Background: Ferritin is a protein that is critical for storing iron. Ferritin has recently been shown to play a role in iron homeostasis, immunomodulation, inflammation, and antioxidation. Previously, it was believed that ferritin was exclusively an intracellular peptide. However, there is significant evidence that ferritin is also in the serum, cerebral spinal fluid, and synovial fluid. Summary: Within the brain, ferritin can bind to oligodendrocytes adjacent to the blood-brain barrier to allow a docking point for ferritin to be engulfed by microglia in the brain parenchyma. When iron supplies in the brain are low, the lysosomal-autophagy pathway is activated to degrade ferritin and mobilize iron. Iron is critical in the brain for the formation of myelin and used during cellular respiration. If this sequestration and degradation of iron are impaired, the oxidative effects of iron may leave the brain vulnerable to neurotoxic effects. Subarachnoid hemorrhage (SAH) causes hemolysis of erythrocytes leading to the release of iron. Subsequently, a rise in ferritin is observed which promotes the neurologic insult following SAH. The degree to which ferritin is elevated post-SAH may correlate with the downstream neurotoxicity. Key Messages: The literature seems to point to a critical balance in ferritin levels. Ferritin is protective against further oxidative effects of iron, but ferritin also contributes to neurotoxic outcomes. In this review, we will discuss the role of ferritin in the brain. Specifically, we will address cerebral ferritin iron uptake and ferritin clearance. This homeostatic process influences the development and progression of SAH.

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Introduction

Ferritin is a ubiquitous protein most recognized for its role in iron storage [1]. It was first measured in a clinical assay in the 1970s and has since been utilized as a primary measure of physiologic iron stores [2, 3]. In recent years, research has demonstrated its expanded physiologic roles, including systemic iron homeostasis, immunomodulation, inflammation, and antioxidation [2-5].

Apoferritin is the protein shell of the ferritin molecule and is composed of 24 subunits [6]. Specifically, it is composed of both H- and L-subunits whose ratios vary based on tissue subtype [6]. The H-ferritin subunit contains a ferroxidase which converts iron to its inactive ferric (Fe3+) form. This conversion allows iron to be sequestered in the hollow protein core. L-Ferritin serves as a site of nucleation enabling iron core formation (Fig. 1) [6]. Within the hollow center, apoferritin can store up to 4,500 iron atoms and when bound to iron, forms the molecule ferritin [6].

Fig. 1.

Ferritin is composed of an apoferritin protein shell which consists of 24 subunits of H- and L-ferritin. The ratio of these subunits depends on the tissue the ferritin molecule is found. H-Ferritin contains ferroxidase which converts ferrous (Fe2+ form) into ferric (Fe3+ form). The apoferritin protein shell contains a hollow center capable of holding 4,500 iron atoms, and when iron is present inside the core, the molecule is known as ferritin. Created with BioRender.com.

Despite its well-known presence intracellularly, ferritin is also found extracellularly within the serum, cerebrospinal fluid (CSF), and synovial fluid [7]. Its extracellular presence implicates a role in systemic iron regulation and iron delivery [2, 4]. Although transferrin is traditionally considered the primary protein mediating iron delivery, increasing evidence demonstrates that alternative mechanisms must exist [8-11]. For example, hypotransferremic mice continue to demonstrate adequate levels of iron uptake in multiple organs [8].

In this review, we will discuss the surge in ferritin that occurs following subarachnoid hemorrhage (SAH) and the downstream neurotoxic effects. We will also discuss preclinical and clinical data that demonstrate therapeutic efficacy of ferritin clearance applying lessons learned from hereditary ferritinopathies. Altogether, this review serves to inform on the pathologic effects of ferritin in the setting of SAH and identify mechanisms through which ferritin can be targeted to improve outcomes in these patients.

