Iron Dysregulation in Parkinson's Disease: Focused on the Autophagy

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Review

Iron dysregulation in Parkinson’s disease: focused on the autophagy-lysosome pathway Lei-Lei Chen, Yu-Jv Huang, Jun-tao Cui, Ning Song, and Junxia Xie ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00390 • Publication Date (Web): 27 Dec 2018 Downloaded from http://pubs.acs.org on January 2, 2019

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ACS Chemical Neuroscience

Iron dysregulation in Parkinson’s disease: focused on the autophagy-lysosome pathway Lei-Lei Chen, Yu-Jv Huang, Jun-tao Cui, Ning Song* & Junxia Xie* Institute of Brain Science and Disease, Shandong Provincial Collaborative Innovation Center for Neurodegenerative Disorders, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders, Qingdao University, Qingdao, Shandong, China

*Corresponding author: Junxia Xie, Ph.D., Tel: 86-532-85955891, Email: [email protected] / [email protected] Ning Song, Ph.D., Tel: 86-532-83780035 Email: [email protected]

Mailing address: No. 308, Ningxia Road, Institute of Brain Science and Disease, Qingdao University, Qingdao 266071, China

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Abstract Parkinson’s disease (PD) is the second most common neurodegenerative disease and is characterized by dopaminergic neuron loss in the substantia nigra pars compacta

(SNc).

Although

both

iron

accumulation

and

a

defective

autophagy-lysosome pathway contribute to the pathological development of PD, the connection between these two causes is poorly documented. The autophagy-lysosome pathway not only responds to regulation by iron chelators/channels but also participates in cellular iron recycling through the degradation of ferritin and other iron-containing components. Previously, ferritin has been posited to be the bridge between iron accumulation and autophagy impairment in PD. In addition, iron directly interacts with α-synuclein in Lewy bodies, which are primarily digested through the autophagy-lysosome pathway. These findings indicate that some link exists between iron deposition and autophagy impairment in PD. In the present review, the basic mechanisms of the autophagy-lysosome pathway and iron trafficking are introduced, and then their interaction under physiological conditions is explained. Finally, we finish by discussing the dysfunction of iron deposition and autophagy in PD, as well as their potential relationship, which will provide some insight for further study.

Keywords: Parkinson’s disease; iron; transferrin receptor; autophagy-lysosome pathway; ferritin; α-synuclein.


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Introduction Iron is an essential micronutrient required for adequate erythropoietic function. In the peripheral organs, 70% of iron is stored in red blood cells or muscle cells in the form of hemoglobin or myoglobin, which are responsible for the transport and exchange of oxygen or carbon dioxide. Twenty-five to thirty percent of iron is stored in the liver, bone marrow or spleen in the form of ferritin or hemosiderin. In addition, a small portion of iron composes the active sites of some enzymes, including aconitase, cytochrome c, and ribonucleotide reductase (1). In the brain, the highest concentration of iron is found in the basal ganglia, especially the substantia nigra pars compacta (SNc). Due to the high energy demand of the brain, high iron levels are required for the generation of ATP by the electron transport chain in the mitochondria. Normally,

iron-dependent

neurotransmitters,

enzymes

including

are

dopamine,

responsible

for

noradrenalin,

the

synthesis

adrenaline

of and

5-hydroxytryptamine. Additionally, iron is involved in the process of myelination(2). However, excess iron usually causes oxidative stress and cellular damage, which are hallmarks of neurodegenerative diseases, such as Parkinson’s disease (PD) and Alzheimer's disease. Recent reviews regarding the pathophysiology of PD have highlighted that the death of dopaminergic neurons in the SNc is related to the accumulation of iron in this region (3-6). The autophagy-lysosome pathway is the primary pathway for the degradation of long-lived proteins, such as the disease-causing proteins α-synuclein and huntingtin, and the only pathway that clears out entire organelles, such as impaired mitochondria. Defective autophagy has been suggested to account for α-synuclein accumulation, which leads to dopaminergic neuron death in PD (7, 8). Autophagy also plays critical roles in maintaining cellular iron homeostasis via ferritin degradation. On the other hand, iron overload causes alterations in both the size and number of lysosomes, in addition to impairing autophagic flux(9). Although both iron deposition and autophagy impairment are suggested to account for the development of PD, the relationship between these two causes is poorly understood. In this review, we



summarize the current knowledge of the autophagy-lysosome pathway and cellular iron metabolism, as well as their interaction under physiological conditions. Finally, we review the current evidence regarding the impairment of iron deposition and autophagy in PD, and we discuss their potential connections.

