**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 [1–3]. Among them, silver nanoparticles (AgNPs) are one of the most widely used in medical and commercial products for their unique antibacterial functions [4–10]. 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 [15–17]. Recently, researchers have begun to explore the potential neurotoxicity of ENMs such as AgNPs in cellular and animal models [18–21]. 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<sup>+</sup> alone, depending on size and coating [24–26].

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

*Impact of Silver Nanoparticles on Neurodevelopment and Neurodegeneration DOI: http://dx.doi.org/10.5772/intechopen.101723*

**Figure 1.**

*AgSC inhibited neurogenesis and promoted gliogenesis. A. AgSC inhibited neuronal rosette formation. Scale bar = 100* μ*m. B. Quantification of SOX2 and nestin relative intensities (fold of control), ratio of intensity between SOX2 and nestin from immunofluorescent staining images. C. BDAccuri C6 flow cytometer analysis the neuronal progenitor population. D. Ratio of nestin+ /SOX2− and nestin+ from flow cytometry result. Data is presented as mean ± SEM, \*p < 0.05, or \*\*p < 0.01 vs. control.*

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

#### **Figure 2.**

*AgSC significantly altered gene expression A. Total DEGenes of 1.0* μ*g/mL AgSCs treated group compared with control group. The significant genes (P* ≤ *0.05) were labeled with red color. B. Quantitative real-time PCR to examine selected genes. FOXG1, NeuroD6 and NTS were significantly down-regulated. MT1E was significantly up-regulated. Data is presented as mean ± SEM, \*p < 0.05, or \*\*p < 0.01 vs. control. C. The clustered by GO biological processes. Result was shown as –log10(P) value. D. KEGG pathway and colored with –log10 (P) value. Min overlap* ≥*3, p-value* ≤*0.01 and min enrichment* ≥*1.5 were used for significant enrichments [25].*

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

#### *Impact of Silver Nanoparticles on Neurodevelopment and Neurodegeneration DOI: http://dx.doi.org/10.5772/intechopen.101723*

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**).

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


#### **Table 1.**

*AgSC mediated up-graduated differential expressed genes.*


#### **Table 2.**

*AgSC-mediated down-graduated differential expressed genes.*

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

#### *Impact of Silver Nanoparticles on Neurodevelopment and Neurodegeneration DOI: http://dx.doi.org/10.5772/intechopen.101723*

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 AgSPstreated 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

#### **Figure 3.**

*AgNP promoted ROS production, induced astrocyte activation and synapse protein loss. A. ROS was generated in a dose-dependent manner in (A–C) AgSP-treated neurons. (E–G) AgSC-treated neurons produced a higher amount of ROS compared to (D) the untreated neurons (ctrl). (H) the inset image of hGNs treated with 5 mg/ml AgSC showed the interneuronal accumulation of ROS. Scale bar 100 mM. B. Immunofluorescent staining images showed AgSC promoted astrocyte activation. C. Effect of AgSC on the excitatory synaptic protein, vGlu1 and PSD95 expression. The co-localization of vGlut1(red) and PSD95 (green) in the controls. Exposure to AgSC (5 mg/ml) significantly diminished the vGlut1and PSD95 expression and co-localization [24, 27].*

#### **Figure 4.**

*The molecular mechanisms of the AgNP induced neurotoxicity. A. Immunoblotting of glutamate receptors NR2A/B, phosphorylated GSK-3*α*/*β *and Tau46 after espousing the AgNPs at three different concentrations for 72 h.* β*-Actin was used as a loading control. B. Immunostaining with Tau46/Map2 indicated that the effect of AgNPs on microtubule assembly proteins expression and axon outgrowth. C. Potential molecular mechanisms underlying AgNP induced neurotoxicity [24].*

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 *N-*methyl-D-aspartate receptor (NMDAR) plays a key role in synaptic plasticity, which is linked to a form of long-term depression (LTD) as well as neuron survival. The dysregulation of NMDAR in neurons will trigger an apoptosis-associated increase in caspase-3 activit*y.* The immunoblotting results showed that AgSCs reduced the expression levels of the post-glutamate receptor subunits NR2A and NR2B and increased the phosphorylation of GSK3α/βTyr216/279, whereas AgSPs had similar effects, but only at a higher concentration (5 μg/ml) (**Figure 4A**). GSK3 α/β phosphorylation has been shown to be associated with neural apoptosis in many neurodegenerative disorders. An increase in GSK-3β activity via GSK3α/βTyr216/279 phosphorylation can lead to Tau phosphorylation (pTau) [37, 38]. Our immunoblotting results confirmed that the AgNPs can increase GSK3 α/β phosphorylation and increase Tau phosphorylation at serine 396 in a dose-dependent manner, whereas AgSPs had no effect on Tau phosphorylation (**Figure 4A**). Tau is involved in the loss of neuronal dendrites and the axonal network by disrupting microtubule assembly. The result of Tau46/MAP2 double immunostaining showed that AgSC treatment caused the reduction of both protein expression and axon outgrowth (**Figure 4B**). **Figure 4D** presents a model of molecular mechanisms for AgNPs induced neurodegeneration. We suggested that phosphorylation of GSK3a/bTyr216/279could be the potential biomarker for AgNPs neurotoxicity testing.
