The interplay between iron accumulation, mitochondrial dysfunction, and inflammation during the execution step of neurodegenerative disorders

1Iron is an essential element necessary for the normal development of brain functions. Enzymes involved in neurotransmitter synthesis that possess iron as a prosthetic group are recognized targets of iron deficiency: monoamine oxidases A and B involved in dopamine catabolism, tryptophan hydroxylase, required for serotonin synthesis, tyrosine hydroxylase, required for dopamine and norepinephrine synthesis, glutamate decarboxylase involved in GABA synthesis and glutamate transaminase involved in L-glutamate synthesis, all belong to this group.

2Abundant evidence shows that iron accumulation in particular areas of the brain is a hallmark of several neurodegenerative disorders, although it is uncertain whether iron accumulation is a primary cause of the disorder or a consequence of a previous dysfunction. Increased levels of iron promote cell death via hydroxyl radical formation, which enhances lipid peroxidation, protein aggregation, glutathione consumption, and nucleic acid modification. We recently put forward the hypothesis that iron accumulation, a process initiated by mitochondrial dysfunction, and the ensuing oxidative damage is part of the execution step, i.e., the death process of affected neurons.

3Mitochondrial dysfunction has long been associated with several neurodegenerative diseases that include Alzheimer’s disease (AD), Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Friedrich’s Ataxia (FA;;; ). Mitochondrial dysfunction results in decreased ATP synthesis, as well as in decreased synthesis of iron–sulfur clusters (ISCs) and heme prosthetic groups. An association between mitochondrial dysfunction and mitochondrial iron accumulation has been found only in FA, although evidence for mitochondrial iron accumulation has been reported in experimental models of PD.

4Inflammation in the central nervous system (CNS) is a condition strongly associated with neuronal death in several neurodegenerative disorders including PD and AD. Inflammation is characterized by the occurrence of reactive microglia and a massive production of pro-inflammatory cytokines. These inflammatory processes trigger a chain of events including increased production of ROS and reactive nitrogen species (RNS), disruption of iron metabolism and mitochondrial dysfunction, finally leading to neurodegeneration.

5The ability of iron to exchange readily one electron underlies its insertion in numerous catalytic processes found in living matter. The iron atom has octahedral coordination chemistry; therefore, it has six possible coordination bonds. Seminal work by Graf and associates demonstrated that iron is redox-inactive only if all its six coordination sites are stably bound. If one of the sites is free or loosely bound, iron is redox-active and competent of undertaking one-electron exchange reactions. It is noteworthy that Fe3+ complexes with the chelators desferrioxamine, DTPA or phytate at 1:10 (mol:mol) ratio result in redox-inactive iron whereas Fe3+ chelation with NTA, EDTA, EGTA, ATP, CDTA or bleomycin results in redox-active iron at the same 1:10 molar ratio.

6Iron is a paramagnetic element with two stable oxidation states: 2+ and 3+. As mentioned above, both Fe2+ and Fe3+ establish coordination complexes with a great variety of ligands. Iron complexes display a variety of reduction potentials, ranging from very positive to negative values because of a basic concept in coordination chemistry that establishes that the ligand modifies the electron cloud surrounding the metal, thus modifying its reduction potential. This versatility in reduction potential allows for fine-tuning between iron reduction potential and the electron transfer process it catalyzes. It is estimated that the predominant reduction potential for iron in the intracellular milieu of the cell is near zero V. Many in vitro experiments confirm iron-mediated production of the hydroxyl radical (•OH), which arises from the following reactions:

7The thermodynamic sum of reactions 1–3 gives reaction 4:

8The intracellular environment provides abundant reducing power in the form of GSH (mM) and Asc (μM), which reduces Fe3+ to Fe2+:

9Changes in free energy were calculated applying the equation δG = -nFE0 (Joule/mol), in which n is the number of electrons exchanged and F the Faraday constant. Reaction 1 values were from; Reaction 2, the half-cell potential for H2O2 dismutation was considerer 0.45 V and the reduction potential of the Fe3+/Fe2+ half-cell was considered 0 V; Reaction 3 (Fenton reaction): E0 half-cell values from. Half-cell potentials for reaction 5 were obtained from. GSH: reduced glutathione; GSSG: oxidized glutathione; Asc: ascorbate; Asc•: ascorbate free radical.