Basic mechanisms of the autophagy-lysosome pathway Autophagy is an essential, conserved cellular catabolic pathway for the degradation of soluble/aggregated proteins, damaged organelles, lipids and pathogens via lysosomes. Based on the ways in which substrates reach the lysosome, the autophagy-lysosome pathway is divided into three types: macroautophagy (generally called autophagy), microautophagy and chaperone-mediated autophagy (CMA). During the process of autophagy, the isolation membrane (phagophore) initially elongates and engulfs portions of the cytoplasmic components to form the double-membraned autophagosome. Once completely formed, autophagosomes are transported to dock with lysosomes and form autolysosomes, which digest the enclosed cargos (10). Autophagosome completion Autophagosome completion is a critical step in the progression of the autophagy-lysosome pathway. This process is regulated by a conserved group of 16 autophagy-related genes (ATGs). ATGs were first identified in yeast(11), and their homologs in eukaryotes have also been discovered and extensively characterized. All these ATG proteins are functionally categorized into six groups: (1) the Atg1/ULK complex, (2) the class III PI3K complex, (3) the Atg2-Atg18/WIPI complex, (4) the Atg12 conjugation system, (5) the Atg8/LC3 conjugation system, and (6) Atg9 vesicles(12). In mammalian cells, overexpression of an inactive mutant of Atg4B, which is a protease that processes pro-LC3 paralogues, inhibits the lipidation of LC3 paralogues and causes a significant accumulation of unclosed autophagosomes (13). In mouse embryonic stem cells, the Atg12-Atg5 conjugate system, which localizes in the isolation membranes, was found to be associated with the elongation of the isolation membranes(14). In mutant mice that are unable to encode the specific E2


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enzyme of the Atg8 system, loss of Atg3 caused malformation of the autophagosomes, indicating an essential role of the Atg8 system in the proper development of autophagic isolation membranes(15). This accumulation of unclosed autophagosomes in the event of a defective ATG-conjugation system indicates the function of this system in the formation of autophagosomes. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is assembled from R-SNAREs and Q-SNAREs. Autophagosomal syntaxin 17 (STX17) usually serves as the Q-SNARE, which is recruited from the mitochondria and endoplasmic reticulum. In the

absence

of

the

ATG-conjugation

systems,

the

STX17-positive

autophagosome-like structures could be generated at a reduced rate and further fused with lysosomes. However, the degradation of the inner autophagosomal membrane was significantly delayed, indicating that ATG-conjugation systems are important for the efficient degradation of the inner autophagosomal membrane but not indispensable for the formation of autolysosomes (16). In mammals, the six members of the Atg8 family are divided into the LC3 and GABARAP (gamma-aminobutyric acid type A receptor (GABAAR)-associated protein) subfamilies. In HeLa cells with knockout of the LC3 and/or GABARAP subfamilies, the GABARAP subfamily was found to govern autophagosome-lysosome fusion, and neither GABARAP nor LC3 is required for autophagosome biogenesis, which is inconsistent with previous conclusions. In this study, even if all the Atg8 family members were lost, smaller autophagosomes could still be formed, indicating the dispensable role of Atg8 family in autophagosome formation(17). Autophagosome-lysosome fusion After the completion step, an autophagosome moves to fuse with a lysosome. First, the single lysosome membrane fuses with the outer autophagosomal membrane. Then, the lysosomal contents enter the space between the outer and inner membranes of the autophagosome, and the inner autophagosomal membrane is degraded in an LC3-dependent manner. Finally, the autophagosomal cargos are degraded by lysosomal enzymes, and the degradation products, including irons, sugars and amino acids, are transported out of the autolysosome through lysosome efflux transporters



