Mechanism of miR-132-3p Promoting Neuroinflammation and Dopaminergic Neurodegeneration in Parkinson’s Disease

Visual Abstract


Introduction
Parkinson's disease (PD) is a neurodegenerative disease in elderly caused by degeneration of dopaminergic neurons mainly in the substantia nigra pars compacta (SNc), a region of the midbrain (Sveinbjornsdottir, 2016;Alizadeh et al., 2019). Severe locomotor deficit, such as freezing of gait, is a typical phenomenon of PD, which refers to intermittent walking disturbance during walk initiation and turning (Strubberg and Madison, 2017). The etiology of PD is probably multifactorial and currently there is no available treatment that can attenuate the neurodegenerative process of the disease. Therefore, a clearer understanding of mechanizing driving PD progression would be beneficial for the proposal of therapeutic approach. A variety of molecular mechanisms that likely contribute to neuronal cell death have been described previously, including a-synuclein aggregation, mitochondrial dysfunction and noxious oxidant stress (Milanese et al., 2021). In addition, neuroinflammation is one of the hallmarks of PD and may induce the degeneration of midbrain dopamine neurons (Krashia et al., 2019). Therefore, therapeutic intervention would be a potential strategy to alleviate the progression of PD by interfering with neuroinflammation and degeneration of dopaminergic neurons.
About 1900 miRNAs that can be encoded by human genome (Strubberg and Madison, 2017). Among these miRNAs, miR-132 has been frequently mentioned in many researches for its increased expression in neurons and for its implication in various neurodegenerative disorders. For example, the enhanced expression of miR-132-3p is related to chronic neuropathic pain (Leinders et al., 2016). MiR-132/Nurr1 axis was reported to have certain relationship with PD progression (Yang et al., 2019). Moreover, neuronal inflammation-induced epilepsy may be attenuated by miR-132 by targeting TRAF6, along with inactivation of NF-k B and MEK/ERK pathways (Ji et al., 2018). MiR-132 is positively associated with dopaminergic neuronal death (Qazi et al., 2021). Evidence in previous study pointed out that microglial cells medicated neuroinflammation triggered the cascade of inflammatory events leading to neuronal degeneration (Hirsch and Hunot, 2009;Bassani et al., 2015;Ye et al., 2018). However, it remains unclear how miR-132-3p is involved in neuroinflammation and dopaminergic neurodegeneration in PD.
GLRX is a small protein that catalyzes the glutathionedependent disulfide oxidoreduction reactions in a coupled system . In models of PD, deficiency of GLRX aggravates neurodegeneration (Johnson et al., 2015). Meanwhile, previous study addressed that suppression of GLRX contributes to PD-relevant motor deficits and dopaminergic degeneration in mice (Verma et al., 2020). Therefore, we speculated GLRX also has certain role to play in neuroinflammation and dopaminergic neurodegeneration in PD. Online software predicted that miR-132-3p was identified as an upstream regulatory factor of GLRX. In this regard, this study aims to investigate the mechanism by which miR-132-3p regulates neuroinflammation and dopaminergic neuron degeneration in PD. Hence, exploring the interactions between miR-132-3p/GLRX, dopaminergic neurodegeneration and neuroinflammation may be of great importance for the proposal of a latent therapeutic alternative for PD.
The main aims of the present study were to determine (1) whether miR-132-3p expression level is significantly altered in patients with PD as compared with healthy controls; (2) whether miR-132-3p is responsible for microglial activation and neuronal injury; (3) whether miR-132-3p affects the dopaminergic neuron degeneration and neuroinflammation in PD mouse models; and (4) whether miR-132-3p intensifies PD by inhibiting GLRX.

Materials and Methods
Collection of clinical brain tissues The study was conducted according to the Declaration of Helsinki. The study protocol concerning human was approved by the Ethics Committee of Hunan Provincial People 's Hospital (No. 202004), and written informed consent from family members of included subjects was obtained. This study was not preregistered and no sample calculation was performed. After death, the midbrain tissues were obtained from five patients with PD (three males and two females, 55.8 6 7.09 years old) and five healthy controls (three males and two females, 59.8 6 8.07 years old) matched for age. The brain tissues were collected and stored in liquid nitrogen, in which the total RNA and total protein were stored at À80°C refrigerator. The diagnosis of PD was performed by at least two or more experienced neurologists based on the clinical diagnostic criteria proposed by International Parkinson and Movement Disorder Society (Postuma et al., 2018). The included PD patients were excluded from secondary PD, tumor, or metabolic disturbance. The healthy controls were excluded from disorders related to nervous system.

