
==== Front
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Environ Int
Environ Int
Environment international
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10.1016/j.envint.2024.108914
nihpa2018504
Article
Developmental neurotoxicity of PFOA exposure on hiPSC-derived cortical neurons
Wu Shichen a
Xie Junkai a
Zhao Han a
Zhao Xihui b
Sánchez Oscar F. a
Rochet Jean-Christophe cd
Freeman Jennifer L. e
Yuan Chongli ad*
a Davidson School of Chemical Engineering, Purdue University, West Lafayette, IN, 47907, USA
b Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, 47907, USA
c Department of Medicinal Chemistry and Molecular Pharmacy, Purdue University, West Lafayette, IN, 47907
d Purdue Institute of Integrated Neuroscience, Purdue University, West Lafayette, IN, 47907
e School of Health Sciences, Purdue University, West Lafayette, IN, 47907
* Corresponding author. cyuan@purdue.edu (C. Yuan).
24 8 2024
8 2024
26 7 2024
17 9 2024
190 108914108914
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
PFOA is a legacy Per- and Polyfluorinated Substances (PFAS), a group of chemicals widely used in various industrial applications and consumer products. Although there has been a voluntary phase out of PFOA since 2005, it is still widely detected in various water supplies. A growing body of evidence suggests an association between PFOA exposure, particularly during developmental stages, with increased risks of neurodegenerative diseases (NDs). The neurotoxic mechanism of developmental PFOA exposure, however, remains poorly understood. Utilizing human induced-pluripotent stem cell (hiPSC)-derived cortical neurons, we investigated the effect of PFOA exposure prior to differentiation and assessed changes in neuronal characteristics, transcriptome, and neurodegeneration markers mimicking a Developmental Origin of Health and Disease (DoHAD) paradigm. Exposure to PFOA before neuron differentiation resulted in persistent alterations in nuclear morphology, neuronal network, and calcium activity. RNA sequencing analysis further revealed transcriptomic changes aligning with Alzheimer’s Disease (AD) after PFOA exposure. These observations were further corroborated by alterations in tau phosphorylation markers, the presence of fibrillar tau, an increase in liquid droplets, and a decrease in RNA translational efficiency characterized using a battery of biochemical assays. Taken together, our results revealed persistent deficits of key neuronal characteristics induced by pre-differentiation PFOA exposure, suggesting impairments in several AD-related pathways that can together contribute to the elevation of AD risk after pre-differentiation PFOA exposure.

PFOA exposure
PFAS
hiPSC-derived cortical neurons
Alzheimer’s Disease
Neurotoxicity
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pmc1. Introduction

Per- and poly-fluoroalkyl substances (PFASs) are highly fluorinated aliphatic compounds that have multifaceted applications in different industrial sectors, including nonstick cookware, firefighting foams, and water-repellent clothing. PFAS has great environmental persistence due to the perfluoroalkyl moiety (Giesy and Kannan, 2001); and exceptional hydrophobic and lipophilic properties due to long perfluoroalkyl chains (de Voogt et al., 2012; Buck et al., 2011). Perfluorooctanoic acid (PFOA) is one of the legacy PFASs that can be found in high abundance in our drinking water, soil, and food supplies (Paustenbach et al., 2006), generating significant public health concerns affiliated with PFOA exposure.

National Health and Nutrition Examination Survey identified mean PFOA level in the serum of US population be ~ 1.42 parts per billion (ppb) during year 2017–2018 (CDC, 2015). PFOA has also been detected in drinking water with a concentration between 0.02 and 0.35 ppb (Hu et al., 2016; Crone et al., 2019; Kaboré et al., 2018). The primary route of human exposure to PFOA in the US is via consumption of contaminated drinking water (Garnick et al., 2021; ATSDR, 2017). Although many PFAS production companies have participated in voluntary phaseout efforts since the early 2000 s, the EPA reports that PFOA stocks may still be in use, and PFOA might still be present in imported articles (EPA, 2010). Furthermore, the long half-life of legacy PFAS has resulted in their slow elimination from the environment; and it is estimated that it will take more than 92 years to completely eliminate PFOA from water (Li et al., 2018). Human exposure to PFOA is thus likely to be a persistent issue affecting multiple generations, warranting detailed studies on the long-term health implications of legacy PFOA.

Exposure to PFOA has been associated with impairments in immune, hepatic, and endocrine systems (Steenland et al., 2010). Accumulating literature evidence, however, suggests that the central nervous system (CNS) can also be a sensitive target of PFOA. PFOA has been identified in different brain regions of human and wildlife (Di Nisio et al., 2022; Greaves et al., 2013; Pedersen et al., 2015). Specifically, a PFOA level as high as 32.28 ppb was detected in human cerebellum (Di Nisio et al., 2022). Findings from laboratory animal models further suggest that PFOA can enter the developing brain and lead to adverse neurological outcomes later in life. For example, developmental PFOA exposure in rodents has been associated with alterations in motor function (Goulding et al., 2017; Sobolewski et al., 2014) and cognitive performance (Wang et al., 2015). Zebrafish with embryonic exposure to 2000 ppb PFOA exhibited hyperactive locomotor function at 14 days post fertilization (Jantzen et al., 2016; Jantzen et al., 2016). A few population studies suggest an association between PFAS exposure and neurodegenerative diseases. For example, a mortality study in the Veneto Region in Italy suggested an association of higher mortality rates from Alzheimer’s Disease (AD)-related causes and PFAS-contaminated drinking water containing PFOA and perfluorooctane sulfonate (PFOS) (Mastrantonio et al., 2017), the two most abundant legacy PFASs. Another study found that individuals with clinical cognitive impairments had elevated concentrations of PFASs in their cerebrospinal fluid (CSF) (Delcourt et al., 2023). Collectively, these studies support a potential association between PFOA exposure and neurodegeneration. However, the causal relationship between PFOA exposure and Alzheimer’s disease risk remains to be established, and the neurotoxic mechanism of PFOA is poorly understood.

