Role of GSK3β/α-synuclein axis in methamphetamine-induced neurotoxicity in PC12 cells

Abstract
Methamphetamine（METH）is well-known as a potent psychostimulant of abuse worldwide. METH administration can cause neurotoxicity and neurodegenerative injury, which is similar as two prevalent neurodegenerative disorders of Alzheimer’s disease (AD) and Parkinson’s disease (PD). Recent results suggested METH exposure increased level of α-synuclein (α-syn) as possible causes of neurotoxicity. However, the mechanism of METH-induced neurodegeneration remains unclear. This study aims to examine the effects of glycogen synthase kinase3β (GSK3β), α-syn and tau on METH-induced neurotoxicity. Our results showed that P-GSK3β (Tyr216), P-Tau (Ser396), α-syn and P-α-syn (Ser129) levels were increased after METH administration in dose and time-dependent manners. While inhibiting GSK3β activity with LiCl or GSK3β-siRNA, these protein expressions were significantly decreased. We observed LiCl protected cells from METH-caused cytotoxicity by weakening cell morphological damage, preventing cell apoptosis and death. We also found P-GSK3β colocalizated with P-Tau and α-syn by immunofluorescence method. Further, METH disrupted cellular autophagy by upregulations of LC3-II and P62 proteins, which was restored by LiCl and GSK3β-siRNA. The expressions of α-syn specific degradative enzyme of glucocerebrosidase (GCase) with its regulator lysosomal integral membrane protein type-2 (LIMP-2) decreased inversely to the doses of METH treatment. The GCase inhibitor of conduritol-β-epoxide (CβE) increased α-syn levels, and LiCl restored GCase and LIMP-2 expressions injured by METH treatment. In summary, we conclude that GSK3β plays key roles in METH-induced neurotoxicity and neurodegenerative injury by promoting abnormal protein phosphorylation and α-syn accumulation, blocking autophagy-lysosomal degradation pathway, and finally leading to cell apoptosis and death. GSK3β may be a potential target to prevent METH-induced neurodegeneration.

Introduction
Methamphetamine (METH) developed as an amphetamine derivative, is a highly addictive psychostimulant drug that is widely abused in the world, whose hydrochloride is called “Ice”.1 METH has adverse effects on each organ, especially for the central nervous system. A great quantity of evidences indicate that METH abuse leads to significant neurotoxicity via multiple events, including oxidative stress, hyperthermia, neuroinflammatory responses, mitochondrial dysfunction, endoplasmic reticulum stress and so on, which converge to mediate METH-induced terminal degeneration and neuronal apoptosis. 2-4 Epidemiological studies suggest that METH abuse is associated with increased risk of PD and may predispose users to develop PD.5 METH abuse can result in PD-like Lewy bodies (LBs) in the substantia nigra and striatum of rats.7,8 Thus, one of the major outcomes for METH toxicity is neurodegenerative injury, but the underlying mechanism is unclear.Pathological accumulation of α-syn in the brain, forming Lewy bodies, is a typical neuropathological hallmark of Parkinson’s disease (PD),9 and increasing evidences suggest that α-syn is a common pathogenic molecule in several neurodegenerative diseases referred to synucleinopathies.10 Previous studies in our Lab have manifested METH exposure induced overexpression of α-syn and led to oxidative stress in vivo and vitro, and suppression of α-syn expression could significantly attenuate METH-induced toxicity.11-13 Although α-syn aggregation is observed in METH-treated PC12 cells, the mechanism of α-syn abnormal modification/accumulation remains unclear.13 Cystoskeleton destruction is another important mechanism of neurodegeneration. Microtubule-associated protein tau has been classically linked to Alzheimer’s disease(AD), which also plays important roles in the pathogenesis of PD and other related disorders, known as tauopathies.14,15 In AD and PD, hyperphosphorylation of tau leads to intracellular accumulation of this protein and the formation of neurofibrillary tangles (NFTs).

