Neurotoxicity Linked to Dysfunctional Metal Ion Homeostasis and Xenobiotic Metal Exposure: Redox Signaling and Oxidative Stress
与金属离子稳态功能失调和异生金属暴露相关的神经毒性：氧化还原信号和氧化应激

Copper 铜

Cu is present in biological systems as Cu⁺ (cuprous ion) and Cu²⁺ (cupric ion) (Supplementary Table S1). Cu is a redox-active metal and a cofactor of many enzymes involved in cellular respiration, radical detoxification, as well as biosynthesis of neurotransmitters, neuropeptides, and hormones. For example, Cu is required as cofactor of several important enzymes in the brain, such as peptidylglycine monooxygenase (PHM), dopamine β-monooxygenase (DBM), tyrosinase (TYR), and cytochrome C oxidase (COX). Cu can activate O₂ for reduction and although its high reactivity with O₂ is essential for life, if uncontrolled it can promote oxidative stress and cellular damage. Cu⁺ can react with H₂O₂ to produce highly reactive ^•OH. Cu also induces microglial activation and mitochondrial ROS formation (137).
Cu 在生物系统中以 Cu ⁺ （亚铜离子）和 Cu ²⁺ （铜离子）的形式存在（补充表 S1 ）。 Cu 是一种氧化还原活性金属，也是许多参与细胞呼吸、自由基解毒以及神经递质、神经肽和激素生物合成的酶的辅助因子。例如，铜是大脑中几种重要酶的辅因子，如肽基甘氨酸单加氧酶 (PHM)、多巴胺 β-单加氧酶 (DBM)、酪氨酸酶 (TYR) 和细胞色素 C 氧化酶 (COX)。 Cu 可以激活 O ₂进行还原，虽然它与 O ₂的高反应性对于生命至关重要，但如果不加控制，它会促进氧化应激和细胞损伤。 Cu ⁺能与H ₂ O ₂反应生成高活性的^· OH。 Cu 还诱导小胶质细胞激活和线粒体 ROS 形成 ( 137 )。

The control of Cu homeostasis in the brain requires a close interrelationship between the BBB, neurons, and astrocytes (300) (Fig. 1). Astrocytes regulate the properties of the BBB, which is the entry point for Cu into the brain from the blood stream, where Cu is bound to albumin or ceruloplasmin (Cp) (58) (Fig. 1a). At the same time, neurons require Cu as a cofactor and neuromodulator, while astrocytes are key players in synaptic transmission and Cu homeostasis (58). The Cu trafficking machineries of the BBB endothelial cells, neurons, and astrocytes resemble those of other extensively studied mammalian cells (Fig. 1a–c). Extracellular Cu is primarily transported into cells as Cu⁺ via the Cu transporter 1 (CTR1) (58). Cu²⁺ reduction to Cu⁺, and Cu uptake from Cp via CTR1, has been proposed to involve a reduction step but no Cu²⁺ reductase has been identified (281). CTR1-independent mechanisms have also been proposed. The divalent metal transporter 1 (DMT1) seems to play a compensatory role for Cu uptake under certain conditions such as in the absence of CTR1 or under low Fe conditions (147, 231). Interestingly, DMT1 loss promotes brain Cu accumulation and oxidative stress (122). Other potential candidates recently proposed to mediate Cu uptake are the Zrt (Zn-regulated transporter)- or Irt (Fe-regulated transporter)-like protein 4 (ZIP4) (11, 29).
大脑中铜稳态的控制需要 BBB、神经元和星形胶质细胞之间密切的相互关系 ( 300 )（图 1 ）。星形胶质细胞调节 BBB 的特性，BBB 是 Cu 从血流进入大脑的入口点，其中 Cu 与白蛋白或铜蓝蛋白 (Cp) 结合 ( 58 )（图 1a ）。同时，神经元需要铜作为辅助因子和神经调节剂，而星形胶质细胞是突触传递和铜稳态的关键参与者（ 58 ）。 BBB内皮细胞、神经元和星形胶质细胞的铜运输机制与其他广泛研究的哺乳动物细胞类似（图1a-c ）。细胞外 Cu 主要通过Cu 转运蛋白 1 (CTR1) 以 Cu ⁺形式转运到细胞内 ( 58 )。 Cu ²⁺还原为 Cu ⁺以及通过CTR1 从 Cp 摄取 Cu 已被提议涉及还原步骤，但尚未鉴定出 Cu ²⁺还原酶 ( 281 )。还提出了独立于 CTR1 的机制。二价金属转运蛋白 1 (DMT1) 似乎在某些条件下（例如缺乏 CTR1 或低 Fe 条件下）对 Cu 吸收发挥补偿作用 ( 147 , 231 )。有趣的是，DMT1 缺失会促进大脑铜积累和氧化应激 ( 122 )。最近提出的介导铜吸收的其他潜在候选蛋白是 Zrt（锌调节转运蛋白）或 Irt（铁调节转运蛋白）样蛋白 4 (ZIP4) ( 11 , 29 )。

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FIG. 1. 如图。 1.

Cu homeostasis in the brain and its redox control. Regulation of Cu homeostasis is a complex process requiring intercommunication between endothelial cells of the BBB, astrocytes, and neurons. These three types of cells have the same protein machinery as other mammalian cells: CTR1 and DMT1 for Cu uptake; Atox1, CCS1, and GSH for Cu delivery to ATP7(A/B), SOD3, and MT, respectively; COX17 and COX19 for Cu delivery to mitochondria, where SCO1, SCO2, and COX11 load Cu into COX; while ATP7 (A/B) participates in Cu export through the secretory pathway (a–c). (a) Cp- and albumin- bound Cu enters into the brain by crossing endothelial cells of BBB, which are in tight communication with astrocytes. (b) Astrocytes serve as an interface to deliver Cu to (c) neurons. (d) Recently, a redox control of Cu efflux was elucidated in neurons during differentiation: the redox state of Atox1 controls Cu delivery to the secretory pathway. ATP7A, ATPase copper transporting alpha; ATP7B, ATPase copper transporting beta; Atox1, antioxidant protein 1; BBB, blood-brain barrier; CCS1, copper chaperone for superoxide dismutase 1; COX, cytochrome C oxidase; Cp, ceruloplasmin; CTR1, copper transporter 1; Cu, copper; DMT1, divalent metal transporter 1; Glu, glutamate; GSSG, glutathione disulfide; GSH, glutathione; MT, metallothionein; SCO, cytochrome c oxidase assembly protein; SOD, superoxide dismutase.
大脑中的铜稳态及其氧化还原控制。 Cu 稳态的调节是一个复杂的过程，需要 BBB 内皮细胞、星形胶质细胞和神经元之间的相互通讯。这三种类型的细胞与其他哺乳动物细胞具有相同的蛋白质机制：CTR1 和 DMT1 用于铜的吸收； Atox1、CCS1 和 GSH 分别用于将 Cu 递送至 ATP7(A/B)、SOD3 和 MT； COX17 和 COX19 用于将 Cu 递送至线粒体，其中 SCO1、SCO2 和 COX11 将 Cu 加载到 COX 中；而 ATP7 (A/B) 通过分泌途径(a–c)参与 Cu 输出。 (a) Cp 和白蛋白结合的 Cu 通过穿过与星形胶质细胞紧密联系的 BBB 内皮细胞进入大脑。 (b)星形胶质细胞充当将铜传递至(c)神经元的界面。 (d)最近，在神经元分化过程中阐明了 Cu 流出的氧化还原控制：Atox1 的氧化还原状态控制 Cu 向分泌途径的输送。 ATP7A，ATP酶铜转运α； ATP7B，ATP酶铜转运β； Atox1，抗氧化蛋白1； BBB，血脑屏障； CCS1，超氧化物歧化酶 1 的铜伴侣； COX，细胞色素C氧化酶； Cp，铜蓝蛋白； CTR1，铜转运蛋白1；铜，铜； DMT1，二价金属转运蛋白1；谷氨酸、谷氨酸； GSSG，二硫化谷胱甘肽；还原型谷胱甘肽（GSH）、谷胱甘肽； MT，金属硫蛋白； SCO，细胞色素c氧化酶组装蛋白； SOD，超氧化物歧化酶。

Intracellular Cu distribution depends on the relative concentration and metal affinity of chaperones or chelators (18) (Fig. 1b, c). The antioxidant protein 1 (Atox1), the Cu chaperone for superoxide dismutase 1 (CCS1), and GSH have been proposed to take Cu⁺ from CTR1 (197). Chaperones not only bind Cu but they also deliver it to specific targets. CCS1 transfers Cu to SOD1, where its reactivity with O₂ is required for SOD1 maturation via the formation of a disulfide bridge (17). Copper chaperones COX19 and COX17 deliver Cu to the COX assembly proteins (SCO1 and SCO2) and COX11. Finally, Atox1 transports Cu to the ATPase copper transporting alpha (ATP7A) and beta (ATP7B) in the secretory pathway, where cuproenzymes such as SOD3 are metalized (Fig. 1b, c). Upon an excess in cytosolic Cu levels, vesicles in the secretory pathway are loaded with Cu and trafficked to the plasma membrane, where Cu is released into the extracellular space (196, 197).
细胞内 Cu 分布取决于分子伴侣或螯合剂的相对浓度和金属亲和力 ( 18 )（图 1b、c ）。抗氧化蛋白 1 (Atox1)、超氧化物歧化酶 1 (CCS1) 的 Cu 分子伴侣和 GSH 已被提议从 CTR1 中获取 Cu ⁺ ( 197 )。分子伴侣不仅能结合铜，还能将其递送至特定目标。 CCS1 将 Cu 转移至 SOD1，其中其与 O ₂的反应是 SOD1通过形成二硫桥成熟所必需的 ( 17 )。铜伴侣 COX19 和 COX17 将铜递送至 COX 组装蛋白（SCO1 和 SCO2）和 COX11。最后，Atox1在分泌途径中将Cu转运至ATP酶铜转运α（ATP7A）和β（ATP7B），其中SOD3等铜酶被金属化（图1b，c ）。当胞质铜水平过量时，分泌途径中的囊泡会装载铜并运输到质膜，其中铜被释放到细胞外空间 ( 196 , 197 )。

Strikingly, although GSH has a lower affinity for Cu, compared to Atox1 and CCS1, the rate of Cu entry into the cell via CTR1 is affected by GSH, but not by Atox1 or CCS1 depletion, likely due to the higher concentration of GSH (208). Thus, GSH is the most important cytosolic first acceptor of Cu from CTR1, providing a tight link between cellular Cu uptake and cellular redox homeostasis. GSH in turn is known to transfer Cu ions to metallothioneins (MTs), small Cys-rich proteins that play a major role as scavengers for metal ions (93) (Fig. 1b, c). Three distinct isoforms of MTs are expressed in the human brain. MT-I, MT-II, and MT-III are found in astrocytes, while MT-III is the main isoform expressed in neurons. MTs can be secreted, playing a crucial role in modulating Cu homeostasis and protecting the cell from oxidative damage (299).
引人注目的是，尽管与 Atox1 和 CCS1 相比，GSH 对 Cu 的亲和力较低，但 Cu通过CTR1 进入细胞的速率受 GSH 影响，但不受 Atox1 或 CCS1 消耗的影响，这可能是由于 GSH 浓度较高（ 208 ）。因此，GSH 是 CTR1 最重要的 Cu 胞质第一受体，在细胞 Cu 摄取和细胞氧化还原稳态之间提供紧密联系。众所周知，GSH 又可将 Cu 离子转移至金属硫蛋白 (MT)，这是一种富含 Cys 的小蛋白质，在金属离子清除剂中发挥着重要作用 ( 93 )（图 1b、c ）。人脑中表达三种不同的 MT 亚型。 MT-I、MT-II 和 MT-III 存在于星形胶质细胞中，而 MT-III 是神经元中表达的主要异构体。 MT 可以被分泌，在调节铜稳态和保护细胞免受氧化损伤方面发挥着至关重要的作用 ( 299 )。

Chaperones use Cys residues to coordinate Cu⁺ in their reduced state. Thus, Cys oxidation affects Cu dynamics. Atox1 coordinates Cu using a CysXXCys motif that can form a disulfide bond, which can be reduced directly by GSH or by Grx1 in a GSH-dependent manner (Fig. 1d) (41, 127). During neuronal differentiation, the GSH/GSSG ratio increases promoting a more reductive environment, which in turn reduces Cys residues at the Cu binding site of Atox1. These events enable Cu transport from Atox1 to ATP7A/B and enhance Cu availability to load the active sites of newly synthetized cuproenzymes (128) (Fig. 1d). After neuronal differentiation, both Cu and MT-III levels increase (249).
伴侣分子使用 Cys 残基来协调还原态的 Cu ⁺ 。因此，Cys 氧化会影响 Cu 动力学。 Atox1 使用可以形成二硫键的 CysXXCys 基序来配位 Cu，该二硫键可以直接被 GSH 或以 GSH 依赖性方式被 Grx1 还原（图 1d ）（ 41 , 127 ）。在神经元分化过程中，GSH/GSSG 比率增加，促进还原性更强的环境，从而减少 Atox1 Cu 结合位点的 Cys 残基。这些事件使铜能够从 Atox1 转运到 ATP7A/B，并增强铜的可用性以装载新合成的铜酶的活性位点 ( 128 )（图 1d ）。神经元分化后，Cu 和 MT-III 水平都会增加 ( 249 )。

The recent development of fluorescent sensors has revealed new important roles of intracellular Cu in neuronal activity (78); for example, in the spine neck of hippocampal neurons, Cu is essential for the control of the dendritic actin cytoskeleton (269). Cu export by ATP7A has been reported to be triggered by the activation of glutamate (Glu)/N-methyl-d-aspartate receptors (NMDARs) (301) (Fig. 2a). At the synapse, Cu can modulate many neurotransmitter receptors (66). For example, Cu inhibits NMDAR activity by Cys nitros(yl)ation, a neuroprotective mechanism associated with neuronal plasticity that requires the participation of the cellular prion protein (PrP^C) (110) (Fig. 2b). Accordingly, selective depletion of ATP7A in neurons and glia increases the susceptibility to NMDA seizures (132).
荧光传感器的最新发展揭示了细胞内铜在神经元活动中新的重要作用（ 78 ）；例如，在海马神经元的脊柱颈中，Cu 对于控制树突状肌动蛋白细胞骨架至关重要 ( 269 )。据报道，ATP7A 的 Cu 输出是由谷氨酸 (Glu)/N-甲基-d-天冬氨酸受体 (NMDAR) 的激活触发的 ( 301 ) (图 2a )。在突触处，铜可以调节许多神经递质受体（ 66 ）。例如，Cu 通过 Cys 亚硝基（基）化作用抑制 NMDAR 活性，这是一种与神经元可塑性相关的神经保护机制，需要细胞朊病毒蛋白（ ^PrPC ）的参与（ 110 ）（图 2b ）。因此，神经元和神经胶质细胞中 ATP7A 的选择性消耗会增加 NMDA 癫痫发作的易感性 ( 132 )。

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FIG. 2. 如图。 2.