Furthermore, the developing brain is a particularly sensitive target to toxicant exposures (Costa et al., 2004). As suggested by the Developmental Origins of Health and Disease (DOHaD) hypothesis, environmental exposure during critical developmental stages can significantly influence both short- and long-term health (Barker, 2007; Heindel and Vandenberg, 2015). 0.01–0.05 ppb PFOA was detected in breast milk (Zheng et al., 2021), while 1.6 ppb PFOA was found in umbilical cord sera (Apelberg et al., 2007), suggesting that prenatal exposure to PFOA is highly common in the human population. Exposure to PFOA was shown to significantly alter the expression of proteins critical for brain development. For instance, in neonatal mouse pups, a single dose of PFOA (8695 ppb) administered on postnatal day (PND) 10 changed the expression of CaMKII, GAP-43, and synaptophysin in the hippocampus, as well as synaptophysin and tau in the cerebral cortex (Johansson et al., 2009). Childhood PFOA exposure was linked to a decrease in mental developmental indices among 6-month female infants (Goudarzi et al., 2016) as well as increased risk for attention deficit/hyperactivity disorder (ADHD) and reduced executive functioning (Oulhote et al., 2016), but there is inconsistency in the literature (Liew et al., 2015; Ode et al., 2014; Quaak et al., 2016; Liew et al., 2018) with several factors (e.g., doses, age and sex) potentially influencing these discrepancies. To the best of our knowledge, no current studies have evaluated neurodegenerative risks after pre-differentiation PFOA exposure following a DOHaD paradigm in a cell culture model. Studies on PFOS, another legacy PFAS structurally similar to PFOA, suggest that PFOS can accumulate in human brains after prenatal exposure (Cao and Ng, 2021). Zhang et al evaluated the effects of pre- and post-natal PFOS exposure in rats and found aberrant changes in AD pathological markers, including increased expression of tau, phosphorylated tau (ptau) (S199, T231 and S396), APP, and Aβ42 at PND 90 (Zhang et al., 2016). No similar assessments of AD-related biomarkers have been performed on developmental PFOA exposure in preclinical animal models to the best of our knowledge. Taken together, accumulating literature suggests that developmental PFOA exposure can alter brain functions at adolescence exhibiting a strong association with elevated risk of ADHD like PFOS (Kim et al., 2023). Given the structural similarity between PFOA and PFOS, we hypothesize that developmental PFOA exposure may lead to similar AD pathological changes and subsequently increased AD risk that we will investigate in this work.

Here, we adopted a neuronal culture model derived from human induced pluripotent stem cells (hiPSCs) to evaluate the effects of low-dose PFOA exposure prior to differentiation on neuronal integrity, and to assess AD-related pathological changes. hiPSC-derived cortical neurons have been widely adopted as a culture model to study neurodevelopmental (Ardhanareeswaran et al., 2017; Mariani et al., 2015; Marchetto et al., 2017) and neurodegenerative (Israel et al., 2012; Muratore et al., 2014; Li et al., 2018; Rivetti di Val Cervo et al., 2021) diseases; and neurotoxicity of various chemicals (Tukker et al., 2018) supporting the validity of applying this culture model in our study. We used 0.04 and 0.4 ppb PFOA before neuron differentiation in our study following a DOHaD exposure paradigm. Differentiated neurons were assayed for changes in critical neuronal characteristics including nuclear morphology, network complexity, neuron activity, as well as AD-related pathological markers. Our study thus offers insights into the molecular mechanisms underpinning PFOA-induced neurotoxicity.

2. Materials and methods

2.1. Differentiation and culturing of cortical neurons derived from hiPSC

Cortical neurons were differentiated from hiPSCs following our established protocol (Xie et al., 2023). Briefly, we started with hiPSC Kolf 2.1 J (JAX laboratory, JIPSC1000) in StemFlex medium (Thermo-Fisher, A3349401). A dual SMAD inhibition approach was used to form embryoid bodies (EBs), followed by replating, rosette selection and formation of neural progenitor cells (NPCs). NPCs were then cultured in forebrain neuron differentiation medium (Stemcell Technologies, 08600) for 8 days before switching to a cortical neuron maturation medium consisting of Brainphys (Stemcell Technologies, 05790), 1 × N2 (Gibco, 17502048), 1 × B27+ (Gibco, A3582801), 20 ng/ml BDNF (PeproTech, 450–02), 20 ng/ml GDNF (PeproTech, 450–10), 1 % Penicillin-Streptomycin (Gibco, 15140122), 1 × GlutaMAX (Gibco, 35050061), 1 × NEAA (Corning, 25–025-CI), 10 μM dibutyryl cAMP sodium salt (Sigma, D0627) and 200 μM Ascorbic Acid (Stemcell Technologies, 72132) as described in prior literature (Que et al., 2021). Neurons were maintained in the maturation medium until assessments. The identities of NPCs were verified by immunostaining for Nestin and PAX6 (for NPCs) as shown in Fig. S1A (Supporting Information), while the identities of the differentiated neurons were verified by NeuN (for postmitotic neurons) as shown in Fig. S1C (Supporting Information), as well as MAP2 (for mature neurons) and VGLUT1 (for glutamatergic neurons) in Fig. S1D (Supporting Information).

