MicroRNA-212-5p Prevents Dopaminergic Neuron Death by Inhibiting SIRT2 in MPTP-Induced Mouse Model of Parkinson’s Disease

Recently, emerging evidences show that sirtuins (SIRTs) modulate aging progress and affect neurodegenerative diseases. For example, inhibition of SIRT2 has been recognized to exert neuroprotective effects in Parkinson’s disease (PD). However, current SIRT2 inhibitors are lack of selective property distinguished from its homolog. In this study, we found that SIRT2 protein level was highly increased in PD model, which was negatively regulated by miR-212-5p. In detail, miR-212-5p transfection reduced SIRT2 expression and inhibited SIRT2 activity. In vivo study, miR-212-5p treatment prevented dopaminergic neuron loss and DAT reduction by targeting SIRT2, which means miR-212-5p shows neuroprotective effect in PD. Mechanismly, we found nuclear acetylated p53 was up-regulation according to p53 is a major deacetylation substrate of SIRT2. Furthermore, decreased cytoplasmic p53 promoted autophagy in PD model, which was showed as autophagosomes, autophagic flux, LC3 B and p62 expression. Meanwhile, we also found miR-212-5p treatment somehow alleviated apoptosis in PD model, which might have some underlying mechanisms. In conclusions, our study provides a direct link between miR-212-5p and SIRT2-mediated p53-dependent programmed cell death in the pathogenesis of PD. These findings will give us an insight into the development of highly specifically SIRT2 inhibitor of opening up novel therapeutic avenues for PD.


INTRODUCTION
Parkinson's disease (PD) is one of the most common neurodegenerative diseases, which is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra compacta (SNc) and aggregation of Lewy bodies in neurons (Savitt et al., 2006). Although aging, genetic factors, oxidative damage, neuroinflammation, and programmed cell death (PCD) play vital roles in the pathogenesis of PD, the precise etiology remains unclear (Dexter and Jenner, 2013). Emerging evidences show that sirtuins (SIRTs) modulate the course of aging and affect neurodegenerative diseases (Jesko et al., 2017). It is well recognized that SIRT1 displays neuroprotective properties in PD (Herskovits and Guarente, 2014;Kida and Goligorsky, 2016). For instance, SIRT1 decreases α-synuclein-caused neuro toxicity by deacetylating HSP70  and inhibits activation of microglia by p53/caspase 3-dependent apoptosis (Ye et al., 2013). Unlike SIRT1 plays neuroprotective effects, SIRT2 aggravates the pathological damage induced by MPTP (Liu et al., 2014). In addition, inhibition of SIRT2 reduces DA neurons loss and blocks the α-synuclein toxicity (Outeiro et al., 2007). However, existing SIRT2 inhibitors, such as AK-1, AK-7, and AGK-2, are lacking of high specificity for SIRT2 over SIRT1, which restricts their pharmacological effects in PD therapy (Cui et al., 2014).
Seven SIRTs (SIRT1-7) are present in mammals. They target distinct protein substrates and are located in distinct subcellular compartments. SIRT2 not only is primarily cytosolic but also may enter the nucleus during the G2/M phase (Houtkooper et al., 2012). Thus, SIRT2 participates in the modulation of multiple and diverse biological processes, such as cell cycle, microtubule dynamics, metabolic networks and autophagy by deacetylating lysines on diverse proteins (Outeiro et al., 2007;Yu and Auwerx, 2009;Zhao et al., 2010;Beirowski et al., 2011;Maxwell et al., 2011). The tumor suppressor protein 53 (p53), one of the important substrates of SIRT2 (van Leeuwen et al., 2013), plays a dual role in autophagy according to its sub-location. Nuclear p53 induces autophagy, and cytoplasmic p53 acts instead as a repressor of autophagy (Levine and Abrams, 2008;Tasdemir et al., 2008). It is reported that SIRT2 inhibited lysosome-mediated autophagic turnover by interfering with aggresome formation in neuro cytotoxicity model (Gal et al., 2012). However, there is lack of study to explore the link between SIRT2 and p53-dependent autophagy in PD model. In addition, silence of SIRT1 decreases acetylation of p53 and inducing autophagy (Lee et al., 2015), which indicates that p53 is enigmatic in autophagy regulation. Thus, researchers are trying to develop highly selective drug-like inhibitors or agonists that show a unique mechanism (Rumpf et al., 2015). According to perturbation of micro RNAs (miRNAs) expression and specific targets in the pathophysiology of PD, miRNA-based therapy has been taken insights to PD treatment (Ma et al., 2013;Maciotta et al., 2013).
miRNAs are essential small RNA molecules (20-24 nt) that negatively regulate the expression of target genes at the post-transcriptional level. Substantial evidences suggest that miRNAs are critical in PD pathogenesis. For instance, miR-7 represses toxicity and cell death by targeting α-synuclein (Junn et al., 2009;Doxakis, 2010). In our previous work, we found miR-7 also enhances neurogenesis and inhibits neuroinflammation by targeting NLRP3 (Fan et al., 2016;Zhou et al., 2016). Studying miRNA involvement in PD might also could provide targets for innovative therapy. According to the development of miRNA-targeting oligonucleotides with improved pharmacological activity and optimized pharmacokinetic properties, miRNAs would be recognized as therapeutic agents against disease (Junn and Mouradian, 2012). Our preliminary data showed that miR-212-5p was decreased in PD model and might be a potential regulator of SIRT2 by bioinformatics prediction. In this study, we prepared MPTP-induced PD mice model in vivo and MPP + stimulation in vitro with miR-212-5p gene therapy, so as to explore its functional and therapeutic role in PD model. We found SIRT2 expression in protein level remarkably increased without alteration in RNA level in the PD experimental model, inhibiting of SIRT2 by miR-212-5p could prevent DA neurons loss via promotes cytoplasmic p53-dependent autophagy. Meanwhile, we also found miR-212-5p treatment somehow alleviated apoptosis in PD model, which might have some underlying mechanisms. Moreover, miR-212-5P highly selectively inhibited SIRT2 expression over SIRT1. These findings give us an insight into the potential development of miR-212-5p-based SIRT2 inhibitor in therapeutic avenues for PD.