Cu at synapse and redox signaling. (a) After activation of NMDAR, Cu is released at the synapse through the secretory pathway. Several Cu binding proteins are present in the synaptic cleft, including PrP^C, APP, Aβ peptides, and NKB. (b) The Cu-PrP^C interaction is particularly important for the modulation of NMDAR activity via S-nitros(yl)ation. In turn, NO production is dependent on the activation of NMDAR and its interaction with nNOS through the PSD-95. (c) While NKB can assist Cu uptake into astrocytes, Cu distribution among Cu-binding proteins at the synapse likely depends on their relative concentration and affinities for Cu. (d) On the contrary, α-synuclein is an important presynaptic Cu-binding protein found in neurons; it is an IDP that can interact with membranes when it acquires α-helix structure at N-terminal region. Interestingly, Cu⁺ binding to the N-Terminal of α-synuclein enhances its α-helical conformation. Aβ, amyloid beta; APP, amyloid precursor protein; Cu⁺, cuprous ion; IDP, intrinsically disordered protein; NKB, neurokinin B; NMDAR, glutamate/N-methyl-d-aspartate receptor; nNOS, neural nitric oxide synthetase; PrP^C, cellular prion protein; PSD-95, postsynaptic density protein 95.
突触和氧化还原信号传导中的铜。 (a) NMDAR 激活后，Cu 通过分泌途径在突触处释放。突触间隙中存在多种 Cu 结合蛋白，包括^PrPC 、APP、Aβ 肽和 NKB。 (b) Cu-PrP ^C相互作用对于通过S-亚硝基(基)化调节 NMDAR 活性特别重要。反过来，NO 的产生依赖于 NMDAR 的激活及其通过 PSD-95 与 nNOS 的相互作用。 (c)虽然 NKB 可以帮助星形胶质细胞摄取铜，但突触处铜结合蛋白之间的铜分布可能取决于它们的相对浓度和对铜的亲和力。 (d)相反，α-突触核蛋白是神经元中发现的一种重要的突触前铜结合蛋白；它是一种 IDP，当它在 N 端区域获得 α 螺旋结构时，可以与膜相互作用。有趣的是，Cu ⁺与 α-突触核蛋白 N 端的结合增强了其 α-螺旋构象。 Aβ，β淀粉样蛋白； APP，淀粉样前体蛋白； Cu ⁺ ，亚铜离子； IDP，本质无序蛋白质； NKB、神经激肽B； NMDAR，谷氨酸/N-甲基-d-天冬氨酸受体； nNOS，神经一氧化氮合成酶； PrP ^C ，细胞朊病毒蛋白； PSD-95，突触后密度蛋白 95。

Cu trafficking at the synapse is complex and involves several Cu binding proteins, such as the membrane-bound PrP^C, the amyloid precursor protein (APP), and the amyloid beta (Aβ) peptide from neurons, or the neurokinin B (NKB) peptide from astrocytes (Fig. 2). PrP^C has several Cu binding sites and it might be involved in Cu sensing and transport into neurons (Fig. 3a). During synaptic transmission, the extracellular Cu concentration may reach ∼100 μM. Cu binding to PrP^C induces its endocytosis, possibly contributing to Cu delivering into the cytosol (263). APP has been reported to regulate Cu efflux, as the APP knockout mice display higher levels of Cu in the brain and in neurons, while APP overexpression leads to decreased intracellular Cu concentrations (28, 330, 375). In contrast, Aβ peptides produced from the proteolytic cleavage of APP have been proposed to act as Cu scavengers (264), particularly those cleaved to yield 4–40 peptides that have higher affinity for Cu and make up to 50% of the Aβ in plaques (374). NKB has been suggested to compete for Cu from PrP^C and transport it into astrocytes by endocytosis (Fig. 2c) (308). PrP^C and Aβ contain intrinsically disordered regions with Cu binding sites that have the capacity to adopt different Cu coordination modes, some of which have been proposed to activate O₂ and produce ROS (Fig. 3a) (184, 350).
突触处的 Cu 运输非常复杂，涉及多种 Cu 结合蛋白，例如膜结合^PrPC 、淀粉样前体蛋白 (APP) 和来自神经元的淀粉样 β (Aβ) 肽或神经激肽 B (NKB) 肽来自星形胶质细胞（图2 ）。 PrP ^C有几个 Cu 结合位点，它可能参与 Cu 传感和转运到神经元中（图 3a ）。在突触传递过程中，细胞外铜浓度可能达到~100 μM 。 Cu 与 PrP ^C结合诱导其内吞作用，可能有助于 Cu 输送到细胞质中 ( 263 )。据报道，APP 可以调节铜的流出，因为 APP 敲除小鼠的大脑和神经元中的铜含量较高，而 APP 过度表达会导致细胞内铜浓度降低 ( 28 , 330 , 375 )。相比之下，APP 蛋白水解裂解产生的 Aβ 肽可作为 Cu 清除剂 ( 264 )，特别是那些裂解产生 4-40 个肽的肽，这些肽对 Cu 具有更高的亲和力，占斑块中 Aβ 的 50% （ 374 ）。有人建议 NKB 与 PrP ^C竞争 Cu 并通过内吞作用将其转运到星形胶质细胞中（图 2c ）（ 308 ）。 PrP ^C和 Aβ 含有具有 Cu 结合位点的本质无序区域，这些区域能够采用不同的 Cu 配位模式，其中一些已被提议激活 O ₂并产生 ROS（图 3a ）（ 184 , 350 ）。

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FIG. 3. 如图。 3.

Structural features and redox properties of relevant metal-protein complexes in neurodegenerative diseases. (a) Cu can interact with PrP^C, Aβ, and α-synuclein. PrP^C has three coordination sites: histidine (His) His96, His111, and an octarepeat. Cu²⁺ coordination to His96 and His111 yields very similar complexes different to those generated upon Cu⁺ binding to His111. The octarepeat region coordinates up to four Cu²⁺ ions depending on the Cu²⁺/PrP^C stoichiometric ratio. At low Cu:protein ratios, four His residues coordinate Cu²⁺, while at high ratios each His can bind one Cu²⁺ ion. The different coordination chemistry of these Cu-PrP^C complexes is reflected in their redox properties. Similarly, Cu²⁺ coordination to Aβ (1–40/42) yields two different coordination modes that display different redox properties. Recently, coordination to Aβ (4–40/42) has also been described; this site displays higher affinity for Cu²⁺ than Aβ (1–40/42), and it yields a redox silent complex. On the contrary, nonacetylated α-synuclein has two high-affinity binding sites for Cu²⁺: site one involving residues at the N-terminal, and site two at His50. Acetylation of α-synuclein, as described to occur in vivo, abolishes Cu²⁺ binding at site 1, although both forms of α-synuclein can bind Cu⁺. (b) Fe²⁺ can interact with Aβ, but the redox properties of Fe-Aβ complexes remain unclear. Interestingly, recent studies propose that heme can bind to Aβ, yielding a heme (Fe)-Aβ complex with peroxidase activity. (c) Finally, Zn²⁺ can bind to the octarepeat region of PrP^C and with Aβ. Cu²⁺, cupric ion; Fe, iron; Fe²⁺, ferrous; Zn, zinc.
神经退行性疾病中相关金属-蛋白质复合物的结构特征和氧化还原特性。 (a) Cu 可以与 PrP ^C 、Aβ 和 α-突触核蛋白相互作用。 PrP ^C具有三个配位位点：组氨酸 (His) His96 、His111 和八重复序列。 Cu ²⁺与 His96 和 His111 配位产生非常相似的复合物，不同于 Cu ⁺与 His111 结合时生成的复合物。八重区域可协调多达四个 Cu ²⁺离子，具体取决于 Cu ²⁺ /PrP ^C化学计量比。在低 Cu: 蛋白质比率下，四个 His 残基与 Cu ²⁺配位，而在高比率下，每个 His 残基可结合 1 个 Cu ²⁺离子。这些 Cu-PrP ^C配合物的不同配位化学反映在它们的氧化还原性质上。类似地，Cu ²⁺与 Aβ ( 1–40 / 42 ) 配位产生两种不同的配位模式，表现出不同的氧化还原特性。最近，还描述了与 Aβ ( 4–40 / 42 ) 的配位；该位点对 Cu ²⁺的亲和力比 Aβ ( 1–40 / 42 ) 更高，并且产生氧化还原沉默复合物。相反，非乙酰化的 α-突触核蛋白对 Cu ²⁺有两个高亲和力结合位点：位点一涉及 N 末端残基，位点二涉及 His50。正如所描述的在体内发生的那样，α-突触核蛋白的乙酰化消除了位点 1 处的 Cu ²⁺结合，尽管两种形式的 α-突触核蛋白都可以结合 Cu ⁺ 。 (b) Fe ²⁺可以与 Aβ 相互作用，但 Fe-Aβ 复合物的氧化还原性质仍不清楚。有趣的是，最近的研究表明血红素可以与 Aβ 结合，产生具有过氧化物酶活性的血红素 (Fe)-Aβ 复合物。 (c)最后，Zn ²⁺可以与 PrP ^C的八重复区域和 Aβ 结合。 Cu ²⁺ ，铜离子；铁、铁； Fe ²⁺ ，亚铁；锌，锌。

A dysfunction in Cu homeostasis is reported to alter neuronal function and lead to disease progression, including neurodegeneration (Supplementary Table S1). Menkes disease and Wilson disease are caused by mutations or partial deletions in ATP7A and ATP7B, respectively. These Cu transporters have different patterns of expression in the CNS, explaining the distinct pathological features of each disease. ATP7B is found in the visual cortex, anterior cingulate cortex, caudate, putamen, substantia nigra (SN), and cerebellum. ATP7A is detected in astrocytes and neurons from the hippocampus and cerebellum, the BBB and choroid plexus, and during neural development (338). Wilson patients display parkinsonism, underscoring the importance of Cu homeostasis in the motor controlling systems. On the contrary, the ubiquitous expression of ATP7A has challenged mechanistic investigations in Menkes disease. However, a recent study shows that depleting ATP7A in neurons and glia does not lead to neurodegeneration, but to an increased susceptibility to NMDA seizures (132), underscoring its neuroprotective role as described above (Fig. 2a,b). Although neurodegeneration is clearly linked to alterations in Cu homeostasis in Menkes and Wilson diseases, the mechanisms are still unknown.
据报道，铜稳态功能障碍会改变神经元功能并导致疾病进展，包括神经变性（补充表S1 ）。门克斯病和威尔逊病分别由ATP7A和ATP7B的突变或部分缺失引起。这些铜转运蛋白在中枢神经系统中具有不同的表达模式，解释了每种疾病的独特病理特征。 ATP7B 存在于视觉皮层、前扣带皮层、尾状核、壳核、黑质 (SN) 和小脑中。 ATP7A 在海马和小脑、血脑屏障和脉络丛的星形胶质细胞和神经元中以及神经发育过程中被检测到 ( 338 )。威尔逊患者表现出帕金森症，强调了铜稳态在运动控制系统中的重要性。相反，ATP7A 的普遍表达对门克斯病的机制研究提出了挑战。然而，最近的一项研究表明，消耗神经元和神经胶质细胞中的 ATP7A 不会导致神经退行性变，而是会增加对 NMDA 癫痫发作的易感性（ 132 ），这强调了其如上所述的神经保护作用（图 2a，b ）。尽管神经变性显然与门克斯病和威尔逊病中铜稳态的改变有关，但其机制仍不清楚。

Neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Parkinson's disease (PD), and Alzheimer's disease (AD) have been associated with alterations in Cu homeostasis, but a link to specific genetic alterations in Cu transport or handling is missing (Supplementary Table S1). These neurodegenerative diseases are associated with the formation of amyloid aggregates composed of proteins that are either a Cu-dependent antioxidant enzyme, such as SOD1 in ALS, or Cu-binding proteins, such as Aβ and α-synuclein in AD and PD, respectively.
肌萎缩侧索硬化症 (ALS)、亨廷顿病 (HD)、帕金森病 (PD) 和阿尔茨海默病 (AD) 等神经退行性疾病与铜稳态的改变有关，但与铜转运或处理的特定基因改变有关缺失（补充表S1 ）。这些神经退行性疾病与淀粉样蛋白聚集体的形成有关，淀粉样蛋白聚集体由铜依赖性抗氧化酶蛋白（例如 ALS 中的 SOD1）或铜结合蛋白（例如 AD 和 PD 中的 Aβ 和 α-突触核蛋白）组成。

ALS is characterized by the degeneration of motor neurons, and mouse models show increased intracellular Cu levels and the formation of protein aggregates composed of SOD1 and Cu transport proteins such as Ctr1, CCS, Atox1, and Cox17 (344, 346). SOD1 aggregation has been associated with an alteration in protein stability, which is impacted by metallation and Cu-dependent dimerization. Although SOD1 plays an important role in clearing O₂^•−, studies have demonstrated that the mechanism is likely other than the alteration in the antioxidant capacity of SOD1 (312). Increased intracellular Cu levels have been reported in ALS models (346). Consistently, Cu chelators or MT-I overexpression extend the life span and slow disease progression of the SOD1 (G93A) ALS mouse model (345).
ALS 的特点是运动神经元变性，小鼠模型显示细胞内 Cu 水平增加以及由 SOD1 和 Cu 转运蛋白（如 Ctr1、CCS、Atox1 和 Cox17）组成的蛋白质聚集体的形成 ( 344 , 346 )。 SOD1 聚集与蛋白质稳定性的改变有关，而蛋白质稳定性的改变受到金属化和 Cu 依赖性二聚化的影响。尽管SOD1 在清除O ₂ ^{•− 方面}发挥着重要作用，但研究表明该机制很可能与SOD1 抗氧化能力的改变不同( 312 )。据报道，ALS 模型中细胞内铜水平增加 ( 346 )。一致地，Cu 螯合剂或 MT-I 过表达可延长SOD1 (G93A) ALS 小鼠模型的寿命并减缓疾病进展 ( 345 )。

HD is an autosomal dominant genetic disorder caused by polyglutamine (polyQ) repeat expansions near the N-terminus of the huntingtin (Htt) protein. HD is characterized by movement dysfunction, psychiatric and cognitive alterations linked to the degeneration of striatal spiny neurons. Cu accumulation in the striatum of HD transgenic mice has been reported (102). Mutant Htt forms toxic aggregates, but the mechanisms of toxicity are still unclear. A putative Cu binding site in Htt involving Met8 and His82 has been identified, and this interaction promotes polyQ aggregate formation (102, 381), which is reduced by MT-III overexpression (123).
HD 是一种常染色体显性遗传性疾病，由亨廷顿蛋白 (Htt) 蛋白 N 末端附近的聚谷氨酰胺 (polyQ) 重复扩增引起。 HD 的特点是运动功能障碍、精神和认知改变，这些改变与纹状体棘神经元的退化有关。据报道，HD 转基因小鼠的纹状体中存在 Cu 积累 ( 102 )。突变体Htt形成有毒聚集体，但毒性机制仍不清楚。已鉴定出 Htt 中涉及 Met8 和 His82 的推定 Cu 结合位点，这种相互作用促进了 polyQ 聚集体的形成 ( 102 , 381 )，而 MT-III 过表达可减少这种聚集体的形成 ( 123 )。

PD is characterized by the degeneration of dopaminergic neurons in the SN, which are rich in Cu. PD patients show decreased Cu content in the SN (70) without changes in Cu levels in serum, plasma, and cerebrospinal fluid (CSF) (203). Accordingly, decreased levels of CTR1 (70) have been reported in the SN, while MT-I and MT-II levels in active astrocytes are increased, reflecting a glial response to the loss of Cu homeostasis in PD (226). In contrast, occupational exposure to Cu has been linked to an increased risk to develop PD (115).
PD 的特征是 SN 中富含铜的多巴胺能神经元变性。 PD患者表现出SN（ 70 ）中的Cu含量降低，而血清、血浆和脑脊液（CSF）中的Cu水平没有变化（ 203 ）。因此，据报道，SN 中 CTR1 水平降低 ( 70 )，而活性星形胶质细胞中 MT-I 和 MT-II 水平升高，反映了神经胶质细胞对 PD 中铜稳态丧失的反应 ( 226 )。相比之下，职业接触铜与患帕金森病的风险增加有关（ 115 ）。

The accumulation of intracellular protein inclusions (Lewy bodies), where α-synuclein is the main protein component, is another hallmark of PD (362). α-Synuclein is a small intrinsically disordered protein (IDP) enriched in presynaptic terminals and nucleus that can interact with cytoskeleton components and lipid membranes (170). IDPs are proteins that can adopt different conformations, and thus respond to changes in their biochemical environment. This property allows them to engage in interactions with multiple protein targets (23). Cu²⁺ and Cu⁺ ions are capable of binding to α-synuclein at three different sites (20, 33, 228, 361) (Fig. 3a). Interestingly, the H50Q SNCA/α-synuclein mutation linked to hereditary PD abolishes one Cu binding site altering Cu-induced α-synuclein aggregation (361). Moreover, Cu binding to the high-affinity Cu-binding site at the N-terminal region of α-synuclein accelerates its amyloid aggregation in vitro (20, 33); although this effect is abolished in acetylated α-synuclein (234), which is found in Lewy bodies (10).
细胞内蛋白质内含物（路易体）的积累（其中 α-突触核蛋白是主要蛋白质成分）是 PD 的另一个标志 ( 362 )。 α-突触核蛋白是一种小的本质无序蛋白 (IDP)，富含突触前末梢和细胞核，可以与细胞骨架成分和脂质膜相互作用 ( 170 )。 IDP 是可以采用不同构象的蛋白质，从而对其生化环境的变化做出反应。这一特性使它们能够与多个蛋白质靶标相互作用（ 23 ）。 Cu ²⁺和Cu ⁺离子能够在三个不同位点(20、33、228、361 )结合α-突触核蛋白(图3a ) 。有趣的是，与遗传性 PD 相关的 H50Q SNCA /α-突触核蛋白突变消除了一个铜结合位点，改变了铜诱导的 α-突触核蛋白聚集 ( 361 )。此外，Cu 与 α-突触核蛋白 N 末端区域的高亲和力 Cu 结合位点的结合加速了其体外淀粉样蛋白的聚集 ( 20 , 33 )；尽管这种效应在乙酰化 α-突触核蛋白 ( 234 ) 中被消除，而乙酰化 α-突触核蛋白存在于路易体 ( 10 ) 中。

While several studies have suggested that Cu-induced aggregation of α-synuclein is directly linked to its neurotoxicity, recent studies suggest a lack of correlation between protein aggregation and cytotoxicity (361); in fact, it has been demonstrated that α-synuclein potentiates the toxicity of Cu in dopaminergic cells in the absence of enhanced accumulation of protein aggregates (8). Clearly, further investigations are needed to completely understand the structural impact of Cu-α-synuclein interactions and their role in PD. On the contrary, acetylation and Cu⁺ binding to α-synuclein are two synergistic events that turn the intrinsically disordered N-terminal region into an α-helix conformation (228) (Fig. 2d), which displays higher affinity for membranes (75). These observations suggest a potential link between Cu⁺-α-synuclein interactions and the proposed function of α-synuclein in vesicle trafficking; a link that might be perturbed in PD.
虽然一些研究表明铜诱导的 α-突触核蛋白聚集与其神经毒性直接相关，但最近的研究表明蛋白质聚集和细胞毒性之间缺乏相关性 ( 361 )；事实上，已经证明，在不增加蛋白质聚集体积累的情况下，α-突触核蛋白会增强多巴胺能细胞中铜的毒性 ( 8 )。显然，需要进一步研究才能完全了解 Cu-α-突触核蛋白相互作用的结构影响及其在 PD 中的作用。相反，乙酰化和 Cu ⁺与 α-突触核蛋白结合是两个协同事件，将本质上无序的 N 末端区域转变为 α-螺旋构象 ( 228 )（图 2d ），这对膜表现出更高的亲和力 ( 75 ) 。这些观察结果表明 Cu ⁺ -α-突触核蛋白相互作用与 α-突触核蛋白在囊泡运输中的拟议功能之间存在潜在联系； PD 中可能受到干扰的链接。

