Effects of Short-Term Exposure to Polystyrene Nanoplastics on the Nervous System: Calcium Homeostasis, BDNF and Synaptic Plasticity

TL;DR

This study explores how short-term exposure to polystyrene nanoplastics affects the nervous system, particularly through changes in calcium signaling and brain-derived neurotrophic factor.

Contribution

The study reveals a novel mechanism by which nanoplastics may impair synaptic plasticity via calcium signaling and BDNF modulation.

Findings

PS-NPs interfere with calcium ion signaling and reduce CaMKIIα activity.

PS-NPs downregulate CREB expression and modulate BDNF levels, impacting synaptic plasticity.

Abstract

(1) Background: The increasing environmental concentration of polystyrene nanoplastics (PS-NPs) may pose a risk of human exposure and health threats. Previous studies have demonstrated that exposure to PS-NPs poses a threat to neural synaptic plasticity, yet the underlying mechanisms remain unclear. (2) Methods: Hippocampal astrocytes and neurons were co-cultured, exposed to PS-NPs at concentrations of 10, 50, and 100 μg/mL, and cytotoxicity was assessed. We investigated PS-NPs-induced impairment of synaptic plasticity by regulating the brain-derived neurotrophic factor (BDNF). (3) Results: Calmodulin-dependent protein kinase II (CaMKII) is a central molecular organizer of synaptic plasticity, learning, and memory, and its activity is intrinsically linked to intracellular calcium ion concentration. Our research indicates that PS-NPs may interfere with calcium ion signaling and CaMKIIα activity, thereby reducing CaMKIIα activity. This subsequently downregulates the expression of cAMP response element-binding protein (CREB), modulates BDNF expression, and impacts synaptic plasticity. (4) Conclusions: In summary, this study primarily focused on the effects of PS-NPs exposure on hippocampal synaptic plasticity.

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Full text

1. Introduction

Micro- and nanoplastics are widely recognized as novel pollutants that contribute to the ecological sustainability crisis and are found in various environmental media [1,2]. Microplastics (<5 mm) and nanoplastics (<1 μm) are produced by the degradation of plastics in the environment and are highly mobile in the biosphere, with increasing concentrations in human organs such as the blood, liver, kidneys, and brain [3,4]. Research indicates that microplastics and nanoplastics ingested or inhaled via the respiratory tract can induce systemic inflammatory responses and oxidative stress [5], leading to damage across multiple organ systems [6].

Nanoplastics can cross the blood–brain barrier and cause altered synaptic function in the nervous system and mitochondrial dysfunction in the nerve cells. Existing research suggests that nanoplastic pollutants entering the brain can exacerbate the aggregation of alpha-synuclein, with potential links to the development of Parkinson’s disease and other neurodegenerative disorders [7,8]. Neurodevelopmental alterations in offspring after prenatal exposure to PS-NPs include increased hippocampal postsynaptic density and widened synaptic clefts [9]. Liang et al. found that exposure to PS-NPs caused disturbances in energy metabolism and impaired mitochondrial quality control in brain cells, accompanied by astrocyte and microglial dysfunction and altered synaptic function [10].

Ca^2+^ maintains normal nervous system function by regulating neurotransmitter release, participating in synaptic plasticity, and influencing neuronal excitability and development. Research indicates that intracellular Ca^2+^ influx can promote BDNF expression by activating the CaMKIIα/CREB pathway, thereby enhancing synaptic responsiveness [11]. CaMKII is one of the most abundant postsynaptic density (PSD) proteins and is responsible for decoding synaptic calcium signaling [12]. The Ca^2+^ concentration regulates CaMKIIα shuttling between PSD subcompartments, and the activation of CaMKIIα further promotes PSD assembly [13]. CaMKIIα triggers the phosphorylation of the cAMP response element-binding protein (CREB), thereby influencing BDNF transcription. A positive feedback loop exists among CaMKIIα, CREB, and BDNF [14,15]. Therefore, the steady-state regulation of calcium ions is crucial for maintaining nervous system health.