for cellular recycling. Tethering factors, SNAREs and lipids are documented to play core roles in the fusion machinery between autophagosomes and lysosomes. Tethering factors include the HOPS (homotypic fusion and vacuolar protein sorting) complex, RAB7 and adaptors. In Drosophila, the entire HOPS complex, including VPS11, VPS16, VPS18, VPS33A, VPS39 and VPS41, is critical for the fusion of autophagosomes with lysosomes (18). The HOPS complex specifically interacts with STX17 on autophagosomes and facilitates autophagosome-lysosome fusion mediated by the trans-SNARE complex (18, 19). RAB7, which is a small GTPase and can bind to membranes and membrane-anchored proteins, binds to HOPS through VPS39 and VPS41 and forms the RAB7-HOPS complex (20), which contributes to the membrane fusion(21, 22). The SNARE complex is another key component required for autophagosome-lysosome fusion. The autophagosomal STX17 functions as Q-SNARE and interacts with SNAP29 and VAMP8, which is a lysosomal R-SNARE, to form the trans-SNARE complex and contributes to the autophagosome-lysosome fusion (23). During this process, ATG14 binds to STX17 and stabilizes the STX17-SNAP29 binary SNARE complex on autophagosomes and then promotes autophagosome-lysosome fusion (24). Lipids including PtdIns3P, PtdIns4P and PtdIns(3,5)P2, which are phosphorylated at the 3-, 4-, or 5-position of the inositol ring phosphatidylinositol, have been found to be involved in autophagosome-lysosome fusion(25-27). In neuronal cells, the decreased levels of PtdIns(3,5)P2 on lysosomes that is mediated by INPP5E (inositol polyphosphate-5-phosphatase E) actives CTTN (cortactin) and enhances the stabilization of actin filaments, which facilities autophagosome-lysosome fusion (25). Lysosomal TECPR1 reportedly binds to PtdIns3P only when it forms a complex with the Atg12-Atg5 conjugate, which promotes the fusion between autophagosomes and lysosomes (28). In addition to PtdIns3P, the generation of PtdIns4P on the autophagosomes, which is mediated by PI4KⅡα, is also essential for fusion with lysosomes (27).

Cellular & subcellular iron metabolism


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Iron transfer from the periphery into the brain occurs through brain capillary endothelial cells of the blood-brain barrier and the choroid plexus epithelium, both of which are characterized by tight junctions and function as a physical barrier for molecule exchange. Comprehensive reviews of the process of iron uptake from the periphery have been written by Hare et al.(2) and Biasiotto et al.(29). Cellular iron homeostasis depends on the process of iron import, storage and iron export (Figure 1). The major mechanism of cellular iron uptake is through the transferrin-transferrin receptor (Tf-TfR) system. Briefly, two Fe3+ ions are loaded onto a single unit of Tf to form a Tf-Fe2 complex, which then binds to TfRs on the cell surface and facilitates endocytosis. At neutral pH, Tf dissociates from the TfRs and returns to iron circulation. To date, two receptors, transferrin receptor 1 (TfR1) and transferrin receptor 2 (TfR2), have been identified. TfR1 is a ubiquitously expressed receptor protein that has a 30-fold higher affinity for Tf-Fe2 than TfR2 has. TfR2 shares 45% amino acid identity with TfR1 and shows expression in dopaminergic neurons, hepatocytes, duodenal crypt cells, and erythrocytes (2, 30). In a condition of iron overload when the capacity of Tf to bind iron is saturated, a very small proportion of iron, consisting of non-transferrin-bound iron, is transported by low-molecular-weight ligands, such as ascorbate ions and citrate, and by the recycling of ATP, albumin and ferritin(31). In the brain, iron is transported in a cell-specific manner. In neurons, which express both TfR1 and divalent metal transporter 1 (DMT1), iron is imported mainly through receptor-mediated endocytosis, and a small proportion

of

iron

is

from

non-transferrin-bound

iron.

