Iron toxicity, ferroptosis and microbiota in Parkinson’s disease: Implications for novel targets

Abstract

Parkinson’s Disease (PD) is a progressive neurodegenerative disease characterized by loss of dopaminergic neurons in substantia nigra pars compacta (SNpc). Iron (Fe)-dependent programmed cell death known as ferroptosis, plays a crucial role in the etiology and progression of PD. Since SNpc is particularly vulnerable to Fe toxicity, a central role for ferroptosis in the etiology and progression of PD is envisioned. Ferroptosis, characterized by reactive oxygen species (ROS)-dependent accumulation of lipid peroxides, is tightly regulated by a variety of intracellular metabolic processes. Moreover, the recently characterized bi-directional interactions between ferroptosis and the gut microbiota, not only provides another window into the mechanistic underpinnings of PD but could also suggest novel interventions in this devastating disease. Here, following a brief discussion of PD, we focus on how our expanding knowledge of Fe-induced ferroptosis and its interaction with the gut microbiota may contribute to the pathophysiology of PD and how this knowledge may be exploited to provide novel interventions in PD.

1. Introduction

Ferroptosis, due to iron toxicity, is a novel form of cell death characterized by reactive oxygen species (ROS)-dependent accumulation of lipid peroxides (Stockwell, 2022; Sun et al., 2023). It is characterized by mitochondrial dysfunction including shrinkage, reduction, disappearance of cristae, and increase in its membrane density (Javadov, 2022; Oh et al., 2022; Fu et al., 2023). It is tightly regulated by a variety of intracellular metabolic processes and has been implicated in a variety of diseases including age-related neurological diseases such as Parkinson’s disease (PD) (Lin et al., 2022; Xu et al., 2023; Xiao et al., 2024). Moreover, recent evidence establishes a bidirectional interaction between ferroptosis and microbiome, the latter considered as having significant influence on overall physiological homeostasis (Yao and Li, 2023; Liu et al., 2024). Here, following a brief discussion of PD including role of glia, we focus on how our expanding knowledge of iron (Fe)-induced ferroptosis and its interaction with gut microbiome may contribute to the pathophysiology of PD and how this knowledge may be exploited to provide novel interventions in this devastating disease.

2. Parkinson’s disease

PD is an age-related progressive neurodegenerative condition, associated with loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). It affects 1% of people older than 60 years and 4% of people older than 85 years. Although PD etiology remains unsettled, it is believed to be a multifactorial disease, with old age being the biggest risk factor. In the Western world, the mean age of onset is in the early-to-mid 60s, but in 3–5% of cases symptoms may start decades earlier, before the age of 40, and in some rare cases as early as age 21. In Japan, however, due to genetic susceptibility, percentages of early onset PD may reach as high as 10–14% (Post et al., 2020). Interestingly, clinical differences are also observed in symptom manifestation such as in dystonia and levodopa (L-DOPA)-induced dyskinesias (discussed below), which are more common in early onset PD (Post et al., 2020; Hua et al., 2022). In addition, exposure to environmental toxicants such as pesticides, herbicides, and heavy metals such as iron (discussed in detail below) also increase the risk of PD (Anderson et al., 2021; McKnight and Hack, 2020; Tizabi et al., 2023b).

The neurodegeneration in PD results in motor deficits characterized by akinesia, rigidity, resting tremor, and postural instability. Non-motor symptoms such as emotional changes, particularly depression, cognitive impairment, sleep perturbations (e.g., insomnia/hypersomnia), gastrointestinal (GI) (primarily constipation but also nausea and dysphagia or, difficulty in swallowing) and autonomic dysfunction such as bladder disturbances, orthostatic hypotension, sweating, sensory symptoms (e.g., pain, visual and olfactory deficits), are also common occurrences (Dinter et al., 2020). Additionally, the patients suffer from inability to produce facial expression or recognize other’s verbal and nonverbal cues (Prenger et al., 2020). The most common treatment for PD is focused on dopamine (DA) replacement (e.g., L-Dopa), which provides symptomatic relief and does not address the progression of neurodegeneration. Moreover, L-Dopa losses its efficacy over months or years and can induce severe dyskinesia (Tizabi et al., 2021a; Yan et al., 2023). Hence, more efficacious interventions without such adverse effects are urgently needed (Tizabi et al., 2021b).

To date, more than 20 pathogenic genes associated with PD, including mutations in genes such as leucine-rich repeat kinase 2 (LRRK2), Parkin RBR E3 ubiquitin protein ligase (PARK2), Parkinson disease protein 7 (PARK7), PTEN-induced putative kinase 1 (PINK1), and SNCA have been identified. Moreover, polymorphism in DA receptor D2 gene (DRD2Taq1A), as well as epigenetic changes, specifically DNA methylation, have also been implicated in PD pathology (Yazar et al., 2023). Alpha- synuclein (α-syn) protein, encoded by SNCA gene, has received the most attention because of its crucial role in PD pathology. Indeed, intraneuronal inclusions containing aggregates of misfolded α-syn are a key pathological hallmark of PD (Wood, 2022). Several cellular and molecular events including accumulation of misfolded proteins, failure of protein clearance, mitochondrial damage, oxidative stress, neuroinflammation, immune dysregulation, apoptosis, excitotoxicity, Ca²⁺ dysregulation as well as autophagy, and more recently ferroptosis (discussed below) have been implicated in neuronal degeneration in PD (Tizabi et al., 2021a; Talman and Safarpour, 2023; Li et al., 2023b; Ding et al., 2023; Yang et al., 2023).