MATERIALS AND METHODS
The study protocol was approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.

Animal Model
Twelve-week-old male C57BL/6 mice were randomly divided into four groups: negative control with saline-treated group, negative control with MPTP-treated group, miR-212-5p with saline-treated group, and miR-212-5p with MPTP-treated group. All animals were kept in cages with constant temperature (25 • C) and humidity and were exposed to a 12/12-h light-dark cycle with unrestricted access to tap water and food. Mice received 25 mg/kg MPTP (Sigma, St. Louis, MO, United States) subcutaneously once a day for 7 days. Saline control mice were treated with the same volume of saline. Animals were sacrificed at 5 days after the last injection of MPTP or saline.

Transmission Electron Microscopic Analysis
Mice were perfused with 2.5% glutaraldehyde and 2% paraformaldehyde. A small portion (∼1 mm 3 ) of the hippocampus was sectioned and incubated for 2 h at 4 • C in the same fixative. Specimens were postfixed in 1% osmium tetroxide, stained in aqueous uranyl acetate, and then dehydrated and embedded in epoxy resin. Ultrathin sections were stained using lead citrate and examined with transmission electron microscope (JEM-1010, Tokyo, Japan). All experiments and photographs of TEM were supported by the grant from the center of forecasting and analysis of Nanjing Medical University.

Immunofluorescence, Unbiased Stereology, and TUNEL Staining
For frozen samples, mice were perfused transcardially with 4% paraformaldehyde. Brains were extracted, post-fixed, dehydrated, embedded in OCT (Tissue-Tek), and cryosectioned at 30 µm per slice. For immunofluorescence, slides were incubated with the indicated primary antibodies at 4 • C overnight, then washed and incubated in secondary fluorescent antibodies, followed by mounting in Prolong Gold Antifade with DAPI (Life Technologies, Cat P36931) before imaging. For in vivo cell quantification studies, the number of TH + neurons in the SNpc of the midbrain was assessed using the optical fractionator (Stereo Investigator 7, MBF Bioscience, Williston, VT, United States) as previously reported (Han et al., 2018). All stereological analyses were performed under the 200× objective of an Olympus BX52 microscope (Olympus America Inc., Melville, NY, United States). The brain slices and cells were sampled to measure TUNEL positive with a TUNEL BrightGreen Apoptosis Detection Kit (Vazyme) following the manufacturer's instructions. Briefly, 30 µm frozen sections were incubated with proteinase K (20 mg/ml) for 10 min at room temperature. The sections were incubated in the TdT buffer including BrightGreen Labeling Mix and Recombinant TdT Enzyme for 1 h at 37 • C. Images were captured by Nikon fluorescence microscope (TE2000-S).