BDNF plays a crucial role in neuronal survival and synaptic plasticity in the nervous system [16]. BDNF and its precursor, proBDNF, exert completely opposite effects in the nervous system. ProBDNF is a key factor in neurodegenerative diseases, whereas BDNF exerts positive effects [17,18]. An imbalance in the proBDNF:BDNF ratio may also lead to neurological abnormalities. Research by Martina Albini et al. revealed that astrocytes can internalize and recycle proBDNF and BDNF at synapses, thereby contributing to synaptic plasticity [19]. Therefore, we hypothesized that altered BDNF expression in astrocytes and neurons may influence synaptic morphology and function.

In summary, we exploited the regulatory effects of Ca^2+^ on BDNF to explore the effects of PS-NPs on BDNF expression under the co-exposure of astrocytes and neuronal cells, and that PS-NPs exposure affects the homeostasis and stabilization of the synaptic plasticity of astrocytes to neurons. This provides a theoretical basis for our study on the effects of PS-NPs exposure on synaptic plasticity.

2. Materials and Methods

2.1. Characterization of PS-NPs

The 50 nm PS-NPs suspension (PS000050, density = 1.05 g/mL) used in this study was purchased from Beijing Zhongke Leiming Biotechnology Co., Ltd. (Beijing, China). The research team conducted a comprehensive preliminary characterization of PS-NPs, including particle size distribution, hydrodynamic diameter, distribution state, and surface potential. Prior to the experiment, the PS-NPs suspension underwent 30 min of ultrasonic treatment using an ultrasonic cleaner (As One Shanghai Corporation, Shanghai, China). The experimental results confirmed that the purity, particle size, and distribution state of the nanomaterials met the experimental requirements, ensuring the scientific validity of subsequent intervention studies. Detailed material information is provided in Reference [9].

2.2. Cell Co-Culture Exposure Model

H19-7 rat hippocampal neuronal cells (Zhongkedisheng, Tianjin, China, PX240101) at a density of 5 × 10^5^ cells (placed in the upper layer) and CTX TNA2 Cell rat astrocytes (Zhongkedisheng, Tianjin, China, PX240102) at 1 × 10^5^ cells (lower layer) in complete cell culture medium (90% DMEM + 10% FBS) in 24-well Transwell nested chambers. The cells were incubated at 37 °C and 5% CO_2_, and the medium was changed the following day. The cells were passaged at a 1:2 ratio into two nested chambers. The cells used in the experiment were at the 3rd to 5th passage (in a stable proliferation phase). The design incorporated three independent experimental batches that effectively controlled systematic errors arising from batch effects. The number of cells cultured was determined based on published cell culture ratios and preliminary experimental data [20,21].

Previous studies have reported the effects of PS-NPs on environmental and neural cell exposures [20,22,23].

2.3. Western Blot

Rat hippocampal neuronal cells and astrocytes were collected and lysed using RIPA lysis buffer containing protease inhibitors (Solarbio, Beijing, China, R0020) to extract the total protein. Protein concentrations were quantified using a BCA Protein Concentration Assay Kit (Beyotime, P0011; Shanghai, China). Proteins were then separated using an SDS-PAGE gel preparation kit (Solarbio, P1200) for hierarchical separation, followed by membrane transfer. The membranes were blocked for 1 h at room temperature with 5% skim milk (Beyotime, Shanghai, China, P0216-1500 g) and incubated overnight at 4 °C with specific primary antibodies, including Cav3.1 (1:2000, NBP2-59322, Novus, Lone Tree, CO, USA), CaMKIIα (1:2000, 3357S, CST, Danvers, MA, USA), CREB (1:2000, 12208-1-AP, Proteintech, Rosemont, IL, USA), BDNF (1:1000, ab226843, abcam, Cambridge, UK), PSD95 (1:1000, 2507S, CST), SYN (1:2000, 17785-1-AP, Proteintech), ProBDNF (1:1000, 28205-1-AP, Proteintech), and GAPDH (1:2000, UM4002, YouKang, Tianjin, China). The membrane was then incubated with secondary antibodies, sheep anti-rabbit IgG-HRP (1:4000, S0001, Affinity Biosciences, Jiangsu, China) and sheep anti-mouse IgG-HRP (1:4000, S0002, Affinity) for 1 h at room temperature. Finally, the membrane was immersed in ECL solution (Beyotime, P0018M) for 1 min and exposed to a fully automated chemiluminescence imaging analysis system (Tanon, Shanghai, China). The bands were analyzed for gray values using the gray value software Gelpro32 (Tanon, Shanghai, China). GAPDH was used as an internal reference.