Besides neurons, it is now evident that PD pathology also involves astroglia and microglia activity. Hence, we include a brief discussion of glial cells and their contribution to PD.

3. Glial cells

Different types of cells comprise glia, which are greater in number (between five and ten times more) than neurons in CNS. These include microglia, astrocytes, and oligodendrocytes. These cells exert a profound effect on neuronal development by providing trophic support essential for neuronal survival and are involved in neuronal migration, axon, and dendrite outgrowth, and in synaptogenesis (Jäkel and Dimou, 2017). Below, the role of microglia and astrocytes, the two major glial cells in PD and their specific relation to alpha-synuclein and later to ferroptosis are discussed in more detail.

3.1. Microglia—PD

Microglia, considered the innate immune cells of CNS, act as brain macrophages, and are mainly found in subventricular and subgranular zones, where under physiological conditions self-renew over an organism’s entire lifespan. Microglia are not uniformly distributed in the brain as a large number is present in the hippocampal dentate gyrus, parts of the basal ganglia, and SNpc. Interestingly, olfactory telencephalon in mice has the largest microglial population and curiously, loss of smell appears to precede motor symptoms in PD (Wei and Li, 2022; Raber et al., 2023; Yu et al., 2023). However, whether microglia dysfunction contributes to this sensory abnormality is not known.

SNpc contains the largest proportion of microglia (about 12%) compared to 5% in the cortex and corpus callosum. The regional heterogeneity is attributed to the residential environment, especially interactions with neurons or neural progenitor cells, as well as intrinsic mechanisms. Microglia also differ in size and ramification patterns within and between different brain regions. They are critical for regulating the neuronal network as they support the development, maintenance, homeostasis, and repair of the brain by wiping out cell debris and phagocytizing viruses and bacteria (Janda et al., 2018; Zhu et al., 2022).

There are several stages in microglia morphology and function. For instance, during the resting state, microglia are sensitive to environmental stimuli such as stress that can activate aberrant microglia functioning and lead to neurodegenerative and psychiatric disorders. Hence, pro-inflammatory microglia (M1-activated state) secrete proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and inducible nitric oxide synthase (iNOS), which typically lead to neuronal dysfunction following chronic activation. In contrast, neuroprotective microglia (M2 state) phagocytose cell debris and misfolded proteins, promote tissue repair and support neuron survival mediated by neurotrophic factors (Yu et al., 2020, 2023; Raber et al., 2023).

Microglia activation could lead to both neuronal damage and onset/progression of several neurodegenerative and neurodevelopmental disorders such as PD. In addition to pro-inflammatory cytokines, other bioactive substances released from overactivated microglia, including ROS and glutamate can also be playing a prominent role in microglia-dependent neuronal loss (Czapski and Strosznajder, 2021; Muzio et al., 2021). Interestingly, α-Syn-induced neuronal loss might also be mediated through microglial activation (Wood, 2022). Thus, mechanisms that activate the reparative and regenerative functions of microglia can be exploited to provide selective therapies in PD (Jurcau et al., 2023; Raber et al., 2023).

3.2. Astrocytes—PD

Like microglia, astrocytes are also involved in cleaning the extracellular environment, facilitate neuronal communication, and help in maintenance of homeostasis. They also participate in many vital CNS functions including cognitive behavior, and through production of antioxidant and anti-inflammatory proteins provide protection in CNS. However, in contrast to microglia, astrocytes are brain cells that mainly control metabolic and redox homeostasis. Due to their swift response to brain pathology in the initial stages of the disease, their activation and differentiation are implicated in the pathogenesis of multiple neurodegenerative diseases, including PD (Qian et al., 2023; Valles et al., 2023). Astrocytes play an important role in synaptic function, K⁺ buffering, neuronal metabolism, and blood brain barrier (BBB) maintenance, disruption of which could accelerate the disease progression. Reactive astrocytes, evident during PD progression, refer to astrocytes that undergo morphological, molecular, and functional remodeling in response to pathological stimuli. Indeed, it is now well established that astrocytes afford a protective role in PD by supplying neurotrophic factors such as nerve growth factor and brain-derived neurotrophic factor (BDNF), and that these protective effects are decreased in advanced PD (Miyazaki and Asanuma, 2020; Valles et al., 2023).

During PD progression, reactive astrogliosis occur. Structural alterations in PD astrocytes including swollen endfeet and soma shrinkage contribute to disruption in vascular integrity at capillary and arterioles levels. Astrocyte endfeet enwrap the entire vascular tree within the central nervous system, where they perform important functions in regulating the BBB, cerebral blood flow, nutrient uptake, and waste clearance (Díaz-Castro et al., 2023). Therefore, it has been suggested that manipulation of astrocytes and/or astrocytic biomarkers could be developed in diagnosis and/or treatment of PD (Kim et al., 2023). Also, like microglia, astrocytes are skewed into proinflammatory and oxidative profiles with increased secretions of vasoactive mediators inducing endothelial disruption and immune cell infiltration (Yue et al., 2022a,b). Since astrocytes are involved in brain metabolism such as glucose and lipid metabolism, neurovascular coupling, synapses, and synaptic plasticity, targeting them might be an option in retarding or stopping the neurodegenerative process (Yue et al., 2022a,b; Kim et al., 2023).

