Parkinson’s disease affects millions of people worldwide, causing progressively worse symptoms like stiffness, tremors and difficulty moving. These issues result from the death of neurons in the brain that produce the neurotransmitter dopamine. While most cases have no known cause, 10 to 15 per cent are due to inherited gene mutations. This includes mutations in the genes that code for the proteins PINK1 and Parkin which are essential for maintaining healthy mitochondria, the powerhouse of the cell.

Mutations in this quality control system affect a protein called CISD1, which sits within the outer surface of the mitochondria. CISD1 contains a cluster of iron and sulfur ions, and is involved in regulating iron levels and mitochondrial energy production. However, its role in inherited cases of Parkinson’s disease, particularly those related to mutations in PINK1 and Parkin, is poorly understood.

To understand the impact of CISD1, Bitar et al. studied genetically modified fruit flies and dopamine-producing neurons from Parkinson’s patients with PINK1 mutations. This revealed that losing PINK1 activity led to higher levels of CISD1 proteins which lacked the iron-sulfur cluster due to a bond forming between two CISD1 molecules.

Reducing levels of the CISD1-equivalent protein in the flies helped to alleviate most of the symptoms caused by PINK1 and Parkin gene mutations, such as difficulties climbing and impaired wing posture. These findings suggest that iron-depleted CISD1 contributes to the symptoms associated with Parkinson’s disease, underscoring its potential as a drug target.

Drugs that target CISD1 already exist, which could ease the way for further research. Recent studies have shown that cases of Parkinson’s related to mutations in PINK-1 share features with some non-inherited instances of the disease, suggesting that this approach could potentially benefit many patients.

Parkinson’s disease (PD) is the fastest growing neurodegenerative disorder clinically characterized by rigor, tremor, and bradykinesia. These motor symptoms stem from the progressive degeneration of dopaminergic neurons in the midbrain’s substantia nigra pars compacta (reviewed in Pickrell and Youle, 2015). While most cases are sporadic, familial forms of PD are linked to mutations in genes that encode proteins involved in mitochondrial quality control like PTEN-induced kinase 1 (PINK1) and Parkin (PRKN), providing valuable insights into the pathophysiology of PD (Shulman et al., 2011). In healthy cells, PINK1 is continuously imported into mitochondria, where it is rapidly degraded (Narendra et al., 2008). However, in unfit mitochondria with a reduced mitochondrial membrane potential (Δψ_m), PINK1 import stalls. When PINK1 accumulates on damaged mitochondria, it phosphorylates various proteins, including the E3 ubiquitin ligase PRKN. This phosphorylation activates PRKN, enabling it to attach ubiquitin to mitochondrial proteins. This ubiquitination acts as a signal for damaged mitochondria to be identified and engulfed by autophagosomes, initiating their degradation through mitophagy. This selective elimination of compromised mitochondria safeguards overall mitochondrial health and function (reviewed in Youle and Narendra, 2011). Much of this mechanistic understanding has been elucidated in the fruit fly Drosophila melanogaster where Pink1 and Prkn loss of function results in profound changes of the mitochondrial ultrastructure causing mitochondrial dysfunction, impaired flight and climbing ability, and a shortened lifespan (Clark et al., 2006; Park et al., 2006).

In addition to disturbances in mitochondrial quality control, disruptions in iron metabolism contribute significantly to the pathogenesis of PD (Ma et al., 2021). PD patients exhibit elevated iron levels within the substantia nigra pars compacta, and this iron accumulation correlates with the severity of the disease (Dexter et al., 1987; Hirsch et al., 1991). Notably, this iron dysregulation is apparent even in asymptomatic carriers of PRKN mutations (Pyatigorskaya et al., 2015), suggesting that it is an early event and correlates with mitochondrial dysfunction. Furthermore, human dopaminergic neurons lacking PINK1 display inhibition of the iron-dependent mitochondrial aconitase, leading to accumulation of NAD⁺ and depletion of essential metabolites (Bus et al., 2020). In flies, Pink1 deficiency similarly results in inactivation of mitochondrial aconitase by impairing its labile iron-sulfur cluster, leading to oxidative distress and mitochondrial swelling (Esposito et al., 2013; Wan et al., 2020). These findings highlight the relevance of iron dysregulation in the PD development and progression.