2.4. Cytotoxicity Assays

Next, 100 µL of cell suspension was added to a 96-well plate (seeding 1 × 10^5^ neuronal cells in the lower chamber and 2 × 10^4^ astrocytes in the upper chamber per well). The mixture was incubated at 37 °C in a 5% CO_2_ incubator. After treatment, neuronal cell viability was determined using a Cell Counting Kit (K009-1000T, Menlo Park, CA, USA) according to the manufacturer’s instructions. Next, 10 µL of CCK-8 solution was added to each well. The cells were incubated at 37 °C in a 5% CO_2_ incubator for 2 h, and the absorbance was measured at 450 nm using a microplate reader.

2.5. Measurement of Calcium

The total calcium content in the cell samples was determined using the cresolphthalein copper complex colorimetric method with a calcium assay kit (PB1181W96, Perseebio, Shanghai, China) according to the manufacturer’s instructions. Cells (5 × 10 ^6^) were sonicated (water bath, 200 W, 3 s sonication, 10 s interval, repeated 30 times), centrifuged at 12,000 rpm for 10 min, and the supernatant was collected for further analysis. Blank, standard, and test tubes (three replicate wells each) were prepared, and 2.5 μL of the solution was added to each tube (interval of 10 s, repeated 30 times) and centrifuged at 12,000 rpm for 10 min. The supernatant was collected for further analysis. Blank, standard, and test tubes (three replicate wells each) were prepared for the experiment. Next, 2.5 μL of the corresponding reagent (distilled water, 2.5 mmol/L calcium standard, and sample supernatant) was added to each tube, followed by the addition of 200 μL of the working solution. The mixture was incubated at room temperature for 2 min. After preheating the microplate reader, the wavelength was set to 575 nm. The instrument was zeroed using distilled water, and the absorbance of each well was measured using a microplate reader. The calcium content was calculated using the following formula: For cell samples, the mass and protein concentrations were corrected as necessary.

Cells were seeded into a 96-well plate using the Fluo-4 calcium detection kit (S1060; Beyotime). The lower and upper chambers were seeded with 5 × 10^5^ neuronal cells and 1 × 10^5^ astrocytes. After trypsin digestion, the cells were resuspended and washed once with PBS. Each sample contained 10^6^ cells. Subsequently, 1 mL of pre-prepared Fluo-4 staining solution was added to the cell pellet and incubated at 37 °C in the dark for 30 min. After incubation, the cells were centrifuged at 250–1000× g at room temperature for 5 min to pellet. The supernatant was removed, and the cells were resuspended in 0.5 mL detection buffer per sample and analyzed by flow cytometry (NoVoCyte 2060R, Agilent Technologies, Santa Clara, CA, USA) using Fluo-4 AM as the green fluorescent marker (Ex/Em = 490/525 nm).

2.6. ELISA

Conditioned neuronal cell culture medium was collected and analyzed for NMDAR receptors (Mouse Anti-N-methyl-D-aspartate Receptor ELISA Kit, CB11301-Ra, Shanghai Keaibo Biological Technology Co., Ltd., Shanghai, China) and lactate dehydrogenase (LDH) (Lactate Dehydrogenase ELISA Kit, G0804W, Grace Biotechnology, Suzhou, China) were analyzed according to the respective kits’ instructions. After incubation, horseradish peroxidase (HRP)-conjugated detection antibodies were added to form a sandwich immunoassay. The substrate solution was then added, and HRP catalyzed the substrate to produce a color change in the solution. A stop solution was then added to terminate the enzymatic reaction, and the absorbance was measured at 450 nm within 15 min.

