A proposed experiment on rats using rotenone and iron from iron carbonyl
The paper in a nutshell. In Parkinson’s disease there are problems with iron but the problems are due to dysregulation of iron utilization not high systematic iron levels which are not present in Parkinson’s disease. Iron utilization can be re-regulated in Parkinson’s disease by supplementation with iron from iron carbonyl three times a day. Iron from iron carbonyl is slowly absorbed. Iron carbonyl given three times a day will keep iron absorption constant and keep systematic iron utilization constant which could prevent Parkinson’s disease. This idea must be first tested out in rats prior to patients with Parkinson’s disease taking iron from iron carbonyl. Patients with Parkinson’s disease now supplementing with iron from iron carbonyl is strongly advised against.
Thomas Berry and Ahmed Moustafa
Ahmed Moustafa – School of Psychology, Western Sydney University, Sydney, New South Wales, Australia, Marcs Institute for Brain and Behaviour, Western Sydney University, Sydney, New South Wales, Australia, Department of Human Anatomy and Physiology, the Faculty of Health Sciences, University of Johannesburg, South Africa
Iron deposits are associated with neuronal cell death in substantia nigra resulting in Parkinson’s disease (PD), however, serum iron levels are not associated with PD. Ferroptosis can occur when there are shortages of glutathione, which leads to cell death and development of PD. Cystine uptake into cells is regulated by the cystine/glutamate antiporter which is regulated by cytosolic aconitase (ACO1). ACO1 upon losing an iron-sulfur cluster becomes IRP1. An increase in iron leads to inactivation of IRP1, increased activity of ACO1 and increased cystine uptake by the cystine/glutamate antiporter. Cystine is rapidly metabolized to L-cysteine upon entering cells. With cysteine in cells glutathione is synthesized which stops ferroptosis. Rotenone induces Parkinson’s disease in rats and mice. In PD there is loss of control of iron metabolism. Keeping iron levels constant in the gut can keep systematic iron levels constant as iron levels are regulated by absorption. Iron from iron carbonyl given three times a day to rats by gavage would keep iron absorption constant in the gut whereby iron levels and iron utilization systematically could be kept constant. Rotenone adversely affects complex 1 iron-sulfur proteins. Iron, however, can increase iron-sulfur cluster formation. Aconitase 1 levels, which becomes IRP1, upon loss of an iron-sulfur cluster are increased by supplemental iron. With IRP1 levels kept a constant but at lower levels iron utilization will be constantly regulated stopping dysregulation of complex 1 and stopping the neural damage done by rotenone.
Key words: Parkinson’s disease (PD); iron dysregulation; ferroptosis; aconitase 1 – ACO1; iron regulatory protein 1 – IRP1; cystine/glutamate antiporter.
Parkinson’s disease (PD) affects 2-3% of the population who are 65 years of age or older with PD being the second most common neurodegenerative disease (Poewe et al.,2017, Moustafa et al., 2016; Caligiore et al., 2016). In 2016 6.1 million individuals globally had Parkinson’s disease compared to 2.5 million individuals in 1990 where increased rates of PD were not due solely to an aging population as age-standardized prevalence rates of Parkinson’s disease also increased in the time period (Dorsey et al., 2018). In 2016, Parkinson’s disease caused 211, 296 deaths (95% UI 167,771-265,160) (Dorsey et al., 2018).
PD is a chronic neurodegenerative disorder with an inexorably progressive course (Poewe and Mahlknecht, 2009). Symptoms of PD can be treated, however, treatment does not slow the progression of PD (Poewe & Mahlknecht, 2009). Levodopa is the platinum standard for the treatment of symptoms of PD (LeWitt, 2015). The on-off effect, where periods of immobility and incapacity alternate with periods of improvement have been associated with levodopa therapy (Lees, 1989; Krishna et al., 2014; Moustafa et al.,2013; Moustafa, 2011; 2011; Phillips., et al., 2016). However, levodopa is not necessarily the only factor underlying the wearing off effect. For example, research on individuals in Ghana with PD that lack access to levodopa indicate that motor fluctuations in PD are associated with disease duration and increased dosages of levodopa but not with use of levodopa (Cilia et al., 2014). Further, one study found that long-term administration of levodopa is not toxic to normal human substantia nigra (Rajput et al., 2015). The on-off effect in PD is then at least partly due to basic biological dysregulations in PD rather than only use of levodopa.
