Role of heavy metals (copper (Cu), arsenic (As), cadmium (Cd), iron (Fe) and lithium (Li)) induced neurotoxicity
重金属（铜 (Cu)、砷 (As)、镉 (Cd)、铁 (Fe) 和锂 (Li)）诱导神经毒性的作用

1. Introduction 一、简介

Parkinson's disease (PD) is a neurodegenerative disorder distinguished by the dopaminergic decline in the substantia nigra pars compacta of basal ganglia (Jayaramayya et al., 2020; Venkatesan et al., 2021b). The approximate incidence of PD in urbanized countries is 1% in people aged above 60 years and 3% in ≥80 years (Dhivya et al., 2016; Ullah et al., 2021). PD increasingly affects motor control symptoms like tremor, rigidity, bradykinesia, and postural imbalance, as well as non-motor issues like olfactory impairment, constipation, and insomnia (Mahalaxmi et al., 2021; Mohana Devi et al., 2020; Venkatesan et al., 2021a). Although multiple factors and mechanistic approaches have been ascertained, proper conclusive etiology of PD is not yet stated. Studies have reported that the genetic background of PD accounts for only 5–10% while in most cases environmental factors may be the influencer for the disease condition (Bjorklund et al., 2018; Dhivya and Balachandar, 2017; Iyer et al., 2021). According to an epidemiological survey heavy metals such as mercury, lead, manganese, copper, iron, aluminium, bismuth, thallium and zinc have a pivotal role in the pathogenesis of PD (Bjorklund et al., 2018). Metals are generally characterized as essential and non-essential metals which involves in biological processes such as protein modification, electron transport, oxygen transport, redox reactions, cell adhesion, to name a few. Excessive levels of metals could induce detrimental effects such as oxidative stress, protein misfolding, mitochondrial dysfunction, autophagy dysregulation and apoptosis (Wright and Baccarelli, 2007). According to a recent study, the authors have stated that these metals can also trigger the expression of genes which might result into neurodegeneration (Ullah et al., 2021). These molecular shortcomings lead to neurodegeneration which directs to motor abnormalities, cognitive disabilities, memory and learning disabilities (Chen et al., 2016; Devi et al., 2021; Venkatesan et al., 2020). Metals can contribute for diseases either through metal toxins or by decline in essential metals (Piao et al., 2017; Renu et al., 2021). Various studies have shown noteworthy relation between PD and metals exposure (Coon et al., 2006; Gorell et al., 1997). The main routes of exposure are occupational, medications, pollution and in dental metals restorations (Dadar et al., 2014, 2016; Petersen et al., 2008). Here, in this present review we address the neurotoxic potential of arsenic (As), cadmium (Cd), copper (Cu), lithium (Li) and iron (Fe) metals in neurodegeneration and its probable mechanisms in PD pathogenesis.
帕金森病(PD) 是一种神经退行性疾病，其特征是基底神经节黑质致密部多巴胺能下降（ Jayaramayya 等，2020 ； Venkatesan 等，2021b ）。在城市化国家，60 岁以上人群的 PD 发病率约为 1%，≥80 岁人群的 PD 发病率为 3%（ Dhivya 等，2016 ； Ullah 等，2021 ）。 PD 越来越多地影响运动控制症状，如震颤、僵硬、运动迟缓和姿势不平衡，以及非运动问题，如嗅觉障碍、便秘和失眠（ Mahalaxmi 等，2021 ； Mohana Devi 等，2020 ； Venkatesan 等）等，2021a ）。尽管多种因素和机制方法已被确定，但帕金森病的确切病因尚未明确。研究表明，PD 的遗传背景仅占 5-10%，而在大多数情况下，环境因素可能是疾病状况的影响因素（ Bjorklund 等，2018 ； Dhiv​​ya 和 Balachandar，2017 ； Iyer 等，2021） ）。 根据流行病学调查，汞、铅、锰、铜、铁、铝、铋、铊和锌等重金属在帕金森病的发病机制中发挥着关键作用( Bjorklund et al., 2018 )。金属通常分为必需金属和非必需金属，涉及生物过程，例如蛋白质修饰、电子传输、氧传输、氧化还原反应、细胞粘附等。金属含量过高可能会引起有害影响，例如氧化应激、蛋白质错误折叠、线粒体功能障碍、自噬失调和细胞凋亡（ Wright 和 Baccarelli，2007 ）。根据最近的一项研究，作者指出，这些金属还可以触发可能导致神经变性的基因表达（ Ullah 等人，2021 ）。这些分子缺陷会导致神经变性，从而导致运动异常、认知障碍、记忆和学习障碍（ Chen et al., 2016 ； Devi et al., 2021 ； Venkatesan et al., 2020 ）。 金属可能通过金属毒素或必需金属的减少而导致疾病（ Piao 等，2017 ； Renu 等，2021 ）。各种研究表明局部放电和金属暴露之间存在值得注意的关系（ Coon 等人，2006 年； Gorell 等人，1997 年）。主要暴露途径是职业、药物、污染和牙科金属修复（ Dadar 等，2014，2016 ； Petersen 等，2008 ）。在这篇综述中，我们探讨了砷 (As)、镉 (Cd)、铜 (Cu)、锂 (Li) 和铁 (Fe) 金属在神经变性中的潜在神经毒性及其在 PD 发病机制中的可能机制。