Different redox modifications of α-synuclein are found in Lewy bodies, including Met oxidation, Tyr nitration (67), and formation of di-Tyr-linked α-synuclein dimers. Cu⁺-α-synuclein complexes have been implicated in these modifications, as they are capable of activating O₂, leading to Met oxidation and di-Tyr bond formation (5, 227) (Fig. 3a). In contrast, a recent report suggests that Cu⁺ complexes with oligomeric or fibrillar α-synuclein reduce metal-catalyzed ROS formation (264). Cu has also been shown to potentiate oxidative damage induced by the dopamine (DA) analog 6-hydroxydopamine (63). Cu also interacts with the early-onset PD recessive genes (protein) PARK7 (DJ-1) and PARK2(Parkin). Cu binding to DJ-1 protects against metal toxicity, possibly acting as a chaperone for SOD1 (35, 114); while Parkin mutations have been reported to increase the cytotoxic effect of heavy metals, including Cu (1).
在路易体中发现了 α-突触核蛋白的不同氧化还原修饰，包括 Met 氧化、Tyr 硝化 ( 67 ) 以及二-Tyr 连接的 α-突触核蛋白二聚体的形成。 Cu ⁺ -α-突触核蛋白复合物参与了这些修饰，因为它们能够激活 O ₂ ，​​导致 Met 氧化和二-Tyr 键形成 ( 5 , 227 )（图 3a ）。相反，最近的一份报告表明，Cu ⁺与寡聚或纤维状 α-突触核蛋白的复合物可减少金属催化的 ROS 形成 ( 264 )。 Cu 还被证明可以增强多巴胺 (DA) 类似物 6-羟基多巴胺 ( 63 ) 引起的氧化损伤。 Cu 还与早发性 PD 隐性基因（蛋白质） PARK7 (DJ-1) 和PARK2 (Parkin) 相互作用。 Cu 与 DJ-1 结合可防止金属毒性，可能充当 SOD1 的伴侣 ( 35 , 114 )；据报道，Parkin 突变会增加重金属（包括 Cu）的细胞毒性作用 ( 1 )。

AD is a neurodegenerative disease associated with the degeneration of hippocampal and cortical neurons and eventual loss of memory and progressive dementia (326). Decreased levels of Cu are found in AD brains (42), while the total Cu content and labile nonprotein-bound Cu fraction are increased in the plasma of AD patients (325). Interestingly, polymorphisms in ATP7B have been linked to AD (326). AD is associated with the formation of extracellular amyloid plaques composed of Cu bound to Aβ peptides 1–40 and 1–42 fragments produced by cleavage of the APP, as well as N-truncated forms 4–40 and 4–42, and to a lesser extent 11–40/42 (210, 273, 364). The Cu binding features of Aβ 4–40/42 and 11–40/42 are different from those of 1–40/42, leading to the formation of Cu-Aβ complexes with distinct redox properties (Fig. 3a) (229). Different aggregation properties have also been described, as illustrated by the faster fiber assembly rate of Aβ 11–40/42 when compared with 1–40/42 (21). Substoichiometric Cu²⁺ concentrations trigger Aβ aggregation through a different pathway that involves the formation of oligomers more neurotoxic than those generated by the peptide alone (25, 204). On the contrary, the redox activity of Cu-Aβ complexes has also been proposed to lead to the generation of ROS (52, 214), but contradictory results exist as well (264). Interestingly, interaction of Cu with N-truncated 4–40 and 4–42 peptides yields redox-inactive Cu-Aβ complexes (229, 350). A dysfunction in Cu homeostasis in AD is also evidenced by decreased levels of MT-III (390). MTs are capable of exchanging Cu²⁺ with Aβ1–40/42, reducing Cu and stabilizing it. This Cu exchange by MTs has been proposed as a redox-silencing mechanism that prevents ROS formation by Cu-Aβ complexes (223).
AD 是一种神经退行性疾病，与海马和皮质神经元退化以及最终记忆丧失和进行性痴呆相关（ 326 ）。 AD 大脑中铜的水平降低 ( 42 )，而 AD 患者血浆中的总铜含量和不稳定的非蛋白质结合铜分数则增加 ( 325 )。有趣的是， ATP7B的多态性与 AD 相关（ 326 ）。 AD 与细胞外淀粉样斑块的形成有关，该斑块由与 APP 裂解产生的 Aβ 肽 1-40 和 1-42 片段以及 N-截短形式 4-40 和 4-42 以及与较小范围 11–40/42 ( 210 , 273 , 364 )。 Aβ 4–40/42 和 11–40/42 的 Cu 结合特征与 1–40/42 不同，导致形成具有不同氧化还原性质的 Cu-Aβ 复合物（图 3a ）（ 229 ）。还描述了不同的聚集特性，如与 1-40/42 相比，Aβ 11-40/42 的纤维组装速率更快 ( 21 )。亚化学计量的 Cu ²⁺浓度通过不同的途径触发 Aβ 聚集，该途径涉及形成比单独肽产生的寡聚物更具神经毒性的寡聚物 ( 25 , 204 )。相反，Cu-Aβ复合物的氧化还原活性也被认为会导致ROS的产生( 52 , 214 )，但也存在矛盾的结果( 264 )。有趣的是，Cu 与 N 截短的 4-40 和 4-42 肽相互作用产生氧化还原无活性的 Cu-Aβ 复合物 ( 229 , 350 )。 MT-III 水平降低也证明了 AD 中铜稳态的功能障碍 ( 390 )。 MT 能够将 Cu ²⁺与 Aβ1–40/42 交换，还原 Cu 并使其稳定。 MT 的这种 Cu 交换被认为是一种氧化还原沉默机制，可防止 Cu-Aβ 复合物形成 ROS ( 223 )。

The impact of dysfunctional Cu homeostasis in AD might go beyond the neurotoxicity of Cu-Aβ complexes. APP, whose mutations are associated with AD, is a type 1 transmembrane protein that displays three Cu binding sites in its extracellular domain (22, 140). One Cu-binding site is located in the growth factor-like domain, and has been implicated in Cu-induced dimerization of APP, a process that would be important in cell adhesion and signaling (22). Cu was found to induce APP phosphorylation at Thr668 promoting its localization to the axonal membrane, suggesting an important link between Cu and APP functions at the synapse that might be perturbed in AD. Aβ peptides can also interfere with Cu-PrP^C interactions implicated in the regulation of NMDAR activity (Fig. 2b) (389). In addition, Cu has been reported to promote the degradation of the low-density lipoprotein receptor-related protein 1 (LRP1) via Tyr nitration and proteasomal degradation, which was linked to a decrease of Aβ clearance and its resultant accumulation in brain vasculature (319). Clearly, Cu plays important roles in neuromodulation and signaling processes, which would be perturbed in AD.
AD 中 Cu 稳态功能失调的影响可能超出 Cu-Aβ 复合物的神经毒性。 APP 的突变与 AD 相关，是一种 1 型跨膜蛋白，在其胞外结构域中显示三个 Cu 结合位点 ( 22 , 140 )。一个 Cu 结合位点位于生长因子样结构域中，并且与 Cu 诱导的 APP 二聚化有关，这一过程对于细胞粘附和信号转导非常重要 ( 22 )。研究发现 Cu 可诱导 APP 在 Thr668 处磷酸化，促进其定位至轴突膜，这表明 Cu 与突触处 APP 功能之间的重要联系可能在 AD 中受到干扰。 Aβ 肽还可以干扰与 NMDAR 活性调节有关的 Cu-PrP ^C相互作用（图 2b ）（ 389 ）。此外，据报道，Cu 可通过Tyr 硝化和蛋白酶体降解促进低密度脂蛋白受体相关蛋白 1 (LRP1) 的降解，这与 Aβ 清除率降低及其在脑血管系统中的积累有关（ 319 ）。显然，铜在神经调节和信号传导过程中发挥着重要作用，而这些过程在 AD 中会受到干扰。

Iron 铁

Fe is found in biological systems primarily as ferrous (2+) and ferric (3+) ions (Supplementary Table S1). Fe is a redox-active metal involved in several redox reactions that catalyze the formation of ROS. Fe is tightly bound to Fe storage and transport proteins, while <5% is present as labile redox-active Fe bound to low-affinity molecules. Fe is required as cofactor of several important enzymes for respiration and synthesis of neurotransmitters, including tryptophan hydroxylase (serotonin) and Tyr hydroxylase (norepinephrine and DA), cholesterol, and fatty acids; the latter particularly important for nerve myelination (161, 342). Electron transfer in many Fe enzymes, including the mitochondrial respiratory complexes, is facilitated by heme and Fe/S clusters (291). Fe is also important as cofactor for peroxidases and catalases, which are important for cellular redox homeostasis (6).
Fe 在生物系统中主要以亚铁 (2+) 和三价铁 (3+) 离子的形式存在（补充表 S1 ）。 Fe 是一种氧化还原活性金属，参与多种催化 ROS 形成的氧化还原反应。 Fe与Fe储存和运输蛋白紧密结合，而<5 id=453>161, 342 )。许多铁酶（包括线粒体呼吸复合物）中的电子转移是由血红素和铁/硫簇促进的（ 291 ）。 Fe 作为过氧化物酶和过氧化氢酶的辅助因子也很重要，这对于细胞氧化还原稳态很重要 ( 6 )。

Fe is heterogeneously distributed in the brain; it is highly concentrated in the SN, hippocampus, striatum, interpeduncular nuclei (125), and myelin (329) (Supplementary Table S1). Fe homeostasis is regulated by communication between the BBB and astrocytes. In the blood stream, Fe³⁺ is found coordinated to transferrin (Tf) or ferritin (Ft), and as heme. Fe uptake into the BBB can occur by two pathways: (a) by direct transport of Fe²⁺ into the cytosol via DMT1; or (b) by endocytosis of Tf-bound Fe³⁺ via the Tf receptor (TfR), where the low pH of the endosome causes the release of Fe³⁺ from Tf. In both cases, Fe³⁺ is reduced to Fe²⁺ by the duodenal cytochrome b (Dcytb) or by the six transmembrane epithelial antigen of the prostate 2 (Steap2) ferrireductases and then transported by DMT1 (Fig. 4a) (215, 218).
Fe在大脑中的分布不均匀；它高度集中在 SN、海马、纹状体、脚间核 ( 125 ) 和髓磷脂 ( 329 )（补充表 S1 ）。 Fe 稳态由血脑屏障和星形胶质细胞之间的通讯调节。在血流中，Fe ³⁺与转铁蛋白 (Tf) 或铁蛋白 (Ft) 配位，并形成血红素。 Fe 吸收到 BBB 中可以通过两种途径发生： (a)通过DMT1 将 Fe ²⁺直接转运到细胞质中；或（b）通过Tf受体（TfR）内吞Tf结合的Fe ³⁺ ，其中内体的低pH导致Tf释放Fe ³⁺ 。在这两种情况下，Fe ³⁺均被十二指肠细胞色素 b (Dcytb) 或前列腺六跨膜上皮抗原 2 (Steap2) 铁还原酶还原为 Fe ²⁺ ，然后由 DMT1 转运（图 4a ）（ 215 , 218） ）。

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FIG. 4. 如图。 4.

Fe transport and redox signaling. (a) Fe transport across the BBB occurs via DMT1 or by endocytosis of Tf- or Ft- bound Fe³⁺; followed by Fe release via the concerted action of Fpn with a ferroxidase, either Hp or sCp. Astrocytes modulate Fe transport through the production of sCp, sAPP, and hepcidin. sAPP stabilizes Fpn, while hepcidin induces its internalization. (b) Mitochondria are the organelles with highest demand of Fe. Although Fe transport into mitochondria is not fully understood, the recently demonstrated interaction between mitochondria and TfR-containing endosomes supports the “kiss and run” hypothesis. Inside the mitochondria, Fe crosses the inner membrane by Mfrn2. (c) In hippocampal neurons, NMDAR activation induces Ca²⁺ entry and NO^• production by nNOS, causing S-nitros(yl)ation of Dexras1, which in turn induces Fe uptake through DMT1 and TfR. (d) Increased intracellular Fe also induces H₂O₂ production, inducing Ca²⁺ release from ER via RyR, which is important for neuronal plasticity. Dexras1, Ras-related dexamethasone induced 1; ER, endoplasmic reticulum; Fe³⁺, ferric; Fpn, ferroportin; Ft, ferritin; H₂O₂, hydrogen peroxide; Hp, hephaestin; Mfrn2, mitoferrin-2; NO^•, nitric oxide; PAP7, peripheral benzodiazepine receptor-associated protein 7; RyR, ryanodine receptor; sAPP, soluble fragment of APP; sCp, soluble ceruloplasmin; Tf, transferrin; TfR: transferrin receptor.
Fe 运输和氧化还原信号传导。 (a)通过DMT1 或通过 Tf- 或 Ft- 结合的 Fe ³⁺的内吞作用发生穿过 BBB 的 Fe 转运；随后通过Fpn 与亚铁氧化酶（Hp 或 sCp）的协同作用释放 Fe。星形胶质细胞通过产生 sCp、sAPP 和铁调素来调节铁转运。 sAPP 稳定 Fpn，而铁调素则诱导其内化。 (b)线粒体是铁需求最高的细胞器。尽管 Fe 转运到线粒体的情况尚不完全清楚，但最近证明的线粒体和含有 TfR 的内体之间的相互作用支持“亲吻并奔跑”的假设。在线粒体内，Fe 通过 Mfrn2 穿过内膜。 (c)在海马神经元中，NMDAR 激活诱导 Ca ²⁺进入和 nNOS 产生 NO ^• ，导致 Dexras1 的 S-亚硝基化，进而通过 DMT1 和 TfR 诱导 Fe 吸收。 (d)细胞内 Fe 的增加也会诱导 H ₂ O ₂的产生，从而诱导 Ca ²⁺通过RyR 从 ER 中释放，这对于神经元的可塑性很重要。 Dexras1，Ras相关地塞米松诱导1； ER，内质网； Fe ³⁺ 、三价铁； Fpn，铁转运蛋白； Ft，铁蛋白； H ₂ O ₂ 、过氧化氢； Hp，火铁黄素； Mfrn2，线粒体铁蛋白-2； NO ^• ，一氧化氮； PAP7，外周苯二氮卓受体相关蛋白7； RyR，兰尼碱受体； sAPP，APP的可溶性片段； scp，可溶性铜蓝蛋白； Tf，转铁蛋白； TfR：转铁蛋白受体。

Fe efflux from endothelial cells to the interstitial space occurs via the coordinated activity of the Fe²⁺ transporter ferroportin (Fpn) and the Cu-dependent ferroxidases hephaestin (Hp) and soluble ceruloplasmin (sCp), which oxidize Fe²⁺ to Fe³⁺. Astrocytes regulate the release of Fe from BBB by either secretion of sCp, which stimulates Fe release, or by production of hepcidin, a peptide that induces internalization and ubiquitination of Fpn and thus, decreased Fe efflux (216). Astrocytes also express a glycosylphosphatidylinositol (GPI)-anchored form of Cp, which interacts with Fpn and participates in Fe efflux (Fig. 4a) (144). In general, oligodendrocytes, astrocytes, microglia, and neurons have the same machinery for Fe efflux, involving the concerted action of Fpn with Cp or Hp (60).
Fe 从内皮细胞流出到间质空间是通过Fe ²⁺转运蛋白铁转运蛋白 (Fpn) 和 Cu 依赖性亚铁氧化酶铁菲蛋白 (Hp) 和可溶性铜蓝蛋白 (sCp) 的协调活性发生的，这些酶将 Fe ²⁺氧化为 Fe ³⁺ 。星形胶质细胞通过分泌 sCp（刺激 Fe 释放）或通过产生铁调素（一种诱导 Fpn 内化和泛素化的肽，从而减少 Fe 流出）来调节 BBB 中 Fe 的释放 ( 216 )。星形胶质细胞还表达糖基磷脂酰肌醇（GPI）锚定形式的 Cp，它与 Fpn 相互作用并参与 Fe 流出（图 4a ）（ 144 ）。一般来说，少突胶质细胞、星形胶质细胞、小胶质细胞和神经元具有相同的 Fe 流出机制，涉及 Fpn 与 Cp 或 Hp 的协同作用 ( 60 )。

