ACSL4 and the lipoxygenases 15/15B are pivotal for ferroptosis induced by iron and PUFA dyshomeostasis in dopaminergic neurons.

Ferroptosis, an iron-dependent regulated cell death triggered by high lipid peroxide levels, has been implicated in several neurodegenerative diseases, including Parkinson's disease (PD). Brain regions such as the striatum are highly rich in both peroxidation susceptible PUFAs and iron, which accumulate at a greater rate than age in PD. The exact molecular pathways and patho-physiological conditions promoting cell death in the dopaminergic neurons that are particularly susceptible in PD remain elusive. In the current work, we show that modifying the PUFA composition in membranes of dopaminergic neurons using arachidonic acid (AA) can determine ferroptosis susceptibility. Furthermore, cotreatment with iron (Fe), increases AA-containing phospholipid association and synergistically promotes high lipid peroxidation to facilitate ferroptosis. Ex vivo analysis with organotypic brain slices, confirm that AA + Fe induces cell death in the nigrostriatal pathway and can be rescued by the anti-ferroptotic drug Ferrostatin-1. Prevention of ferroptotic AA + Fe induced cell death through inhibition of ACSL4, ALOX15 or ALOX15B provides mechanistic support of this lipid peroxidation pathway being involved in dopaminergic neuronal death and novel potential pharmacological targets for neuroprotective strategies in PD.

Alpha synuclein determines ferroptosis sensitivity in dopaminergic neurons via modulation of ether-phospholipid membrane composition.

There is a continued unmet need for treatments that can slow Parkinson's disease progression due to the lack of understanding behind the molecular mechanisms underlying neurodegeneration. Since its discovery, ferroptosis has been implicated in several diseases and represents a therapeutic target in Parkinson's disease. Here, we use two highly relevant human dopaminergic neuronal models to show that endogenous levels of α-synuclein can determine the sensitivity of dopaminergic neurons to ferroptosis. We show that reducing α-synuclein expression in dopaminergic neurons leads to ferroptosis evasion, while elevated α-synuclein expression in patients' small-molecule-derived neuronal precursor cells with SNCA triplication causes an increased vulnerability to lipid peroxidation and ferroptosis. Lipid profiling reveals that ferroptosis resistance is due to a reduction in ether-linked phospholipids, required for ferroptosis, in neurons depleted of α-synuclein (α-syn). These results provide a molecular mechanism linking α-syn levels to the sensitivity of dopaminergic neurons to ferroptosis, suggesting potential therapeutic relevance.

Synthesis, physicochemical characterization and neuroprotective evaluation of novel 1-hydroxypyrazin-2(1H)-one iron chelators in an in vitro cell model of Parkinson's disease.

Iron dysregulation, dopamine depletion, cellular oxidative stress and α-synuclein protein mis-folding are key neuronal pathological features seen in the progression of Parkinson's disease. Iron chelators endowed with one or more therapeutic modes of action have long been suggested as disease modifying therapies for its treatment. In this study, novel 1-hydroxypyrazin-2(1H)-one iron chelators were synthesized and their physicochemical properties, iron chelation abilities, antioxidant capacities and neuroprotective effects in a cell culture model of Parkinson's disease were evaluated. Physicochemical properties (log ß, log D7.4, pL0.5) suggest that these ligands have a poorer ability to penetrate cell membranes and form weaker iron complexes than the closely related 1-hydroxypyridin-2(1H)-ones. Despite this, we show that levels of neuroprotection provided by these ligands against the catecholaminergic neurotoxin 6-hydroxydopamine in vitro were comparable to those seen previously with the 1-hydroxypyridin-2(1H)-ones and the clinically used iron chelator Deferiprone, with two of the ligands restoring cell viability to ≥89% compared to controls. Two of the ligands were endowed with additional phenol moieties in an attempt to derive multifunctional chelators with dual iron chelation/antioxidant activity. However, levels of neuroprotection with these ligands were no greater than ligands lacking this moiety, suggesting the neuroprotective properties of these ligands are due primarily to chelation and passivation of intracellular labile iron, preventing the generation of free radicals and reactive oxygen species that otherwise lead to the neuronal cell death seen in Parkinson's disease.

