Heavy metals and adult neurogenesis

Abstract

With extensive use in industrial and agriculture applications, overexposure to heavy metals has become a global public health concern. The nervous system is vulnerable to many heavy metals, including cadmium, lead, and mercury. However, the knowledge about the underlying mechanisms of these metals’ neurotoxicity is still very limited. Adult neurogenesis is a process of generating functional neurons from adult neural progenitor/stem cells (aNPCs), which plays an important role in cognitive function and olfaction. The studies of adult neurogenesis provide new insights into mechanisms of heavy metal neurotoxicity. This review summarizes the current research about the effects of heavy metals on adult neurogenesis and discusses their importance in understanding the mechanisms of heavy metals neurotoxicity, as well as challenges and future directions.

Graphical Abstract

Introduction

Heavy metals are generally defined as naturally occurring metallic elements with relatively high densities compared to water (> 5 g/cm³) [1]. Due to their wide use in industrial, agricultural, and technological applications, heavy metals have become global environmental pollutants of major public health concern [2]. Heavy metals consist of both biologically essential metals and non-essential metals. Biologically essential heavy metals, including iron, manganese, and zinc, are essential nutrients required for multiple physiological functions. Others, such as arsenic, cadmium, lead, and mercury, are categorized as non-essential metals [3]. Most heavy metals can induce damage in different organs or systems, even at low exposure levels [3]. Among them, cadmium, lead, mercury, and arsenic, are some of the most toxic metals, ranking among the Top 10 on the Substance Priority List of the Agency for Toxic Substances and Disease Registry (ATSDR) [4].

Neurotoxicity is one major toxic effect of heavy metals. However, the underlying mechanisms of heavy metal neurotoxicity are not completely understood. In recent years, adult neurogenesis has become a hot topic in neuroscience research [5], providing exciting new mechanistic insights concerning heavy metal neurotoxicity. In this review, we aim to present the findings on the effects of heavy metal exposure on adult neurogenesis and highlight their implications on the function of the central nervous system (CNS) and neurodegenerative disease (Table 1).

Table 1.

Studies investigating the effects of heavy metals on adult neurogenesis.

