MiR-124 Regulates Apoptosis And Autophagy Process In MPTP Model Of Parkinson’s Disease By Targeting To Bim

BackgroundParkinson’s disease (PD) is the most prevalent movement disorder and the second most common chronic and systemic neurodegenerative disorder after Alzheimer’s disease. The most common motor symptoms of PD are tremor, bradykinesia and myotonia, and some patients with non-motor symptoms of olfactory dysfunction, constipation, sleep disorder and depression, etc. There are family PD and sporadic PD, which family PD is associated with gene mutation, such as LRRK2、SNCA、VPS35、EIF4G1、PARK2、PINK1、DJ-1. However, the pathogenesis of sporadic PD is still unclear. Age is the most important risk factor of PD, the prevalence increases steadily with age. In addition, some occupational expose also will induce PD, such as Insecticides, herbicides, and heavy metal. Although L-dopa substantially improves quality of life and movement function of PD patient, it’s still a replacement therapy and could not stop the pathogenesis progress of PD. The pathological hallmark of PD is loss of neurons within the substantia nigra pars compacta (SNpc) and Lewy bodies aggregation, which is related to mitochondrial dysfunction, oxidative stress and protein aggregation supported by extensive evidence. Correspondingly, mitochondrial dysfunction resulting in mitochondrial outer membrane permeabilization will induce apoptosis process, while protein aggregation always associated with impaired autophagy leading to autophagic cell death. Therefore, strategies aimed at reducing the loss of SNpc DA cells might be an effective neuroprotection for PD.MicroRNAs (miRNAs) are endogenous highly conserved 21-25nt non-coding RNAs, which modulate transcription of multiple genes through binding to 3’ untranslated regions (3"UTR). To date, several lines of evidence demonstrate that microRNA machinery plays an important role in multiple cellular and biological progresses, such as development, differentiation, inflammation and cancer. In addition, the expression profile of microRNA varies among development stage, tissue and pathological states. Especially, some microRNA participate in dopamine neuron biology and Parkinson’s disease. MiR-7, miR-153, miR-34 and miR-34c are downregulated in Parkinson’s disease, while some microRNA regulate genes related to Parkinson’s disease, such as miR-7 and miR-153 both target to alpha-synuclein. Thus, microRNA might be a sensitive biomarker for Parkinson’s disease and regulation on microRNA network would damper PD’s pathogenesis.MiR-124 is highly expressed in the central nervous system with abundance (more than 100 times) higher than in other organs. Convincing researches have demonstrated the neuroprotection of miR-124 in some animal model of CNS diseases, such as experimental autoimmune encephalomyelitis, stroke and focal cerebral ischemia. However, it’s still unclear whether miR-124 could attenuate the loss of dopaminergic neuron in PD.Thus, we hypothesized that miR-124 might play a protective role in the pathology of PD. To be specific, we investigate the expression profile of miR-124 in MPTP-induced PD mice, and the mechanism of miR-124 neuroprotection on apoptotic and autophagic cell death in MPTP-treated PD and MPP+-exposed SH-SY5Y cells.First chapter:expression profile of miR-124 in MPTP-induced PD modelObjective:to investigate expression profile of miR-124 in MPTP-treated mice and MPP+-exposed SH-SY5Y cells.Methods: ①the mice received one intraperitoneal injection MPTP per day for five consecutive days. Control mice received saline injections only. Mice were executed at 0 (immediately after the last MPTP injection),1,2,4,7, and 21 d after the last MPTP administration and midbrain tissue were collected. ②SH-SY5Y cells were exposed to different concentration of MPP+(0.25mM,0.5mM, 1mM and 2.5mM) for 24 hour, or 1mM MPP+ for different duration (0,6,12,24 and 48 hour). ③ To investigate expression level of miR-124, total RNA were extracted from midbrain tissue or SH-SY5Y cells, reversed and amplified by PCR, and analyzed by methods of 2-△△Ct.