Core symptoms of PD are difficulties in movement where there is shaking, rigidity, slowness of movement, postural instability and difficulties in walking (Opara et al., 2017; Crouse et al., 2016; Crouse and Moustafa, 2015 2015; Moustafa, 2014; Muralidharan et al., 2014, Muralidharan et al.,2017; Shine et al., 2013; Shine et al., 2013). In PD there is a loss of dopaminergic neurons in the substantia nigra that gives rise to movement disorders (Davie, 2008; Chakravarthy and Moustafa, 2018). PD is not exclusively a brain disease. There are gastrointestinal symptoms in PD where there can be abnormal salivation, dysphagia, nausea, constipation, and defecatory dysfunction (Edwards et al., 1991). There are aberrant α-synuclein aggregates in both the substantia nigra and the enteric system (Yan et al., 2018). Non-motor symptoms arise before motor symptoms in PD (Lebouvier et al., 2009). There have been various hypotheses about how PD spreads from the gut to the brain. Braak’s hypothesis holds that that in PD a pathogen enters through the nasal cavity, spreads to the gut and then via the enteric system spreads to the brain (Rietdijk et al., 2017). Another hypothesis holds that α-synuclein spreads prion-like from the gut to the brain (Kujawska and Jodynis-Liebert, 2018).
There are iron abnormalites in PD (Lingor et al., 2017). A meta-analysis shows that there are increased levels of iron in the substantia nigra of Parkinsonian autopsied brains (Wang, et al.,2016). There is increased iron (III) and total iron content in substantia nigra of autopsied brains of individuals who had PD (Sofic et al., 1988). While there are increased deposits of iron in brains of individuals who had PD a meta-analysis indicates that increased levels of iron in serum are associated with decreased risks of developing PD (Pichler et al., 2013). Another meta-analysis indicates there that there is no association between serum iron levels and PD (Mostile et al., 2017). The connection of iron to PD requires elucidation.
This paper proposes that ferroptosis in PD is due to low activity of aconitase 1 (ACO1) which decreases glutathione levels in cells leads to ferroptosis in PD. ACO1, an enzyme in tricarboxylic acid cycle (TCA cycle), is a dual function protein than upon a loss of a 4Fe-4S iron-sulfur cluster becomes iron regulatory protein 1 (IRP1) (Artymiuk & Green, 2006). The cystine/glutamate antiporter regulates levels of glutathione levels in cells (Lewerenz et al., 2013; Bridges et al., 2012). Increased activity of ACO1 increases synthesis of 2-oxoglutarate and glutamate in cytosols. The cystine/glutamate antiporter then transports glutamate out of cells and transports cystine into cells. Increasing activity of ACO1 with iron results in transport of cystine into the cells by the cystine/glutamate antiporter which increases levels of glutathione in cells (Lall et al., 2008; Harned et al., 2010). With increased levels of glutathione in cells ferroptosis can be stopped.