Immunocytochemistry
Cells plated on coverslips were fixed in 4% paraformaldehyde in PBS for 10 min. Cells were then blocked with 5% BSA containing 0.1% Triton X for 1 h. After that, the cells were incubated with primary antibodies (anti-SIRT2, Abcam; anti-LC3, Cell Signaling Technology; anti-p53, Invitrogen) overnight at 4 • C. Afterward, cells were incubated with the Alexa 488 and 555 coupled secondary antibodies (Invitrogen) for 1 h at room temperature followed by counterstained with DAPI. Images were captured by Nikon fluorescence microscope (TE2000-S).

qRT-PCR Analysis of mRNA and MicroRNA Expression
Total RNA was prepared using TRIZOL reagent (Invitrogen Life Technologies, United States). Reverse transcription of total RNA using TaKaRa Master Mix (TaKaRa, Japan). The primers were purchased from GENEray (Shanghai, China).
MicroRNA specific primers were purchased from GeneCopoeia. The relative expression was calculated using the CT method as described elsewhere and normalized to uniformly expressed U6. All qRT-PCRs were performed in triplicates, and the data are presented as mean ± standard error (SEM).

Dual Luciferase Reporter Assays
Luciferase reporter assays were performed using the psiCHECK2-3 UTR vector described previously (Fan et al., 2016). Briefly, cells were grown to 50% confluence in 24-well plates and co-transfected with psiCHECK2-SIRT2-3 UTR or Mut-SIRT2-3 UTR (GENEray Biotechnology, Shanghai, China) plus miR-212-5p mimics or negative control mimics. Cells were incubated with a transfection complex for 24 h followed by luciferase reporter assay using the Dual Luciferase Assay System (Promega, Madison, WI, United States). Renilla luciferase activity was normalized to firefly luciferase activity. Cell lysates were subjected to luciferase activity measurement according to the manufacturer's instructions.

Cell Viability Assay
Cells were cultured in the 96-well plate and two extra wells were set for each group. After treatment, we discarded the medium and performed according to the instruction of EnoGeneCell TM Counting Kit-8 (CCK-8). 10 µL CCK-8 solution was added in one well and the plate was put in the incubator for about 3 h. We measured the wavelength of each well at 450 nm by microplate reader when time was over.

Lactate Dehydrogenase (LDH) Assay
Cells were planted in 96-well plate at 5,000 cells/well, and treated as described above. Culture medium was collected to measure LDH levels with an assay kit (Nanjing Jiancheng Bioengineering Institute) following the manufacturer's instructions. The samples were quantified at 440 nm with a spectrophotometric plate reader.

Hoechst Staining
To quantify apoptotic SH-SY5Y cells, monolayer cells were fixed and stained with Hoechst 33342 (Sigma, St Louis, MO, United States). The morphological features of apoptosis (cell shrinkage, chromatin condensation, and fragmentation) were monitored by fluorescence microscopy (Olympus BX 60, Tokyo, Japan). At least 300 cells from 12 randomly selected fields per dish were counted, and each treatment was performed in triplicate.

SIRT2 Activity Assay
The SIRT2 activity was determined by the instrument of SIRT2 direct fluorescent screening assay kit (Cayman, United States).
Generally speaking, Cayman's SIRT2 direct fluorescent screening assay provides a fluorescence-based method for screening SIRT2 inhibitor or activators. The procedure requires only two steps. In the first step, the substrate is incubated with human recombinant SIRT2 along with its co-substrate NAD + . Deacetylation sensitizes the substrate such that treatment with the developer in the second step release a fluorescent product. The fluorophore can be analyzed with an excitation wavelength of 350 nm and an emission wavelength of 465 nm. Subtract each inhibitor sample value from the 100% initial activity sample value. Divide the result by the 100% initial activity value and then multiply by 100 to give the percent of inhibition.

Statistics
The data are presented as mean ± SEM. Statistical differences were evaluated using Student's t-test for comparisons of two groups or analysis of variance (ANOVA) and appropriate post hoc analyses for comparisons of more than two groups. P < 0.05 was considered significant.

SIRT2 Protein Expression Is Increased in PD Experimental Model
To examine the expression of SIRT2 in PD model, we detected SIRT2 protein and mRNA levels both in vivo and in vitro. Compared to the saline-treated mice, MPTP injection highly increased the expression of SIRT2 in the midbrain (Figures 1A,B). Similarly, SIRT2 protein level was markedly up-regulated with MPP + stimulation in SH-SY5Y cells (Figures 1C,D). Consistent with the result of Western blotting, immunocytochemistry staining showed SIRT2 was highly expressed in SH-SY5Y cells by MPP + stimulation ( Figure 1E). However, there was no significant difference in SIRT2 mRNA level both in vivo and in vitro (Figures 1F,G), which indicated there might have post-transcriptional regulation of SIRT2 expression.

