Despite concerted efforts to reduce heavy metal pollution over the past few decades, exposure to heavy metals still poses a significant threat to public health [1]. Due to natural activities or anthropogenic industrial activities, large amounts of heavy metals have been discharged into the environment. Humans are exposed to heavy metals through food, water, and air ^[2][3][2,3]. Heavy metals accumulate in multiple target organs, and even exposure to low levels may cause health effects [4].

Manganese (Mn) is an essential trace element involved in the synthesis and activation of many enzymes, including glutamine synthetase, pyruvate decarboxylase, manganese superoxide dismutase (MnSOD), and arginase [5]. Mn is involved in the regulation of amino acid, lipid, and carbohydrate metabolism [6]. Mn enters the human body mainly through the respiratory and digestive tracts. Under normal circumstances, an adult’s daily intake of Mn is in the order of 2–6 mg, of which 1–5% is absorbed by the duodenum. It enters the enterohepatic circulation through the portal vein and then enters the gastrointestinal tract via biliary secretions, and it is finally excreted in the feces [7].

Mn deficiency and intoxication are both associated with adverse neuropsychiatric effects, but Mn deficiency is extremely rare [8]. Excessive exposure to Mn is mainly related to occupational exposures, including welding, mining, smelters, paints, dry batteries, and Mn-rich agricultural chemicals ^[9][10][11][9,10,11]. The high concentrations of Mn in the environment are due to its abundance in the Earth’s crust, the release of Mn-containing exhaust gases and wastewater into the environment by anthropogenic activities, and the use of gasoline additives (methylcyclopentadienyl manganese tricarbonyl (MMT)) and fungicides ^{[12][13][14][15]}[12,13,14,15]. Infant Mn exposure is mainly associated with excess Mn supplementation in parenteral nutrition [16].

The brain is the most sensitive target organ for Mn toxicity. Mn exists in 11 oxidation states from −3 to +7, among which Mn²⁺ and Mn³⁺ are commonly found in biological systems [17]. Mn enters the brain through the blood–brain barrier (BBB) and blood–cerebrospinal fluid barrier (BSCFB), primarily in the form of Mn²⁺, Mn-citrate, Mn³⁺-transferrin, or alpha-2- macroglobulin [18]. Mn mainly accumulates in the basal ganglia (striatum, pallidum, and substantia nigra), hippocampus, thalamus, and cortex [19]. Excessive exposure to Mn can lead to manganism, which is characterized by symptoms similar to Parkinson’s disease (PD), including gait disturbance, dystonia, and postural tremor [20]. In addition, Mn exposure has been associated with cognitive deficits, attention loss, and neuropsychological abnormalities [21]. Moreover, excessive exposure to Mn early in life can lead to learning disabilities, hyperactivity, and attention deficit disorders, and may increase the risk of neurodegenerative diseases later in life ^{[22][23][24][25]}[22,23,24,25].

In addition to manganism, multiple studies demonstrated a significant association between Mn and neurodegenerative diseases. Elevated Mn levels were observed in patients with PD [26] and amyotrophic lateral sclerosis (ALS) [27], but Mn deficiency has been observed in Huntington’s disease (HD) [28], and the relationship between Mn levels and Alzheimer’s disease (AD) remains controversial [29]. However, due to the excretion of Mn, the time point and biomarkers of assessment may affect the results of these studies.