2. An overview of heavy metals role on neurodegeneration
2. 重金属对神经退行性变的作用概述

The ideal levels of metals and their homeostasis in each organ is necessary for sustaining vital functions. Nutritional deficits and metabolic disorders display plausible cause-and-effect association in many detrimental conditions. Though many metal ions are essential but its excess accumulation can be extremely toxic and probably fatal. Neurodegeneration is considered to be the most frequent manifestation of metal toxicity in many neurological disorders which functions in the onset and progression of disorders including PD, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), stroke and Wilson's disease (WD) (Jomova et al., 2010). The role of metals in brain disorders is supported by the evidences from biochemical studies, molecular genetics and imaging. The studies have encouraged in understanding the function of metals as well as in the development of therapeutic approaches in treating brain disorders (White et al., 2015). The prominent metal which is involved in neurodegenerative disorders that causes substantial change is the Fe. Studies on Fe transport and its role in neurotransmission, dyshomeostasis, oxidative stress and reactive oxygen species (ROS) formation have been explored widely showing the genetic insights on altered Fe in neurodegeneration (Hare et al., 2013; Mariani et al., 2013; Muhoberac and Vidal, 2013). Other metals such as Cu have a probable role in neuronal survival and synaptic function (Castro et al., 2014; Dringen et al., 2013). Zinc (Zn) is also described to exacerbate neuronal death either by Zn influx or its deficiency (Szewczyk, 2013). Similar to Cu, Zn and Fe, metals such as manganese (Mn), mercury (Hg) have also intoxicated with high doses in the brain resulting with neurodegeneration (Chen et al., 2016).
每个器官中金属的理想水平及其稳态对于维持重要功能是必要的。营养缺乏和代谢紊乱在许多有害条件下显示出看似合理的因果关系。尽管许多金属离子都是必需的，但其过量积累可能会产生剧毒，甚至可能致命。神经退行性被认为是许多神经系统疾病中金属毒性最常见的表现，其在帕金森病、阿尔茨海默病 (AD)、肌萎缩性脊髓侧索硬化症 (ALS)、中风和威尔逊病 (WD) 等疾病的发作和进展中起作用（ Jomova等人，2010 ）。生化研究、分子遗传学和成像证据支持金属在脑部疾病中的作用。这些研究鼓励人们了解金属的功能以及开发治疗脑部疾病的治疗方法（ White et al., 2015 ）。参与神经退行性疾病并引起实质性变化的主要金属是铁。关于铁转运及其在神经传递、稳态失衡、氧化应激和活性氧（ROS）形成中的作用的研究已得到广泛探索，显示了神经退行性变中铁改变的遗传见解（ Hare等人，2013年； Mariani等人，2013年）, 2013 ;穆霍贝拉克和维达尔，2013 ）。其他金属（例如 Cu）可能在神经元存活和突触功能中发挥作用（ Castro 等人，2014 ； Dringen 等人，2013 ）。锌 (Zn) 也被描述为因锌流入或缺乏而加剧神经元死亡( Szewczyk, 2013 )。与铜、锌和铁类似，锰（Mn）、汞（Hg）等金属在高剂量时也会使大脑中毒，导致神经退行性变（ Chen et al., 2016 ）。

