Norepinephrine causes a change in REST expression and subcellular localization in cerebellar interneurons
Publication Date
July 2019
Location
LSU Health Medical Education Building
Document Type
Abstract
Start Date
26-7-2019 9:00 AM
End Date
26-7-2019 12:00 PM
Description
Our lab has previously shown that stress induces a change in AMPA receptor (AMPAR) subunit composition in molecular layer interneurons of the cerebellar cortex. This involves the upregulation of the AMPAR subunit GluA2 and induces a phenotypic switch in synaptic AMPAR subtype from GluA2-lacking to GluA2-containing. Such a switch alters neurotransmission within the cerebellar cortex and cerebellar output to other brain regions, with important implications for behavior and cognition. We therefore explored the mechanism by which stress may induce transcription, since the change in GluA2 is a transcriptional-dependent increase. RE1-Silencing Transcription factor (REST) is a transcriptional repressor of many neuronal genes, including GluA2. Our results have shown that stress reduces the suppression of GluA2 transcription by REST. We explored the possibility that the expression of REST was downregulated after stress. To mimic stress on the cellular level, cerebellar neurons in culture were treated with norepinephrine (NE), a hormone that increases in the presence of stress. Previous in vitro experiments have shown that NE signaling is both required and sufficient to induce an upregulation of synaptic GluA2 and induces an overall decrease in REST expression in cerebellar interneurons. Here we examined a second mechanism underlying a stress-induced decrease in REST function, in which NE causes REST to translocate from the nucleus to the cytoplasm. This type of redistribution is seen in neurodevelopmental diseases, such as Huntington’s, where REST gets trapped outside the nucleus and it cannot repress gene expression allowing for GluA2 to transcribe. The current study is focused on analyzing REST expression and subcellular localization in an in-vitro model of stress. REST expression was analyzed by taking cerebellar cultures from 7 day old wildtype or GAD-GFP mice. Cultures were then maintained in vitro for 14-18 days. After incubation in the presence or absence of norepinephrine for 3 hours, cerebellar neurons were immunostained for REST and parvalbumin to label inhibitory interneurons. Immunofluorescence images were acquired using a confocal microscope and a 10-plane z-stack image was taken of each cell. The immunofluorescence intensity of REST (REST-ir) in the nucleus and cytoplasm were quantified in GFP- or PV-expressing interneurons. The nucleus to cytoplasmic ratio was calculated to evaluate the distribution of REST in the cell.
Recommended Citation
Fong, Jordyn T., "Norepinephrine causes a change in REST expression and subcellular localization in
cerebellar interneurons" (2019). Summer Research Internship Program. 13.
https://digitalscholar.lsuhsc.edu/srip/2019/undergrad/13
Norepinephrine causes a change in REST expression and subcellular localization in cerebellar interneurons
LSU Health Medical Education Building
Our lab has previously shown that stress induces a change in AMPA receptor (AMPAR) subunit composition in molecular layer interneurons of the cerebellar cortex. This involves the upregulation of the AMPAR subunit GluA2 and induces a phenotypic switch in synaptic AMPAR subtype from GluA2-lacking to GluA2-containing. Such a switch alters neurotransmission within the cerebellar cortex and cerebellar output to other brain regions, with important implications for behavior and cognition. We therefore explored the mechanism by which stress may induce transcription, since the change in GluA2 is a transcriptional-dependent increase. RE1-Silencing Transcription factor (REST) is a transcriptional repressor of many neuronal genes, including GluA2. Our results have shown that stress reduces the suppression of GluA2 transcription by REST. We explored the possibility that the expression of REST was downregulated after stress. To mimic stress on the cellular level, cerebellar neurons in culture were treated with norepinephrine (NE), a hormone that increases in the presence of stress. Previous in vitro experiments have shown that NE signaling is both required and sufficient to induce an upregulation of synaptic GluA2 and induces an overall decrease in REST expression in cerebellar interneurons. Here we examined a second mechanism underlying a stress-induced decrease in REST function, in which NE causes REST to translocate from the nucleus to the cytoplasm. This type of redistribution is seen in neurodevelopmental diseases, such as Huntington’s, where REST gets trapped outside the nucleus and it cannot repress gene expression allowing for GluA2 to transcribe. The current study is focused on analyzing REST expression and subcellular localization in an in-vitro model of stress. REST expression was analyzed by taking cerebellar cultures from 7 day old wildtype or GAD-GFP mice. Cultures were then maintained in vitro for 14-18 days. After incubation in the presence or absence of norepinephrine for 3 hours, cerebellar neurons were immunostained for REST and parvalbumin to label inhibitory interneurons. Immunofluorescence images were acquired using a confocal microscope and a 10-plane z-stack image was taken of each cell. The immunofluorescence intensity of REST (REST-ir) in the nucleus and cytoplasm were quantified in GFP- or PV-expressing interneurons. The nucleus to cytoplasmic ratio was calculated to evaluate the distribution of REST in the cell.
Comments
Mentors: Jessica Fawcett-Patel and Siqiong June Liu, Department of Cell Biology and Anatomy