Cell culture
The BV-2 microglial cells, human neuroblastoma cell line SH-SY5Y and human embryonic kidney (HEK)293T cells were supplied by American type Culture Collection (ATCC). The BV-2 and HEK293T cells were soaked in DMEM (Invitrogen), and SH-SY5Y cells were immersed in DMEM/Nutrient Mixture F-12 (DMEM/F12, Invitrogen). The culture medium contained 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cell culture was kept at 37°C in a humidified atmosphere containing 5% CO 2 . The inflammation of BV-2 cells was induced by 0.1 mg/ml lipopolysaccharide (LPS) for 24 h. The cell passage of cell lines shall not exceed 10 times.

Cell transfection
The miR-132-3p mimic (miRNA mimic refers to a sequence that can simulate specific endogenous miRNA), mimic negative control (NC), miR-132-3p inhibitor (miRNA inhibitor refers to a sequence that can interfere with miRNA), inhibitor NC, overexpressing GLRX (GLRX) and pcDNA3.1 were synthesized and purchased from GenePharma. Cell transfection was conducted by using the Lipofectamine 3000 reagent (Invitrogen). The transfection dose of overexpression plasmids was 2 mg, and the dose of mimic and inhibitor was 50 nM. Cells were treated by LPS for 24 h before following experiments were conducted.

qRT-PCR
TRIzol (Invitrogen) was employed to extract the total RNA of tissues or cells, and the reverse transcription was conducted by using the reverse transcription kit (TaKaRa). The expression of gene was quantitated by LightCycler 480 fluorescence quantitative PCR instrument (Roche), and reaction condition was instructed by the fluorescence quantitative PCR kit (SYBR Green Mix, Roche Diagnostics). The thermal cycle parameters were 95°C for 10 s, followed by 45 cycles of 95°C for 5 s, 60°C for 10 s, and 72°C for 10 s. A final extension was conducted at 72°C for 5 min. The quantification of mRNA was normalized to b -actin and miRNA to U6. The fold changes were calculated by the 2 -DDCt method. The formula is as follows: DDCt = [Ct (target gene) -Ct (reference gene) ] experimental group -[Ct (target gene) -Ct (reference gene) ] control group . All primers are shown in Table 1.

Western blotting
For collection of protein samples, RIPA lysis buffer (Beyotime Biotech) was used to treat cells or tissues. Following determination of protein concentration with a BCA kit, the proteins in the corresponding volume were mixed with loading buffer (Beyotime) and subjected to denaturation in a boiling-water bath for 3 min. Electrophoresis was embarked at 80 V for 30 min, and then for 1-2 h at 120 V when bromphenol blue reached the separation gel. The   proteins were transferred onto membranes at 300 mA for 60 min in an ice bath. Then, the membranes were rinsed 1-2 min with washing solution and inactivated in the blocking solution at room temperature for 60 min, or sealed overnight at 4°C. Following incubation with the primary antibodies against b -actin (ab8226, 1 mg/ml) and GLRX (ab45953, 1:250; Abcam) at room temperature in a shaking table for 1 h, the membranes were washed with the washing solution for 3 Â 10 min and incubated with the secondary antibody for 1 h at room temperature. The membranes were washed thrice for 10 min and exposed to developing liquid for color development. Then, the membranes were observed in chemiluminecence imaging analysis system (Gel Doc XR, Bio-Rad).

ELISA
The ELISA kit (R&D Systems) was adopted to determine the contents of TNF-a, IL-6, and IL-1b in the cell supernatant. All operations were performed in accordance with the instructions of the ELISA kit.

Coculture of BV-2 and SH-SY5Y cells
The effect of microglial activation on SH-SY5Y cells was studied by coculture of BV-2 cell supernatant and SH-SY5Y cells. The supernatant of BV-2 cells in each group was collected and filtered with a 0.45-mm filter. SH-SY5Y cells were seeded onto the six-well plates for cell culture till the density of SH-SY5Y cells reached 70%. The supernatant of BV-2 cells and DMEM/F12 containing 10% FBS were mixed at a ratio of 1:1, and the mixture was co-cultured with SH-SY5Y cells for 24 h.

