Studies On Molecular Mechanisms Of Akt/AMPK-mTOR And PP5/PP2A-MAPK Signaling And Its Regulation In The Process Of Neuronal Apoptosis/degeneration

The present study, using cellular and molecular biology techniques and methods including cell culture, trypan blue exclusion, TUNEL and DAPI staining, MDC staining, fluorescent protein labeling, RNA interference, DNA cleavage,immunohistochemistry, Western Blotting etc.; and employing PC12 cells and murine primary neurons, C57BL mice and SD rats as experimental subjects, systematically studied the mechanisms of neuronal apoptosis and/or autophagy inhibition in in vivo and/or in vitro models of Parkinson’s disease (PD), as well as in in vitro model of cadmium (Cd) exposure, and deeply dissected the role and its regulative molecular mechanisms of Akt/AMPK-mTOR and PP5/PP2A-MAPK signaling in neuronal apoptosis. The detailed results were summarized as follows:1. Neuronal apoptosis in SNc and hippocampus of brain is involved in activation of AMPK and inactivation of Akt as well as inhibition of mTOR pathway activity in animal models of PDMPTP injection-induced C57BL mouse model of subacute PD and rotenone gavage-elicited SD rat model of PD were established. Expressions of Akt, AMPK,mTOR, caspase-3 and PARP in brain tissues were detected by Western blotting. In SNc and hippocampus of brain,the manifestation of TH+ cells and apoptosis were assayed using immunohistochemistry and DAPI/TUNLE staining, respectively. The results showed inhibition of mTOR signaling pathway,significantly increased cleavages of caspase-3 and PARP, increased AMPKα phosphorylation and decreased Akt phosphorylation in brain tissues in animal models of PD induced by MPTP and rotenone. The number of TH+ cells in the SNc was profoundly reduced, and there existed marked difference of apoptotic neurons in PD model group compared to control group. These findings suggest that MPTP and Rotenone-induced murine PD model,brain activation of AMPK and inactivation of Akt as well as inhibition of mTOR pathway contribute to neuronal apoptosis/degeneration in brain of PD development.2. Crosstalk between Ca2+/CaMKⅡ signaling and mitochondrial H2O2 inhibits mTOR pathwaycontributing to apoptosis in neuronal cellular model of PDPC 12 cells and primary neurons, PC 12 cells infected with shRNA 4E-BP1,shRNA CaMKⅡ and shRNA GFP (as controls), or PC 12 cells infected with Ad-mTOR-wt, Ad-S6K1-ca and Ad-GFP (as controls), respectively, were treated with different concentration of rotenone (0-1 μM) for 24 h, or treated with 1 μM rotenone for different time (0-24 h), or were pretreated with/without BAPTA/AM (30 μM),EGTA (100 μM), KN93 (10 μM), catalase (CAT) (350 U/ml) or Mito-TEMPO (10μM) for 1 h and then exposed to rotenone (1 μM) for 24 h, or co-treated with rotenone(1 μM) in the presence or absence TTFA (10 μM) or Antimycin A (50 μM) for 24 h.The intracellular Ca2+ was measured by Ca2+ fluorescent probe, Fluo-3/AM, and intracellular H2O2 level was analyzed by H2DCFDA probe, cell apoptosis was assessed using TUNEL staining. Expression of CaMKII, mTOR, 4E-BP1, S6K1 and caspase-3 was detected by Western blotting. The results showed that rotenone induced elevation of intracellular free calcium ion ([Ca2+]i) level, which mediated CaMKⅡphosphorylation, thereby leading to inhibition of mTOR pathway and neuronal apoptosis. Rotenone-induced mitochondrial H2O2 overproduction elicited [Ca2+]i elevation, and thus stimulating CaMKII phosphoryltion, leading to inhibition mTOR pathway and neuronal apoptosis. Moreover, overexpression of mTOR-wt or S6K1-ca,or silencing 4E-BP1 strengthened the inhibitory effects of CAT, Mito-TEMPO,BAPTA/AM or EGTA on rotenone-induced [Ca2+]i elevation. Our results suggest that crosstalk between Ca2+ signaling and mitochondrial H2O2 is required for rotenone inhibition of mTOR signaling pathway leading to neuronal apoptosis.3. Autophagy inhibition involves a mechanism of AMPK activation that does not rely on mTOR pathway in neuronal cellular model of PDPC12 cells and primary neurons, or PC12 cells infected with Ad-dn-AMPKa,Ad-LacZ and Ad-GFP-LC3,were treated with different concentration of 6-OHDA(0-240 μM), MPP+ (0-1.5 mM) or rotenone (0-1 μM) for 24 h, or treated with 6-OHDA (120 μM), MPP+ (1 mM) or rotenone (1 μM) for different time (0-24 h), or pretreated with/without Compound C (10 μM) for 1 h and then exposed to 6-OHDA(120 μM), MPP+ (1 μM) or rotenone (1 μM) for 24 h. MDC staining showed intracellular acidic protein, GFP-LC3 labeled the puncta of autophagy, Expression of AMPKa, mTOR, 4E-BP1, S6K1, a-Synuclein, LC3, p62/SQSTMI and cleaved-caspase-3 were detected by Western blotting. The results showed that 6-OHDA, MPP+ and rotenone activated AMPKa, inhibited mTOR pathway in a concentration- and time-dependent manner, resulting in an decrease in autophagic marker protein LC3-Ⅱ and an increase in autophagic receptor p62 protein expression.The findings suggest that autophagy inhibition involves a mechanism of AMPK activation that does not rely on mTOR pathway in neuronal cellular model of PD.4. Autophagy inhibition is attributed to blockage of Ulk1-Beclin 1 and Atg5-Atg12 