From Neural Stem Cells to Children: Secreted Phosphoprotein 1 in Lead Neurotoxicity

Abstract

Lead (Pb) exposure in the earliest stages of neurodevelopment leads to lasting deficits in cognitive function and behavior. Neural stem cells (NSCs) are multipotent stem cells and the first cells of the central nervous system, but little is known of their molecular response to Pb exposure. We exposed human NSCs to 1μM Pb for 24 hours and performed RNA sequencing. 16 of 19 differentially expressed transcripts are upregulated, and of these 10 are known targets of NRF2, the master transcriptional regulator of the cellular antioxidant response. One of the top Pb-induced genes, Secreted Phosphoprotein 1 (SPP1), was not a previously known NRF2 target. We show SPP1 is induced following activation of NRF2 by other known activators and by knockdown of its negative regulator KEAP1. In addition, the induction of SPP1 by Pb was attenuated after siRNA-mediated knockdown of NRF2. We identified a putative Antioxidant Response Element in the SPP1 promoter and confirmed its function by Chromatin immunoprecipitation (ChIP) of NRF2. To further investigate the role of SPP1 in neurodevelopment, we examined SPP1 single nucleotide polymorphisms (SNPs) in a longitudinal birth cohort of infants profiled at 24 months for the mental and psychomotor development indices (MDI and PDI, respectively) of the Bayley Scales of Infant Development. Sixteen single nucleotide polymorphisms (SNPs) in and near SPP1 were tested for main effect and effect modification. For main effect on MDI, eight SNPs are associated at p<0.05, but none pass multiple testing correction. In PDI analyses, SNP rs4693923 shows statistically significant effect modification on the PDI response to second trimester Pb exposure. A-allele carriers of this SNP show a positive correlation between Pb exposure and PDI, which is not seen among GG homozygotes. Together our studies show SPP1 is a novel target of NRF2 and suggest a critical role for SPP1 in modulating prenatal Pb neurotoxicity.