4. Iron (Fe)

Heavy metals in general, and iron (Fe) in particular, play crucial roles in various biological functions. Specifically, Fe is a critical component of a variety of enzymes or co-enzymes, including catalases and cytochromes that mediate cellular processes and drug metabolism. Because catalase neutralizes hydrogen peroxide (by converting it into oxygen and water), it provides protection against oxidative stress (Hansberg, 2022).

4.1. Fe deficiency

Fe is well recognized as an essential element in hemoglobin synthesis where its deficiency is reflected in wide-spread Fe-deficiency anemia leading to fatigue, headaches, heart problems, pregnancy complications, developmental delays in children, and increased susceptibility to infection. Moreover, Iron deficiency occurs in many chronic inflammatory conditions, including congestive cardiac failure, chronic kidney disease and inflammatory bowel disease (Kumar et al., 2022). Interestingly, heart failure may occur due to low bioavailability of Fe, irrespective of anemia (Augusto and Martens, 2023; Shamsi et al., 2023). Anemia is also a common condition in patients with Alzheimer’s disease (AD) and its comorbidities such as obesity, depression, and type 2 diabetes mellitus (Fehsel, 2023). These conditions are believed to be due to reduced glucose import and hence reduced neuronal energy (Fehsel, 2023). Low Fe levels in the brain are associated with cognitive impairment and curiously with restless legs syndrome as well (Earley et al., 2022; Baringer et al., 2023).

4.2. Fe overload

On the other hand, excessive exposure to Fe may have serious detrimental consequences due to severe neurotoxicity including precipitation of PD. The latter is because Fe selectively damages the dopaminergic neurons in the substantia nigra (Dexter et al., 1989; Riederer et al., 1989; Lu et al., 2023; Mateo et al., 2023). It is of relevance to note that parkinsonism may also be caused by one or more small strokes; in which case it is referred to as vascular parkinsonism, which is also characterized by motor syndrome that manifests as rigidity, tremors, and bradykinesia, like what is observed in PD. However, a major distinction between the two is that unlike PD, parkinsonism is not progressive (Shrimanker et al., 2022). Nonetheless, the search for slowing or stopping the progression of PD is an ongoing challenge as its etiology remains elusive.

Since excess accumulation of trace elements such as Fe have been suggested to play a role in PD etiology (Andrade et al., 2017; Vaccari et al., 2017), drugs that might inhibit or block the toxicities of this heavy metal may not only be of use in detoxification of this element, but might point to novel therapeutics for PD. Curiously, reduction of Fe content via clioquinol, an anti-infective or antiparasitic drug, was associated with a remarkable improvement of the motor and non-motor deficits in an MPTP-induced monkey model of PD (Shi et al., 2020). MPTP (1-methyl4-phenyl-1,2,3,6-tetrahydropyridine), an analog of the opioid analgesic meperidine gets metabolized into MPP+ (1-methyl-4-phenylpyridinium), which is a potent neurotoxin as it inhibits mitochondrial complex-I and selectively damages the dopaminergic cells in SNpc (Langston, 2017; Fujita et al., 2020). For this reason, MPTP models of PD are commonly used to investigate the mechanism of neurotoxicity and/or development of novel therapeutics (Schneider et al., 2021; Yao et al., 2023).

It was reported recently that toxicity to the neuroblastoma-derived dopaminergic SH-SY5Y cells induced by Fe could be prevented by pretreatment with nicotine (Getachew et al., 2019). Moreover, both nicotine and butyrate, a short chain fatty acid (SCFA) produced by the GM, provided protection against salsolinol (SALS), a selective dopaminergic toxin, in the same cell line (Copeland et al., 2005; Getachew et al., 2020). Butyrate, acting as an energy source for colonic epithelial cells, has anti-inflammatory, enteroendocrine and epigenetic effects that not only can influence colonic and systemic health, but can also affect the brain functions (Cantu-Jungles et al., 2019; Duan et al., 2023; Jayashankar et al., 2023). Indeed, a few studies indicate beneficial effects of butyrate in animal models of PD (Liu et al., 2017; Cavaleri and Bashar, 2018; Kovács et al., 2023; Tizabi et al., 2021a) as well as in PD itself (Karunaratne et al., 2020). Interestingly, nicotine and butyrate may act synergistically to protect against Fe toxicity in SH-SY5Y cells (Tizabi et al., 2023b).

4.3. Fe homeostasis

Thus, Fe homeostasis, particularly in the brain is crucial for neurological health as fluctuations in brain Fe levels may result in pathological conditions associated with a variety of neurological disorders. Fe entry into the brain is regulated via transferrin and H-ferritin. It is proposed that the movement of Fe through endothelial cells into the brain can be divided into three distinct processes: uptake, transcytosis, and release, each of which is under external and internal influences (Baringer et al., 2023). Moreover, intracellular Fe homeostasis is vital in maintaining inflammatory homeostasis (Lee and Hyun, 2023). Cellular Fe levels are under Fe regulatory mechanisms that include primarily hepcidin-ferroportin axis, divalent metal transporter 1 (DMT1)-transferrin, and ferritin-nuclear receptor coactivator 4 (NCOA4), iron-regulatory protein (IRP)/iron-responsive element (IRE) system and nuclear factor erythroid 2-related factor 2 (Nrf2) (Lee and Hyun, 2023).