Due to the significant effects of Mn exposure on the nervous system, the mechanisms of Mn-induced neurotoxicity have been extensively studied, including neuroinflammation, protein homeostasis [30], mitochondrial function and REDOX homeostasis [31], calcium homeostasis [32], neurotransmitter metabolism [33], microRNA (miRNA) function [34], and metal homeostasis [35]. At present, the understanding of the underlying mechanisms of Mn neurotoxicity is rapidly developing. Several studies revealed the insulin-mimicking effects of Mn, which can activate several of the same metabolic kinases and directly increase peripheral and neuronal insulin and insulin-like growth factor (IGF)-1 levels in rodent models ^{[36][37][38][39]}[36,37,38,39]. As an important cofactor of many kinases and phosphatases, Mn plays a key role in cell signaling pathways. Mn can activate mitogen-activated protein kinase (MAPK), protein kinase B (PKB/Akt), ataxia telangiectasia mutated (ATM), and mammalian target of rapamycin (mTOR) both in vivo and in vitro. Since these kinases regulate transcription factors, such as cyclic adenosine monophosphate (cAMP), cAMP response element-binding protein (CREB), p53, nuclear factor κB (NF-κB), and forkhead box O (FOXO), Mn can also regulate cellular function at the transcriptional level ^[40][41][42][40,41,42]. Therefore, it is important to study the role of Mn homeostasis and related signaling pathway in Mn essentiality and toxicity. However, it is not fully understood which Mn-dependent enzymes are most sensitive to changes in Mn homeostasis and the relationship between Mn and these signaling pathways.

2. Insulin and Insulin-like Growth Factor (IGF) Signaling Pathway

Insulin and IGF are homologous growth hormones that regulate cell metabolism, and their main function is to increase anabolism and reduce catabolism. Insulin has short-term metabolic effects, such as increasing glucose and amino acid transport, inducing glycolysis, glycogenesis, lipogenesis, and protein synthesis, and inhibiting gluconeogenesis, lipolysis, and protein degradation. However, IGF has chronic effects that determine cell fate, such as inducing proliferation, inhibiting apoptosis, and inducing differentiation [43]. In the brain, insulin and IGF are necessary for synaptic maintenance and activity, neurogenesis, neurite growth, and mitochondrial function [28]. Therefore, the dysregulation of these neurotrophic factors is believed to be associated with neurodegenerative diseases, and abnormal IGF/ insulin levels and insulin-related signaling changes have been observed in neurodegenerative diseases, including PD [44], AD [45], HD [28], and ALS [46]. Upon the binding of insulin or IGF receptor (IR/IGFR) on the target cell, the receptor’s tyrosine kinase is activated to phosphorylate several specific substrates, particularly insulin receptor substrates (IRSs) and Src homologous collagen (SHC) [47]. Phosphorylated tyrosine residues of these substrates are recognized by various signaling molecules containing the Src Homology 2 (SH2) domain. For example, 85 kDa regulatory subunits (p85) of phosphatidylinositol 3-kinase (PI3K), growth factor receptor binding 2 (GRB2), and protein tyrosine phosphatase 2 containing sh2 (SHP2/Syp) [48]. These bindings activate downstream signaling pathways, the PI3K pathway, and the Ras mitogen-activated protein kinase (MAPK) pathway [49]. Activation of these well-known signaling pathways is required to induce the various biological activities of IGF [50]. Mn has been shown to activate several of the same pathways as insulin/IGF, including AKT, MAPK, and mTOR, and even the IR/IGFR itself [28]. Several in vivo studies found that Mn deficiency caused glucose intolerance and reduced insulin secretion, manifested by decreased circulating IGF-1 and insulin, and increased IGFBP3 ^{[36][37][38][39][51]}[36,37,38,39,51]. Mn supplementation improves glucose intolerance and prevents diet-induced diabetes by increasing insulin secretion and the expression of IGFR and IGF-1 in the hypothalamus, as well as increasing the expression of MnSOD ^[52][53][54][52,53,54]. These results are consistent with reports of diabetic patients responding to oral Mn therapy, as well as reduced blood Mn levels in diabetic patients ^[55][56][55,56]. However, few studies focused on the role of the IGF receptor itself in Mn toxicity. Tong and his colleagues found that Mn exposure reduced the expression of ATP and insulin/IGF receptors [57]. In addition, Srivastava and his colleagues found that Mn stimulated hypothalamic IGF-1 dose–dependent release and increased p-IGF-1R expression in vitro and affected hypothalamic development in adolescent rats through IGF-1/Akt/mTOR pathways [54]. Other studies demonstrated that Mn could regulate two important downstream signaling pathways of the IGF, PI3K/Akt, and MAPK signaling pathways.