2.2. PFOA treatment

PFOA (Sigma Aldrich, Cas # 335–67-1) was dissolved in UltraPure water (Gibco, Cat # 10977015) at a concentration of 4 parts per million (ppm). The stock solution was further diluted with neural progenitor medium to a concentration of 0.04 or 0.4 ppb and then used for treating NPCs for 48 h prior to differentiation. After 48 h incubation, NPCs were washed 3 times with 1 × DPBS (Gibco, 14190144) to remove any residual PFOA before induction of differentiation. We chose 0.4 ppb for exposure based on PFOA serum levels suggesting ~ 1.4 ppb in the general US population, with serum levels up to 227.6 ppb in exposed communities (ATSDR, 2024), and PFOA level as high as 32.28 ppb detected in human cerebellum (Di Nisio et al., 2022). We also included a lower concentration of 0.04 ppb because PFOA levels of 0.01–0.05 ppb have been detected in breast milk (Zheng et al., 2021), underscoring the relevance of human developmental exposure.

2.3. MTT assay

MTT assays were conducted using an MTT assay kit (Abcam, 211091) following the manufacturer’s protocol. Briefly, NPCs exposed to PFOA for 48 h were rinsed with 1 × DPBS three times and treated with a solution mixture with 50 % serum-free medium and 50 % MTT reagents. After a 3 h incubation, MTT solvent was added into each well followed by incubation at RT for 15 min. We then recorded the absorbance at 580 nm using a SpectraMax microplate reader (Molecular Devices).

2.4. Immunocytochemistry and fluorescence microscopy

Cells were fixed and immunostained following our established protocols (Xie et al., 2023; Wu et al., 2023), and then analyzed using fluorescence microscopy. All fluorescent and differential interference contrast (DIC) images were acquired using an ImageXpress Confocal system (Molecular Device). Objectives used for image acquisition included Nikon Plan Apo 20 × /0.75NA air and 60 × /1.2NA water objective. For confocal images, a z step-size of 1 μm was utilized. For FRET imaging, we used a Nikon Plan Apo 20 ×/0.75NA objective where the sample was excited at 395/25 nm and emission was collected using a 536/40 nm emission filter. All antibodies were validated prior to usage. Primary antibodies used here include anti-Synapsin1 (SySy, 106011), anti-Homer1 (SySy, 160003), anti-MAP2 (SySy, 188004), anti-Phospho-tau (Thr181) (AT270) (Invitrogen, MN1050), anti-Phospho-tau (Ser202, Thr205) (AT8) (Invitrogen, MN1020), anti-puromycin, clone 12D10 (Sigma, MABE343). Secondary antibodies used were anti-mouse Alexa-488 (Invitrogen, A11001), anti-rabbit Alexa-488 (Invitrogen, A11008), anti-mouse Alexa-568 (Invitrogen, A11004), anti-rabbit Alexa-568 (Abcam, ab175471), anti-guineapig Alexa-594 (Invitrogen, A11076) and anti-guinea-pig Alexa647 (Invitrogen, A21450). We also used DAPI (Sigma, D9542) to stain cell nuclei.

2.5. Microelectrode array (MEA)

A Maestro Pro MEA system (Axion) was used to record neuronal activities. Differentiating neurons were plated into CytoView MEA 48 well plates (Axion, m768-tMEA-48 W) coated with 0.1 % polyethyleneimine (PEI) at a density of 1 × 105 cell / well and maintained in the maturation medium until assessments. Before recording, the plate with neurons was equilibrated in the Maestro Pro chamber for 20 min at 37 °C with 5 % CO2. We used the spontaneous recording setting in AxIS Navigator software (Axion Biosystems) to record neuronal activity for 10 min. The AxIS Neural Metric tool (Axion Biosystems) was used for the identification and analysis of firing frequency, burst frequency and number of spikes per burst. An adaptive threshold of 6 times the standard deviation of the estimated noise on each electrode was used for spike detection. A burst was identified as a group of > 5 spikes with an interspike interval (ISI) of < 100 ms.

2.6. Calcium recording

AAV1 packaged hsyn-jGCaMP7s (Addgene, 104487-AAV1) was added into differentiated cortical neurons with and without PFOA treatment and incubated for 3 days prior to a complete medium exchange. Neurons expressing jGCaMP7s were imaged using the ImageXpress Micro Confocal system after transduction. Time-lapse imaging was carried out utilizing a Nikon Plan Apo 20×/0.75NA objective within an environment-controlled chamber. Images were captured at a sampling rate of 4 Hz for a duration of 60 s. The fluorescence intensity (F) of a selected region of interest (ROI) was quantified using the intensity measurement tool in ImageJ (NIH). The corresponding baseline fluorescence (F0) value was determined by measuring the intensity of a background region with the same area as the selected ROI and subtracted from F to determine ΔF. The dynamics of calcium flux were established as ΔF/F0. The “Find Peaks” function in OriginPro (2023) was employed to identify the number of peaks and their respective amplitudes.

2.7. FRET based tau-reporter cell line

An HEK293T tau biosensor cell line (provided by Dr. Marc Diamond, University of Texas Southwestern) (Holmes et al., 2014; Hitt et al., 2021) was applied to detect and quantify tau fibrils in the culture medium. This established cell line has stable integration of DNA carrying tau repeat-domain (RD) variants bearing the P301S mutation and fused with either mClover3 or mCerulean3. These variants aggregate upon exposure to internalized tau preformed fibrils (PFFs), leading to the generation of a strong Förster resonance energy transfer (FRET) signal, which exhibits a positive correlation with the concentration of tau fibrils. Reporter cells were seeded into a 96-well TC plate at a confluency of 40 % 24 h prior to the addition of collected culture medium. Lipofectamine 3000 (Invitrogen, L3000015) was used to stimulate the uptake of tau PFFs as described previously (Holmes et al., 2014; Hitt et al., 2021). The transfection mixture for each well of 96-well plate (~1.5 × 104 cells) contained 40 μl of harvested neuronal medium, 1.25 μl of Lipofectamine 3000, and 10 μl of Opti-MEM (Gibco, 31985070). Cells were incubated with the transfection mixture for 24 h prior to FRET imaging.