According to studies it is reported that protein profiling especially in metals like Pb, AS and methylmercury (MeHg) can induce oxidative stress, RNA splicing and ubiquitin which can lead to alterations in mitochondrial dysfunction resulting into neurodegenerative diseases (Karri et al., 2020). Pb has been linked to neurodegeneration, apoptosis, epigenetic alterations, and essential metal disturbances in the brain (Bakulski et al., 2020). According to a previous study, it was found that due to accumulation of Hg in the nerve endings, ganglia and central nerve cells triggers PD pathogenesis (Bjorklund et al., 2018). Metal exposure on dopaminergic neurons enhanced lipid peroxidation and mitochondrial dysfunction in the substantia nigral region and other brain parts, indicating oxidative stress in PD (Cheng et al., 2021). In the present review the following section discusses the role of heavy metals on dopamine (DA) receptors in PD.
据研究报道，蛋白质分析，尤其是 Pb、AS 和甲基汞(MeHg) 等金属的蛋白质分析，可以诱导氧化应激、 RNA 剪接和泛素，从而导致线粒体功能障碍的改变，从而导致神经退行性疾病（ Karri 等人，2020 ） 。 Pb 与大脑中的神经退行性变、细胞凋亡、表观遗传改变和必需金属紊乱有关（ Bakulski 等，2020 ）。根据先前的研究发现，由于汞在神经末梢、神经节和中枢神经细胞中积累，引发PD发病机制( Bjorklund et al., 2018 )。多巴胺能神经元的金属暴露增强了黑质区和其他大脑部分的脂质过氧化和线粒体功能障碍，表明 PD 中存在氧化应激（ Cheng et al., 2021 ）。在本综述中，以下部分讨论了重金属对 PD 中多巴胺 (DA) 受体的作用。

3. Role of heavy metals on DA receptors
3.重金属对DA受体的作用

4. Influence of heavy metals in PD
4.重金属对PD的影响

4.1. As role in PD
4.1.作为PD中的角色

The structural mechanism alterations hypothesized for As stimulate neurotoxicity by increasing the oxidative pressure with an elevated level of active oxygen species, lipid peroxides, decline in superoxide dismutase, and decreased glutathione levels which leads to As induced neurotoxicity (Piao et al., 2005). As vulnerability is believed to alter the existence of multiple synapses such as monoamines, acetylcholine, corrosive gamma amino butyric acid, and glutamate (Singh et al., 2011). A huge decrease in monoamines like epinephrine, nor epinephrine, DA and serotonin has been noticed in the striatum, brain and hippocampus regions of the brain on a persistent vulnerability to As causing a decrease in the level DA leading to symptoms similar to those of PD (Yadav et al., 2010). As exposures have been shown to alter brain neurotransmitter levels (Tolins et al., 2014). As exposure declines DA levels and its metabolite in the brain due to an increase in ROS levels resulting into neurodegeneration (Nagaraja and Desiraju, 1993). As-initiated oxidative pressure in the brain causes oxidative DNA impairment and consequent death of the brain cell and prompts dopaminergic neuron degeneration (Singh et al., 2011). As is metabolized by omega1 glutathione transferase (GSTO1) and As (III) methyltransferase (AS3MT) which involve As methylation through the metabolism of a carbon by sadenosyl methionine (SAM) as the methyl donor by reducing GSH as the donor electron in the reductase reaction bringing about Parkinson-like manifestations (Ríos et al., 2009). As has an unexpected function with α-synuclein (αSyn) accumulation and few epidemiological studies have linked high As exposure to neuronal cell degeneration, neuronal cell death, and neurobehavioral deficits in PD (Jacobson et al., 2012; Lau et al., 2013; Mochizuki, 2019). Chronic As exposure can inhibit the mechanism of TH by inhibiting αSyn oligomers and aggregation in various parts of the brain, leading to ubiquitination increase and expression of markers such as gamma -glutamytcysteine synthetase (GCS), protein glutathione (GSH) binding, microtubule-associated protein 1A/1B-light chain 3-I and 3-II (LC3-I and LC3-II), NAD(P) Quinone Dehydrogenase-I (NQO1) and sequestosome 1 (P62) (Cholanians et al., 2016). In omics-based cell model, the impact of As, (Pb) and MeHg induced oxidative stress, mitochondrial dysfunction, ubiquitin protein system (UPS) dysfunction (Karri et al., 2018, 2020). In Saccharomyces cerevisiae model, the toxicity of As and Cd induced the aggregation and distribution of αSyn (Lorentzon et al., 2021). In SH-SY5Y model, As toxicity resulted with oxidative stress, αSyn aggregation and autophagy (Cholanians et al., 2016; Shavali and Sens, 2008). Irrigated farms with high As water increased the level of As in soil which reported to have significant association with PD prevalence in Taiwan population (Lee et al., 2021). As also induced autophagy-dependent apoptosis through Akt inactivation and AMP-activated protein kinase (AMPK) activation signalling pathways which resulted in neuronal death (Fu et al., 2021).
假设砷的结构机制改变通过增加氧化压力、活性氧、脂质过氧化物水平升高、超氧化物歧化酶下降和谷胱甘肽水平降低来刺激神经毒性，从而导致砷诱导的神经毒性（ Piao等，2005 ）。人们认为，脆弱性会改变多种突触的存在，例如单胺、乙酰胆碱、腐蚀性γ氨基丁酸和谷氨酸（ Singh et al., 2011 ）。人们注意到，在纹状体、大脑和海马区域，肾上腺素、去甲肾上腺素、DA 和血清素等单胺类物质的大幅减少，因为 As 持续脆弱，导致 DA 水平下降，从而导致类似于 PD 的症状。亚达夫等人，2010 ）。由于暴露已被证明会改变大脑神经递质水平（ Tolins 等人，2014 ）。 由于ROS水平增加，接触导致大脑中 DA 水平及其代谢物下降，导致神经变性（ Nagaraja 和 Desiraju，1993 ）。大脑中初始氧化压力会导致 DNA 氧化损伤，进而导致脑细胞死亡，并促进多巴胺能神经元变性( Singh et al., 2011 )。 As 由 omega1 谷胱甘肽转移酶 (GSTO1) 和 As (III) 甲基转移酶 (AS3MT) 代谢，其中通过在还原酶反应中还原作为供体电子的谷胱甘肽 (GSH) 作为甲基供体，以腺苷甲硫氨酸(SAM) 进行碳代谢，从而实现 As甲基化带来帕金森样表现（ Ríos et al., 2009 ）。 As 对 α-突触核蛋白 (αSyn) 积累具有意想不到的功能，并且很少有流行病学研究表明高砷暴露与 PD 中的神经元细胞变性、神经元细胞死亡和神经行为缺陷有关（ Jacobson 等，2012 ； Lau 等，2013）望月，2019 ）。 慢性砷暴露可通过抑制αSyn寡聚物和在大脑各部位的聚集来抑制TH机制，导致泛素化增加和标记物的表达，例如γ-谷氨酰胺半胱氨酸合成酶（GCS）、蛋白谷胱甘肽（GSH）结合、微管相关蛋白 1A/1B-轻链 3-I 和 3-II（LC3-I 和 LC3-II）、NAD(P)醌脱氢酶-I (NQO1) 和多螯体 1 (P62)（ Cholanians 等，2016 ）。在基于组学的细胞模型中，As、(Pb) 和 MeHg 的影响会诱导氧化应激、线粒体功能障碍、泛素蛋白系统 (UPS) 功能障碍 ( Karri et al., 2018 , 2020 )。在酿酒酵母模型中，As和Cd的毒性诱导了αSyn的聚集和分布( Lorentzon et al., 2021 )。在SH-SY5Y模型中， As毒性导致氧化应激、αSyn聚集和自噬( Cholanians等，2016 ； Shavali和Sens，2008 )。使用高砷水的灌溉农场增加了土壤中砷的含量，据报道这与台湾人口的帕金森病患病率显着相关（ Lee et al., 2021 ）。 As 还通过 Akt 失活和 AMP 激活蛋白激酶 (AMPK) 激活信号通路诱导自噬依赖性细胞凋亡，从而导致神经元死亡( Fu et al., 2021 )。