The mechanisms of Fe uptake differ between brain cell types. Fe, Tf, and Ft are primarily found in oligodendrocytes (61). Most Tf in the brain is synthesized and secreted by oligodendrocytes as Fe-free Tf or apo-Tf and is required for Fe mobilization within the interstitial fluid brain (87). Although oligodendrocytes and astrocytes can accumulate high levels of Fe, they do not express TfR. Fe uptake into oligodendrocytes has been recently proposed to involve the internalization of H-Ft by a mucin-domain containing protein (Tim-2) (343). In astrocytes, ascorbate-dependent Fe²⁺ uptake is mediated by DMT1 (167) and transient-receptor potential channels (265).
不同类型的脑细胞吸收铁的机制有所不同。 Fe、Tf 和 Ft 主要存在于少突胶质细胞中 ( 61 )。大脑中的大多数 Tf 由少突胶质细胞合成并分泌为无铁 Tf 或 apo-Tf，并且是脑间质液内铁动员所必需的 ( 87 )。尽管少突胶质细胞和星形胶质细胞可以积累高水平的 Fe，但它们不表达 TfR。最近有人提出，少突胶质细胞对 Fe 的吸收涉及含有粘蛋白结构域的蛋白质 (Tim-2) 对 H-Ft 的内化 ( 343 )。在星形胶质细胞中，抗​​坏血酸依赖性 Fe ²⁺摄取由 DMT1 ( 167 ) 和瞬时受体电位通道 ( 265 ) 介导。

In neurons, the mechanisms involved in Fe uptake are still unclear. DMT1 is found in human neurons and its expression levels are negatively regulated by Fe exposure via ubiquitination and degradation (134). However, DMT1 seems to colocalize in cytoplasmic vesicles with TfR and to primarily contribute to Tf-bound Fe uptake (232, 266). The ZIP8 and ZIP14 members of the ZIP family of metal transporters have also been demonstrated to mediate Fe²⁺ uptake (188) and to be expressed in the brain (113). A recent report demonstrates that ZIP8 at the plasma membrane is the primary transporter involved in non-Tf-bound Fe into neurons (145).
在神经元中，铁吸收的机制仍不清楚。 DMT1 存在于人类神经元中，其表达水平通过泛素化和降解受到 Fe 暴露的负调节 ( 134 )。然而，DMT1 似乎与 TfR 共定位于细胞质囊泡中，并且主要促进 Tf 结合的 Fe 吸收 ( 232 , 266 )。金属转运蛋白 ZIP 家族的 ZIP8 和 ZIP14 成员也已被证明可以介导 Fe ²⁺吸收 ( 188 ) 并在大脑中表达 ( 113 )。最近的一份报告表明，质膜上的 ZIP8 是参与非 Tf 结合 Fe 进入神经元的主要转运蛋白 ( 145 )。

In brain cells, the ferrireductases Dcytb and stromal cell-derived receptor, SDR2, are expressed in astrocytes (192, 351), while SDR2 and Steap2 are found in neurons (145). In all cases, it is important to note that Fe uptake and efflux always require redox cycling between Fe²⁺ and Fe³⁺ oxidation states.
在脑细胞中，铁还原酶 Dcytb 和基质细胞衍生受体 SDR2 在星形胶质细胞中表达 ( 192 , 351 )，而 SDR2 和 Steap2 在神经元中表达 ( 145 )。在所有情况下，重要的是要注意 Fe 的吸收和流出始终需要 Fe ²⁺和 Fe ³⁺氧化态之间的氧化还原循环。

Cytosolic Fe is stored by Ft, which is a protein complex formed by 24 subunits of heavy-ferritin (H-Ft) and light-ferritin (L-Ft) chains. Ft genes are regulated by Nrf2 (272). While H-subunits have ferroxidase activity and participate in Fe uptake, L-subunits are involved in Fe mineralization and long-term storage. Ft can store ∼4500 Fe ions in its core. However, the mechanism by which Fe is released from Ft remains unclear (47). Although Fe is mainly stored in the cytosol, mitochondria are the organelles with the highest Fe demand, as they require Fe-S clusters and heme groups for electron transfer during respiration. Similarly, mitochondria are also considered the main sources of ROS under physiological conditions. Therefore, mitochondrial Fe homeostasis must be tightly regulated to prevent uncontrolled ROS production.
胞质铁由 Ft 储存，Ft 是由重铁蛋白 (H-Ft) 和轻铁蛋白 (L-Ft) 链的 24 个亚基形成的蛋白质复合物。 Ft 基因受 Nrf2 调节 ( 272 )。 H 亚基具有亚铁氧化酶活性并参与铁的吸收，而 L 亚基则参与铁的矿化和长期储存。 Ft 的核心可储存 ∼4500 个 Fe 离子。然而，Fe 从 Ft 中释放的机制仍不清楚 ( 47 )。虽然Fe主要储存在细胞质中，但线粒体是Fe需求量最高的细胞器，因为它们需要Fe-S簇和血红素基团在呼吸过程中进行电子传递。同样，线粒体也被认为是生理条件下ROS的主要来源。因此，必须严格调节线粒体 Fe 稳态，以防止不受控制的 ROS 产生。

The mechanism(s) involved in mitochondrial Fe uptake are still unclear. Fe is delivered by a direct “kiss and run” interaction between the endosomes containing Tf-bound Fe and mitochondria (69). Subsequently, the metal crosses the inner membrane via the Fe importer mitoferrin-2 (Mfrn2) (Fig. 4b). In the mitochondrial matrix, Fe is either used for the biogenesis of prosthetic groups, Fe-S clusters, or heme, or it is stored by mitochondrial ferritin (FtMt), which is homologous to H-Ft, and it displays the same Fe uptake efficiency but lower ferroxidase activity. Fe distribution between mitochondria and cytosol depends on FtMt expression and the export of Fe prosthetic groups (106, 176).
线粒体铁吸收的机制仍不清楚。 Fe 通过含有 Tf 结合 Fe 的内体与线粒体之间的直接“亲吻和奔跑”相互作用来传递 ( 69 )。随后，金属通过Fe 输入端线粒体铁蛋白-2 (Mfrn2) 穿过内膜（图 4b ）。在线粒体基质中，Fe 要么用于假体基团、Fe-S 簇或血红素的生物合成，要么由线粒体铁蛋白 (FtMt) 储存，FtMt 与 H-Ft 同源，并且表现出相同的 Fe 吸收效率较高，但亚铁氧化酶活性较低。线粒体和细胞质之间的 Fe 分布取决于 FtMt 表达和 Fe 辅基的输出 ( 106 , 176 )。

Heme is a component of globins, a superfamily of heme-containing proteins involved in binding and/or transporting O₂, and a cofactor for cytochromes, catalase, NOX, NOS, and myeloperoxidase, which is also found in microglia (59, 151, 198). Hemoglobin (Hb) is involved in O₂, NO, and carbon dioxide (CO₂) transport in cells of erythroid lineage, but Hbα and β transcripts have been found in dopaminergic neurons, oligodendrocytes, and cortical or hippocampal astrocytes as well (32). Brain Hb levels are altered during neurodegeneration (94), but their functional consequences are unclear. Neuroglobin is another globin expressed in the CNS and PNS and found in both neurons and astrocytes. Neuroglobin has a higher affinity for O₂ than Hb and exerts a protective effect against oxidative and ischemic insults (7, 43, 81, 179, 331, 368). Oxidative stress has the potential to release heme from Hb, and labile heme can induce oxidative damage via Fenton reactions or NOX activation (165, 239). Heme oxygenases (HOs) catalyze heme degradation. HO-1 transcription is induced by oxidative stress, inflammation, hypoxia, and metal exposure, while HO-2 is constitutively expressed. HO activity has both antioxidant and pro-oxidant effects that relate to the ability of the cell to detoxify labile Fe released from heme (286, 369).
血红素是球蛋白的组成部分，球蛋白是参与结合和/或运输 O ₂的含血红素蛋白的超家族，也是细胞色素、过氧化氢酶、NOX、NOS 和髓过氧化物酶的辅助因子，也存在于小胶质细胞中 ( 59 , 151 , 198 ）。血红蛋白 (Hb) 参与红系细胞中的 O ₂ 、NO 和二氧化碳 (CO ₂ ) 转运，但在多巴胺能神经元、少突胶质细胞以及皮质或海马星形胶质细胞中也发现了 Hbα 和 β 转录本 ( 32 ) 。大脑 Hb 水平在神经退行性变期间发生改变 ( 94 )，但其功能后果尚不清楚。神经球蛋白是另一种在 CNS 和 PNS 中表达的球蛋白，在神经元和星形胶质细胞中都有发现。神经球蛋白对 O _{2 的}亲和力比 Hb 更高，并且对氧化和缺血性损伤发挥保护作用 ( 7 , 43 , 81 , 179 , 331 , 368 )。氧化应激有可能从 Hb 中释放血红素，而不稳定的血红素可以通过芬顿反应或 NOX 活化诱导氧化损伤 ( 165 , 239 )。血红素加氧酶 (HO) 催化血红素降解。 HO-1 转录由氧化应激、炎症、缺氧和金属暴露诱导，而 HO-2 则持续表达。 H2O 活性具有抗氧化和促氧化作用，与细胞解毒血红素释放的不稳定铁的能力有关 ( 286 , 369 )。

Cellular Fe trafficking is controlled by the iron regulatory proteins (IRP) IRP1 and IRP2 which regulate translation of proteins involved in Fe storage (H-Ft and L-Ft), Fe uptake (TfR), and Fe efflux (Fpn). Under conditions of Fe depletion, IRPs can bind to messenger RNAs (mRNAs), decreasing the levels of Ft and Fpn and promoting the translation of TfR via the stabilization of its mRNA. Conversely, Fe overload prevents mRNA binding to IRPs, promoting Ft and Fpn translation while reducing TfR levels. The ability of IRP1 and IRP2 to bind mRNAs exerts an important redox control via two distinct mechanisms. IRP1 has two conformations: (a) a closed one, triggered via an Fe-S cluster that prevents mRNA binding and has aconitase activity; and (b) an open conformation that is favored when NO^•, O₂, and H₂O₂ cause dissociation of the Fe-S cluster. In contrast, the ability of IRP2 to bind mRNAs is controlled by proteasomal degradation, involving the ubiquitin ligase F-box/LRR-repeat protein (FBXL5), which is also an Fe and O₂ sensing protein. FBXL5 has a binuclear nonheme Fe site, and it is stabilized on Fe and O₂ binding, promoting IRP2 degradation. Together, these two mechanisms illustrate the redox control of cellular Fe trafficking (164).
细胞铁运输由铁调节蛋白 (IRP) IRP1 和 IRP2 控制，它们调节参与铁储存（H-Ft 和 L-Ft）、铁吸收（TfR）和铁流出（Fpn）的蛋白质的翻译。在 Fe 耗尽的条件下，IRP 可以与信使 RNA (mRNA) 结合，降低 Ft 和 Fpn 的水平，并通过稳定其 mRNA 来促进 TfR 的翻译。相反，Fe 超载会阻止 mRNA 与 IRP 结合，促进 Ft 和 Fpn 翻译，同时降低 TfR 水平。 IRP1 和 IRP2 结合 mRNA 的能力通过两种不同的机制发挥重要的氧化还原控制作用。 IRP1 有两种构象：(a) 闭合构象，通过Fe-S 簇触发，阻止 mRNA 结合并具有乌头酸酶活性； (b)当NO ^· 、O ₂和H ₂ O ₂导致Fe-S簇解离时有利的开放构象。相比之下，IRP2 结合 mRNA 的能力受到蛋白酶体降解的控制，涉及泛素连接酶 F-box/LRR 重复蛋白 (FBXL5)，该蛋白也是一种 Fe 和 O ₂传感蛋白。 FBXL5 具有双核非血红素 Fe 位点，它在 Fe 和 O ₂结合上稳定，促进 IRP2 降解。这两种机制共同说明了细胞 Fe 运输的氧化还原控制 ( 164 )。

In neurons, Fe uptake is induced by a redox signaling cascade that starts with the activation of the NMDAR (Fig. 4c). Increased intracellular Ca²⁺ induces NO^• production by the neuronal (n) NOS, leading to S-nitros(yl)ation of the small GTPase Dexras1 (Ras-related dexamethasone induced 1), which in turn induces Fe uptake through DMT1 and TfR (51). In hippocampal neurons, NMDAR activation increases intracellular Fe and H₂O₂ production, which activates the redox-sensitive ryanodine receptor (RyR) and promotes Ca²⁺ release from the ER (237) (Fig. 4d). Thus, Fe uptake is clearly part of a redox signaling mechanism that might be important for neuronal plasticity.
在神经元中，Fe 的吸收是由氧化还原信号级联诱导的，该级联始于 NMDAR 的激活（图 4c ）。细胞内 Ca ²⁺增加诱导神经元 (n) NOS 产生 NO ^• ，导致小 GTP 酶 Dexras1（Ras 相关的地塞米松诱导 1）发生 S-亚硝基（基）化，进而通过 DMT1 和 TfR 诱导 Fe 摄取（ 51 ）。在海马神经元中，NMDAR 激活增加细胞内 Fe 和 H ₂ O ₂的产生，从而激活氧化还原敏感的兰尼碱受体（RyR）并促进 Ca ²⁺从 ER 释放（ 237 ）（图 4d ）。因此，铁的吸收显然是氧化还原信号机制的一部分，可能对神经元可塑性很重要。

Cell death induced by an increase in the labile Fe pool within cells has been defined as a specific entity named ferroptosis. Ferroptotic cell death is a necrotic-like cell death characterized by Fe-dependent lipid peroxidation due to either the formation of ^•OH and H₂O₂ via Fenton-like reactions or the activation of lipoxygenases. As such, ferroptosis is counteracted by Fe-chelators and the GSH/GPX4 system. GPX4 knockout in neurons induces motor neuron degeneration, paralysis (53), and cognitive impairment (121). During ferroptosis, Tf-dependent Fe uptake and release of Fe from lysosomal compartments have been shown to act as important sources for Fe. Interestingly, lysosomal permeabilization is a common phenomenon observed in a number of neurodegenerative disorders, including PD (40, 73). However, during pathological conditions such as neurodegeneration and hemorrhagic stroke, cell death is likely to involve a combination of different pathways and a complex balance between them, including apoptosis, necrosis, autophagic cell death, and ferroptosis as well (77, 143, 357, 398).
由细胞内不稳定铁池增加引起的细胞死亡被定义为一种称为铁死亡的特定实体。铁死亡细胞死亡是一种类似坏死的细胞死亡，其特征是由于通过类芬顿反应形成^· OH 和H ₂ O ₂或脂氧合酶的激活而导致Fe 依赖性脂质过氧化。因此，Fe 螯合剂和 GSH/GPX4 系统可以抵消铁死亡。神经元中的GPX4敲除会导致运动神经元变性、瘫痪 ( 53 ) 和认知障碍 ( 121 )。在铁死亡过程中，Tf依赖性铁的吸收和溶酶体区室中铁的释放已被证明是铁的重要来源。有趣的是，溶酶体通透是在许多神经退行性疾病中观察到的常见现象，包括 PD ( 40 , 73 )。然而，在神经退行性疾病和出血性中风等病理情况下，细胞死亡可能涉及不同途径的组合以及它们之间的复杂平衡，包括细胞凋亡、坏死、自噬性细胞死亡和铁死亡（ 77、143、357 、 398 ）。

During aging, accumulation of Fe in the frontal lobes and striatum is associated with motor dysfunction, loss of myelin sheaths, and memory decline (2, 327). Inflammation, a common hallmark of many brain disorders, increases the expression of DMT1 and hepcidin (354).
在衰老过程中，额叶和纹状体中铁的积累与运动功能障碍、髓鞘损失和记忆力下降有关 ( 2 , 327 )。炎症是许多脑部疾病的常见标志，会增加 DMT1 和铁调素的表达 ( 354 )。

Abnormal accumulation of Fe is a common feature of many neurodegenerative diseases, including a group of twelve diseases known as neurodegeneration with brain iron accumulation (NBIA) (225). The most common clinical features of NBIA are movement disorders, such as ataxia, parkinsonism, and dystonia. Although in NBIA Fe is usually accumulated in the globus pallidus, and in some cases in the SN and cerebellum, only two types of NBIA involve the dysfunction of a protein that participates in Fe trafficking: aceruloplasminemia (lack of Cp) and neuroferritinopathy (loss of function mutations in L-Ft) (65, 261, 290, 360). Another neurodegenerative disease associated with disturbed Fe homeostasis is Friederich ataxia, which is linked to mutations in frataxin, an Fe-chaperone involved in Fe-S cluster biogenesis (55).
Fe 的异常积累是许多神经退行性疾病的共同特征，其中包括 12 种称为脑铁积累神经退行性疾病 (NBIA) 的疾病 ( 225 )。 NBIA 最常见的临床特征是运动障碍，例如共济失调、帕金森症和肌张力障碍。尽管在 NBIA 中，Fe 通常积聚在苍白球中，在某些情况下积聚在 SN 和小脑中，但只有两种类型的 NBIA 涉及参与 Fe 运输的蛋白质功能障碍：铜蓝蛋白血症（缺乏 Cp）和神经铁蛋白病（缺乏 Cp）。 L-Ft的功能突变） （ 65、261、290、360 ）。另一种与 Fe 稳态紊乱相关的神经退行性疾病是 Friederich 共济失调，它与 frataxin 的突变有关，frataxin 是一种参与 Fe-S 簇生物发生的 Fe 伴侣 ( 55 )。