Hepcidin, a protein encoded by HAMP gene, is considered the master regulator of intracellular Fe content. It negatively regulates ferroportin, a transmembrane protein that transports Fe form the inside to the outside of the cell. Ferroportin, also known as Fe-regulated transporter, is encoded by SLC40A1 gene, and is the only known Fe exporter. It allows the dietary Fe that is absorbed into the cells of small intestine to be transported out of these cells into the bloodstream. Ferroportin also mediates the efflux of recycled Fe from macrophages in the spleen and liver. It is tightly regulated by hepcidin, which binds ferroportin and limits its Fe-efflux activity, resulting in reduced Fe delivery to blood. Hepcidin also acts by internalization and degradation of the ferroportin receptor (Billesbølle et al., 2020). Thus, during conditions in which hepcidin level is abnormally high, such as inflammation, plasma Fe falls due to Fe trapping within macrophages and liver cells. Moreover, there is a decrease in Fe absorption from the gut, all of which lead to anemia. On the other hand, when hepcidin level is abnormally low (e.g., hemochromatosis), there is an Fe overload due to increased ferroportin-mediated Fe efflux from storage as well as increased gut Fe absorption (Galy et al., 2023; Miozzo et al., 2023). Nonetheless, the above-mentioned complex mechanisms operate to increase Fe availability during Fe deficiency and decrease Fe level during overload, to maintain homeostasis. However, excess Fe leads to ROS formation, which reacts with essential cellular components such as lipids, proteins, and DNA, causing cellular dysfunction and/or cell death (Kontoghiorghes, 2023; Lee and Hyun, 2023). Moreover, ROS may promote neuroinflammation by activating the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and inducing pro-inflammatory cytokines such as IL-6, TNF-α, and IL-1β (Kontoghiorghes, 2023; Lee and Hyun, 2023).

In short, excess Fe leads to oxidative stress through generation of free radicals that is mediated by Fenton reactions. Numerous studies confirm that brain diseases, especially stroke and neurodegenerative diseases such as PD are closely related to an imbalance in Fe homeostasis (Long et al., 2023).

4.4. Fe—ferroptosis

Ferroptosis, is a term coined in 2012 to replace “oxytosis,” which referred to a form of neuronal cell death that shared some similarities with ferroptosis and was triggered by the excitotoxin glutamate due to cysteine depletion. Ferroptosis, which is an Fe-dependent regulated cell death was originally identified by screening RSL (RAS-selective lethal) compounds. The key characteristic of ferroptosis is an extensive accumulation of lipid peroxides, making ferroptosis genetically, morphologically, and biochemically distinct from other types of cell death such as apoptosis, necrosis, and autophagy (Stockwell, 2022; Sun et al., 2023). However, oxytosis and ferroptosis share several key characteristics including their gene expression patterns, high activity of lipoxygenases, and high accumulation of ROS (Sun et al., 2023). Thus, oxidation of fatty acids in the membrane results in ROS generation that triggers ferroptosis, hence promoting cell death (Cerasuolo et al., 2023). Interestingly, numerous recent studies have shown an interaction between ferroptosis and other cell death processes. Thus, ferroptosis and necroptosis may have a synergistic effect on tissue damage during acute organ failure (Wu et al., 2023).

Distinct morphological characteristics of ferroptosis include shrunken mitochondria, increased membrane density, and significantly diminished or absent mitochondrial cristae (Javadov, 2022; Oh et al., 2022; Fu et al., 2023). Major biochemical characteristics include increased oxidative stress (lipid peroxidation) and depleted antioxidative defense. In mammalian cells, ferroptosis is regulated mainly by Fe homeostasis, lipid metabolism, and glutathione-dependent redox balance (Sun et al., 2023). It was revealed for example, that ferroptosis, activated by concomitant increase in ROS and decrease of mitochondrial membrane potential led to cell death in nerve growth factor (NGF)-induced PC12 cells. Furthermore, treatment of cells with MPP⁺, a cellular model of PD, resulted in mitochondrial shrinkage, decreased expression of glutathione peroxidase 4 (Gpx4) and ferritin heavy chain (FTH1), and increased divalent metal transporter (DMT1) and transferrin receptor 1 (TfR1) expression levels (Zeng et al., 2021). Interestingly cellular death in this paradigm could be inhibited by an Fe chelator (Zeng et al., 2021). It is of relevance to note that Gpx4 is considered as the master regulator and a key ferroptosis biomarker (Lin et al., 2022). Indeed, impaired GPX4 function has been implicated in not only neurodegeneration, but also in tumorigenesis, infertility, inflammation, immune disorders, and ischemia-reperfusion injury (Xie et al., 2023a,b).

In addition, oxidized polyunsaturated fatty acids-phospholipids (PUFA-PLs) are propagated in the presence of Fe²⁺, causing membrane damage and/or the generation of reactive lipid-derived electrophiles, which are a hallmark of ferroptosis. Notably, Fe²⁺ induced promotion of PUFA-PL hydroperoxides can be eliminated by their reduction to non-toxic lipid alcohols by GPx4, utilizing cysteine-derived GSH as a cofactor. Thus, reduction in GPx4-activity results in the generation of lipid hydroperoxides and subsequent reactive products of degradation such as Malondialdehyde (MDA) or 4-Hydroxynonenal (4-HNE). Hence, lipid peroxides, considered hallmark of ferroptosis, disrupt the thickness, permeability, and structure of membrane bilayers, resulting in significant cellular damage (Mortensen et al., 2023).