10The hydroxyl radical is considered one of the most reactive species generated in biological systems, since its reaction rate is only limited by diffusion, with rate constants in the 109–1012 Mol-1 s-1 range. This molecule induces irreversible damage to DNA, RNA, proteins, and lipids. Indeed, the hydroxyl radical is believed to be the etiological agent for several diseases and may be involved in the natural process of aging.

11The main components of cell iron homeostasis are the divalent metal transporter 1 (DMT1), a Fe2+ transporter that brings iron into the cell, the transferrin receptor 1 (TfR1) that brings iron in through the endocytosis of Ferro-transferrin, the iron export transporter ferroportin 1 (FPN1) and the cytosolic iron storage protein ferritin. The expression of these proteins is transductionally regulated by the iron responsive element/iron regulatory protein (IRE/IRP) system, which is activated when cells have low iron levels, resulting in increased DMT1 and TfR1 levels and decreased FPN1 and ferritin expression.

12In cells, iron in the 0.2–1.5 μM range is weakly complexed to low-molecular weight substrates such as citrate, carboxylates, amines, phosphate, nucleotides, GSH, and other molecules conforming the “cytosolic labile iron pool” (cLIP;;;; ). Iron in this pool is redox-active, cycling between the Fe+2 and Fe+3 forms, with prevalence of the reduced form because of the reductive cytosol environment. This redox-active pool is suitable to experimental detection by the fluorophore calcein, which has higher affinity for Fe3+ than for Fe2+ but since the reduction potential for iron in the Fe-calcein complex is low, Fe3+ bound to calcein is readily reduced in the intracellular environment, resulting in decreased calcein fluorescence. In cultured neuroblastoma cells the LIP represents about 3% of total cellular iron under basal culture conditions, but this percentage increases 3–4 fold, to μM concentrations, after exposure of cells to high extracellular iron concentrations. In cell models, iron overload generates increased lipid peroxidation, protein modifications and damage to DNA, consistent with the production of the hydroxyl radical.

13Postmortem tissues from patients with AD, PD, HD, ALS or FA show oxidative damage in the affected brain regions. The association between inflammation and oxidative damage is mediated by the release of RNS and ROS during the inflammatory process. In particular, activated microglia have high levels of nitric oxide synthase (NOS) and NADPH oxidase (NOX), two enzyme systems that mediate the increase in the oxidative tone induced by inflammation.

14Microglia, the brain-resident immune cells, are essential for the generation of the inflammatory response. They are activated by distress signals released from neighboring cells, initiating an innate response characterized by the production of pro-inflammatory cytokines and, incidentally, phagocytosis. Indeed, many cases of AD and PD are accompanied by a dramatic proliferation of reactive amoeboid macrophages and activated microglia in the substantia nigra (SN) or frontal cortex, together with high expression of pro-inflammatory cytokines.

15Inducible NOS (iNOS, also called NOS-2), which is scarcely expressed in the brain is induced during gliosis in pathological situations including AD and PD. Up-regulation of iNOS and of cyclo-oxygenase-1 and cyclo-oxygenase-2 in amoeboid microglia occurs in the SN of human PD patients. A study on the levels of iNOS mRNA in postmortem PD basal ganglia found a significant increase in iNOS expression in the dorsal two-thirds of the striatum and in the medial medullary lamina of the globus pallidus, accompanied by a reduction in iNOS mRNA expression in the putamen.

16Inflammatory mediators, including LPS and some cytokines (TNF-α, IL-1β, and IFN-γ) induce the transcriptional activation of the iNOS gene in astrocytes and microglia via activation of the transcription factors STAT1 and NF-kB. These factors translocate to the nucleus and bind to response elements present in the iNOS coding sequence. Upregulation of microglial iNOS expression is also observed after administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP;;;; ). Interestingly, administration of MPTP produces significantly less neuronal loss in mice deficient in iNOS compared to their wild type counterparts. In the 6-hydroxidopamine (6-OHDA) model, iNOS activity in the striatum induces neurodegeneration in rats. Pretreatment with the iNOS inhibitor L-NAME blocks amphetamine-induced rotations and significantly restores striatal dopamine (DA) levels in 6-OHDA treated rats. In neuroinflammatory models of PD, iNOS also participates in nigral neurodegeneration. Injection of LPS induces iNOS expression in the SN in a time- and dose-dependent manner; iNOS is present mainly in fully activated microglia with the characteristic amoeboid morphology. Furthermore, LPS-induced loss of dopaminergic neurons decreases significantly by administration of an iNOS inhibitor.

