Comparative Proteomics Highlights that GenX Exposure Leads to Metabolic Defects and Inflammation in Astrocytes

Abstract

Exposure to PFAS such as GenX (HFPO dimer acid) has become increasingly common due to the replacement of older generation PFAS in manufacturing processes. While neurodegenerative and developmental effects of legacy PFAS exposure have been studied in depth, there is a limited understanding specific to the effects of GenX exposure. To investigate the effects of GenX exposure, we exposed Drosophila melanogaster to GenX and assessed the motor behavior and performed quantitative proteomics of fly brains to identify molecular changes in the brain. Additionally, metabolic network-based analysis using the iDrosophila1 model unveiled a potential link between GenX exposure and neurodegeneration. Since legacy PFAS exposure has been linked to Parkinson's disease (PD), we compared the proteome data sets between GenX-exposed flies and a fly model of PD expressing human α-synuclein. Considering the proteomic data- and network-based analyses that revealed GenX may be regulating GABA-associated pathways and the immune system, we next explored the effects of GenX on astrocytes, as astrocytes in the brain can regulate GABA. An array of assays demonstrated GenX exposure may lead to mitochondrial dysfunction and neuroinflammatory response in astrocytes, possibly linking non-cell autonomous neurodegeneration to the motor deficits associated with GenX exposure.

Keywords: Drosophila; HFPO-DA; astrocytes; behavior; nontargeted proteomics.

Figure 1

GenX exposure leads to an age-dependent reduction in motor behavior in flies. (A) Dose-dependent decreases in climbing activity in female flies exposed to 500 and 1000 ppm of GenX over 20 days. (B) Dose-dependent decreases in climbing activity in male flies exposed to 500 and 1000 ppm of GenX over 20 days. (C) Locomotor activity assay for female flies exposed to GenX at two different doses, 1000 and 500 ppm. (D) Locomotor activity assay for male flies exposed to GenX at two different doses, 1000 and 500 ppm. (E) Percentage of female flies exposed to different doses of GenX that are experiencing seizure like phenotypes over a 20-day period. (F) Percentage of male flies exposed to different doses of GenX that are experiencing seizure-like phenotypes over a 20 day period.

Figure 2

Quantitative proteomics of GenX-exposed fly brains identifies changes in molecular signatures in fly brains. (A) Volcano plot showing that protein abundance is significantly altered after 7 days of GenX exposure in female flies. (B) Heat map showing top 50 proteins that are upregulated or downregulated. (C) Enriched terms (DAVID) analysis of proteins that were affected by GenX exposure. (D) IPA showing pathways that are associated with toxic effects of the proteins. (E) IPA showing pathways commonly found altered with exposure to toxicants, that are also altered with GenX exposure. (F) IPA showing upstream regulators of the proteins that are altered post GenX exposure.

Figure 3

General flowchart of the metabolic network analyses. First approach is based on in silico identification of GenX-induced fly phenotypes with PANGEA tool and regulated reactions with ΔFBA using the differentially expressed proteins. Second approach is iMAT-based reaction activity analysis applied for extending the list of ΔFBA-predicted differential reactions. The genes and metabolites in the combined list of the differential reactions are characterized in terms of enriched terms.

Figure 4

Metabolic network-based analyses of the differential metabolism induced by GenX. (A) The analysis of differentially abundant proteins (P-value <0.05) revealed the modulated fly phenotypes (motor and nonmotor properties). (B) Using network-based analyses, the genes and metabolites in differential reactions were determined for the GenX treatment. In this process, gene and metabolite lists were first created using two independent methods (ΔFBA and iMAT-based reaction activity analysis). Then, the lists derived from either method were combined to obtain the final regulated gene and metabolite lists, as in the simplified illustration. The final lists including a total of 155 genes and 343 metabolites were characterized in the next analyses. (C) Enrichment analysis of the GenX-induced regulated genes determined significantly enriched biological processes and metabolic pathways (FDR < 0.05). The enriched prominent terms are displayed by the bar plot and the bar sizes are proportional to the number of genes in each term. (D) GABA-related regulated pathways and the associated genes derived from the differential proteomic analysis and network-based analyses. The genes derived from each method are labeled by red and blue colors. (E) Enrichment analysis of the regulated metabolites in response to the GenX treatment was also applied to identify the over-represented metabolic pathways (FDR < 0.05). The enriched prominent terms are illustrated, and the bar sizes of the plot are proportional to the number of metabolites in each term.

Figure 5

Comparative proteomics and integrating GenX with human PD genetics identifies Glial genes that are altered by GenX exposure. (A) Overlap of protein expression changes between flies exposed to GenX and those expressing human αSyn. (B) Comparing MAGMA analysis results between PD MAGMA genes in flies exposed to GenX and α synuclein, respectively. (C) MAGMA analysis results showing proteins altered in both GenX and α synuclein (highlighted in green). Highlighted in red are the proteins expressed in glia, in blue are the proteins expressed in neurons, and the proteins highlighted in black are expressed ubiquitously in all cell types.

Figure 6

GenX exposure leads to metabolic dysregulation in cell culture. (A) MTS assay showing the change in metabolic activity of human astrocytic cell line, U373, after treatment with different doses of GenX for 24 h. (B) MitoStress test using a Seahorse Xfe96 bioanalyzer on U373 cells treated with 1 ppm GenX for 24 h. (C) The effect of treating U373 cells with 1 ppm GenX for 24 h on ATP production. (D) The effect of treating U373 cells with 1 ppm GenX for 24 h on basal respiration. (E) The effect of treating U373 cells with 1 ppm GenX for 24 h on proton leak. (F) The effect of treating U373 cells with 1 ppm GenX for 24 h on nonmitochondrial respiration. All data are analyzed using Student’s t-test. N = 3–4. *p < 0.05, **p < 0.01, ***p < 0.005.

Figure 7

GenX exposure leads to enhanced markers of A1 astrocytes in cell culture. (A–C) qPCR analysis showing (A) C3, (B) IL-1β, and (C) IL-6 mRNA fold change in U373 astrocytes treated with 1 ppm GenX for 24 h, (D–G) qPCR analysis of mouse primary astrocytes treated with different doses of GenX for 24 h, showing change in (D) C3, (E) Serpin 1, (F) IL-1β, and (G) IL-6 mRNA levels relative to control. All data are analyzed using Student’s t-test. N = 3–4. *p < 0.05, **p < 0.01, ***p < 0.005.