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. 2020 May 18;12(10):9515-9533.
doi: 10.18632/aging.103225. Epub 2020 May 18.

Clioquinol improves motor and non-motor deficits in MPTP-induced monkey model of Parkinson's disease through AKT/mTOR pathway

Liangqin Shi  1 , Chao Huang  1   2 , Qihui Luo  1   2 , Yu Xia  1 , Wentao Liu  1   2 , Wen Zeng  3 , Anchun Cheng  2 , Riyi Shi  4 , Chen Zhengli  1   2
Affiliations

Clioquinol improves motor and non-motor deficits in MPTP-induced monkey model of Parkinson's disease through AKT/mTOR pathway

Liangqin Shi et al. Aging (Albany NY). .

Abstract

Despite decades of research into the pathology mechanisms of Parkinson's disease (PD), disease-modifying therapy of PD is scarce. Thus, searching for new drugs or more effective neurosurgical treatments has elicited much interest. Clioquinol (CQ) has been shown to have therapeutic benefits in rodent models of neurodegenerative disorders. However, it's neuroprotective role and mechanisms in PD primate models and PD patients, especially in the advanced stages, are not fully understood. Furthermore, issues such as spontaneous recovery of motor function and high symptom variability in different monkeys after the same toxic protocol, has not been resolved before the present study. In this study, we designed a chronic and long-term progressive protocol to generate a stabilized PD monkey model showed with classic motor and non-motor deficits, followed by treatment analysis of CQ. We found that CQ could remarkably improve the motor and non-motor deficits, which were based on the reduction of iron content and ROS level in the SN and further improvement in pathology. Meanwhile, we also showed that ferroptosis was probably involved in the pathogenesis of PD. In addition, the study shows a positive effect of CQ on AKT/mTOR survival pathway and a blocking effect on p53 medicated cell death in vivo and in vitro.

Keywords: Parkinson’s disease; clioquinol; ferroptosis; iron; monkey.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Experimental design of the study and behavior test by Kurlan rating scale. (A) A progressive intoxication protocol of MPTP from 0-22w, MPTP withdraw period from 22-25w, and treatment period from 25-29w. IM: Intramuscular injection; IV: Intravenous injection. (B) Longitudinal evaluation (in weeks) of motor function by Kurlan rating scale for each monkey during the MPTP intoxication and withdraw period.
Figure 2
Figure 2
CQ improved clinical phenotypes after long-term of MPTP intoxication. (A) Evaluation of motor function for each group by Papa scale during the treatment period. Motor deficits mitigated by both LD and CQ treatment displayed with decreased Papa scale. (B) The comparison of Papa scores in each group before and after treatment. Papa scores were significantly declined by both LD and CQ treatment. (C–J) The comparison of spirit evaluation, including grooming score, checking behavior score, interactivity after stimulation score, spontaneous interactivity score, defense reaction score, hypersomnia score, facial expression score, and vocalizing score in each group, before and after treatment, respectively. (K) Representative images showed constipation after MPTP intoxication, and which was relieved by both LD and CQ treatment. Data expressed as the mean ± SD. *P<0.05, **P<0.01, indicate significant difference.
Figure 3
Figure 3
CQ improved the pathology of monkeys in the SN after MPTP intoxication. (A) Representative images of Nissl and immunofluorescence staining of TH (biomarkers of DA neurons) showed cell loss and cell atrophy in the SN after long-term of MPTP injection, and which were mitigated by both LD and CQ treatment. (B) Stereology analysis of cell count of DA neurons in the SN. (C, D) Western bolts and quantification showed increased protein expression of TH, DAT, and D3R in the SN of MPTP+LD and MPTP+CQ group in comparison to MPTP group. (E) Quantification showed the mRNA level of TH, DAT, and D3R in each group. (F, G) Western bolts and quantification showed increased protein expression of NF-M and MBP in the SN of MPTP+LD and MPTP+CQ group in comparison to MPTP group. Data expressed as the mean ± SD. *P<0.05, **P<0.01, indicate significant difference. Scale bar=200μm.
Figure 4
Figure 4
CQ suppressed iron content and oxidative stress in the SN after MPTP intoxication. (A) Iron content in the striatum, SN and organized in overall (total) of each group tested by flame atomic absorption spectrometry in each group. (B–I) Quantification showed the mRNA level of iron metabolism related genes, including TFR2, FPN1, H-Fn, L-Fn, IRP1, IRP2, TF, and HO-1 in each group, respectively. (J) Representative images of Prussian blue staining showed iron distribution in the SN of each group. (K) Quantification showed GSH levels in the SN in each group, respectively. (L–N) Quantification showed SOD, GSH, and MDA levels in serum of each group, respectively. (O–P) Western bolt and quantification showed increased 4-HNE expression after MPTP intoxication, and which was decreased by both LD and CQ treatment. (Q) Representative images of immunofluorescence staining of 4-HNE in each group. Data expressed as the mean ± SD. *P<0.05, **P<0.01, indicate significant difference. Scale bar=200μm.
Figure 5
Figure 5
CQ attenuated MPTP toxicity by decreasing ROS level in vitro. (A, E) Cell viability was determined for SK-N-SH cells treated with different concentration of H2O2 (0, 10, 20, 50, 100, 200, 500μM) or MPP+ (0, 0.1, 0.2, 0.5, 1, 2, 5mM) for 24h, respectively. (B, F) Low dose of CQ (5μM) increased cell viability in the absence or presence of H2O2 (200μM) or MPP+(2mM) tested by CCK-8 kit, respectively. (C, G) CQ decreased cell death tested by Hoechst staining after the exposure of H2O2 (200μM) or MPP+(2mM) for 24h, respectively. (D, H) Representative images of Hoechst staining for SK-N-SH cells treated with H2O2 (200μM) or MPP+(2mM), respectively. (I–K) CQ decreased ROS in the absence or presence of MPP+(2mM) tested by flow cytometry, microplate reader, and microscope, respectively. Data expressed as the mean ± SD. *P<0.05, **P<0.01, indicate significant difference. Scale bar=50μm.
Figure 6
Figure 6
CQ play neuroprotective effect by improving AKT/mTOR survival pathway and blocking p53 medicated cell death. (A–D) Western bolts and quantification showed decreased expression of phosphor-AKT (S473 and T308), phosphor-mTOR (S2448) and Bcl-2, as well as increased p-p53 and Bax in the SN of monkeys, and which were reversed by both LD and CQ treatment. (E–H) Western bolts and quantification showed decreased expression of phosphor-AKT (S473 and T308), phosphor-mTOR (S2448) and Bcl-2, as well as increased p-p53 and Bax in SK-N-SH cells, and which were reversed by CQ treatment. Data expressed as the mean ± SD. *P<0.05, **P<0.01, indicate significant difference.
Figure 7
Figure 7
A summary diagram is shown. In the brain both glial and DA neurons are involved with iron dysfunction during the development of PD. Specifically, both increased levels of ROS and lipid peroxidation (main features of ferroptosis) were happened in the MPTP-induced monkey model, while which could be reversed by low dosage of CQ treatment. So ferroptosis dysfunction probably be involved in the pathogenesis of PD. Meanwhile, the protection effect of CQ was depending on the activation of the AKT/mTOR survival pathway and the prevention of p53-medicated cell death.
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