2.8. ELISA assay for Aβ quantification

ELISA assay kits for Aβ40 (Invitrogen, KHB3481) and Aβ42 (Invitrogen, KHB3441) were used to quantify the concentration of two isoforms of Aβ secreted by neurons. The assays were performed following the manufacturer’s protocol with 25 μl of neuronal culture medium used for each well. All absorbance measurements were performed using a SpectraMax iD3 plate reader (Molecular Device).

2.9. Lipid droplet analysis

Lipid droplets were visualized using a LipidSpot 488 fluorescent dye (Biotium, 70065-T) following the manufacturer’s protocol. Briefly, cortical neurons were incubated in cortical neuron maturation medium supplemented with LipidSpot 488 (diluted to 1x from a 1000x stock solution) for 30 min and imaged with the ImageXpress Confocal system.

2.10. Puromycin incorporation assay

Puromycin (Sigma, P8833) was dissolved in UltraPure water at a concentration of 25 mg/mL and further diluted in neuron maturation medium to 1 μg/mL. Cortical neurons were incubated in the diluted puromycin solution for 10 min, fixed with 4 % formaldehyde, and analyzed immunocytochemically.

2.11. RNA-sequencing

Differentiated neurons were prepared as described in our previous work (Xie et al., 2023). RNA samples with RNA integrity number (RIN) scores exceeding 6.0 (characterized by an Agilent Bioanalyzer 2100) were used for library preparation followed by sequencing using HiSeq (Illumina) at Novogene Inc. with an average sequencing depth of 6G raw data. The reference genome employed was Homo sapiens (GRCh38/hg38). EdgeR was used to determine differentially expressed genes (DEGs) using an adjusted q value of 0.05. Gene Ontology (GO) was utilized to conduct pathway analysis. Ingenuity Pathway Analysis (IPA) was used to identify AD- and tauopathy-related genes for the generation of DEG heatmaps. Four independent biological samples from each group were sequenced and analyzed.

2.12. Data analysis and statistics

Neurite analysis was performed using the Neurite Outgrowth Module embedded in MetaXpress software (Molecular Device) with DIC images. The chromatin condensation parameter (CCP) was calculated following a published protocol (Irianto et al., 2014) utilizing a customized MetaXpress software module. To identify synapses and analyze synaptic density, SynQuant (Wang et al., 2019), a Fiji (NIH) plugin, was applied. Quantification results were reported as mean ± standard error (SE). OriginPro 2021 was used to perform statistical analyses and prepare quantitative illustrations. Statistical differences were determined using one-way ANOVA followed by Tukey’s post-hoc test with p-value < 0.05 considered statistically significant. AxIS Neural Metric tool (Axion Biosystems) was used to prepare raster plots of MEA recordings. BioRender was used to prepare illustrative figures.

3. Results

3.1. Pre-differentiation exposure of PFOA induced morphological changes

We exposed NPCs to 0.04 and 0.4 ppb PFOA for 48 h before differentiation, then differentiated the NPCs in neuron differentiation medium for 8 days before switching to maturation medium. The cells were cultured for an additional 37 (Day 45) to 52 (Day 60) days before assessments were conducted as illustrated in Fig. 1A. No significant alteration in NPC viability was observed after 48 h exposure as determined by an MTT assay (Fig. S1B) (Supporting Information). We further differentiated NPCs into cortical neurons and assessed their nuclear morphology (Fig. 1B–C) as well as neuronal network properties (Fig. 1D–E) upon the completion of differentiation. We stained nuclei of cortical neurons by DAPI and assessed their CCP (which quantifies the percentage of condensation state of chromatin), nuclear area as well as roundness. The shape factor was used as an indicator of nuclear roundness and was calculated as shapefactor = 4πA/P2, where P and A refers to the perimeter and area of nuclei. We only observed a notable increase in nuclear area and a decrease in CCP (but no change in nuclear roundness) after 0.4 ppb PFOA prior exposure (Fig. 1C top and bottom), whereas 0.04 ppb pre-differentiation exposure to PFOA had no effect on nuclear morphology. To assess neuronal network complexity, we further stained neurons for MAP2, a microtubule-binding protein enriched in dendrites, and characterized the total neurite outgrowth as well as the number of processes and branches of neurons with and without prior PFOA exposure as summarized in Fig. 1E. Processes refer to neurite outgrowths connected to cell bodies, branches denote junctions where processes divide into more outgrowths, and total outgrowth represents the cumulative length of skeletonized outgrowths in the neuronal culture. A significant decrease in all three parameters was noted after exposure to either 0.04 or 0.4 ppb PFOA, among which the number of processes had the largest fold decrease after PFOA exposure (~35 % for 0.04 ppb and ~ 40 % for 0.4 ppb). Significantly larger decreases in neurite outgrowth, process numbers, and branch numbers were observed after exposure to 0.4 ppb PFOA compared to 0.04 ppb.

3.2. PFOA exposure prior to differentiation altered neuronal activity

One crucial feature of neurons is their ability to generate electrical signals, facilitating the transmission of signals enabled by synapses that also partake in memory formation and consolidation. Synapses, composed of presynaptic terminals and postsynaptic boutons, function as junctions where the transmission and processing of neuronal signals take place (Südhof, 2021; Kandel et al., 2000). The dysfunction of synapses is commonly observed in AD patients (Tzioras et al., 2023). To quantify synaptic characteristics, we stained neurons for Synapsin1 and Homer1 (Fig. 2A), markers of pre- and post-synapse, respectively, and analyzed for pre-synaptic, post-synaptic and synaptic density via SynQuant (ImageJ) (Wang et al., 2019), with the results summarized in Fig. 2B. Significant decreases in pre-synaptic and synaptic density were observed in 0.4 ppb PFOA-treated neurons, while interestingly, an increase in post-synaptic density was also noted. 0.04 ppb PFOA-treated neurons only had an increase in post-synaptic density but showed no significant changes in either pre-synaptic density or synaptic density.