The Role of Ferritin in the Brain

Experimental studies continue to implicate a role for ferritin in iron delivery to the brain. Both in vitro and in vivo studies have demonstrated uptake of ferritin into the brain, as well as transport across the blood-brain barrier, in a process that appears to be receptor-mediated and favor H-ferritin. In cell culture models of the blood-brain barrier, H-ferritin has been shown to bind to endothelial cells and subsequently undergo clathrin-mediated endocytosis [8]. In vivo, ferritin can bind the vessels of the rat brain with subsequent evidence of uptake into brain parenchyma [8]. Although the exact receptor to which ferritin binds continues to be investigated, studies have identified a ferritin receptor, T-cell immunoglobulin mucin domain-2, on the surface of oligodendrocytes [12, 13]. Oligodendrocytes are glial cells of the white matter and contain high concentrations of iron despite being deficient in transferrin receptors [10]. However, T-cell immunoglobulin mucin domain-2 is not found on human oligodendrocytes, and the analogous receptor on human oligodendrocytes TIM-1 has demonstrated ferritin binding [14]. Altogether, such evidence demonstrates that ferritin can bind at the blood-brain barrier and glial cell surface to be trafficked into the brain parenchyma and intracellular environment of the central nervous system.

Once inside the cell, ferritin functions as a mediator of iron storage and iron homeostasis [12]. In order for iron to be released from ferritin for cell utilization, it has been proposed that the degradation of ferritin is required [8, 15]. In particular, evidence suggests that the lysosomal-autophagy pathway is critical to ferritin degradation and iron release [12]. When iron is low, the lysosomal-autophagy pathway is activated and nuclear receptor coactivator-4 is mobilized to bind H-ferritin. Ferritin is then targeted to the lysosome where autophagy-mediated degradation occurs and iron is released into the cytoplasm (Fig. 2) [12]. Alternatively, ferritin that is iron-poor may be degraded via a proteosome pathway [12, 16]. Studies suggest that microglia may utilize this pathway to clear ferritin during times of oxidative stress (Fig. 2) [17]. Within the brain, ferritin can also be secreted by astrocytes and microglia via proposed mechanisms such as a nonclassical lysosomal pathway or exosomal-mediated secretion [4, 12]. However, such mechanisms have primarily been studied in peripheral cells and further investigation is needed to clarify secretory mechanisms within the brain.

Fig. 2.

The release of iron is critical for homeostasis. When iron is low in the cell, the lysosome-autophagy pathway is activated to release iron from iron-rich ferritin. Nuclear receptor coactivator-4 (NRCOA4) binds to H-ferritin. The action of NRCOA4 targets ferritin to the lysosome. In the lysosome, autophagy-mediated degradation of ferritin occurs where iron is freed from ferritin and released into the cytoplasm of the cell. Ferritin that is iron-poor can be degraded via the proteosome pathway. The purpose of this pathway is to clear excess ferritin during oxidative stress. Created with BioRender.com.

Within the brain, ferritin may assume additional physiologic roles apart from iron storage and homeostasis, such as glial cell differentiation and antioxidation [12, 18, 19]. Iron itself is the most abundant mineral in the brain and is an essential enzymatic cofactor in numerous biological reactions [20, 21]. Specifically, it is essential for myelin formation and cellular respiration and can alter neurodevelopment when deficient [22-26]. However, iron is also a key cofactor in the nonenzymatic production of reactive oxidative species (ROS) via the Fenton reaction and can be highly toxic if not properly regulated [27]. Thus, ferritin is proposed to serve as an important antioxidant, protecting tissues from iron’s deleterious oxidative properties within the brain and systemically [28, 29].

In recent years, research has continued to reveal an association between elevated ferritin and various neuropathologies, such as neurovascular, neurodegenerative, and neuroautoimmune diseases [12, 30-32]. In such diseases, ferritin may become overwhelmed and no longer able to maintain proper homeostasis within the brain [27]. As a consequence, iron may be improperly sequestered, leaving the brain vulnerable to its neurotoxic affects.

Following SAH, there is a rise in CSF ferritin and iron that appear to be related to neurotoxic cascades following subarachnoid bleeds [31, 33, 34]. Of interest, there is evidence to demonstrate that elevations in ferritin correlate with severity of subarachnoid bleeds [31]. Such elevations could be reflective of dysregulated iron homeostasis and downstream oxidative events. Specifically, in the setting of SAH, oxidative stress can lead to both direct neurotoxicity and vasospasm. In the context of neural toxicity, ROS production from the Fenton reaction can cause DNA damage and trigger downstream apoptotic signaling pathways, while chronic intracellular ferritin elevations may lead to neural cell death [35, 36]. In the setting of vasospasm, this ROS production may also cause vasoconstriction leading to vasospasm and delayed cerebral ischemia [34, 37, 38]. Vasospasm is relatively common sequelae of SAH and can increase the risk of delayed cerebral ischemia and associated morbidity [39, 40]. Interestingly, in this context, ferritin has been postulated to serve a protective role, sequestering redox-active iron, thus preventing vasospasm and subsequent delayed cerebral ischemia [34, 41]. Ultimately, ferritin likely plays a dynamic role in the setting of SAH with both protective and neurotoxic properties – and its fluid role must be carefully addressed when considering it as a therapeutic target.