④ the miR-124 expression in dopaminergic neuron were determined by in situ hybridization combined with Immunofluorescence.Results:comparing with control mice, expression of miR-124 decreased after intraperitoneal injection of MPTP in all groups. By using in situ hybridization, the miR-124 expression in SNpc dopaminergic neurons was downregulated. Comparing to control SH-SY5Y cells, all the concentration of MPP+treatment for 24h reduced miR-124 level. For treatment duration, 1mM MPP+treatment does not significantly reduce miR-124 level until 12h.Conclusion:miRNA-124 was downregulated in MPTP-induced model of PD.Second Chapter:exogenous delivery of miR-124 exhibits neuroprotection on MPTP-treated miceObjective:to investigate the neuroprotection of miR-124 in MPTP-treated mice.Methods: ①a stereotactic catheter was surgically implanted into the right lateral ventricle of mice. After one week of recovery, mice were given one treatment of agomir or negative control through the catheter per day for five consecutive days. The treatment of agomir was performed two days prior to injection of MPTP. Then mice received one intraperitoneal injection MPTP per day for five consecutive days. Control mice received saline injections only. ② Using immunohistochemical staining of TH to determine the number of dopaminergic neurons in SNpc. ③ Midbrain dopamine level was determined by High Performance Liquid Chromatography.Results:the ventricle injection of miR-124 agomir significantly upregulate expression of miR-124. Comparing with negative control in the group of MPTP-treated mice, miR-124 level was significantly upregulated by agomir, Id after last injection of MPTP (0.3850±0.065 vs 0.7550±0.045,95%CI:0.05951±0.6805), 4d after last injection of MPTP (0.4050±0.025 vs 0.7450±0.055,95%CI:0.02951± 0.6505),21d after last injection of MPTP (0.475±0.075 vs 0.820±0.09,95%CI: 0.03451±0.6555). Comparing with negative control group, the density and number of TH-positive neurons in the miR-124 agomir group was higher at 21 days after treatment of MPTP (2397.667±616.694 vs 6266±455.566,95%CI:1295±6442), and the loss of striatal dopamine was significantly less pronounced in the miR-124 agomir group (31.08±4.184 vs 75.53±9.209,95%CI:2.404±86.49).Conclusion:miR-124 attenuates the loss of dopaminergic neurons within SNpc and the loss of striatal dopamine in the MPTP-treated mice.Third chapter:miR-124 targets to BimObjective:to predict and confirm a target of miR-124.Methods: ①using Target Scan software to predict targets of miR-124. ②HEK293 cells were cotransfected with plasmids expressing either wild-type 3’UTR of Bim or mutant 3’UTR of Bim of miR-124 binding sites and miR-124 mimics or control. A Dual-Luciferase Reporter Assay System was performed to determined luciferase activity.