Glycogen synthase kinase 3 (GSK3) proteins are multifunctional serine/threonine kinases originally identified as key enzymes in glycogen metabolism.16 In mammals, there are two closely related isoforms, GSK3α and GSK3β, the latter isoform is highly enriched in the nervous system.17 Researches show that absence or misregulation of GSK3 functions has been involved in several CNS diseases such as AD and PD.18,19 GSK3β can phosphorylate a majority of sites on tau, especially Ser396 hyperphosphorylation seems to play a pivotal role for its function and in particular destabilizes microtubules. 20,21 Alpha-syn may act as a connecting mediator for GSK3β and tau, resulting in GSK3β-mediated tau phosphorylation.22-24 Thus, GSK3β may act as an important signaling molecule for regulating α-syn and tau through phosphorylation during the pathogenic process of neurodegenerative diseases. Related research showed METH-induced neurotoxicity in PC12 cells was associated with the Akt/GSK3β/mTOR pathway.25 Increased GSK3β activity was found in the NAc core after chronic METH administration.26 Here, we hypothesized that GSK3β could mediate METH-induced neurotoxicity via regulating α-syn and tau in our model. Therefore, one of the objectives of this study was to investigate the variety of GSK3β activity and its regulation effects on α-syn and tau, and the role of GSK3β in METH-induced cytotoxicity in PC12 cells. Recently, impairment of autophagy-lysosomal pathways (ALPs) is regarded as another important pathogenic event in neurodegenerative diseases. Since α-syn can be degraded by the enzyme GCase and autophagy-lysosomal pathway27,28 and GSK3β is also involved in autophagy regulation,29-31 we proposed our second presumption whether GSK3β regulates α-syn expression through ALPs? Our next goal was to investigate the roles of GSK3β in lysosomal GCase/α-syn and autophagy pathways during METH-induced neurotoxicity.

Differentiated PC12 cells, a rat adrenal medulla pheochromocytoma cell line obtained from the Cell Bank of Shanghai Institute for Biological Science, Chinese Academy of Science, were cultured in highglucose DMEM medium containing 10% fetal bovine serum and incubated at 37℃ in a humidified atmosphere containing 5% CO2. The cells were passaged every 2–3 days. Once cell cultures reached 70% confluency in 6-well plates or 96-well plates, cells were treated with METH at differentconcentrations of 0, 0.5, 1.0, 1.5, 2.0 mM for 24 h or exposed to 2.0 mM METH for 0 h, 2 h, 6 h, 12 h, 24 h, or pretreated with 10 mM LiCl for 30min; 100µM CβE for 2 h prior to being treated with METH (2.0 mM). After treatment, cell morphological characteristics was observed under inverted microscope. Cell culture media was collected and total protein was isolated from the cells and used for further analysis.Cell viability assayCell viability was measured by CCK8 assay. Briefly, PC12 cells were seeded in 96-wells culture plates at a density of 5 × 103 cells per well, at the end of exposure, 10 ul CCK-8 was added to each well, and then cells were sulfated at 37 °C in a humidified atmosphere of 5 % CO2 for 2 h. Enzyme standard instrument was used to determine the absorbance plate values at 450 nm.Annexin V apoptosis staininginstructions of Annexin V-FITC apoptosis detection kit (Keygen, Nanjing, China), PC12 cells were seeded on six well plates at a density of 5 × 105/well. Cells were treated with METH and LiCl as described before, and harvested using trypsin.

Then, cells were centrifuged with 2000 rpm for 5 min to remove the medium, washed twice with 4 °C PBS, and stained with Annexin V-FITC and propidiumiodide (PI). The percentage of apoptotic cells was quantified with flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA, USA).siRNA and transient transfectionPieces of siRNA sequences that targeting GSK3β and α-syn were designed by GenePharma (Shanghai, China), as shown below: GSK3β siRNA:No. 1 (Rat: 5′-GGAGAGCCCAAUGUUUCAUTT-3′), No. 2 (Rat: 5′-GGGAGCAAAUUAGAGAAAUTT-3′), and No. 3 (Rat: 5′-GGGACCCAAAUGUCAAA CUTT-3′). α-syn siRNA: No.1(Rat: 5′-GCUGUACAGUGUAUUUCAATT-3′), No.2 (Rat: 5′-CCCU AGCAGUGAGGCUUAUTT-3′), No. 3 (Rat: 5′-AGAACAAGUGACAAAUGUUTT-3′). ScrambledsiRNA sequence (5′-UUCUCCGAACGUGUCACGUTT-3′) was used as control (siNC). siRNAs were dissolved in DEPC water at a concentration of 20 µM. Cells were placed on a 6-well plate at a density of 4 × 105 cells/well. When cells reached 70% confluence, 5 µl Lipfectamine 2000 Reagent (Invitrogen, Carlsbad, CA) and 20 µmol siRNA were added to Opti-MEM medium (Gibco BRL, Paisley, UK). The mixed solution was incubated at room temperature for 20 min, and then siRNA/Lipofectamine 2000 complex was added to the cells. This medium was replaced after 6 h incubation at 37 °C with the same volume of fresh DMEM medium. After 48 h, cells were changed to non-serum medium prior to the treatment of 2.0 mM METH for another 24 h. Total protein was extracted from PC12 cells and protein expressions were determined by western blot.