However,

non-transferrin-bound iron contributes the major source of iron in astrocytes due to their lack of TfR1. In oligodendrocytes, iron is also imported in a TfR1-independent manner, and it participates in the synthesis of myelin (32, 33). Both in mice and in primary cultured oligodendrocytes, reduced iron uptake was found after knockdown/ or knockout of DMT1 expression, suggesting that DMT1 is required for the iron uptake of oligodendrocytes (33). The only iron export pathway is through ferroportin 1, which transports ferrous iron out of cells. As only ferric iron can be bound by Tf and return to circulation, a ferroxidase is necessary in this progress to oxidize the



Page 8 of 26

ferrous iron to ferric iron (2). In addition to the classical ferroxidases ceruloplasmin and hephaestin, amyloid precursor protein (APP) was also found to act as a ferroxidase and oxidize ferrous iron (34, 35). In mice with ablation of hephaestin or ceruloplasmin, disordered iron homeostasis was found in the brain, indicating the distinct role of ferroxidase in maintaining iron homeostasis in the brain (36). The storage of cellular iron is mainly dependent on ferritin, which removes excess iron from the cytoplasm or releases iron for the cellular synthesis of iron-containing structures

(37).

Iron

release

from

ferritin

occurs

mainly

through

the

autophagy-lysosome pathway and the ubiquitin-proteasome system (details will be discussed below) (38). Both lysosomes and mitochondria play critical roles in cellular iron metabolism, and iron metabolism in mitochondria has been thoroughly discussed in our previous review paper (5). In the present review, we mainly focus on iron metabolism in lysosomes. Lysosomes contain a large amount of iron, which is recycled from the degradation of iron-containing substrates (such as ferritin and mitochondrial components) via autophagy or from the endocytosis of red blood cells by macrophages. Because of the acidic and reducing environment, iron usually exists as a ferrous form with strong reductive activity in the lysosome, which can catalyze the Fenton reaction that metabolizes hydrogen peroxide and results in the generation of reactive oxygen species (ROS). Following permeabilization of the lysosomal membrane, an increase in cellular oxidative stress is observed (39, 40). In lung epithelial cancer cells, the autophagy-lysosome pathway was hyperactivated by the application of iron oxide nanoparticles, which is correlated with ROS generation and mitochondrial damage (41). The relocation of iron from lysosomes to the cytoplasm is mainly mediated by DMT1 and TRPML1 (mucolipin-1/MCOLN1) (42), and the primary manner of iron relocation likely depends on the source of iron. For example, when the iron is transported through Tf-TfR recycling, the release of iron from the lysosome occurs primarily via DMT1, which is localized in the early or late endosome (2). TRPML1 is a novel endolysosomal Fe2+ release channel that is primarily localized on the late endosome and lysosome membranes. In TRPML1-deficient cells,


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the level of iron available for chelation is reduced, accompanied by evidence of decreased cytosolic Fe2+ levels but increased intralysosomal Fe2+ levels and accumulated lipofuscin-like molecules (42). On the other hand, TRPML1 and natural resistance-associated macrophage protein 1 (Nramp1), which is another release channel that participates in the recycling of senescent red blood cells (43), might manage the release of iron from the degradation of the Ft-Fe2 complex or iron-containing cargos through the autophagy-lysosome pathway. Of course, the mechanisms of iron relocation mediated by DMT1 and TRPML1 might coexist, just as they do in neurons. In cells with dual expression of DMT1 and TRPML1, the inhibition of DMT1-mediated endosomal iron release might be tolerated because of the complementary role of TRPML1 in the iron entering the late endocytic pathway (44, 45). TPCNs (two-pore channels) are a family of intracellular cation channels consisting of TPCN1 and TPCN2, which are uniquely localized to endolysosomal structures and are well known to be permeable to calcium and sodium(46). Recently, TPCNs were also found to mediate iron release from endolysosomes, which is modulated by NAADP-AM and Ned-19 (9).

