Developmental neurotoxicity of PFOA exposure on hiPSC-derived cortical neurons

Abstract

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.

Keywords: Alzheimer’s Disease; Neurotoxicity; PFAS; PFOA exposure; hiPSC-derived cortical neurons.

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.