4.1.1. Induction of αSyn accumulation in PD by As
4.1.1. As 诱导 PD 中 αSyn 积累

Aggregation of αSyn protein is a pathological hallmark in PD where As exposure induces αSyn secretion which might connect with the alterations in ubiquitin-proteasome, oxidative injury, or dysfunction of mitochondria to promote neurodegeneration and apoptosis (Cholanians et al., 2016). High levels of As exposure induces αSyn oligomerization and accumulation which are accelerated by DA. Low-levels of As exposure will expand αSyn oligomerization along with cell proteotoxicity and autophagy corresponding with a hindrance of autophagic motion, which further leads to cell death (Cholanians et al., 2016). The mechanisms in which As contribution to neurodegeneration are multifaceted, and it is advancing. The oxidative role of As is complex and its function in the destruction of mitochondria by transforming oxygen (O₂) to superoxide anion (O₂ ^-) results in oxidative stress and thereby causing ROS (Xu et al., 2017). The increase in ROS causes αSyn protein misfolding and aggregation. Aggregation causes decreased expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and nuclear factor erythroid 2–related factor 2 (NRF₂) thus resulting in mitochondrial dysfunction and activation of nuclear factor kappa light chain enhancer of activated B cells (NF-κB) signalling pathway. Finally, NF-κB upregulates IFN-1, IL-1β and IL-18 which ends up in neuroinflammation and apoptosis (Rasheed et al., 2021). The increase in inflammatory cytokines leads to DA degeneration in PD. This probable mechanism is depicted in Fig. 1.
αSyn 蛋白的聚集是 PD 的病理标志，砷暴露会诱导 αSyn 分泌，这可能与泛素蛋白酶体的改变、氧化损伤或线粒体功能障碍有关，从而促进神经变性和细胞凋亡 ( Cholanians 等，2016 )。高水平的 As 暴露会诱导 αSyn寡聚和积累，而 DA 会加速这一过程。低水平的砷暴露会扩大 αSyn 寡聚化以及细胞蛋白毒性和自噬，从而阻碍自噬运动，从而进一步导致细胞死亡 ( Cholanians 等，2016 )。砷对神经退行性变的影响机制是多方面的，而且还在不断发展。 As 的氧化作用很复杂，其通过将氧 (O ₂ ) 转化为超氧阴离子 (O ₂ ^- ) 来破坏线粒体，导致氧化应激，从而产生ROS ( Xu et al., 2017 )。 ROS 的增加导致 αSyn蛋白错误折叠和聚集。聚集导致过氧化物酶体增殖物激活受体 γ 共激活剂 1-α (PGC-1α) 和核因子红细胞 2 相关因子 2 (NRF ₂ )的表达减少，从而导致线粒体功能障碍和激活 B 的核因子 kappa 轻链增强子的激活细胞(NF-κB) 信号通路。 最后，NF-κB 上调 IFN-1、IL-1β 和 IL-18，最终导致神经炎症和细胞凋亡 ( Rasheed et al., 2021 )。炎症细胞因子的增加导致 PD 中 DA 变性。这种可能的机制如图 1所示。

