AgSC mediated up-graduated differential expressed genes.
Abstract
Silver nanoparticles (AgNPs) are one of the most highly commercialized nanoparticles, having been used extensively as an antimicrobial agent in cosmetics, textiles, foods, and the treatment of diseases. However, the impact of AgNPs on human mental health has not yet been well characterized. Using the human pluripotent stem cell (hPSC) neuronal differentiation cellular model to assess AgNPs neurotoxicity has several benefits. First, hPSCs neuronal differentiation process can faithfully recapitulate stages of neural development from neuronal progenitors to mature neurons which can provide an excellent platform for neurodevelopment and neurodegeneration toxicity testing. Furthermore, it can limit the amount of animal use for toxicity studies. With this cellular model, we examined citrate-coated AgNPs (AgSCs) and Polyvinylpyrrolidone-coated (AgSP) mediated neurotoxicity. Our results suggested that AgNP induced neurotoxicity exhibited a coating and dose-dependent manner. AgSC had high neurotoxicity compared with AgSP. AgSC significantly up-graduated Metallothionein (1F, 1E, 2A) proteins, a metal-binding protein that plays an essential role in metal homeostasis, heavy metal detoxification, and cellular anti-oxidative defense. Transcriptome analysis indicated that AgSC inhibited neurogenesis and axon guidance, promoted gliogenesis and neuronal apoptosis through oxidative stress. Supplementation with ascorbic acid can act as an antioxidant to attenuate AgNP-mediated neurotoxicity.
Keywords
- silver nanoparticles (AgNP)
- human pluripotent stem cell-derived neuronal network
- transcriptome analysis
- oxidative stress
- neurogenesis and gliogenesis
- neurodegeneration
1. Introduction
Engineered nanomaterials (ENMs) are ultra-fine materials (ranging from 1 to 100 nm in length or diameter) that are currently being developed for diverse applications due to their unique optical, electrical, and thermal properties [1, 2, 3]. Among them, silver nanoparticles (AgNPs) are one of the most widely used in medical and commercial products for their unique antibacterial functions [4, 5, 6, 7, 8, 9, 10]. The AgNP market is expected to reach USD 2.45 billion by 2022 (Globe Newswire, San Francisco, 2015). Furthermore, over the next decade, Nanotechnological approaches will continue to play a vital role in neuroscience, not just in the development of highly specific and sensitive imaging probes and biosensor interfaces, but also potential tools for treatment strategies [11, 12]. For example, molecules will be nano-engineered to cross the blood-brain barrier to target specific cell or signaling systems or act as vehicles for gene delivery [13, 14].
Although the translation of nanotechnology into the treatment of human neurological disorders is very promising, the biocompatibility of these materials is still a primary concern [8]. A wealth of data demonstrates that ENMs have the potential to induce inflammation, oxidative stress, and DNA damage, which point towards potential health risks for humans, including cardiovascular diseases, pulmonary diseases, impairment of brain function, and developmental toxicity [15, 16, 17]. Recently, researchers have begun to explore the potential neurotoxicity of ENMs such as AgNPs in cellular and animal models [18, 19, 20, 21]. These studies showed that AgNPs can accumulate in the central nervous system (CNS) through the upper respiratory tract via the olfactory bulb or through crossing the blood-brain barrier, and thus induce neurodegeneration [10, 22, 23]. Furthermore, studies showed that AgNP exposure impairs neurodevelopment in PC12 cells and stem cell-derived neuronal networks and alters the expression of genes involved in neuronal function that are distinct from those of Ag+ alone, depending on size and coating [24, 25, 26].
So far, there has been limited information regarding the impact of AgNPs on neuronal development and neurodegeneration both in vivo and in vitro. hPSCs neuronal differentiation protocol evaluates the impact of AgNPs on multiple stages of differentiation ranging from neuronal progenitors to mature neuron and astrocyte networks [24, 25, 27]. This cellular model will help us to understand the mechanisms behind AgNP-mediated neuronal toxicity and identify the molecular markers to assess mental health risks associated with products containing EMNs. This book chapter is a summary of our recent studies regarding AgNP mediated neurotoxicity.