Iron Uptake in the Brain

While iron functions to support important processes in the brain, excess neuronal iron has been shown to cause oxidative stress linked to multiple neurological disorders. To prevent this, microglia and macrophages scavenge iron-containing debris and dying inflammatory cells which results in iron uptake [42, 43]. Iron uptake by microglia can occur through two different pathways. One pathway is the M1 polarization (classical) pathway. In the M1 polarization pathway, the expression of divalent metal transporter-1 is upregulated in response to pro-inflammatory molecules. The M1 polarization pathway also increases the uptake of nontransferrin-bound iron [44]. Activation of toll-like receptor and the interferon-gamma signaling pathway activates the M1 polarization pathway. As a result, there is secretion of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin 1-beta, interleukin-12, and C-C motif chemokine ligand 2 [45]. The second pathway is known as the M2 (alternative) pathway and can occur when an anti-inflammatory stimulus increases transferrin receptor levels to upregulate transferrin-bound iron uptake through receptor-mediated endocytosis [46, 47]. Anti-inflammatory cytokines in this pathway include interleukin-10 and transforming growth factor-beta [48].

CSF ferritin has been associated with a multitude of conditions, including SAH and cerebrovascular events like infarction and infection. Interestingly, the accumulation of ferritin in various cells and tissues in the body has been observed with aging. Mitochondrial dysfunction and weakened antioxidant defenses that advance with aging can lead to excessive iron release, and abnormal iron deposition may be an indirect indicator of progression and severity of neurodegenerative diseases like Alzheimer’s disease and Parkinson’s disease [49-52]. Ferritin is the main extracellular transporter of iron, which is one of the major contributors to vasospasm and toxicity following SAH [37]. In SAH, erythrocytes hemolyze, leading to a degradation of hemoglobin and subsequent release of iron. This suggests that the major source of CSF ferritin is from damaged cells [31].

Both ferrous (Fe2+) and Fe3+ iron can generate hydroxyl radicals, very destructive ROS, through the Fenton reaction and Haber-Weiss cycle. ROS production by free iron may disrupt the blood-brain barrier, leading to increased edema and intracranial pressure. Additionally, ROS are mediators in the mitochondria, DNA repair enzymes, and transcription factors, which may lead to cell injury and necrosis [53]. Immune cells play an important role in regulation and distribution of iron in the CNS, since they engulf and recycle dying cells. Iron deposition by macrophages can lead to neuronal damage due to oxidative stress, free radical formation, and the promotion of pro-inflammatory mediators [54]. Macrophages that accumulate iron show an increase in TNF-α, which could contribute to the neuroinflammation observed in SAH [55, 56]. Indeed, inflammatory cytokines like TNF-α and IL-6 induce the expression of iron transporter receptors and promote the accumulation of iron in neurons and microglia (Fig. 3). A positive feedback cycle between inflammation, iron accumulation, and mitochondrial dysfunction has recently been proposed [57], suggesting that there is a synergistic effect between all these factors.

Fig. 3.

Fe2+ and Fe3+ are both capable of oxidative effects leading to reactive oxygen species (ROS) such as hydroxyl radicals (OH−). These ROS are created through the Fenton reaction and the Haber-Weiss cycle. The ROS that are created damage the blood-brain barrier (BBB) leading to increased permeability to fluid and solutes. Increased BBB permeability leads to intracranial edema and increased intracranial pressures (ICP). Additionally, macrophages are capable of phagocytosing iron. Macrophages that internalize iron have been shown to release inflammatory cytokines like TNF-α and IL-6. This demonstrates the importance of ferritin in eliminating excess iron within the brain. Created with BioRender.com.