We further applied MEA (Axion), a high-throughput platform for the evaluation of extracellular neuronal activities, to measure the electrical activity of neuronal networks with and without PFOA exposure. Fig. 2C shows typical raster plots of differentiated neurons with 0 (top), 0.04 (middle) and 0.4 (bottom) ppb PFOA exposure prior to differentiation, respectively. Spike and burst events were identified using Neural Metric Tool (Axion), the frequencies of which are summarized in Fig. 2D. We found a decrease in both mean firing rate and burst frequency after PFOA treatment, although the decreasing trend is only statistically significant in mean firing rate of 0.4 ppb PFOA treated neurons (Fig. 2D top).

Calcium (Ca2+) is an important secondary messenger in neurons, the activity of which is closely associated with neuron firing and synapse activities (Brini et al., 2014). jGCaMP7s is a genetically encoded probe that can fluoresce upon binding to Ca2+, enabling measurement of Ca2+ activity based on imaging techniques (Zhang et al., 2023). We utilized AAV packaging to introduce a plasmid encoding jGCaMP7s driven by the neuron-selective hSyn promotor into cortical neurons and recorded Ca2+ activity at 4 Hz. Fig. S2 (Supporting Information) shows a typical image of cortical neurons transduced with AAV-packaged jGCaMP7s, and Fig. 2E shows typical traces of Ca2+ transients in neurons with and without prior PFOA exposure. The frequency and amplitude representing Ca2+ flux dynamics are summarized in Fig. 2F. After pre-differentiation exposure to 0.04 and 0.4 ppb PFOA, a significant decrease in Ca2+ frequency was observed only in 0.4 ppb PFOA treated cells, while there was a significant decrease in the amplitude of both 0.04 and 0.4 ppb treated cells. The results along with the decreased extracellular activity and altered synaptic density after PFOA exposure indicate impairment in neuronal activity after prior PFOA treatment, particularly in 0.4 ppb treated cells.

3.3. Transcriptomic changes associated with tauopathy and AD after PFOA exposure

To further investigate the potential disease-related alterations induced by prior PFOA exposure, we proceeded to RNA-sequencing (RNA-seq) to evaluate post-exposure transcriptomic changes. Volcano plots exhibiting deferentially expressed genes (DEGs) with prior PFOA exposure of 0.04 and 0.4 ppb are shown in Fig. S3A and Fig. S3B (Supporting Information), respectively. RNA-seq identified a large set of DEGs in both 0.04 and 0.4 ppb PFOA-treated neurons, with 1162 shared DEGs as shown in the Venn diagram in Fig. 3A. We further performed gene enrichment analysis using the DEGs shared between 0.04 and 0.4 ppb treatments via Gene Ontology (GO) and summarized the top enriched pathways in biological processes (BPs) (Fig. 3B), cellular compartments (CCs) (Fig. 3C) and molecular functions (MFs) (Fig. 3D). Of note, positive regulation of tau-protein kinase activity was identified in BPs, synapse-associated extracellular matrix was identified in enriched CCs, and phosphatidate phosphatase activity was identified in enriched MFs, which are closely associated with tauopathy, synaptic function and phospholipid metaboblism, respectively. In BPs, enriched pathways associated with cell–cell adhesion include positive regulation of integrin-mediated signaling pathway, regulation of basement membrane organization, and cell–cell adhesion mediated by integrin; cellular response to progesterone stimulus was noted as well. CCs responsible for cell adhesion including protein complex involved in cell-matrix adhesion, laminin complex, alphav-beta3 integrin-HMGB1 complex and collagen type IX trimer were also enriched. Nuclear retinoic acid receptor binding enriched in MFs can regulate inflammatory responses via suppressing cytokines and chemokines (Shudo et al., 2009). Apolipo-protein binding enriched in MF plays a critical role in Aβ delivery. Low-density lipoprotein receptors (LDLR) serve as targets for cholesterol-carrying lipoprotein particles, the binding of which may be related to Aβ and tau uptake (Sagare et al., 2012; Rauch et al., 2020). Insulin-like growth factor I binding in MF also has a suggested correlation with AD risk (Williams et al., 2018).

We used IPA to further explore the association between PFOA exposure and AD (Fig. 3E). Similar analysis was carried out between PFOA exposure and tauopathy as shown in Fig. 3F. Transcriptional changes in AD-related genes were observed. For example, we observed a decrease in APOE and an increase in APP transcription, both of which are strongly associated with both AD- and tauopathy- related pathways. Furthermore, we noted decreases in GRIA2 expression (Fig. 3E), a subunit of the glutamate ionotropic receptor AMPA, which is important in modulating synaptic plasticity; and increases in GSK3B (Fig. 3F), a kinase that catalyzes tau phosphorylation. Hierarchical clustering further verified PFOA-dose dependent changes in the transcriptional level of AD- and tauopathy genes. Collectively, our results suggest that PFOA exposure before differentiation can contribute to AD pathology potentially via transcriptional alterations leading to tauopathy in cortical neurons.

3.4. Pre-differentiation exposure to PFOA induced tauopathy but not Aβ pathology in cortical neurons

The tau protein plays a pivotal role in the structural integrity of the neuronal (axonal) cytoskeleton through its microtubule-stabilizing function. pTau (T181), a tau variant phosphorylated on threonine residue 181, is frequently observed in early-stage AD patients (Braak I-IV) (Shen et al., 2021), whereas pTau (Ser202, Thr205), a tau variant phosphorylated at serine 202 and threonine 205, is associated with late stage Ads (Barthélemy et al., 2020).