pathways in neuronal cellular model of PDPC 12 cells and primary neurons were treated with different concentration of 6-OHDA (0-240 μM), MPP+ (0-1.5 mM) or rotenone (0-1 μM) for 24 h, or treated with 6-OHDA (120 μM), MPP+ (1 mM) or rotenone (1 μM) for different time (0-24 h).PC12 cells infected with Ad-mCherry-GFP-LC3 or Ulkl-downregulated PC12 cells using CRISPR/Cas9, respectively, were treated with 6-OHDA (120 μM), MPP+ (1 mM) or rotenone (1 μM) for 24 h. The autophagy was analyzed by serialization of mCherry-GFP-LC3 labeled puncta. Western blot was used to detect Ulkl, Beclin-1,Ambra1, Atg5 and Atg12 expression or immunoprecipitation analysis the relationship between Beclin-1 and Ambral. Our results showed that 6-OHDA, MPP+ and rotenone inhibited phosphorylation of Ulkl (Ser757 and 556) and Beclin 1 (Ser14) in a concentration- and time-dependent fashion, and relieved expression of Atg5-Atg12 complex. The results suggest that in neuronal cellular model of PD, suppression of Ulkl-Beclin 1 pathway leads to the reduction of basal autophagy, and the lack of Atg5-Atg12 complex makes LC3 esterification blocked was related to the autophagy inhibition of neuronal cells.5. Resveratrol protects from Cd-induced apoptotic cell death in neuronal cellsPC 12 cells and primary neurons were seeded in 6-well plates at a density of 5×105/well. The cells were pretreated with/without 25, 50,100 μM resveratrol for 1h and then exposed to 10 and 20 μM Cd for 8 h or 24 h. Cell morphology and the number of live cells were determined by microscopic imaging and trypan blue exclusion, respectively. Cell apoptosis was assessed by DAPI and TUNEL staining,Western blotting detected the expression of cleaved-caspase-3. The results showed that 100μM resveratrol significantly inhibited Cd-induced cell shrinkage, cell viability reduction,increases cleaved-caspase-3 and apoptosis in PC 12 cells and primary neurons. It is means that resveratrol protects from Cd-induced apoptotic cell death in neuronal cells.6. Resveratrol resists Cd-induced neuronal apoptosis through inhibiting both mTORC1 and mTORC2 signaling pathwaysPC 12 cells and/or primary neurons, or PC 12 cells infected with shRNA mTOR,shRNA Rictor and shRNA Raptor, shRNA S6K1 and shRNA GFP, or PC12 cells infected with Ad-4EBP1-5A, Ad-dn-Akt, Ad-myr-Akt and Ad-Lac Z, respectively, were pretreated with/without resveratrol (100 pM) for 1 h, or pretreated with PP242 (1 μM)or Akt inhibitor X (20 μM) for 1 h and then pretreated with/without resveratrol (100 μM)for 1 h, followed by exposure to Cd (10 μM) for 8 h or 24 h. The number of live cells was analyzed by trypan blue exclusion,cell apoptosis was assessed by TUNEL staining,and expression of related proteins was detected using Western blotting. The results showed that down-regulates of mTOR strengthened resveratrol’s prevention against Cd-induced neuronal apoptosis. Pretreatment with PP242 enhanced resveratrol’s inhibition of Cd-induced neuronal apoptosis. Down-regulation of rictor, raptor and/or rictor/raptor strongly reversed Cd-induced neuronal apoptosis and enhanced the inhibitive activity of resveratrol. Silencing S6K1 or ectopic expression of 4EBP1-5A potentiated the inhibitory effects of resveratrol on Cd-induced apoptosis. Akt inhibitor X or expression of dominant negative Akt (dn-Akt) also enhanced resveratrol’s resistance to Cd neurotoxicity, and expression of constitutively active Akt (myr-Akt)resisted resveratrol’s inhibition of Cd-induced apoptosis. The results suggest that resveratrol protects from neuronal apoptosis by inhibiting Cd activation of mTORC1-mediated S6K1 and 4E-BP1 pathway and mTORC2-mediated Akt pathway.7. Resveratrol prevents Cd activation of Erk1/2 and JNK pathways from neuronal apoptosis via protein phosphatases 2A and 5PC 12 cells and primary neurons, or PC 12 cells infected with Ad-dn-c-Jun,Ad-MKK1-K97M, Ad-PP2A, Ad-PP5 and Ad-GFP (as controls), were pretreated with/without resveratrol (25, 50 and/or 100 μM) for 1 h, or pretreated with/without U0126 (5 μM) or SP600125 (20 μM) for 1 h and then pretreated with/without resveratrol (100 p.M) for 1 h, followed by exposure to Cd (10 and/or 20 μM) for 4 h or 24 h. The number of live cells was analyzed by trypan blue exclusion, cell apoptosis was assessed by DAPI staining, and expression of related proteins was detected using Western blotting. The results showed that resveratrol inhibits Cd-induced activation of JNK, Erk1/2 and p38 pathways. Pretreatment with JNK inhibitor SP600125 or Erk1/2 inhibitor U0126 strengthened resveratrol’s inhibition of Cd-induced neuronal apoptosis. This is further supported by the finding that ectopic expression of dn-c-Jun or MKK1-K97M enhanced the inhibitory effects of resveratrol on Cd-induced cell apoptosis. Resveratrol reversed Cd inactivation of PP2A and PP5. Overexpression of PP2A or PP5 could potently enhance resveratrol’s inhibition of Cd-induced activation of JNK and/or Erk1/2 pathways and neuronal apoptosis. Our findings strongly support the notion that resveratrol blocks Cd-activated JNK and Erk1/2 pathways aganist neuronal apoptosis by preventing Cd from inactivation of PP2A and PP5.