A total of 80 male C57BL/6 mice (7–8 weeks, ~25 g) were obtained from the Shanghai Experimental Animal Center of Chinese Academy of Sciences (Shanghai, China). Each individual cage contained 4 mice, with free access to water and food. All of the animal rooms were maintained at a temperature of 21–23°C and humidity of 40–60%, with a 12-h light-dark cycle. To develop the MPTP PD model, male C57BL/6J mice were injected intraperitoneally (i.p.) with 1-methyl-4-phenyl-1,2,3,6-tetrahydrapyridine (MPTP) at 20 mg/kg (body weight; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) twice a week, plus 250 mg/kg probenecid injected 0.5 h before. Administration of the treatment was carried out for 5 weeks with feeding of 0.2% lithium carbonate (Sigma-Aldrich; Merck KGaA) by chow. On the 36th day, the mice were subjected to behavioral tests. Upon completion, mice were anesthetized with 250 mg/kg avertin intraperitoneally and perfused transcardially with sterile saline. Brains were quickly harvested, and one hemibrain was immediately frozen in liquid nitrogen for biochemical studies while the other hemibrain was post-fixed in 4% paraformaldehyde for immunohistochemical stains. All animal procedures complied with the current ethical considerations of the Shanghai University of Traditional Chinese Medicine's Animal Ethics Committee, which is in accordance with the National Research Council criteria. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai University of Traditional Chinese Medicine and were performed in accordance with the relevant guidelines and regulations as well as approved by the guidelines on ethical standards for investigation of PD in conscious animals. Animals were sedated or anesthetized using carbon dioxide prior to cervical dislocation. Cervical dislocation to euthanize mice was performed by trained research personnel after the approval of the IACUC of Shanghai University of Traditional Chinese Medicine's Animal Ethics Committee and the method was performed in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2013 Edition).

The open field activity was assessed in a 27×27×38 cm chamber with 50 lux (lx) illumination. Fifteen minutes of free movement was tracked by TruScan Open Field version 2.04 software 2.04 (Coulbourn Instruments, Holliston, MA, USA). Total movement times and distances were scored to assess locomotor activity while a percentage of movement time in the margin area (the area within 8.76 cm from the chamber wall) was calculated for scrutinizing the autonomous activity (13).

Rotarod training was performed concurrently for 10 min during 5 consecutive days: On the first day, the mice were placed on the rotating rod at 1.5 × g for 5 min. Every 30 sec, the rod speed was increased by 0.4 × g up to 5.5 × g, and then maintained at this speed for 1 min. On the second day, a 1.5 × g speed was used for 1.5 min. The rod speed was increased by 0.4 × g every 30 sec until it reached 7.3 × g, where it was maintained for 10 min. On the third, fourth, and fifth days, the latencies of falling off the rod with a linear increase in rod speed from 1.5 × g up to 15 × g for 5 min were measured 3 times and averaged. The actual test protocol was the same for the last 3 days of the training protocol.

