MiR-107 overexpression attenuates neurotoxicity induced by 6hydroxydopamine both in vitro and in vivo

Li Suna, Tingting Zhanga, Wenna Xiua, Wenhui Caoa, Mengfei Heb, Wenqiang Sunb, Weina Zhaob,∗

Abstract

Alzheimer’s disease (AD), the most common form of dementia, is a neurodegenerative disease characterized by neuronal atrophy in various brain regions. The expression of miR-107 is down-regulated in AD patients and target genes of miR-107 have been shown to directly involved in AD. In this study, we aimed to investigate the potential neuroprotective effects of miR-107. We first assessed brain activity in health controls and patients with AD. Then we examined miR-107 expression in SH-SY5Y and PC12 cells treated with 6-hydroxydopamine (6OHDA), and investigated its function in cytotoxicity induced by 6-OHDA. We predicted a potential miR-107 target and assessed its role in miR-107 mediated effects and explored the intracellular signaling pathways downstream of miR-107. Finally, we assessed the function of miR-107 in the mouse model insulted by 6-OHDA. We found that 6-OHDA suppressed miR-107 expression and miR-107 played neuroprotective effects against 6OHDA mediated cytotoxicity. We showed that miR-107 targeted programmed cell death 10 (PDCD10). MiR-107 suppressed PDCD10 expression and exogenous expression of PDCD10 inhibited miR-107 mediated neuroprotection. Additionally, we found that Notch signal pathway was downstream of miR-107/PDCD10. Finally, we found that 6-OHDA treatment suppressed miR-107 in mice and restoration of miR-107 alleviated motor disorder in the mouse model. Our study shows that miR-107 plays important neuroprotective roles against neurotoxicity both in vitro and in vivo by inhibiting PDCD10. Our findings confirm that miR-107 may be involved in AD pathogenesis and may be a therapeutic target for the treatment of AD-related impairments.

Keywords:
Alzheimer’s disease miR-107
Notch signaling pathway
Programmed cell death 10 (PDCD10)
Neurotoxicity

1. Introduction

One of the most common neurodegenerative diseases, Alzheimer’s disease (AD) is histopathologically manifested by presence of amyloid plaques and Tau tangles that lead to neuronal and synaptic loss and subsequently impaired cognitive function of varying degrees in the elderly patients [1,2]. While the etiology of AD is yet to be determined, several risk factors have been identified including aging with a rising prevalence associated with age, genetics, gender and exposure to environmental toxins, and abnormally regulated microRNAs have been shown to be involved in AD pathogenesis.
MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression [3]. MiRNAs are critically involved in multiple fundamental biological processes such as differentiation, apoptosis, proliferation, development and inflammation and are associated with the pathogenesis of multiple disorders, such as cardiovascular diseases, cancer and neurodegenerative diseases including AD and Parkinson’s disease (PD) [4–6]. MiR-107 has been implicated in multiple diseases including cancer, neural injury and neurodegenerative diseases [7–9]. Upregulation of miR-107 is involved in osthole mediated reduction of betaamyloid levels in AD, increased cell survival and suppressed cell toxicity [9]. However, it is not clear whether miR-107 plays any other functions related to AD. Here, we examined the protective effects of miR-107 against the neurotoxicity caused by 6-hydroxydopamine (6OHDA). 6-OHDA is a toxin that specifically kills dopaminergic neurons when injected into the striatum of the adult rats [10]. Furthermore, we tried to identify the underlying mechanisms for such protection. Notch signaling pathway is a highly conserved pathway playing critical role in normal physiology as well as the pathogenesis of various diseases including neurodegenerative diseases [11]. While Notch is required for embryonic neurogenesis, elevated levels of Notch were detected in AD and Pick’s disease patients in the hippocampus of postmortem specimens [12]. Our study established an important connection among miR107, programmed cell death 10 (PDCD10) and Notch signaling pathway and proposes that this molecular network may be an ideal target for the development of therapeutic strategies for AD.

2. Materials and methods

2.1. Subjects and fMRI tasks

Patients were recruited in Mudanjiang Medical University, Affiliated Hongqi Hospital. Gender- and age-matched healthy controls were recruited simultaneously. All participants signed informed consent. All procedures were approved by the ethics committee of Mudanjiang Medical University, Affiliated Hongqi Hospital. Brain activity changes in AD patients were assessed by functional magnetic resonance imaging (fMRI) as described previously [13]. All participants were asked to perform motor imagery test. The activities of the brain were recorded.

