Papers of the Month
By Robin Arnette, David Banks, Deacqunita Diggs, Simone Otto, and Qing Xu
New NTP assay for chemicals that affect neurological development
Researchers in the National Toxicology Program (NTP) led a collaborative team that, for the first time, screened 80 compounds with a high-throughput, high content assay using human neurons derived from induced pluripotent stem cells (iPSC). The screened compounds ranged from those associated with developmental neurotoxicity and neurotoxicity, to those with unknown potential, such as polycyclic aromatic hydrocarbons and flame retardants.
The results suggest the screen may narrow down the number of compounds that would undergo more comprehensive in vivo testing. Since previous assays were only able to screen small numbers of neurotoxic compounds, the iPSC studies represent an advance in developing reliable tools to identify environmental chemicals that could affect neurological development.
The scientists treated neurons for 72 hours in 384-well plates, using six different concentrations of each compound. They evaluated compound-specific effects on neurite growth by quantifying total outgrowth, branches, and processes. Simultaneously, they quantified cell viability, as a marker of nonspecific cell toxicity. Among the 80 compounds tested, 38 decreased neurite outgrowth or cell viability, with 16 specifically inhibiting neurite outgrowth. Twelve of the 16 compounds were associated with developmental neurotoxicity or neurotoxicity. Three were polycyclic aromatic hydrocarbons, and one was a flame retardant. Independent repeat studies of these 16 compounds verified six compounds that specifically inhibited neurite outgrowth more than cell viability. (RA)
Ryan KR, Sirenko O, Parham F, Hsieh JH, Cromwell EF, Tice RR, Behl M. 2016. Neurite outgrowth in human induced pluripotent stem cell-derived neurons as a high-throughput screen for developmental neurotoxicity or neurotoxicity. Neurotoxicology 53:271-281.
Maternal folate levels linked to altered DNA methylation in newborns
NIEHS researchers have found that higher levels of maternal folate, an essential vitamin for fetal development, are associated with altered newborn DNA methylation, an epigenetic mark that controls gene expression. Since folate is central to a cellular pathway that provides methyl groups for DNA methylation, the connection between maternal folate and newborn DNA methylation was explored in this study.
Physicians recommend that pregnant women take folic acid supplements to protect against neural tube birth defects. To ensure that levels are high enough in women before they become pregnant, the United States and many other countries add folic acid, the synthetic form of folate, to flour, grains, and other foods. However, some are concerned that increasing folate levels in an entire population may result in adverse health effects.
The researchers examined the blood folate levels in mothers during pregnancy, and methylation of CpG sites in DNA from newborn cord blood. After combining results from two European population groups, the scientists concluded that higher levels of maternal folate were associated with methylation of 443 CpGs, which localized to 320 genes. Many of these genes were not previously associated with folate and are involved in neurological functions, developmental diseases, and tumor progression. Identification of these genes provides possible clues to the mechanistic effects of folate supplementation on human health. (QX)
Joubert BR, den Dekker HT, Felix JF, Bohlin J, Lighthart S, Beckett E, Tiemeier H, van Meurs JB, Uitterlinden AG, Hofman A, Haberg SE, Reese SE, Peters MJ, Kulle Andreassen B, Steegers EA, Nilsen RM, Vollset SE, Midttun O, Ueland PM, Franco OH, Dehghan A, de Jongste JC, Wu MC, Wang T, Peddada SD, Jaddoe VW, Nystad W, Duijts L, London SJ. 2016. Maternal plasma folate impacts differential DNA methylation in an epigenome-wide meta-analysis of newborns. Nat Commun 7:10577.
Protein may protect mice from inflammatory diseases
NIEHS researchers have demonstrated the ability of increased levels of endogenous tristetraprolin (TTP), a protein that facilitates turnover of specific mRNA transcripts, to protect against inflammatory diseases.
