Cloning of human DING: Developmental expression and downregulation by EtOH in-utero

An estimated 15-20% of women consume alcohol (EtOH) during pregnancy. Women with alcohol use in early pregnancy are likely to have a child with fetal alcohol spectrum disorders (FASD). Recently, we reported neuroprotective effects of human DING (a member of the DING family of phosphatases) against EtOH-mediated toxicity in rats and in human fetal cortical neurons in vitro. Now, we report the sequencing and developmental expression patterns of endogenous DING in human fetal brain.

DING cDNA was cloned from human U87MG astrocytoma cells with primers specific to the plant DING gene and known prokaryotic DING genes. This cDNA was used to prepare antibodies. The full-length human DING gene p38hu (1095 nucleotide bases) is flanked by the first initiating codon, ATG, and the last, stop codon, TAA. Post-mortem fetal tissues and maternal blood were collected during pregnancy between 8 and 37 weeks' gestation. The developmental, spatial, and temporal expression of DING protein in fetal brain tissue was analyzed by immunohistochemistry. Developmental expression of DING in fetal brain and placenta was quantified by qWestern blots. DING promoter expression was assayed by ddPCR. Statistical analysis included ANOVA.

Sequencing revealed different-sized genomic DNA clones. The anti-DING antibody detected proteins ranging in size from 35 to 40 kDa, and high molecular weight precursor protein in fetal brain and placenta. DING protein was present in fetal brain at early stages and its level was increased at later gestational ages. The DING promoter was expressed in fetal brain, neurospheres, and fetal brain-derived exosomes. DING levels were reduced in samples exposed to maternally consumed alcohol.

Because DING is neuroprotective, its reduced expression in fetuses exposed to alcohol may suggest a mechanism that contributes to the pathogenesis of FASD, which could lead to the development of therapeutic tools aimed at preventing, ameliorating or reversing this prevalent group of syndromes that are implicated in as many as 5% of births world-wide.