For the bisulfite modification, the EZ DNA Methylation-Gold™ Kit (Zymo Research Corp., Irvine, CA, USA) was used. In this technique, unmethylated cytosines are converted to uracil while methylated cytosines remain unchanged in bisulfite-treated DNA. Thus, the methylation status of the DNA sample can be analyzed by PCR amplification, called ‘methylation specific-PCR.’ DNA (~1 µg) was used for bisulfite conversion, and then MSP was carried out using specific primers for both bisulfite converted methylated and unmethylated DNA samples in a total 25 µl mix containing 1X PCR buffer, 15 mM MgCl₂, 200 µM of each dNTP, 0.4 µM of each primer, <1 µg template DNA, and 2.5 U HotStarTaq DNA Polymerase (Qiagen, Inc., Valencia, CA, USA). After the initial denaturation step at 94°C for 15 min, 35 cycles were performed for the regions of SNCA using the following conditions: denaturation at 94°C for 1 min, at annealing temperature for each PCR bisulfide conversion-specific primer pair for 1 min, extension at 72°C for 1 min, and then final extension at 72°C for 10 min. Bisulfite-treated DNA was amplified using primers SNCA-PromF, 5′-AAAATTTTGAAGATATTTGAATTAAAG-3′ and SNCA-PromR, 5′-CTAATCCTCCTCCTTCTCCTTCTC-3′; SNCA-IntF, 5′-GGAGTTTAAGGAAAGAGATTTGATT-3′ and SNCA-IntR, 5′-CAAACAACAAACCCAAATATAATAA-3′, specifically designed for the bisulfite-treated DNA. The PCR products SNCA (−926/-483; intron 1) were cloned into pMD 18-T Vector (Takara Biotechnology Co., Ltd., Dalian, China) following the manufacturer's instructions. Plasmid DNA was isolated from at least 10 clones per region (Wizard Plus SV Minipreps; Promega Corporation, Madison, WI, USA), sequenced using vector-specific primers and the Big Dye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and analyzed on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.). Quality control for DNA methylation was performed using BiQ (software tool for DNA methylation analysis; http://biq-analyzer.bioinf.mpi-inf.mpg.de/).

Total RNA, including mRNA and miRNA, was isolated from samples using an RNeasy Mini Kit (Qiagen, Inc.). Next, cDNA was synthesized using a PrimeScript™ RT reagent kit (Takara Biotechnology Co., Ltd.) and subsequently used as templates for qPCR. The primers were designed using Primer 5.0 (Premier Biosoft International, Palo Alto, CA, USA). Primers of real-time PCR were SNCA F, 5′-GGACCAGTTGGGCAAGAATG-3′ and R, 5′-GGGCACATTGGAACTGAGCAC-3′; GAPDH F, 5′-CGGAGTCAACGGATTTGGTC-3′ and R, 5′-TTCTCCATGGTGGTGAAGAC-3′; miR-148a F, 5′-TCAGTGCACTACAGAACTTTGT-3′ and R, 5′-GCTGTCAACGATACGCTACG-3′; U6 F, 5′-CTTCGGCAGCACATATAC-3′ and R, 5′-GAACGCTTCACGAATTTGC-3′. qPCR was performed in triplicate with SYBR-Green (Takara Biotechnology Co., Ltd.) on an Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The reaction parameters were set as follows: 95°C for 5 min, followed by 40 cycles of 10 sec for 95°C and 1 min for 60°C. For miRNA RT-qPCR, 1 mg of isolated RNA was reverse-transcribed with stem-loop primers from a BioTNT miRNA qPCR Detection Primer Set (BioTNT Biotechnologies Co, Ltd., Shanghai, China). All reactions were repeated in triplicate. The relative mRNA and miRNA expression levels were separately analyzed using the 2^−∆∆Cq method (14) with GADPH and U6 as the endogenous controls.

Total RNA (including miRNAs) was isolated from the brain tissue of the substantia nigra using TRIzol^® (Thermo Fisher Scientific, Inc.) with slight modification. Polyacryl carrier was added to improve RNA recovery. The global miRNA expression profiles were obtained using the Agilent SurePrint Mouse miRNA Microarrays (Agilent Technologies, Inc., Santa Clara, CA, USA) containing 1,881 mouse miRNAs based on Sanger miRBase release 12.0. RNA samples were processed, labeled, and hybridized onto the microarrays according to the manufacturer's protocols. Microarray slides were then scanned using the G3 High Resolution Scanner, and microarray data were extracted by Feature Extraction software version 10.7.3.1 (Agilent Technologies, Inc.).