Previous studies have shown that mice deficient in TTP developed a chronic inflammatory syndrome caused by overproduction of a proinflammatory protein called tumor necrosis factor. After generating a new mouse model in which an instability sequence within the TTP transcript was deleted, researchers observed a marked increase in TTP transcript half-life and protein levels throughout the body. As a result of enhanced TTP availability, these mice were protected from collagen antibody-induced arthritis, showed reduced inflammation in imiquimod-induced dermatitis, and resisted experimental autoimmune encephalomyelitis induction when compared to normal mice. These treatment conditions model rheumatoid arthritis, psoriasis, and multiple sclerosis, respectively.
Current therapies for the diseases modeled in this study require administering agents that bind directly to proinflammatory molecules after they have been produced and circulated. Here, the scientists showed that increasing levels of an endogenous molecule by genetic means could point to novel therapeutic approaches to these diseases. (DB)
Patial S, Curtis AD 2nd, Lai WS, Stumpo DJ, Hill GD, Flake GP, Mannie MD, Blackshear PJ. 2016. Enhanced stability of tristetraprolin mRNA protects mice against immune-mediated inflammatory pathologies. Proc Natl Acad Sci U S A 113(7):1865-1870. [Story]
How CA2 hippocampal neurons respond to changes in the environment
Scientists at NIEHS and the University of Texas at Austin established a possible mechanism used by neurons in the hippocampal area of the brain to create memories of novel and social information. They looked at neuronal activity in the CA2 portion of the hippocampus and found that, like in other areas of the hippocampus, CA2 used groups of cells, called place cells, to represent spatial information. However, the place cell properties in CA2 changed in response to other animals and novel objects in the environment.
The researchers used fluorescent in-situ hybridization to look at genes that switch on when neurons are active, as well as recordings of individual neurons in rats, to see whether CA2 neurons became more active during social conditions, such as a male rat socializing with a female rat. Rather than becoming more active in response to changes in the environment, the data demonstrated that CA2 neurons instead updated their maps where place cells were active.
Understanding the mechanism behind the formation of social memories is integral to eventually solving the puzzle of how to help those with altered social capacities, such as individuals with schizophrenia and autism. (SO)
Alexander GM, Farris S, Pirone JR, Zheng C, Colgin LL, Dudek SM. 2016. Social and novel contexts modify hippocampal CA2 representations of space. Nat Commun 7:10300. [Story]
GDNF is needed for undifferentiated spermatogonial cell development
Scientists at NIEHS are the first to provide genetic evidence that glial cell line-derived neurotrophic factor (GDNF) produced by peritubular myoid (PM) cells is required for development of undifferentiated spermatogonial cells. By generating mice with PM cells lacking the Gdnf gene, researchers were able to observe adverse effects in male fertility.
The absence of GDNF resulted in a decrease in fertility in male mice, by a reduction in sperm count and a defect in spermatogenesis. Testes of mice lacking PM cell GDNF exhibited a marker for undifferentiated spermatogonia. Scientists were able to show there was a decrease in undifferentiated spermatogonia, but not in the level of differentiated spermatogonia, in the testes of one-week-old mice or of spermatocytes in four-week-old mice lacking PM cell GDNF.
The results show the importance of PM cell GDNF in undifferentiated spermatogonial cell development in vivo, and support the hypothesis of its importance in spermatogonial stem cell maintenance. These results are a step toward determining one underlying cause of male infertility. (DD)
Chen LY, Willis WD, Eddy EM. 2016. Targeting the Gdnf gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development. Proc Natl Acad Sci U S A 113(7):1829-1834.
(Robin Arnette, Ph.D., is a science writer and editor in the NIEHS Office of Communications and Public Liaison. David Banks is a postbaccalaureate Intramural Research and Training Award (IRTA) fellow in the NIEHS Intracellular Regulation Group. Deacqunita Diggs, Ph.D., is an Oak Ridge Institute for Science and Education fellow in the U.S. Environmental Protection Agency Developmental Toxicity Branch. Simone Otto, Ph.D., is an IRTA fellow in the NIEHS Ion Channel Physiology Group. Qing Xu is a biologist in the NIEHS Metabolism, Genes, and Environment Group.)