2.2. Cell culture

SH-SY5Y cells, a human neuroblastoma cell line and rat pheochromocytoma PC12 cells were obtained from Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China) and verified by Short Tandem Repeat (STR) analysis. Cells were cultured in DMEM medium containing 100 U/ml penicillin and 100 μg/ml streptomycin, as well as 10% fetal bovine serum (FBS). Cells were placed in a humidified chamber containing 5% CO2 at 37 °C. To determine the effects of 6-OHDA on miR-107 expression, following 48 h of culture, 6OHDA was added into the culture medium at indicated concentration for 24 h.

2.3. Cell transfection

To modulate miR-107 expression in 6-OHDA treated cells, cells were transfected with 1 μg of a negative control miR-mimic (mimics NC), miR-107 mimics, an anti-miR-NC and the miR-107 inhibitor anti-miR107 (Ruibo Biological Technology, Guangzhou, China) respectively using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). To assess the regulation of PDCD10 on miR-107 mediated cellular effects, cells were also transfected with a PDCD10 expressing plasmid (p-PDCD10) or control plasmid (p-NC) with or without miR-107.

2.4. Quantitative real time polymerase chain reaction (qPCR)

Levels of miR-107 expression were determined by qPCR according to a previous procedure [14]. Briefly, a Qiagen miRNA easy mini kit (Qiagen, Valencia, CA, USA) was used to isolate total RNA. cDNA was synthesized from an equal amount of total RNA using the Qiagen miScript reverse transcription kit. A miRNA reverse transcription kit (Qiagen) was used to determine the levels of miR-107 expression. U6 was used as an internal control. Primers were listed in Table 1.

2.5. Cell viability assay

MTT assay was used to determine cell viability as described previously [14]. Briefly, 72 h after indicated transfection, 100 μM 6OHDA was added to the medium and cells were cultured for additional 24 h. Then cells were treated with 20 μl MTT at 5 mg/ml for 4 h at 37 °C. Cell viability was determined by the absorbance at 490 nm using a microplate reader (SpectraMax M5, Molecular Devices). Results were relative to mimics NC transfected cells.

2.6. ELISA detection

Cell damage markers including Caspase-3 activity, lactate dehydrogenase (LDH) release, levels of superoxide dismutase (SOD) and production of reactive oxygen species (ROS) were measure by respective ELISA kit (Invitrogen, Waltham, MA USA) as described previously [15].

2.7. Dual-luciferase reporter assay

The association between miR-107 and PDCD10 was determined by a dual-luciferase reporter assay as described previously [14]. Briefly, the 3′-UTR harboring putative miR-107 binding sites in PDCD10 was cloned into the luciferase promotor vector pGL3 (Promega, Madison, WI, USA) to generate a PDCD10 wild type reporter (PDCD10 wt). In the meantime, a mutant reporter was generated in which the miR-107 putative binding sites were mutated (PDCD10 mut). PDCD10 wt or mutant reporters were transfected into SH-SY5Y cells along with mimics NC or miR-108 mimics. The corresponding luciferase activities were assessed by a dual-luciferase reporter assay kit (Promega, Madison, WI, USA).

2.8. Western blot

Western blot analysis was used to determine protein expression in SH-SY5Y cells as described previously [14]. Briefly, miR-107 mimics, anti-miR-107 and respective control or different plasmids were transfected into SH-SY5Y and PC12 cells as indicated for 48 h and treated with 6-OHDA for 24hr. Cells were then homogenenized with a lysis buffer. An equivalent amount of proteins was separated by electrophoresis. After blocking in 5% skim milk in PBS supplemented with 0.1% Tween-20 (PBST), membranes transferred with proteins were incubated with primary antibodies overnight at 4 °C, washed in PBST and then incubated in secondary antibodies. Enhanced chemiluminescence was used to visualize protein expression which was then quantified by ImageJ densitometry analysis. Antibodies used in this study were as following: anti-PDCD10 (1:500, Abcam, USA), anti-Notch-1 (1:500, Abcam, USA), anti-Hes 1 (1:500, Cell Signaling Technology, USA) and anti β-actin (Sangon, Shanghai, China). Levels of protein expression were normalized to β-actin.