CCK-8 assay
The SH-SY5Y cells were seeded onto 96-well plates, and cells in each well received 100-ml prediluted cell suspension (1 Â 10 5 cells/ml). Twenty-four hours later, SH-SY5Y cells were grown in conditioned medium of BV-2 cells for 24 h. The experiment was designed with three replicates. Ten microliters of CCK-8 solution (Dojindo) was added to each well for 2 h of incubation. The optical density (OD) at 450-nm wavelength was assessed.

Flow cytometry
After SH-SY5Y cells (10 5 cells/ml) were incubated with conditioned medium of BV-2 cells for 24 h, 3-ml cell suspension from each sample was transferred into a 10-ml centrifuge tube for 5 min of centrifugation at 500 rpm. After removal of culture medium, cells were washed with PBS and centrifuged at 500 rpm for 5 min. The supernatant was discarded. Then, cells were resuspended in 100 ml of binding buffer, and then gently mixed with 5-ml Annexin V-fluorescein isothiocyanate (FITC) and 5 ml PI for incubation for 15 min in the dark. The fluorescence of FITC and PI was examined by flow cytometry, and the apoptosis rate was analyzed.

RNA immunoprecipitation (RIP)
Following wash twice with precooled PBS, cells were centrifuged at 1500 rpm for 5 min and lysed with equivoluminal RIP lysis buffer. The magnetic beads were resuspended in 100-ml RIP Wash buffer followed by 30 min of incubation with 5-mg antibody against Ago2 (ab186733, 1:30, Abcam) or IgG (ab172730, negative control) at room temperature. Cells in the centrifuge tube were placed on a magnetic separation rack to discard the supernatant. Following incubation with 500-ml RIP Wash buffer for vortex oscillation twice, cells were given 500 ml of RIP Wash buffer for vortex oscillation and placed on ice. The magnetic bead tube was transferred to the magnetic separation rack, and the supernatant was removed. After that, cells in each tube received 900 ml of RIP immunoprecipitation buffer. Cell lysates were centrifuged at 14,000 rpm at 4°C for 10 min, and 100 ml of supernatant was pipetted into the magnetic bead-antibody complex for incubation overnight at 4°C. The complex processed centrifugation with supernatant removed. Then, the centrifuge tube received 500 ml of RIP Wash buffer for vortex oscillation and  cell supernatant was abandoned before the sediments were washed for six times. The magnetic bead-antibody complex was resuspended in 150 ml of Proteinase K buffer and incubated at 55°C for 30 min. Then, the samples were put in the magnetic separation rack to remove the supernatant. The gene expression was analyzed by qRT-PCR after RNA extraction.

Dual-luciferase reporter assay
The binding site of miR-132-3p and GLRX was predicted by the online prediction software StarBase (http://starbase.sysu.edu.cn/). The mutated type and wild-type sequences in the binding sites were designed and cloned into pGL3-Promoter luciferase plasmid (Promega), namely mut-GLRX and wt-GLRX. Then, mut-GLRX or wt-GLRX was cotransfected with miR-132-3p mimic or miR-132-3p inhibitor, respectively, into HEK-239T cells or pRL-TK (Promega). After that, Renilla luciferase activity and Firefly luciferase activity were determined by dual-luciferase reporter gene assay kit (Promega). Renilla luciferase activity was deemed as the internal control, and the ratio between the activities of Firefly luciferase and Renilla luciferase was calculated as the relative activity.

PD mouse model
Six-month-old male C57BL/6J mice (n = 24) were purchased from the Shanghai SLAC Laboratory Animal Co, Ltd. All animal handling and experimental procedures were approved by the Animal Care and Use Committee of Hunan Provincial People's Hospital (No. 202004). Mice were allowed food and water ad libitum and housed in rooms maintained at 24 6 1°C and 60-80% humidity using a 12-h dark cycle. The following experiments were conducted after one week of feeding. The study consisted of four groups of six mice each (random grouping by an Excel random number generator): the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), saline, miR-132-3p antagomir and antagomir NC groups. Mice in the MPTP group were intraperitoneally injected with 30 mg/kg MPTP (Sigma-Aldrich) every day for five consecutive days (Hu et al., 2019), and the mice of the Saline group were intraperitoneally injected with the same amount of Saline every day for five consecutive days. Mice in the MPTP1miR-132-3p antagomir group or MPTP1antagomir NC group were injected with MPTP the next day after stereotactic injection of miR-132-3p antagomir or antagomir NC (20 nM, total volume of 5 ml, GenePharma) into targeted brain areas of mice.