In sum, it may be concluded that ROS plays an important role in generation of ferroptosis, which in turn can further induce ROS through lipid peroxidation and therefore result in a vicious cycle of cellular damage.

5. Ferroptosis—diseases

The list of diseases where ferroptosis has been implicated in their etiology keeps expanding. To date, these include cancer, cardiovascular, renal, hepatic, pulmonary, osteoporosis, autoimmunity, rheumatologic, immunologic, hematologic (including sickle cell disease), and ophthalmologic disorders (Han et al., 2020; Menon et al., 2022; dos Santos et al., 2023; Patel et al., 2023; Shen et al., 2023; Shi et al., 2023; Wang et al., 2023a). Moreover, emerging role of ferroptosis in female reproductive disorders (Liu et al., 2023), neuropsychiatric diseases (Sousa et al., 2023; Wang et al., 2023a), mitochondrial diseases (Ahola and Langer, 2023). diabetic retinopathy (He et al., 2023), multi-organ complications in COVID-19 (Li et al., 2023a,b), stroke and ischemia reperfusion diseases (Tang and Kang, 2023) and neurodegenerative diseases (discussed below) have been documented.

6. Ferroptosis—neurodegenerative diseases

Neurodegenerative diseases such as Parkinson’s disease, multiple sclerosis, Huntington’s disease, Alzheimer’s disease, prion diseases such as Creutzfeldt-Jakob’s disease, and amyotrophic lateral sclerosis, are progressive and irreversible pathologies, characterized by neuron vulnerability, loss of structure or function of neurons leading to functional and clinical maladies including cognitive dysfunction and movement disorders (Cerasuolo et al., 2023).

Fe accumulation has been confirmed as a hallmark feature in many neurodegenerative diseases (Ndayisaba et al., 2019; Dusek et al., 2022). In these diseases, the brain Fe content is significantly increased in vulnerable regions, resulting in mitochondrial alterations and lack of antioxidant defenses. Fe interacts with glucose metabolism reciprocally. Overall, Fe and ferroptosis play significant roles, particularly in the context of cognitive declines. Indeed, Fe chelators and ferroptosis inhibitors have already shown promise in ameliorating cognitive decline and hence may provide novel targets in neurodegenerative diseases (Cerasuolo et al., 2023; Wang et al., 2023c). In this context, pharmacological inhibition of ferroptosis by bioactive small-molecule compounds referred to as ferroptosis inhibitors, could be effective for treatments of these diseases (Ryan et al., 2023; Wang et al., 2023d). Effectiveness of ferroptosis inhibitors is believed to be due to enhancement of antioxidative capacity, which can be achieved through augmentation of Gpx4 and the coenzyme Q10 (CoQ10) (Lin et al., 2022; Dar et al., 2023). Reduced function of Gpx4, common in degenerative disorders, is considered as a critical trigger of ferroptosis (Dar et al., 2023). In this regard, considerable effort has been devoted in developing in-vitro and in-vivo models to allow identification of novel targets for ferroptosis inhibition and eventual transition to clinical studies. These models include differentiated SH-SY5Y and PC12 cells for in-vitro, and rodents and invertebrate animals, such as Drosophila melanogaster, Caenorhabditis elegans, and zebrafish, for in-vivo investigations (Costa et al., 2023).

7. Ferroptosis—PD

Ample evidence confirms a key role for ferroptosis in pathophysiology of PD. A major reason for this contention is that Fe can be selectively sequestered in dopaminergic neurons of SNpc, leading to neuronal death in this critical area. As mentioned above and elaborated in more detail below, this toxicity is due to Fe-induced ferroptosis, the knowledge of which can be exploited for targeted therapy in PD.

Overall, molecular mechanisms in PD involve a complex interplay between genetic, environmental, and cellular factors that disrupt cellular homeostasis, and ultimately lead to degeneration of dopaminergic neurons in SNpc. Alpha-synuclein (α-syn), a protein intimately involved in PD pathology, can trigger ferroptosis and hence lead to generation of ROS and lipid peroxides within cellular membranes (Mahoney-Sánchez et al., 2021). Indeed, α-syn has been functionally connected to Fe and lipid metabolism, suggesting that dysregulated α-syn may also interact with other PD clinical traits associated with ferroptosis (Thapa et al., 2022). In addition, pathological changes closely linked with ferroptosis have been seen in the brain tissues of PD patients, including alterations in Fe metabolism, lipid peroxidation, and increased levels of ROS (Lin et al., 2022; Ding et al., 2023; Yang et al., 2023; Xiao et al., 2024).

Another pathway where ferroptosis may contribute to progression of PD pathology is through its damage to BBB. For this reason, it is suggested that by increasing endothelial GSH activity, damage to BBB may be prevented (Lin et al., 2022). GSH (L-γ-glutamyl-L-cysteinyl-glycine), is the most abundant nonprotein thiol in mammalian cells acting as a major reducing agent and antioxidant defense by maintaining a tight control of the redox status (Franco and Cidlowski, 2012).

The role of ferroptosis in the main cells of the CNS, including glial cells, and in the interaction of glia and neurons in relation to neurodegenerative diseases including PD has been recently reviewed (Li et al., 2022). In this regard, it is of importance to note that the there is a reciprocal interaction between glial cells and Fe metabolism and disturbance in each may aggravate the dysfunction of the other and hence lead to a vicious cycle in PD pathology. Hence, manipulation of these interactions may also offer a novel therapeutic intervention (Song et al., 2018; Li et al., 2022; Lin et al., 2022; Xu et al., 2023).