2.9. Flow cytometry analysis

Flow cytometry was performed to assess apoptosis as described previously [14]. Briefly, following indicated transfection and treatment, Annexin V conjugated to FITC and propidium iodide (PI) were used to label apoptosis cells and cells then underwent flow cytometry analysis. Annexin V positive cells were considered undergoing apoptosis.

2.10. Animal model

Mouse model was established by introducing 6-OHDA into the right ventral tegmental area and the right median forebrain bundle using stereotactic injection following anesthesia according to a previous procedure [16]. Mice were randomly divided into 4 groups: mice in control group received no treatment, mice with 6-OHDA treatment along, mice treated with 6-OHDA and lenti-NC, and mice treated with 6-OHDA and lenti-miR-107. Negative control lentivirus (lenti-miR-NC) and the recombinant lentivirus expressing miR-107 (lenti-miR-107) were provided by Genepharma Company and were introduced into the mice of corresponding groups through tail vein injection for 10 days at the dose of 1 × 109 TU/ml, 150 μl as described previously [17]. Expression level of miR-107 in each group was determined by qPCR.

2.11. Rota-rod test

The motor ability of the mice was assessed by the Rota-rod test as described previously [18]. The residence time was determined by the duration of each mouse remaining to a rolling rod at 10 rpm.

2.12. Data analysis

The SPSS software was used for data analysis. One-way ANOVA analysis followed by a Tukey’s post hoc test was performed to determine the statistical difference. Difference was considered significant with P<0.05.

3. Results

3.1. AD patients had reduced brain activation in fMRI tasks

Previously it has been shown that AD patients had differential brain activation during fMRI tasks. Here we also assessed brain activation in healthy controls and AD patients by fMRI. We found that consistent with previous reports, although both healthy controls (Fig. 1, left panels) and AD patients (Fig. 1, right panels) had a similar overall activation pattern of different brain regions during motor imagery test, the activities in the brains of the AD patients were significantly lower than that of controls (Fig. 1).

3.2. 6-OHDA suppressed miR-107 expression

As an attempt to dissect out the molecular mechanism underlying the neurotoxic effect of AD, we treated SH-SY5Y and PC12 cells with 6OHDA that induces an in vitro cytotoxicity [19]. We then assessed miR107 expression under control and 6-OHDA treatment by qPCR. We found that while a low dose at 25 μM did not alter the miR-107 expression compare to control group, higher doses of 6-OHDA at 50 μM, 75 μM and 100 μM significantly suppressed miR-107 expression in both SH-SY5Y (Fig. 2A) and PC12 cells (Fig. 2B).

3.3. MiR-107 promoted SH-SY5Y and PC-12 cell viability following 6-OHDA treatment

To investigate how miR-107 affects viability of cells treated with 6OHDA, we modulated miR-107 expression by transfecting cells. We found that, compared to the mimics NC group, transfection of miR-107 mimics significantly increased the miR-107 levels in both SH-SY5Y cells (Fig. 3A) and PC12 cells (Fig. 3B). Similarly, transfection of anti-miR107 significantly reduced miR-107 levels compared to anti-miR-NC in both cell types.
We then assessed the viability of 6-OHDA treated cells with upregulated or downregulated miR-107 levels by the MTT assay. We found that, compared to mimics NC, miR-107 mimics transfection significantly increased viability of SH-SY5Y (Fig. 3C) and PC12 cells (Fig. 3D). On the other hand, transfection of anti-miR-107 significantly reduced cell viability. These results indicate that higher miR-107 levels correspond to better viability of SH-SY5Y and PC12 cells.

3.4. MiR-107 overexpression alleviated cytotoxicity induced by 6-OHDA

We investigated whether miR-107 played any role in 6-OHDA induced cytotoxicity. We modulated intracellular miR-107 expression by either overexpressing miR-107 mimics or introducing an anti-miR-107 to suppress its expression, and treated cells with 6-OHDA. Cell toxicity was assessed by levels of Caspase-3 activity which is associated with apoptosis, levels of released LDH which is released upon cell damage, and levels of SOD and ROS production which are indicators of oxidative stress. Strikingly, we found that compared to mimics NC transfected cells, miR-107 overexpression significantly reduced Caspase-3 activity (Fig. 4A and E), suppressed LDH release (Fig. 4B and F), increased SOD levels (Fig. 4C and G) and decreased ROS levels (Fig. 4D and H) in both SH-SY5Y and PC12 cells. In contrary, inhibition of miR-107 results in increased caspases-3 activity, enhanced LDH release, reduced SOD levels as well as increased ROS levels. Our finding suggests that 6-OHDA induced cytotoxicity is alleviated upon miR-107 overexpression and exacerbated upon miR-107 inhibition.