Altered Fe homeostasis and mitochondrial dysfunction are also hallmarks of PD. While no significant differences in Fe levels have been found in the blood, serum, and CSF (203), Fe is increased in the SN of PD patients (365). Neuromelanin is an Fe-rich pigment found in the dopaminergic neurons targeted in PD (A9) and it has been suggested that its presence makes this neuronal population vulnerable to oxidative damage (91). Decreased levels of serum Cp or its oxidation has also been proposed to exacerbate Fe accumulation in PD (149, 251). DMT1 is found increased in the SN of PD brains (294). Interestingly, Parkin regulates DMT1 expression levels via ubiquitination and proteasomal degradation (289).
Fe 稳态改变和线粒体功能障碍也是 PD 的标志。虽然血液、血清和脑脊液中的 Fe 水平没有发现显着差异 ( 203 )，但 PD 患者的 SN 中的 Fe 却增加了 ( 365 )。神经黑色素是在 PD (A9) 靶向的多巴胺能神经元中发现的一种富含铁的色素，有人认为它的存在使该神经元群体容易受到氧化损伤 ( 91 )。血清 Cp 水平降低或其氧化水平也被认为会加剧 PD 中 Fe 的积累 ( 149 , 251 )。发现 PD 大脑 SN 中的 DMT1 增加 ( 294 )。有趣的是，Parkin通过泛素化和蛋白酶体降解调节 DMT1 表达水平 ( 289 )。

Fe accumulation in SN might be associated with a dysfunctional delivery of Fe to mitochondria through the “kiss and run” interaction mentioned above (Fig. 4b) (69). Indeed, a Tf/TfR2-dependent mechanism for Fe transport into the mitochondria of dopaminergic neurons has been described (211), while a role for Tf and TfR2 genes in Fe accumulation and mitochondrial dysfunction in PD has been implicated as well (285).
SN 中 Fe 的积累可能与通过上述“亲吻和奔跑”相互作用向线粒体输送 Fe 的功能失调有关（图 4b ）（ 69 ）。事实上，已经描述了铁转运到多巴胺能神经元线粒体中的 Tf/TfR2 依赖性机制 ( 211 )，同时也涉及Tf和TfR2基因在 PD 铁积累和线粒体功能障碍中的作用 ( 285 )。

Mitochondrial dysfunction in PD might lead to decreased synthesis of Fe-S clusters, which in turn would activate IRP1 binding to mRNAs, resulting in augmented Fe accumulation (142). Accordingly, knockdown of mitochondrial Grx2 impairs Fe-S cluster biogenesis in dopaminergic cells, decreases the activity of Complex I and aconitase, and increases the activation of IRP1 (171). Loss of PINK1/PARK6, another autosomal early-onset PD-related gene, has also been reported to inactivate Fe-S clusters via O₂^•− formation. As such, overexpression of FtMt exerts a protective effect on mitochondrial dysfunction and oxidative stress induced by loss of PINK1 (88).
PD 中的线粒体功能障碍可能导致 Fe-S 簇合成减少，进而激活 IRP1 与 mRNA 的结合，导致 Fe 积累增加 ( 142 )。因此，线粒体 Grx2 的敲低会损害多巴胺能细胞中 Fe-S 簇的生物发生，降低复合物 I 和乌头酸酶的活性，并增加 IRP1 的激活 ( 171 )。据报道，PINK1/ PARK6 （另一种常染色体早发性PD相关基因）的缺失也会通过O ₂ ^{•− 的}形成使Fe-S簇失活。因此，FtMt 的过度表达对 PINK1 缺失引起的线粒体功能障碍和氧化应激具有保护作用 ( 88 )。

HO-1 protects against neuronal cell death induced by PD-related mitochondrial toxins and α-synuclein, suggesting a role of heme in dopaminergic cell loss. However, HO-1 becomes toxic at high levels, but this effect is counteracted by FtMt (391). Interestingly, the pathogenic PINK1 mutation G309D impairs the induction of HO-1 on oxidative stress (56).
HO-1 可防止 PD 相关线粒体毒素和 α-突触核蛋白诱导的神经元细胞死亡，表明血红素在多巴胺能细胞损失中发挥作用。然而，HO-1 在高浓度时会变得有毒，但这种效应会被 FtMt 抵消 ( 391 )。有趣的是，致病性 PINK1 突变 G309D 会损害 HO-1 对氧化应激的诱导 ( 56 )。

Fe²⁺ can interact with the negatively charged C-terminal region of α-synuclein, accelerating its amyloid aggregation (34). Conversely, α-synuclein overexpression also enables Fe accumulation (254). In PD, oxidative stress has been linked to intracellular Fe levels due to the redox activity of Fe-DA and Fe-neuromelanin complexes (400). Furthermore, overexpression of FtMt protects against neuronal cell death induced by 6-hydroxydopamine (313). Interestingly, a recent report demonstrates that depletion of Fpn has no consequence on the survival of dopaminergic neurons. In contrast, loss of TfR causes Fe deficiency and a PD-like neurodegeneration in mice (212). Previous findings have also suggested a link between Fe deficiency and predisposition to PD, while Fe overload seems to be protective (157, 190, 271). Thus, the exact role of Fe homeostasis in PD is still far from being understood.
Fe ²⁺可以与 α-突触核蛋白带负电荷的 C 末端区域相互作用，加速其淀粉样蛋白聚集 ( 34 )。相反，α-突触核蛋白过度表达也会促进 Fe 积累 ( 254 )。在 PD 中，由于 Fe-DA 和 Fe-神经黑色素复合物的氧化还原活性，氧化应激与细胞内 Fe 水平有关 ( 400 )。此外，FtMt 的过度表达可防止 6-羟基多巴胺诱导的神经元细胞死亡 ( 313 )。有趣的是，最近的一份报告表明，Fpn 的消耗对多巴胺能神经元的存活没有影响。相反，TfR 的缺失会导致小鼠缺铁和 PD 样神经变性 ( 212 )。先前的研究结果还表明，铁缺乏与帕金森病易感性之间存在联系，而铁过载似乎具有保护作用（ 157、190、271 ）。因此，Fe 稳态在 PD 中的确切作用尚不清楚。

A role of Fe in AD has also been proposed based on observations showing that Fe levels are decreased in the serum of AD patients (324), and increased in AD-related brain areas such as the hippocampus, neocortex, and basal ganglia (168). Mitochondrial dysfunction is also observed in AD, which might be related to a disruption in Fe homeostasis. Accordingly, overexpression of FtMt protects against the toxicity of Aβ (380). Fe is accumulated in amyloid plaques in the AD brain (38, 194), consistent with the observation that Aβ is able to bind Fe²⁺ in vitro (Fig. 3b) (39). Fe bound to Aβ plaques catalyzes H₂O₂ formation (321).
Fe 在 AD 中的作用也被提出，基于观察结果显示 AD 患者血清中的 Fe 水平降低 ( 324 )，而在与 AD 相关的大脑区域，如海马体、新皮质和基底神经节中 Fe 水平升高 ( 168 ) 。 AD 中也观察到线粒体功能障碍，这可能与铁稳态的破坏有关。因此，FtMt 的过度表达可以防止 Aβ 的毒性 ( 380 )。 Fe 积聚在 AD 大脑中的淀粉样斑块中 ( 38 , 194 )，这与 Aβ 能够在体外结合 Fe ²⁺的观察结果一致 (图 3b ) ( 39 )。与 Aβ 斑块结合的 Fe 催化 H ₂ O ₂的形成 ( 321 )。

A novel mechanism for controlling Fe efflux from the BBB was recently discovered, involving the soluble fragment of APP (sAPP), which interacts with Fpn, stabilizing its membrane location (219) and counteracting the effects of hepcidin (216). APP expression is also negatively regulated by IRP1 (57), and production of sAPP and Aβ is increased in AD. Thus, the proposed role of sAPP in Fe movement from BBB into the brain is consistent with the accumulation of Fe in AD brains and the observation of higher levels of Aβ plaques surrounding the brain blood vessels. Indeed, BBB damage is a common feature in AD (217).
最近发现了一种控制 Fe 从 BBB 流出的新机制，涉及 APP (sAPP) 的可溶性片段，它与 Fpn 相互作用，稳定其膜位置 ( 219 ) 并抵消铁调素 ( 216 ) 的影响。 APP 表达也受到 IRP1 ( 57 ) 的负向调节，并且 AD 中 sAPP 和 Aβ 的产生增加。因此，sAPP 在 Fe 从 BBB 移动到大脑中的作用与 AD 大脑中 Fe 的积累以及脑血管周围 Aβ 斑块水平较高的观察结果一致。事实上，BBB 损伤是 AD 的一个常见特征（ 217 ）。

Heme metabolism also contributes to AD. APP binds and inhibits HO-1 activity and this effect is enhanced by pathogenic APP mutations (334). In addition, Aβ can form a complex with heme that displays peroxidase activity (16, 112). Conversely, neuroglobin has been proposed to protect against the toxicity of 1–42 Aβ peptides (180). Overall, disruption of Fe homeostasis in AD is likely linked to oxidative stress, and it may also impair NMDAR- and Fe- dependent redox signaling mechanisms (Fig. 4d) impacting neuronal plasticity and memory.
血红素代谢也有助于 AD。 APP 结合并抑制 HO-1 活性，致病性 APP 突变会增强这种效应 ( 334 )。此外，Aβ 可以与血红素形成复合物，显示出过氧化物酶活性 ( 16 , 112 )。相反，神经球蛋白被认为可以防止 1-42 Aβ 肽的毒性 ( 180 )。总体而言，AD 中 Fe 稳态的破坏可能与氧化应激有关，并且还可能损害 NMDAR 和 Fe 依赖性氧化还原信号机制（图 4d ），从而影响神经元可塑性和记忆。

Manganese 锰

Mn is an essential metal required for the activity of a plethora of enzymes, including hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases, involved in diverse metabolic functions such as amino acid (arginase and glutamine synthetase [GS]), lipid, protein, and carbohydrate metabolism (phosphoenolpyruvate decarboxylase), as well as protein glycosylation, energy production, and redox homeostasis (SOD2). The main source for Mn intake is food, but occupational/environmental exposures also occur associated with mining, smelting, welding, alloy, battery, pesticide, and electrical industries. Ingestion and inhalation are the primary routes of Mn exposure. Importantly, inhalation can transfer Mn directly to the brain. The brain is a major target for chronic Mn intoxication (manganism) where Mn is accumulated in nonheme Fe-rich regions. Manganism is defined as a parkinsonism that results in dystonia, hypokinesia, and rigidity as a consequence of impaired neurotransmitter function (133, 352).
锰是多种酶活性所需的必需金属，包括水解酶、异构酶、连接酶、裂合酶、氧化还原酶和转移酶，参与多种代谢功能，如氨基酸（精氨酸酶和谷氨酰胺合成酶 [GS]）、脂质、蛋白质和碳水化合物代谢（磷酸烯醇丙酮酸脱羧酶），以及蛋白质糖基化、能量产生和氧化还原稳态 (SOD2)。锰摄入的主要来源是食物，但职业/环境暴露也发生在采矿、冶炼、焊接、合金、电池、农药和电气行业。摄入和吸入是锰暴露的主要途径。重要的是，吸入可以将锰直接转移到大脑。大脑是慢性锰中毒（锰中毒）的主要目标，其中锰积聚在非血红素铁丰富的区域。锰中毒被定义为一种帕金森病，由于神经递质功能受损而导致肌张力障碍、运动功能减退和强直（ 133、352 ）。

Mn can be transported via Tfr and DMT1 as it competes with Fe for its binding sites (333). Other proposed Mn transporters include the Zn carriers ZIP-8 and ZIP-14, voltage-regulated, store-operated, and ionotropic Glu receptor Ca²⁺ channels, and the Mn-citrate complex shuttle (Fig. 5a) (352). Mn is efficiently detoxified by Fpn at the plasma membrane. In addition, Mn is also detoxified by sequestration in the Golgi via the solute carrier family 30 member 10 or human Zn transporter 1 (SLC30A10/hZnT1) transporter whose mutations are directly associated with manganism (178). Alternatively, the Ca²⁺/Mn²⁺ ATPases SPCA1 and SPCA2 also detoxify Mn via the secretory pathway (356). Finally, the autosomal recessive early-onset PD-related gene ATP13A2/PARK9 mediates sequestration of Mn in lysosomes (Fig. 5b) (336). Recently, direct comparison of different detoxification proteins demonstrated that hZnT1 and SPCA1, but not ATP13A2, are involved in Mn detoxification and resistance (246).
Mn 可以通过Tfr 和 DMT1 运输，因为它与 Fe 竞争其结合位点 ( 333 )。其他提出的 Mn 转运蛋白包括 Zn 载体 ZIP-8 和 ZIP-14、电压调节、存储操作和离子型 Glu 受体 Ca ²⁺通道，以及 Mn-柠檬酸复合物穿梭（图 5a ）（ 352 ）。 Mn 在质膜上被 Fpn 有效解毒。此外，Mn 还通过溶质载体家族 30 成员 10 或人锌转运蛋白 1 ( SLC30A10 /hZnT1) 转运蛋白在高尔基体中隔离而解毒，其突变与锰中毒直接相关 ( 178 )。或者，Ca ²⁺ /Mn ²⁺ ATP酶SPCA1和SPCA2也通过分泌途径使Mn解毒( 356 )。最后，常染色体隐性早发性 PD 相关基因 ATP13A2/ PARK9介导溶酶体中 Mn 的隔离（图 5b ）（ 336 ）。最近，不同解毒蛋白的直接比较表明，hZnT1 和 SPCA1，而不是 ATP13A2，参与锰解毒和抵抗 ( 246 )。

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FIG. 5. 如图。 5.

Mn homeostasis and toxicity. (a) Mn can be transported via Tf and DMT1, as well as via Zn carriers, ion channels, and shuttle systems. (b) Mn is detoxified by Fpn at the plasma membrane or by its sequestration in the secretory system via the ZnT1 transporter or the Ca²⁺/Mn²⁺ ATPases SPCA1/2. ATP13A2 mediates sequestration of Mn in lysosomes. (c) Mn enhances ROS generation via Mn-catalyzed autoxidation of DA that involves the redox cycling of Mn²⁺ and Mn³⁺ and the generation of ROS and DA-o-quinones. (d) Mn accumulates in the mitochondria via the MCU and increases the accumulation of labile Fe. (e) Mn-induced neurotoxicity is linked to oxidative damage and mitochondrial dysfunction, and also to a decrease in Glu uptake by the EAATs in astrocytes (arrow in EAAT indicates reversal of Glu transport) leading to excitotoxicity in neurons, as well as the induction of inflammation. DA, dopamine; EAAT, excitatory amino acid transporter; Glu, glutamate; MCU, mitochondrial Ca²⁺ uniporter; Mn, manganese; ROS, reactive oxygen species; SPCA, secretory pathway Ca²⁺ ATPase; ZnT, zinc transporter.
锰稳态和毒性。 (a) Mn 可以通过Tf 和 DMT1 以及 Zn 载体、离子通道和穿梭系统运输。 (b) Mn 在质膜上被 Fpn 解毒，或通过ZnT1 转运蛋白或 Ca ²⁺ /Mn ²⁺ ATP 酶 SPCA1/2 在分泌系统中隔离。 ATP13A2 介导溶酶体中 Mn 的螯合。 (c) Mn通过Mn 催化的 DA 自氧化作用增强 ROS 的生成，其中涉及 Mn ²⁺和 Mn ³⁺的氧化还原循环以及 ROS 和 DA-o-醌的生成。 (d) Mn通过MCU 在线粒体中积累，并增加不稳定 Fe 的积累。 (e)锰诱导的神经毒性与氧化损伤和线粒体功能障碍有关，也与星形胶质细胞中 EAAT 摄取 Glu 的减少有关（EAAT 中的箭头表示 Glu 转运的逆转），导致神经元的兴奋性毒性，以及诱导炎症。 DA，多巴胺； EAAT，兴奋性氨基酸转运蛋白；谷氨酸、谷氨酸； MCU，线粒体 Ca ²⁺单向转运蛋白；锰，锰； ROS，活性氧； SPCA，分泌途径Ca ²⁺ ATP酶； ZnT，锌转运蛋白。

