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The Zinc Ionophore Clioquinol Reduces Parkinson’s Disease Patient-Derived Brain Extracts-Induced Neurodegeneration

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Abstract

Parkinson’s disease (PD) is pathologically characterized by intracellular α-synuclein-rich protein aggregates, named Lewy bodies (LB), and by the progressive loss of dopaminergic neurons in the substantia nigra. Several heavy metals, including zinc (Zn), have been suggested to play a role in PD progression, although the exact role of Zn in neurodegeneration remains to be fully elucidated. To address this gap, we investigated the effects of Zn modulation on the progression of degeneration in mice injected with PD patient-derived LB-extracts carrying toxic α-synuclein aggregates. Zn modulation was achieved using either a clioquinol-enriched diet, a Zn ionophore that redistributes cellular Zn, or a Zn-enriched diet that increases Zn levels. Clioquinol treatment significantly prevented dopaminergic neurodegeneration and reduced α-synuclein-associated pathology in LB-injected mice, while no differences were observed with Zn supplementation. Biochemical analyses further demonstrate that the expression levels of vesicle-specific Zn transporter ZnT3 in the striatum of LB-injected mice treated with clioquinol were decreased, suggesting an intracellular redistribution of Zn. Additionally, we found that clioquinol modulates the autophagy-lysosomal pathway by enhancing lysosomal redistribution within the neuronal compartments. Collectively, we found that in vivo pharmacological chelation of Zn, by dampening Zn-mediated cytotoxicity, can result in an overall attenuation of PD-linked lysosomal alterations and dopaminergic neurodegeneration. The results support zinc chelation as a disease-modifying strategy for treating PD.

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Data availability

All data generated or analyzed during this study are included in Table 1 of supplementary data; further inquiries can be directed to the corresponding author.

Abbreviations

AD:

Alzheimer’s disease

ALP:

Autophagy-lysosomal pathway

ClQ:

Clioquinol

CNS:

Central nervous system

ELISA:

Enzyme-linked immunosorbent assay

PD:

Parkinson’s disease

α-syn:

α-Synuclein

LB:

Lewy bodies

LC3:

MAP1LC3B, microtubule-associated protein 1 light chain 3 β

MTF1:

Metal transcription factor 1

PK:

Proteinase K

SNpc:

Substantia nigra pars compacta

SOD1:

Superoxide dismutase 1

TH:

Tyrosine hydroxylase

Zn:

Zinc

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Acknowledgements

We thank Guillaume Dabée and Christelle Martin for animal care, as well as Dr. Federico N. Soria for the script to analyze the TH-LAMP2 perinuclear fractions. The synchrotron Diamond Light Source is acknowledged for provision of I18 beam time (exp. SP28279). We would also like to thank Tina Geraki for scientific support. MT was supported by a Ministère de l’Enseignement Supérieur et de la Recherche fellowship (France). The human samples were obtained from the Brain Bank GIE NeuroCEB (BRIF number 0033-00011), funded by France Alzheimer, France Parkinson, ARSEP, and Connaître les Syndromes Cérébelleux, to which we express our gratitude.

Funding

The University of Bordeaux and the Centre National de la Recherche Scientifique provided infrastructural support. This study received financial support from the French government in the framework of the University of Bordeaux’s IdEx “Investments for the Future” program/GPR BRAIN_2030. Animal experiments were performed at the Animal Facilities of the University of Bordeaux, supported by the Région Nouvelle-Aquitaine.

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Authors and Affiliations

  1. Univ. Bordeaux, CNRS, IMN, UMR 5293, F-33000, Bordeaux, France

    Margaux Teil, Evelyne Doudnikoff, Marie-Laure Thiolat, Erwan Bezard & Benjamin Dehay

  2. Synchrotron Radiation for Biomedicine (STROBE), Univ. Grenoble Alpes, Inserm, UA7, 38000, Grenoble, France

    Sylvain Bohic

  3. Institut des Maladies Neurodégénératives, Université de Bordeaux, CNRS UMR 5293, Centre Broca Nouvelle-Aquitaine, 146 rue Léo Saignat, 33076, Bordeaux cedex, France

    Benjamin Dehay

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  1. Margaux Teil
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Contributions

BD isolated LB fractions. MT performed the surgeries. MT and ED performed the animal termination. MT performed histologic and immunohistochemical analysis of the data. MT and MLT performed the biochemistry experiments. MT, SB, and BD performed the synchrotron analysis. MT prepared the figures. MT, EB, and BD wrote the paper with input from all authors. BD and EB conceptualized the study and designed experiments. EB and BD secured funding.

Corresponding author

Correspondence to Benjamin Dehay.

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Ethics Approval and Consent to Participate

Animals. Experiments were performed following the European Union directive of September 22, 2010 (2010/63/EU) on the protection of animals used for scientific purposes. The Institutional Animal Care and Ethical Committee of Bordeaux University (CE50, France) approved experiments accepted by the ministry under references APAFIS#17592–2018111914281699.