P53 Transfection Abolished the SIRT2-Related Programmed Cell Death in SH-SY5Y Cells
To demonstrate whether the function of miR-212-5p is dependent on p53, we transfected pcDNA3.0-Flag-p53 into SH-SH5Y cells. We adopted three different plasmid concentrations (0.5, 1, and 2 µg) and found that 1 µg plasmid was efficient to increase p53 expression (Figures 5A-C) without impact FIGURE 4 | MiR-212-5p regulates p53 sub-localization via SIRT2 activity in PD model. (A,B) Western blotting and quantitative analysis showed that the total p53 expression was highly increased by MPP + stimulating, which was somehow inhibited by miR-212-5p mimic treatment. (D,E) In contrast, miR-212-5p inhibitor indeed enhanced the increased total p53 expression. Since p53 is a key deacetylation substrate of SIRT2, we detected the acetylation of p53 by Western blotting. Results showed that miR-212-5p regulated the level of acetylated p53 (C,F). (G) SIRT2 activity assay revealed that miR-2125p could suppress the deacetylation potential of SIRT2. (H) Double immunofluorescence of SIRT2 (green) and p53 (red). Scale bar: 40 µm. Consistent with previous results, SIRT2 and p53 were highly increased under MPP + treatment, which was inhibited by miR-212-5p transfection. (I-K) Western blotting and quantitative analysis showed that p53 was up-regulated both in cytoplasm and nucleus. miR-212-5p transfection suppressed the expression of p53, especially in cytoplasm. * p < 0.05, * * p < 0.01 versus control or NC-control group; # p < 0.05, ## p < 0.01 versus NC plus MPP + group.
These data further suggests that miR-212-5p is closely related to accelerating neuronal apoptosis in the pathological process of PD.