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Fig. 1. Mechanistic behaviour of As in neurodegeneration process.
图1 . As 在神经变性过程中的机制行为。

As enters the cell membrane and it transforms oxygen (O₂) to superoxide anion (O₂ ^-) which results in ROS that leads to increase in αSyn aggregation. The protein accumulation causes decrease in PGC-1α and NRF₂ which ends in mitochondrial dysfunction, neuroinflammation and apoptosis.
当进入细胞膜时，它将氧 (O ₂ ) 转化为超氧阴离子 (O ₂ ^- )，从而产生 ROS，从而导致αSyn聚集增加。蛋白质积累导致 PGC-1α 和 NRF ₂减少，最终导致线粒体功能障碍、神经炎症和细胞凋亡。

As: Arsenic; ROS: reactive oxygen species; αSyn: alpha-synuclein; PGC-1α: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; NRF₂: nuclear factor erythroid 2–related factor 2; NF-κB: nuclear factor kappa light chain enhancer of activated B cells; IL-1β: interleukin-1 beta; IL-18: interleukin 18.
如：砷； ROS：活性氧； αSyn : α-突触核蛋白； PGC-1α：过氧化物酶体增殖物激活受体γ共激活剂1-α； NRF ₂ ：核因子红细胞2相关因子2； NF-κB：活化B细胞的核因子κ轻链增强子； IL-1β：白介素-1β； IL-18：白细胞介素18。