2. The impact of AgNP on neurogenesis
Neurogenesis is a series of developmental events leading to the formation of new neurons and astrocyte support cells. Neurogenesis is not only the most active process during the pre-natal stage but also happens in certain regions of the brain, such as the subgranular layer of the hippocampal dentate gyrus throughout life in mammals. Studies found that adult brains are more plasticity than previously thought. The process of neurogenesis is tightly regulated and influenced by both intrinsic genetic factors and extrinsic environmental factors. The process involves transitions from proliferation to differentiation, accompanied sequentially by the expression of the transcriptional factors such as Pax6, Tbr2, NeuroD, and Tbr1 [28]. If these gene expressions are altered, the neurogenesis events will be disrupted, which can lead to neuropsychiatric diseases such as anxiety, learning and memory, and Alzheimer’s disease (AD) [29, 30].
Our study indicates that when citrate-coated AgNP (AgSC) were administered to the media during stem cell neuronal differentiation, neuronal progenitor rosettes were immunostained with neuronal progenitor markers: sex-determining region Y-box 2 (SOX2) and VI intermediate filament protein (Nestin). The results showed that AgSC exposure disrupted neuronal tube-like rosette formation and reduced neuronal progenitor population (Figure 1A). Quantification of SOX2 and Nestin relative fluorescence intensity showed that AgSC reduced SOX2 expression and increased Nestin expression in a concentration-dependent manner (Figure 1B). The alternation of the expression level of Sox2 and Nestin will change the neural progenitor fate. Furthermore, flow cytometric analysis for the population of neuronal progenitors with SOX2 and Nestin markers indicated that the percentage of SOX2+ and Nestin+ neuronal progenitors decreased from 54.3.3% to 20.9%, while SOX2− and Nestin− cells which would be unable to differentiate into neurons increased from 20.19% to 47.7% at 1.0 μg/mL AgSC exposure compared to untreated sample. In contrast, SOX2− and Nestin+ progenitors, which potentially could develop into astrocytes, increased from 23.3% to 26.1% with the same treatment (Figure 2A). The ratio of Nestin+/SOX2− and Nestin+ elevated to 1.45 at 1.0 μg/mL AgSC exposure, while the control group is 0.43. Those data support our hypothesis that AgSC inhibited neurogenesis and promoted gliagenesis. Lower concentrations of AgSC (0.1 μg/mL) slightly reduced SOX2 and Nestin expression, but the impact is insignificant. Supplements of AA partially reduced the effects (Figure 1C).
To further understand the molecular mechanisms of AgSC neuronal toxicity, a transcriptome analysis was performed., Total RNA was extracted from 3 replicates of 1.0 μg/ml AgSC exposure groups and untreated control groups to make libraries for sequencing. Significant differential expression (SDE) was cut off by padj <0.05 and |log2foldChange| > = 1. Among 322 SDE genes, 134 were up-regulated and 188 were down-regulated upon AgSC exposure (Figure 2A). The topmost up-regulated genes Metallothioneins 1F; Metallothioneins 1E; Metallothioneins 2A (45, 52, and 24 times), and frizzled class receptor 10 (FZD10) (Table 1). There are four main isoforms of cysteine-rich proteins Metallothioneins (MTs) which have the capacity to bind heavy metals such as zinc, copper, selenium, cadmium, mercury, silver, through the thiol group of its cysteine residues. MTs play important roles in metal homeostasis and protect against heavy metal toxicity, DNA damage, and oxidative stress. The other up-graduated gene is FZD10, a key regulator of the WNT signaling pathway. FZD10 plays acritical role in the neuronal pattern specification process, gliagenesis, and neurite outgrowth [31]. In addition, transcriptional factors NeuroD6, FOXG1, and NTS are among the top 20 significantly down-regulated genes (Table 2). Those genes play an important role in regulating neuronal differentiation, synaptogenesis, and axon extension during brain development [32]. The selected genes MT1E, NeuroD6, FOXG1, and NTS mRNA expression levels were examined with qPCR, respectively, and confirmed by RNA-seq data (Figure 2B).