8. Gut microbiota (GM)

Microbiota refers to the collection of microorganisms that colonize the GI tract in what is termed the ‘gut microbiota=GM’. GM has an intricate and symbiotic relationship with the host organism, both of which have evolved over thousands of years (Bäckhed et al., 2005; Tizabi et al., 2023a). Although microbiota and humans have comparable number of cells, the genomic contents of microbiota, however, offers greater coding potential (Sender et al., 2016). Thus, GM is considered as a new metabolic ‘organ’ due to its immense impact on maintaining physiological homeostasis via influence on critical functions including host metabolism, nutrition, and immune function, where the latter also maintains a symbiotic relationship with GM (Quan et al., 2023; VanEvery et al., 2023).

8.1. GM—enteric nervous system

The enteric nervous system (ENS) that is within the wall of the GI tract plays a crucial role in controlling many of the functions of the digestive system. Although ENS receives input from the CNS and there is a bi-directional communication between the two, it is referred to as the “second brain” as it can operate independently. Nonetheless, ENS integrity is maintained by GM. Moreover, the enteric glia, located along nerve fibers in the gut mucosa, can influence the gut epithelium in maintaining the barrier integrity, ion transport and self-renewal capacity (Prochera and Rao, 2023). The enteric glial system also plays an important role in sustaining homeostasis. This it does by interacting with the endocrine and immune cells within the intestinal wall (Seguella et al., 2023). Overall, it is now well recognized that GM not only maintains ENS survival, but also influences diverse CNS functions including mood regulation and cognition. Thus, it has been proposed that GM manipulation may offer novel treatment for neuropathies of ENS (Vicentini, et al., 2021) as well as other conditions (Doroszkiewicz et al., 2021; Escobar et al., 2022; Queiroz et al., 2022; Liang et al., 2022). Moreover, GM exploitation in neurodegenerative diseases in general, and PD in particular is a subject of intense interest that is elaborated upon below.

8.2. GM—neurotransmitters—short chain fatty acids (SCFAs)

GM, by encoding genes for specific enzymes that catalyze the conversion of some substrates into neurotransmitters can indirectly influence the functioning of CNS. A variety of neurotransmitters such as serotonin, dopamine, noradrenaline, acetylcholine, GABA, glutamate that the brain uses to regulate variety of functions are also produced by GM (Chen et al., 2021). Indeed, many bacteria can produce large quantities of mammalian neurotransmitters. For example, some strains of Escherichia coli (E. coli K12) produce dopamine, noradrenaline, and serotonin whereas Bacillus mycoides and Bacillus subtilis produce mainly dopamine and noradrenaline. This modulatory effect of the GM on CNS has been exploited for the development of specific probiotics that can improve several CNS disorders including PD (Parra et al., 2023; Xie et al., 2023a,b; Liu et al., 2024).

GM can also affect the function of the brain via SCFAs, metabolites that are produced by the microbiota in the large intestine through the anaerobic fermentation of indigestible polysaccharides such as dietary fiber and resistant starch (Silva et al., 2020; Zhang et al., 2023a,b). SCFAs have been associated not only with CNS development and maturation but also many other physiological processes including maintenance of the integrity of the blood brain barrier (BBB) (Tang et al., 2020; Parker et al., 2020; Doroszkiewicz et al., 2021), and GI and immune functions (van de Wouw et al., 2017; Zhang et al., 2023a,b). Indeed, decreased levels of SCFAs have been associated with degenerative diseases such as PD (Unger et al., 2016).

8.3. GM—microglia—PD

As alluded to earlier, it is now well-recognized that microglia, considered the immune cells of the brain, play a critical role in the inflammatory response, and that alterations in GM composition can exacerbate central inflammatory response via a variety of mechanisms, including the vagus nerve and circulating cytokines (Mayer et al., 2014; Sharon et al., 2016). Interestingly, in an animal model of PD, where ageing mice were given oral rotenone, there was an alteration in GM along with microglial activation and iron deposition, selectively in SNpc, prior to loss of motor coordination and dopaminergic neurons (Ma et al., 2023). The same effects could be observed with injection of, C-X-C motif chemokine ligand-1 (CXCL1), possibly via PPARγ signaling inhibition. Moreover, the effects of rotenone could be fully blocked by elimination of microglia with a formulated diet. The authors concluded that altered gut microbiota induced by rotenone, resulted in neuroinflammation and iron deposition (ferroptosis) in SNpc of ageing mice, prior to motor symptom manifestation (Ma et al., 2023). Thus, prevention of dysbiosis might be a viable novel intervention in PD.

9. GM—ferroptosis—PD

An intimate association between GM and ferroptosis has recently been suggested. This is because: first, gut microbes influence the level of hepcidin, a primary regulator of Fe homeostasis, second, disturbed GM can induce ferroptosis within the GI, and lastly, supplementation of probiotics can prevent ferroptosis (Yao and Li, 2023). However, the precise microbes and/or small molecules that modulate ferroptosis and the temporal window during which they exert their effects remain elusive. Thus, extensive research to identify the microbial genera and genes associated with ferroptosis is being carried out. Nonetheless, since involvement of GM and ferroptosis in pathophysiology of PD is well-established, interaction between GM and ferroptosis suggests that therapeutic targeting of either one is likely to be involving the other mechanism as well. Interestingly, it was recently revealed that a bacterial metabolite from streptomyces venezuelae known as aerugine (C₁₀H₁₁NO₂S; 2-[4-(hydroxymethyl)-4,5-dihydro-1,3-thiazol-2-yl]phenol), triggered selective dopaminergic neurotoxicity in human cell lines as well as in C-elegans model. Moreover, this neurotoxicity was completely prevented by Fe chelators (Ückert et al., 2023).

