We previously found that the widely used disinfectants, benzalkonium chlorides (BACs), alter cholesterol and lipid homeostasis in neuronal cell lines and in neonatal mouse brains. Here we investigate the effects of BACs on neurospheres, an in vitro three-dimensional model of neurodevelopment. Neurospheres cultured from mouse embryonic neural progenitor cells (NPCs) were exposed to increasing concentrations (1 to 100 nM) of a short-chain BAC (BAC C12), a long-chain BAC (BAC C16), and AY9944 (a known DHCR7 inhibitor). We found that the sizes of neurospheres were decreased by both BACs, but not by AY9944. Furthermore, we observed potent inhibition of cholesterol biosynthesis at the step of DHCR7 by BAC C12, but not by BAC C16, suggesting that cholesterol biosynthesis inhibition is not responsible for the observed reduction in neurosphere growth. Using immunostaining and cell cycle analysis, we found that both BACs induced apoptosis and decreased proliferation of NPCs. To explore the mechanisms underlying their effect on neurosphere growth, we carried out RNA sequencing on neurospheres exposed to each BAC at 50 nM for 24 hr, which revealed activation of the integrated stress response by both BACs. Overall, these results suggest that BACs affect neurodevelopment by inducing the integrated stress response in a manner independent of their effects on cholesterol biosynthesis.

Neurospheres exposed to BAC C12 (50 nM), BAC C16 (50 nM), or vehicle control from DIV 4 to DIV 5 in 4 wells of a 6-well plate were pelleted. Total RNA was extracted from each sample and RNA concentration was quantified using a microplate spectrophotometer. RNA integrity was evaluated by formaldehyde agarose gel electrophoresis to visualize the 18S and 28S rRNA bands. RNA integrity and purity were further confirmed by Novogene (Chula Vista, CA) using an Agilent 2100 BioAnalyzer (Agilent Technologies Inc., Santa Clara, California). Samples with RNA Integrity Number (RIN) of 10.0 or above were submitted for RNA sequencing.  Novogene performed the cDNA library construction and sequencing using the Illumina NovaSeq 6000 platform (150 base pairs paired-end, with sequencing depth above 20 million reads per sample). Raw RNA sequencing reads in FASTQ format were mapped to the mouse genome using HISAT (https://ccb.jhu.edu/software/hisat/index.shtml; Last accessed 8/14/2019) and format conversions were performed using Samtools. Cufflinks (http://cufflinks.cbcb.umd.edu/; Last accessed 8/14/2019) was used to estimate relative abundances of transcripts from each RNA sample. Cuffdiff, a module of Cufflinks, was used to determine differentially expressed genes (DEGs) between control and BAC C12, as well as control and BAC C16. DEGs met the criterion of an adjusted P value < 0.05 (corresponding to the allowed false discovery rate of 5%).

This excel workbook contains 3 sheets. The first sheet, titled FPKM, contains 11 columns. Three biological replicates per treatment were used for transcriptomics analyses. Rows contain FPKM values of each transcript, signified by Ensembl IDs and Gene Names. The second and third sheets, titled DEGs_BAC C12 and DEGs_BAC C16, respectively, contain 3 columns. Rows contain the fold change and adjusted P value of each transcript, signified by Gene Names.