3.5. MiR-107 directly targets PDCD10

Next, we explored potential miR-107 targets. Prediction by TargetScan showed that the 3′ untranslated region (UTR) of the PDCD10 gene contains specific binding site for miR-107 (Fig. 5A). To determine whether PDCD10 is a miR-107 target, we examined miR-107 and PDCD10 3′ UTR interaction by a dual –luciferase assay in SH-SY5Y cells treated with 6-OHDA. Compared to mimics NC, we found that miR-107 mimics transfection significantly suppressed wild type (wt) PDCD10 3’ UTR luciferase activity, and mutation in this region (Fig. 5A) completely abolished the changes in luciferase activity (Fig. 5B). To further confirm the regulation of PDCD10 by miR-107, we determined PDCD10 mRNA and protein levels in miR-107 overexpressed or inhibited cells. Our results showed that, consistently, increased miR-107 lead to suppressed of both the mRNA (Fig. 5C) and protein (Fig. 5D) levels of PDCD10, and on the other hand, inhibition of miR-107 significantly upregulated PDCD10 mRNA and protein expression. These results suggest that PDCD10 is a direct miR-107 target.

3.6. PDCD10 overexpression attenuated miR-107 induced protection against cytotoxicity induced by 6-OHDA

To investigate the role of PDCD10 in miR-107 mediated neuroprotection, we first determined whether PDCD10 overexpression alone had any effects on the viability of 6-OHDA treated cells. Western blot analysis (Fig. 6A) showed that transfection of PDCD10 expressing plasmid p-PDCD10 significantly increased PDCD10 levels compared to that of control plasmid transfected cells (Fig. 6B). We found that overexpression of PDCD10 lead to reduced cell viability (Fig. 6C) and increased apoptosis (Fig. 6D and E) in both SH-SY5Y and PC12 cells. Next, we investigated whether miR-107 induced protection was blocked by PDCD10 levels. We found that while miR-107 overexpression along increased cell viability (Fig. 6F) and suppressed apoptosis (Fig. 6G, H and 6I) in both SH-SY5Y and PC12 cells, overexpression of both miR-107 and PDCD10 showed reduced viability and increased apoptosis. These results suggest that miR-107 protects against 6-OHDH induced cytotoxicity through inhibiting PDCD10 expression.

3.7. MiR-107/PDCD10 axis modulates Notch signaling pathway in SH-SY5Y and PC12 cells treated with 6-OHDA

Notch signaling pathway is implicated in 6-OHDA mediated neuronal death [20]. To further determine the molecular mechanism underlying miR-107/PDCD10 mediated regulation in 6-OHDA induced cytotoxicity, we investigated whether Notch signaling pathway is involved in this regulation. We examined Notch-1 and its target Hes1 expression in SH-SY5Y (Fig. 7A and B) and PC12 cells (Fig. 7C and D). Our results showed that miR-107 overexpression significantly inhibited Notch-1 and Hes1 expression in both cell types. Importantly, this miR107 overexpression induced suppresses of Notch-1 and Hes1 was restored by simultaneous overexpression of PDCD10. These results suggest that the Notch signaling pathway may be associated with miR-107/ PDCD10 mediated effects on cytotoxicity induced by 6-OHDA.

3.8. MiR-107 overexpression alleviates motor disorder in a mouse model

Finally, we investigated the role of miR-107 in vivo using a mouse model in which we treated the mice with 6-OHDA and lentivirus expressing miR-107. We found that mice treated with 6-OHDA along or 6OHDA together with control lentivirus significantly suppressed miR107 expression compared to that of the untreated mice (Fig. 8A). On the other hand, mice treated with miR-107 significantly elevated miR-107 expression level. We then investigated the effects of 6-OHDA and miR107 expression on motor function of the mice. As expected, we found that 6-OHDA treatment alone or 6-OHDA together with control lentivirus significantly impaired the motor function of the mice (Fig. 8B). Importantly, our results showed that overexpression of miR-107 completely suppressed 6-OHDA induced motor disorder.