2.7. Cell Reactive Oxygen Species (ROS) Fluorescence Detection Kit

Neuronal cells (5 × 10^5^) were inoculated into the lower chamber and astrocytes (1 × 10^5^) were inoculated into the upper chamber of a 6-well plate. After the intervention, ROS levels were measured using a ROS Fluorescence Assay Kit (GY0169W48, Grace Biotechnology, China). Neuronal cells were collected by centrifugation at 1000 rpm for 5 min, washed twice, and the cell pellet was retained for further analysis. The fluorescence microplate reader (MB-530, Shenzhen Huisong Technology Development Co.,Ltd., Shenzhen, China) was preheated for 30 min, the excitation wavelength was set to 488 nm, and the emission wavelength was set to 525 nm. Reagent 1 was thawed to room temperature, and a mixture of reagents 1 and 2 was prepared in a 1:100 ratio. Light exposure was avoided during the experiment. Next, 1 mL of the mixture was added to the cell pellet, centrifuged at 1000× g for 5 min, the supernatant was discarded, and 1 mL of Reagent 2 was added. The mixture was incubated at 37 °C for 30 min (mixed thoroughly every 3–5 min). A blank tube was simultaneously prepared under the same conditions. Then, 200 μL of the sample and blank solutions were transferred to a black 96-well plate. Fluorescence values were measured, and the fluorescence intensity was calculated.

2.8. Statistical Analysis

We used an independent-samples t-test to assess the statistical differences between the two datasets. A one-way analysis of variance was conducted to evaluate distinctions among multiple groups, followed by Tukey’s multiple comparison test for intergroup disparities arising from the different treatments. The analysis was performed using IBM SPSS Statistics 25 (IBM, Armonk, NY, USA) and GraphPad Prism 9.0 (GraphPad Software, Inc., Boston, MA, USA). The results are expressed as mean ± SD. Statistical significance was defined as * p < 0.05 or ** p < 0.01, *** p < 0.001, **** p < 0.0001.

3. Results

3.1. Toxicity Study of PS-NPs Exposure on Hippocampal Neurons

In the present study, we simulated possible forms of exposure in real brain environments by exposing co-cultured hippocampal astrocytes and hippocampal neuronal cells to PS-NPs and examined neuronal activity. The 10, 50, and 100 μg/mL concentration intervention groups showed a concentration-dependent decrease in hippocampal neuronal activity compared to that of the control group (Figure 1A, p < 0.0001), with neuronal activity decreasing to approximately 40% in the 50 μg/mL concentration group. This suggests that exposure to PS-NPs may cause hippocampal neuronal damage in mice.

3.2. Effects of PS-NPs Exposure on Synaptic Plasticity in Hippocampal Neurons

Western blot analysis revealed significant alterations in the levels of synapse-associated proteins in hippocampal neurons exposed to PS-NPs. Specifically, compared to the control group, neurons treated with PS-NPs exhibited a marked decrease in postsynaptic density protein 95 (PSD95) expression (Figure 1C, p < 0.001). The expression of synapsin (SYN), a presynaptic marker for synaptogenesis, was also significantly decreased (Figure 1D, p < 0.01). This suggests that PS-NPs exposure causes synaptic damage and impairs synaptic vesicle formation and, consequently, exocytosis.

3.3. PS-NPs Exposure Mediates the Effects of BDNF on Neuroprotection

In a co-culture system of neurons and astrocytes, we measured the protein levels of proBDNF and BDNF in astrocytes. We found that ProBDNF expression was significantly increased (Figure 2A, p < 0.001), whereas the BDNF levels significantly decreased (Figure 2B, p < 0.01). ProBDNF promotes neuronal apoptosis and long-term depression, whereas BDNF promotes neuronal survival and synaptic plasticity [24]. Following PS-NPs intervention, BDNF expression decreased and ProBDNF expression increased in astrocytes, indicating disruption of the ProBDNF/BDNF balance.

3.4. Role of BDNF in the Mechanistic Pathway of Synaptic Plasticity Damage

Our experimental results (Figure 2) showed that hippocampal astrocytes exposed to PS-NPs exhibited an imbalance in the ratio of ProBDNF to mBDNF. As BDNF is transported from astrocytes to neurons to exert its effects and influence CREB expression, we examined the protein expression levels of CREB, BDNF, and other related proteins in the hippocampal neurons. Compared to the control group, the protein expression levels of CREB and BDNF were significantly reduced in the experimental group (Figure 3B, p < 0.001; Figure 3C, p < 0.01).