4.2. Fe role in PD
4.2. Fe 在 PD 中的作用

Fe accumulation is said to be one of the PD pathophysiology were its decreasing levels lead to DA and 5-hydroxytryptamine (5-HT) reduction in PD with restless legs syndrome (Piao et al., 2017; Xuan et al., 2017). A previous study conducted by Sofic et al. (1988) found an elevated level of Fe in the post-mortem substantia nigra and other areas of the brain of parkinsonian patients when compared to control subjects (Sofic et al., 1988). In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism in monkeys resulted in dopaminergic cell death and nigral degeneration due to Fe deposition in the substantia nigra (He et al., 2003; J. Li et al., 2020). In an African Green monkey total free Fe was examined with unilateral MPTP-induced hemi-parkinsonism with degenerating DA cells as well as the encompassing matrix and glial cells (Temlett et al., 1994). In human brain, Fe deposition was measured in PD subjects with elevated levels of Fe associated with gene expression providing the progression of neurodegeneration (Thomas et al., 2021). In catecholaminergic a-differentiated (CAD) cells and PTEN-induced kinase 1 (PINK−1) Drosophila melanogaster PD models, Fe influenced nigrostriatal pathway with exacerbating motor deficits with increased ROS (Ohiomokhare et al., 2020). In C. elegans model, Fe deposition induced neurodegeneration with ATP13A2 mutation (Anand et al., 2020). In human brain, increased Fe deposition in substantia nigra has proved to be a potential biomarker in PD (He et al., 2020). Studies have also focused on treating metals homeostasis by using derivatives of Fe chelators as a neuroprotective therapy for PD (Devos et al., 2014; Grolez et al., 2015; Martin-Bastida et al., 2017; Ortega-Arellano et al., 2021; Zhu et al., 2007).
Fe 积累被认为是 PD病理生理学之一，其水平下降会导致 PD不宁腿综合征中 DA 和 5-羟色胺 (5-HT) 减少（ Piao 等，2017 ； Xuan 等，2017 ）。 Sofic 等人之前进行的一项研究。 (1988)发现与对照组相比，帕金森病患者死后黑质和大脑其他区域的铁含量升高 ( Sofic 等，1988 )。在 1-甲基-4-苯基-1,2,3,6-四氢吡啶 (MPTP) 诱导的猴子帕金森病中，由于铁沉积在黑质中，导致多巴胺能细胞死亡和黑质变性（ He et al., 2003 ； J. Li 等人，2020 ）。在非洲绿猴中，用单侧 MPTP 诱导的偏侧帕金森病以及退化的 DA 细胞以及周围的基质和神经胶质细胞检查了总游离铁（ Temlett 等人，1994 ）。在人脑中，在 PD 受试者中测量了铁沉积，铁水平升高与基因表达相关，导致神经退行性变的进展（ Thomas 等人，2021 ）。 在儿茶酚胺能α分化 (CAD) 细胞和 PTEN 诱导激酶 1 (PINK−1)果蝇PD 模型中，Fe 影响黑质纹状体通路，并随着 ROS 的增加而加剧运动缺陷 ( Ohiomokhare 等人，2020 )。在秀丽隐杆线虫模型中，Fe 沉积诱导伴有ATP13A2突变的神经变性（ Anand 等人，2020 ）。在人脑中，黑质中铁沉积的增加已被证明是PD的潜在生物标志物（ He et al., 2020 ）。研究还集中于通过使用 Fe螯合剂衍生物作为PD 的神经保护疗法来治疗金属稳态（ Devos 等人，2014 年； Grolez 等人，2015 年； Martin-Bastida 等人，2017 年； Ortega-Arellano 等人，2017 年）。 ，2021 ；朱等人，2007 ）。

4.2.1. Effect of Fe chelation on ROS generation
4.2.1. Fe 螯合对 ROS 生成的影响

Oxidative stress is caused by Fe accumulation in neurons through Fenton's reaction, which generates ROS. ROS is produced in cells from a variety of sources, including Fe and its derivatives, which are required by the ROS-producing enzymes (J. Li et al., 2020). Increase in ROS causes severe damage to phospholipids, nucleic acids, and proteins, which are thought to lead to neuronal death (Carocci et al., 2018; Subramaniam et al., 2020). There are two major ROS-mediated mechanisms associated with the presence of Fe chelators: (i) formation of free radicals and (ii) lipid peroxidation, both of which may occur during chelating therapy. Thus, combining chelators and antioxidants may be beneficial in the treatment of certain Fe-related neurodegenerative diseases (Jomova and Valko, 2011). It has been proposed that neuromelanin is normally 50% saturated with Fe, which preserves its chelation capacity and protects neurons from Fe-catalysed ROS generation (Ma et al., 2012). Other studies tested Fe chelators with D2/D3 dopamine receptor agonists to treat motor symptoms using a multifunctional approach in Fe chelation and oxidative stress in MPTP treated PD model (Muñoz et al., 2016). Fe chelation process interacts with hydrogen peroxide (H₂O₂) with an increase in the formation of hydroxyl free radicals (OH, OH). Furthermore, Fe chelators may influence the stability and transcriptional activation of Fe-dependent hypoxia-inducible factor (HIF)-1 through a class of prolyl-4-hydroxylases (PHDs), which target HIF-1α for hydroxylation and degradation (Weinreb et al., 2013). In PD, overload of Fe decreases the activity of HIF-1α which declines the level of TH and DA thereby causing degradation of dopaminergic neurons (Fig. 2).
氧化应激是通过芬顿反应，Fe在神经元中积累，产生ROS而引起的。细胞中产生的 ROS 有多种来源，包括铁及其衍生物，这是产生 ROS 的酶所必需的（J. Li 等人，2020 ）。 ROS 的增加会对磷脂、核酸和蛋白质造成严重损害，这被认为会导致神经元死亡（ Carocci et al., 2018 ； Subramaniam et al., 2020 ）。有两种主要的 ROS 介导机制与 Fe螯合剂的存在相关：(i)自由基的形成和 (ii)脂质过氧化，这两种机制都可能在螯合治疗期间发生。因此，结合螯合剂和抗氧化剂可能有益于治疗某些与铁相关的神经退行性疾病（ Jomova 和 Valko，2011 ）。有人提出，神经黑色素通常被 Fe 饱和 50%，这保留了其螯合能力并保护神经元免受 Fe 催化的 ROS 生成（ Ma 等，2012 ）。 其他研究测试了 Fe 螯合剂与 D2/D3多巴胺受体激动剂在 MPTP 治疗的 PD 模型中使用Fe 螯合和氧化应激的多功能方法治疗运动症状（ Muñoz 等，2016 ）。 Fe 螯合过程与过氧化氢 (H ₂ O ₂ ) 相互作用，增加羟基自由基(OH, OH) 的形成。此外，Fe 螯合剂可能通过一类脯氨酰 4-羟化酶 (PHD )影响 Fe 依赖性缺氧诱导因子 (HIF)-1 的稳定性和转录激活，该酶靶向 HIF-1α 进行羟基化和降解（ Weinreb 等人） .，2013 ）。在PD中，Fe超载会降低HIF-1α的活性，从而降低TH和DA的水平，从而导致多巴胺能神经元退化（图2 ）。

