Acute gene expression changes in the mouse hippocampus following a combined Gulf War toxicant exposure

Abstract

Aims: Approximately 30% of the nearly 700,000 Veterans who were deployed to the Gulf War from 1990 to 1991 have reported experiencing a variety of symptoms including difficulties with learning and memory, depression and anxiety, and increased incidence of neurodegenerative diseases. Combined toxicant exposure to acetylcholinesterase (AChE) inhibitors has been studied extensively as a likely risk factor. In this study, we modeled Gulf War exposure in male C57Bl/6J mice with simultaneous administration of three chemicals implicated as exposure hazards for Gulf War Veterans: pyridostigmine bromide, the anti-sarin prophylactic; chlorpyrifos, an organophosphate insecticide; and the repellant N,N-diethyl-m-toluamide (DEET).

Main methods: Following two weeks of daily exposure, we examined changes in gene expression by whole transcriptome sequencing (RNA-Seq) with hippocampal isolates. Hippocampal-associated spatial memory was assessed with a Y-maze task. We hypothesized that genes important for neuronal health become dysregulated by toxicant-induced damage and that these detrimental inflammatory gene expression profiles could lead to chronic neurodegeneration.

Key findings: We found dysregulation of genes indicating a pro-inflammatory response and downregulation of genes associated with neuronal health and several important immediate early genes (IEGs), including Arc and Egr1, which were both reduced approximately 1.5-fold. Mice exposed to PB + CPF + DEET displayed a 1.6-fold reduction in preference for the novel arm, indicating impaired spatial memory.

Significance: Differentially expressed genes observed at an acute timepoint may provide insight into the pathophysiology of Gulf War Illness and further explanations for chronic neurodegeneration after toxicant exposure.

Keywords: Arc; Chlorpyrifos; DEET; Gene expression; Gulf War; Hippocampus; Immediate early genes; Pyridostigmine bromide; RNA-Seq.

Fig. 1.

RNA-Seq analysis workflow with CLC Genomics Workbench and Ingenuity Pathway Analysis. Whole transcriptome sequencing was performed using mouse hippocampal RNA isolates collected 2–4 h after final exposure. Gene expression tracks were analyzed using the Differential Expression for RNA-Seq tool with RPKM >10.0, |FC| ≥ 1.2, and p < 0.1 as criteria for significance. GO enrichment analysis was performed on subset of genes that were significantly dysregulated. Data for significant genes was exported to Ingenuity Pathway Analysis to assess canonical pathways, molecules, diseases and functions, and other relevant information.

Fig. 2.

(a) Preference for novel arm, (b) number of entries per arm, and (c) distance travelled during trial phase of Y-maze. Hippocampal-dependent spatial memory was assessed by performance on a Y-maze task 2–4 h after final exposure. (a) Preference for the novel arm was significantly lower in mice receiving PB + CPF + DEET (mean = −0.12 ± 0.099) compared to control mice (mean = 0.21 ± 0.073) (t(9.18) = 2.63, p = 0.027). (b) The number of entries into the novel arm was also significantly lower in mice exposed to PB + CPF + DEET (mean = 15.2 ± 1.15) compared to controls (mean = 22.7 ± 1.52) (t(9.31) = 3.95, p = 0.0031). (c) Distance travelled during the test stage did not significantly differ between conditions (PB + CPF + DEET: mean = 26.6 ± 2.32, control: mean = 30.2 ± 0.74, t(6.00) = 1.51, p = 0.18). All results are graphed as mean ± SEM.

Fig. 3.

Differentially expressed genes identified by RNA-Seq analysis. Sequence counts from the RNA samples were evaluated with CLC Genomics Workbench and Ingenuity Pathway Analysis software. 158 dysregulated genes were identified in mice exposed to PB + CPF + DEET vs. controls. Genes were considered to be significantly dysregulated if they met the following criteria: RPKM >10.0, |fold change| ≥ 1.2, p < 0.1. Green = downregulated; red = upregulated.