Iron and the autophagy-lysosome pathway Iron recycling through the autophagy-lysosome pathway Although the areas of iron metabolism and the autophagy-lysosome pathway have both been extensively investigated, the relationship between them is poorly explored (47). Recently, the autophagy-lysosome pathway was suggested to play a critical role in maintaining iron homeostasis for the normal physiological functions of cells (48). As mentioned above, iron release from ferritin occurs mainly through lysosomal digestion (Figure 1), with the ferritin entering the lysosomes through autophagy (49). Proteasomes are also involved in cytosolic ferritin degradation, and the primary pathway of ferritin degradation depends on the depletion of cellular iron. When cytosolic iron was depleted via expression of iron exporter ferroportin or membrane-permeable iron chelators (deferasirox, DFX; deferiprone, DP), ferritin was degraded through the proteasome (50). Alternatively, ferritin degradation that is



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induced by a poorly permeable iron chelator (deferoxamine, DFO) occurs in lysosomes. After inhibition of the autophagy-lysosome pathway via 3-methyladenine, incubation of DFO resulted in degradation of ferritin by proteasome (38, 51), indicating a compensatory role of proteasomes in the iron release from ferritin. In addition to ferritin, iron-binding proteins, such as metallothioneins and Hsp70, also function as endogenous iron chelators to keep labile iron at a minimum (52, 53). They enter lysosomes through autophagocytosis and temporarily chelate intralysosomal redox-active iron, which process will protect cells from Fenton reactions and lysosomal membrane integrity (53-55). Therefore, the autophagocytosis of iron-binding protein is suggested to increase the concentration of iron in lysosomes, which play an important role in balancing the redox status of cells (47). Because the current knowledge of the autophagy-lysosome pathway in iron metabolism is still limited, mainly focus on the degradation of iron storage or binding proteins, further study directed at these experimental questions is desirable. Regulation of autophagy by iron chelator/channel Chelating excessive iron is a direct and effective strategy to reduce the alterations induced by iron overload, including oxidative stress, protein aggregation, and protein misfolding.

Several

iron

chelators

have

been

reported

to

regulate

the

autophagy-lysosome pathway. In SH-SY5Y cell models of Parkinson's disease treated with rotenone, when the gene of HIF-1α was inhibited, DFO-induced autophagy was suppressed, indicating that DFO induces autophagy in a HIF-1α-dependent manner (56). In myeloma cells, both DFO and DFX induce autophagy through the mTOR pathway, with evidence of decreased activity of p70S6 kinase, which is downstream of mTOR (57). However, in breast cancer cells, DFO was found to inhibit the degradation step of the autophagy-lysosome pathway, with evidence of increased LC3-Ⅱ and autophagosome numbers (58). These distinct effects of DFO on autophagy may depend on the origin of cells. Dp44mT (di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone) is another iron chelator, which is more effective relative to DFO and forms a redox-active complex with iron and copper. Increased


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LC3-Ⅱ and accumulation of autophagic substrates and receptor p62 indicated that Dp44mT induced autophagosome formation; however, autophagosome degradation and fusion with lysosome was inhibited by Dp44mT (47, 58). Dp44mT was also found to activate AMPK, which is vital for autophagic activation (59). Recently, through a spectrophotometry assay, curcumin, a well-investigated autophagy inducer, was found to form a complex with ferric ammonium citrate (FAC), with evidence of decreased absorbance at 430 nm when FAC was added to neutral solution, indicating an iron-chelating property of curcumin. Under iron deprivation conditions, TfR1 is usually induced via iron regulatory protein 1 (IRP1)-mediated mRNA stabilization (60). In castration-resistant prostate cancer cells with curcumin treatment, increased TfR1 and active IRP1 reflected the reduction of intracellular iron levels, which was a result of iron deprivation by curcumin (61). These findings suggest that iron chelators are involved in the regulation of autophagy. As we discussed under cellular/subcellular iron metabolism, TRPML1 and TPCNs that function as iron release channels are also recognized to be involved in the autophagy-lysosome pathway. In TRPML1-deficient fibroblasts, which are from mucolipidosis type IV patients, the formation of autophagosomes is increased; however, the fusion of autophagosomes with lysosomes is delayed, indicating the requirement of TRPML1 for the fusion of autophagosomes with lysosomes (62). As the binding of p62/SQSTM1 to ubiquitin and LC3 Ⅱ is required for both the formation