Mn²⁺ is the predominant species found in cells that can be oxidized to the more reactive and toxic species Mn³⁺. Neither Mn²⁺ nor Mn³⁺ can generate free radicals via Fenton-type reactions. However, it has been proposed that Mn enhances ROS generation via the Mn-catalyzed autoxidation of DA that involves the redox cycling of Mn²⁺ and Mn³⁺ and the generation of ROS and DA-o-quinone (Fig. 5c) (79, 89). Mn accumulates in the mitochondria via the mitochondrial Ca²⁺ uniporter (MCU) (111) and increases the accumulation of labile Fe. Both mitochondrial dysfunction and Fe lead to ROS formation and oxidative damage (Fig. 5d) (54, 206). Mn specifically generates H₂O₂ but not O₂^•− in the mitochondria via complex II (92, 186, 322). Furthermore, Mn impairs oxidative phosphorylation and ATP production.
Mn ²⁺是细胞中发现的主要物质，可以被氧化成更具活性和毒性的物质Mn ³⁺ 。 Mn ²⁺和Mn ³⁺都不能通过芬顿反应产生自由基。然而，有人提出，Mn通过Mn 催化的 DA 自氧化来增强 ROS 的生成，其中涉及 Mn ²⁺和 Mn ³⁺的氧化还原循环以及 ROS 和 DA-o-醌的生成（图 5c ）（ 79 ， 89 ）。 Mn通过线粒体 Ca ²⁺单向转运蛋白 (MCU) ( 111 ) 在线粒体中积累，并增加不稳定 Fe 的积累。线粒体功能障碍和 Fe 都会导致 ROS 形成和氧化损伤（图 5d ）（ 54 , 206 ）。 Mn通过复合物 II 在线粒体中专门生成 H ₂ O ₂但不生成 O ₂ ^•− ( 92 , 186 , 322 )。此外，Mn 还会损害氧化磷酸化和 ATP 的产生。

Astrocytes seem to have a high capacity to accumulate Mn (14). Mn-induced neurotoxicity has been linked to a decrease in Glu uptake by astrocytes (156) leading to excitotoxicity in neurons, as well as the induction of inflammation and increased activity of NOS (Fig. 5e) (95, 185).
星形胶质细胞似乎具有很高的积累 Mn 的能力 ( 14 )。 Mn 诱导的神经毒性与星形胶质细胞 ( 156 ) 摄取 Glu 的减少有关，导致神经元兴奋性毒性，以及诱导炎症和 NOS 活性增加 (图 5e ) ( 95 , 185 )。

Arsenic 砷

As is naturally present in air, water, and soil and is the 20th most abundant element in the earth's crust and 12th in the human body. This metal is named inorganic As (iAs) when found combined with other elements such as O₂, chlorine (Cl), and sulfur (S). Combined with carbon (C) and hydrogen (H) is referred to as organic As. In the environment and within the human body, iAs predominantly exists in two oxidation states: arsenite +3 (or AsIII, found as arsenic trioxide [As₂O₃], sodium arsenite [NaAsO₂], and arsenic trichloride [AsCl₃]), and arsenate +5 (or AsV found as arsenic pentoxide [As₂O₅], arsenic acid [H₃AsO₄], and arsenates [PbHAsO₄, Ca₃(AsO₄)₂]).
天然存在于空气、水和土壤中，是地壳中第 20 位最丰富的元素，在人体中排名第 12 位。当这种金属与 O ₂ 、氯 (Cl) 和硫 (S) 等其他元素结合时，被称为无机砷 (iAs)。与碳（C）和氢（H）结合称为有机As。在环境和人体内，iAs 主要以两种氧化态存在：亚砷酸盐 +3（或 AsIII，以三氧化二砷 [As ₂ O ₃ ]、亚砷酸钠 [NaAsO ₂ ] 和三氯化砷 [AsCl ₃ ] 形式存在） ，和砷酸盐+5（或以五氧化二砷[As ₂ O ₅ ]、砷酸[H ₃ AsO ₄ ]和砷酸盐[PbHAsO ₄ , Ca ₃ (AsO ₄ ) ₂ ]形式发现的AsV）。

iAs has been widely used as a therapeutic agent to treat leukemia. Currently, iAs compounds are predominantly used in pesticides, herbicides, cotton desiccants, wood preservatives, alloys for batteries, and in semiconductors and light-emitting diodes. Millions of individuals are currently exposed to iAs across the world due to natural groundwater contamination. Fish and crustaceans contain very high levels of organic arsenobetaine but no toxicity has been reported in vivo (382). The concentration of iAs in natural surface and groundwater is generally about 1 parts per billion (ppb) of water but it may exceed 1000 ppb in contaminated areas or where iAs soil levels are high (118, 200) (Supplementary Table S1).
iAs已被广泛用作治疗白血病的治疗剂。目前，砷化合物主要用于杀虫剂、除草剂、棉花干燥剂、木材防腐剂、电池合金、半导体和发光二极管。由于天然地下水污染，目前全世界有数百万人暴露于无机砷。鱼类和甲壳类动物含有非常高水平的有机砷甜菜碱，但尚未有体内毒性的报道（ 382 ）。天然地表和地下水中 iAs 的浓度通常约为十亿分之 1 (ppb)，但在受污染地区或 iAs 土壤含量较高的地方可能会超过 1000 ppb ( 118 , 200 )（补充表 S1 ）。

While As is considered a carcinogen, in the brain, acute exposure to iAs can induce encephalopathy, with symptoms such as confusion, hallucinations, reduced memory, and emotional lability (exaggerated changes in mood or affect). Long-term exposure to lower levels of iAs can lead to the development of peripheral neuropathies.
虽然砷被认为是一种致癌物质，但在大脑中，急性接触砷会诱发脑病，症状包括精神错乱、幻觉、记忆力下降和情绪不稳定（情绪或情感的过度变化）。长期接触较低水平的 iAs 可导致周围神经病变的发生。

There are reports of neurobehavioral alterations (cognitive function, verbal abilities, long-term memory, and motor skills) in children exposed to As concentrations ranging from 5 to 50 ppb in water, in Bangladesh (260), Mexico (45, 287), and in the United States (370). Although scientific understanding of the developmental neurotoxicity of As is still evolving, epidemiological and toxicological studies clearly show that As is a developmental neurotoxicant that affects intellectual function. Moreover, exposures even below current safety guidelines are associated with decrements in full-scale intelligence quotient (IQ) and memory (90, 347). Evidence in experimental models, including mice, rats, Caenorhabditis elegans (worm), and Danio rerio (zebrafish), has replicated many of the observations in humans supporting the notion that As can lead to cognitive, locomotor, and neurological impairment (85). Gestational exposure to NaAsO₂ leads to a significant iAs accumulation in the mice offspring's brain (280). As neurotoxicity has been linked to changes in neurotransmitter metabolism and synaptic transmission (85, 276, 280). However, the mechanisms involved remain unclear.
有报告称，孟加拉国 ( 260 )、墨西哥 ( 45 , 287 )、墨西哥 (45, 287) 的儿童接触砷浓度为 5 至 50 ppb 的水中，神经行为发生改变（认知功能、语言能力、长期记忆和运动技能）。以及美国（ 370 ）。尽管对砷的发育神经毒性的科学认识仍在不断发展，但流行病学和毒理学研究清楚地表明砷是一种影响智力功能的发育神经毒物。此外，即使低于当前安全准则的暴露也会导致全面智商 (IQ) 和记忆力的下降 ( 90 , 347 )。实验模型（包括小鼠、大鼠、秀丽隐杆线虫（线虫）和斑马鱼）中的证据已经在人体中复制了许多观察结果，支持砷可导致认知、运动和神经功能障碍的观点（ 85 ）。妊娠期接触NaAsO ₂会导致小鼠后代大脑中iAs 大量积累( 280 )。因为神经毒性与神经递质代谢和突触传递的变化有关（85、276、280 ） 。然而，所涉及的机制仍不清楚。

In the environment, oxygenated water contains iAsV species, while in reducing environments iAsIII species are prevalent. iAsV enters cells through phosphate transporters to be subsequently reduced to iAsIII, while iAsIII is transported via aqua(glycerol)porins (AQP), organic anion transporters, and glucose transporters (GLUT) (Fig. 7a) (44, 187, 348). Once in the cytoplasm, iAsIII is methylated by different mechanisms. Oxidative methylation (Fig. 7b) is mediated by arsenite methyltransferase (AS3MT) that uses S-adenosylmethionine (AdoMet) as a cosubstrate. AS3MT methylates iAsIII to monomethylarsonic acid or arsonate (MMAV) that is reduced to monomethylarsonous acid (MMAIII) before being methylated again to dimethylarsinic acid (DMAV) by AS3MT (353, 372). Finally, DMAV is reduced generating dimethylarsinous acid (DMAIII). The reduction of pentavalent arsenicals (iAsV, MMAV, and DMAV) in this pathway is now well recognized to be mediated by the Trx/TR system, but GSH seems to increase the methylation rates by an unknown mechanism (76).
在环境中，含氧水含有 iAsV 物种，而在还原环境中 iAsIII 物种普遍存在。 iAsV 通过磷酸盐转运蛋白进入细胞，随后被还原为 iAsIII，而 iAsIII通过水（甘油）孔蛋白（AQP）、有机阴离子转运蛋白和葡萄糖转运蛋白（ GLUT ）转运（图 7a ）（ 44,187,348 ）。一旦进入细胞质，iAsIII 就会通过不同的机制被甲基化。氧化甲基化（图7b ）由亚砷酸甲基转移酶（AS3MT）介导，该酶使用S-腺苷甲硫氨酸（AdoMet）作为共底物。 AS3MT将iAsIII甲基化为一甲基胂酸或胂酸盐(MMAV)，其在被AS3MT再次甲基化为二甲基胂酸(DMAV)之前还原为一甲基胂酸( MMAIII )( 353、372 )。最后，DMAV 被还原生成二甲基胂酸 (DMAIII)。现在人们普遍认为该途径中五价砷（iAsV、MMAV 和 DMAV）的减少是由 Trx/TR 系统介导的，但 GSH 似乎通过未知机制增加甲基化率 ( 76 )。

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FIG. 7. 如图。 7.

iAs biochemistry, redox signaling, and neurotoxicity. (a) iAsIII species prevail in anaerobic conditions, while aerobic environments contain iAsV species. iAsIII enters cells through AQP9/7 and GLUT, while iAsV uses phosphate transporters. Three mechanisms for iAs methylation have been proposed. (b) Oxidative methylation refers to the progressive reduction of AsV to AsIII metabolites by the Trx/TR system and the oxidative methylation of AsIII species by AS3MT. (c) The GSH conjugation pathway is based on the formation of As(GS)₃ by the GSTs. As(GS)₃ is subsequently methylated by AS3MT. (d) The protein thiol conjugation mechanism suggests that instead of As(GS)₃, iAsIII binds to protein thiols and is methylated while still being conjugated to proteins. iAsIII methylation is likely to alter epigenetic signatures by alterations in AdoMet, a cosubstrate required for methyl group transfer. (e) Methylated metabolites and GSH/protein complexes are highly reactive and are exported through the MRPs. (f) DMA(GS) can form DMAH and react with O₂ to form DMAH^• and DMAOO^•. DMAIII also reacts with O₂ and forms DMAOOH. These reactive species lead to lipid peroxidation and protein carbonylation (oxidative stress). (g) AsV can replace phosphate in several metabolic pathways (arsenylation) where the end product is reduced AsIII. AsV binds ADP via ATP-synthase and uncouples oxidative phosphorylation and ATP formation in the mitochondria. AsV is also arsenylated by GAPDH to generate iAsV-3-P-glycerate from G3P. (h) iAsIII toxicity generates ER stress likely via thiol depletion and alterations in redox balance. (i) AsIII binds to thiol containing molecules (coenzyme A, DLA, GSH) and protein thiols inactivating enzymatic function. (j) iAsIII toxicity is counteracted by Nrf2 and MTF1 that mediate the transcriptional-dependent induction of antioxidant systems and MTs. AdoMet, S-adenosylmethionine; AQP, aqua(glycerol)porin; AS3MT, arsenite methyltransferase; AsIII, arsenite +3; As(SG)₃, arsenic triglutathione; AsV, arsenate +5; DLA, dihydrolipoamide; DMA(GS), dimethylarsinic GSH; DMAH, dimethylarsine; DMAH^•, DMAH radical; DMAIII, dimethylarsinous acid; DMAOO^•, dimethylarsine peroxyl radical; DMAOOH, dimethylated arsenic peroxide; G3P, glyceraldehyde 3-phosphate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GLUT, glucose transporters; GSTs, glutathione-S transferases; iAs, inorganic As; iAsV-3-P-glycerate, 1-arsenato-3-phospho-D-glycerate; MRPs, multidrug resistance proteins; MTF1, metal-responsive transcription factor-1; Nrf2, nuclear factor erythroid-2-related factor 2; O₂, molecular oxygen; Trx, thioredoxin.
iAs 生物化学、氧化还原信号传导和神经毒性。 (a) iAsIII 物种在厌氧条件下占主导地位，而有氧环境中则含有 iAsV 物种。 iAsIII 通过 AQP9/7 和 GLUT 进入细胞，而 iAsV 使用磷酸盐转运蛋白。已经提出了 iAs 甲基化的三种机制。 (b)氧化甲基化是指 Trx/TR 系统将 AsV 逐步还原为 AsIII 代谢物，以及 AS3MT 将 AsIII 物质氧化甲基化。 (c) GSH 缀合途径基于 GST 形成 As(GS) ₃ 。 As(GS) ₃随后被 AS3MT 甲基化。 (d)蛋白质硫醇缀合机制表明，iAsIII 不是 As(GS) ₃ ，而是与蛋白质硫醇结合，并在仍与蛋白质缀合的同时被甲基化。 iAsIII 甲基化可能通过改变 AdoMet（甲基转移所需的共底物）来改变表观遗传特征。 (e)甲基化代谢物和 GSH/蛋白质复合物具有高反应性，并通过 MRP 输出。 (f) DMA(GS)可以形成DMAH并与O ₂反应形成DMAH ^·和DMAOO ^· 。 DMAIII 还与O ₂反应并形成DMAOOH。这些活性物质导致脂质过氧化和蛋白质羰基化（氧化应激）。 (g) AsV 可以在多种代谢途径（砷化）中取代磷酸盐，最终产物被还原为 AsIII。 AsV通过ATP 合酶结合 ADP，并解开线粒体中的氧化磷酸化和 ATP 形成。 AsV 还被 GAPDH 砷化，从 G3P 生成 iAsV-3-P-甘油酸。 (h) iAsIII 毒性可能通过硫醇消耗和氧化还原平衡的改变产生内质网应激。 (i) AsIII 与含有硫醇的分子（辅酶 A、DLA、GSH）和蛋白质硫醇结合，使酶功能失活。 (j) iAsIII 毒性被 Nrf2 和 MTF1 抵消，Nrf2 和 MTF1 介导抗氧化系统和 MT 的转录依赖性诱导。 AdoMet，S-腺苷甲硫氨酸； AQP，水（甘油）孔蛋白； AS3MT，亚砷酸甲基转移酶； AsIII，亚砷酸盐+3； As(SG) ₃ ，三谷胱甘肽砷； AsV，砷酸盐+5； DLA，二氢硫辛酰胺； DMA(GS)，二甲基砷GSH； DMAH，二甲基胂； DMAH ^• ，DMAH 基团； DMAIII，二甲基胂酸； DMAOO ^• ，二甲基胂过氧自由基； DMAOOH，二甲基过氧化砷； G3P，3-磷酸甘油醛； GAPDH，3-磷酸甘油醛脱氢酶；过剩，葡萄糖转运蛋白； GST、谷胱甘肽-S 转移酶； iAs，无机砷； iAsV-3-P-甘油酸酯、1-砷酸-3-磷酸-D-甘油酸酯； MRP，多药耐药蛋白； MTF1，金属响应转录因子-1； Nrf2，核因子红细胞2相关因子2； O ₂ ，​​分子氧； Trx，硫氧还蛋白。

Developmental exposure to As alters the methylation patterns of genes involved in neuroplasticity likely due to changes in AdoMet, but its long-term implications are unclear (207). Recent in vivo studies demonstrated that the alterations in synaptic plasticity (LTP), memory, and learning induced by gestational exposure to iAs were associated with an increase in extracellular Glu levels and downregulation of AMPAR subunits (244).
发育过程中接触 As 会改变神经可塑性相关基因的甲基化模式，这可能是由于 AdoMet 的变化所致，但其长期影响尚不清楚 ( 207 )。最近的体内研究表明，妊娠期暴露于 iAs 引起的突触可塑性 (LTP)、记忆和学习的改变与细胞外 Glu 水平的增加和 AMPAR 亚基的下调相关 ( 244 )。

As methylation via the GSH conjugation mechanism is based on the formation of GSH complexes with iAsIII resulting in arsenic triglutathione [As(SG)₃] (Fig. 7c). Conjugation of iAsIII with GSH has been proposed to occur nonenzymatically, but enzymatically as well by the activity of glutathione-S transferases (GST isoforms GSTO1, GSTM1, or GSTP1) (173, 372). As(GS)₃ is subsequently methylated by AS3MT to form monomethylarsinic diglutathione [MMA(GS)₂] and then again to generate dimethylarsinic GSH [DMA(GS)]. At low GSH levels, As(GS) conjugates are hydrolyzed and then oxidized to generate MMAV and DMAV (372). A third mechanism for iAsIII methylation has been recently proposed, where instead of As(GS) conjugate formation, iAsIII binds to protein-Cys (thiol) and is methylated while still being conjugated to proteins (Fig. 7d). This hypothesis is supported by the preferential binding of iAsIII to protein-Cys when compared to GSH (284).
因为通过GSH缀合机制的甲基化是基于GSH与iAsIII复合物的形成，从而产生砷三谷胱甘肽[As(SG) ₃ ]（图7c ）。 iAsIII 与 GSH 的结合被认为是非酶促地发生，但也通过谷胱甘肽-S 转移酶（GST 同工型 GSTO1、GSTM1 或 GS​​TP1）的活性以酶促方式发生（ 173 , 372 ）。 As(GS) ₃随后被 AS3MT 甲基化，形成单甲基胂二谷胱甘肽 [MMA(GS) ₂ ]，然后再次生成二甲基胂 GSH [DMA(GS)]。在低 GSH 水平下，As(GS) 缀合物被水解，然后被氧化生成 MMAV 和 DMAV ( 372 )。最近提出了 iAsIII 甲基化的第三种机制，其中 iAsIII 与蛋白质-Cys（硫醇）结合，而不是形成 As（GS）缀合物，并在仍与蛋白质缀合的同时被甲基化（图 7d ）。与 GSH 相比，iAsIII 优先与蛋白质-Cys 结合支持了这一假设 ( 284 )。