17The iNOS enzyme is a relevant factor in the neurodegenerative process associated to AD. Early observations reported increased iNOS and nitrotyrosine protein modifications in AD brains, mainly in neurofibrillary tangle-bearing neurons and neuropil threads as well as in astrocytes. Studies in transgenic mice overexpressing amyloid beta precursor protein (APP) demonstrated that several pathological changes such as vessel lesions, amyloid deposition and mitochondrial DNA deletions, are associated with the degree of NOS overexpression. Nevertheless, the APPsw/iNOS(-/-) mice, which express human APP mutations on an iNOS knockout background, show increased appearance of tau pathology, neuronal death, neuroinflammation and behavioral deficits compared with the parental APPsw mice. This evidence indicates that in AD, the production of NO can be protective or damaging, depending on the levels of NO production.

18The phagocyte NOX is the main regulated source of ROS generation. The catalytic component of the NOX complex is composed by a family of multiple-pass transmembrane proteins, named NOX1–4. The most studied, NOX2, also known as gp91phox or phagocyte oxidase (PHOX), is highly expressed in innate immune cells including microglia and it is most likely the predominant NOX isoform expressed in astrocytes, while neurons express both NOX2 and NOX4. NOX2 forms a complex with p67phox, p47phox, p40phox, and p22phox subunits. Several stimuli induce NOX2 complex priming, including pro-inflammatory cytokines (TNF-α, IL-1β) and Toll-like receptor (TLR) agonists like LPS, peroxynitrite and proteases. The primed NOX2 complex requires yet additional activation to initiate substantial ROS production. PKC activators, growth factors, complement protein C5a and G protein-coupled receptor agonists generate a fully active NOX complex.

19Activation of NOX also occurs in experimental models of PD and AD. Treatment with MPTP results in increased synthesis of the proinflammatory cytokine IL-1β and increased membrane translocation of p67phox that is prevented by minocycline, a tetracycline derivative that exerts multiple anti-inflammatory effects. In addition, aging mice treated with MPTP display an increase in gp91phox and 3-nitrotyrosine. In agreement, gp91phox-/- mice display decreased degeneration of dopaminergic neurons induced by MPTP compared to wild type mice. The unilateral injection of 6-OHDA into the right striatum of rats induces an increase of NOX1 and NOX2 both in the striatum and the SN. In concordance, dopaminergic neuronal and TNF-α and IFN-γ induction triggered by 6-OHDA are abrogated in the gp91phox-/- or minocycline treated mice. Additionally, striatal injection of 6-OHDA increases NOX1 expression in dopaminergic neurons in rat SN, and also increases 8-oxo-dG content, a marker of DNA oxidative damage. Moreover, NOX1 knockdown reduces 6-OHDA-induced oxidative DNA damage and dopaminergic neuronal degeneration.

20Microglia of AD subjects display activated NOX2, resulting in the formation of ROS that are toxic to neighboring neurons. In conjunction, an increment in NOX1 and NOX3 mRNA levels in the frontal lobe tissue from AD brains was reported, suggesting the participation of other NOX family members in AD neuropathology. Recently, increased NOX-dependent ROS production in the superior/middle temporal gyri at the earliest clinical manifestations of disease, but not in late-stage AD, was reported. Genetic inactivation of NOX2 in 12- to 15-month-old mice overexpressing the APPsw mutation (Tg2576 mice) results in reduced oxidative damage and rescues both the vascular and behavioral alterations observed in Tg2576 mice. Studies done in cell cultures replicated the postmortem and animal findings on oxidative damage driven by NOX activation. Experiments using co-cultures of neuronal and glial cells found that Aβ acts preferentially on astrocytes but causes neuronal death. The Aβ peptide causes transient increases in cytoplasmic calcium in astrocytes, associated with increased ROS generation, glutathione depletion and mitochondrial depolarization. Neuronal death after Aβ exposure was reduced both by NOX inhibitors and in the gp91phox knockout mice. These data are consistent with a sequence of events in which Aβ activates NOX in astrocytes by increasing cytoplasmic calcium, generating an oxidative burst that causes the death of neighboring neurons.