Metal	Model	Exposure	Effect	Reference
Cadmium	C57BL/6 mice; Nestin-CreER™:caMEK5-eGFPloxP/loxP (caMEK5) mice	0.6 mg/L Cd as CdCl2 through drinking water; start at 12-week-old for 16–18.5 weeks. (C57BL/6); start at 15–17-week-old for 38 weeks (caMEK5)	No effect on cell proliferation (2h BrdU+); impaired neuronal differentiation (2.5-week BrdU+/NeuN+, 2.5-week BrdU+/DCX+) and dendritic complexity of immature neurons in SGZ. Inducible and conditional expression of caMEK5 in aNPCs (stimulating adult neurogenesis) rescued animals from Cd-induced impairments of adult hippocampal neurogenesis and related spatial memory (novel object location test, contextual fear memory test)	[8]
Cadmium	C57BL/6 mice; Nestin-CreER™:caMEK5-eGFPloxP/loxP (caMEK5) mice	0.6 mg/L Cd as CdCl2 through drinking water; start at 12-week-old for 16–18.5 weeks. (C57BL/6); start at 15–17-week-old for 38 weeks (caMEK5)	No effect on cell proliferation (2h BrdU+) in SVZ; impaired neuronal differentiation (2.5-week BrdU+/NeuN+, 2.5-week BrdU+/DCX+) in OB. Inducible and conditional expression of caMEK5 in aNPCs (stimulating adult neurogenesis) rescued animals from Cd-induced impairments of SVZ neurogenesis and related olfactory memory (short term olfactory memory test, odor-cued associative learning and memory test)	[9]
Cadmium	C57BL/6 mice	3 mg/L Cd as CdCl2 through drinking water; start at 8-week-old for 20 weeks	Impaired hippocampus-dependent spatial working memory in novel object location test and T-maze test; impaired contextual fear memory. Impaired olfactory memory in short-term olfactory memory test and odor-cued associative learning and memory test.	[17]
Cadmium	SGZ aNPCs; C57BL/6 mice	0–0.45 μΜ CdCl2 (in vitro); 3 mg/L Cd as CdCl2 through drinking water; start at 8-week-old for 13 weeks (in vivo).	Induced apoptosis, inhibited cell proliferation (BrdU+) and spontaneous neuronal differentiation (β-ΙΙΙ tubulin+); induced activation of JNK and p38 MAP kinase pathway in vitro. Decreased cell survival (5-week BrdU+) without impairment of cell proliferation (Ki67+); Impaired cell differentiation (5-week BrdU+/DCX+) and maturation (5-week BrdU+/NeuN+) in DG; decreased dendritic complexity in immature DG neurons in vivo	[18]
Cadmium	SVZ aNPCs	0–0.45 μΜ CdCl2	Induced apoptosis, impaired cell proliferation (BrdU+); induced activation of JNK and p38 MAP kinase pathway	[19]
Cadmium	ApoE3/ApoE4-KI mice	0.6 mg/L CdCl2 through drinking water; start at 8-week-old for 14 weeks.	Impaired spatial working memory in novel object location test; deficits manifested earlier in ApoE4 mice than ApoE3 mice within the same sex and earlier in males within the same genotype. Impaired neuronal differentiation of adult-born neurons in hippocampus of male ApoE4 mice	[20]
Cadmium	Swiss Albino mice	2.5 mg/kg/day oral dose Cd; start from 4-week-old for 60 days	Impaired spatial learning and memory in Morris water maze test; impaired novel object recognition memory. Induced oxidative stress, reduced proteins associated with neurogenesis (hippocampus BDNF, synapsin II, DCX, CREB).	[21]
Cadmium	Murine neural stem cells	0.75, 1.5 μΜ CdCl2	Decreased differentiation into immature neurons, increased differentiation into astrocytes.	[22]
Lead	ApoE3/ApoE4-KI mice	0.2% Lead acetate through drinking water; start from 8-week-old for 12 weeks	Impaired proliferation (2h BrdU+), neuronal differentiation (3-week BrdU+/DCX+), and maturation (3-week BrdU+/NeuN+) of aNPCs in DG; decreased dendritic complexity of immature neurons in SGZ of female ApoE4 mice. Impaired hippocampus-dependent spatial memory in novel object location test in all mice but manifested first in female ApoE4-KI mice; reduced contextual fear memory in all animals; decreased spontaneous alternation in T-maze in female ApoE4 mice.	[10]
Lead	SGZ aNPCs	0–2 μΜ Lead as Lead acetate	Induced apoptosis, inhibited proliferation (BrdU+), and impaired spontaneous neural differentiation (β-ΙΙΙ tubulin+); induced activation of JNK and p38 MAP kinase pathway.	[25]
Lead	Long Evans rats	0.2% Lead acetate through drinking water; start rom gestation day 16	Decreased survival of adult-born cells (BrdU+ ;28 days after last BrdU dosing, postnatal day 114); did not affect cell proliferation (BrdU+;24 h after last BrdU dosing, postnatal day 86) in SGZ.	[26]