An important target protein of PINK1/PRKN crucial for iron homeostasis and redox homeostasis is CISD1, also known as mitoNEET (reviewed in Mittler et al., 2019). CISD1 is a homodimeric protein N-terminally inserted into the outer mitochondrial membrane facing the cytosol (Wiley et al., 2007a). CISD1 is ubiquitinated by PRKN (Sarraf et al., 2013) and its levels are reduced upon dissipation of Δψ_m (Chan et al., 2011; Cunningham et al., 2015; Lazarou et al., 2013; McWilliams et al., 2018; Narendra et al., 2012; Okatsu et al., 2012). Moreover, CISD1 forms a complex with PRKN, and this interaction is markedly increased by activation of PINK1 and ubiquitination (Narendra et al., 2013; Sarraf et al., 2013). In Drosophila, the homolog of CISD1 is the most strongly ubiquitinated protein in response to Prkn overexpression in vivo (Martinez et al., 2017).

Functionally, CISD1 exists as a homodimer, and each monomer of CISD1 harbors an 2Fe/2S (2 iron/2 sulfur) cluster coordinated by three cysteines (Cys72,74,83) and one histidine (His87). In contrast, most conventional 2Fe/2S clusters are coordinated by four cysteines or two cysteines and two histidines (Wiley et al., 2007b). This peculiarity renders the CISD1 2Fe/2S cluster more susceptible to oxidation. It was thus proposed that the transfer of the iron-sulfur cluster from CISD1 to stress-sensitive Fe/S proteins constitutes a cellular adaptive response that helps recovery from oxidative injury (reviewed in Mittler et al., 2019). In support of this hypothesis, it has been demonstrated that the oxidized 2Fe/2S cluster of CISD1 can be transferred to iron regulatory protein 1 (IRP1) (Ferecatu et al., 2014), a bifunctional protein that can switch between two distinct enzymatic activities based on the availability of iron. When iron levels are sufficient, IRP1 binds an 2Fe/2S cluster, converting it into a functional aconitase enzyme. When iron levels are low, IRP1 loses its 2Fe/2S cluster and undergoes a conformational change. In this state, known as the ‘apo’ form, IRP1 functions as an RNA-binding protein and binds to specific RNA sequences called iron-responsive elements (IREs) located in the untranslated regions of target mRNAs (reviewed by Rouault and Maio, 2017). CISD1 is capable of recycling the cytosolic apo-IRP1 to holo-aconitase by donating its 2Fe/2S cluster (Camponeschi et al., 2017). The fate of the remaining apo-CISD1 is unknown. In addition to its role in iron metabolism and defense against oxidative distress, CISD1 is also involved in mitochondrial bioenergetics and in the regulation of mitochondrial morphology. Cardiac myocytes from Cisd1 knockout (KO) mice exhibit a reduced mitochondrial oxidative capacity (Wiley et al., 2007a), akin to KO mouse embryonic fibroblasts (MEFs) where this dysfunction is attributed to a reduction in the total cellular mitochondrial volume (Vernay et al., 2017).

In Drosophila, there is a single ortholog of CISD1 initially named Cisd2 because it is slightly more similar to the mammalian CISD2 than to CISD1 (Jones et al., 2014) protein. Human CISD2 is located at the membrane of the endoplasmic reticulum (ER) (Wang et al., 2014). Overexpression of Cisd2 (this publication used the alternative name Dosmit) in fly muscles is detrimental and results in mitochondrial enlargement and the formation of double-membraned vesicles containing cytosolic proteins within mitochondria (Chen et al., 2020), implicating Cisd2 in the regulation of mitochondrial morphology. Similar to human CISD1, fly Cisd2 is an Fe/S protein and all amino acids linked to cluster coordination are conserved. In addition, fly Cisd2 (this publication used the alternative name MitoNEET) interacts with IRP1 (Huynh et al., 2019) further linking Cisd2 to iron homeostasis. Together these findings suggest that fly Cisd2 rather resembles the human mitochondrial homolog CISD1 and to avoid confusion, we will henceforth use Cisd for the fly ortholog. Based on the involvement of mammalian CISD1 in mitochondrial bioenergetics and quality control, iron metabolism and defense against oxidative distress – all hallmarks of PD – we aimed to investigate CISD1’s role in PD using both fly and mammalian model systems.