To assess the potential effect of prior PFOA exposure towards tauopathy, we stained cortical neurons for pTau (Thr181) with antibody AT270 (Fig. 4A) and pTau (Ser202, Thr205) with antibody AT8 (Fig. 4D), respectively, with typical images of neurites shown in Fig. 4B and Fig. 4E. We quantified the intensity of pTau (Thr181) (Fig. 4C) and pTau (Ser202, Thr205) (Fig. 4F) in neurites and found a significant increase in pTau (Thr181) in neurites of cells exposed to 0.4 ppb PFOA, while pTau (Ser202, Thr205) levels significantly decreased after exposure to 0.04 and 0.4 ppb PFOA. We further assessed the accumulation of pTau (Ser202, Thr205) in cell soma (Fig. 4G) and found a significant increase in soma pTau (Ser202, Thr205) levels in 0.04 ppb PFOA treated neurons. We did not observe pTau (Thr181) accumulation in cell soma.

In addition to intracellular pTau assessment, we also performed extracellular tau fibril quantification utilizing an HEK293T biosensor cell line that generates a FRET signal upon exposure to tau fibrillar seeds47,51,52. We collected culture medium from exposed neurons and acquired FRET signals via the reporter line with results summarized in Fig. 4H. Representative images of tau biosensor cells treated with medium collected from PFOA-exposed cells are shown in Fig. S4 (Supporting Information). A significant increase in FRET signal was noted in 0.4 ppb PFOA-treated cells, indicating higher extracellular levels of tau fibrils. Taken together, our observations suggest that PFOA exposure before differentiation stage can induce tauopathy (Xie et al., 2023; Holmes et al., 2014; Hitt et al., 2021).

Abnormal Aβ aggregation is another important pathological marker of AD. We therefore assessed the expression of Aβ40, Aβ42 and their ratio using culture medium (Fig. S5) (Supporting Information) and found no significant difference between PFOA-treated and untreated cells.

3.5. Pre-differentiation exposure to PFOA altered lipid homeostasis

PFAS are highly hydrophobic and thus can preferentially partition into lipids, which are key components of membranes of multiple organelles in cells. Lipid-related abnormalities were among the first pathological observations made by Alois Alzheimer, underscoring their critical role in AD (Alzheimer et al., 1995). Lipid droplets (LDs) are organelles that store intracellular neutral lipids. LDs have been identified alongside AD pathologies such as Aβ accumulation in the brains of AD patients (Gómez-Ramos and Asuncion Moran, 2007). LDs accumulate in AD postmodern brain as well as in the brain of 3xTg-AD mice (Hamilton et al., 2015). We therefore characterized changes in LDs after prior PFOA exposure in neurons utilizing a live cell-compatible dye (LipidSpot 488, see Fig. 5A), and quantified the number and size distribution of LDs per cell as in Fig. 5B and 5C, respectively. We found that cells with prior exposure to 0.04 ppb PFOA had increased numbers of LDs per cell. PFOA exposure also resulted in significant shifts in LD size distributions suggesting the formation of smaller LDs.

3.6. Pre-differentiation exposure of PFOA affects RNA translation efficiency in cortical neurons

RNA regulation plays a key role in neuronal activity. The dysregulation of RNA translation has been correlated with AD (Ghosh et al., 2020). We therefore assessed RNA translation efficiency of neurons by incorporating puromycin into nascent polypeptides of live cells followed by visualization with anti-puromycin antibody using an established protocol (López-Erauskin et al., 2018). Typical cells exposed to PFOA and stained with DAPI or with puromycin and MAP2 antibodies are shown in Fig. 5D, with zoomed-in views of neurites shown in Fig. 5E. We quantified puromycin intensity per cell (Fig. 5F top) and neurites (Fig. 5F bottom). A significant decrease in total puromycin intensity was observed in neurons treated with PFOA (0.04 and 0.4 ppb), while no significant differences were observed in neurites. This finding suggests possible impairment of RNA translation efficiency after PFOA exposure.

4. Discussion

In this work, we adopted hiPSC-derived cortical neurons to study the effects of PFOA exposure before differentiation based on its human relevance and the feasibility of applying low-dose exposure at a progenitor stage, an intermediate stage of hiPSC-to-neuron differentiation representing progenitor cells in the developing brain of the fetus (Yin et al., 2013), to mimic the exposure window during CNS development. Differentiated cortical neurons consist primarily of glutamatergic neurons and are thus suitable for studying PFOA effects on AD risks. We chose exposure doses of 0.04 and 0.4 ppb based on PFOA concentrations detected in US drinking water (0.02–0.35 ppb) (Hu et al., 2016) and breast milk (0.01–0.05 ppb) (Zheng et al., 2021), and also the PFOA serum level ~ 1.4 ppb in the general US population (ATSDR, 2024).

We observed significant morphological alterations in neurons, such as increases in nuclear area and decreases in chromatin condensation after exposure to 0.4 ppb PFOA. Assessment of neuronal network complexity showed decreases in neurite outgrowth, process number and branch number in cortical neurons after prior PFOA exposure, indicating dystrophic neurons as a signature marker of neurodegeneration. These observations are consistent with prior studies using SH-SY5Y cells after pre-differentiation exposure to PFOA (Zhao et al., 2022). Nuclear size enlargements of neurons have been documented as an adaptive response associated with the aging process in humans (Navarro and Gonzalo, 1991). An increase in neuronal nuclear size in AD patients has also been reported (Ishunina et al., 2019). De-condensation of heterochromatin has been observed in AD animal models and postmortem brain tissue of AD patients (Frost et al., 2014).

Neurons with pre-differentiation exposure of PFOA also exhibited altered activities, including changes in synaptic densities and firing patterns. Despite an increase in post-synaptic density after PFOA exposure, we observed decreases in mean firing rate and a trend towards a decrease in burst frequency after prior PFOA exposure, suggesting potential disruptions in synaptic communication and network activities. The changes observed here are consistent with prior work using hiPSC-derived glutamatergic/astrocyte co-cultures after an acute exposure to high dose PFOA (414 - 41400 ppb) (Tukker et al., 2020). Also, alterations in synaptic densities and neuronal firing frequency are commonly observed in the brain of AD patients (Scheff et al., 2015; Scheff et al., 1990; Scheff and Price, 2006) and rodent AD models (Sanchez et al., 2012; Jacobsen et al., 2006).