Amyloidogenic processing of Alzheimer's disease ß-amyloid precursor protein induces cellular iron retention.

The proteolytic cleavage of ß-amyloid precursor protein (APP) to form the amyloid beta (Aß) peptide is related to the pathogenesis of Alzheimer's disease (AD) because APP mutations that influence this processing either induce familial AD or mitigate the risk of AD. Yet Aß formation itself may not be pathogenic. APP promotes neuronal iron efflux by stabilizing the cell-surface presentation of ferroportin, the only iron export channel of cells. Mislocalization of APP can promote iron retention, thus we hypothesized that changes in endocytotic trafficking associated with altered APP processing could contribute to the neuronal iron elevation and oxidative burden that feature in AD pathology. Here, we demonstrate, using genetic and pharmacological approaches, that endocytotic amyloidogenic processing of APP impairs iron export by destabilizing ferroportin on the cell surface. Conversely, preferential non-amyloidogenic processing of APP at the cell surface promotes ferroportin stabilization to decrease intraneuronal iron. A new Aß-independent hypothesis emerges where the amyloidogenic processing of APP, combined with age-dependent iron elevation in the tissue, increases pro-oxidant iron burden in AD.

Conservative iron chelation for neurodegenerative diseases such as Parkinson's disease and amyotrophic lateral sclerosis.

Focal iron accumulation associated with brain iron dyshomeostasis is a pathological hallmark of various neurodegenerative diseases (NDD). The application of iron-sensitive sequences in magnetic resonance imaging has provided a useful tool to identify the underlying NDD pathology. In the three major NDD, degeneration occurs in central nervous system (CNS) regions associated with memory (Alzheimer's disease, AD), automaticity (Parkinson's disease, PD) and motor function (amyotrophic lateral sclerosis, ALS), all of which require a high oxygen demand for harnessing neuronal energy. In PD, a progressive degeneration of the substantia nigra pars compacta (SNc) is associated with the appearance of siderotic foci, largely caused by increased labile iron levels resulting from an imbalance between cell iron import, storage and export. At a molecular level, α-synuclein regulates dopamine and iron transport with PD-associated mutations in this protein causing functional disruption to these processes. Equally, in ALS, an early iron accumulation is present in neurons of the cortico-spinal motor pathway before neuropathology and secondary iron accumulation in microglia. High serum ferritin is an indicator of poor prognosis in ALS and the application of iron-sensitive sequences in magnetic resonance imaging has become a useful tool in identifying pathology. The molecular pathways that cascade down from such dyshomeostasis still remain to be fully elucidated but strong inroads have been made in recent years. Far from being a simple cause or consequence, it has recently been discovered that these alterations can trigger susceptibility to an iron-dependent cell-death pathway with unique lipoperoxidation signatures called ferroptosis. In turn, this has now provided insight into some key modulators of this cell-death pathway that could be therapeutic targets for the NDD. Interestingly, iron accumulation and ferroptosis are highly sensitive to iron chelation. However, whilst chelators that strongly scavenge intracellular iron protect against oxidative neuronal damage in mammalian models and are proven to be effective in treating systemic siderosis, these compounds are not clinically suitable due to the high risk of developing iatrogenic iron depletion and ensuing anaemia. Instead, a moderate iron chelation modality that conserves systemic iron offers a novel therapeutic strategy for neuroprotection. As demonstrated with the prototype chelator deferiprone, iron can be scavenged from labile iron complexes in the brain and transferred (conservatively) either to higher affinity acceptors in cells or extracellular transferrin. Promising preclinical and clinical proof of concept trials has led to several current large randomized clinical trials that aim to demonstrate the efficacy and safety of conservative iron chelation for NDD, notably in a long-term treatment regimen.