Animals were anaesthetized with ether and perfused through the heart with 4% paraformaldehyde in 0.1 mM phosphate buffer pH 7.4. Brains were removed, post-fixed overnight in the same fixative, and then washed in buffered 18% sucrose until they sunk. Sections were cut with a freezing microtome at 30-µm thickness, and then permeabilized for 20 min with phosphate-buffered saline (PBS) containing 0.1% Triton X-100, before a 20-min incubation in methanol containing 0.3% H₂O₂ to quench endogenous peroxidase activity. Subsequently, the sections were incubated for 30 min in PBS containing 2% normal goat serum to block non-specific binding sites. For immunohistochemical localization of tyrosine hydroxylase (TH), the TH antibody was used at a dilution of 1:1,000 (cat. no. sc-136100, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Antibody exposure was performed overnight at 4°C, followed by a 90-min incubation at room temperature with a horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) secondary antibody (cat. no. A0216, Beyotime Institute of Biotechnology, Haimen, China). The immunoreaction was visualized using DAB Horseradish Peroxidase Color Development Kit (Beyotime Institute of Biotechnology). Digital images were captured using a Leica DM2000 Microscopic imaging system, and TH-positive neuronal counts and the integrated optical density value (IOD) of α-synuclein-positive fibers were estimated within the substantia nigra by ImageJ software (NIH) as previously described (15).

The substantia nigra of wild-type, MPTP-treated, and lithium-treated mice (n=3), were dissected, pooled, and homogenized in lysis buffer (Beyotime Institute of Biotechnology) on ice plus 1:100 volume of phenylmethylsulfonyl fluoride (PMSF), before final centrifugation at 14,000 × g for 5 min to remove debris. The BCA kit (Beyotime Institute of Biotechnology) was used to assess the protein concentrations. After heating at 100°C with loading buffer for 4 min, the proteins (30 µg) were resolved by SDS-PAGE (12% for a-synuclein, 6% for DNMT1) and transferred to nitrocellulose membranes (Amersham; GE Healthcare, Chicago, IL, USA) at 200 mA for 40 min. Tris-buffered saline and Tween-20 (TBST) containing 5% skimmed milk powder was used to block the membranes at room temperature for 1 h, and then membranes were washed with TBST prior to incubation with antibodies against α-synuclein (ab27766, 1:2,000; Abcam, Cambridge, MA, USA) and DNMT1 (ab19905, 1:2,000; Abcam) overnight at 4°C. After washing thrice with TBST, the membranes were incubated with horseradish peroxidase conjugated anti-rabbit (cat. no. A0208) or anti-mouse IgG (cat. no. A0216, Beyotime Institute of Biotechnology) at a dilution of 1:2,000 at room temperature for 45 min. After washing for three further times with TBST, the proteins were visualized using an enhanced chemiluminescence kit (EMD Millipore, Billerica, MA, USA), and then quantified using Quantity One software version 4.4.6 (Bio-Rad Laboratories, Inc., Hercules, CA, USA), normalized to GAPDH.

The raw data were analyzed by Origin 8.5 software (OriginLab, Northampton, MA, USA). Results of data analysis were expressed as the means ± standard error of the mean (SEM) with the number of experiments indicated in the figure legends. Behavioral experiments were statistically analyzed by one-way ANOVA. Tukey's test was employed to compare the differences between all the groups. For all statistical tests, a value of P<0.05 was considered to indicate a statistically significant difference.

To examine the effects lithium had on MPTP-induced Parkinson mice, behavioral tasks were performed after 5 weeks of lithium treatment. The results revealed a significant reduction in the locomotor activity in the MPTP-treated mice and lithium treatment relieved this effect satisfactorily. As revealed in Fig. 1A and B, the average movement distance was 534.23±48.01 cm and the average activity time was 246.33±6.48 sec. Following impairment, the movement distance and activity time values were reduced to 315.09±25.59 cm (MPTP group vs. CTRL group, P<0.01) and 194.80±40.79 sec (MPTP group vs. CTRL group, P<0.01), respectively, however, after lithium administration for 5 weeks, those values rebounded to 423.40±33.21 (lithium group vs. MPTP group, P<0.05) cm and 242.75±8.05 sec (lithium group vs. MPTP group, P<0.05), respectively. Furthermore, in rotarod tests, the compound also prolonged the drop time of MPTP-treated mice, resulting in an increase in the drop time for MPTP-treated mice from 148.59±13.03 sec (MPTP group vs. CTRL group, P<0.05) to 204.70±23.27 sec (lithium group vs. MPTP group, P<0.05) bringing it close to that of the control group results 228.62±15.79 sec (lithium group vs. CTRL group, P>0.05; Fig. 1C).