PC12 cells exposed to vehicle or METH were lysed in ice-cold RIPA buffer with protease inhibitors and phosphatase inhibitors. Protein concentrations were determined with the BCA-100 Protein Quantitative Analysis kit (Biocolors, Shanghai, China). Protein samples were separated by 8–12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred onto polyvinylidene-difluoride (PVDF) membranes (Millipore, USA). The membranes were incubated overnight at 4℃ orat room temperature for 2 h in blocking buffer (5% nonfat dry milk or 5 % BSA in TBST buffer). After blocking, membranes were incubated with primary antibodies (1:1000 for all): anti-GSK3β, anti-P-GSK3β (Tyr216), anti-P-Tau (Ser396), anti-P-α-syn (Ser129), anti-GCase (Abcam, Cambridge,UK); anti-tau, anti-P62, anti-LC3-II (CST, Boston, MA, USA); anti-α-syn, anti-LIMP-2 (Santa Cruz, California, USA), and anti-β-actin overnight at 4 °C. Membranes were washed three times with TBST followed by incubation with horseradish peroxidase conjugated secondary antibody (1:10,000) for 1 h at room temperature. The blot membranes were washed as above and developed with Chemiluminescence ECLPlus Western Blotting detection reagents (Thermo Scientific, Waltham, MA, USA). Proteins of interest were quantified with the Gel-Pro analyzer software.PC12 cells were placed on slides and fixed with 4% paraformaldehyde in PBS for 30 min at room temperature and then washed three times in PBS. The cells were permeabilized in 0.1% Triton-X-100 for 10 min, blocked in 3% goat serum for 1 h at room temperature, and subsequently labeled with P-GSK3β (Tyr216), P-Tau (Ser396) and α-syn antibodies (1:200 for all). After washing with PBS three times, the cells were incubated with Cy3-goat anti-rabbit IgG or FITC-goat anti-mouse IgG (1:00) for 1 h at room temperature, followed by mounting with DAPI (4′,6- diamidino-2-phenylindole) for nuclear counter staining. Fluorescently stained cells were analyzed using a confocal laser scanning microscope (Nikon, Tokyo, Japan).Data were summarized as the mean ± SD of three independent replicates at least. Statistical analyses were performed using SPSS 20.0 software. The parametric test contained one-way ANOVA and the post hoc test was done with the Tukey HSD method. The value of p < 0.05 was considered statistically significant.

Results
The activity of GSK3β was evaluated by examining the level of phosphorylated tyrosine 216 residue (Tyr216) of this protein. As shown in Fig. 1A, B and Fig. 2A, B, there were time-dependent anddose-dependent elevation of P-GSK3β expression levels after 0.5 mM～2.0 mM METH treatment, butthe expressions of total GSK3β were unchanged. The P-GSK3β expression level (P-GSK3β/GSK3β protein ratio) was about 2.8-fold higher in the 2.0 mM METH-treated group than in the control. After treated with 2.0 mM METH for 2 h, the activation of GSK3β was observed, which lasted up to 24 h with significant increase (p < 0.01, Fig. 2A, B). Similarly, P-Tau, α-syn and P-α-syn expression ratioswere increased in the dose-dependent manner after METH exposure, with the maximal elevation of about 2-fold, 5-fold and 2.7-fold respectively (p < 0.01) (Fig. 1A, C, D). After exposure to 2.0 mM METH for various durations, we observed the gradually elevating levels of α-syn, while P-α-syn levels, similar to the expression trend of P-Tau, increased at 6 h and lasted up to 24 h (Fig. 2A, C, D). Taken together, these results reveal METH exposure can activate GSK3β, induce α-syn accumulation, upregulate tau and α-syn phosphorylation.Inhibition of GSK3β activity attenuated METH-induced protein overexpression and phosphorylationTo assess the effects of GSK3β activation on the expressions of α-syn and tau, and their phosphorylation levels, PC12 cells were pretreated with LiCl, an inhibitor of GSK3β. Results showedLiCl effectively inhibited GSK3β activity, and decreased the ratio of P-GSK3β/GSK3β by ～35% inPC12 cells exposed to LiCl alone (Fig. 3A, B). After pretreated with LiCl, METH-mediatedphosphorylation of GSK3β, tau and α-syn were all significantly prevented (p < 0.01, Fig. 3). In the same way, total tau and GSK3β expressions had no changes.