4. Discussion

Here, we confirmed reduced brain activations in AD patients by fMRI analysis. As an effort to search for molecular signaling that are dysregulated in AD, we used a 6-OHDA induced neuronal toxicity in vitro and examined the levels of miR-107 expression in cells treated with 6-OHDA. This study showed that 6-OHDA dose-dependently suppressed miR-107 expression, with higher dose resulting lower miR-107. We also showed that miR-107 is an important suppressor of 6-OHDA mediated cell toxicity. Exogenous overexpression of miR-107 significantly increased cell viability, reduced cell toxicity as indicated by reduced caspase-3 activity, reduced LDH release, increase SOD levels and decreased ROS levels. Prediction of miR-107 targets revealed PDCP10 as a potential miR-107 target and our study showed that miR107 regulated PDCP10 expression. Importantly, we found that miR-107 mediated neuronal protection from toxicity induced by 6-OHDA through suppressing PDCD10 expression. We also identified Notch signaling pathway to be down stream of miR-107/PDCD10. Finally, miR-107 overexpression in mice rescued 6-OHDA induced motor dysfunction in mice. We thus confirmed important roles of miR-107 and also dissected out the molecular mechanism underlying miR-107 mediated suppression of neurotoxicity.
MiR-107 is highly expressed in some tumors and is involved in tumor progression, playing significant roles in invasion, migration and cell growth [7]. On the other hand, decreased miR-107 expression was found in the brains of patients with AD and was involved in AD progression [21,22]. While the exact mechanism on whether and how miR107 in involved in AD pathogenesis is not clear, a previous study has suggested that miR-107 expression level is negatively correlated with the expression of the β-site APP cleaving enzyme (BACE1), an enzyme that is critically involved in the production of Aβ, whose accumulation is a hallmark of AD. During AD pathogenesis, decreased levels of miR107 are associated with increased BACE1 levels and miR-107 may regulate BACE1 expression by binding to the 3′UTR sequence of BACE1 [9]. It is not clear whether miR-107 regulates other targets during AD progression and how miR-107 is involved in the AD mediated cytotoxicity and behavioral dysfunction. Consistent with potential involvement of miR-107 in AD, here, our study confirmed that miR-107 expression was reduced in a cellular model of AD. Importantly, we revealed that miR-107 was negatively associated with neuronal toxicity induced by 6-OHDA in both SH-SY5Y and PC12 cells. Increased miR107 expression suppressed caspase 3 activity, a marker for apoptosis, LDH release, a marker for cell toxicity and oxidative stress as manifested by increased SOD levels and decreased ROS levels, and as a result promoted cell survival. These intriguing results leading us to hypothesize that increasing miR-107 expression in neurons may make them less vulnerable in patients with AD. Although this is beyond the scope of this study, in the future, it will be interesting as well as important to characterize the functions of miR-107 in patients with AD.
This study further identified PDCD10 to be a miR-107 target in mediating neuroprotective effects of miR-107 against 6-OHDA induced toxicity. Our study revealed that miR-107 inhibited PDCD10 expression and restoration of PDCD10 in miR-107 overexpressing cells abolished the protective effects of miR-107. PDCD10, or the cerebral cavernous malformation 3 (CCM3), mutation of which causes human Cerebral Cavernous Malformations, is ubiquitously expressed in mouse neurons although the expression level has not been carefully characterized [23]. PDCD10 is required for neuronal apoptosis and conditional neural deletion of PDCD10 increases cell survival and proliferation [24,25]. The role of PDCD10 has not been shown to be directly involved in AD pathogenesis, although one study showed that a CCM2 variant was found in a family with non-progressive cognitive complaints [26]. Here, in this study, we attempted to characterize the role of PDCD10 in neuronal viability. Consistent with these previous studies, our study also showed an anti-survival role of PDCD10. Overexpression of PDCD10 significantly suppressed cell viability and increased apoptosis in SH-SY5Y and PC12 cells treated with 6-OHDA, indicating that PDCD10 may be a neuronal death regulator in patients with AD. Although beyond the scope of this study, additional research is required to characterize PDCD10 functions in AD.
In search for the intracellular molecular pathways mediating the effects of miR-107 and PDCD10 in 6-OHDA induced neuronal toxicity, our study identified Notch signaling pathway to be potentially downstream of miR-107/PDCD10. Overexpression of miR-107 in 6-OHDA treated cells enhanced Notch-1 and the Notch-1 downstream transcription factor Hes 1 expression and this was restored by simultaneous overexpression of PDCD10. Notch signaling pathway is crucial for the development of nervous system and is abnormally regulated in patients with AD. Our study suggests that Notch signaling pathway is downstream of miR-107/PDCD10. In the future, we will further characterize the functions of this pathway in the neurotoxicity of AD.
Finally, in an attempt to determine whether restoring miR-107 expression was sufficient to rescue the behavioral abnormality in individuals, we used a mouse model in which we induced motor dysfunction by injection of 6-OHDA. Our finding that overexpression of miR-107 suppressed by 6-OHDA induced motor abnormality in the Rota-rod test. Although beyond the scope of our current study, in the future, it will be interesting to investigate whether and how miR-107 in involved in other pathological and behavioral manifestations in such animal model.
It is worth noting that there are several limitations in our study. First, it would be more convincing if genetic animal model of AD could be examined. Second, the improvement of cognitive impairments could be further validated to prove the protective effects of miR-107 overexpression in the animals.