Changes in cortical protein markers of iron transport with gender, major depressive disorder and suicide.

Objectives: The objective of this study was to determine whether a breakdown in proteins regulating cortical iron homeostasis could be involved in the pathophysiology of mood disorders.Methods: Levels of select proteins responsible for cortical iron transport were quantitated by Western blotting of Brodmann's (BA) areas 6 and 10 from patients with major depressive disorder (n = 13), bipolar disorder (n = 12) and age/sex matched controls (n = 13).Results: We found the inactive form of ceruloplasmin was lower in BA 6 from males compared to females. Levels of copper containing ceruloplasmin was lower in BA 6 from suicide completers whilst levels of amyloid precursor protein, TAU and transferrin were higher in BA 10 from those individuals. The level of prion protein was lower in BA 6 from subjects with major depressive disorder.Conclusions: Our data suggests that perturbation in cortical iron transport proteins is not prevalent in mood disorders. By contrast, our data suggests changes in iron transport proteins in BA 6 and BA 10 are present after suicide completion. If these changes were present before death, they could have had a role in the genesis of the contemplation and completion of suicide.

Recapitulating Parkinson's disease pathology in a three-dimensional human neural cell culture model.

Extensive loss of dopaminergic neurons and aggregation of the protein α-synuclein into ubiquitin-positive Lewy bodies represents a major neuropathological hallmark of Parkinson's disease (PD). At present, the generation of large nuclear-associated Lewy bodies from endogenous wild-type α-synuclein, translationally regulated under its own promoter in human cell culture models, requires costly and time-consuming protocols. Here, we demonstrate that fully differentiated human SH-SY5Y neuroblastoma cells grown in three-dimensional cell culture develop Lewy-body-like pathology upon exposure to exogenous α-synuclein species. In contrast to most cell- and rodent-based PD models, which exhibit multiple diffuse α-synuclein aggregates throughout the cytoplasm, a single large nuclear inclusion that is immunopositive for α-synuclein and ubiquitin is rapidly obtained in our model. This was achieved without the need for overexpression of α-synuclein or genetic modification of the cell line. However, phosphorylation of α-synuclein within these inclusions was not observed. The system described here provides an ideal tool to screen compounds to therapeutically intervene in Lewy body formation, and to investigate the mechanisms involved in disease progression in synucleinopathies.

Post Translational Modulation of ß-Amyloid Precursor Protein Trafficking to the Cell Surface Alters Neuronal Iron Homeostasis.

Cell surface ß-Amyloid precursor protein (APP) is known to have a functional role in iron homeostasis through stabilising the iron export protein ferroportin (FPN). Mechanistic evidence of this role has previously only been provided through transcriptional or translational depletion of total APP levels. However, numerous post-translational modifications of APP are reported to regulate the location and trafficking of this protein to the cell surface. Stable overexpressing cell lines were generated that overexpressed APP with disrupted N-glycosylation (APPN467K and APPN496K) or ectodomain phosphorylation (APPS206A); sites selected for their proximity to the FPN binding site on the E2 domain of APP. We hypothesise that impaired N-glycosylation or phosphorylation of APP disrupts the functional location on the cell surface or binding to FPN to consequentially alter intracellular iron levels through impaired cell surface FPN stability. Outcomes confirm that these post-translational modifications are essential for the correct location of APP on the cell surface and highlight a novel mechanism by which the cell can modulate iron homeostasis. Further interrogation of other post-translational processes to APP is warranted in order to fully understand how each modification plays a role on regulating intracellular iron levels in health and disease.

Amyloid Precursor Protein Mediates Neuronal Protection from Rotenone Toxicity.