RNA-seq revealed significant alterations in the transcriptome after pre-differentiation exposure to PFOA, among which we noted dose-dependent changes in AD pathways. Pathway enrichment analysis underscores alterations in tauopathy, a phenomenon strongly implicated in the pathology of AD. Our analysis revealed increases in pTau (Thr181) expression in neurites and pTau (Ser202, Thr205) in the soma. A tau reporter line was used to further demonstrate elevation in extracellular fibrillar tau after PFOA exposure. These findings align with tau hyperphosphorylation and aggregation in AD. Particularly, pTau (Thr181) is an early-stage AD marker associated with cognitive decline (Pereira et al., 2021) and the progression towards dementia onset (Palmqvist et al., 2021; Karikari et al., 2021). AT8 specifically probes pTau at serine 202 and threonine 205 sites that are prone to form PHF-tau, which is commonly observed in the brains of AD patients (Braak and Del Tredici, 2013). We observed significant decreases of AT8 levels in neurites after prior PFOA exposure to both 0.04 and 0.4 ppb, which can possibly be explained by PHF-tau transport into cell soma (supported by the observed increase in pTau (Ser202, Thr205) soma levels after 0.04 ppb PFOA exposure) and tau fibril release into the extracellular space (supported by the observed increase in levels of fibrillar tau in culture medium of 0.4 ppb PFOA-treated cells). hiPSC-derived neurons from AD patients also exhibited elevated pTau (Thr181) and pTau (Ser202, Thr205) levels similar to our observation (Ochalek et al., 2017). Interestingly, we did not observe significant changes in extracellular concentrations of Aβ40, Aβ42 and the Aβ42/Aβ40 ratio. Our findings thus suggest that PFOA exposure may lead to an altered neuronal phenotype consistent with AD primarily via tauopathy.

LDs originate from ER, where they synthesis neutral lipids and bud to the cytosol as they grow via incorporation of more neutral lipids (Olzmann and Carvalho, 2019). LD accumulation can be induced by cellular stress, such as ER stress (Fei et al., 2009), hypoxia (de la Rosa Rodriguez et al., 2021) and mitochondrial dysfunction (Lee et al., 2013). The size of LD reflect their storage capacity and is thus associated with lipolysis; it is therefore widely used as an indicator of LD functionality (Yang et al., (2012). In neurons, cellular stress can induce the formation of LDs (Ioannou et al., 2019), which can cause neuronal damage when they accumulate (Zhao et al., 2023). PFOA-exposed neurons showed increased numbers of LD but with decreased sizes along with transcriptional alterations in APOE4, a major component in lipoprotein transport. These observations are consistent with PFOA-induced LD accumulation in prior work using mouse hepatic cells (Wang et al., (2013) and in C. elegans (Lin et al., 2022). Strong associations between PFAS and increased blood lipid levels have been reported in several epidemiological studies (Ference et al., 2017; Leritz et al., 2016; Piepoli et al., 2016). Of note, elevated levels of LDs have also been observed in AD patients (Gómez-Ramos and Asuncion Moran, 2007).

We noticed a decrease in global RNA translation efficiency in neurons with PFOA exposure before differentiation, suggesting impaired RNA translation and protein synthesis, which can potentially lead to weakened responses to neurotransmission and subsequently altered synaptic plasticity (Oliveira and Klann, 2021). RNA translation dysregulation has also been implicated in AD patients. For example, Langstrom et al. identified a decrease in mRNA translational activity in AD patient brain (Langstrom et al., 1989).

To date, studies on the neurotoxicity of PFOA mainly focus on immediate effects of PFOA exposure, and many of them mainly utilize MTT assay as an indicator for neurotoxicity in cell culture models. Due to PFOA’s bioaccumulative ability in the human CNS and its long half-life, it is particularly important to determine whether exposure to PFOA before differentiation would lead to significant effects at a later stage. Our work is thus innovative in focusing on the latent effect of PFOA exposure towards cortical neurons following a DOHaD paradigm and assessing its possible impact as a potential neurotoxin, specifically on features of neurodegenerative diseases.

5. Conclusion

PFOA exposure before differentiation elicited neuron-specific characteristic changes, including alterations in nuclear morphology, neuronal network complexity and activity, as well as changes in network activities. RNA sequencing further revealed transcriptomic changes in genes closely associated with AD which were further verified using ICC. In addition to pathological features consistent with tauopathy, we also noted alterations in lipid droplet accumulation and RNA translation efficiency. Taken together, our results revealed persistent deficits in neurons induced by low-dose PFOA exposure before differentiation consistent with an AD phenotype, suggesting potential neurotoxicity of PFOA exposure before differentiation via tauopathy.

Supplementary Material

1

Acknowledgement

This work was supported by grants from Purdue Institute of Integrative Neuroscience (PIIN), National Science Foundation (NSF, EF-1935226) and National Institute of Health (R01ES035429).

Data availability

Data will be made available on request.

Fig. 1. (A) A schematic illustration of PFOA exposure mimicking developmental exposure during a progenitor stage. (B) Representative images of DAPI-stained cortical neurons previously treated with 0, 0.04 and 0.4 ppb PFOA. Scale bar = 5 μm. (C) Relative changes in nuclear features of cortical neurons, including nuclear area (top) and nuclear roundness (middle) and chromatin condensation parameter (CCP) (bottom). n > 3000 cells from N=3 independent differentiations. (D) Typical images of 0, 0.04 and 0.4 ppb PFOA treated cortical neurons stained with DAPI (blue) and MAP2 antibodies (red). Scale bar = 50 μm. Neurite characteristics including processes (yellow) and branches (white) are indicated by arrows. (E) Relative changes of neurite morphology parameters including total neurite outgrowth (top), process number (middle) and branches (bottom). n ≥ 59 views of Day 45 neurons from N=6 independent differentiations. Data = Mean ± S.E. N.S.: not significant. *: p < 0.05. ***: p < 0.001.