21Inflammatory conditions such as those found in neurodegenerative diseases also affect iron homeostasis through transcriptional modification of iron transporters. In this context, the observation that the transcription factor NFκB induces DMT1 expression is highly relevant for understanding the relationship between inflammation and iron homeostasis. We recently reported that the pro-inflammatory cytokines TNF-α, IL-6 and the TLR4 agonist LPS directly regulate DMT1mRNA and protein levels and induce a transient decrease in FPN1 protein, thus generating an increment of iron content in neurons and microglia. Supporting the results described above, a recent study using primary cultures of ventral mesencephalic neurons demonstrated that TNF-α or IL-1β induce an increment in DMT1 and TfR1 protein levels, together with a reduction of FPN1 levels, resulting in an increase in ferrous iron influx and decreased iron efflux in neurons. These findings were replicated in systemic tissues. Treatment of mouse splenocyte with LPS down-regulates the expression of FPN1 through a signaling mechanism mediated by TLR4. Moreover, stimulation of macrophage cell lines with IFN-γ, TNF-α or LPS results in increased IRE-binding activity of IRP1 and IRP2, and increased DMT1 mRNA expression.

22Considering that NFκB activation takes place downstream of TNF-α, IL-1 and LPS signaling pathways, inflammatory stimuli may induce DMT1 expression via NFκB activation. Indeed, TNF-α was detected in glial cells in the SN of PD patients but not in control subjects, together with immunoreactivity for TNF-α receptors in dopaminergic neurons of both control and PD patients. These findings are suggestive of a circuit in which activation of nigral microglia results in TNF-α secretion, which might increase iron uptake by dopaminergic neuron via NF-κB-induced DMT1 expression. Indeed, an increase in the nuclear immunoreactivity of NFκB has been observed in PD brains or in animal models for this disease, so it is possible that activation of NF-κB via inflammatory stimuli contributes to iron accumulation in PD. Accordingly, inflammation could induce the production of hydroxyl radical trough the activation of two parallel pathways: (i) through DMT1-mediated increase of intracellular iron levels and (ii) through increased hydrogen peroxide levels mediated by NOX activation.

23A positive feedback loop can be established between ROS/RNS and inflammatory cytokines. ROS induce intracellular signaling pathways that result in the activation of transcriptional factors like NF-kB, AP-1 and Nrf-2, which regulate the expression of pro-inflammatory mediators such as Cox-2, MCP-1, IL-6, TNF-α, IL-1α, and IL-1β. These cytokines and chemokines, in turn, stimulate a cascade of events leading to increased oxidative stress via iNOS and NOX activation.

24The study of the relationship between inflammation and mitochondrial activity in the CNS is incipient. Intrastriatal injection of LPS induces mitochondrial dysfunction, microgliosis, iron accumulation and progressive degeneration of the dopamine nigro-striatal system, as observed in PD pathology. Similarly, cytokines such as IL-1β decrease mitochondrial activity through the production of NO in cardiomyocytes.

25Several reports indicate that TLRs regulate mitochondrial activity. Activation of TLR3 results in reduction of mitochondrial oxygen consumption mediated by opening of the permeability transition pore. In co-cultures of cortical neurons with microglial cells, the TLR4 agonist LPS promotes decreased oxygen consumption and oxidative stress, with the subsequent nigral dopaminergic neuronal death in a rat model of inflammation. Although these studies strongly suggest a link between TLRs and mitochondria dysfunction, further studies should clarify the molecular mechanisms involved and its relevance to particular neurodegenerative processes.

26The production of ROS and RNS affects mitochondrial activity through destabilization of the ISCs. The free radical superoxide damages and/or oxidizes 4Fe-4S clusters, which results in the formation of the “null” 3Fe-4S center form. Additionally, NO reacts with 4Fe-4S clusters generating [(NO)2Fe(SR)2] type complexes that inactivate several mitochondrial iron–sulfur enzymes including proteins which compose the electron transport chain (; see below). The above data are consistent with the notion that inflammation, ROS/RNS production, and mitochondrial dysfunction are linked processes.