Mitochondrial complex I dysfunction is the most common respiratory chain defect in human disorders and a hotspot for neurodegenerative diseases. Amyloid precursor protein (APP) and its non-amyloidogenic processing products, in particular soluble APP α (sAPPα), have been shown to provide neuroprotection in models of neuronal injury; however, APP-mediated protection from acute mitochondrial injury has not been previously reported. Here, we use the plant-derived pesticide rotenone, a potent complex I-specific mitochondrial inhibitor, to discover neuroprotective effects of APP and sAPPα in vitro, in neuronal cell lines over-expressing APP, and in vivo, in a retinal neuronal rotenone toxicity mouse model. Our results show that APP over-expression is protective against rotenone toxicity in neurons via sAPPα through an autocrine/paracrine mechanism that involves the Pi3K/Akt pro-survival pathway. APP-/- mice exhibit greater susceptibility to retinal rotenone toxicity, while intravitreal delivery of sAPPα reduces inner retinal neuronal death in wild-type mice following rotenone challenge. We also show a significant decrease in human retinal expression of APP with age. These findings provide insights into the therapeutic potential of non-amyloidogenic processing of APP in complex I-related neurodegeneration.

Marked Age-Related Changes in Brain Iron Homeostasis in Amyloid Protein Precursor Knockout Mice.

Proteolytic cleavage of the amyloid precursor protein (APP) into the Aß peptide has been an extensively researched mechanism for Alzheimer's disease, but the normal function of the protein is less understood. APP functions to regulate neuronal iron content by stabilizing the surface presentation of ferroportin-the only iron exporter channel of cells. The present study aims to quantify the contribution of APP to brain and peripheral iron by examining the lifetime impact on brain and liver iron levels in APP knockout mice. Consistent with previous reports, we found that wild-type mice exhibited an age-dependent increase in iron and ferritin in the brain, while no age-dependent changes were observed in the liver. APP ablation resulted in an exaggeration of age-dependent iron accumulation in the brain and liver in mice that was assessed at 8, 12, 18, and 22 months of age. Brain ferroportin levels were decreased in APP knockout mice, consistent with a mechanistic role for APP in stabilizing this iron export protein in the brain. Iron elevation in the brain and liver of APP knockout mice correlated with decreased transferrin receptor 1 and increased ferritin protein levels. However, no age-dependent increase in brain ferritin iron saturation was observed in APP-KO mice despite similar protein expression levels potentially explaining the vulnerability of APP-KO mice to parkinsonism and traumatic brain sequelae. Our results support a crucial role of APP in regulating brain and peripheral iron, and show that APP may act to oppose brain iron elevation during aging.

iPSC-derived neuronal models of PANK2-associated neurodegeneration reveal mitochondrial dysfunction contributing to early disease.

Mutations in PANK2 lead to neurodegeneration with brain iron accumulation. PANK2 has a role in the biosynthesis of coenzyme A (CoA) from dietary vitamin B5, but the neuropathological mechanism and reasons for iron accumulation remain unknown. In this study, atypical patient-derived fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs) and subsequently differentiated into cortical neuronal cells for studying disease mechanisms in human neurons. We observed no changes in PANK2 expression between control and patient cells, but a reduction in protein levels was apparent in patient cells. CoA homeostasis and cellular iron handling were normal, mitochondrial function was affected; displaying activated NADH-related and inhibited FADH-related respiration, resulting in increased mitochondrial membrane potential. This led to increased reactive oxygen species generation and lipid peroxidation in patient-derived neurons. These data suggest that mitochondrial deficiency is an early feature of the disease process and can be explained by altered NADH/FADH substrate supply to oxidative phosphorylation. Intriguingly, iron chelation appeared to exacerbate the mitochondrial phenotype in both control and patient neuronal cells. This raises caution for the use iron chelation therapy in general when iron accumulation is absent.

Amyloid precursor protein drives down-regulation of mitochondrial oxidative phosphorylation independent of amyloid beta.