Fig. 2. (A) Representative images of neurites stained for the pre-synaptic marker (Synapsin1), the post-synaptic marker (Homer1), and the microtubule-binding protein MAP2 after prior exposure of the neurons to 0, 0.04 and 0.4 ppb PFOA. Scale bar = 10 μm. (B) Quantification of relative changes in pre-synaptic, post-synaptic and synapse density after PFOA exposure. (C) Typical raster plots of differentiated neurons with prior exposure to 0, 0.04 and 0.4 ppb PFOA. Each row corresponds to the spikes detected by a single electrode over a duration of 300 s. Each tick on the row signifies a spontaneous event, and clusters of blue ticks indicate instances of bursting activity. (D) Mean firing rate and burst frequency of neurons with 0, 0.04 and 0.4 ppb PFOA prior exposure. N≥6 independent differentiations of Day 45 neurons. (E) Typical traces of calcium (Ca2 + ) activity from neurons expressing GCaMP7s. (F) Quantification of the frequency and amplitude of synaptic Ca2 + activity. n ≥ 10 traces from N=3 independent differentiations of Day 45 neurons. Data = Mean ± S.E. N.S.: not significant. *: p < 0.05. **: p < 0.01.

Fig. 3. (A) A Venn diagram illustrating shared DEGs between 0.04 and 0.4 ppb PFOA exposed neurons. (B-D) Top biological processes (BPs) (B), cellular compartments (CCs) (C), and molecular functions (MFs) (D) altered by PFOA exposure identified with the shared gene list via Gene Ontology (GO) analysis. (E-F) Heatmaps of genes enriched in AD pathways (E) and tauopathy pathways (F) affected by PFOA exposure identified via IPA. Standardized gene expression levels were presented using calculated average z-scores. Hierarchical cluster analysis was performed on AD-and tauopathy- related DEGs in 0, 0.04 and 0.4 ppb PFOA-treated groups as shown in the dendrogram. N=4 independent differentiations of Day 45 neurons.

Fig. 4. (A) Typical images of neurons stained with DAPI and AT270 antibodies. Scale bar = 50 μm. (B) Typical images of neurites stained with AT270 and MAP2 antibodies. Scale bar = 10 μm. (C) Relative changes in pTau (Thr181) expression in neurites of cells with prior exposure to 0, 0.04 and 0.4 ppb PFOA indicated by AT270 intensity. n ≥ 68 neurites from N=3 independent differentiations. (D) Typical images of neurons stained with DAPI and AT8 antibodies. Scale bar = 50 μm. (E) Typical images of neurites stained with AT8 and MAP2 antibodies. Scale bar = 20 μm. (F) Relative changes in pTau (Ser202, Thr205) expression in neurites of cells exposed to 0, 0.04 and 0.4 ppb PFOA previously indicated by AT8 intensity change. n ≥ 92 neurites from N=3 independent differentiations. (G) Relative changes in AT8 expression in soma of neurons with prior PFOA exposure. n ≥ 93 cells from N=3 independent differentiations of Day 45 neurons. (H) Relative changes in FRET intensity of tau biosensors treated with culture medium collected from 0, 0.04 and 0.4 ppb PFOA-treated neurons. n ≥ 73 views of images from N=4 independent differentiations of Day 45 neurons. Data = Mean ± S.E. *: p < 0.05. ***: p < 0.001. N.S.: not significant.

Fig. 5. (A) Typical images of neurons exposed to 0, 0.04 and 0.4 ppb PFOA before differentiation and stained with LipidSpot 488, a live cell-based lipid droplet (LD) dye. Scale bar = 10 μm. (B) Quantification of LD number/cell in neurons with prior exposure to 0, 0.04 and 0.4 ppb PFOA. n ≥ 35 views from N=3 independent differentiations. (C) Quantification of LD size distribution in neurons previously exposed to 0, 0.04 and 0.4 ppb PFOA. n > 7000 LDs from N=3 independent differentiations of Day 60 neurons. Statistical test of size distribution variation was performed using the Mann-Whitney test. (D) Typical images of neurons treated with puromycin for 10 min before fixation and staining with DAPI, puromycin and MAP2. Scale bar = 50 μm. (D) Typical images of neurons treated with puromycin for 10 min before fixation and staining with DAPI or antibodies specific for puromycin and MAP2. Scale bar = 50 μm. (E) Typical images of neurites stained with puromycin and MAP2 antibodies. Scale bar = 20 μm. (F) Relative changes in puromycin intensity of cells (top) and neurites (bottom) with prior exposure to 0, 0.04 and 0.4 ppb PFOA. n ≥ 28 views from N=3 independent differentiations of Day 60 neurons. Data = Mean ± S.E. ***: p < 0.001. N.S.: not significant.

CRediT authorship contribution statement

Shichen Wu: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Formal analysis, Data curation, Conceptualization. Junkai Xie: Writing – review & editing, Visualization, Methodology, Formal analysis, Conceptualization. Han Zhao: Visualization, Methodology, Formal analysis. Xihui Zhao: Writing – review & editing, Visualization, Formal analysis. Oscar F. Sánchez: Writing – original draft. Jean-Christophe Rochet: Writing – review & editing, Resources, Methodology. Jennifer L. Freeman: Writing – review & editing, Supervision, Resources, Conceptualization. Chongli Yuan: Writing – review & editing, Writing – original draft, Supervision, Methodology, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.envint.2024.108914.
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