A reduction in TH-positive neurons and an increase in expression of α-synuclein are both characteristic of PD pathology. Corresponding pathological changes were also observed in MPTP-impaired mice, and lithium treatment was able to mitigate these alterations as well. Using immunohistochemical staining, a significant loss of TH⁺ neurons in the substantia nigra regions of MPTP-impaired mice at 6 weeks (48.60±0.022% reduction; P<0.01 compared to control) was revealed. Conversely, the number of TH⁺ neurons rebounded considerably after lithium treatment (32.20±0.029% increased; P<0.01 compared to MPTP group; Fig. 2). In addition, the integrated optical density value (IOD) of α-synuclein-positive fibers in MPTP-impaired mice increased from 54,370±699 to 74,790±736 (37.5% increased; P<0.01 compared to the control), however, after lithium treatment it decreased to 68,958±654 (11.3% increased; P<0.05 compared to the MPTP group; Fig. 2).

To investigate whether epigenetic changes may contribute to the dysregulation of SNCA expression in the MPTP-induced PD animal model, DNA from the substantia nigra regions of each group was analyzed. Significantly fewer methylated CpG sites in the DNA of PD mice were revealed. There was a significant decrease in the mean methylation rate of SNCA (−926/-483; intron 1) in PD mouse substantia nigra (2.05±0.41%, P<0.01) when compared with that determined in the control group (3.62±0.58%). Although the methylation rate was lower (2.38±0.46%) in the lithium-treated group when compared to the control group, the opposite was revealed with comparison to the MPTP group, which resulted in a significant increase in the methylation rate (P<0.05; Fig. 3).

The role of miRNAs in the pathogenesis of Parkinsonism has attracted increasing attention. Furthermore, the recent discovery of miRNA expression and regulation has led to the consideration of lithium as a novel mechanism of neuroprotection. SurePrint Mouse miRNA Microarrays (Agilent Technologies, Inc.) were utilized to profile changes in the miRNA expression of MPTP-impaired mice. The results revealed alterations in the expression levels of 39 miRNAs >1.5-fold in the substantia nigra of PD mice (data not shown). The results also revealed an upregulation in the expression of miR-148a in PD mice, an effect which was reversed after lithium treatment, resulting in a downregulation of this particular miRNA. RT-qPCR confirmed this result (Fig. 4A). Previous research has indicated that miR-148a could be considered as a potential inhibitor of DNMT1, a DNA methylase, and thus play a role in the establishment and regulation of tissue-specific patterns of methylated cytosine residues. Further RT-qPCR results revealed decreased expression of DNMT1 in the substantia nigra of PD mice compared to the control, and a significant difference was recorded when compared with the lithium-treated group (Fig. 4B). These results were also confirmed by western blot analysis, which presented a significant reduction in the expression of DNMT1 protein in PD mice compared with the control group. Furthermore, lithium treatment resulted in a significant increase in DNMT1 expression in PD mice (Fig. 4C).

Aberrant aggregation of α-synuclein is considered to be the major neuropathological characteristic of PD, and α-synuclein has been detected in insoluble inclusions within Lewy bodies and neurites. In the present study, a significant increase was observed in the expression of α-synuclein in the substantia nigra of mice. However, the increase of α-synuclein in PD mice could be attenuated through administration of lithium (Fig. 4D).