and

degradation

of

polyubiquitinated

bodies

through

the

autophagy-lysosome pathway, upregulated p62 has been demonstrated to be a result of impaired autophagy. In TRPML1-deficient mouse neurons, both alterations in LC3-II clearance via lysosomal hydrolases and increases in p62/SQSTM1 indicate that the function of the autophagosome-lysosome pathway is abnormal (63). Meanwhile, the loss of TRPML1 contributes to the impairment of CMA. The interaction between TRPML1 and the lysosomal chaperone complex is required for the function of CMA (63). The activity of TPCN2, a newly identified endolysosomal iron release channel, is modulated by RAB7A, which participates in the trafficking steps of the autophagy-lysosome pathway, indicating a potential involvement of the



TPCN2 channel in the autophagy-lysosome pathway (9).

Impairment of the autophagy-lysosome pathway in PD Aberrant aggregation of α-synuclein in Lewy bodies has been identified as a pathological trait of PD. α -Synuclein is a natively unfolded protein that is enriched at the presynaptic terminals in the central nervous system and participates in vesicular release (64). In its native form, α-synuclein can be degraded by both the autophagy-lysosome pathway and the ubiquitin-proteasome system (65). However, oligomers and aggregates of α-synuclein are mainly degraded through the autophagy-lysosome pathway because they cannot pass through the proteasome barrel (66-68). In the 15-amino-acid sequence of α-synuclein, there is an overlapping variation of the KFERQ motif, which can be recognized by Hsc70 for CMA. After binding to Hsc70, α-synuclein binds to lysosomal-associated membrane protein type 2A (LAMP-2A) and be transported into the lysosome, where degradation occurs. Impairment of the autophagy-lysosome pathway is suggested to account for the abnormal aggregation of α-synuclein in PD, which was found in the postmortem brains of PD patients and in in PD animal models, with evidence of autophagosome accumulation and lysosomal malfunction (69-71). α-Synuclein blocks autophagy by reducing the activity of RAB1A, which functions as a GTPase and participates in the early secretory pathway (72). The inhibition of RAB1A disturbed ATG9 localization and autophagosome formation. Although wild-type α-synuclein was selectively transported into lysosomes and degraded by the CMA pathway, mutants of α-synuclein (A53T and A30P) can occupy lysosomal membrane receptors and reduce the degradation of other CMA substrates, followed by accumulation and cytotoxicity (73). These mutants strongly bind to lysosomal receptors, and they cannot pass through the membrane and therefore cannot be degraded by CMA. In addition to α-synuclein, several mutations related to familial PD, including ubiquitin carboxy terminal hydrolase L1 (UCHL1), leucine-rich repeat kinase 2 (LRRK2), and DJ-1, were also found to block or reduce the activity of CMA (74). Mutant UCHL1 binds to the lysosome-associated membrane protein (LAMP)-2A and inhibits CMA activity.


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The formation of the CMA translocation complex was found to be disrupted by mutant LRRK2 (75). The autophagy-lysosome pathway is the only pathway that clears our entire organelles, including impaired mitochondria, and the process of eliminating damaged mitochondria via autophagy is called mitophagy. PD-associated proteins, including PINK1 and Parkin, also regulate mitophagy. PINK1 is located in the outer membrane of mitochondria. Parkin is an E3 ubiquitin ligase that is encoded by PARK2. When the mitochondrial membrane potential is depolarized, PINK1 is activated, and Parkin is recruited to mitochondria, where it ubiquitinates the outer mitochondrial membrane proteins, which promotes the degradation of damaged mitochondria through autophagy. However, this process was impaired by mutations in PINK1 or Parkin (76). Mutations in PINK1 reduced its binding activity to Parkin, resulting in the failure of mitochondrial translocation by Parkin and the accumulation of damaged mitochondria, which may contribute to the development of PD. Both pharmacological and genetic enhancement of autophagy promotes the clearance of α-synuclein and reduces PD pathology (77-79). These findings indicate the important roles and involvement of the autophagy-lysosome pathway in the pathological progress of PD.

