5p was observed to be downregulated in Aβ1-42-treated microglia compared with that in the control group

Discussion
AD is an aging-related neurodegenerative disease featured with cognitive dysfunction and neuron loss [43]. Available reports have suggested that Aβ cleaved from APP by β-secretase (BACE1) and γ-secretase plays a pivotal role in triggering complicated pathological cascades leading to AD [44]. The formation of amyloid plaque caused by Aβ extracellular accumulation is the hallmark of AD [45]. Mouse model of AD with Tau pathology or amyloid plaques shows hippocampal memory impairments [46]. APP/PS1 mice have been reported to establish classic mouse model of AD in various studies [47][48][49]. Here, pathological changes of hippocampus in APP/PS1 mice were evaluated by H&E staining and Nissl staining, which showed that APP/PS1 mice presented the loss of neurons and Nissl bodies in hippocampus. Moreover, high level of Aβ-positive granules in the hippocampal tissues of APP/PS1 mice was examined by immunohistochemical staining. All the results revealed that APP/PS1 mice in the study possess AD pathological features. Aβ accumulation induces apoptosis of neurons, which plays a pivotal role in AD development [50]. Hence, inhibiting apoptotic behavior of neurons is often considered as an approach for AD treatment [50]. Similar to previous studies [51], in vitro cell model of AD was established by using Aβ1-42 to treat microglial cells. In our study, Aβ1-42 stimulation decreased microglial cell viability and promoted microglial cell apoptosis, suggesting that Aβ1-42 induces microglial cell injury. Additionally, Aβ1-42 administration inhibited BACE1 and Tau-5 protein levels in microglial cells. BACE1 and Tau-5 is regarded as AD marker proteins in many studies focusing on AD investigation [52,53]. The above results indicated that in vitro cell model of AD was successfully established.
Furthermore, Map3k12 was identified as the target gene of miR-191-5p in this study. Map3k12, also known as Dual leucine zipper kinase (DLK), can trigger neuronal stress response that modulates acute neuronal injury and neurodegeneration in models of chronic neurodegenerative diseases including Parkinson's disease, AD and amyotrophic lateral sclerosis [62]. Pharmacological Map3k12 suppression relieves neuronal injury response and provides potent protection for neuronal cells against degeneration in response to neuronal insults [63,64]. Genetic Map3k12 deletion demonstrates benefits in mouse model of AD by inhibiting APP and Tau [64]. In this study, Map3k12 was negatively regulated by miR-191-5p in Aβ1-42-treated microglial cells. Moreover, all effects of miR-191-5p upregulation on the viability, apoptosis rate, BACE1 and Tau-5 protein levels in Aβ1-42-administrated microglia were offset by Map3k12 elevation. Thus, miR-191-5p mitigates microglial cell injury by targeting Map3k12.
Subsequently, the downstream signaling mediated by Map3k12 was investigated. Map3k12 (DLK) is a serine/threonine kinase that acts as an upstream activator of the MAPK pathways and plays a key role in cell differentiation, apoptosis and neuronal response to injury [65]. MAPKs are serine/threonine protein kinases that transmit extracellular signals to the nucleus and induce cell proliferation and differentiation [4,66]. The MAPK signaling is closely associated with AD neurodegeneration. p38 MAPK inhibitor can inhibit the activity of caspase-3-like and suppress cellular apoptosis [67]. Since the ERK1/2 pathway is engaged in cellular survival mechanisms, its activation attenuates cognitive impairments in AD [68]. Numerous studies have substantiated the involvement of the MAPK signaling in AD development. For example, Qingxin kaiqiao fang treatment restricts cellular apoptotic behaviors by regulating phosphorylation of p38 MAPK and ERK1/2, thus exerting neuroprotective effects on AD development [33]. MiR-132 inhibits oxidative stress and hippocampal inducible nitric oxide synthase (iNOS) expression by suppressing the MAPK signaling to alleviate cognitive deficits of rats with AD [32]. Herein, miR-191-5p overexpression inhibited protein levels of p-ERK1/2 and p-p38 in Aβ1-42-treated microglial cells, and the inhibitory effect on p-ERK1/2 and p-p38 was reversed by Map3k12 upregulation. The results suggested that miR-191-5p inactivates the MAPK pathway by targeting Map3k12 in microglia.
In conclusion, miR-191-5p alleviates Aβ1-42induced microglia cell injury by targeting Map3k12 to inactivate the MAPK signaling, which may provide promising therapeutic biomarkers for AD treatment. In addition to the MAPK signaling, other pathways, such as the mTOR signaling [69] and the Wnt/β-catenin signaling [70], were reported to be associated with AD development. Studies on whether the miR-191-5p/ Map3k12 axis can regulate these signaling pathways in AD pathogenesis will be carried out in the future. Moreover, further animal experiments will be designed to explore the signaling pathways in vivo.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The work was supported by Wuhan Municipal Health and Family Planning Commission (NO. WZ18Q07).