Gene NAME | log2FoldChange |
---|---|
Metallothionein 1F (MT1F) | 5.733703827 |
Frizzled class receptor 10 (FZD10) | 5.63545845 |
Metallothionein 1E (MT1E) | 5.55542047 |
Vestigial like family member 3 (VGLL3) | 5.257650459 |
Pentraxin 3 (PTX3) | 5.249022406 |
Metallothionein 2A (MT2A) | 4.634538775 |
Cyclin O (CCNO) | 4.614228599 |
FZD10 antisense RNA 1 (head to head) (FZD10-AS1) | 3.906526795 |
Canopy FGF signaling regulator 1 (CNPY1) | 3.102452269 |
NAD(P)H quinone dehydrogenase 1 (NQO1) | 2.932494502 |
Sodium voltage-gated channel beta subunit 4 (SCN4B) | 2.863730164 |
Transcription factor AP-2 beta (TFAP2B) | 2.822828878 |
Zic family member 1 (ZIC1) | 2.792907219 |
Actin, alpha 2, smooth muscle, aorta (ACTA2) | 2.779703608 |
Actin, gamma 2, smooth muscle, enteric (ACTG2) | 2.726196918 |
Alpha-2-macroglobulin (A2M) | 2.584555887 |
Crumbs 2, cell polarity complex component (CRB2) | 2.561792956 |
Zic family member 4 (ZIC4) | 2.479236853 |
Neuronal pentraxin 2 (NPTX2) | 2.443739583 |
Protein tyrosine kinase 2 beta (PTK2B) | 2.421852257 |
Vasoactive intestinal peptide receptor 2 (VIPR2) | 2.404813991 |
Collagen type I alpha 2 chain (COL1A2) | 2.330235258 |
Zinc finger DHHC-type containing 22 (ZDHHC22) | 2.286038754 |
Iroquois homeobox 1 (IRX1) | 2.283963197 |
EF-hand and coiled-coil domain containing 1 (EFCC1) | 2.280818988 |
Gene name | log2FoldChange |
---|---|
CREBATF bZIPtranscription factor (CREBZF) | −3.554019527 |
LIM domain 7 (LMO7) | −3.064914689 |
SRSF protein kinase 2 (SRPK2) | −2.924926145 |
Myocyte enhancer factor 2C (MEF2C) | −2.81151306 |
Transmembrane protease, serine 13 (TMPRSS13) | −2.794313859 |
Neuritin1 (NRN1) | −2.770934839 |
Ring finger protein 175 (RNF175) | −2.724670035 |
G1 to S phase transition 2 (GSPT2) | −2.674801389 |
Methylsterol monooxygenase 1 (MSMO1) | −2.634382164 |
Coiled-coil domain containing 171 (CCDC171) | −2.583794397 |
Meis homeobox 2 (MEIS2) | −2.540498698 |
Gamma-aminobutyric acid type A receptor gamma2 subunit (GABRG2) | −2.503960528 |
Src-like-adaptor (SLA) | −2.478947195 |
calcium binding protein 1 (CABP1) | −2.341485603 |
semaphorin 3F (SEMA3F) | −2.336576641 |
fatty acid binding protein 6 (FABP6) | −2.333357057 |
B-cell CLLlymphoma 11A (BCL11A) | −2.268903493 |
neuronal differentiation 6 (NEUROD6) | −2.2525029 |
neurotensin (NTS) | −2.251646035 |
DLG associated protein 1 (DLGAP1) | −2.233930932 |
zinc finger CCCH-type containing 15 (ZC3H15) | −2.227494717 |
cerebellar degeneration related protein 1 (CDR1) | −2.217658748 |
neurotensinreceptor 1 (NTSR1) | −2.214928804 |
forkheadbox G1 (FOXG1) | −2.185129942 |
chromosome 12 open reading frame 65 (C12orf65) | −2.176721998 |
These significantly differentially expressed genes were analyzed by metascape (http://metascape.org) for functional annotation clustering. Based on gene ontology analysis, in response to AgSC exposure, the most significant impact on the biological processes were regulation of neuron differentiation, brain development, synapse organization, pattern specification processes, gliogenesis, and cholesterol biosynthetic processes (Figure 2B). The KEGG analysis results showed that the affected genes were enriched in C5 isoprenoid biosynthesis, axon guidance, neuron apoptotic progress lysosomes, MAPK, WNT, Hedgehog, and Notch signaling pathways (Figure 2D). In conclusion, our data suggest that AgSCs interfere with metal homeostasis and cholesterol biosynthesis which induces oxidative stress, reduces neurogenesis and axon guidance and promotes gliogenesis and apoptosis.