In our study, we identified CISD1, particularly its iron-depleted apo form, as a downstream mediator of the PD pathology induced by the loss of Pink1 and Prkn function. Apo-CISD1 accumulates in PINK1 patient-derived human dopaminergic neurons and apo-Cisd in mutant Pink1 flies. Complete loss of Cisd rescues all detrimental effects of Pink1 loss of function on climbing ability, wing posture, dopamine levels, lifespan, and mitochondrial ultrastructure. In Prkn mutant flies, Cisd deficiency ameliorates climbing ability and wing posture impairments, but does not mitigate the reduction in lifespan. These findings strongly suggest that the buildup of iron-depleted CISD1 may contribute to the pathophysiology of both familial and sporadic PD.

We here identified the mitochondrial protein CISD1, specifically its iron-depleted form, as a potential downstream mediator of the pathophysiological effects arising from the loss of PINK1 function, and to a certain extent, Parkin loss of function.

Our investigation revealed that both dopaminergic neurons from familial PD patients with PINK1 mutations and Pink1 mutant flies exhibit increased levels of a slower-migrating form of CISD1/Cisd on immunoblots. The presence of this slow migrating form of CISD1/Cisd was age-dependent in flies and apparent in human dopaminergic neurons after 28 days of differentiation. We did not assess whether this increased even more in older cultures but such an age-dependent increase would be in line with the fact that PD usually becomes symptomatic at an older age. The slower-migrating form likely does not represent a ubiquitinated form, as treatment with the deubiquitinase USP2 had no effect on fly Cisd dimerization (Martinez et al., 2024). Additionally, it is improbable that the slower-migrating Cisd form is an unrelated protein, as under non-reducing conditions, fly Cisd shifted to the dimeric form (Martinez et al., 2024) while harsh reducing conditions shifted Cisd to the monomeric form (Figure 3E). Based on our experiments, we conclude that the slower-migrating form of CISD1/Cisd corresponds to a dimer caused by the formation of an intermolecular disulfide bond between two molecules of CISD1/Cisd. The dimeric form has been described before even in recombinant CISD1. Under non-reducing conditions, recombinant CISD1 ran as a monomeric and a dimeric band while under reducing conditions using beta-mercaptoethanol only the monomeric band was detected (Roberts et al., 2013). Using proteomic analysis, it was determined that the disulfide bond forms between cysteine C83 and either C72 or C74 of CISD1 (Roberts et al., 2013). Formation of the disulfide bond therefore results in loss of the Fe/S cluster because the residues involved in bond formation also coordinate the Fe/S cluster of CISD1. It is unclear what comes first, loss of the Fe/S cluster followed by disulfide bond formation or vice versa disulfide bond formation followed by loss of the Fe/S cluster. However, CISD1 C83S, a genetically engineered CISD1 incapable of binding the iron-sulfur cluster and probably also incapable of forming a disulfide bond (Roberts et al., 2013), has a higher propensity for dimer formation when analyzed in a cellular assay based on complementation of split luciferase (Figure 2A). This suggests that Fe/S cluster loss comes first and results in an altered structure that facilitates the formation of the disulfide bond. The iron-sulfur cluster of CISD1 is easily lost because it is coordinated in an unconventional manner by one histidine (H) and three cysteines (C) instead of the more common 2H/2C or 4C coordination (Wiley et al., 2007b). This serves to transfer the iron-sulfur cluster to other proteins as part of a cellular adaptive response that helps recovery from oxidative injury (reviewed in Mittler et al., 2019).