Methylated (and maybe unmethylated) As metabolites are exported through the multidrug resistance proteins (MRP1, MRP2, or MRP4) (173, 317, 388) (Fig. 7e). AS3MT is ubiquitously expressed in all brain regions, and animal studies have shown that the iAs that crosses the BBB is methylated and accumulated across the brain, with the highest accumulation observed in the pituitary gland (297). Interestingly, knockout mouse for P-glycoprotein accumulates more As in the brain (183). Endothelial cells and astrocytes feet surrounding capillaries are the first barrier of detoxification of xenobiotics entering from the circulation. We (unpublished data) and others have observed that the resistance of astrocytes to iAsIII is mediated by MRPs (332).
甲基化（并且可能未甲基化）作为代谢物通过多药耐药蛋白（MRP1、 MRP2或MRP4 ）输出（ 173、317、388 ）（图7e ）。 AS3MT 在所有大脑区域中普遍表达，动物研究表明，穿过 BBB 的 iAs 被甲基化并在整个大脑中积累，其中在垂体中观察到积累量最高 ( 297 )。有趣的是，P-糖蛋白基因敲除小鼠的大脑中积累了更多的 As ( 183 )。毛细血管周围的内皮细胞和星形胶质细胞是从循环中进入的外源物质解毒的第一道屏障。我们（未发表的数据）和其他人观察到星形胶质细胞对 iAsIII 的抵抗是由 MRP 介导的（ 332 ）。

iAs generates ROS and dimethylarsenic or peroxyl radicals that in turn lead to lipid peroxidation and the accumulation of oxidized by-products (MDA and HNE) (Fig. 7f). Importantly, MMAIII and DMAIII are proposed to be more potent toxicants than iAsIII due to their increased ability to generate radicals (392). Oxidative stress has been reported in brain regions of different animal models and in neurons and glial cell cultures exposed to As compounds (48, 107, 108, 243, 394).
iAs 产生 ROS 和二甲基砷或过氧自由基，进而导致脂质过氧化和氧化副产物（MDA 和 HNE）的积累（图 7f ）。重要的是，MMAIII 和 DMAIII 被认为是比 iAsIII 更有效的毒物，因为它们产生自由基的能力更强 ( 392 )。据报道，不同动物模型的大脑区域以及暴露于砷化合物的神经元和神经胶质细胞培养物中存在氧化应激( 48,107,108,243,394 )。

Mitochondria have been proposed to be a primary source for ROS formation by iAs (Fig. 7g) (97, 150). Chronic iAs exposure generates mitochondrial oxidative stress in the rat brain by impairment of mitochondrial complexes I, II, and IV activities followed by increased ROS generation, lipid peroxidation, and protein carbonylation (275). Mitochondrial pyruvate dehydrogenase is also directly inhibited by iAs (136). In addition, iAs reduces the levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), downstream targets Nrf1 and Nrf2, and the mitochondrial transcription factor A (TFAM) decreasing mitochondrial biogenesis (274). ER stress has also been shown to contribute to iAs toxicity, but the mechanisms involved remain unclear (Fig. 7h) (182).
线粒体被认为是 iAs 形成 ROS 的主要来源（图 7g ）（ 97 , 150 ）。慢性 iAs 暴露会损害线粒体复合物 I、II 和 IV 活性，进而导致 ROS 生成、脂质过氧化和蛋白质羰基化增加，从而在大鼠大脑中产生线粒体氧化应激 ( 275 )。线粒体丙酮酸脱氢酶也直接被 iAs 抑制 ( 136 )。此外，iAs 降低过氧化物酶体增殖物激活受体 γ 共激活剂 1-α (PGC-1α)、下游靶标 Nrf1 和 Nrf2 以及线粒体转录因子 A (TFAM) 的水平，从而减少线粒体生物发生 ( 274 )。 ER 应激也已被证明会导致 iAs 毒性，但所涉及的机制仍不清楚（图 7h ）（ 182 ）。

iAs toxicity has also been attributed to the ability of AsV to replace phosphate in several metabolic pathways (arsenylation) where the end product is the reduction of AsV to AsIII, because the arsenylated by-product is more readily reduced than AsV itself (Fig. 7g). AsV uncouples oxidative phosphorylation and ATP formation in the mitochondria by binding to ADP via ATP synthase. Replacement of phosphate in glycolysis also impairs carbon flux and ATP production. Reaction of AsV with glucose generates glucose 6-arsenate, an analog of glucose 6-phosphate that is suggested to act as an inhibitor of hexokinase. AsV is also arsenylated by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to produce the unstable product 1-arsenato-3-phospho-D-glycerate (iAsV-3-P-glycerate). Purine nucleoside phosphorylase, glycogen phosphorylase, and mitochondrial ornithine carbamoyl transferase (OCT) have also been shown to arsenylate AsV (139, 245, 340). Thus, energy failure, alterations in central carbon metabolism, and mitochondrial dysfunction are consequences of AsV toxicity.
iAs 毒性也归因于 AsV 在几种代谢途径（砷化）中取代磷酸盐的能力，其中最终产物是将 AsV 还原为 AsIII，因为砷化副产物比 AsV 本身更容易被还原（图 7g） ）。 AsV通过ATP 合酶与 ADP 结合，解开线粒体中的氧化磷酸化和 ATP 形成。糖酵解中磷酸盐的替代也会损害碳通量和 ATP 产生。 AsV 与葡萄糖反应生成葡萄糖 6-砷酸盐，这是葡萄糖 6-磷酸盐的类似物，被认为可作为己糖激酶的抑制剂。 AsV 还可被甘油醛 3-磷酸脱氢酶 (GAPDH) 砷化，产生不稳定的产物 1-砷酸-3-磷酸-D-甘油酸酯 (iAsV-3-P-甘油酸酯)。嘌呤核苷磷酸化酶、糖原磷酸化酶和线粒体鸟氨酸氨基甲酰基转移酶 (OCT) 也已被证明可使 AsV 砷化 ( 139 , 245 , 340 )。因此，能量衰竭、中心碳代谢的改变和线粒体功能障碍是 AsV 毒性的后果。

AsIII binds to thiol containing molecules (coenzyme A, GSH, and dihydrolipoamide also known as dihydrolipoic acid [DLA]) and protein-Cys thiols inactivating enzyme function (Fig. 7d, i). AsIII has higher affinity for dithiols than monothiols as demonstrated by the transfer of AsIII from the GSH-adduct to 2,3-dimercaptosuccinic acid (DMSA) a sulfhydryl-containing metal chelator used to treat heavy metal toxicity. In addition, AsIII conjugated with GSH has the ability to bind protein thiols, which highlights the importance of detoxification of GSH-As adducts from the cell (230). Dithiol molecules such as the cofactor DLA and dithiol oxidoreductases Trxs, Trx reductase (TrxR), Prxs (except for monothiol Prx6), Grx, and GR, as well as proteins with adjacent Cys (MT), have been reported to avidly bind AsIII (Fig. 7d, j) (50, 279, 311, 393, 397). In addition to binding AsIII, Zn finger domains have been shown to be oxidized upon As binding (396). Binding of AsIII to DLA (Fig. 7i) is expected to interfere with the TCA cycle and energy production as DLA is a cofactor for the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes that catalyze the synthesis of acetyl-CoA and succinyl-CoA, respectively. DLA reverses protein oxidation and loss of protein-SHs in the brains of rats exposed to high levels of iAs (82, 296, 315, 316).
AsIII与含有硫醇的分子（辅酶A、GSH和二氢硫辛酰胺，也称为二氢硫辛酸[DLA]）和蛋白质-Cys硫醇结合，使酶功能失活（图7d，i ）。 AsIII 对二硫醇的亲和力比单硫醇更高，这一点通过 AsIII 从 GSH 加合物转移到 2,3-二巯基丁二酸 (DMSA)（一种用于治疗重金属毒性的含巯基金属螯合剂）来证明。此外，与 GSH 缀合的 AsIII 具有结合蛋白质硫醇的能力，这凸显了细胞中 GSH-As 加合物解毒的重要性 ( 230 )。据报道，二硫醇分子，例如辅因子 DLA 和二硫醇氧化还原酶 Trxs、Trx 还原酶 (TrxR)、Prxs（单硫醇 Prx6 除外）、Grx 和 GR，以及具有相邻 Cys (MT) 的蛋白质，可强烈结合 AsIII。图7d、j ) ( 50、279、311、393、397 ) 。除了结合 AsIII 之外，Zn 指结构域已被证明在 As 结合时被氧化 ( 396 )。 AsIII与DLA的结合（图7i ）预计会干扰TCA循环和能量产生，因为DLA是丙酮酸脱氢酶和2-酮戊二酸脱氢酶复合物的辅助因子，分别催化乙酰辅酶A和琥珀酰辅酶A的合成。 DLA 可逆转暴露于高水平 iAs 的大鼠大脑中的蛋白质氧化和蛋白质-SH 的损失 ( 82 , 296 , 315 , 316 )。

As activates the cystine/Glu exchanger system (xCT) in microglia to increase extracellular Glu levels (320), while in astrocytes it decreases the expression levels and activity of GS and Glu transporters (GLAST/excitatory amino acid transporter (EAAT) 1 and GLT-1/EAAT2) (48, 395). Importantly, these effects were linked to an increase in GSH levels and Nrf2 activity (Fig. 7i), but not oxidative stress (48). Accordingly, activation of Nrf2 by As seems to involve a noncanonical pathway where inhibition of autophagy leads to the accumulation of the ubiquitin-binding protein/adaptor p62 that sequesters Keap1 (169). iAsIII toxicity is also counteracted by the transcriptional regulation of MT via MTF1 (Fig. 7j) (129).
As 激活小胶质细胞中的胱氨酸/Glu 交换系统 (xCT)，以增加细胞外 Glu 水平 ( 320 )，而在星形胶质细胞中，它会降低 GS 和 Glu 转运蛋白（GLAST/兴奋性氨基酸转运蛋白 (EAAT) 1 和 GLT）的表达水平和活性-1/EAAT2) ( 48 , 395 )。重要的是，这些效应与 GSH 水平和 Nrf2 活性的增加有关（图 7i ），但与氧化应激无关（ 48 ）。因此，As 激活 Nrf2 似乎涉及一种非经典途径，其中自噬的抑制导致泛素结合蛋白/接头 p62 的积累，从而隔离 Keap1 ( 169 )。 iAsIII 毒性还可以通过MTF1 的 MT 转录调节来抵消（图 7j ）（ 129 ）。

Lead 带领

Inorganic Pb remains one of the most studied toxic elements due to several reasons. To begin with, human contact with Pb started very early in human civilization, and its toxic effects were also known since then. However, more importantly, Pb is neurotoxic leading to lower IQ even at lower doses than those recommended by the World Health Organization (10 ppb in drinking water). Human exposure to Pb not only occurs occupationally but also environmentally. The presence of Pb in the environment has multiple sources such as gasoline, industrial processes, paint, water pipes, and solder in canned food. It is present in air, household dust, soil, water, and food (Supplementary Table S1). Environmental Pb levels have fortunately decreased, especially in those countries where the Pb addition to gasoline and paints was banned. This prohibition was enforced after several studies associated the presence of high blood levels of Pb with impaired or diminished cognitive functions. Epidemiological studies have clearly shown that exposure to Pb in early stages of development is associated with significant deficits in neurobehavioral performance, including lower IQ, attention deficits, and aggressiveness later in life. Despite all the enforced restrictions, Pb contamination is still a major public health concern. For example, in November 2000 in Washington DC, there was a “lead drinking water crisis” triggered by a change in the disinfectant used to clean the water, this contamination affected hundreds of kids for 3 years. The health consequences of the recent crisis of Pb-contaminated water in Flint Michigan (United States, 2015) are still to be revealed in the future (68, 84, 124, 298).
由于多种原因，无机铅仍然是研究最多的有毒元素之一。首先，人类在人类文明的早期就开始接触铅，并且从那时起人们就知道了铅的毒性作用。然而，更重要的是，铅具有神经毒性，即使在低于世界卫生组织建议的剂量（饮用水中 10 ppb）的情况下也会导致智商降低。人类接触铅不仅发生在职业中，而且发生在环境中。环境中存在的铅有多种来源，例如汽油、工业过程、油漆、水管和罐头食品中的焊料。它存在于空气、家庭灰尘、土壤、水和食物中（补充表S1 ）。幸运的是，环境中的铅含量已经下降，特别是在那些禁止在汽油和油漆中添加铅的国家。这项禁令是在多项研究将血液中高浓度铅与认知功能受损或减弱联系起来之后实施的。流行病学研究清楚地表明，在发育早期接触铅与神经行为表现的显着缺陷相关，包括智商较低、注意力缺陷和晚年的攻击性。尽管有所有强制限制，铅污染仍然是一个主要的公共卫生问题。例如，2000年11月，华盛顿特区因清洁水所用消毒剂的变化引发了一场“铅饮用水危机”，这种污染影响了数百名儿童长达3年。最近密歇根州弗林特铅污染水危机（美国，2015）的健康后果仍有待未来揭晓（ 68,84,124,298 ）。

Inhalation and ingestion of Pb and Pb-containing particles or products are the main routes of Pb entry into the body. Young children are especially vulnerable because they show higher gastrointestinal absorption than adults. Inhaled Pb particles are quickly absorbed in alveoli and distributed to other organs through the circulation. Thus, blood lead levels (BLL) are reliable biomarkers of exposure and risk. However, BLL do not reflect the total Pb body burden because Pb is absorbed in bones where it can be stored for several years (68). Currently, the acceptable BLL for children is lower than 10 μg/dl (0.48 μM) (49, 376), but due to the devastating effects that might occur later in life, there is a consensus to recommend efficient surveillance methods for children protection to reduce BLL to the lowest possible level (141). Pb binds with high affinity to erythrocytes' δ-aminolevulinic acid dehydratase (ALAD) that catalyzes the second step in the porphyrin and heme biosynthetic pathway, causing the accumulation of aminolevulinic acid (ALA) in both plasma and urine, which is used as a biomarker of exposure (248).
吸入和摄入铅及含铅颗粒或制品是铅进入体内的主要途径。幼儿尤其容易受到伤害，因为他们的胃肠吸收能力比成人更高。吸入的铅颗粒很快被肺泡吸收，并通过循环分布到其他器官。因此，血铅水平（BLL）是暴露和风险的可靠生物标志物。然而，BLL 并不反映体内铅的总负荷，因为铅被吸收在骨骼中，可以储存数年 ( 68 )。目前，儿童可接受的 BLL 低于 10 μg/dl (0.48 μM ) ( 49 , 376 )，但由于可能在以后的生活中发生破坏性影响，人们一致认为推荐有效的监测方法来保护儿童将BLL降低到尽可能低的水平（ 141 ）。 Pb 以高亲和力与红细胞的 δ-氨基乙酰丙酸脱水酶 (ALAD) 结合，催化卟啉和血红素生物合成途径的第二步，导致血浆和尿液中氨基乙酰丙酸 (ALA) 的积累，可用作生物标志物曝光量（ 248 ）。

Pb can cross the BBB and cell membrane because of its ability to mimic Ca²⁺ and Fe²⁺ ions (Fig. 8a) (205, 298). In children, due to a more permeable BBB and a lower bone storage capacity for Pb, the amount of Pb passing into the nervous system is higher than in adults. The highest accumulation of Pb has been reported in the hippocampus, amygdala (116), and choroids plexus (201). The PNS may accumulate considerably more Pb than the CNS. Animals chronically exposed to Pb had impaired dendritic spines and synapse formation (306). Developmental exposure to Pb impacts the prefrontal cerebral cortex, hippocampus, and cerebellum regions, which can lead to neurological disorders, mental retardation, behavioral problems, and nerve damage (242). Early life exposure to Pb has also been linked to neurodegenerative diseases such as AD and PD (62, 209).
Pb 可以穿过 BBB 和细胞膜，因为它能够模拟 Ca ²⁺和 Fe ²⁺离子（图 8a ）（ 205 , 298 ）。在儿童中，由于血脑屏障的渗透性更强，骨铅储存能力较低，因此进入神经系统的铅量比成人更高。据报道，海马体、杏仁核 ( 116 ) 和脉络丛 ( 201 ) 中铅的积累量最高。 PNS 可能比 CNS 积累更多的 Pb。长期接触铅的动物树突棘和突触形成受损（ 306 ）。发育过程中接触铅会影响前额叶大脑皮层、海马体和小脑区域，从而导致神经系统疾病、智力障碍、行为问题和神经损伤（ 242 ）。生命早期接触铅也与 AD 和 PD 等神经退行性疾病有关 ( 62 , 209 )。

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FIG. 8. 如图。 8.