Amyloid precursor protein (APP) and its extracellular domain, soluble APP alpha (sAPPα) play important physiological and neuroprotective roles. However, rare forms of familial Alzheimer's disease are associated with mutations in APP that increase toxic amyloidogenic cleavage of APP and produce amyloid beta (Aß) at the expense of sAPPα and other non-amyloidogenic fragments. Although mitochondrial dysfunction has become an established hallmark of neurotoxicity, the link between Aß and mitochondrial function is unclear. In this study we investigated the effects of increased levels of neuronal APP or Aß on mitochondrial metabolism and gene expression, in human SH-SY5Y neuroblastoma cells. Increased non-amyloidogenic processing of APP, but not Aß, profoundly decreased respiration and enhanced glycolysis, while mitochondrial DNA (mtDNA) transcripts were decreased, without detrimental effects to cell growth. These effects cannot be ascribed to Aß toxicity, since higher levels of endogenous Aß in our models do not cause oxidative phosphorylation (OXPHOS) perturbations. Similarly, chemical inhibition of ß-secretase decreased mitochondrial respiration, suggesting that non-amyloidogenic processing of APP may be responsible for mitochondrial changes. Our results have two important implications, the need for caution in the interpretation of mitochondrial perturbations in models where APP is overexpressed, and a potential role of sAPPα or other non-amyloid APP fragments as acute modulators of mitochondrial metabolism.

Post translational changes to α-synuclein control iron and dopamine trafficking; a concept for neuron vulnerability in Parkinson's disease.

Parkinson's disease is a multifactorial neurodegenerative disorder, the aetiology of which remains elusive. The primary clinical feature of progressively impaired motor control is caused by a loss of midbrain substantia nigra dopamine neurons that have a high α-synuclein (α-syn) and iron content. α-Syn is a neuronal protein that is highly modified post-translationally and central to the Lewy body neuropathology of the disease. This review provides an overview of findings on the role post translational modifications to α-syn have in membrane binding and intracellular vesicle trafficking. Furthermore, we propose a concept in which acetylation and phosphorylation of α-syn modulate endocytic import of iron and vesicle transport of dopamine during normal physiology. Disregulated phosphorylation and oxidation of α-syn mediate iron and dopamine dependent oxidative stress through impaired cellular location and increase propensity for α-syn aggregation. The proposition highlights a connection between α-syn, iron and dopamine, three pathological components associated with disease progression in sporadic Parkinson's disease.

Oxidation of Iron under Physiologically Relevant Conditions in Biological Fluids from Healthy and Alzheimer's Disease Subjects.

Ferroxidase activity has been reported to be altered in various biological fluids in neurodegenerative disease, but the sources contributing to the altered activity are uncertain. Here we assay fractions of serum and cerebrospinal fluid with a newly validated triplex ferroxidase assay. Our data indicate that while ceruloplasmin, a multicopper ferroxidase, is the predominant source of serum activity, activity in CSF predominantly derives from a <10 kDa component, specifically from polyanions such as citrate and phosphate. We confirm that in human biological samples, ceruloplasmin activity in serum is decreased in Alzheimer's disease, but in CSF a reduction of activity in Alzheimer's disease originates from the polyanion component.

Parkinson's disease iron deposition caused by nitric oxide-induced loss of ß-amyloid precursor protein.

Elevation of both neuronal iron and nitric oxide (NO) in the substantia nigra are associated with Parkinson's disease (PD) pathogenesis. We reported previously that the Alzheimer-associated ß-amyloid precursor protein (APP) facilitates neuronal iron export. Here we report markedly decreased APP expression in dopaminergic neurons of human PD nigra and that APP(-/-) mice develop iron-dependent nigral cell loss. Conversely, APP-overexpressing mice are protected in the MPTP PD model. NO suppresses APP translation in mouse MPTP models, explaining how elevated NO causes iron-dependent neurodegeneration in PD.

ß-Amyloid precursor protein does not possess ferroxidase activity but does stabilize the cell surface ferrous iron exporter ferroportin.