One protein that can accept the Fe/S cluster from holo-CISD1 is IRP1, as previously demonstrated (Ferecatu et al., 2014). Also in flies, Cisd physically interacts with holo-IRP1, and its deficiency renders flies more susceptible to iron depletion (Huynh et al., 2019), as also observed by us for Pink1 mutant flies. How apo-CISD1 is then reconstituted is rather unclear. A recent report suggested that CISD1 can receive its Fe/S cluster from its homolog CISD3 (aka MiNT) in the mitochondrial matrix via the porin VDAC1 (Karmi et al., 2022). So it appears possible that either this transfer is inhibited/attenuated or that the apo-CISD1/Cisd observed in PINK1 mutant dopaminergic neurons and Pink1 mutant flies constitutes a permanently fixed apo-CISD1/Cisd that cannot be reconstituted. This disruption could result in a defective recycling of cytosolic apo-IRP1 into holo-IRP1, leading on the one hand to a deficiency in cytosolic aconitase activity. Knockdown of CISD1 in cells indeed significantly reduces the cytosolic aconitase activity (Tan et al., 2016). On the other hand, apo-IRP1 not only lacks aconitase activity but also binds to IREs in the mRNA of iron-responsive genes (Haile et al., 1992a; Haile et al., 1992b). Association with IREs in the 5’ untranslated region of ferritin mRNA inhibits ferritin translation resulting in less bound iron and an increase in free iron (Chen et al., 1997) while association with IREs in the 3’ untranslated region of the mRNA encoding the iron importers transferrin receptor 1 and SLC11A2 stabilizes the mRNAs and facilitates their translation resulting in increased iron import (Galy et al., 2010). Together the transcriptional changes caused by the presence of apo-IRP1 result in iron overload which is an important factor in the pathology associated with PD (recently reviewed in Ma et al., 2021). Our study also revealed increased total iron levels in Pink1 mutant flies. Notably, KO of PINK1 in flies (Esposito et al., 2013), in mice (Gautier et al., 2008), and in human dopaminergic neurons (Bus et al., 2020) also results in a reduced aconitase activity in mitochondria. Mitochondrial aconitase contains an IRE in its 5’ untranslated region (Kim et al., 1996), making its expression subject to repression by apo-IRP1. The inactivation of mitochondrial aconitase caused by Pink1 loss of function triggers increased superoxide production, oxidative stress, and mitochondrial swelling (Esposito et al., 2013). Others reproduced these findings and reported that overexpression of mitoferrin, a mitochondrial iron transporter, mitigates the reduced mitochondrial aconitase activity, abnormal wing posture, flight deficits, and mitochondrial morphology defects associated with Pink1 loss of function by elevating mitochondrial bioavailable iron levels (Wan et al., 2020). Interestingly, KO of IRP2, a protein highly homologous to IRP1 but lacking aconitase activity, results in iron accumulation and Parkinsonism in mice (LaVaute et al., 2001). An interaction of CISD1 with IRP2 has not been reported so far but appears likely. In summary, these results strongly implicate altered mitochondrial iron homeostasis and redox dysregulation in fly and human models of PD and link it to CISD1 dysfunction. The accumulation of apo-CISD1/Cisd and the subsequent inability to repair defective IRP1/2 would place CISD1 upstream of the changes in iron homeostasis observed in PD and models of PD (Ma et al., 2021).

Our results also suggest that apo-CISD1 exhibits a toxic gain of function possibly independent of the effects on iron homeostasis. Ubiquitous overexpression of Cisd and apo-Cisd in flies aggravated known phenotypes of Pink1 mutation as a reduced climbing ability and lifespan. This was evident in WT and Pink1 mutant flies and overexpression of apo-Cisd was associated with a more profound reduction in climbing speed and resulted in an inability to completely inflate their wings. Interestingly, prolonged exposure of MEFs to the iron-depleting agent 2,2′-bipyridine results in downregulation of Cisd1 (Key et al., 2020) possibly as a compensatory mechanism to limit the abundance of such a toxic iron-depleted CISD1. In line with a toxic function of Cisd and apo-Cisd, removing fly Cisd by KO of the coding sequence completely protected flies against Pink1 depletion-induced changes in climbing ability, abnormal wing posture, dopamine levels, and lifespan but ameliorated only the climbing ability and wing posture in Prkn mutant flies. Previous work demonstrated that ectopic overexpression of Cisd in muscle decreased the lifespan of flies and resulted in enlarged mitochondria containing numerous intramitochondrial vesicles surrounded by a double membrane (Chen et al., 2020). Our data now indicate that this process might contribute to the pathophysiology resulting from the loss of Pink1/Prkn-mediated mitochondrial quality control.