DISCUSSION
Parkinson's disease is an age-associated neurodegenerative disorder primarily known as a motor disorder due to the loss of dopaminergic neurons from the substantia nigra in the brain. Although there is still no cure or treatment available for PD, what is certain is that aging is the major risk factor. Therefore, any regimen that delays or interferes with the age-related decline in brain function would delay or prevent neurodegenerative diseases. SIRTs, a family of class III histone deacetylases, have beneficiary effects against age-related diseases by modulating a myriad of cellular processes, including energy metabolism, stress response, cell/tissue survival and malignancy (Donmez, 2012). In mammals, there are seven SIRTs (SIRT1-7), which are in different subcellular locations, and enzymatic activities, including the nucleus (SIRT1, SIRT6, and SIRT7), cytosol (SIRT2), and mitochondria (SIRT3, SIRT4, and SIRT5). Among them, SIRT1, SIRT2, and SIRT3 are robust deacetylases (Donmez, 2012;Duan, 2013). To date, SIRT1 has been extensively investigated due to its initial connection with lifespan extension and involvement in important biological processes. SIRT1 is protective in cell culture and animal models of PD, as long as SIRT1 deacetylates heat shock factor 1 (HSF1), peroxisome proliferators activated receptor gamma co-activator 1 alpha (PGC1α) and affects α-synuclein toxicity (Herskovits and Guarente, 2014). Unlike SIRT1, SIRT2 is present primarily in the cytoplasm, co-localizes with microtubules and deacetylates the major component of microtubules, α-tubulin at lysine 40 (North et al., 2003). In addition, SIRT2 deacetylates forkhead transcription factors of class O (FOXO) (Wang et al., 2007;Pais et al., 2013;Akbulut et al., 2015) and NF-κB (Li et al., 2013), as results of affecting apoptosis and inflammation. Moreover, sirtuin 2 is reported to exacerbate alpha-synuclein toxicity in models of Parkinson's disease (de Oliveira et al., 2017). Consistent with the previous finding, we observe SIRT2 protein expression is highly increased in PD model. Meanwhile, SIRT1 protein expression is obviously decreased. Although SIRT2 inhibitor has been verified to play neuroprotective effects on PD (Outeiro et al., 2007;Li et al., 2013;Chen et al., 2015), these inhibitors lacked the desired isotype selectivity (Rumpf et al., 2015). Future work will be necessary to identify more potent and selective inhibitors/activators of SIRTs for neurodegeneration and may provide avenues for therapeutic intervention.
Recent studies have provided important insights into the autophagy underlying PD. It is reported that α-synuclein is predominantly degraded by the lysosomal pathways, particularly via chaperone-mediated autophagy (CMA) (Vogiatzi et al., 2008). In addition, autophagy progress regulates PD-related proteins like, such as LRRK2, PINK1, Parkin, and ATP13A2 (Martinez-Vicente, 2015). Furthermore, blocking autophagy could also trigger apoptosis (Venderova and Park, 2012). Thus, impaired autophagy causes accumulation of pernicious PD-related proteins and loss of neuron cells. In this study, we observe that p53 is highly increased in PD experimental models. As well known, p53 activates autophagy via damage-regulated autophagy modulator (DRAM) as a transcriptional target (Crighton et al., 2006). However, p53 also inhibits this process depending on its subcellular localization (Tasdemir et al., 2008) Acetylation of p53 exerts stability and transcription factor activity, which is regulated by SIRTs. Recent study reported that SIRT1 inhibited dopaminergic neurons injury via p53-caspase-3-dependent mechanism of apoptosis (Ye et al., 2013). As mentioned before, SIRT2 plays diverse role from SIRT1 in PD. In the present study, we reveal that inhibition of SIRT2 decrease the cytoplasmic p53 expression by increasing acetylation level of p53. It is well established that cytoplasmic p53 inhibit autophagy, meanwhile, the nuclear p53 promotes apoptosis. These results indicate that p53 is required for SIRT2-mediated autophagy in DA neurons, which exerts the critical role in the pathogenesis of PD. Therefore, searching therapeutic intervention for SIRT2/p53/autophagy pathway is impending.
Emerging evidences suggest that multiple miRNAs participate in the pathology of AD and PD by targeting specially genes and exhibit the therapeutic potential in neurodegenerative diseases. Identified by microarray and verified by qPCR methods, several miRNAs are highly expressed and altered in PD, such as let-7, miR-7, miR-9, miR-29, miR-34 (Qiu et al., 2014). Here, we found that miR-212-5p decreased in PD experimental model. We further revealed miR-212-5p directly and specifically regulated SIRT2 expression. There is lack of miR-212-5p study besides a recently study reported that miR-212-5p was related to fungal infections (Dix et al., 2017). Recently, besides the potential of miRNAs as biomarkers, miRNAs are also gaining attention for their therapeutic potential with far-reaching roles in neurodegenerative diseases (Zi et al., 2015). Here, we found stereotactic injection of miR-212-5p mimics into the midbrain of mice could prevent dopaminergic neuron damage and loss. An elegant study demonstrated the therapeutic feasibility and safety of miRNA in a primate disease model. The locked-nucleic-acids-modified oligonucleotides targeting miR-122 (SPC3649) method is currently being used in Phase I clinical trials for hepatitis C virus infection, probably becoming the first miRNA therapeutic target in humans (Lanford et al., 2010). However, miRNA-based therapies pose many unsolved challenges, including the specificity of miRNA function, the specific non-invasive delivery to the central nervous system, and evaluate the toxicity of therapeutic small oligonucleotides.
Taken together, as shown in Figure 8, we demonstrate that the miR-212-5p is downregulated and specifically regulate the translation of SIRT2 in the midbrain or SH-SY5Y cells of PD experimental models. Furthermore, inhibition of SIRT2 promotes autophagy by decreases cytoplasmic p53 expression, according to the deacetylation of p53 by SIRT2. Most notably, stereotactic injection of miR-212-5p mimics into midbrain significantly improves the impairment of dopaminergic neuron by targeting SIRT2. Our study provides a direct link between SIRT2 suppression and MPTP-induced neurodegeneration. These findings will give us an insight into the potential of miR-212-5p in opening up novel therapeutic avenues for PD.

ETHICS STATEMENT
Animal welfare and experimental procedures in this study were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, the United States) and the related ethical regulations of Nanjing Medical University.

AUTHOR CONTRIBUTIONS
GH and SS designed the research. XH and SS performed cotransfections for the Western blotting assays, neuropathological study, RT-PCR and drafted the manuscript. XL and MJ performed the immunohistochemistry study and statistical analysis. XL and QS helped to perform the Western blotting assays and contributed materials and analysis tools. JD and ML discussed the project and gave valuable suggestions to this project. All authors read and approved the final manuscript.