Ferroptosis is a form of regulated cell death which occurs due to an increase in lipid peroxidation and reactive oxygen species (Xie et al., 2016; Cao and Dixon, 2016). Ferroptosis can be triggered by inhibition of cystine uptake by the cystine/glutamate antiporter (Dixon et al., 2012; Zhang et al., 2019). Inhibition of cystine uptake by the cystine/glutamate antiporter results in decreased levels of glutathione. L-cysteine is the rate-limiting amino acid in the synthesis of glutathione (Taniguchi et al., 1989). Inactivation of glutathione peroxidase 4 by depletion of glutathione results in ferroptosis (Yang et al., 2014). Ferroptosis has been linked to cell death in PD (Guiney et al. 2017). Ferroptosis can kill dopaminergic neurons (Do Van et al., 2016)
Autopsied brains of individuals who had Parkinson’s disease showed almost a virtual absence of glutathione (Perry et al., 1982). There is a 40% decrease is glutathione levels in substantia nigra neurons in Parkinson’s disease (Sian et al., 1994). A meta-analysis shows that glutathione levels are significantly reduced in Parkinson’s disease (Wei et al., 2018). Decreased glutathione levels in PD could result in ferroptosis in PD killing dopaminergic neurons in the substantia nigra.
Aconitase 1/iron regulatory protein 1
ACO1 is a dual function protein where ACO1 upon losing a 4Fe-4S iron cluster becomes IRP1 (Artymiuk and Green, 2006). Increased levels of iron switch IRP1 to ACO1 whereas decreased levels of iron switch ACO1 to IRP1 (Haile et al., 1992; Henderson, 1996, Hentze and Kühn, 1996) Mammalian mitochondrial aconitase 2 (ACO2) has a functional iron-responsive element in the 5′-untranslated region of ACO2 mRNA which attenuates translation of ACO2 upon being bound by !RP1 (Kim et al., 1996). High levels of IRP1 decrease activity of both ACO1 and ACO2 and decrease activity of the TCA cycle. Increases in levels of iron, by switching IRP1 to ACO1, will increase energy production by the TCA cycle (see Figure 1 for explanation).
ACO1 increases levels of glutathione intracellularly by increasing cystine uptake by the cystine/glutamate antiporter (Lall et al., 2008). With increased activity cytosolic ACO1 due to increases in iron, there is increased synthesis of glutamate and increased secretion of glutamate (McGahan et al., 2006). With increased secretion of glutamate, there is increased import of cystine by cystine/glutamate antiporter which leads to increases in glutathione synthesis in cells. On-off symptoms in PD (discussed above) could be due to high but fluctuating levels of IRP1 which dysregulate iron regulated proteins and dysregulates the TCA cycle.
Iron Transporters in PD
In the small intestine iron is transported across the apical membrane of enterocytes via the divalent metal transporter 1 (DMT1) (Knutson, 2017). mRNA of DMT1 has an iron responsive element in the 3’untransaled region which increases stability of mRNA transcripts when the iron responsive element is bound by IRP1 (Gunshin et al., 2001). DMT1 is upregulated in the substantia nigra of PD patients (Salazar et al., 2008). In 6-OHDA-intoxicated mice IRP1 and DMT1 are upregulated while ferroportin is downregulated (Xu et al., 2018). Upregulation of DMT1 by 6-OHDA intoxication is dependent on iron response elements and iron regulatory proteins (Jiang et al., 2010).
Ferroportin is the only known iron exporter (Ward and Kaplan, 2012). mRNA of ferroportin has an iron-responsive element in the 5’ untranslated region which destabilizes mRNA of ferroportin decreasing translation of ferroportin when bound by IRP1 (Chen et al., 2005; McKie et al., 2000). Increased levels of DMT1 and decreased levels of ferroportin in PD are consistent with there being high levels of IRP1 in PD. Supplementation with iron would inactivate IRP1 (Henderson,1996).
IRP1 regulation of HIF2α
Hypoxia-inducible factor (HIF2α) mRNA transcripts have a 5’ iron regulatory element whereby HIF2α mRNA transcripts are destabilized by IRP1 (Anderson et al., 2013). The translation of ferroportin is induced by HIF2α (Taylor et al., 2011). HIF2α induces duodenal cytochrome b reductase (DCYTB) which is a ferric reductase involved in iron absorption (Anderson et al., 2013). HIF2α also induces the gene for DMT1 (Shah, et al. 2009). High activity of IRP1 in PD in the gut by decreasing translation of HIF2α will decrease transcription of ferroportin, decrease transcription of DMT1 and decrease transcription of duodenal cytochrome b reductase in the gut all of which could contribute to a systematic loss of control over iron metabolism.