Lead	Long Evans rats	1500 ppm Lead acetate in diet; from 10 days before breeding to postnatal day 50 for proliferation study; to postnatal day 78 for survival study	Impaired cell proliferation (24h BrdU+) and survival (4-week BrdU+) of aNPCs; reduced dendritic length of newly born granule cells, did not affect cellular fate of newly born cells (Brdu+/DCX+ or BrdU+/GFAP+) in DG.	[27]
Lead	Wistar rats	0.2% Lead acetate through drinking water; from postnatal day 1 to postnatal day 30	Reduced cell proliferation (24h BrdU+) and neuronal differentiation (3-week BrdU+/Calbindin+) in SGZ; did not affect survival of adult-born cells in SGZ. Impaired contextual fear memory.	[28]
Mercury	Sprague-Dawley rats	0.6 μg/g or 5 μg/g Methylmercury as Methylmercury chloride; subcutaneous injection, single dose	Induced caspase-dependent apoptosis; reduced proliferating cells (BrdU+) in DG. Impaired hippocampus-dependent spatial memory in Morris water maze test.	[11]
Mercury	Sprague-Dawley rats	0.4 mg/kg Methylmercury as Methylmercury chloride; intraperitoneal injection; once a day from postnatal day 5-day 33	Decreased number of immature neurons (DCX+) in the SGZ. Impaired hippocampus-dependent spatial memory in Morris water maze test.	[12]
Mercury	Murine-derived neural stem cell line C17.2	0–2 μΜ Methylmercury	Induced apoptosis through activation of caspase-3 and Bax.	[31]
Mercury	Human neural progenitor cells (ReNcell CX cells)	0, 10 and 50 nM Methylmercury	Deceased mitochondrial function, induced apoptosis, and induced ROS generation.	[32]
Mercury	Sprague-Dawley rats	5 μg/kg Dimethylmercury; intraperitoneal injection, once a day for 36 days	Reduced number of proliferating cells and immature neurons in SGZ. Impaired hippocampus-dependent spatial memory in Morris water maze test and novel object recognition test.	[33]
Arsenic	Kunming mice	4 mg/L As2O3 through drinking water; group1: 4 months; group2: 4 months + 2 months recover	Reduced number of adult-born (BrdU+) cells and adult-born mature neurons (BrdU+/NeuN+); did not induce apoptosis in hippocampus; deficits were reversible after arsenic removed. Wnt3 mRNA levels decreased in the treated group.	[37]
Arsenic	C57BL/6 mice	50 ppb Arsenic through drinking water; from breeding to Postnatal day 23–25.	Reduced total number of 4-week-old adult-born immature (BrdU+/DCX+) and mature (BrdU+/NeuN+) neurons; did not affect number of proliferating cells (12h-labeling BrdU+/Ki67+) in SGZ. Alteration in expression levels of neurogenesis-related genes, such as Dcx, Fgf2, and Nf1.	[39]
Arsenic	C57BL/6 mice	50 ppb Arsenic through drinking water; from 10 days before mating to Postnatal day 23–25.	Affected histone modification, including H3K4me3 and H3k9ac, along with protein expression of their chromatin modifiers in DG.	[40]
Arsenic	Swiss Albino mice	2 mg/kg As2O3; oral gavage for 45 days	Impaired spatial working memory in Morris water maze test.	[42]
Arsenic	Swiss Albino mice	100 mg/L Sodium arsenite; through drinking water for 60 days	Impaired spatial working memory in Morris water maze test.	[43]
Manganese	SVZ aNPCs	0–800 μΜ Manganese as MnCl2	Reduced total neurites length of differentiated SVZ aNPCs; cytoskeletal reorganization; impaired neuronal differentiation (DCX+).	[47]
Manganese	Sprague-Dawley rats	6 mg/kg as MnCl2; intraperitoneal injection, once per day, 5 days per week for 4 weeks	Increased proliferation (4h BrdU+) of aNPCs in SVZ; decreased survival rate of adult-born neurons (BrdU+/NeuN+) in OB.	[48]
Manganese	Sprague-Dawley rats	6 mg/kg Manganese as MnCl2; intraperitoneal injection, once per day, 5 days per week for 4 weeks	Inhibited proliferation of aNPCs; impaired survival rate of adult-born cells (2-week and 4-week BrdU+); reduced number of adult-born immature (BrdU+/DCX+) and mature neurons (BrdU+/NeuN+) in SGZ.	[49]
Manganese	Sprague-Dawley rats	6.55 mg/kg Manganese as MnCl2 for 4, 8, and 12 weeks	Impaired hippocampus-dependent spatial memory in Morris water maze test.	[50]
Manganese	Mice	100 mg/kg Manganese as MnCl2; intraperitoneal injection; once per day for 3 days with 3 days interval	Impaired hippocampus-dependent contextual fear memory.	[51]