In addition, administration of Lactococcus lactis (MG1363-pMG36e-GLP-1), a probiotic strain, in a PD animal model reduced 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP)-induced motor disorders, α-synuclein aggregation, as well as histopathological alterations in these mice (Yue et al., 2022a,b). These effects were associated with normalization of dysbiosis as well as with an increase in glucagon-like peptide 1 (GLP-1), a bioactive protein synthesized by enteroendocrine cells. GLP-1 not only possesses neurotrophic effects, but also improves the integrity of the BBB, thus delaying dopaminergic neuronal death (Yue et al., 2022a,b). Importantly, Lactococcus lactis also suppresses ferroptosis via activation of Keap1-Nrf2-GPX4 pathway, a primary antioxidant and defense mechanism against oxidative stress (Yue et al., 2022a,b). Interestingly, Fe absorption and release were also reduced by Lactococcus lactis due to suppression of DMT1 and restoration of TfR1 (Yue et al., 2022a,b).

In summary, targeting GM and/or ferroptosis by probiotics or other compounds may offer advantageous intervention in PD (Wang et al., 2023b).

10. Ferroptosis as drug target

10.1. Inhibition of ferroptosis

Preclinical research has demonstrated neuroprotective qualities of certain Fe chelators and antioxidants (Thapa et al., 2022; Mahoney-Sánchez et al., 2021; Yang et al., 2023). Thus, significant improvement in MPTP model of PD was obtained by Fe chelators (Ding et al., 2023). Moreover, in the same mouse model, morroniside, a natural glycoside product found in Lonicera japonica, was shown to improve motor deficits and boost dopamine synthesis by activating the Nrf2/ARE pathway, hence, enhancing antioxidant capacity, and curbing Fe accumulation (Li et al., 2023b). Similarly, α-lipoic acid (ALA) enhances antioxidant defenses, regulates iron metabolism, and suppresses Fe-dependent ferroptosis, ameliorating PD symptoms (Zheng et al., 2023).

Ferroptosis antagonists have been shown to help alleviate ferroptosis-related diseases such as ischemia/reperfusion (I/R)-induced damage, inflammatory diseases, as well as neurodegenerative diseases including PD (Ko et al., 2021; Sun et al., 2023). Ferroptosis inhibitors via GPX4 axis and the coenzyme Q10 (CoQ10)/FSP1 pathway enhance antioxidative capacity and hence counter the oxidative stress from lipid peroxidation (Lin et al., 2022). CoQ10, also known as ubiquinone, is an important electron carrier in the mitochondrial electron transfer chain (ETC) and acts as a lipophilic free-radical-scavenging antioxidant in the cytosol and intracellular membranes. The mitochondrial ETC utilizes a series of electron transfer reactions to generate cellular ATP through oxidative phosphorylation, a consequence of which is the generation of ROS (Nolfi-Donegan et al., 2020). Interestingly, CoQ10 requires assistance from other oxidoreductases to resume its reduced state to stop the propagation of lipid peroxides. One such oxidoreductase is ferroptosis suppressor protein 1 (FSP1), which complemented CoQ10 in suppressing ferroptosis that was induced by genetic deletion of GPX4 (Lin et al., 2022).

As evident, ferroptosis may also be hindered by maintaining Fe homeostasis. Hence targeted therapies for Fe accumulation can significantly affect pathophysiological pathways of relevance to PD. As mentioned above, clioquinol (CQ) by regulating Fe homeostasis and inhibiting ferroptosis as well as its interaction with AKT/mTOR pathway improves motor and non-motor deficits in MPTP-induced monkey model of PD (Shi et al., 2020; Ding et al., 2023). In this regard, it would be of interest to investigate whether ferroptosis interaction with this molecular pathway may also be involved in PD pathology. Curiously, such an interaction was recently reported in relation to ischemia–reperfusion induced inflammation (Zhang et al., 2023b).

Several other mechanisms to inhibit ferroptosis, including Fe chelation, reducing lipid ROS production, and blocking lipid peroxidation have been suggested (Lin et al., 2022). In addition, inhibition of ferroptosis by Ferrostatin-1 (fer-1), an aryl alkyl amine with scavenging properties directed to the Fe²⁺-induced fatty acid derivate alkoxyl radicals, was shown to be much more efficient than phenolic antioxidants (Miotto et al., 2020).

Targeting of ferroptosis in astrocytes and microglia is another option. Hence, treatment with N-acetylcysteine (NAC), a precursor of GSH, natural plant products, Cu (II)ATSM, a Cu²⁺ complex with radical-trapping activity, curcumin, melatonin, selenium, CoQ10, vitamins D, E, and K, flavonoids including quercetin have been proposed (Bellavite, 2023; Jiang et al., 2023; Landis, 2024). Curiously, quercetin may have a dual role in that it may deactivate ferroptosis in neurodegenerative diseases but might have the opposite and desirable effect of inducing ferroptosis in diseases such as cancer (Cruz-Gregorio and Aranda-Rivera. 2023). Recently, two other flavonoids, ginkgetin and biochanin A, have been suggested for PD treatment due to their multifunctional effects (García-Beltrán et al., 2023). Since both nicotine and butyrate have been indicated as novel therapies in metal toxicities and PD (Tizabi et al., 2021a, 2023b), it would be of interest to determine whether any of these effects are also mediated via ferroptosis inhibition.