Colorimetric assays for Ca^2+^ detection revealed a significant decrease in neuronal Ca^2+^ content (Figure 3D, p < 0.0001). Moreover, the levels of Cav3.1, a calcium channel protein regulating intracellular Ca^2+^ concentration, and CaMKIIα were significantly reduced (Figure 3E, p < 0.0001; Figure 3F, p < 0.01). The reduced Cav3.1 expression further decreased the total intracellular Ca^2+^ content. Subsequently, detection of free cytoplasmic calcium using the Fluo-4 calcium detection kit revealed that PS-NPs intervention enhanced intracellular calcium influx in a time-dependent manner (Figure 4, p < 0.0001), while calcium overload may inhibit CaMKIIα activity. This suggests that PS-NPs may influence synaptic transmission and plasticity by altering the activity and content of Cav3.1, Ca^2+^, and CaMKIIα in neurons.

3.5. BDNF Ameliorates PS-NPs-Induced Neuronal Synapse Damage

Next, we performed an upregulating intervention on BDNF in the PS-NPs exposure group by exogenously adding recombinant BDNF protein (RPA011Ra01) (PS-NPs + BDNF group). Compared with the PS-NPs intervention group, the PS-NPs + BDNF group exhibited upregulation of endogenous BDNF expression in neurons (Figure 5F, p < 0.0001), and significantly enhanced neuronal activity (Figure 5A, p < 0.0001). Compared with the control group, both the reactive oxygen species (ROS) levels and lactate dehydrogenase (LDH) release were significantly elevated in the PS-NPs intervention group. The PS-NPs + BDNF group effectively alleviated oxidative stress-induced damage and membrane structural damage in hippocampal neurons induced by PS-NPs (Figure 5B,C; p < 0.001), with reductions of approximately 50% compared to the untreated group.

Analysis of synaptic functional markers revealed that the PS-NPs + BDNF group exhibited enhanced expression levels of the postsynaptic density protein PSD95 and presynaptic protein SYN compared to the PS-NPs intervention group. Using the Fluo-4 calcium detection kit to measure intracellular free calcium, it was observed that the PS-NPs + BDNF group exhibited reduced intracellular free calcium levels compared to the PS-NPs intervention group (Figure 4, p < 0.0001). NMDAR receptor detection results revealed that the PS-NPs + BDNF group effectively alleviated the PS-NPs-induced elevation of neuronal NMDAR receptor expression (Figure 5D, p < 0.0001). Moreover, our experiments indicated that compared to the PS-NPs intervention group, the expression level of CaMKIIα, a key kinase for synaptic plasticity, significantly increased (Figure 5E, p < 0.01), suggesting the effective restoration of synapse-associated proteins. These findings suggest that exogenous BDNF supplementation reverses PS-NPs-induced dysfunction in the CaMKIIα/CREB/BDNF signaling pathway, reactivating its regulatory role in neuronal survival and in synaptic plasticity.

4. Discussion

PS-NPs can penetrate the blood–brain barrier and enter the brain parenchyma, and short-term exposure can potentially cause neuronal damage. Short-term exposure to PS-NPs in early adulthood leads to synaptic loss and neuronal degeneration in middle-aged mice [25]. Building upon existing research elucidating the cellular and molecular mechanisms of nanoplastics’ neurotoxicity [6], our study co-cultured hippocampal astrocytes and neurons for joint exposure to PS-NPs. The PS-NPs exposure concentration was based on environmentally accessible concentrations and referenced previous experimental data [23,26], providing a basis for assessing the risks associated with long-term exposure to low concentrations of PS-NPs. In the nervous system, astrocytes regulate neuronal activity and provide nutritional support to neurons [19]. Co-culture systems can simulate the brain environment and clarify the roles of different cell types in neuronal injury. Existing research indicates that astrocytes have a superior capacity to internalize and accumulate PS-NPs compared to neurons. However, when the intracellular accumulation of PS-NPs exceeds a critical threshold, astrocytes may lose their inherent neuroprotective function [27]. The inflammatory transformation of astrocytes following PS-NPs uptake may further exacerbate the harmful microenvironment surrounding neurons [21], offering new insights into the selective toxicity of nanoplastics in complex brain tissue.

