| The main pathological change was thought to be apoptosis of dopaminergic neurons in Parkinson’s disease(PD). However, previous studies have indicated that axonal injury might be more critical in the PD models. The therapeutic effect was also not satisfactory just by decreasing the apoptosis of dopaminergic neurons in PD. It has been recognized that the disaggregation of microtubule lead to axonal degeneration because the disaggregated microtubules were more likely to be degraded than the aggregated microtubules.The Akt signaling pathway was involved in protection of cells from various stresses. It promots an anti-apoptotic signal in PC12 cells treated with MPP+.As one of downstream targets of Akt, glycogen synthase kinase-3β(GSK-3β)/collapsin response mediator protein 2(CRMP-2) could regulated axonal-dendritic polarity and microtubule-disaggregating. However, whether Akt/ GSK-3b/CRMP-2 pathway is involved in disaggregation of microtubule and axonal degeneration induced by MPTP is unclear. In this study, we observed the axonal degeneration and neuronal injury in N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) treated C57 black(C57BL)mice and 1-methyl-4-phenylpyridiniumion(MPP+) treated dopaminergic neurons by scanning electron microscope, Fluoro-Jade-C stains(FJC), tyrosine hydroxylase stains(TH).At thesame time, we detected the expression of total and phosphorylation of Akt, GSK-3β and CRMP-2, as well as the aggregated microtubules.Part 1. Identified dopaminergic axonal degeneration induced by MPP+in vitro. Objective We cultured dopaminergic neurons of C57 BL mice to observe the axonal degneration induced by MPP+ in vitro.Methods C57/BL6 mice, embryonic age of which was 14 days were used for this study. These neurons were cultured with a density of 0.5 ×106 cells/L in poly-L-lysine coated 24-well plates for the immunohistochemistry.The medium of these neurons was Neurobasal medium supplemented with B27, glutaminate, penicillin and streptomycin. The culture medium was semi-refreshed every 2d. From the eighth day, a sublethal concentration MPP+(10μM) was added to the neurons. TH, TUNEL and APP staining were used to detect the injury of axon and cell bodies. Results Treated by a sublethal concentration MPP+(10μM),the axons appeared abnormal varicosities after 2h, apparent string-of-beads change became all round after 6h while the cell bodies were intact. After 24 h, we specifically found that most of the axons and dendrites disappeared completely and the number of dopaminergic neurons decreased strikingly, the shape of the survival dopaminergic neurons became irregular and axonal root were observed in some dopaminergic neurons. During the first 12 h, although the axons were shorter and more fragmented than control group, the number of dopaminergic neurons had no obvious different with the control group while the number of axons started to decrease at 4h.After 24 h, there was a 40% decrease in the number of neurons and a 71% decrease in the number of axons. This phenomenon suggests that the axons may be more sensitive to MPP+ and the injury of axons may lead to antidromic death of dopaminergic neurons. TUNEL staining showed compared to control group, there was no significant change in the number of apoptotic cells(P> 0.05) after 12 h administration of MPP +, while after 24 h, the number of apoptotic neurons increased by 29% compared with the control group, APP staining showed APP expression gradually increased, and it was elevated in the local accumulation of axons when the cell bodies were normal. Conclusion The axonal injury occurred earlier than the cell bodies, furthermore, if this injury was not prohibited, it might induce ‘dying-back’ degeneration of neurons. Therefore, the loss of dopaminergic neurons may result not only from the direct effect of MPTP, but also fromaxonal degeneration.So the axonal injury may be an active response to damage like apoptosis and a key cause of PD.Part 2. Identified dopaminergic axonal degeneration induced by MPTP in vivo. Objective To observe the axonal degneration induced by MPTP in vivo.Certify the degeneration of axons and cell bodies with the morphological description and verify the degeneration of axons and cell bodies with quantitative analysis. Methods Male C57 black(C57BL) mice weighing 25 –27 g and aged 8 weeks were used. MPTP(Sigma) was dissolved in normal saline,animals(n=100)were injected intraperitoneally with MPTP 30mg/kg at 3-days intervals for 10 times. 