2.1. PI3K/Akt Signaling Pathway

PI3K/Akt can be activated by a variety of growth factors and plays a central role in cell growth regulation, proliferation, metabolism, and cell survival, as well as neuroplasticity [58]. The interaction between the p85 and IGF-IR substrate leads to the activation of PI3K. Activated PI3K phosphorylates PIP2 to produce PIP3. PIP3, in turn, activates PDK1. PDK1 phosphorylates Akt threonine 308 residues and mTORC2 phosphorylates Akt serine 473 residues, resulting in complete Akt/PKB activation. Activated Akt phosphorylates a variety of Akt substrates, including AS160, Bad, forkhead box-containing protein, O subfamily (FoxO1), Tsc1, and glycogen synthase kinase 3β (GSK3β) [43]. PI3K inhibition reduces Mn uptake in mouse striatal cell lines (STHdh) [59]. In a Huntington’s disease cell model, exposure to both physiological (1nM) and over-physiological (10 nM) Mn levels enhanced p-IGFR/IR-dependent AKT phosphorylation, and more than 70% of Mn-induced p-Akt signaling was dependent on p-IGFR rather than other upstream and downstream effectors [60]. Researchers' previous studies have shown that exposure to toxic doses of Mn can increase the phosphorylation levels of Akt in rat hippocampal tissue [61] and PC12 cells [62]. Moreover, pretreatment of PC12 cells with LY294002, a PI3K/Akt inhibitor, further increased apoptosis, suggesting that the Mn-activated PI3K/Akt signaling pathway played a protective role. Bae et al. [63] reported that the phosphorylation level of Akt in BV2 microglia increased after 500 μM Mn treatment for 1 h. Short-term (postnatal day (PND) 8–12) exposure to Mn increased Akt phosphorylation levels in rat striatum [23]. Mn exposure also increased Akt expression levels in C. elegans [64]. However, another study reported that Mn exposure inhibited the PI3K/Akt signaling pathway in cortical neurons, and the apoptosis induced by Mn can be improved by increasing the level of the PI3K/Akt signaling pathway [65]. Brain-derived neurotrophic factor (BDNF) is a key nutrient factor involved in neurobiological mechanisms of learning and memory, and both population studies and animal experiments have confirmed that Mn exposure can reduce BDNF levels ^[66][67][66,67]. Mn exposure can increase α-Synuclein (α-Syn) expression in mice, and the Mn-dependent enhancement of α-Syn expression can further exacerbate the decrease in BDNF protein level and inhibit TrkB/Akt/Fyn signaling, and thus interfere with FYN-mediated phosphorylation of NMDA receptor GluN2B subtyrosine [68]. Whether Mn exposure activates or inhibits the PI3K/Akt signaling pathway may be due to different doses and/or timing of exposure and the use of different animals or cell lines, but in general, activation of the PI3K/Akt signaling pathway protects against Mn-induced neurotoxicity. Transcription factor FOXO family proteins, substrates of Akt, can be phosphorylated by active Akt and excluded from the nucleus ^[69][70][69,70], thereby inhibiting the transcription of genes associated with oxidative stress protection; for example, MnSOD-2, catalase, and anti-apoptotic effects (Bim and Fas ligands) ^{[71][72][73][74][75]}[71,72,73,74,75]. Mn exposure can increase FOXO3a levels ^[62][76][62,76]. In C. elegans, resistance in AKT-1/2-deficient mutant strains to Mn toxicity was higher than in wild type N2 strains, possibly because the loss of AKT-1/2 reduced the inhibition of DAF-16, and thus increased the antioxidant response [77]. In contrast, another study [40] found that treating astrocytes with 100 or 500 μM of Mn resulted in increased FOXO (dephosphorylation and phosphorylation) levels, and p-FoxO disappeared from the cytoplasm after Mn exposure, while AKT phosphorylation levels remained unchanged, but MAPK phosphorylation levels, especially p38 and ERK, and PPARγ coactivator 1 (PGC-1) levels were elevated, suggesting that FOXO may be regulated by MAPK rather than the Akt signaling pathway.