Regarding Fe chelator intervention, more studies are needed as two studies published last year did not find benefit or Fe chelation in early PD (Devos et al., 2022; Galasko and Simuni, 2022). It was suggested that deferiprone, the Fe-chelator used, could also inhibit tyrosine hydroxylase (TH), a rate-limiting enzyme in DA synthesis, which uses Fe as a cofactor, thereby reducing DA availability (Devos et al., 2022). More recently, it was suggested that in addition to this hypothesis, disruption of Fe homeostasis in mitochondria could be a reason for ineffectiveness of deferiprone in early PD (Levi and Volonté, 2023). However, it should be noted that a combination of deferiprone and L-Dopa had shown effectiveness in PD (Martin-Bastida et al., 2017).

10.2. Induction of ferroptosis

In a few cases, rather than inhibition of ferroptosis such as in neurodegenerative conditions discussed above, induction of ferroptosis to induce selective cell toxicity and damage may be desirable. Thus, in variety of cancers including blood-related cancers, multiple solid tumors such as lung, breast, pancreatic, liver, colorectal, esophageal squamous cell carcinoma, and gastric cancers as well as in melanoma, induction of ferroptosis has been advocated (Chen et al., 2023). The postulated mechanisms include increasing lipid peroxidation, ${Xc}^{-}$ blockade, reducing GSH components, or directly inhibiting the Gpx4 enzyme (Yu et al., 2022; Cruz-Gregorio and Aranda-Rivera. 2023; Landis, 2024).

11. GM-ferroptosis as targets for PD

As alluded to earlier, use of probiotics may offer a novel intervention in PD. This is significant as it adds a new dimension in PD therapeutics. Moreover, combination of drugs that can affect different targets simultaneously (e.g., microbiome and ferroptosis) can be investigated in eventual treatment of not only the symptoms but also the underlying cause of PD. Greater insights into mechanisms that govern the reciprocal drug–gut microbiota interactions will facilitate the design of microbiome‐targeted dietary or pharmacological interventions, and combined with specific targeting of ferroptotic events, can enhance novel drug development with increased efficacy and reduced side effects in treatment of variety of diseases including PD (Fig. 1).

Fig. 1.

Schematic diagram showing how iron toxicity, ferroptosis and gut microbiota (GM) may contribute to Parkinson’s Disease (PD) pathology and provide targets for intervention. Environmental factors such as chemicals and medications may also lead to iron dysregulation which can result in ferroptosis. Both ferroptosis and dysbiosis of GM may directly or indirectly through neuroglial dysregulation lead to neuroinflammation and neurodegeneration and ultimately PD.

12. Conclusion

Fe-dependent programmed cell death known as ferroptosis, plays a crucial role in incidence and progression of neurodegenerative diseases including PD. Ferroptosis, characterized by ROS-dependent accumulation of lipid peroxides, is associated with mitochondrial dysfunction (e.g., shrinkage, disappearance of mitochondrial cristae, etc.), and is tightly regulated by a variety of intracellular metabolic processes. Since SN_pc is particularly vulnerable to Fe toxicity, a central role for ferroptosis in etiology and progression of PD is envisioned. Similarly, a disruption in GM, commonly referred to as dysbiosis, can lead to neuronal degeneration in CNS. This is likely mediated via activation of microglia and the ensuing neuroinflammation. Furthermore, the intimate relationship between GM and ferroptosis can now be fully exploited to develop novel therapeutics in a plethora of diseases including PD.

Abbreviations

α-syn

alpha, synuclein

4-HNE

4-Hydroxynonenal

AD

Alzheimer’s disease

ALA

α-lipoic acid

BBB

blood brain barrier

BDNF

brain-derived neurotrophic factor

CNS

central nervous system

CoQ10

coenzyme Q10

CQ

Clioquinol

DA

dopamine

DMT1

divalent metal transporter 1

ENS

enteric nervous system

ETC

electron transfer chain

Fe

Iron

Fer-1

ferrostatin-1

FSP1

ferroptosis suppressor protein 1

FTH1

ferritin heavy chain

GI

gastrointestinal

GLP-1

glucagon-like peptide 1

GM

gut microbiota

Gpx4

glutathione peroxidase 4

GSH

glutathione

IL

interleukin

iNOS

nitric oxide synthase

IRE

iron-responsive element

IRP

iron-regulatory protein

LRRK2

leucine-rich repeat kinase 2

MDA

malondialdehyde

NAC

N-acetylcysteine

NCOA4

ferritin-nuclear receptor coactivator 4

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

NGF

nerve growth factor

Nrf2

nuclear factor erythroid 2-related factor 2

PARK2

parkin RBR E3 ubiquitin protein ligase

PARK7

Parkinson’s disease protein 7

PD

Parkinson’s Disease

PINK1

PTEN-induced putative kinase 1

PPAR

peroxisome proliferator-activated receptor

PUFA-PLs

polyunsaturated fatty acids-phospholipids

ROS

reactive oxygen species

SALS

salsolinol

SCFA

short-chain fatty acid

SNpc

substantia nigra pars compacta

TfR1

transferrin receptor

TH

tyrosine hydroxylase

TNF-α

tumor necrosis factor-α