Stereotactic injection
After anesthesia of mice with ketamine (100 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection, the head of mouse was fixed to expose the skull. The intracerebral injection was performed on the following coordinates: À2.8 mm anteroposterior, À1.2 mm mediolateral, and À4.3 mm dorsoventral. Five microliters of miR-132-3p antagomir suspension or antagomir NC suspension was injected into SNc by using a 10-ml stereotactic catheter (1 ml/5 min). The needle remained in place for 5 min after complete injection then slowly removed. The mice were placed on a pad until recovery from the anesthesia. The healthy conditions of mice were monitored on the following 5 d, during which mice were subjected to acesodyne and local disinfection. The activity of mice after injection was recorded. No mouse was died during the whole experiments.

Behavioral tests
One week after establishment of PD mouse models, the behavioral tests were commenced. Motor coordination ability of experimental animals was investigated with the rotarod test. Before the experiments, animals were placed on rotating lanes for 5 min. Then, mice were trained for 2 min at a fixed speed of 4 rpm. After training, the rotational speed was accelerated uniformly from 4 rpm to 40 rpm within 60 s. The time of mice falling off the rotating rod was recorded. The open field test was conducted to evaluate the autonomous and exploratory behaviors of experimental animals in novel environments. Mice were individually placed into the center of an open field box (38 Â 38 cm) in a noise and light-controlled room. The spontaneous locomotor activities (central-area distance and whole-area distance) of each mouse were recorded and analyzed. The relative time of mouse falling from the rotating rod and the relative distance of mouse staying in the open field were recorded. Relative time and relative distance are the ratio of time or distance of experimental mouse/control mouse.

Brain tissue collection
Approximately 24 h after behavior test, ketamine (100 mg/kg) and xylaafine (10 mg/kg) were given to mouse for anesthesia through intraperitoneal injection. The heart was exposed and mouse (n = 6) in each group was perfused with 200 ml of normal saline through ventriculus sinister. The skull was opened and the brain tissues were collected and isolated. Part of the brain tissues was stored at À80°C refrigerator for qRT-PCR and the rest brain tissues were fixed in 4% triformol for 48 h for fluorescence in situ hybridization (FISH), immunofluorescence and immunohistochemistry.

FISH
The 4-mm paraffin sections were de-paraffinized with xylene and gradient alcohol (xylene soak for 10 min, refresh xylene for another 10 min, 50% xylene soaking for 10 min, absolute ethanol for 5 min, refresh absolute ethanol for another 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, and 70% ethanol for 5 min) before PBS wash. Sections were digested with 37°C protease K for 15 min, washed with PBS for 2 -Â 5 min, prehybridized at 55°C constant temperature, and incubated with digoxigenin-labeled (Exiqon) miR-132-3p probes overnight at incubator with 55°C constant temperature. Then, sections were subsequently washed with 5 Â SSC buffer, 1 Â SSC, and 0.2 Â SSC buffer for 2 Â 5 min at 55°C, followed by 5 min of wash with 0.2 Â SSC buffer at room temperature, 10 min of inactivation with 0.3% hydrogen peroxide-methanol solution, and 3 Â 5 min of PBS wash. After that, sections underwent three times of incubation each for 1 h: first blocked with normal serum blocking buffer at room temperature, second probed with mouse anti-DIG at room temperature, and then incubated with polymer anti-mouse. After each incubation, sections were washed three times with PBS for 5 min. Sections were stained with DAB for 5-10 min and washed with tap water for 10 min, before 2 min of hematoxylin counterstaining, hydrochloric ethanol differentiation and 10 min of tap water wash. These sections were sealed with neutral balata for observation under a microscope after dehydration and permeabilization. The ratio of positive cell numbers to total number of cells was calculated. DAPI was used for staining of cell nucleus and Iba1 was used to labeled microglial cells.