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Fig. 2. Effect of Fe chelation on DA degeneration.
图2 . Fe 螯合对 DA 变性的影响。

In normal neuronal cell, Fe chelators interact with H₂O₂ and increases OH level. Meanwhile, Fe chelators activate HIF-1 and PHDs which targets HIF1 for degradation. Whereas in PD, Fe overload decreases HIF-1 activity which thereby reduces the expression of TH and DA.
在正常神经细胞中，Fe螯合剂与 H ₂ O ₂相互作用并增加 OH 水平。同时，Fe螯合剂会激活 HIF-1 和PHD ，从而靶向 HIF1 进行降解。而在 PD 中，Fe 超载会降低 HIF-1 活性，从而降低 TH 和 DA 的表达。

Fe: iron; H₂O₂: hydrogen peroxide; OH: free radicals; HIF-1: hypoxia-inducible factor-1; PHDs: prolyl-4-hydroxylases; TH: tyrosine hydroxylase; DA: dopamine.
Fe：铁； H ₂ O ₂ ：过氧化氢； OH：自由基； HIF-1：缺氧诱导因子-1； PHD ：脯氨酰-4-羟化酶； TH：酪氨酸羟化酶； DA：多巴胺。

4.2.2. Probable role of epigenetic modifications due to Fe transport in PD
4.2.2. Fe 转运导致的表观遗传修饰在 PD 中的可能作用

Fe is required for many processes in the brain (D'Mello and Kindy, 2020), were its accumulation depends on age which is yet to be identified. Recently, possible role of Fe in αSyn aggregation, oxidative stress and ubiquitin-proteasome system (UPS) in PD has been explained (Ullah et al., 2021). It was proposed that increased Fe levels in specific brain areas could be caused by alterations in brain vascularization during neurodegeneration (Weinreb et al., 2013). Transportation of Fe occurs through divalent metal transporter 1 (DMT1) which contributes Fe exchange and distribution during ageing and neurodegenerative disorders. In a study, excess Fe contributed on oxidizing DA to DA o-quinone which results in reduction of intracellular dopamine levels (Shen and Dryhurst, 1998). Although Fe homeostasis with dopamine had been investigated, mounting evidences has proposed Fe overload can constrain histone acetylation. Fe accumulation in the brain will decrease the level of H3K9 acetylation in the hippocampus which leads to memory impairment and neurodegenerative diseases (Silva et al., 2012). In a study, decrease in histone deacetylase upregulated histone acetylation in DA neurons which resulted in neurodegeneration (Park et al., 2016). There are no conclusive evidences on Fe causing histone modifications in PD hence Fe regulation in epigenetic mechanism might aid to find a cure in PD (Fig. 3).
大脑中的许多过程都需要铁（ D'Mello 和 Kindy，2020 ），铁的积累取决于年龄，但尚未确定。最近，Fe 在 PD 中的 αSyn 聚集、氧化应激和泛素蛋白酶体系统 (UPS) 中的可能作用已得到解释 ( Ullah et al., 2021 )。有人提出，特定大脑区域中铁水平的增加可能是由神经退行性变期间脑血管化的改变引起的（ Weinreb 等，2013 ）。 Fe 的运输通过二价金属转运蛋白 1 (DMT1) 进行，二价金属转运蛋白 1 在衰老和神经退行性疾病期间有助于 Fe 的交换和分布。在一项研究中，过量的 Fe 有助于将 DA 氧化为 DA 邻醌，从而导致细胞内多巴胺水平降低（ Shen 和 Dryhurst，1998 ）。尽管已经研究了多巴胺与铁的稳态，但越来越多的证据表明铁过载可以限制组蛋白乙酰化。大脑中铁的积累会降低海马中 H3K9乙酰化水平，从而导致记忆障碍和神经退行性疾病（ Silva et al., 2012 ）。在一项研究中，组蛋白脱乙酰酶的减少会上调 DA 神经元中的组蛋白乙酰化，从而导致神经变性（ Park 等人，2017）。，2016 ）。没有确凿的证据表明 Fe 会导致 PD 中的组蛋白修饰，因此表观遗传机制中的 Fe 调节可能有助于找到 PD 的治疗方法（图 3 ）。