Adult neurogenesis

Neurogenesis is the process of neural stem and progenitor cell proliferation, differentiation, migration, and integration into the nervous system. While neurogenesis is ubiquitous in the developing brain, the process is restricted in the adult [6]. In the adult mammalian brain, the neural progenitor/stem cell (aNPC) population is restricted to two neurogenic regions: the subventricular zone (SVZ), which generates new inhibitory neurons in the olfactory bulb (OB) through migration via the rostral migratory stream, and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG), which generates excitatory granular cells via a short migration into the granular cell layer (GCL) of the DG. While SVZ adult neurogenesis can regulate olfaction, SGZ adult neurogenesis is important for hippocampus-dependent learning and memory [6].

Adult neurogenesis can be affected by many external stimuli. For instance, stress and irradiation reduce adult neurogenesis, whereas environmental enrichment, as well as activities such as mating and exercise enhance the process [6]. Since perturbation of adult neurogenesis is associated with different neurodegenerative diseases [7], an increasing body of literature has accumulated in recent years suggesting that certain environmental neurotoxicants can induce neurotoxicity by affecting adult neurogenesis [8–12].

Cadmium

Cadmium is a heavy metal widely used in commercial and industrial applications. The main sources of exposure for the general population are food and smoking [13]. Increasing studies suggest Cd is a neurotoxicant. For example, epidemiological studies have found associations between blood or urine Cd levels and cognitive and olfactory deficits, as well as increased Alzheimer’s disease mortality in adults [14–16]. However, the full spectrum of its neurotoxicity is not established.

A series of studies from our group showed that cadmium exposure can impair cognition and olfactory memory by disturbing adult neurogenesis. Cadmium exposure (3 mg/L) through drinking water impaired hippocampus-dependent learning and memory, olfactory memory, and key processes of neurogenesis such as adult-born cell survival, neuronal differentiation and maturation in adult mice [17,18]. In addition, cadmium exposure impaired survival, inhibited proliferation, and induced apoptosis in primary cultured SVZ and SGZ aNPCs isolated from adult mice [18,19]. We recently reported impaired cognition, olfaction, and adult neurogenesis in mice following 0.6 mg/L cadmium exposure, which yielded blood cadmium concentrations comparable to levels found in the general US population. These data suggest that cadmium levels found in the general population may be sufficient to impair cognition and olfactory memory [8,9]. Furthermore, genetic and conditional induction of adult neurogenesis successfully rescued cadmium impairments in hippocampus-dependent spatial working memory and olfactory memory, providing the first evidence for a causal relationship between cadmium impairments of adult neurogenesis and cognitive and olfactory deficits [8,9].

Interestingly, there is a gene-environmental interaction (GxE) between ApoE4 and cadmium on cognition, as well as adult hippocampal neurogenesis [20]. Mice expressing the human ApoE4 gene, the strongest known genetic risk factor for Alzheimer’s disease, were more susceptible to the adverse effects of cadmium on hippocampus-dependent spatial memory and adult hippocampal neurogenesis than ApoE3-KI (genotype control) mice. These results further suggest that cadmium-impaired adult neurogenesis may underlie accelerated or exacerbated cognitive decline associated with neurodegeneration and/or aging.

Several other studies also support the notion that cadmium exposure impairs adult neurogenesis. One study found that cadmium exposure induced impairments in spatial learning and memory and a significant reduction of levels of proteins associated with neurogenesis (Synapsin II, DCX, and CREB) in mice [21]. Recently, by using single-cell RNA sequencing, Song et al. revealed that cadmium exposure decreased neuronal differentiation and increased astrocytic differentiation in primary cultured mouse neural stem cells [22].

