Armstrong discusses endocrine disruption of synaptic plasticity
By Negin Martin
The Integrated Toxicology and Environmental Health Program (ITEHP) at Duke University hosted a Jan. 13 presentation by NIEHS senior investigator David Armstrong, Ph.D., reporting on new findings about the regulation of synaptic plasticity by endocrine modulators. Synaptic plasticity is the ability to change the strength of neuronal connections in brain to facilitate learning and memory formation.
The presentation, hosted by neurotoxicologist Edward Levin, Ph.D., was part of the ITEHP weekly seminar series, supported in part by the NIEHS Superfund Research Program.
Armstrong’s presentation focused on endocrine disruption of oxytocin and thyroid hormone signaling pathways. Disruption of thyroid hormone signaling by environmental toxicants that resemble thyroid hormone in structure, such as PCBs, flame retardants, and dioxin, could interfere with proper development of neuronal maturation and cause learning disorders. Oxytocin is known as a social neuropeptide, because blocking its secretion or mutations in its receptor are associated with social disorders, such as autism, depression, and impaired social bonding.
Regulation of ion channels
Armstrong, chief of the Laboratory of Neurobiology and the head of the Membrane Signaling Group at NIEHS, is an expert electrophysiologist who studies the regulation of ion channels by endocrine signaling. Ion channels regulate membrane potential that controls neuronal signaling and secretion of hormones. By monitoring the effects of chemicals on channel activity and synaptic function in cultured cells and mouse brain slices, Armstrong and his colleagues have discovered molecular mechanisms that govern neuronal signaling and designed a high throughput assay to screen for endocrine disruption.
A capacity audience of young toxicologists in training at Duke attended the lecture to gain insight into how to exploit detailed knowledge of molecular pathways in the design of effective toxicological screens.
Disruption of oxytocin signaling by flame retardant TDCPP
Although fluorescent indicators of intracellular calcium have been available for decades, only recently have scientists at Howard Hughes Medical Institute’s Janelia Farm (http://www.hhmi.org/janelia/) Research Campus developed an indicator, GCAMP3, that can be stably expressed in neurons and reliably report individual calcium transients without killing the cells. Armstrong’s lab used GCAMP3 to create a stable human cell line that can be used to measure oxytocin signaling. Oxytocin regulates neuronal function through a calcium signaling cascade that is initiated by a G protein-coupled receptor.
The GCAMP3 cell line has been used to develop a high throughput assay on a fluorescent plate reader for identifying environmental toxicants that disrupt oxytocin signaling. The initial chemicals used to disrupt oxytocin signaling and to test the system were flame retardants provided by environmental chemist Heather Stapleton, Ph.D., (http://fds.duke.edu/db/Nicholas/esp/faculty/stapleto) a collaborator at Duke and the recipient of an Outstanding New Environmental Scientist award from NIEHS. “To our great surprise,” said Armstrong, “two out of the first eight compounds that we tested disrupted oxytocin signaling.”
In the process of confirming this result on single cells using confocal microscopy, they discovered that the flame retardants block calcium entry after store depletion. Oxytocin’s potentiation of neuronal synapses depends on calcium entry, and is completely blocked by 50 micrometers of Tri(2,3-dichloropropyl) phosphate (TDCPP). “Fortunately, no one is exposed to such high levels of TDCPP,” Armstrong noted, “but we have only tested eight chemicals so far.”
Currently, the predictive toxicology consortium Tox21 is using the new cell line to screen over 1,500 chemicals for their effects on oxytocin signaling.
(Negin Martin, Ph.D., is a biologist in the NIEHS Laboratory of Neurobiology Viral Vector Core Facility.)
Mutant thyroid receptor blocks potentiation of excitatory response in neurons
In a separate project in Armstrong’s lab, researchers have discovered a cytoplasmic signaling pathway involving phosphatidylinositol 3-kinase (PI3K) that is activated by TR-beta, a nuclear receptor for thyroid hormone. Thyroid hormone also potentiates synaptic responses in the hippocampus, but through a calcium independent mechanism.
The Armstrong laboratory has created a strain of mice with mutated thyroid hormone receptor that cannot stimulate PI3K, but still regulates transcription. Electrophysiological studies performed on mouse hippocampal brain slices by Fengxia Mizuno, Ph.D., a research fellow in Armstrong’s group, have revealed that blocking thyroid hormone-dependent stimulation of PI3K by mutating the receptor prevents potentiation of excitatory response in hippocampal neurons.
“Now that we have shown that some of the effects of thyroid hormone on brain development are mediated by signaling through PI3K,” Armstrong concluded, “we would like to develop a similar cell line for high throughput screening of endocrine disruptors of thyroid hormone signaling through PI3K.”