Pb neurotoxicity and synaptic signaling. (a) Pb enters cells due to its ability to mimic Ca²⁺ and Fe²⁺ ions. (b) Inside the cell, Pb can trigger oxidative stress by different mechanisms, including the accumulation of ALA; the formation of Pb²⁺–O₂^•− complexes; and the release of labile Fe²⁺ from Ft. (c) Pb can also form complexes with GSH [Pb(GSH_2–3)] and bind to protein thiols. (d) Mitochondrial dysfunction and oxidative stress induced by Pb have also been linked to MCU. (e) Pb can displace Zn²⁺ and impair Zn²⁺ binding protein function. Furthermore, Pb interferes with the Se-dependent redox processes. (f) Finally, Pb can prevent CaM activation and thus reduce NOS activity. ALA, aminolevulinic acid; CaM, Ca²⁺/calmodulin; NOS, nitric oxide synthase; O₂^•−, superoxide anion radical; Pb, lead; Se, selenium.
铅的神经毒性和突触信号传导。 (a) Pb 由于其模拟 Ca ²⁺和 Fe ²⁺离子的能力而进入细胞。 (b)在细胞内部，Pb 可通过不同机制引发氧化应激，包括 ALA 的积累； Pb ²⁺ –O ₂ ^•−络合物的形成；以及从 Ft 中释放不稳定的 Fe ²⁺ 。 (c) Pb 还可以与 GSH [Pb(GSH _2–3 )] 形成复合物并与蛋白质硫醇结合。 (d) Pb 引起的线粒体功能障碍和氧化应激也与 MCU 有关。 (e) Pb 可以取代 Zn ²⁺并损害 Zn ²⁺结合蛋白功能。此外，Pb 会干扰 Se 依赖性的氧化还原过程。 (f)最后，Pb 可以阻止 CaM 激活，从而降低 NOS 活性。 ALA，氨基乙酰丙酸； CaM、Ca ²⁺ /钙调蛋白； NOS，一氧化氮合酶； O ₂ ^•− ，超氧阴离子自由基； Pb、铅；硒，硒。

The mechanisms of Pb toxicity include the ability of Pb to bind SH groups of proteins Cys and to mimic or compete with Ca²⁺, Fe²⁺, and Zn²⁺ (Fig. 8b, c, e, f) (99, 283). Zn deficiency increases the toxicity of Pb (4). The generation of oxidative damage by Pb in vitro and in vivo suggests that ROS also participate in Pb toxicity. For example, Pb acetate induces the opening of the mitochondrial permeability transition pore in human neuroblastoma SH-SY5Y cells via ROS (384). Pb can form a Pb²⁺–O₂^•− complex with higher oxidizing capacity than O₂^•− (3). In addition, accumulated δ-ALA by Pb-induced ALAD inhibition can be subsequently oxidized to generate O₂^•−, ^•OH, and H₂O₂. Pb per se has been reported to stimulate Fe²⁺-initiated lipid peroxidation (Fig. 8b) (337). Early postnatal exposure of rats to Pb leads to a higher accumulation of oxidative DNA damage in the cerebral cortical tissue when compared with aged controls or aged mice exposed acutely to Pb (36).
Pb 毒性的机制包括 Pb 结合蛋白质 Cys 的 SH 基团以及模拟或竞争 Ca ²⁺ 、 Fe ²⁺和 Zn ²⁺的能力（图 8b、c、e 、f）（ 99 , 283 ）。缺锌会增加铅的毒性 ( 4 )。 Pb在体外和体内产生的氧化损伤表明，ROS 也参与了 Pb 毒性。例如，乙酸铅通过ROS 诱导人神经母细胞瘤 SH-SY5Y 细胞中线粒体通透性转换孔的打开 ( 384 )。 Pb可形成Pb ²⁺ –O ₂ ^•−络合物，其氧化能力高于O ₂ ^•− ( 3 )。此外，通过Pb诱导的ALAD抑制而积累的δ-ALA随后可以被氧化生成O ₂ ^•− 、 ^• OH和H ₂ O ₂ 。据报道，Pb本身会刺激 Fe ²⁺引发的脂质过氧化（图 8b ）（ 337 ）。与老年对照或急性暴露于 Pb 的老年小鼠相比，出生后早期暴露于 Pb 的大鼠会导致大脑皮质组织中氧化 DNA 损伤的积累更高（ 36 ）。

Perinatal exposure to Pb acetate inhibits the activity of brain acid and alkaline phosphatases, catalase, acetylcholinesterase, and ATPases (12). Similar observations have been made for the activities/levels of SOD1, GPX1, and GPX4 in the hippocampus, and for mitochondrial SOD2 and GSH, both in the cortex and hippocampus (19). Antioxidant nutrients such as vitamin E, vitamin C, vitamin B6, β-carotene, and DLA, as well as metal chelators such as DMSA, or replenishment of displaced metals has been shown to be beneficial against Pb-induced oxidative stress in the brain (98, 236, 256, 258, 277, 367). Diet supplementation with Zn and Se, which participates in the regulation of the GSH and Trx antioxidant systems, can effectively outcompete Pb binding to Zn- and Se-binding sites (Fig. 8e) (135).
围产期接触醋酸铅会抑制脑酸性和碱性磷酸酶、过氧化氢酶、乙酰胆碱酯酶和 ATP 酶的活性 ( 12 )。对于海马中 SOD1、GPX1 和 GPX4 的活性/水平，以及皮层和海马中线粒体 SOD2 和 GSH 的活性/水平也进行了类似的观察 ( 19 )。维生素 E、维生素 C、维生素 B6、β-胡萝卜素和 DLA 等抗氧化营养素以及 DMSA 等金属螯合剂或补充置换金属已被证明有助于对抗铅引起的大脑氧化应激。 98、236、256、258、277、367 ） 。​​​饮食中补充锌和硒参与 GSH 和 Trx 抗氧化系统的调节，可以有效地竞争 Pb 与 Zn 和 Se 结合位点的结合（图 8e ）（ 135 ）。

Pb interferes with and disrupts Ca²⁺ signaling and homeostasis leading to excitotoxicity. In addition, Glu potentiates Pb-induced cell death in PC12 cells (267). Recently, oxidative stress induced by Pb has been shown to be linked to changes in the levels of MCU (Fig. 8d) (383). Other important intracellular targets of Pb in the brain are both neural NOS and endothelial NOS due to an impairment in their Ca²⁺/calmodulin (CaM)-dependent activation (Fig. 8f) (241). Importantly, Pb amplifies Glu-induced oxidative stress in a Ca²⁺-independent manner, but neither Ca²⁺ nor ROS seem to be essential for the enhanced cytotoxicity of combined exposure to Glu and Pb (191, 238).
Pb 干扰并破坏 Ca ²⁺信号传导和稳态，导致兴奋性毒性。此外，Glu 还可增强 PC12 细胞中 Pb 诱导的细胞死亡 ( 267 )。最近，Pb 诱导的氧化应激已被证明与 MCU 水平的变化有关（图 8d ）（ 383 ）。大脑中 Pb 的其他重要细胞内靶标是神经 NOS 和内皮 NOS，因为它们的 Ca ²⁺ /钙调蛋白 (CaM) 依赖性激活受损（图 8f ）（ 241 ）。重要的是，Pb 以不依赖 Ca ²⁺的方式放大 Glu 诱导的氧化应激，但 Ca ²⁺和 ROS 似乎对于联合暴露于 Glu 和 Pb 增强的细胞毒性都不是必需的 ( 191 , 238 )。

Mercury 汞

Hg is a transition metal that exists as elemental, inorganic, and organic Hg (Fig. 9a). Hg is ubiquitously found in the environment as sulfide compounds generated from volcanic activity and erosion, or released by anthropogenic sources such as fuel combustion, waste disposal, and industrial activities (Supplementary Table S1). Elemental or metallic Hg (Hg⁰) used in thermometers and amalgams is primarily absorbed via inhalation, while inorganic mercury (Hg^{1+ or 2+}) used in medicine and everyday life products is partially absorbed through the gut. Organic Hg (ethylmercury [EtHg or C₂H₅Hg] and methylmercury [MeHg or CH₃Hg]) is originated from atmospheric sources that are deposited in water body surfaces to be biomethylated and magnified in the food chain (Fig. 9a). Around 95% of MeHg is absorbed by the gastrointestinal tract making it the most toxic Hg species. Neurotoxic signs of Hg intoxication are vast and include ataxia, dizziness, insomnia, speech impairment, arthralgia, cognitive and behavioral changes, seizures, fatigue, and sensory disruption. While there has been an association between Hg exposure and neurodegeneration or autism, the neurological effects of chronic exposure to Hg are largely unclear. However, research has clearly demonstrated that Hg impairs neuronal development, communication, and myelination (89).
Hg 是一种过渡金属，以元素汞、无机汞和有机汞的形式存在（图 9a ）。汞在环境中普遍存在，以火山活动和侵蚀产生的硫化物形式存在，或由燃料燃烧、废物处理和工业活动等人为来源释放（补充表S1 ）。温度计和汞合金中使用的元素或金属汞 (Hg ⁰ ) 主要通过吸入吸收，而药物和日常生活用品中使用的无机汞 (Hg ^{1+ 或 2+} ) 部分通过肠道吸收。有机汞（乙基汞[EtHg或C ₂ H ₅ Hg]和甲基汞[MeHg或CH ₃ Hg]）源自大气源，沉积在水体表面，被生物甲基化并在食物链中放大（图9a ）。大约 95% 的甲基汞被胃肠道吸收，使其成为毒性最强的汞种类。汞中毒的神经毒性症状多种多样，包括共济失调、头晕、失眠、言语障碍、关节痛、认知和行为改变、癫痫发作、疲劳和感觉障碍。虽然汞暴露与神经退行性疾病或自闭症之间存在关联，但长期暴露于汞对神经系统的影响尚不清楚。然而，研究清楚地表明汞会损害神经元发育、交流和髓鞘形成 ( 89 )。

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FIG. 9. 如图。 9.

Hg redox-related toxicity mechanisms in brain cells. (a) Hg species are highly transformed (oxidation or methylation) before they are absorbed into organisms. MeHg and EtHg form a complex with Cys (CH₃HgCys) and are transported across the membranes via LAT. (b) MeHg suffers dealkylation and thiol exchange to form Hg²⁺. (c) Thiol exchange reactions of CH₃HgCys complexes with low-molecular-weight thiols (GSH) and protein thiols alter the cellular redox balance. MeHg also has a strong affinity for selenol groups. (d) GSH-MeHg adduct formation (CH₃HgGS) is catalyzed by the GSTs and adducts are detoxified by MRPs. (e) MeHg also induces mitochondrial ROS and energy failure. Furthermore, MeHg-induced neurotoxicity has been ascribed to a reduction in Glu uptake by astrocytes via EAAT triggering neuronal excitotoxicity (arrow in EAAT indicates reversal of Glu transport). Cys, cysteine; Hg, mercury; EtHg, ethylmercury; Hg¹⁺, inorganic mercurous ions; Hg²⁺, inorganic mercuric ions; LAT, L-type neutral amino acid transporter; MeHg, methylmercury.
脑细胞中汞氧化还原相关的毒性机制。 (a)汞物质在被生物体吸收之前经过高度转化（氧化或甲基化）。 MeHg 和 EtHg 与 Cys (CH ₃ HgCys) 形成复合物，并通过LAT 跨膜转运。 (b) MeHg经过脱烷基化和硫醇交换形成Hg ²⁺ 。 (c) CH ₃ HgCys 复合物与低分子量硫醇 (GSH) 和蛋白质硫醇的硫醇交换反应改变细胞氧化还原平衡。 MeHg 对硒醇基团也有很强的亲和力。 (d) GSH-MeHg 加合物形成 (CH ₃ HgGS) 由 GST 催化，加合物由 MRP 解毒​​。 (e) MeHg 还会诱导线粒体 ROS 和能量衰竭。此外，MeHg 诱导的神经毒性归因于星形胶质细胞通过EAAT 减少 Glu 摄取，从而触发神经元兴奋性毒性（EAAT 中的箭头表示 Glu 转运的逆转）。 Cys，半胱氨酸；汞、汞； EtHg，乙基汞； Hg ¹⁺ ，无机汞离子； Hg ²⁺ ，无机汞离子； LAT，L型中性氨基酸转运蛋白； MeHg，甲基汞。

Most of the studies regarding the mechanisms involved in Hg neurotoxicity have been done using MeHg. MeHg and EtHg are potent electrophiles that form a complex with Cys (CH₃HgCys or C₂H₅HgCys), and then transported across the BBB and into neuronal cells via L-type neutral amino acid transporters (LAT1 and 2) (Fig. 9a) (318, 385, 399).
大多数有关汞神经毒性机制的研究都是使用甲基汞进行的。 MeHg 和 EtHg 是有效的亲电子试剂，它们与 Cys（CH ₃ HgCys 或 C ₂ H ₅ HgCys）形成复合物，然后通过L 型中性氨基酸转运蛋白（LAT1 和 2）穿过 BBB 转运到神经元细胞中（图 1）。 9a )(318、385、399 ) 。

A high percentage of Hg in individuals intoxicated with MeHg is found as Hg²⁺, suggesting that dealkylation of MeHg is an important mechanism for the high persistence of Hg in the brain (Fig. 9b) (72). Thiol exchange from CH₃HgCys to low-molecular-weight thiols (GSH) and protein thiols has been proposed to be central mechanisms by which MeHg induces GSH depletion, inhibition of thiol-dependent antioxidant systems, and alters the activity or function of proteins with redox-sensitive Cys (signaling proteins, metabolic enzymes, neurotransmitter receptors, and transporters) (Fig. 9c) (89). MeHg also has a stronger affinity for selenol groups (selenohydryl groups in selenocysteines) compared with thiol groups. As such, selenoproteins are important targets for direct electrophilic attack of MeHg or transfer from thiol adducts (CH₃HgCys, CH₃HgGS, or CH₃HgPS [protein-Cys adduct]) (Fig. 9c) (104, 221). GSTs have been proposed to mediate the formation of CH₃HgGS adducts, which are detoxified by MRP1-mediated transport (Fig. 9d). GSH synthesis, GST, and MRP1 levels are regulated transcriptionally by the Nrf2 antioxidant system (152, 292, 349).
在甲基汞中毒个体中，高比例的汞以 Hg ²⁺形式存在，这表明甲基汞的脱烷基化是汞在大脑中高持久性的重要机制（图 9b ）（ 72 ）。从 CH ₃ HgCys 到低分子量硫醇 (GSH) 和蛋白质硫醇的硫醇交换已被认为是 MeHg 诱导 GSH 消耗、抑制硫醇依赖性抗氧化系统以及改变蛋白质活性或功能的核心机制。氧化还原敏感的半胱氨酸（信号蛋白、代谢酶、神经递质受体和转运蛋白）（图 9c ）（ 89 ）。与硫醇基团相比，MeHg 对硒醇基团（硒代半胱氨酸中的硒氢基团）也具有更强的亲和力。因此，硒蛋白是MeHg直接亲电攻击或从硫醇加合物（CH ₃ HgCys、CH ₃ HgGS或CH ₃ HgPS [蛋白质-Cys加合物]）转移的重要靶标（图9c ） （ 104、221 ）。已提出GST介导CH ₃ HgGS加合物的形成，其通过MRP1介导的运输解毒（图9d ）。 GSH 合成、GST 和 MRP1 水平由 Nrf2 抗氧化系统进行转录调节 ( 152 , 292 , 349 )。

MeHg induces mitochondrial ROS and energy failure (175, 233). Neurotoxicity induced by MeHg has also been ascribed to its inhibitory effect on Glu uptake by astrocytes, triggering neuronal excitotoxicity (15, 235) (Fig. 9e).
MeHg 会诱导线粒体 ROS 和能量衰竭 ( 175 , 233 )。 MeHg 引起的神经毒性也归因于其对星形胶质细胞摄取 Glu 的抑制作用，从而引发神经元兴奋性毒性 ( 15 , 235 ) (图 9e )。

Hg⁰ absorbed through the respiratory tract is oxidized to inorganic mercurous (Hg¹⁺) and mercuric ions (Hg²⁺) (Fig. 9a). While inorganic Hg ions have limited access to the CNS, they induce profound neurotoxic alterations that seem to be mediated as well by their binding to thiol groups (89). Accordingly, MTs exert protective effects against Hg⁰-induced neurotoxicity (387) (Fig. 9b).
通过呼吸道吸收的Hg ⁰被氧化成无机汞(Hg ¹⁺ )和汞离子(Hg ²⁺ )(图9a )。虽然无机汞离子进入中枢神经系统的机会有限，但它们会引起深刻的神经毒性改变，而这种改变似乎也是通过它们与硫醇基团的结合来介导的（ 89 ）。因此，MT对Hg ⁰诱导的神经毒性发挥保护作用( 387 )(图9b )。