Lead

Although its use has reduced significantly, lead remains a ubiquitous environmental contaminant. Lead exposure mainly occurs through ingestion of contaminated food, water, and inhalation of contaminated dust. The CNS is the prime target of lead in development. Although lead has historically been associated with peripheral nervous system effects in adults, increasing epidemiology studies are linking lead exposure with impairments of cognition [23] and olfaction [24].

Lead exposure is toxic to adult neurogenesis. Using SGZ aNPCs collected from adult mice, our group has demonstrated that lead induced apoptosis, inhibited proliferation, and impaired spontaneous neuronal differentiation in vitro [25]. In this study, activation of JNK and p38 MAP kinase pathway was found to mediate lead toxicity in SGZ aNPCs. In another study, adult lead exposure was sufficient to impair hippocampus-dependent learning and memory, as well as adult hippocampal neurogenesis in mice, and these deficits occurred much earlier or were more severe in female ApoE4-KI mice [10]. Together, these data suggest that impaired adult neurogenesis may contribute to lead-induced cognitive deficits, and GxE between ApoE4 and lead exposure, as well as sex differences may contribute to the lead toxicity on adult neurogenesis.

Developmental lead exposure can also affect adult neurogenesis. Perinatal lead exposure decreased the survival of adult-born cells without affecting cell proliferation in the adult rat hippocampus [26]. One study found developmental lead treatment impaired proliferation and survival of aNPCs, reduced the dendritic length of newly born granule cells, but did not affect the cellular fate of adult-born cells in the rat DG [27]. However, another study discovered developmental lead exposure impaired contextual fear memory, reduced cell proliferation and neuronal differentiation of aNPCs, but did not affect the survival of adult-born cells in the DG of adult rat [28]. Although findings have not been entirely consistent, which may be due to the differences in dose, exposure route and time, and animal strains, these results still support the conclusion that lead exposure can impair adult neurogenesis.

Mercury

Mercury is a heavy metal in the environment and exists in three forms (elemental, inorganic, and organic) with variable toxicity. General exposure to mercury is mostly caused by ingestion of contaminated food. Neurotoxicity is the major toxic effect from exposure to mercury, especially methylmercury [29]. In humans, mercury exposure can induce detrimental effects on cognition, memory, and attention [30].

Several in vitro studies have reported that neuronal stem cells are susceptible to methylmercury toxicity. Methylmercury can induce apoptosis in a neural stem cell line (C17.2 cell) via Bax activation, cytochrome c release, as well as caspase and calpain activation [31]. In ReNcell CX cells (human neural progenitor cells), low-dose methylmercury can induce apoptosis by impairing mitochondrial function and causing oxidative stress [32].

Furthermore, findings from different research groups suggest that methylmercury exposure can impair spatial memory through affecting adult hippocampal neurogenesis. Several studies have found that developmental methylmercury exposure can induce impairments of hippocampal neurogenesis and hippocampus-dependent spatial learning and memory in rats [11,12]. In addition, a recent study [33] showed that methylmercury-treated adult rats exhibited impaired adult hippocampal neurogenesis and cognitive deficits.

Arsenic

Arsenic is a prevalent toxic heavy metal. For the general population, contaminated food and water are the major sources of arsenic exposure [34]. Arsenic can impair the nervous system and contribute to the development of different neurological disorders. Epidemiological studies of people living in the vicinity of arsenic sources have shown that cognitive functions are negatively associated with arsenic exposure in adults [35,36].