Simultaneously, α-syn overexpression induced by METH was also observably attenuated by LiCl (Fig. 3 A, D).We further used RNA knock-down technique to silence GSK3β expression and detected these protein levels again. Compared with the negative control group, transfection with siRNA-1/2/3 led to significant decreases of GSK3β expression, and siRNA-1 inhibited it most efficiently (Fig. 4 A, B). Thus, we selected siRNA-1 to carry out our next experiment. As shown in Fig. 4 C-F, the results were consistent with that of LiCl treatment. Knocking down GSK3β also significantly decreased α-syn expression, and effectively prevented hyperphosphorylation of tau and α-syn caused by METH. These date demonstrate that both LiCl and siGSK3β block GSK3β activity, and inhibition of GSK3β attenuates METH-induced α-syn accumulation and phosphorylation of tau and α-syn.especially siRNA-1 (A and B). Then the siRNA-1 was selected for next experiments. Cells were exposed to vehicle, GSK3β siRNA, METH, and METH+GSK3β siRNA. Western blot (C) and quantitative analyses (D to F) showed METH-induced upregulations of P-GSK3β, P-Tau, α-syn, and P-α-syn were significantly blocked by siGSK3β. β-actin was used as a loading control. Data were expressed as means ± SD (n = 3/group). All data were analyzed with one-way ANOVA followed by Tukey HSD post-hoc test. *p < 0.01 vs. control group. #p < 0.01 vs. METH group.Cell morphological alteration, cell survival rates and cell apoptosis were examined to further explore the role of GSK3β in METH-induced cytotoxicity. As shown in Fig. 5A, we could see slender dendrites and neuron-like reticular formation in the normal PC12 cells and the LiCl-only treatment cells.

After METH exposure, cell bodies shrinked and became round with the disruption of the dendrites and disappearance of cell reticular formation. Adding LiCl in METH-treated cells, we found the above cell morphological damages were weakened and part of slender dendrites could still be seen clearly. Cell apoptosis is one of important mechanisms of METH-induced neurotoxicity. The flow cytometry results showed that 2.0 mM METH exposure alone significantly increased the percentage of PC12 cell apoptosis compared to the control group (26.07 ± 1.63 vs. 6.73 ± 0.51, p < 0.01), while the percentageof cell apoptosis was reduced to 15.03 ± 1.36 after pretreated with LiCl (Fig. 5C, D，p < 0.01).Similarly, LiCl reversed METH-caused decreasing of cell survival rates (62.45 ± 3.48 vs. 80.19 ± 2.30, p < 0.01) (Fig. 5B). Taken together, these data suggest that LiCl plays a protective role in METH-induced cytotoxicity, inhibition of GSK3β could prevent cell apoptosis and death.To further investigate the possible interaction between P-GSK3β, α-syn and P-Tau in our METH model, we used double labeling immunofluorescence method with confocal microscopy to observe their subcellular localizations. Our results showed that both P-GSK3β and P-Tau expressions localizated in the cytoplasm, but increased levels of them with no changes of subcelluar localization after 2.0 mM METH treatment (Fig. 6(A2-B4)). In contrary, α-syn expression mainly located in the cell nuclei of the control group, while exposure to METH for 24 h, it translocated into the cytoplasm and colocalizated with P-GSK3β obviously (Fig. 6C2-D4). These results indicate that these three proteins might interactwith each other in the PC12 cells, METH administration may promote their interaction. GSK3β phosphorylation may play a role as an intermediary agent in α-syn accumulation and aggregation after METH injury.