Iron absorption and systematic iron utilization
There are no controlled mechanisms for the excretion of iron rather iron levels are is regulated by absorption, utilization, and recycling (Wallace, 2016) What is being postulated in PD is a loss of control over iron absorption and iron utilization. Imposing a tight constant control over iron absorption in the gut can result in re-regulation of iron utilization systematically. IRP1, which regulates DMT1 (Gunshin et al., 2001), ferritin (Rouault, et al., 2006) and ferroportin (Chen et al., 2005; McKie et al., 2000), is regulated by iron levels. Constant systematic levels of iron obtained by tight constant control of iron absorption can result in constant tight systematic control of iron utilization.
Rotenone inhibits complex I of the electron transport chain by dysregulating iron-sulfur clusters of complex I (Palmer, et al., 1968). Rotenone increases IRP1 levels (Mena et al., 2011). Silencing of IRP1 protects cells from death induced by complex I inhibition (Urrutia, et al., 2017). With iron abosrption tightly controlled IRP1 could be regulated both in the gut and systematically whereby deleterious effects of rotenone could be inhibited. Upon gaining an iron-sulfur cluster due to increases in iron IRP1 becomes aconitase 1 (Volz, et al. 2008). Dysregulation of ACO1/IRP1 by rotenone, which is an iron-sulfur protein, could be as important to the effects of rotenone as inhibition of complex I. The goal of supplemental iron would not be high systematic levels of iron but rather constant systematic levels of iron.
Blood levels of iron could be misleading as to the status of iron-sulfur proteins. Iron in blood would be iron in heme. Heme proteins could be normal is PD while iron-sulfur proteins could be dysregulated.
The TCA Cycle in PD
Postmortem brain tissue from PD patients shows increased IRP1 activity when compared to controls (Salazar, et al., 2006). With increased levels of IRP1 in PD there will be decreased levels of ACO1 which is an enzyme in the TCA cycle. Dysregulation of ACO1 could lead to dysregulation of the KGDHC. Activity of the enzymes in the TCA cycle enzymes are decreased is PD. The alpha-ketoglutarate dehydrogenase complex (KGDHC) was decreased by 50.5% in cerebellums of Parkinson’s brains compared to controls (Gibson, et al., 2003). The KGDHC in PD is more vulnerable to degeneration than Complex II, Complex III and Complex IV (Mizumo, et al.,1994). Decreases in activity of the KGDHC result in decreased energy production and can lead to neurodegeneration (Berndt, et al., 2012). A very prominent feature of Parkinson’s disease is on-off symptoms. Dysregulations of the TCA cycle and ATP production could result in deficits in energy which could result in on-off symptoms.
Chemically lesioned rats and mice and iron regulatory proteins
6-hydroxydopamine (6-OHDA) increases the levels of IRP1 in 6-OHDA lesioned rats while also upregulating DMT1 and downregulating ferroportin (Xu et al, 2018). 6-OHDA upregulates IRP1 (Zhang, et al., 2013; Jiang et al., 2010; Song et al., 2010). The inhibition of IRP1 is protective in 6-OHDA lesioned rats (Zhang et al., 2014). Decreasing levels of IRP1 and IRP2 in MES23.5 cells markedly blocked 6-OHDA-induced ferroportin down-regulation (Song et al., 2010). The most straightforward way to decrease IRP1 levels is to provide supplemental iron. Iron rapidly inactivates IRP1 (Henderson, 1996). Increased levels of iron in serum are associated with decreased risks of developing PD (Pichler et al., 2013). ACO1/IRP1 are iron-sulfur proteins. Dysregulation of ACO1/IRP1 could arise from difficulties is synthesizing iron-sulfur clusters, however, supplementation with iron could compensate for such difficulties resulting in increased activity of ACO1 and decreased activity of IRP1.