Very recent work, published during the revision of this manuscript, discovered that both genetic and pharmacological suppression of CISD1 and Cisd restored increased calcium release from the ER conferred by PINK1 and Parkin mutation in mammalian cells and flies (Ham et al., 2023). However, the specific role of apo-Cisd in this remains to be fully elucidated. Also Martinez et al. very recently reported that Cisd depletion using RNAi protects against Pink1 and Prkn loss of function in flies (Martinez et al., 2024), suggesting that a reduction, instead of a complete KO, of Cisd as used by us is more beneficial in Park²⁵ flies and equally beneficial in Pink1^B9 flies. In their hands, Cisd levels increased in Pink1 and Prkn mutant flies which was not evident in our study. Cisd overexpression was neurotoxic, disrupted mitochondrial morphology, and led to an accumulation of phospho-ubiquitin which they speculate also blocks mitophagy, resulting in the accumulation of more defective mitochondria (Martinez et al., 2024). All three papers propose distinct mechanisms for the toxic effects of CISD1 in PD. While our study suggests a role in redox imbalance and iron dysregulation, particularly implicating the iron-depleted form of CISD1, Ham et al., 2023, describe its impact on ER calcium release, and Martinez et al., 2024, propose interference with autophagy/mitophagy. These mechanisms are not mutually exclusive, as they often intersect. Both studies support our findings, replicating key results, but also underscore the need for further research to fully elucidate the interplay of these proposed mechanisms.

In summary, our results suggest that the mitochondrial iron-sulfur cluster protein Cisd is downstream of the pathophysiological cascade initiated by Pink1 and partially Prkn loss of function in flies and probably also in humans. This sheds light on the pathophysiology of PD and implicates CISD1 as a therapeutic target protein.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Sara Bitar

   University Medical Center of the Johannes Gutenberg-University Mainz, Institute for Molecular Medicine, Mainz, Germany

   Contribution

   Formal analysis, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9369-0500

2. Timo Baumann

   University Medical Center of the Johannes Gutenberg-University Mainz, Institute for Molecular Medicine, Mainz, Germany

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5374-380X

3. Christopher Weber

   University Medical Center of the Johannes Gutenberg-University Mainz, Institute for Molecular Medicine, Mainz, Germany

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

4. Majd Abusaada

   University Medical Center of the Johannes Gutenberg-University Mainz, Institute for Molecular Medicine, Mainz, Germany

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Liliana Rojas-Charry

   University Medical Center of the Johannes Gutenberg-University Mainz, Institute for Molecular Medicine, Mainz, Germany

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Patrick Ziegler

   Institute for Occupational, Social and Environmental Medicine, RWTH Aachen University, Aachen, Germany

   Contribution

   Investigation

   Competing interests

   No competing interests declared

7. Thomas Schettgen

   Institute for Occupational, Social and Environmental Medicine, RWTH Aachen University, Aachen, Germany

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1256-1713

8. Isabella Eva Randerath

   Institute for Occupational, Social and Environmental Medicine, RWTH Aachen University, Aachen, Germany

   Contribution

   Investigation

   Competing interests

   No competing interests declared

9. Vivek Venkataramani

   Comprehensive Cancer Center Mainfranken, University Hospital Würzburg, Würzburg, Germany

   Contribution

   Supervision, Funding acquisition, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

10. Bernhard Michalke

    Research Unit Analytical BioGeoChemistry, Helmholtz Zentrum München-German, Research Center for Environmental Health GmbH, Neuherberg, Germany

    Contribution

    Investigation

    Competing interests

    No competing interests declared

11. Eva-Maria Hanschmann

    Experimental and Translational Research, Department of Otorhinolaryngology, University Hospital Essen, Essen, Germany

    Contribution

    Conceptualization, Resources, Formal analysis, Writing - review and editing

    Competing interests

    No competing interests declared

12. Giuseppe Arena

    University of Luxembourg, Luxembourg Centre for Systems Biomedicine, Esch-sur-Alzette, Luxembourg

    Contribution

    Funding acquisition, Investigation, Writing - review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-2398-5503

13. Rejko Krueger

    1. University of Luxembourg, Luxembourg Centre for Systems Biomedicine, Esch-sur-Alzette, Luxembourg
    2. Luxembourg Institute of Health (LIH), Strassen, Luxembourg
    3. Centre Hospitalier de Luxembourg (CHL), Luxembourg, Luxembourg

    Contribution

    Supervision, Project administration

    Competing interests

    No competing interests declared

14. Li Zhang

    University Medical Center of the Johannes Gutenberg-University Mainz, Institute for Molecular Medicine, Mainz, Germany

    Contribution

    Supervision, Writing - review and editing

    For correspondence

    lizhang2017deu@gmail.com

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-6339-6087

15. Axel Methner

    University Medical Center of the Johannes Gutenberg-University Mainz, Institute for Molecular Medicine, Mainz, Germany

    Contribution

    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

    For correspondence

    axel.methner@gmail.com

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-8774-0057

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