Human tissues. The samples were obtained from brains collected in a Brain Donation Program of the Brain Bank “GIE NeuroCEB” run by a consortium of Patients Associations: ARSEP (association for research on multiple sclerosis), CSC (cerebellar ataxias), France Alzheimer, and France Parkinson. The consent documents were signed by the patients themselves or their next of kin in their name, in accordance with the French Bioethical Laws. The Brain Bank GIE NeuroCEB (Bioresource Research Impact Factor number BB-0033–00011) has been declared at the Ministry of Higher Education and Research and has received approval to distribute samples (agreement AC-2013–1887).

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E.B. owns equity stake in Motac Holding Ltd. and receives consultancy payments from Motac Neuroscience Ltd. All other authors declare no competing interests.

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Supplementary file1 (XLSX 33 KB)

Supplementary file2

Supplementary Figure 1: Clioquinol treatment inhibits the loss of neurons in the substantia nigra, but both dopaminergic fiber density and dopamine transporter expression remain unchanged in LB-injected or Vehicle-injected mice. (A-B) Scatter plots of Tyrosine Hydroxylase (TH) and Nissl-positive neuron count in the substantia nigra of Vehicle and LB-injected mice treated with Clioquinol or zinc. (B) Illustrative images (left) and quantifications (right) of TH staining in the whole striatum of mice injected with Vehicle or LB fractions that received one of three diets. (C) Illustrative images (left) and quantifications (right) of DAT staining in the whole striatum of mice injected with Vehicle or LB fractions that received one of three diets. Comparisons were made using two-way ANOVA followed by Tukey’s post-hoc analysis, n=5 per group. Scale bar =500μm. * p<0.05 vs Veh, Veh ClQ, LB ClQ; # p<0.05 vs Veh, Veh Zn. (PNG 2057 kb)

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Supplementary file3

Supplementary Figure 2: Expression of monomeric α-synuclein remains unchanged in LB-injected mice treated with Clioquinol and zinc. Immunoblot and quantification of α-synuclein expression in the substantia nigra of injected mice. Scatter plots represent the mean protein expression normalized by β-Actin levels in Vehicle and LB mice treated with clioquinol or zinc. Comparisons were made using two-way ANOVA followed by Tukey’s post-hoc analysis, n=4-5 per group. (PNG 282 kb)

High resolution image (TIF 13423 kb)

Supplementary file4

Supplementary Figure 3: Zinc Transporter expression is unaltered in the substantia nigra of mice with modulated zinc diets. Representative immunoblots and histograms of ZnT4 (A), ZIP8 (B) and SOD1 (C) expressions in the substantia nigra. Scatter plots represent the mean protein expression normalized by β-Actin levels in Vehicle and LB mice treated with clioquinol or zinc. Comparisons were made using two-way ANOVA followed by Tukey’s post-hoc analysis, n=4-5 per group. (PNG 531 kb)

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Supplementary file5

Supplementary Figure 4: Clioquinol and zinc treatments induce few alterations in metal concentrations in the SN of LB-injected mice. Levels of zinc (A), calcium (B), iron (C), copper (D), manganese (E), and sulfur (F) were measured using synchrotron X-ray fluorescence in the ipsilateral SN of control and LB-injected mice treated with clioquinol or zinc. Comparisons were made using two-way ANOVA followed by Tukey’s post-hoc analysis, n=5 per group. * p<0.05 vs all (Zn), vs LB (Ca), vs LB ClQ and LB Zn (Fe), # p<0.05 Veh Zn vs LB and LB ClQ. (PNG 151 kb)

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Supplementary file6

Supplementary Figure 5: Clioquinol and zinc induce alterations in expression of autophagy and lysosomal markers. (A-C) Representative images and quantification of LC3-II (A), p62 (B), and LAMP1 (C) immunoblotting in the different experimental groups. Scatter plots represent the mean protein expression normalized by β-Actin levels in Vehicle and LB mice treated with clioquinol or zinc. Comparisons were made using two-way ANOVA followed by Tukey’s post-hoc analysis, n=4-5 per group. * p<0.05 vs Veh, Veh Zn, LB. #p<0.05 vs Veh, LB, LB ClQ. (PNG 745 kb)

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Supplementary file7

Supplementary Figure 6: Zinc supplementation induces alterations in protein ubiquitination in mice. (A-C) Representative images (A) and quantification of ubiquitin (B) and poly-ubiquitin (C) immunoblotting in the different experimental groups. Scatter plots represent the mean protein expression normalized by β-Actin levels in Vehicle and LB mice treated with clioquinol or zinc. Comparisons were made using two-way ANOVA followed by Tukey’s post-hoc analysis, n=4 per group. * p<0.05 vs LB ClQ (ClQ), vs Veh, LB, LB Zn (Zn), vs Veh, LB Zn (Zn polyUb); #p<0.05 vs Veh, LB, $p<0.05 vs LB. (PNG 885 kb)

High resolution image (TIF 23299 kb)

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Teil, M., Doudnikoff, E., Thiolat, ML. et al. The Zinc Ionophore Clioquinol Reduces Parkinson’s Disease Patient-Derived Brain Extracts-Induced Neurodegeneration. Mol Neurobiol 59, 6245–6259 (2022). https://doi.org/10.1007/s12035-022-02974-5

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