Targeting Ferritin Clearance

As ferritin accumulates, the ferritin inclusion bodies materially and energetically abuse cell machinery by hindering their maintenance capacities, transport, and spatially dependent functions [58]. Ultimately, ROS damage proteins and lipids [58]. Observations in hereditary ferritinopathy (HF) studies are useful in observing the impacts of excessive accumulation of ferritin and loss of ferritin clearance. HF is a neurodegenerative disease caused by a rare autosomal dominant mutation in the ferritin light chain defined by the formation of inclusion bodies in glia and neurons, in which the ferritin content within cells is increased between 320 and 470% [58, 59]. In 2016, Garringer et al. [60] used mouse models to understand the systemic impact of iron overload and chelation therapy in HF. As expected, they found reduced cell viability when the HF model mice were exposed to excess iron, and a recovery of viability with treatment of iron chelator deferiprone. Interestingly, their data suggest the iron overload in the HF mode mice was greater in the systemic compartment when compared to the CNS, likely due to the relative independence of the brain [60].

In human studies, elevated iron-containing compounds have been associated with poor neurological outcomes in various conditions. Hemopexin is an abundant plasma protein that binds free heme, which is then rapidly scavenged by CD91. The CD91-hemopexin system is positively correlated with iron deposition in brain tissue, and it has been associated with a higher probability of delayed cerebral ischemia and poorer neurological outcomes after SAH [61]. Preclinical studies have shown a vasoconstrictive effect from iron-containing hemin, and Gomes et al. [34] performed a pilot study showing that levels of nonprotein-bound iron were significantly higher in patients who developed vasospasm after aneurysmal SAH [62]. A recent study by Rajendran et al. [30] used serum ferritin as a prognostic marker in acute hemorrhagic stroke and found that elevated ferritin on admission indicated poor short-term and long-term outcomes using a modified Rankin scale, an extensive stroke scoring system.

Therefore, CNS pathology is likely not due to iron overload, and the ferritin aggregates likely independently lead to significant disease manifestations. This discordance is illustrated throughout the literature, as serum ferritin is not necessarily strictly a measure of iron, as ferritin levels fluctuate in the presence of trauma or inflammation [63]. While deferiprone is an excellent chelator in removing and redistributing iron to prevent ferritin aggregation, it lacks in penetrating the blood-brain barrier [60].

Qin et al. [64] used rat models of SAH to assess the use of deferoxamine (DFX) in reducing the degree of brain injury. DFX treatment significantly reduced ferritin levels and SAH-induced mortality in animal models [33, 65]. This finding suggests the possibility of reducing ferritin levels clinically. Suzuki et al. [41] used ferritin levels in patients with SAH to predict subsequent chronic hydrocephalus. This study showed that elevated CSF ferritin of 300 ng/mL on day 3 to day 4 exhibited a positive predictive value of 100% in consequential chronic hydrocephalus. Intracranial hemoglobin, heme metabolites, bilirubin, and iron measures did not exhibit an association with chronic hydrocephalus, suggesting ferritin is intracranially metabolized versus a measure of blood in the subarachnoid space, and serves as a key protein in SAH morbidity when elevated [41]. Chronic hydrocephalus patients also maintain significantly elevated inflammatory cells, regardless of treatment attempts, and these persistent inflammatory reaction may be the driver of the increase in CSF ferritin [41]. Increased CSF ferritin has been reported in patients with meningitis and positively correlates with disease severity. Additionally, CSF ferritin decreases the occurrence of cerebral vasospasm in SAH patients by detoxifying free iron in the CSF [41, 66].

The elevated CSF ferritin may serve as a protective agent against the prolonged iron-mediated neurotoxicity in SAH patients [67]. Recent data illustrate this protective property of ferritin in cell lines treated with hemoglobin. Toxicity resulting from CNS hemorrhage may be iron-dependent due to hemoglobin degradation and a loss of ferritin due to endogenous hemopexin and haptoglobin responses [68]. Their data suggest endogenous hemopexin inhibits ferritin upregulation in response to hemorrhage, thus leaving neuronal tissue susceptible to hemoglobin toxicity [68]. Further research is needed to understand the clear mechanism of toxicity resulting from SAHs.

The literature suggests ferritin contributes to neurotoxic outcomes and chronic morbidities, while also supporting a protective mechanism from further iron-dependent toxicity. Understanding the role of ferritin in the pathology of SAH outcomes is vital for clinicians as they work to prevent these outcomes in acute settings, as the literature is beginning to suggest the importance of finding a therapeutic balance of ferritin levels. It is vital to use physiological models of elevated ferritin levels, as in HF, while understanding the additional inflammatory component of trauma-induced ferritin levels.