③ SH-SY5Y cells were transfected with miR-124 mimics or negative control for 24h, using western blot and qRT-PCR to determine protein expression and mRNA expression of target gene, respectively.Results: ①there are two conserved miR-124 binding sites at 3’UTR of Bim predicted by Target Scan software. ②Luciferase reporter assay demonstrated that miR-124 binds to the 3’UTR of Bim. Comparing with negative control group, miR-124 mimics significantly reduces luciferase activity in the cells expressing WT-Bim plasmid (1.081 vs 0.3694,95%CI:-1.142--0.2822). ③Comparing with control SH-SY5Y cells, miR-124 mimics reduces both protein (0.61±0.06083, p<0.05) and mRNA level (0.6584±0.04156, p<0.05) of Bim, but not miR-124 negative group (protein:1.033±0.04910, p>0.05; mRNA:1.029±0.1397, p>0.05).Conclusion:Bim is a target of miR-124.Fourth chapter:miR-124 reduces MPTP-induced apoptotic cell death by targeting to BimObjective:to determine whether miR-124 could attenuate apoptosis of dopaminergic neuron of MPTP-treated mice.Methods:stereotactic ventricle injection of miR-124 agomir and MPTP treatment were performed as previously. Total RNA were extracted, reversed and amplified to determined Bim mRNA level. Total and mitochondrial protein were extracted from midbrain of mice at 4d after last injection of MPTP, and analyzed by western blot to determine Bim, Puma, Noxa, Bid and Bax protein expression. Counting the apoptotic cells at SNpc based on morphology and TH immunohistochemistry staining.Results: ヽomparing with control mice, MPTP induced a significantly increase of Bim mRNA, which was counter-regulate by miR-124 agomir (negative control group vs miR-124 agomir group,1.757±0.213 vs 1.226±.118,95%CI:-1.046--0.0153). ②MiR-124 agomir reduced the increase of Bim protein expression in the midbrain induced by the treatment of MPTP (3.106±0.245 vs 1.947±0.344,95%CI:-2.022--0.2966). However, Puma and Noxa expression were not affected by MPTP treatment, and Bid expression, and miR-124 cannot reduce the increase of Bim expression induced by MPTP.③Bax protein translocate to mitochondrial after treatment of MPTP. Within control mice, there is no difference of mitochondrial Bax between miR-124 negative control and miR-124 agomir (1±0 vs 0.957±0.049, 95%CI:-1.042-0.9552), while within MPTP-treated group, mitochondrial Bax level of miR-124 agomir group was significantly lower than miR-124 negative control (3.5±0.45 vs 1.887±0.243,95%CI:-2.612--0.6148). ④There were rare apoptotic cells within control mice (negative control vs miR-124 agomir: 1.333±0.333 vs 1.667±0.882; 95%CI:-7.230-7.896), while within MPTP-treated mice, apoptosis of dopaminergic neuron at SNpc significantly increased, which was reduced by miR-124 agomir injection (29.333±2.963 vs 15.667±2.333,95%CI:-21.23--6.104)Conclusion:exogenous delivery of miR-124 significantly reduces midbrain Bim upregulation caused by MPTP treatment, thus inhibiting Bax translocation to mitochondrial and mitochondrial outer membrane permeabilization. In all, miR-124 attenuates the apoptotic loss of dopaminergic neuron caused by MPTP.Fifth chapter:miR-124 regulates impaired autophagy process in the MPTP-induced PDObjective:to investigate whether and how miR-124 participate in the impaired process of autophagy in the MPTP-induced PD.Methods:CD stereotactic ventricle inj ection of miR-124 agomir and MPTP treatment were performed as previously. ￤estern blot analyzed autophagosome marker LC3Ⅱ and lysosome marker LAMP1 expressed in the midbrain of MPTP-treated mice. In addition, si-Bim or miR-124 mimics were transfected to SH-SY5Y cells 6h prior to treatment of MPP+or PBS.24h later, cells were collected and lysed to determine the LC3Ⅱ and LAMP1 expression. Meanwhile, lysosome were isolated and lysed to investigate Bax protein abundance. In addition, lysotracker-red was used to label lysosome to reflect lysosomal membrane permeabilization. ③ Immunoprecipitation was used to investigate binding between Beclin-1 and Bim in the SH-SY5Y cells with or without treatment of MPP+.Results: ①comparing with control mice, MPTP increased the midbrain LC3Ⅱ expression (1±0 vs 6.407±0.578), and within MPTP-treated mice, miR-124 significantly reduced the increase of LC3II (6.407±0.578 vs 3.7±0.684,95%CI:-4.461--0.9525). Similarly, MPTP reduced 40% LAMP1 expression (-4.461--0.9525), which was counter-regulated by miR-124 agomir (negative group vs miR-124 agomir group,0.620±0.064 vs 0.927±0.047; 95%CI:0.1295±0.4838).