Identification of chemicals that mimic transcriptional changes associated with autism, brain aging and neurodegeneration

Abstract

Environmental factors, including pesticides, have been linked to autism and neurodegeneration risk using retrospective epidemiological studies. Here we sought to prospectively identify chemicals that share transcriptomic signatures with neurological disorders, by exposing mouse cortical neuron-enriched cultures to hundreds of chemicals commonly found in the environment and on food. We find that rotenone, a pesticide associated with Parkinson's disease risk, and certain fungicides, including pyraclostrobin, trifloxystrobin, famoxadone and fenamidone, produce transcriptional changes in vitro that are similar to those seen in brain samples from humans with autism, advanced age and neurodegeneration (Alzheimer's disease and Huntington's disease). These chemicals stimulate free radical production and disrupt microtubules in neurons, effects that can be reduced by pretreating with a microtubule stabilizer, an antioxidant, or with sulforaphane. Our study provides an approach to prospectively identify environmental chemicals that transcriptionally mimic autism and other brain disorders.

Figure 1. Mouse cortical cultures contain all principle brain cell types.

(a–b) Vehicle-treated cortical cultures contain NeuN-positive neurons, Gfap-positive astrocytes and Iba1-positive microglia. n=98,890–145,032 cells per coverslip counted. Values are mean±s.e.m. of four biological replicates. Scale bar, 100 μm. (c) Cortical cultures exhibit expression of marker genes representative of nine cell types found in the brain (y axis RPKM).

Figure 2. Gene expression defines six chemical clusters in cortical neuron cultures.

Median-centred gene expression values for 297 chemicals and vehicle (v; median of 49 replicates, t; topotecan-positive control, median of 32 replicates) were hierarchically clustered across 5,121 variably expressed genes. Genes >100 kb in length are tick marked (right), and mitochondrial health (bottom) was estimated by comparing the fraction of reads that align to the mitochondrial genome for each chemical–vehicle pair (blue indicates negative log₂ fold change with respect to vehicle). mtDNA, mitochondrial DNA.

Figure 3. Cluster 2 chemicals show significant gene set enrichment with autism and other brain diseases.

The enrichment scores of brain disease gene sets that were statistically significant (FDR<0.1) in each chemical cluster were plotted on a scale from −1 (blue) to +1 (red). Author surname and/or gene set name are indicated in parentheses. The composition of these gene sets is shown in Supplementary Data 2.

Figure 4. Cluster 2 chemicals alter expression of a common set of genes.

(a) Fenamidone and (b) pyraclostrobin (c,d) up- and downregulate a largely overlapping set of genes. RNA-seq was performed in quadruplicate after treating cortical cultures with fenamidone (10 μM) or pyraclostrobin (0.1 μM) for 24 h (n=3 biological replicates) and compared with matched vehicle controls using DESeq to detect differentially expressed genes, listed in Supplementary Data 4. Quantitative RT–PCR fold change (mean±s.e.m. across three to four biological replicates) of selected cluster 2 upregulated genes relative to vehicle after 24 h treatment with (e) 10 μM fenamidone or (f) the indicated doses of fenamidone.

Figure 5. Fenamidone causes mitochondrial superoxide production and microtubule destabilization.

(a,b) Superoxide (O₂⁻, MitoSOX fluorescent indicator) and aberrant cell morphology elicited by 2-h treatment with 10 μM fenamidone. Scale bars, 10 μm. (c) O₂⁻ generation and (d) aberrant morphology is dose dependent. RNA-seq dose is denoted by red circles. (e) Pretreatment with vitamin E (10 μM, 2 h) blocked O₂⁻ formation and (f) aberrant morphology elicited by fenamidone (10 μM, 2 h). (g) Microtubule stabilization with paclitaxel pretreatment (10 μM, 2 h) attenuated O₂⁻ formation and (h) aberrant morphology elicited by fenamidone (10 μM, 2 h). All values mean±s.e.m. n=33–256 cells per condition across six biological replicates. Bar graphs display mean±s.e.m. *P<0.05, ***P<0.001, ****P<0.0001.

Figure 6. Sulforaphane attenuated fenamidone-induced transcriptional and cellular responses in cortical cultures.

(a) Genome-wide (RNA-seq) transcriptional changes caused by fenamidone (10 μM, 24 h; n=3 replicates) and fenamidone (10 μM, 24 h) after pretreating with sulforaphane (10 μM, 18 h; n=3 replicates). Gene order is identical to Fig. 2. (b) Fenamidone-induced (10 μM, 2 h) O₂⁻ production and (c) aberrant cell morphology were attenuated by pretreating (18 h) with two concentrations of sulforaphane (SULF). n=175–327 cells per condition across 6–18 biological replicates. Bar graphs display Mean±s.e.m. **P<0.01, ***P<0.001, ****P<0.0001.

Figure 7. Usage trends and environmental fate of cluster 2 chemicals.

(a–i) Left: amount of chemical (alphabetical order) applied in the United States based on United States Geological Survey data. (a–i) Right: the five foods with the highest residue levels and the year of detection based on USDA and FDA data spanning 2008–2012. Red arrows indicate the year each chemical was first registered for use with the EPA. Chemicals approved before 2000 list the registration year in red font below the x axis. Spinach tested for high levels of fenamidone in both the 2009 USDA and FDA surveys (c), hence explaining why ‘Spinach (2009)' is displayed twice.