12 h after each intraperitoneal injection,10 mice were sacrificed.Animals in the control group(n=10) were given intraperitoneal injections of an equivalent volume of normal saline at the same times,12 h after the final injection,all these 10 mice were sacrificed. We observed the axonal degeneration and neuronal injury in MPTP treated C57 black(C57BL)mice by scanning electron microscope, Fluoro-Jade-C stains(FJC), tyrosine hydroxylase stains(TH). Results The tyrosine hydroxylaseimmunoreactive(TH-IR) dopaminergic neurons and axons were found in SNc and VTA. They showed strong fluorescence and had dense distribution, the TH-IR fibers were thick, long and straight in appearance in the control group. The same status was also observed in the group of first MPTP injection. After the second injection of MPTP, the TH-IR axons in SNc became thin, short, and irregularly varicositie, some axons showed string-of-beads change which was the appearance of axoplasmic transport disturbance while the cell bodies of dopaminergic neurons were intact and normal. All these axonal injuries were seen only in SNc and the TH-IR axons in VTA were normal. As the administration of MPTP, the degree of axonal injury became more severe and the injury of cell bodies was also observed.After the tenth injection, only a small quantity of TH-IR dopaminergic neurons remained, the axons and dendrites of which almost disappeared completely. During the whole experiment procedure, there were no significant changes in the TH-IR dopaminergic neurons in VTA.FJC-IR cells were only discovered in SNc after the third MPTP injection with regular bright green soma, no distribution of FJC-IR neurons and their axons or dendrites were found in the control group, the first and second MPTPinjection group. Although the FJC-IR cell remained intact cell bodies, the axons and dendrites almost disappeared, only few neurons exhibited axonal root. In all group, no FJC-IR neurons were found in VTA. Compared with the control group, the number of dopaminergic neurons in SNc had no significantly reduction after the first,second and third MPTP injection. From the fourth injection, the number of TH-IR neurons in SN decreased by a slow progressive procedure(P < 0.05). The TH-IR axonal fibers decreased more severely and earlier than dopaminergic neurons. The optical density of TH-IR fibers in striatum decreased significantly compared with the control group after the second MPTP injection(P < 0.05). After 10 times MPTP injections, there was a 47% decrease in the number of TH-IR neurons in SN while the reduction of TH-IR fibers in striatum was 64%(n=8 for each group). No obvious changes were found in the first injection and control group. After the second injection, although the dopaminergic neurons neurons were normal, the axons already appeared an early appearances of degeneration, for example: local varicosities was seen, the structure of neurofilament and neurotubule was disorder and discontinuation etc.As the administration of MPTP, the axonal injury became more severe: the local swelling was more severe, the number of microtubules and neurofilament was significantly reduced, axonal myelin was breakdown and more degeneration of mitochondria was seen. Conclusion: The axonal injuries were earlier and more severe than dopaminergic neuronal injury.So the axonal degeneration may play a more crucial role in the pathogenesis of PD instead of an accompaniment of neurons death. Axonal injury might be an active and independent response to injury as neuronal apoptosis.Part 3. Pathomechanism for the dopaminergic axonal degeneration induced by MPP+ and MPTP. Objective To explore whether Akt/GSK-3β/CRMP-2 pathway was involved in axonal injury even degeneration in PD. Methods To investigate whether this pathway is relevant to the degeneration of axons induced by MPP+, we evaluated levels of phosphorylation of Akt, GSK-3β and CRMP-2 in MPP+-treated dopaminergic neurons at different time points using Western blot analysis.To detect the role of β-tubulin III in theaxonal injury and the relationship with Akt/GSK-3β/CRMP-2 pathway, we evaluated levels of β-tubulin III by Western blot analysis and