5. Conclusions

This study identified miR-107 to be an important suppressor of neurotoxicity induced by 6-OHDA in a cellular model and animal model by inhibition of PDCD10 expression. Our results suggest that miR-107 and PDCD10 may be important candidates in the investigation of molecular mechanism underlying AD pathogenesis and may represent targets for the development of therapeutic strategies against AD.

References

[1] J. Carter, C.F. Lippa, Beta-amyloid, neuronal death and Alzheimer's disease, Curr. Mol. Med. 1 (2001) 733–737.
[2] G.S. Bloom, Amyloid-beta and tau: the trigger and bullet in Alzheimer disease pathogenesis, JAMA Neurol. 71 (2014) 505–508.
[3] A.M. Mohr, J.L. Mott, Overview of microRNA biology, Semin. Liver Dis. 35 (2015) 3–11.
[4] A. Wojciechowska, A. Braniewska, K. Kozar-Kaminska, MicroRNA in cardiovascular biology and disease, Adv. Clin. Exp. Med. 26 (2017) 865–874.
[5] C. Cogoni, F. Ruberti, C. Barbato, MicroRNA landscape in Alzheimer's disease, CNS Neurol. Disord. - Drug Targets 14 (2015) 168–175.
[6] A. Singh, D. Sen, MicroRNAs in Parkinson's disease, Exp. Brain Res. 235 (2017) 2359–2374.
[7] S. Wang, G. Ma, H. Zhu, C. Lv, H. Chu, N. Tong, D. Wu, F. Qiang, W. Gong, Q. Zhao, G. Tao, J. Zhou, Z. Zhang, M. Wang, miR-107 regulates tumor progression by targeting NF1 in gastric cancer, Sci. Rep. 6 (2016) 36531.
[8] J.D. Jiang, X.C. Zheng, F.Y. Huang, F. Gao, M.Z. You, T. Zheng, MicroRNA-107 Regulates Anesthesia-Induced Neural Injury in Embryonic Stem Cell Derived Neurons, IUBMB Life, 2018.
[9] Y. Jiao, L. Kong, Y. Yao, S. Li, Z. Tao, Y. Yan, J. Yang, Osthole decreases beta amyloid levels through up-regulation of miR-107 in Alzheimer's disease, Neuropharmacology 108 (2016) 332–344.
[10] S. Przedborski, M. Levivier, H. Jiang, M. Ferreira, V. Jackson-Lewis, D. Donaldson, D.M. Togasaki, Dose-dependent lesions of the dopaminergic nigrostriatal pathway induced by intrastriatal injection of 6-hydroxydopamine, Neuroscience 67 (1995) 631–647.
[11] J.D. Lathia, M.P. Mattson, A. Cheng, Notch: from neural development to neurological disorders, J. Neurochem. 107 (2008) 1471–1481.
[12] M.H. Nagarsheth, A. Viehman, S.M. Lippa, C.F. Lippa, Notch-1 immunoexpression is increased in Alzheimer's and Pick's disease, J. Neurol. Sci. 244 (2006) 111–116.
[13] F. Agosta, R. Gatti, E. Sarasso, M.A. Volonte, E. Canu, A. Meani, L. Sarro, M. Copetti, E. Cattrysse, E. Kerckhofs, G. Comi, A. Falini, M. Filippi, Brain plasticity in Parkinson's disease with freezing of gait induced by action observation training, J. Neurol. 264 (2017) 88–101.
[14] R.F. Dong, B. Zhang, L.W. Tai, H.M. Liu, F.K. Shi, N.N. Liu, The neuroprotective role of MiR-124-3p in a 6-hydroxydopamine-induced cell model of Parkinson's disease via the regulation of ANAX5, J. Cell. Biochem. 119 (2018) 269–277.
[15] Y. Song, Y. Liu, X. Chen, MiR-212 attenuates MPP(+)-Induced neuronal damage by targeting KLF4 in SH-SY5Y cells, Yonsei Med. J. 59 (2018) 416–424.
