The Anthelmintic Drug Niclosamide and Its Analogues Activate the Parkinson's Disease Associated Protein Kinase PINK1

Abstract Mutations in PINK1, which impair its catalytic kinase activity, are causal for autosomal recessive early‐onset Parkinson's disease (PD). Various studies have indicated that the activation of PINK1 could be a useful strategy in treating neurodegenerative diseases, such as PD. Herein, it is shown that the anthelmintic drug niclosamide and its analogues are capable of activating PINK1 in cells through the reversible impairment of the mitochondrial membrane potential. With these compounds, for the first time, it is demonstrated that the PINK1 pathway is active and detectable in primary neurons. These findings suggest that niclosamide and its analogues are robust compounds for the study of the PINK1 pathway and may hold promise as a therapeutic strategy in PD and related disorders.


I.
Antibodies and reagents (page 1) II.

I. Antibodies and reagents
The following primary antibodies were used: mouse monoclonal antibodies against Parkin (Santa Cruz), βI-tubulin (Sigma), β-actin (Sigma), GAPDH (Santa Cruz), PSD95 (Cell Signaling), synaptophysin (cell signalling), CISD1 (Proteintech). Horseradish-peroxidase (HRP)-conjugated secondary antibodies (Sigma) were used. Anti-Parkin phospho-Ser65 rabbit monoclonal antibody was raised by Epitomics in collaboration with the Michael J Fox Foundation for Research. Stock solutions of niclosamide (Sigma), Antimycin A (Sigma) Oligomycin A (Sigma) were used for experiments in vitro. Unless otherwise specified, general reagents and chemicals were from Sigma and cell culture reagents were from Invitrogen.

II. Cell culture
HeLa cells stably expressing untagged Parkin WT and PINK 1 KO, were cultured using DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS, 2 mM Lglutamine, 1×penicillin/streptomycin. To uncouple mitochondria, cells were treated with 10 µM Antimycin (Sigma) and 1 µM Oligomycin dissolved in DMSO for 3 h. To express Parkin, cell transfections were performed using polyethylenimine (Polysciences) according to the manufacturer's instruction. Primary cortical neurons were isolated from the brain of WT embryos of either sex at E16.5. Embryonic cortices were collected in HBSS, and cells were dissociated by incubation with trypsin (GIBCO) at 37°C. Cells were then diluted in Neurobasal medium containing B27 supplement, Glutamax, penicillin/streptomycin and plated at a density of 6.0 × 106 cells/well on 6-well plates coated with 0.1 mg/ml poly-L-lysine (PLL; Sigma). Neurons were cultured at 37 °C in a humidified incubator with 5% CO 2 . Every 7 days, the medium was replaced with fresh medium containing B27. Neurons were treated with 30 µM niclosamide and analogues for 1h at 37 °C.

IV. Supplementary figure 1 Supplementary 1. Niclosamide activates PINK1 after 20 minutes treatment. A)
Niclosamide-induced Parkin activation is PINK1 dependent and can be detected after 20 min of in vitro stimulation. HeLa cells transfected with Parkin were stimulated with either a combination of antimycin A and oligomycin A (A/O) for 3 h or with 10 µM of niclosamide (Niclo) for 5, 10, 20, 40 min. Detection of Parkin S65 phosphorylation (pS65Parkin), Parkin, Full length (F/L) and cleaved OPA1, CISD1 ubiquitylation (CISD1-Ub) can be detected in niclosamide stimulated HeLa cells at different time points. GAPDH and HSP60 were used as loading controls. B) Quantitative analysis of Parkin S65 phosphorylation after niclosamide treatment at different time points (5, 10, 20, 40 min). Bars represent the average ratio ± SEM of two independent experiments and the value is a ratio between pS65 Parkin and total Parkin.

V. Supplementary figure 2
Supplementary 2. AM85 activates PINK1 and uncouples mitochondria after 40 minutes of treatment. A) CISD1 ubiquitylation after niclosamide and AM85 treatment is PINK1 dependent. Wildtype (WT) and PINK1 knockout (Pink1 KO) HeLa cells transfected with Parkin were stimulated with a combination of 10 µM antimycin A and 1 µM oligomycin A (A/O) for 3 h, 10 µM niclosamide (Niclo) and 10 µM AM85 for 40 min. CISD1 ubiquitylation (CISD1-Ub) was detected by western blotting. B) Time course of AM85 in HeLa cells. Detection of Parkin S65 phosphorylation (pS65 Parkin), Parkin, Full length and cleaved OPA1 and CISD1 ubiquitylation (CISD1-Ub) upon different time points of AM85 treatment (5, 10, 20, 40 min). C) Quantitative analysis of Serine 65 phosphorylation in response to AM85 treatment at different time points. Bars represent the average ratio ± SEM of 2 independent experiments and the value is a ratio between pSer65 Parkin and total Parkin. D) Niclosamide and AM85 uncouple mitochondria in HeLa cells. Exemplary FACS graph of CMXRos fluorescence intensity in Hela cells treated on site. Hela cells were treated on site with antimycin A and oligomycin A (A/O, green), niclosamide (Niclo, red), AM85 (blue) and the vehicle, DMSO (black).