immunofluorescence. Results: After treated with a sublethal concentration MPP+(10μM), p-Akt protein decreased at 1 h, increased at 2 h and reached a peak at 4 h, then decreased at 8h,12 h and 24 h compared to sham controls. At the same time, we found the total Akt had no significant change compared to sham controls.By Western blot analysis, we found the p- GSK3β decreased at1 h, increased at 2 h and reached the highest at 4 h, returned to baseline at 8h and decreased at 12 h and 24 h compared to sham controls after treated with a sublethal concentration MPP+(10μM). At the same time, we found the total GSK3β had no significant change compared to sham controls.By Western blot analysis, the p-CRMP-2 increased at 1 h, decreased at 2 h, reached the lowest at 4 h and remained at low level at 8h,12 h and 24 h compared to sham controls. At the same time, we found the total CRMP-2 had no significant change compared to sham controls. By Western blot analysis and immunofluorescence,we found Glu-β-tubulin III began to decrease at 1h and the descent was maintained during the whole 24 h compared to sham controls. To explore whether Akt/GSK-3β/CRMP-2 pathway was involved in axonal injury of dopaminergic neurons treated by MPTP and MPP+, LY294002, a direct inhibitor of Akt was injected to the mice before MPTP administration.After 10 times injections, TH-IR and FJC-IR neurons in SN were counted and the optical density of TH-IR fibers in striatum was measured(n=8). We observed that MPTP and LY294002 treatment resulted in a significant loss of TH-IR neurons(about 27%) in SNc and the optical density of TH-IR fibers in striatum(35%)compared to the 10 times MPTP treated group(n=8). In contrast,LY294002, markedly incresaed the FJC-IR neurons in SN compared to the 10 times MPTP treated group(31%).In vitro,we found LY294002 enhanced the axonal injury more severe than neurons. After treatment of a sublethal dose of MPP+ and LY294002,TH-IR neurons and axons were counted. At 4h, there was a 17% decrease in the number of axons while the number of neurons had no significant change compared to MPTP treated group.After 24 h, there was a 22% decrease in the number of neurons and 15% in the number of axons compared to MPTP treated group. We determined the expression of p-Akt,p-GSK3β andp-CRMP-2 at 4h after MPP+ and LY294002 treatment by Western blot analysis. We found that LY294002 significantly decreased the expression of p-Akt and p-GSK3β compared with neurons treated by MPP+; the expression of p-Akt and p-GSK3β increased at 4 h after MPP+ treatment, compared with sham controls, however, LY294002 suppressed the expression of p-Akt and p-GSK3β lead by MPP+. At the same time, we found LY294002 significantly increased the expression of p-CRMP-2 at 4h, compared with compared with neurons treated by MPP+; the expression of p-CRMP-2 decreased at 4 h after MPP+ treatment compared with sham controls and LY294002 up-regulated the expression of p-CRMP-2.However, total Akt,GSK3β and CRMP-2 remained unchanged after normalization with GAPDH. We determined the expression of β-tubulin III at 4h after MPP+ and LY294002 treatment by Western blot analysis. We found that LY294002 significantly decreased the expression of β-tubulin III compared with neurons treated by MPP+. Conclusion: The phosphorylation level of Akt and GSK-3β, and dephosphorylation level of CRMP-2 was simultaneous after the MPTP administration, suggesting that Akt might regulate the activity of GSK-3β and CRMP-2 in axonal injury;the expression of Glu-β-tubulin III began to decrease at 1h after MPP+, suggesting that phosphorylation of CRMP-2 might be associate with disaggregation of neuronal microtubules in prophase of axonal degeneration; the reduction of Glu-β-tubulin III was maintained during the whole 24 h while the p-CRMP-2 began to decrease after 2h, suggesting a protective effect to maintain structural integrity of axons and other intracellular mechanisms might be involved in the regulation of Glu-β-tubulin III as a response to MPP+; LY294002 suppressed the expression of p-Akt and p-GSK-3β,increased expression of p-CRMP-2,decreased the Glu-β-tubulin III and enhanced the axonal injury, suggesting that Akt/GSK-3β/CRMP-2 pathway was involved in axonal injury,especiallly in the early period. |
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