[16] Y.Y. Liu, Y.N. Zhang, Q.S. Yang, Downregulated expression of microRNA-329 inhibits apoptosis of nigral dopaminergic neurons by regulating CDKN2D expression via the FoxO3a signaling pathway in rats with Parkinson's disease, J. Cell. Physiol. 233 (2018) 8617–8629.
[17] N. Tang, Y. Wu, W. Cao, Y. Liang, Y. Gao, L. Hu, Q. Yang, Y. Zhou, F. Tang, J. Xiao, Lentivirus-mediated over-expression of let-7b microRNA suppresses hepatic fibrosis in the mouse infected with Schistosoma japonicum, Exp. Parasitol. 182 (2017) 45–53.
[18] H. Jing, S. Wang, M. Wang, W. Fu, C. Zhang, D. Xu, Isobavachalcone attenuates MPTP-induced Parkinson's disease in mice by inhibition of microglial activation through NF-kappaB pathway, PLoS One 12 (2017) e0169560.
[19] F.M. Lopes, L.L. da Motta, M.A. De Bastiani, B. Pfaffenseller, B.W. Aguiar, L.F. deSouza, G. Zanatta, D.M. Vargas, P. Schonhofen, G.F. Londero, L.M. de Medeiros, V.N. Freire, A.L. Dafre, M.A. Castro, R.B. Parsons, F. Klamt, RA differentiation enhances dopaminergic features, changes redox parameters, and increases dopamine transporter dependency in 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells, Neurotox. Res. 31 (2017) 545–559.
[20] J.S. Mo, J.H. Yoon, E.J. Ann, J.S. Ahn, H.J. Baek, H.J. Lee, S.H. Kim, Y.D. Kim, M.Y. Kim, H.S. Park, Notch1 modulates oxidative stress induced cell death through suppression of apoptosis signal-regulating kinase 1, Proc. Natl. Acad. Sci. U. S. A.110 (2013) 6865–6870.
[21] W.X. Wang, B.W. Rajeev, A.J. Stromberg, N. Ren, G. Tang, Q. Huang, I. Rigoutsos, P.T. Nelson, The expression of microRNA miR-107 decreases early in Alzheimer's disease and may accelerate disease progression Oxidopamine through regulation of beta-site amyloid precursor protein-cleaving enzyme 1, J. Neurosci. 28 (2008) 1213–1223.
[22] P.T. Nelson, W.X. Wang, MiR-107 is reduced in Alzheimer’s disease brain neocortex: validation study, J. Alzheimer’s Dis. 21 (2010) 75–79.
[23] N. Petit, A. Blecon, C. Denier, E. Tournier-Lasserve, Patterns of expression of the three cerebral cavernous malformation (CCM) genes during embryonic and postnatal brain development, Gene Expr. Patterns 6 (2006) 495–503.
[24] C. Lin, S. Meng, T. Zhu, X. Wang, PDCD10/CCM3 acts downstream of {gamma}protocadherins to regulate neuronal survival, J. Biol. Chem. 285 (2010) 41675–41685.
[25] A. Louvi, L. Chen, A.M. Two, H. Zhang, W. Min, M. Gunel, Loss of cerebral cavernous malformation 3 (Ccm3) in neuroglia leads to CCM and vascular pathology, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 3737–3742.
[26] P.E. Cohn-Hokke, H. Holstege, M.M. Weiss, W.M. van der Flier, F. Barkhof,E.A. Sistermans, Y.A. Pijnenburg, J.C. van Swieten, H. Meijers-Heijboer, P. Scheltens, A novel CCM2 variant in a family with non-progressive cognitive complaints and cerebral microbleeds, Am. J. Med. Genet. B Neuropsychiatr. Genet.174 (2017) 220–226.