② Comparing with control SH-SY5Y cells, both si-Bim and miR-124 can reduce mRNA level of Bim (control vs miR-124 mimics, 1±0 vs 0.5068±0.07661,95%CI: 0.1992～0.7871; control vs si-Bim, 1±0 vs 0.3856±0.09973,95%CI:0.3204± 0.9083). Upon treatment of MPP+, Bim expression was upregulated and attenuated by miR-124 mimics and si-Bim (control vs miR-124 mimics,2.185■0.3350 vs 1.232±0.1711,95%CI:0.05941-1.847; control vs si-Bim,2.185±0.3350 vs 0.6610±0.06937,95%CI:0.6301 ～ 2.418). ③ Correspondingly, within control SH-SY5Y cells, LC3II expression was not affected by either miR-124 or si-Bim (control vs miR-124 mimics, 1±0 vs 0.99±0.091,95%CI:-2.691±2.671; control vs si-Bim, 1±0 vs 1.023±0.104,95%CI:-2.658～2.705), while within MPP+-treated group, the aggregation of LC3II was significantly reduced by both miR-124 and si-Bim (control vs miR-124 mimics,11.333±1.068 vs 7.90±0.656,95%CI:-6.115±-0.7521; control vs si-Bim,11.333±1.068 vs 6.933±0.956,95%CI:-7.081～-1.719). Similarly, there is no effect of both si-Bim and miR-124 on LAMP1 expression within control cells (control vs miR-124 mimics, 1±0 vs 1.030±0.113,95%CI:-0.2836-0.3436; control vs si-Bim, 1±0 vs 0.990±0.075,95%CI:-0.3236-0.3036), however, silencing Bim gene by miR-124 or si-Bim both attenuated reduction of LAMP1 caused by MPP+treatment (control vs miR-124 mimics,0.510±0.104 vs 0.833±0.056,95% CI:0.009762 ± 0.6369; control vs si-Bim,0.510±0.104 vs 0.843±0.043,95%CI:0.01976-0.6469). ④Noormally, there is rare Bax expressing in the lysosome, but after treatment of MPP+, Bax expression significantly increased, which was counter-regulated by both si-Bim and miR-124. Within control SH-SY5Y cells, there was no difference of lysosomal Bax expression among control, miR-124 and siBim group (control vs miR-124 mimics,4.367±0.423 vs 2.677±0.339,95%CI:-1.014-1.134; control vs si-Bim,4.367±0.423 vs 2.293±0.300,95%CI:-1.068 to 1.081). But within MPP+-treated SH-SY5Y cells, both si-Bim and miR-124 reduced the increase of lysosomal Bax protein level (control vs miR-124 mimics, 1.000±0.000 vs 1.060±0.089,95%CI:-2.764 ～-0.6156; control vs si-Bim, 1.000±0.000 vs 1.007±0.100,95%CI:-3.148～-0.9989). ⑤The decline of fluorescence of lysosome labeled by lysotracker-red in MPTP-treated cells was reduced by miR-124 and si-Bim. ⑥The treatment of MPP+ disrupted the interaction of Beclin-1 and Bim.Conclusion:The autophagosome aggregation and lysosome deletion in MPTP-induced PD are attenuated by miR-124 through inhibiting Bim expression, which reduces Bax translocation to lysosome.Sixth chapter:miR-124 is responsible for maintaining basal autophagy in SH-SY5Y cellsObjective:to determine the basal autophagy in SH-SY5Y cells when inhibiting expression of miR-124.Methods:basal autophagy in SH-SY5Y cells that transfected with miR-124 inhibitor or negative control was evaluated by western blot analysis of LC3Ⅱ and electron microscopy.Results:comparing with negative group, the Bim expression increased (1±0 vs 1.370±0.07234, p=0.0362), and miR-124 inhibitor significantly reduced the autophagosome marker LC3II expression (1±0 vs 0.68±0.07095, p=0.0458), and electron microscopy showed that there were less autophagosome-like membrane structure in the miR-124 inhibitor group.Conclusion:the basal autophagy level in SH-SY5Y cells decline when miR-124 is inhibited.