Our study found that PS-NPs decreased BDNF protein expression in astrocytes. BDNF plays an important role in the regulation of synaptic plasticity. For example, BDNF enhances synaptic transmission efficacy by binding to the specific TrkB receptor [28]. Second, BDNF affects axonal and dendritic function and morphology by [29] enhancing synaptic maturation and stability [30,31,32]. Astrocytes can uptake and secrete proBDNF and BDNF, and the balance between the two is critical for the role of BDNF in the regulation of synaptic plasticity [33]. Our data suggest that a significant bias in the ProBDNF/BDNF expression profile (upregulation of ProBDNF expression and downregulation of BDNF expression) occurs in astrocytes, disrupting their neuromodulatory homeostasis, and suggesting that this imbalance may be an important causative factor for aberrant synaptic plasticity in neurons.

We detected PSD95, a specific marker for synapses in hippocampal neurons, and SYN, a synaptic vesicle-associated protein that regulates neurotransmitter release. Together, they participate in maintaining normal synaptic function and plasticity. The results showed that the expression of both genes was reduced. These changes indicate that PS-NPs exposure may disrupt the stability and balance of synaptic function, reduce the efficiency of synaptic transmission, and impair learning and memory. Related studies have reported that the NMDAR not only functions as a highly permeable calcium channel but also plays a crucial role in activating CaMKIIα in the hippocampus [34,35]. Consistent with this, our study found that PS-NPs exposure led to an abnormal elevation of NMDAR receptors, accompanied by sustained increases in free Ca^2+^ in the cytoplasm. This resulted in intracellular calcium overload, which inhibited CaMKIIα activity. Exogenous BDNF activation enhances the CaMKIIα pathway and alleviates calcium overload caused by NMDAR receptor dysfunction, partially repairing synaptic damage induced by PS-NPs exposure. This suggests that abnormal BDNF expression disrupts the synaptic homeostasis.

The CaMKIIα/CREB/BDNF pathway plays a crucial role in the formation and stabilization of neuronal synapses. Our findings indicate that both CREB and BDNF expression are reduced in hippocampal neurons. CREB serves as a crucial link between CaMKIIα and BDNF. When phosphorylated by CaMKIIα, CREB can bind to the CRE site within the BDNF promoter region, thereby promoting BDNF gene transcription and protein synthesis [14]. BDNF influences synaptic plasticity in neurons by regulating synaptic homeostasis and function, thereby providing a material basis for learning and memory [36,37]. Total intracellular calcium ions decrease in hippocampal neurons, whereas free cytoplasmic calcium increases in a time-dependent manner, enhancing calcium influx into cells [38]. This calcium overload inhibits CaMKIIα activation, which is consistent with our experimental findings. CaMKIIα is one of the most abundant postsynaptic density proteins in the hippocampus and significantly influences synaptic plasticity and learning/memory [15]. Ca^2+^ can bind to calmodulin to form the Ca^2+^/CaM complex, which binds to and activates CaMKIIα and is expressed in excitatory or inhibitory neurons [39]. Cav3.1 participates in neuronal excitability, synaptic plasticity, and calcium influx [40]. Our detection revealed that PS-NPs exposure led to reduced expression of the calcium channel protein Cav3.1 in hippocampal neurons. This indicates that PS-NPs exposure may induce intracellular calcium overload, leading to reduced CaMKIIα activity and downregulation of CREB transcription, which suppresses BDNF expression and consequently disrupts synaptic plasticity.

5. Conclusions

This study investigated the neurotoxic effects of PS-NPs using an astrocyte-neuron co-culture model that mimics the brain’s microenvironment. The results suggest that PS-NPs may disrupt the CaMKIIα/CREB/BDNF signaling pathway, thereby inhibiting endogenous BDNF synthesis, impairing synaptic plasticity, and causing neuronal damage. Exogenous BDNF supplementation experiments further validated this critical role. In summary, PS-NPs may cause neuroplasticity damage by disrupting the calcium signaling–BDNF pathway axis, with the interruption of BDNF signaling serving as a key downstream target of their neurotoxicity. This study provides an experimental reference for deepening our understanding of the neurotoxicity of nanoplastics and potential intervention strategies.

6. Limitations

We acknowledge the limitations of this study. First, the findings have not yet been validated through animal experiments. Second, the applicability of these conclusions is restricted to the current experimental systems. The extent of damage to neural synapses following human exposure to PS-NPs requires further support from additional human cell-based research.