Immunofluorescence
Sections were incubated for 60 min at 60°C, dewaxed with xylene and washed with distilled water. Following antigen retrieval with 0.01 mol/l sodium citrate, sections were subjected to 10 min of incubation with 3% H 2 O 2 . Then, sections were washed with PBS for 3 Â 5 min, and inactivated with 5% normal goat serum for 30 min at room temperature. Sections were cultured with the primary antibody against tyrosine hydroxylase (ab137869, 1:200, Abcam), Iba1 (ab178846, 1:500, Abcam) or GLRX (ab45953, 1 mg/ml, Abcam) overnight at 4°C, while sections in negative control group contains corresponding antigens and were incubated with PBS. After that, sections were washed three times with PBS and incubated with FITC-labeled secondary antibody (ab6785, 1:1000, Abcam) at room temperature for 1 h. Then, the secondary antibody was removed, and cells were subjected to 5 min of staining with DAPI and 3 Â 5 min of PBS wash. Before pictures were captured by fluorescence microscope, sections were given glycerophosphoric acid for sealing. StereoInvestigator (MBF Bioscience) was used for stereological analysis on the total number of TH positive neurons and Iba1 positive cells for every sixth coronal section through the midbrain. After the the SN pars compacta with a 4Â objective was delineated, cells were counted under Â 60 magnification using ImageJ and following parameters: 8-mm height of an optical dissector, 50 Â 50 mm counting frame, 100 Â 100 mm area of a grid. The error coefficient of ,0.10 was acceptable. All sections were quantified in a blinded manner.

Immunohistochemistry
Following 60 min of bake, sections were dewaxed by xylene and washed with distilled water. Before 30 min of inactivation with normal goat serum at room temperature, sections underwent the following steps: antigen retrieval with 0.01 mol/ l sodium citrate, 10 min of reaction with 3% H 2 O 2 and three times of PBS wash for 5 min. The sections were inactivated with 5% normal goat serum for 30 min at room temperature. After that, sections were incubated with antibody against GLRX (ab45953, 1 mg/ml, Abcam) overnight at 4°C, while sections in negative control group contains corresponding antigens and were incubated with PBS. Sections were then subjected to three times of PBS wash and 1 h of incubation with secondary antibody (ab6785, 1:1000, Abcam). DAB was used for color development, and sections were given three times of PBS wash to terminate the color reaction (1-3 min). The nucleus was stained with hematoxylin for 3 min, and sections were dehydrated, permeabilized, and sealed. The percentage of positive cells was counted. Images were analyzed using ImageJ software (version 1.46, National Institutes of Health).

Statistical analysis
Experiments and statistical analysis were performed by different personnel. Statistical analysis was conducted using GraphPad Prism 7 software, and data are displayed as the mean 6 SD. The normal distribution of data was detected by Kolmogorov-Smirnov test, D'Agostino, Pearson omnibus normality test, or Shapiro-Wilk normality test. All data were complied with normal distribution. The t test was employed for comparisons between two groups. The one-way ANOVA was adopted followed by Tukey's multiple comparison tests for comparisons among multiple groups. P values of significance were those ,0.05 (Tables 2, 3, 4).

Results
Highly expressed miR-132-3p and lowly expressed GLRX in midbrain tissues of patients with PD The expressions of miR-132-3p and GLRX in midbrain tissues of patients with PD and in healthy controls were determined by qRT-PCR and Western blotting. The results of qRT-PCR manifested compared with control group, miR-132-3p in midbrain tissues of patients with PD was increased by 1.45 6 0.33-fold (p , 0.05; Fig. 1A). Furthermore, analyses of qRT-PCR and Western blotting exhibited that the mRNA and protein expressions of GLRX in the tissues of patients with PD were decreased to respectively 0.63 6 0.15-fold and 0.69 6 0.18-fold (p , 0.05, vs the control group; Fig. 1B,C). These finding indicated that miR-132-3p and GLRX may be implicated in the progression of PD.