Iron deposition in PD Iron plays a positive role in oxidation-reduction reactions because of its oxidative nature. However, it is also potentially harmful in an oxygen-rich environment. Therefore, it is necessary to precisely regulate the process of iron metabolism. In 1924, iron deposition was first identified in the basal ganglia of PD patients’ brains (80). Although iron accumulates in the normal aging brain, this process seems to be exacerbated in PD; this change is observable using magnetic resonance imaging (MRI) and could function as a potential diagnostic biomarker (81). According to the MRI results of PD patients, iron deposition occurs earlier than the clinical symptoms, and the relative risk of PD in persons with increased iron in the substantia nigra was almost 17 times higher the risk in normal people (82). Iron injection could result in an almost 95% selective decrease in striatal dopamine and induce dopamine-related



behavioral parkinsonism, including spontaneous movements in a novel space and rearing and ipsilateral rotation in rats (83). In addition, an iron chelator, which can relieve iron overload, is reported to improve the symptoms in PD animal models with iron deposition in the SNc. Enhancing the export of iron via peripheral infusion of ceruloplasmin attenuated neurodegeneration in PD mouse model (84). All of these findings indicate a contribution of iron deposition to the pathogenesis of PD. α-Synuclein is the major component of Lewy bodies in PD and directly interacts with iron. In the SNc neurons of PD patients, the colocalization between redox-active iron and Lewy body was identified. However, redox-active iron is not stained in neocortical Lewy bodies in cases of the Lewy body variant of Alzheimer’s disease (85). With the methods of electrospray mass spectrometry, cyclic voltammetry, and fluorescence spectroscopy, Fe2+ was detected to bind with α-synuclein and form an α-synuclein-Fe2+ complex, which could be oxidized to α-synuclein-Fe3+ complex (86). Both α-synuclein and iron can affect each other simultaneously. A low (micromolar) concentration of ferric iron accelerated the aggregation of α-synuclein and the formation of oligomers (87). When primary rat midbrain or PC12 cell lines were treated with excess iron, overexpression of α-synuclein resulted in an increased level of intracellular iron, as well as iron redistribution from the cytoplasm to the perinuclear region where α-synuclein inclusions are abundant (87). Mutation or dysregulation of iron-related proteins, which participate in the storage/traffic of cellular iron, has been shown to cause iron accumulation in PD (88). The level of ferritin, which acts as a primary iron storage protein, is significantly decreased in the brain of postmortem PD patients, indicating lost capacity of iron storage, which leaves iron in an unbound and potentially toxic state (89). Cellular iron is controlled by iron regulatory proteins 1 and 2 (IRP1/2), which regulate the expression of iron-related proteins. Defects in Ireb2, a gene that regulates the expression of IRP2 lead to iron accumulation in mice (90). Elevated DMT1 is found in the SNc of PD patients, as well as in the PD animal model, facilitating the import of iron by dopaminergic neurons and/or microglia (91-93). A genetic analysis also revealed that the genetic variations in both Tf and TfR2, which form the Tf/TfR2


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complex and mediate iron import into mitochondria, are associated with the risk of PD (94). Impairment of iron export could also result in iron accumulation. Both in cell models and in animal models of PD, the level of ferroportin 1 is downregulated, which is suggested to account for the nigral iron accumulation (95). Reduced activity of ceruloplasmin, which functions as an iron-export ferroxidase, was found in the SNc of idiopathic PD patients (84). Mice that are deficient in ceruloplasmin developed parkinsonism-like symptoms, which were rescued by the administration of an iron-chelating agent. Selectively decreased tau protein, which can transform into APP and mediate iron export, is also found in the SNc of PD patients (96).