27Additionally, recent evidence shows that under certain conditions mitochondria can modulate the immune response. The mitochondrial protein MARCH5 (an ubiquitin E3 ligase constitutively expressed in the mitochondrion outer membrane) positively regulates TLR7 and TLR4 signaling, resulting in NFκB activation and expression of the NFκB-responsive genes IL-6 and TNF-α. In addition, activation of TLR1, TLR2 and TLR4 results in augmented mitochondrial ROS production by inducing translocation to mitochondria of TRAF6 (TLR signaling adaptor, tumor necrosis factor receptor-associated factor 6), which leads to the engagement and ubiquitination of ECSIT (evolutionarily conserved signaling intermediate in Toll pathways), a protein required for efficient assembly of mitochondrial complex I. It remains to be demonstrated whether this mechanism is operative in CNS cells.

28Interestingly, mitochondrial ROS (mtROS) could arguably activate the inflammatory response. In vascular endothelium, mtROS act as intermediate signaling molecules to trigger production of IL-6. In addition, patients with the autoinflammatory disorder TRAPS (tumor necrosis factor receptor-associated periodic syndrome), exhibit altered mitochondrial function with enhanced mtROS generation and increased production of IL-6, TNFα, and IL-1β; decreasing mtROS levels by the general antioxidant N-acetylcysteine effectively reduces inflammatory cytokine production after LPS stimulation. These results point to novel pathways that link inflammation to mtROS production.

29In summary, inflammation induces ROS production and mitochondrial dysfunction generating a self-feeding cycle that could lead to neurodegeneration in diseases where inflammation and oxidative damage are prevalent (Figure 1). In this cycle, [1] inflammation induces ROS and RNS generation by activation of the NOX and iNOS enzymes; [2] in turn, ROS/RNS induce the expression of inflammatory cytokines. [3] Additionally, inflammation induces mitochondrial dysfunction through activation of TLR signaling. [4] ROS in turn induce mitochondrial dysfunction by destabilizing ISCs, which results in the inactivation of several mitochondrial iron–sulfur enzymes. [5] Mitochondrial dysfunction leads to IRP1 activation and increased iron uptake. [6] Iron increases oxidative damage by transforming mild oxidative molecules like superoxide and hydrogen peroxide into the hydroxyl radical. [7] Electron transport chain inhibition increases ROS production by electron leak, and arguably could modulate the innate immune response by TLR signaling regulation [8]. Finally, [9] inflammation is likely to cause iron accumulation through induction of DMT1 expression and transient ferroportin decrease.

30Mitochondria have a key role in iron metabolism in association with the synthesis of ISCs and heme, prosthetic groups that are vital for cell function. Iron complexes are particularly relevant components of the electron transport chain: 12 proteins contain ISCs and eight proteins contain heme in their active centers. Other proteins that have ISCs are the Krebs cycle enzymes aconitase and succinate dehydrogenase, ribonucleotide reductase, an enzyme that catalyzes the formation of deoxyribonucleotides from ribonucleotides, and ferrochelatase, involved in the addition of Fe to porphyrin IX during heme synthesis. We refer the reader to for a comprehensive listing of ISC-containing proteins. Particular attention should be given to cytoplasmic IRP1, which contain a 4Fe-4S cluster in its inactive form and becomes active in the clusterless form.

31Mitochondria have a redox-active iron pool; an increase in this pool directly associates with an increase in oxidative damage and with calcium-dependent changes in the mitochondrial permeability transition pore. Thus, cells must regulate tightly their mitochondrial Fe levels because an iron shortage affects numerous processes that have iron as a co-factor, including the electron transport chain, whereas an excess of redox-active iron promotes the generation of the noxious hydroxyl radical. How mitochondria regulate their iron content and what, if any, is the interplay between cytoplasmic and mitochondrial iron are incipient but highly relevant subjects to understand the mechanisms of mitochondrial dysfunction in neurodegenerative diseases.

32There is increasing evidence that mitochondrial dysfunction plays an important role in the development of neurodegenerative diseases such as AD, HD, FA, and PD. Imbalances in ROS and ATP levels derived from mitochondrial dysfunction affect neurons particularly, given their dependence on ATP to propagate electrical signals, maintain ionic gradients, and facilitate anterograde and retrograde transport along axons. The involvement of mitochondrial dysfunction in the pathophysiology of PD was noted very early in the study of the disease. Evidence of mitochondrial dysfunction in PD began in the eighties, when, after an intravenous injection of illicit drugs, four college students developed marked Parkinsonism. Analysis of the substances injected revealed the presence of MPTP, a compound metabolized by astrocytes into 1-methyl-4-phenylpyridinium (MPP+), which is then released into the extracellular space. MPP+ is taken up selectively by dopaminergic (DA) neurons where it inhibits mitochondrial complex I. Further evidence showed that complex I activity and the number of complex I subunits are decreased in postmortem tissue of idiopathic PD patients. These results strongly suggest that mitochondrial dysfunction is a pathognomonic sign in the pathophysiology of PD. Reduced complex-I activity and an increased susceptibility to MPP+ were also observed in cybrids containing mitochondrial DNA from PD patients, suggesting the presence of mitochondrial DNA-encoded defects in PD. Additionally, in the epidemiology field, the use in farming of the highly lipophilic pesticide rotenone, a potent inhibitor of mitochondrial complex I, has been linked to a higher incidence of PD in agricultural workers.