Arsenic exposure reduced the number of adult-born cells and adult-born mature neurons but did not induce apoptosis in the SGZ of adult mice [37]. In this study, they also found reduced expression level of Wnt3 mRNA in the hippocampus of arsenic treated mice. Since Wnt3/β-catenin signaling pathway regulates adult neurogenesis [38], these data suggest that arsenic may impair adult neurogenesis through affecting the Wnt3/β-catenin pathway. Another study found developmental arsenic exposure decreased the number of adult-born immature and mature neurons but did not affect proliferating cells in mice. They also reported alteration in the expression levels in the DG of 31% of neurogenesis-related genes, such as Dcx, Fgf2, and Nf1, which suggests arsenic exposure impairs differentiation in the adult hippocampus [39]. The same group further discovered that under the same exposure paradigm that impaired adult hippocampal neurogenesis, arsenic can affect the histone modification, along with the protein levels of their chromatin modifiers in the DG [40]. Because adult neurogenesis is highly orchestrated by epigenetic factors [41], their findings indicate that arsenic can impair adult neurogenesis through inducing epigenetic dysregulation.

Several animal behavioral studies have established that arsenic can impair cognition, including hippocampus-dependent learning and memory [42,43]. However, direct evidence that arsenic impairs cognition through affecting SGZ adult neurogenesis is still lacking.

Manganese

Manganese is a biological essential metal. However, overexposure can induce excessive manganese accumulation in the CNS and cause adverse effects. Manganese exposure mostly occurs through food consumption and inhalation from industrial sources [44]. It is known that manganese exposure is associated with learning deficits, neurodegeneration, and olfactory deficits [45,46].

Both in vitro and in vivo studies have shown that manganese can impair adult neurogenesis. Environmentally relevant manganese exposure can reduce total neurite length, induce cytoskeleton reorganization, and impair neuronal differentiation in cultured SVZ aNPCs from young adult mice [47]. In addition, manganese exposure in rats significantly increased cell proliferation, decreased survival of adult-born cells within the SVZ, and reduced the survival rates of the adult-born immature and mature neurons in the OB [48]. In another study, the same group reported that manganese exposure could impair proliferation, survival, and neuronal differentiation of SGZ aNPCs in adult rats [49]. Furthermore, the detrimental effects of manganese on adult neurogenesis-related behavioral tasks, such as hippocampus-dependent learning and memory [50] and contextual fear memory [51], have been addressed by different groups, suggesting affecting adult neurogenesis may be one of the mechanisms of manganese neurotoxicity.

Conclusion

In the past 15 years, an increasing number of studies have been published focusing on the effects of heavy metals on adult neurogenesis and related behavioral deficits. Their findings confirm that exposure to certain heavy metals, such as cadmium, lead, and mercury, can affect critical processes of adult neurogenesis, and impair cognitive function and olfaction. These findings suggest that these heavy metals can induce neurotoxic effects by affecting adult neurogenesis. However, the direct evidence supporting their causal relationship is only reported in cadmium [8,9], utilizing a transgenic mouse model that allows inducible and conditional activation of adult neurogenesis. Given that heavy metals can induce pleiotropic detrimental effects to the CNS, future studies directly examining the causal relationship between effects on adult neurogenesis and cognitive and olfactory deficits are required to establish perturbation of adult neurogenesis as an underlying mechanism of other heavy metal neurotoxicity. Moreover, although the existence of adult neurogenesis in humans is still controversial [52], the success of rescuing mice from cadmium-induced behavioral deficits through activating adult neurogenesis provides exciting translational implications in humans.

Adult neurogenesis is a complex process regulated by the interaction of intrinsic factors from the aNPCs and their progeny, as well as the microenvironment provided by various cells in the niche [53]. Cellular heterogeneity in the neurogenic regions makes it extremely difficult to study the effects of neurotoxicants on adult neurogenesis in vivo, especially in investigating the underlying molecular mechanisms. However, single-cell RNA sequencing can facilitate identification of cell-type-specific effects of heavy metal exposure on adult neurogenesis and measure cell-to-cell expression variability of tens of thousands of genes [54]. Through integration of systems biology techniques and novel toxicology systems (e.g., 3D in vitro models derived from human induced pluripotent cells [55]) with in vivo methods to investigate the effects of toxicants on adult neurogenesis, we see a great opportunity to elucidate the potential mechanisms and provide new insights into the prevention and treatment of heavy metal neurotoxicity in the future.