Autophagy is suggested to be important for α-syn clearance and degradation. To assess whether GSK3β regulates autophagy degradation, the expressions of autophagy makers LC3-II and P62 (representing respectively autophagosomal number and autophagy flux) were measured. Our results showed that METH induced increasing levels of these two proteins in the dose-dependent manner (Fig. 7A, B). The levels of LC3-II and P62 proteins were respectively about 3.5-fold and 2.7-fold higher in the 2.0 mM METH-treated group than in the control. It could be explained that METH triggeredautophagy but inhibited the autophagy flux. Next, we evaluated the relationship between α-syn and the induction of autophagy. Seen from Fig. 7C-F, α-syn siRNA-3 inhibited α-syn expression most effectively, and after pretreated with α-syn siRNA-3, METH-induced LC3-II overexpression was significantly downregulated, implying the induction of autophagy was associated with the triggering of α-syn degradation. When cells were exposed to 2.0 mM METH in presence of LiCl, upregulation ofLC3-II and P62 caused by METH were partly inhibited (METH group vs. METH+LiCl group, p＜0.01,Fig. 7G, H). In consistent with the above results, exposure to METH combined with GSK3β siRNA obviously restored the disruption of autophagy induced by METH via downregulating LC3-II and P62 (Fig. 7 I, J). These data manifest GSK3β participates in the disruption of α-syn autophagy mediated by METH.We detected the decreasing levels of GCase and LIMP-2 after METH treatment in a dose dependent manner, which were α-syn specific degradative molecules (Fig. 8A, B). Compared to the control group,both GCase and LIMP-2 expressions were reduced by ~56% in the 2.0 mM METH group. In contrast with the decreasing levels of GCase, the accumulation of α-syn increased significantly in the METH or CβE group compared with that in the control. After co-exposure of CβE, there were lowest levels of GCase and highest levels of α-syn (Fig. 8C, D). LiCl also restored the decreases of GCase and LIMP2 caused by METH (Fig.8 E, F). From these data, it can be inferred that GSk3β is involved in METH-induced GCase dysfunction, disturbing α-syn clearance.(n = 3/group) and analyzed with one-way ANOVA followed by Tukey HSD post-hoc test. *p < 0.05 vs. control group. #p < 0.05 vs. METH group.

Discussion
Our results showed that METH exposure induced GSK3β phosphorylation which led to increasing levels of P-Tau, P-α-syn and α-syn. LiCl and GSK3β siRNA blocked GSK3β activity and attenuated METH-induced protein phosphorylation and cytotoxicity. P-GSK3β colocalizated with α-syn and P-Tau in the cytoplasm of PC12 cells after METH treatment. Both α-syn autophagy and GCase degradative pathways were disrupted by METH administration, and inhibition of GSK3β could restore these effects. These findings demonstrate that GSK3β is critical for α-syn abnormal phosphorylation and accumulation in METH-induced neurotoxicity. GSK3β is a multifunctional serine/threonine kinase enriched in the nervous system. GSK3β is highly regulated by phosphorylation and phosphorylation of tyrosine 216 residue (Tyr216) leads to GSK3β activation, but phosphorylation of Serine 9 residue (Ser9) inhibits its activity.17,32 Lithium is a known inhibitor of GSK3β. Relative studies show increased GSK3β activity is found in the NAc core after chronic METH administration, which is associated with behavioral sensitization or hyperlocomotor activity, but lithium treatment inhibits GSK3β activity in the NAc and blocks locomotor sensitization induced by METH.26,33 Lithium also is reported to protect against METH-induced neurotoxicity via phosphorylation of Akt/GSK3β/mTOR pathway.25 In our present results, METH exposure increased phosphorylated-GSK3β at tyrosine 216 residue in PC12 cells in a dose-dependent and time-dependent manner, but total GSK3β had no change. When cells pretreated with LiCl, METH-induced GSK3β phosphorylation was inhibited. Thus, it is confirmed that METH administration indeed can activate GSK3β which may be involved in further process during toxicity injury.
METH is the second most widely used illicit drug worldwide, which is a highly addictive drug causing neurodegeneration with unclear mechanism. Tau and α-syn have been classically linked to AD and PD, respectively.