Suppression of miR-132-3p alleviates MPTP-induced neuroinflammation and dopaminergic neurodegeneration in PD mouse models
Mice were subjected to stereotactic injection of miR-132-3p antagomir and given MPTP by intraperitoneal injection to probe the role of miR-132-3p in neuroinflammation and dopaminergic neuron degeneration of MPTP-induced PD mouse. The results of FISH presented that injection with MPTP elevated miR-132-3p expression in SNc of mice by 196.37 6 17.39% (p , 0.001), while the following exposure to MPTP1miR-132-3p antagomir repressed the level of miR-132-3p (p , 0.01, 125.59 6 12.67% vs 179.34 6 14.34%; Fig. 7A). Immunohistochemistry results displayed that there were enhanced expression of GLRX in the MPTP1miR-132-3p antagomir group (p , 0.01, vs the MPTP1antagomir NC group, 87.25 6 12.57% vs 57.16 6 6.28%) and decreased level of GLRX in the MPTP group by 53.47 6 6.39% (p , 0.001, vs the saline group; Fig. 7B). FISH and immunofluorescence were applied to detect the expressions of miR-132-3p and GLRX in microglial cells. The results showed that miR-132-3p expression of SNc of mice in MPTP group was increased by 2.16 6 0.36-fold, while GLRX expression was suppressed by 0.46 6 0.11-fold when compared Figure 5. MiR-132-3p negatively mediates GLRX. qRT-PCR (A) and Western blotting (B) were used to detect the mRNA and protein expressions of GLRX after miR-132-3p knock-down or overexpression in BV-2 cells. RIP experiment was applied to verify the binding of miR-132-3p to GLRX mRNA (C). After LPS or PBS treatment, RIP was applied to detect the GLRX mRNA expression in Ago2 complex (D). The binding site of miR-132-3p to the 39-UTR of GLRX mRNA was predicted by StarBase (E). Dual-luciferase reporter assay was utilized to verify the binding relationship between miR-132-3p and GLRX (F); N (number of independent cell culture preparations) = 3, *p , 0.05, **p , 0.01, ***p , 0.001, Error bars, standard deviation (SD).