33Mitochondrial complex I is a major source of ROS. Complex I from mitochondria of PD patients contain 47% more protein carbonyls localized to catalytic subunits and a 34% decrease in complex I 8-kDa subunit. NADH-driven electron transfer rates through complex I inversely correlate with complex I protein oxidation status and with the reduction in the 8-kDa subunit protein levels.

34Knowledge on the mechanisms that associate mitochondrial dysfunction and iron dyshomeostasis in PD is incipient. Treatment of SH-SY5Y dopaminergic neuroblastoma cells with mitochondrial complex I inhibitors such as rotenone or MPP+ results in ROS production and increased mitochondrial iron uptake. Moreover, inhibition of complex I by rotenone decreases the activity of three ISC-containing enzymes: mitochondrial and cytoplasmic aconitases and xanthine oxidase, and decreases the ISC content of glutamine phosphoribosyl pyrophosphate amidotransferase. The reduction in cytoplasmic aconitase activity is associated with an increase in iron regulatory IRP1 mRNA binding activity and with an increase in the mitochondrial labile iron pool. Since IRP1 activity post-transcriptionally regulates the expression of iron import proteins, ISC synthesis inhibition may result in a false iron deficiency signal with the ensuing iron accumulation.

35Considering the evidence discussed, we propose that inhibition of mitochondrial complex I by endogenous and/or exogenous toxins or by inflammatory processes resulting from trauma or other causes, engage a vicious cycle of increased oxidative stress and increased iron accumulation (Figure 2). In this scheme, inhibition of mitochondrial complex I by endogenous or exogenous toxins, or because of mutations in PD genes Parkin, Pink 1, alpha-synuclein, DJ-1 or LRRK2, results in decreased electron transport chain activity [1] and the ensuing ATP synthesis decrease and ROS increase [2]. Decreased ATP levels impairs ISC synthesis that results in decreased activity of ISC-containing proteins and increased mRNA binding activity of the iron homeostasis protein IRP1. IRP1 activation leads to increased DMT1 and TfR1 expression [3] and the ensuing iron accumulation [4]. Increased ROS and increased redox-active iron promotes the consumption of intracellular reductants such as GSH and ascorbate [5], resulting in a further decrease in mitochondrial activity and ISC synthesis. Another input to this cycle is contributed by inflammatory cytokines liberated by activated microglia and astrocytes [6], which enhance mitochondrial dysfunction [7], increase ROS production [8] and increase iron accumulation by modifying the expression of the iron transporters DMT1 and FPN1 [9]. As discussed in the text, increased ROS back-feed the production of cytokines. Increased ROS levels, in particular increased hydroxyl radical generation, produces increased oxidative damage, which is counteracted by antioxidant defenses [10]. In time, the positive feedback loop of mitochondrial dysfunction, iron dyshomeostasis and inflammation results in alpha-synuclein aggregation, proteasomal dysfunction, changes in mitochondrial fission/fusion dynamics, opening of the mitochondrion PTP, increased cytoplasmic cytochrome c and activation of death pathways [11]. Debris and toxins from dying neurons enhance the activation of glial cells, which contributes to the inflammatory network [12].

36In summary, because of the innate interconnectivity of mitochondrial complex I dysfunction, iron accumulation, oxidative stress, and inflammation, probably the initiation of any one of these factors will induce or enhance the others through the generation of a positive feedback loop that in time will end in apoptotic neuronal death. Still unanswered is the question of why neurons of the SNc are so particularly prone to carry-on this cycle. On examination of this cycle, several therapeutic targets come to mind. Its intervention should result in prolonged life of the affected neurons.

37The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.