Ceruloplasmin is a ferroxidase that interacts with ferroportin to export cellular iron, but is not expressed in neurons. We recently reported that the amyloid precursor protein (APP) is the analogous iron-exporting chaperone for neurons and other cells. The ferroxidase activity of APP has since been called into question. Using a triplex Fe2+ oxidation assay, we analyzed the activity of a soluble form of APP (sAPPα) within a buffer of physiological pH and anionic charge, and determined that iron oxidation originated from phosphate. Using various techniques such as flow-cytometry to measure surface presented proteins, we confirmed that endogenous APP is essential for ferroportin persistence on the neuronal surface. Therefore, despite lacking ferroxidase activity, APP still supports iron export from neurons.

The iron regulatory capability of the major protein participants in prevalent neurodegenerative disorders.

As with most bioavailable transition metals, iron is essential for many metabolic processes required by the cell but when left unregulated is implicated as a potent source of reactive oxygen species. It is uncertain whether the brain's evident vulnerability to reactive species-induced oxidative stress is caused by a reduced capability in cellular response or an increased metabolic activity. Either way, dys-regulated iron levels appear to be involved in oxidative stress provoked neurodegeneration. As in peripheral iron management, cells within the central nervous system tightly regulate iron homeostasis via responsive expression of select proteins required for iron flux, transport and storage. Recently proteins directly implicated in the most prevalent neurodegenerative diseases, such as amyloid-ß precursor protein, tau, α-synuclein, prion protein and huntingtin, have been connected to neuronal iron homeostatic control. This suggests that disrupted expression, processing, or location of these proteins may result in a failure of their cellular iron homeostatic roles and augment the common underlying susceptibility to neuronal oxidative damage that is triggered in neurodegenerative disease.

Ceruloplasmin and ß-amyloid precursor protein confer neuroprotection in traumatic brain injury and lower neuronal iron.

Traumatic brain injury (TBI) is in part complicated by pro-oxidant iron elevation independent of brain hemorrhage. Ceruloplasmin (CP) and ß-amyloid protein precursor (APP) are known neuroprotective proteins that reduce oxidative damage through iron regulation. We surveyed iron, CP, and APP in brain tissue from control and TBI-affected patients who were stratified according to time of death following injury. We observed CP and APP induction after TBI accompanying iron accumulation. Elevated APP and CP expression was also observed in a mouse model of focal cortical contusion injury concomitant with iron elevation. To determine if changes in APP or CP were neuroprotective we employed the same TBI model on APP(-/-) and CP(-/-) mice and found that both exhibited exaggerated infarct volume and iron accumulation postinjury. Evidence supports a regulatory role of both proteins in defence against iron-induced oxidative damage after TBI, which presents as a tractable therapeutic target.

Iron accumulates in Huntington's disease neurons: protection by deferoxamine.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a polyglutamine-encoding CAG expansion in the huntingtin gene. Iron accumulates in the brains of HD patients and mouse disease models. However, the cellular and subcellular sites of iron accumulation, as well as significance to disease progression are not well understood. We used independent approaches to investigate the location of brain iron accumulation. In R6/2 HD mouse brain, synchotron x-ray fluorescence analysis revealed iron accumulation as discrete puncta in the perinuclear cytoplasm of striatal neurons. Further, perfusion Turnbull's staining for ferrous iron (II) combined with transmission electron microscope ultra-structural analysis revealed increased staining in membrane bound peri-nuclear vesicles in R6/2 HD striatal neurons. Analysis of iron homeostatic proteins in R6/2 HD mice revealed decreased levels of the iron response proteins (IRPs 1 and 2) and accordingly decreased expression of iron uptake transferrin receptor (TfR) and increased levels of neuronal iron export protein ferroportin (FPN). Finally, we show that intra-ventricular delivery of the iron chelator deferoxamine results in an improvement of the motor phenotype in R6/2 HD mice. Our data supports accumulation of redox-active ferrous iron in the endocytic / lysosomal compartment in mouse HD neurons. Expression changes of IRPs, TfR and FPN are consistent with a compensatory response to an increased intra-neuronal labile iron pool leading to increased susceptibility to iron-associated oxidative stress. These findings, together with protection by deferoxamine, support a potentiating role of neuronal iron accumulation in HD.