2.2. MAPK Signaling Pathway

Protein kinases ERK1/2, JNK1/2, and p38MAPK are the most important enzymes of the MAPK family ^[78][79][78,79]. ERK1/2 is primarily activated by growth factors and regulates gene expression, embryogenesis, proliferation, cell death/survival, and neuroplasticity ^[80][81][80,81]. JNK1/2/3 and p38MAPK protein kinases are commonly known as stress-activated protein kinases (SAPKs), which can be activated by cytokines and cytotoxic damage and are associated with stress and cell death ^[82][83][82,83]. GRB2 interacts with the IGF1 receptor substrate to activate GRB2-associated SOS guanine nucleotide exchange activity, thereby activating Ras small GTPase, which, in turn, activates MAPK. This kinase cascade is called the Ras-MAPK pathway. Activated MAPK phosphorylates transcriptional activators, leading to the induction of various IGF biological activities [43]. Acute exposure to Mn (3–6 h) at concentrations that did not affect cell viability activated MAPKs (ERK1/2 and JNK1/2) in the hippocampus and striatum sections of immature rats (PND14) [84]. Mn (500 μM) exposure increased iNOS expression in BV2 microglia, and the increase in iNOS protein expression was mediated by the JNK-ERK MAPK and PI3K/Akt signaling pathways, but not by the p38 MAPK signaling pathway [63]. Sub-acute (4 weeks) and sub-chronic (8 weeks) exposure to Mn (15 mg/kg) resulted in an increased expression of p38, p-ERK, and p-JNK in the thalamus of rats. Treatment with sodium para-aminosalicylic acid (PAS-Na), an anti-inflammatory drug, decreased p-JNK and p-P38 levels but did not decrease p-ERK levels [85]. Mn exposure also enhanced the activation of the p38 MAPK and JNK pathways in PC12 cells and mouse brain tissue, but only the activation of p38 MAPK reduced Mn-induced neuronal apoptosis through BDNF regulation [86]. Short-term exposure to Mn (PND8-12) increased the phosphorylation of DARPP-32-Thr-34, ERK1/2, and AKT in the rat striatum. TroloxTM, an antioxidant, reversed the increase in ROS and ERK1/2 phosphorylation but failed to reverse the increase in AKT phosphorylation and motor deficit induced by Mn (20 mg/kg) [23], indicating that MAPK activation is related to oxidative stress but Akt activation is not directly related to oxidative stress. cAMP is the second intracellular messenger. CREB is a target of CAMP-dependent protein kinase A (PKA), activated by the phosphorylation of PKA at Serine-133 (Ser133), and is also regulated by Ca²⁺ and p38 MAPK ^{[87][88][89][90][91]}[87,88,89,90,91]. BDNF is downstream of the cAMP-PKA-CREB signaling pathway [92]. Mn exposure increased CREB and p38 MAPK phosphorylation and apoptosis in PC12 cells, while CREB knockdown decreased BDNF levels and increased Mn-induced apoptosis, suggesting that CREB activation had a protective effect on neuronal apoptosis. In addition, the inhibition of Ca²⁺ and p38 MAPK significantly reduced CREB phosphorylation levels [86]. Mn exposure may also reduce the level of BDNF by inhibiting the CAMP-PKA-CREB signaling pathway in hippocampal and PC12 cells, inducing apoptosis and leading to cognitive impairment ^[66][93][66,93]. Phosphoprotein DARPP-32 is highly expressed in spinous neurons in the striatum. The altered phosphorylation status of Thr-34 or Thr-75 in DARPP-32 gives it the unique properties of dual function as both an inhibitor of protein phosphatase 1 (PP1) and an inhibitor of PKA. Rats exposed to 5 or 10 mg Mn/kg on PND8-12 showed increased phosphorylation of DARPP-32 at Thr-34 in the striatum on PND14 [23].