![]() SNCA WT SH-SY5Y cells were treated with MANF (250 and 500 ng/ml) and Dox (600 ng/ml) for 24 h (a, b) or 48 h (c, d), respectively then, the protein levels of SNCA, LC3-I/II, Beclin-1, and P62 were detected by western blot analysis. (a)–(d) Effects of MANF on the levels of SNCA WT and macroautophagic-related protein expression. MANF inhibits the accumulation of SNCA WT in PD cellular model by macroautophagy activation. combined treatment with negative control Lamp-2A RNAi plasmid, MANF, and Dox group. Data were expressed as mean ± SD from three independent experiments. The levels of LAMP-2A and SNCA WT were detected by western blot analysis. SNCA WT SH-SY5Y cells were treated with Lamp-2A RNAi 2 for 24 h, followed by the incubation of MANF (500 ng/ml) and Dox (600 ng/ml) for another 24 h. (i, j) Lamp-2A knocked down partly abolished MANF-induced SNCA WT clearance. The level of LAMP-2A was detected by western blot analysis. (g, h) SNCA WT SH-SY5Y cells were treated with Lamp-2A RNAi 1-3 for 48 h. Cell lysates were immunoprecipitated with anti-SNCA, and the precipitated proteins were analyzed by immunoblotting with anti-Hsc70. ![]() SNCA WT SH-SY5Y cells were treated with MANF (500 ng/ml) and Dox (600 ng/ml) for 24 h. (e, f) Effects of MANF on the interaction between SNCA WT and Hsc70. SNCA WT SH-SY5Y cells were treated with MANF (250, 500 ng/ml) and Dox (600 ng/ml) for 24 h (a, b) or 48 h (c, d), respectively then, the protein levels were detected by western blot analysis. (a)–(d) Effects of MANF on the levels of SNCA WT, Lamp-2A, and Hsc70. MANF inhibited the accumulation of SNCA WT in PD cellular model by CMA activation. (g, h) RNA-sequence analysis of gene expression in substantia nigra followed by AAV8-MANF injection. Rotenone + PBS group and rotenone + AAV8 − MANF group: n = 5. (e, f) The level of SNCA in substantia nigra was detected using western blot analysis. (c, d) The effect of AAV8-MANF on rotenone-induced DA neuron degeneration was determined by TH staining. Ctrl group: n = 14 rotenone group: n = 14 rotenone + PBS group: n = 15 rotenone + AAV8 − MANF group: n = 13. (b) The motor function of AAV8-MANF on rotenone-induced PD mouse models was evaluated by rotarod analysis. The expression of MANF in DA neurons was detected by double immunofluorescence staining using anti-His tag antibody and anti-TH antibody. (a) AAV8-MANF (with His tag) was injected into the substantia nigra of rat brain. The protective effect of AAV8-MANF on PD model and its role on the expression of lysosome-related genes in substantia nigra. Nrf2 and its role in MANF-mediated degradation may provide new sights that target degradation pathways to counteract SNCA pathology in PD. Hence, our findings suggested that MANF has potential therapeutic value for PD. Using A53T mutant SNCA overexpression cellular model to mimic CMA dysfunction situation, we concluded that macroautophagy rather than CMA was responsible to the degradation of SNCA A53T, and this degradation was mediated by Nrf2 activation. Nuclear factor erythroid 2-related factor (Nrf2) was activated to stimulating macroautophagy activity when CMA pathway was impaired. By establishing wildtype (WT) SNCA overexpression cellular model, we found that chaperone-mediated-autophagy (CMA) and macroautophagy were both participated in MANF-mediated degradation of SNCA WT. In this study, we showed that AAV8-MANF relieved Parkinsonian behavior in rotenone-induced PD model and reduced SNCA accumulation in the substantia nigra. Our previous study showed that mesencephalic astrocyte-derived neurotrophic factor (MANF) facilitated the removal of misfolded SNCA and rescued dopaminergic (DA) neurons, but the underlying mechanisms remain unknown. ![]() Drugs aiming at degrading SNCA may be an efficient therapeutic strategy for PD. ![]() Progressive accumulation of misfolded SNCA/ α-synuclein is key to the pathology of Parkinson's disease (PD).
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