Search

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 (from 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. By 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 h, which revealed the 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.

© 2016 Elsevier Inc. Specific spontaneous heritable neurodegenerative diseases have been associated with lower serum and cerebrospinal fluid α-tocopherol (α-TOH) concentrations. Equine neuroaxonal dystrophy (eNAD) has similar histologic lesions to human ataxia with vitamin E deficiency caused by mutations in the α-TOH transfer protein gene (TTPA). Mutations in TTPA are not present with eNAD and the molecular basis remains unknown. Given the neuropathologic phenotypic similarity of the conditions, we assessed the molecular basis of eNAD by global transcriptome sequencing of the cervical spinal cord. Differential gene expression analysis identified 157 significantly (FDR

Smith-Lemli-Opitz Syndrome (SLOS) is a recessive human disease caused by defective cholesterol (CHOL) synthesis at the level of DHCR7 (7-dehydrocholesterol reductase), which normally catalyzes the conversion of 7-dehydrocholesterol (7DHC) to CHOL. Formation and abnormal accumulation of 7DHC and 7DHC-derived oxysterols occur in SLOS patients and in rats treated with the DHCR7 inhibitor AY9944. The rat SLOS model exhibits progressive and irreversible retinal dysfunction and degeneration, which is only partially ameliorated by dietary CHOL supplementation. We hypothesized that 7DHC-derived oxysterols are causally involved in this retinal degeneration, and that blocking or reducing their formation should minimize the phenotype. Here, using the SLOS rat model, we demonstrate that combined dietary supplementation with CHOL plus antioxidants (vitamins E and C, plus sodium selenite) provides better outcomes than dietary CHOL supplementation alone with regard to preservation of retinal structure and function and lowering 7DHC-derived oxysterol formation. These proof-of-principle findings provide a translational, pre-clinical framework for designing clinical trials using CHOL-antioxidant combination therapy as an improved therapeutic intervention over the current standard of care for the treatment of SLOS.

Cholesterol and cholesterol-derived oxysterols are critical for embryonic development, synapse formation and function, and myelination, among other biological functions. Indeed, alterations in levels of cholesterol, sterol precursors, and oxysterols result in a variety of developmental disorders, emphasizing the importance of cholesterol homeostasis. The ability of xenobiotics to reproduce similar phenotypes by altering cholesterol homeostasis has increasingly become of interest. Therefore, the ability to quantitatively assess alterations in cholesterol homeostasis resulting from exposure to xenobiotics is of value. This unit describes methods for the quantitative assessment of altered post-squalene cholesterol biosynthesis and subsequent oxysterol formation in various sample types using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Understanding alterations in cholesterol homeostasis resulting from xenobiotic exposure can provide key insight into the toxicant's mechanism of action and resulting phenotype. © 2018 by John Wiley & Sons, Inc.