Rotenone, which is used as a pesticide, is an inhibitor of Complex 1 of the oxidative phosphorylation pathway that induces symptoms of Parkinson’s disease with Lewy bodies (Betarbet et al., 2000; Caboni et al., 2004). Intragastric administration of rotenone in mice can induce symptoms of Parkinson’s disease in mice (Pan-Montojo, et al., 2010). Environmental exposure with rotenone can result in symptoms of Parkinson’s disease in mice (Liu et al., 2015). Silencing IRP1 protects cells from death due to rotenone-induced inhibition of complex I (Urrutia et al., 2017). Rotenone increases IRP1 levels and decreases ACO1 levels (Mena et al., 2011).
Systematic administration of rotenone produces both central and enteric system pathology (Murakami et al., 2015).There are many gastrointestinal symptoms in PD, which often appear before motor symptoms (Edwards et al., 1991). ACO1 is highly expressed in the gastrointestinal system. Low activity of ACO1 in the gastrointestinal system in PD could give rise to gastrointestinal difficulties in PD.
Some clinical trials of iron chelators in PD are still ongoing (Martin-Bastida et al.,2017; Grolez et al., 2015). Deferoxamine, an iron chelator, induces IRP1 (Yoo et al., 2008). Iron chelators would decrease activity of the TCA cycle which could negatively affect PD. In vitro co-administration of iron with erastin, which inhibits the cystine/glutamate antiporter, can result in ferroptosis while co-administration of erastin with iron chelators can stop ferroptosis (Dixon et al., 2012), however, in vivo effects of iron supplementation without erastin could be much different. Iron given alone increases cystine uptake by the cystine/glutamate antiporter and increases glutathione levels in cells (Lall et al., 2008; Harned et al., 2010) which could stop ferroptosis. Iron is the proposed experiment is also being given by gavage so iron absorption is regulated by natural mechanisms. The goal of giving iron is not high iron levels but rather is constant absorption of iron and constant tight control over systematic iron utilization.
Proposed experiment where rats are given rotenone by subcutaneous injections and iron from iron carbonyl by gavage.
The experiments being proposed must be differentiated from other experiments with iron and rotenone. Iron carbonyl given by gavage has been given to neonatal rat pups from day 10 to day 17 post-partum with the rat pups injected with rotenone at 1 mL/kg each day beginning at age 14 weeks for 35 days (Yu, et al., 2017). In the experiment proposed rats will be pretreated with iron carbonyl by gavage prior to injections with rotenone where iron carbonyl by gavage will continue during injections with rotenone. In the experiments being proposed a goal, by pretreating rats with iron carbonyl by gavage prior to rotenone treatment and during rotenone treatment, is to regulate IRP1 levels and aconitase 1 during rotenone treatment. That iron in the proposed experiments is given by gavage is important as PD starts in the gut. Levels of iron in the body are controlled by absorption whereby iron metabolism in the brain is affected by iron given in the gut. Iron given by gavage would not bypass natural mechanisms to regulate iron levels. ACO1 and IRP1 must be appropriately regulated in the gut otherwise the TCA cycle and iron metabolism in the gut is dysregulated which can lead to a cascade of systematic effects whereby PD can develop.
The experiment being proposed must also be distinguished from an experiment where rotenone and iron were both given by i.p. injection which induced motor deficits and biochemical and neurotransmitter alterations in experimental rats (Sharma, et al., 2020). In the experiment proposed iron is given by gavage whereby systematic iron levels will be regulated using natural biological mechanisms. Another key difference is that in the proposed experiment levels of ACO1, IRP1 and the TCA cycle are regulated continuously in the gut where PD starts. As iron levels and iron utilization can be controlled by absorption dysregulation of iron metabolism in the gut could have a cascade of effects which could affect brains and lead to the development of PD. The fundamental difficulty in PD could be in iron-sulfur cluster formation, however, giving supplemental iron can increase iron-sulfur cluster formation. Iron must be given through out the day as the basic dysregulation is not being addressed.