Prospects Although both iron deposition and autophagy dysfunction are characteristics of PD, the underlying relationship between them is still elusive. The autophagy-lysosome pathway not only represents the primary pathway for α-synuclein clearance but also takes part in in the recycling of iron through ferritin degradation. It has been hypothesized that ferritinophagy provides a bridge between autophagy impairment and iron homeostasis dysfunction. However, the situation seems to be more complicated. Iron deposition results in defective autophagy, as well as increased α-synuclein levels and ROS, in both primary dopaminergic neurons and SH-SY5Y cells (97). On the other hand, in SH-SY5Y cells with stable expression of both DMT1 and α-synuclein, increased uptake of Fe2+ caused excessive autophagy and cell death (98). Enhanced autophagy is beneficial for the purpose of promoting the clearance of α-synuclein aggregation. However, autophagy promotes ferroptosis by ferritin degradation (99). Autophagy is thus believed to be a double-edged sword in PD. Therefore, attention should be devoted to further investigation of these important and significant questions regarding how autophagy and iron metabolism modulate each other, as well as how they could be targeted to correct iron deposition and the defective autophagy-lysosome pathway in PD.



Abbreviations APP ATG CMA CTTN DFO DFX DMT1 DP Dp44mt

amyloid precursor protein autophagy-related gene chaperone-mediated autophagy cortactin deferoxamine desferasirox divalent metal transporter 1 deferriprone di-2-pyridylketone 4, 4-dimethyl-3-thiosemicarbazone ferric ammonium citrate gamma-aminobutyric acid type A receptor (GABAAR)-associated protein homtypic fusion and protein sorting iron regulatory protein inositol polyphosphate-5-phosphatase E lysosomal-associated membrane protein type 2A leucine-rich repeat kinase 2 magnetic resonance imaging natural resistance-associated macrophage protein 1 Parkinson’s disease substantia nigra pars compacta Soluble N-ethylmaleimide-sensitive factor attachment protein receptor syntaxin 17 transferrin transferrin receptor two-pore channels mucolipin-1 ubiquitin carboxy terminal hydrolase L1

FAC GABARAP HOPS IRP INPP5 LAMP-2A LRRK2 MRI Nramp1 PD SNc SNARE STX17 Tf TfR TPCNs TRPML1/MCOLN1 UCHL1

Author Contributions All authors contributed to writing the manuscript. All authors have given approval to the final version of the manuscript.


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Funding Information This work was supported by grants from National Natural Science Foundation of China (81430024, 31771124, 31871049, 31800893), China Postdoctoral Science Foundation (2017M622128).

The authors declare no competing financial interest.

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(2010)

Neuroprotective

mechanism

of

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Figure legend Figure 1. Cellular and lysosomal iron trafficking and recycling. Iron import into cells occurs mainly through divalent metal transporter-1 (DMT1) or transferrin receptor (TfR1)-mediated endocytosis. The only iron export pathway is through ferroportin-1 (FPN1) in the presence of ferroxidases. Iron in lysosomes can originate from either TfR1-mediated endocytosis or the degradation and recycling of ferritin or mitochondrial components. The relocation of iron from lysosomes to the cytoplasm is achieved by DMT1, TRPML1 (mucolipin-1/MCOLN1) or TPNs (two-pore channels).


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Table of Contents Graphic Cellular and lysosomal iron trafficking and recycling. Iron import into cells occurs mainly through divalent metal transporter-1 (DMT1) or transferrin receptor (TfR1)-mediated endocytosis. The only iron export pathway is through ferroportin-1 (FPN1) in the presence of ferroxidases. Iron in lysosomes can originate from either TfR1-mediated endocytosis or the degradation and recycling of ferritin or mitochondrial components. The relocation of iron from lysosomes to the cytoplasm is achieved by DMT1, TRPML1 (mucolipin-1/MCOLN1) or TPNs (two-pore channels).



Cellular and lysosomal iron traffic and recycle. Iron import into cells is mainly through divalent metal transporter-1 (DMT1) or transferrin receptor (TfR1)-mediated endocytosis. The only iron export pathway is through ferroportin-1 (FPN1) with the presence of ferroxidases. Iron in lysosomes could be either from TfR1mediated endocytosis or recycled from the degradation of ferritin or mitochondrial components. The relocation of iron from lysosomes to cytoplasma is achieved by DMT1, TRPML1 (mucolipin-1/MCOLN1) or TPNs (two-pore channels).


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Iron Dysregulation in Parkinson's Disease: Focused on the Autophagy

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