3. Impact of AgNPs on neurodegeneration
Neurodegeneration is the progressive loss of structure or function of neurons due to aging, diseases, and environmental factors. Free radicals or oxidative stress may damage lipids, nucleic acids, and proteins. The brain is particularly vulnerable to oxidative stress because of its high level of protein and lipid content and low level of antioxidants [33]. Reactive oxygen species (ROS) such as superoxide (O2−) and hydrogen peroxide (H2O2) are typically categorized as neurotoxic molecules associated with decreased synaptic plasticity performances in cognitive function and cell death. ROS can initiate excitotoxicity effects by inducing an intracellular calcium influx that leads to the activation of glutamate receptors and apoptosis [24]. To investigate the molecular mechanisms underlying AgNP-induced neurodegeneration, mature glutamatergic neuronal networks containing astrocytes were generated from iPSC. ROS production were examined with 20 nm citrate-coated AgNPs (AgSCs) and polyvinylpyrrolidone-coated AgNPs (AgSPs) exposure. Our results showed AgNPs-induced ROS production was coating and dose-dependent (Figure 3A). AgSCs-treated neurons produced more ROS compared to the AgSPs-treated samples.
We examined our hypothesis, stating that AgNPs-induced ROS will promote astrocyte activation and neuronal cell death. Astrocytes are the most numerous neuroglial cells in the central nervous system (CNS). Astrocyte vital functions include blood-brain barrier formation, providing structural and metabolic support, and regulating synaptic transmission and water transport [34, 35]. Astrocytes are sensitive to environmental changes. Under the chronic stress condition, astrocytes will undergo significant structural remodeling which reduces process length, branching, and density length [36]. Our results indicated that 0.1 μg/ml dose AgSC exposure increased the number of GFAP positive astrocytes for neuronal protection. At high doses, 5.0 μg/ml AgSC exposure altered astrocyte morphology and induced astrocyte activation. Furthermore, we examined how AgSCs affect synaptic structural and functional components. Neurons were double-stained for the presynaptic vesicle membrane protein Synaptophin (Syn) and the postsynaptic marker PSD-95 (Figure 3B). Untreated control neurons showed extensive neuritis processes co-localized between Syn and PSD-95 (Figure 3B). Exposure with AgSCs at 1.0 and 5.0 μg/mL drastically reduced Syn and PSD-95 expression and their co-localization.
We further investigated the signaling cascade involved in AgNP mediated neurodegeneration with different coatings. Glutamate receptor
4. Conclusion
In our study, neuronal progenitors, mature glutamatergic neurons, and astrocytes were derived from hPSC which were used for testing AgNPs toxicity. The results indicated that citrate-coated AgSCs significantly affected neuronal progenitor proliferation, gliagenesis, neuronal neuritis outgrowth, and cell viability due to up-graduated Metallothionein (1F, 1E, 2A) gene expression and increased ROS production. AgSPs had similar effects but only exhibited the toxicities at higher concentration exposure. In this context, the proper coating can prevent or limit the neurotoxicity associated with the AgNPs exposure. Our study indicates that stem cell-derived neuronal differentiation is an excellent cellular platform for investigating the impact of AgNPs on neuronal development and neurodegeneration and identifying biomarkers for risky assessment. In addition, this cellular model could also be used for different types of nanoparticles such as carbon-based nanoparticles, ceramic nanoparticles, metal nanoparticles, semiconductor nanoparticles, and lipid-based nanoparticles neuronal toxicity assessment in the future.
Acknowledgments
This work was supported by the National Institutes of Environmental Health Sciences (1R15 ES019298-01A1). I would like to thank Aynun Begum, Hao Li, and Neza Repar who had worked on these projects and provided the data for this book chapter.
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