Clinical trials in patients with PD where iron from from iron carbonyl is given are not warranted now. The paper proposes that rats be tested with the treatment. There would be 3 groups of rats. Group 1 would be given a placebo by gavage three times a day and sham injections. Group 2 would be given a placebo by gavage three times a day and subcutaneous injections of rotenone. Group 3 would be given iron from iron carbonyl by gavage three times a day and subcutaneous injections of rotenone. Subcutaneous injection of rotenone induces PD in rats (Zhang, et al., 2017).
The experiment would test whether PD starts from the enteric system and whether tight constant control of iron absorption can tightly regulate systematic iron utilization preventing PD from developing in rats. The iron is being given by gavage. If the environment of the enteric system can be arranged to prevent PD this would strongly indicate that the enteric system is key to the etiology and that the treatment of PD must take into account the environment of the enteric system. Rotenone is given by subcutaneous injection so as to give iron from iron carbonyl a window is which to work in the enteric system.
Rats being treated with iron would be treated with iron the entire course of the experiment and prior to any injections with rotenone. The iron in given three times a day as ACO1 in the gastrointestinal tract must be regulated throughout the day. ACO1 is highly expressed in the gastrointestinal tract (Uhlén, et al., 2015). Iron from iron carbonyl is slowly absorbed (Huebers, et al., 1986) As iron from iron carbonyl is slowly absorbed and as iron carbonyl is given by gavage three times a day iron absorption will be constant and systematic iron utilization will be constant. After rats in Group 2 clearly develop PD then Group 2 rats with PD would be given iron carbonyl by gavage, to see whether the treatment ameliorated already developed PD where on-off symptoms would be a particular focus.
The proposed experimentis designed to do three things. Iron from iron carbonyl given by gavage three times a day will constantly regulate iron absorption and thereby constantly regulate systematic iron utilization. Iron from iron carbonyl given by gavage three times a day will entrain systematic iron utilization. Iron from iron carbonyl given by gavage will, also l keep ACO1 levels constant whereby glutathione levels in cells will be kept constant preventing ferroptosis. There will be no iron overload as iron given by gavage will work with natural mechanisms to control iron absorption. The experiment will also test whether PD spreads from the enteric system and gives an explanation of how this could occur. ACO1 is an iron-sulfur protein that is highly expressed in the gastrointestinal tract. Dysregulation of iron metabolism in the enteric system could result in loss of control of systematic iron utilization. A target symptom to be examined is on-off symptoms which could be due to dysregulations of the TCA cycle.
Ferroptosis can lead to dopaminergic cell death. Ferroptosis in PD could be due to loss of control over iron utilization, leading to low activity of ACO1 and high activity of IRP1 decreasing the import of cystine into cells leading to low levels of glutathione in cells resulting in ferroptosis. Increased activity of ACO1, which is a cytosolic protein, results in increased synthesis of glutamate in the cytosol which is exported out of cells by the cystine/glutamate antiporter which at the same time imports cystine into the cytosol. Cystine in the cytosol is rapidly converted into L-cysteine in the cytosol, where glutathione levels increase preventing ferroptosis. Iron has been shown to result in ferroptosis when the cystine/glutamate transporter is blocked by erastin (Dixon et al., 2012). Results of In vivo experiments where erastin is not employed and iron is given by gavage to experimental animals could be much different than results of in vitro experiments where erastin is employed and experiments are done on cell cultures.
The fundamental difficulty in PD could be with synthesis of iron-sulfur clusters. Effectiveness of rotenone, which inhibits iron–sulfur clusters in complex I, in inducing PD points to iron-sulfur cluster dysregulation as being crucial to the devolpment of PD. While clinical trials in patients with PD, where supplemental iron is given, are not warranted now experiments on rats given rotenone and iron from iron carbonyl could re-orientate research in PD. Iron carbonyl is highly unlikely to be a completely effective treatment in patients with PD as patients could have much neuronal damage prior to being treated with iron carbonyl but the experiment could point to ways to prevent PD.
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