Discussion
Neuroinflammation is a characteristic of neurodegenerative diseases, including PD, in which microglia confer pathogenic and exacerbating effects (Lull and Block, 2010;Zhao et al., 2020). Furthermore, the notable hallmark of PD is the degeneration of dopaminergic neurons in the SNc (Mead et al., 2017). Herein, BV-2 cells and SH-SY5Y were used in current study to explore the effect of miR-132-3p on inflammation of microglial cells and neuronal injury. We have reported that miR-132-3p is abnormally upregulated, a change positively connected with the inflammatory response of microglial cells. We demonstrated that the activated microglial cells caused by miR-132-3p leads to increased cell apoptotic rate and diminished viability of neuroblastoma cells. However, miR-132-3p was also reported to alleviate neuron apoptosis and impairments of learning and memory abilities in Alzheimer's disease (Qu et al., 2021). Alzheimer's disease and PD, both belonging to neuro-degenerative diseases: the former is a neurodegenerative brain pathology formed because of piling up of amyloid proteins, development of plaques, and disappearance of neurons (Tufail et al., 2021), while the latter is caused by the loss of dopaminergic neurons in the substantia nigra (Xu et al., 2021). On parallel, the treatment strategies between our two literatures were also different. In our study, LPS induced BV-2 cells were used as inflammatory cell models and the supernatant of BV-2 cell was co-cultured with SH-SY5Y cells. As for the PD rat models, MPTP treatment was given to rats for consecutively 5 d. This discrepancy may be explained by the difference on the disease background and treatment strategy. Similar to our detection, previous studies identified that miR-132-3p was one of the upregulated cimiRNAs in patients with major depression disorder (van den Berg et al., 2020) and was found to be elevated in the serum of patients with mild cognitive impairment (Xie et al., 2015). In addition, we also revealed that GLRX suppresses activation of microglial cells and ameliorates neuronal injury caused by miR-132-3p. Finally, we found that MPTP-induced neuroinflammation and degeneration of dopaminergic neurons in PD mouse models are dramatically attenuated after miR-132-3p downregulation. Thus, our study not only uncovered novel roles for miR-132-3p and GLRX in the pathologic abnormalities related to PD but also identified their potential application in the treatment for PD.
Microglial cells are macrophages residing in the brain, which originate from early erythro-myeloid precursors in the embryonic yolk sac (Kanthasamy et al., 2019). Activated microglial cells at the inflammation site promote the release of inflammatory cytokines, thereby intensifying the inflammatory response through activation and recruitment of other cells to the brain lesion (Kim and Joh, 2006). Accumulating evidence proposed that in the process of PD, microglial cells are activated, and then trigger the secretion of a variety of proinflammatory factors, including IL-6, IL-1b , and TNF-a (Guan et al., 2017). In an attempt to elucidate the mechanism by which miR-132-3p accelerates the progression of PD, we first investigated whether miR-132-3p affects the activation of microglial cells. Initially, remarkable high expression pattern of miR-132-3p was noticed in midbrain tissues from patients with PD rather than tissues of healthy controls. To this end, LPS was applied to simulate the inflammatory response in BV-2 cells. Herein, results of gain-and lossof-function experiments confirmed that miR-132-3p might likewise contribute to the activation of microglial cells. In our study, knock-down of miR-132-3p can suppress the release of inflammatory cytokines, including TNF-a, IL-1b , and IL-6, while overexpression of miR-132-3p can promote the inflammatory response in BV-2 cells, those results indicated that miR-132 as a driver of microglia proinflammatory responses. Of note, there has been relevant evidence supporting our findings that miR-132 confers a pivotal role in intracerebral hemorrhage by regulating inflammation, which is evident from the activation state of microglial cells and the expression of proinflammatory cytokines . Furthermore, by using CCK-8 assay and flow cytometry, we discovered that LPS can suppress survival rate of microglial cells and increase cell apoptosis, while further treatment by miR-132-3p knock-down partially reverse the LPS induced cell apoptosis and elevate cell survival rate to certain extent. Further measurement showed that activated microglial cells by miR-132-3p may lead to neuronal injury, as evidenced by reinforced cell apoptotic ability and reduced the proliferative ability of SH-SY5Y cells after SH-SY5Y cells were cultured with the conditioned medium of BV-2 cells which were transfected with miR-132-3p mimic, which highlighted the role of miR-132-3p in activation of microglial cells and neuronal injury. Interestingly, former work described that the dysregulation of miR-132 leads to the occurrence and exacerbation of PD (Qian et al., 2017). The BACE1-AS/miR-132-3p axis is responsible for the berberine-mitigated neuronal injury in Alzheimer's disease (Ge et al., 2020). Therefore, miR-132-3p may exert a negative effect on PD by inducing neuroinflammation and neuronal injury.
Subsequently, we are prompted to further look into the molecular actions of miR-132-3p in regulating PD by investigating the downstream target. Based on the comprehensive analysis from StarBase, dual-luciferase reporter assay and RIP assay, we identified GLRX as a direct target of miR-132-3p. GLRX is an indispensable thioltransferase whose main function is to remove protein glutathionylation (Burns et al., 2020). Herein, the lowly expressed GLRX was observed in the midbrain tissues of PD patients. Furthermore, analyses of qRT-PCR, Western blotting, ELISA, CCK-8 assay and flow cytometry elaborated that miR-132-3p interfered with microglial activation and neuronal injury by targeting GLRX. Our data are in agreement with the earlier findings showing that enhancement of GLRX activity in these brain cells would impede the progression of PD (Gorelenkova Miller and Mieyal, 2019). MPTP is a neurotoxin that results in a profound reduction of striatal dopamine levels and specific loss of dopaminergic neurons in animals (Lee et al., 2019). To further shed light into the relationship between miR-132-3p/GLRX and PD, we used a mouse model of PD stimulated by MPTP. Consistently, mice received MPTP injection had increased inflammatory cytokine release and decreased TH positive neurons, indicating the neuron loss in MPTP-treated mice. On parallel, elevated miR-132-3p expression and decreased expression of GLRX were also observed in mice in MPTP group, suggesting the possible implication of miR-132-3p in neuron loss of PD mouse. Here, we showed that miR-132-3p downregulation in PD mouse induces alterations in GLRX expression, and miR-132-3p was responsible for the inflammatory response of brain tissues of PD mouse models by modulating GLRX. Additionally, immunofluorescence of Iba1 on detection of microglial activation and immunofluorescence of tyrosine hydroxylase on assessment of dopaminergic neuron loss revealed that depletion of miR-132-3p may alleviate MPTP-induced dopaminergic neurodegeneration and neuroinflammation of PD mouse models. Simultaneously, these findings were further supported by the rotarod test and open field test.
In conclusion, our data suggest that the deficiency of miR-132-3p contributes to ameliorated PD. MiR-132-3p enhances the activation of microglia cells and promotes the release of inflammatory cytokines by targeting GLRX to exert toxic effect on neurons. These findings suggest that targeting neuroprotective pathways controlled by miR-132-3p may represent a potential therapeutic intervention strategy for PD therapy. Further work is required to ascertain whether the protection from PD observed here by silencing of the miR-132-3p is exerted by GLRX, inhibition of microglial activation and dopaminergic neuron loss or perhaps via modulation of other pathways.