Clinical Investigations in Human Subjects

As previously stated, the hemoglobin breakdown that occurs following intracerebral hemorrhage leads to an accumulation of iron in the brain which mediates neural toxicity. DFO has been shown to be neuroprotective in animal models of intracerebral hemorrhage [65, 69]. A clinical trial out of Beth Israel Deaconess Medical Center (NCT01662895) showed that daily intravenous infusions of DFO up to 62 mg/kg/day were safe in a phase-1 clinical trial.

A phase-2 multicenter, randomized, placebo-controlled, double-blind trial to assess efficacy of the treatment was performed (NCT02175225) (i-DEF trial) [70]. 291 adults with primary, spontaneous intracerebral hemorrhage were randomized to receive 32 mg/kg/day DFO or saline (placebo) infusions for 3 days within 24 h of hemorrhage onset. The outcome assessed was a “good clinical outcome” defined as a modified Rankin scale score of 0–2 at day 90 of follow-up. The study’s results showed that 48/140 (34%) participants in the DFO group and 47/143 (33%) of patients had a modified Rankin scale score of 0–2. The rate of adverse events (27% DFO vs. 33% placebo) and death (7% for both groups) was not statistically significant. The investigators concluded that the drug, DFO mesylate, did not significantly improve the chance of a good clinical outcome.

In a recent post hoc analysis of the i-DEF trial, the authors sought to determine if the clinical usefulness of DFO depended on the hematoma volume [71]. Hematoma volume is a strong determinant of intracerebral hemorrhage mortality and clinical outcome [72, 73]. The 291 subjects were classified according to their hematoma volume where small <10 mL, moderate 10–30 mL, and large >30 mL. The authors hypothesized that DFO would provide little benefit to patients with small or large hematoma volumes.

At 180 days, in patients with moderate hematoma volume, 27/54 (50%) patients in the DFO group had a modified Rankin scale score of 0–2 compared to 13/51 (25.5%) of placebo-treated patients. The difference in good clinical outcomes for small (55.9% DFO vs. 51.9% placebo) and large (4.2% DFO vs. 26.7% placebo) hematoma volume was not significant. Thus, DFO may be of clinical value in patients with moderate hematoma volumes. Additionally, this study raises the question as to whether or not patients with small or large hematoma volumes should be included in clinical trials evaluating treatment effects on intracerebral hemorrhage.

Future Investigation

Understanding the role of ferritin in the pathogenesis of SAH is in its infancy. It is likely that microglia engulf the ferritin once blood breakdown occurs and ferritin transfers across the blood-brain barrier. At this point, little is known about the progression. A working hypothesis is that ferritin eventually overwhelms the normal functioning of microglia. These microglia then go on to release an inflammatory surge that can occur around the vasospasm window. Targeting this pathway may therefore have strong clinical implications in reducing vasospasm and delayed cerebral ischemia. Ongoing work is needed in the preclinical setting to tease apart mechanism and set the stage for a first in human clinical trial. The key will be to understand the timing of when microglia get overwhelmed and more importantly what level is engulfed ferritin is toxic.

Conclusion

Ferritin is crucial in mediating the toxic, oxidative effects of excess iron in the brain. However, significant elevations in ferritin and decreased clearance can lead to neurotoxicity. In SAH, the levels of ferritin are increased. While this may be protective against the free iron produced from hemolysis, increased ferritin levels have been shown to correlate with severe neurotoxic effects. CSF ferritin levels may be an appropriate value in monitoring post-SAH patients. Further study is needed to determine the ideal balance between ferritin and iron.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

No funding.

Author Contributions

Eric James Panther – original manuscript preparation (lead), manuscript editing (lead), figure preparation, and supervision. Rebecca Zelmanovich – original manuscript preparation (supporting) and manuscript editing (supporting). Jairo Hernandez – original manuscript preparation and manuscript editing (supporting). Emma Rose Dioso – original manuscript preparation. Devon Foster – original manuscript preparation. Brandon Lucke-Wold – supervision, concept formation, original manuscript preparation, and manuscript editing.

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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