Tau activity is regulated by phosphorylation/dephosphorylation cycles. Abnormal tau phosphorylation is thought to prevent tau from binding to microtubules, causing an accumulation of hyperphosphorylated tau that leads to PHF formation, microtubule instability, and neurodegeneration.14 GSK3β is a primary kinase for tau phosphorylation which can also directly phosphorylated α-syn at a single site, Ser129.32 In the PD model induced by the rotenone, GSK3β phosphorylates α-syn at Ser129 and lithium decreases phosphorylation of α-syn.24 Our results showed that there were time-dependent and dose-dependent increases of P-Tau (Ser396) and P-α-syn (Ser129) after METH exposure accompanying with P-GSK3β overexpression. It was worth noting that increased expression of P-GSK3β was examined at 2 h, while both P-Tau (Ser396) and P-α-syn (Ser129) were found to increase at 6 h, which were later than P-GSK3β. When GSK3β activity was inhibited by LiCl or GSK3β siRNA, both P-Tau and P-α-syn expressions induced by METH were siginificantly down-regulated. Therefore, we speculate that METH induces tau and α-syn phosphorylation via activating GSK3β. As we know, the major function of tau is stabilization and regulation of microtubule (MT) dynamics necessary for neurite outgrowth, morphogenesis, axonal transport and normal neuronal functions. Since the regulation of tau phosphorylation is closely related to microtubule stability and cytoskeleton maintain, and LiCl inhibited P-Tau expression and weakened cell morphological damage caused by METH, we infer the microtubule and cytoskeleton instability might be the possible mechanism of METH-caused cell morphological changes. In our previous studies, we confirmed that METH induced α-syn expression and abnormal aggregation mediating oxidative stress and apoptosis.11-13 GSK3β inhibition decreased α-syn protein expression has been reported previously.19,24 In present METH-induced injury model, we detected α-syn overexpression in agreement with our previous studies.

Notably, we found GSK3β can regulate not only phosphorylation of α-syn but also α-syn accumulation, as METH-induced increase of α-syn was significantly down -regulated when inhibiting GSK3β with LiCl and GSK3β siRNA. Abnormal aggregation of α-syn is main component of LBs, and the predominance of α-syn phosphorylated at serine 129 (Ser129) in LBs suggests its important pathological role.32 Evidences indicate that phosphorylation at Ser129 enhanced α-syn toxicity, promoting its mis-folding, aggregation, and accumulation.34To sum up, it is well concluded that GSK3β mediates α-syn and tau toxicity of abnormal phosphorylation, aggregation, and accumulation induced by METH.Increasing evidences show there is a strong association between α-syn and hyperphosphorylation of tau. Tau phosphorylation increased at Ser396, Ser202/Thr205 and Thr231 in α-syn-injected brains.35 Neuronal colocalization of tau and α-syn inside LBs has been reported in brains of sporadic PD and dementia with Lewy bodies.36-37 Alpha-syn forms a heterotrimeric complex with phosphorylated tau and GSK3β,19,22 P-GSK3β (Tyr216) colocalized with P-Tau and P-α-syn (Ser129) in TH+ DA-neurons of the midbrain.32 To further investigate how GSK3β interacts with α-syn and tau during METH toxicity, we observed their subcellular localizations with confocal microscopy. The immunofluorescence results showed that P-GSK3β and P-Tau colocalizated in the cytosol regardless of whether cells were exposed to METH or not, however, α-syn translocated from the cell nucleus into the cytosol and colocalized with P-GSK3β after METH exposure. Therefore, we conclude that there is interaction between P-GSK3β, α-syn and P-Tau, and the increased interaction among them stimulated by METH may facilitate its cytotoxicity. However, it is unclear and remains to be investigated that whether α-syn is essential for METH-induced protein phosphorylation, and whether they interact with each other by forming heterotrimeric complex in our model.

It is reported that GSK3β can regulate cell survival via modulation of autophagy and cell death (Yang et al., 2010). PD mimetics such as 6-OHDA (6-hydroxydopamin), Aβ (amyloid β peptide) and MPTP (1-methyl4-phenyl-1,2,3,6-tetrahydropyridine) induce neuronal apoptosis in a GSK3β- dependent manner in SH-SY5Y cells, PC12 cells and dopaminergic neurons.38-40 Thus, GSK3β may be an important apoptosis modulator. Our previous studies have revealed METH can induce cell apoptosis by different signal pathways such as caspase-related pathway, Nupr1/Chop signal axis.4,41 Cell death and apoptosis are usually the terminal events in METH toxicity injury. Our results showed that PC12 cell morphology was severe changed and damaged, with increased cell apoptosis and decreased cell viability after exposed to 2.0 mM METH for 24 h. After inhibition of GSK3β with LiCl, both cell apoptosis and cell survival rate were restored significantly. We considered GSK3β as an important signal regulator in METH-caused cell death and apoptosis, and LiCl functions as a neuroprotective agent. GSK3β-mediated protein phosphorylation and α-syn expression participate in the process of METH toxicity injury.
Impairment of autophagy-lysosomal pathways (ALPs) is increasingly regarded as a major pathogenic event in neurodegenerative diseases, including PD.42,43 Aggregating and nonaggregating α-syn species can be degraded by autophagy,28 impaired ALP in the diseased brain limits intracellular degradation of misfolded proteins, and subsequent accumulation of abnormal α-syn species. LC3-II is the autophagosome marker for measure of autophagosomal number, and P62 is a maker for autophagy flux.44,45 There was a trend for LC3-II levels to be increased in PD.