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Fig. 3. Fe transportation contributing to epigenetic modifications.
图3 .铁的运输有助于表观遗传修饰。

Increase in Fe elevates ROS and αSyn accumulation. Meanwhile, Fe interacts with DMT1 and enters the neuronal cell where αSyn increases the level of Fe inside the neuronal cells and it decreases H3K9 levels, converts DA to DA o-quinone and inhibits histone acetylation. These molecular changes contribute to neurodegeneration.
Fe 的增加会增加 ROS 和 αSyn 的积累。同时，Fe 与DMT1相互作用并进入神经元细胞，其中 αSyn 增加神经元细胞内 Fe 的水平，降低 H3K9 水平，将 DA 转化为 DA 邻醌并抑制组蛋白乙酰化。这些分子变化导致神经变性。

Fe: iron; ROS: reactive oxygen species; αSyn: alpha synuclein; DMT1: divalent metal transporter 1; DA: dopamine.
Fe：铁； ROS：活性氧； αSyn：α突触核蛋白； DMT1 ：二价金属转运蛋白1； DA：多巴胺。

4.3. Cu role in PD
4.3. Cu 在 PD 中的作用

Earlier, biochemical examinations of PD brain samples indicated decreased levels of Cu in the substantia nigra (Dexter et al., 1989; Genoud et al., 2020) and accelerate the aggregation of αSyn which is a PD biomarker (Rasia et al., 2005) and also the level of Cu transporter 1 (Ctr1) drastically reduced, which suggested Ctr1 association with PD (Davies et al., 2014). Recently, Gou and his colleagues accelerated Cu levels with intracellular αSyn inclusions in yeast and mammalian cell models, were they generated Ctr1 knocked-out transgenic mouse model with induced adeno-associated viral human- αSyn into the substantia nigra, which inhibited DA loss, and improved motor function (Gou et al., 2021). In SH-SY5Y cell line and N₂a cells, Cu and Fe exposure for 3 days induced αSyn aggregation (Y. Li et al., 2020). In few studies, Cu and Fe induction in human serum revealed with redox activity, neuroinflammation, Cu dyshomeostasis, ceruloplasmin activity, αSyn aggregation and SNCA polymorphism (Ajsuvakova et al., 2020; Cheng et al., 2015; Sanyal et al., 2020; Tórsdóttir et al., 1999). Intrastriatal administration of Cu with 6-OHDA in rat model for 30 days resulted with oxidative stress and dopaminergic degeneration (Cruces-Sande et al., 2019). A probable mechanism of Cu influence on neurodegeneration is depicted in Fig. 4.
早些时候，PD脑样本的生化检查表明黑质中的Cu水平降低（ Dexter等，1989 ； Genoud等，2020 ）并加速PD生物标志物αSyn的聚集（ Rasia等，2005 ） ），并且铜转运蛋白 1 (Ctr1) 的水平也急剧降低，这表明 Ctr1 与 PD 相关 ( Davies et al., 2014 )。最近，Gou 和他的同事在酵母和哺乳动物细胞模型中通过细胞内 αSyn 内含物加速了 Cu 水平，他们是否生成了 Ctr1 敲除转基因小鼠模型，并将腺相关病毒人类 αSyn 诱导进入黑质，从而抑制 DA 损失，以及改善运动功能（ Gou et al., 2021 ）。在 SH-SY5Y 细胞系和 N ₂ a 细胞中，Cu 和 Fe 暴露 3 天诱导 αSyn 聚集 ( Y. Li et al., 2020 )。在少数研究中，人血清中的 Cu 和 Fe 诱导揭示了氧化还原活性、神经炎症、Cu 稳态失衡、铜蓝蛋白活性、αSyn 聚集和SNCA多态性（ Ajsuvakova 等，2020 ； Cheng 等，2015 ； Sanyal 等，2020）托斯多蒂尔等人，1999 ）。 在大鼠模型中纹状体内注射 6-OHDA 铜 30 天会导致氧化应激和多巴胺能变性（ Cruces-Sande 等人，2019 ）。 Cu 对神经变性影响的可能机制如图 4所示。

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Fig. 4. Effect of Cu on epigenetic alterations.
图4 . Cu 对表观遗传改变的影响。

Cu overloads causes oxidative stress by phosphorylating AMPKα and