Inhibition of autophagy correlates with increased levels of P62, suggesting that steady state levels of this protein reflect the autophagic status.44 GSK3β is also associated with regulation of autophagy.29,31 In present study, we observed the increased protein levels of LC3-II, suggesting that METH treatment triggered or induced autophagy. However, the expression of P62 was also enhanced, which meant autophagy flux was obstructed and the process of autophagy was incompleted (complete process of autophagy including the delivery of cargo to lysosomes, its subsequent breakdown and recycling). Therefore, increased LC3-II levels also represented inhibition of autophagosome clearance. Then, we silenced α-syn expression and found METH-induced LC3-II expression decreased, which hinted autophagy could not be induced, suggesting the autophagy is important to METH-related α-syn degradation. Owing to the obstruction of autophagy flux, α-syn could not be cleared and finally accumulated in the cells. When blocking GSK3β with LiCl and siRNA, both LC3-II and P62 levels decreased and autophagy capabilities were restored. Together, METH inhibited autophagy process by GSK3β activation, then reserved excessive α-syn and finally led to α-syn aggregation and accumulation in the cytoplasm. Therefore, we conclude that GSK3β is involved in autophagy dysfunction, which destroys the balance of α-syn homeostasis after METH treatment.

Except for autophagy pathway to α-syn degradation, there is specific enzyme of glucocerebrosidase (GCase) which makes clearance of α-syn in the lysosome. According, GCase deficiency and abnormal accumulation of α-syn were found in neurodegenerative diseases.27,48 There was newly observation of inhibitions between GCase and α-syn through unique domains in the lysosome.49 GCase is encoded by glucocerebrosidase gene (GBA), whose mutation causes the lysosomal storage disease named Gaucher disease.50 It is interesting that many PD patients carry GBA gene mutation, and Gaucher disease patients have high risk of developing PD, suggesting close relationship between these two diseases.51,52 We thus hypothesized that lysosomal GCase/α-syn metabolic pathway was critical not only in neurodegenerative disease but also in our METH-induced model. In consistent with the prediction, the expression of both GCase and its trafficking receptor LIMP-2 decreased inversely to the doses of METH treatment. GCase is transported from the endoplasmic reticulum to the lysosome by LIMP-2. Thus, METH might disturb the process of GCase delivery by affecting LIMP-2, leading to lysosomal dysfunction. Next, the GCase inhibitor, CβE enhanced both upregulation of α-syn and downregulation of GCase levels induced by METH. Furthermore, LiCl restored the decreasing of GCase and LIMP-2 injured by METH treatment. Moreover, immunofluorescence results showing METH-caused α-syn accumulation in the cytoplasm was in agreement with the outcome of distribution imbalance of α-syn due to GCase dysfunction.50 Therefore, we consider that GSK3β acts as upstream of GCase/α-syn pathway and disruptes α-syn degradation after METH injury.

Conclusion
In conclusion, our results demonstrate that the GSK3β/α-syn axis plays critical roles in METH- induced neurotoxicity. A schematic depicting the novel mechanism is provided in Fig. 9. METH exposure induced GSK3β activation and phosphorylation at tyrosine 216 residue, and targeted its substrates tau and α-syn by phosphorylation. GSK3β also mediated cell morphological alteration, apoptosis and cell death after METH treatment. On the other hand, GSK3β/α-syn axis disturbed lysosomal autophagy and GCase degradative pathways, leading to abnormal α-syn accumulation and aggregation which might explain for the neurodegenerative injury induced by METH. Significantly, as the inhibitor of GSK3β, LiCl played protective effects on METH neurotoxicity. It is imaginable that GSK3β blockers including LiCl might be potential candidates for further therapeutic application of drug abuse, although it needs more convincing Daporinad evidences.