Regulator of G Protein Signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates G protein and MAPK signaling pathways. In the adult mouse brain, RGS14 mRNA and protein are found almost exclusively in hippocampal CA2 neurons. We have shown that RGS14 is a natural suppressor of CA2 synaptic plasticity and hippocampal-dependent learning and memory. However, the protein distribution and spatiotemporal expression patterns of RGS14 in mouse brain during postnatal development are unknown. Here, using a newly characterized monoclonal anti-RGS14 antibody, we demonstrate that RGS14 protein immunoreactivity is undetectable at birth (P0) with very low mRNA expression in the brain. However, RGS14 protein and mRNA are upregulated during early postnatal development, with protein first detected at P7, and both increasing over time until reaching highest sustained levels throughout adulthood. Our immunoperoxidase data demonstrate that RGS14 protein is expressed in regions outside of hippocampal CA2 during development including the primary olfactory areas, the anterior olfactory nucleus and piriform cortex, and the olfactory associated orbital and entorhinal cortices. RGS14 is also transiently expressed in neocortical layers II/III and V during postnatal development. Finally, we show that RGS14 protein is first detected in the hippocampus at P7 with strongest immunoreactivity in CA2 and fasciola cinerea and sporadic immunoreactivity in CA1; labeling intensity in hippocampus increases until adulthood. These results show that RGS14 mRNA and protein are upregulated throughout postnatal mouse development, and RGS14 protein exhibits a dynamic localization pattern that is enriched in hippocampus and primary olfactory cortex in the adult mouse brain.
Pyramidal neurons in hippocampal area CA2 are distinct from neighboring CA1 in that they resist synaptic long-term potentiation (LTP) at CA3 Schaffer collateral synapses. Regulator of G protein signaling 14 (RGS14) is a complex scaffolding protein enriched in CA2 dendritic spines that naturally blocks CA2 synaptic plasticity and hippocampus-dependent learning, but the cellular mechanisms by which RGS14 gates LTP are largely unexplored. A previous study has attributed the lack of plasticity to higher rates of calcium (Ca2+) buffering and extrusion in CA2 spines. Additionally, a recent proteomics study revealed that RGS14 interacts with two key Ca2+-activated proteins in CA2 neurons: calcium/calmodulin and CaMKII. Here, we investigated whether RGS14 regulates Ca2+ signaling in its host CA2 neurons. We found that the nascent LTP of CA2 synapses caused by genetic knockout (KO) of RGS14 in mice requires Ca2+-dependent postsynaptic signaling through NMDA receptors, CaMK, and PKA, revealing similar mechanisms to those in CA1. We report that RGS14 negatively regulates the long-term structural plasticity of dendritic spines of CA2 neurons. We further show that wild-type (WT) CA2 neurons display significantly attenuated spine Ca2+ transients during structural plasticity induction compared with the Ca2+ transients from CA2 spines of RGS14 KO mice and CA1 controls. Finally, we demonstrate that acute overexpression of RGS14 is sufficient to block spine plasticity, and elevating extracellular Ca2+ levels restores plasticity to RGS14-expressing neurons. Together, these results demonstrate for the first time that RGS14 regulates plasticity in hippocampal area CA2 by restricting Ca2+ elevations in CA2 spines and downstream signaling pathways.
Number of tables: 0 Number of multimedia: 0 Number of words for Abstract: 249 Number of words for Significance Statement: 111 Number of words for Introduction: 722 Number of words for Discussion: 1439Acknowledgements: We thank Jaime Richards for preparing organotypic slice cultures and reagents, David Kloetzer for laboratory management, and Drs. Lesley Colgan and Tal Laviv for assistance with two-photon microscopy. AbstractPyramidal neurons in hippocampal area CA2 are distinct from neighboring CA1 in that they resist synaptic long-term potentiation (LTP) at CA3 Schaffer Collateral synapses. Regulator of G Protein Signaling 14 (RGS14) is a complex scaffolding protein enriched in CA2 dendritic spines that naturally blocks CA2 synaptic plasticity and hippocampus-dependent learning, but the cellular mechanisms by which RGS14 gates LTP are largely unexplored. A previous study has attributed the lack of plasticity to higher rates of calcium (Ca 2+ ) buffering and extrusion in CA2 spines. Additionally, a recent proteomics study revealed that RGS14 interacts with two key Ca 2+ -activated proteins in CA2 neurons: calcium/calmodulin, and CaMKII. Here, we investigate whether RGS14 regulates Ca 2+ signaling in its host CA2 neurons. We find the nascent LTP of CA2 synapses due to genetic knockout (KO) of RGS14 in mice requires Ca 2+ -dependent postsynaptic signaling through NMDA receptors, CaMK, and PKA, revealing similar mechanisms to those in CA1. We report RGS14 negatively regulates the long-term structural plasticity of dendritic spines of CA2 neurons. We further show that wild-type (WT) CA2 neurons display significantly attenuated spine Ca 2+ transients during structural plasticity induction compared with the Ca 2+ transients from CA2 spines of RGS14 KO mice and CA1 controls.Finally, we demonstrate that acute overexpression of RGS14 is sufficient to block spine plasticity, and elevating extracellular Ca 2+ levels restores plasticity to RGS14-expressing neurons. Together, these results demonstrate for the first time that RGS14 regulates plasticity in hippocampal area CA2 by restricting Ca 2+ elevations in CA2 spines and downstream signaling pathways. Significance StatementRecent studies of hippocampal area CA2 have provided strong evidence in support of a clear role for this apparently plasticity-resistant subregion of the hippocampus in social, spatial, and temporal aspects of memory. Regulator of G Protein Signaling 14 (RGS14) is a critical factor 4 that inhibits synaptic plasticity in CA2, but the molecular mechanisms by which RGS14 limits LTP remained unknown. Here we provide new evidence that RGS14 restricts spine calcium (Ca 2+ ) in CA2 neurons and that key downstream Ca 2+ -activated signaling pathways are required for CA2 plasticity in mice lacking RGS14. These results define a previously unrecognized role for RGS14 as a natural inhibitor of postsynaptic Ca 2+ signaling in hippocampal area CA2.
Regulator of G Protein Signaling 14 (RGS14) is a complex scaffolding protein that integrates G protein and MAPK signaling pathways. In the adult mouse brain, RGS14 is predominantly expressed in hippocampal CA2 neurons where it naturally inhibits synaptic plasticity and hippocampus-dependent learning and memory. However, the signaling proteins that RGS14 natively engages to regulate plasticity are unknown. Here, we show that RGS14 exists in a high-molecular-weight protein complex in brain. To identify RGS14 neuronal interacting partners, endogenous RGS14 immunoprecipitated from mouse brain was subjected to mass spectrometry and proteomic analysis. We find that RGS14 interacts with key postsynaptic proteins that regulate plasticity. Gene ontology analysis reveals the most enriched RGS14 interactors have functional roles in actin-binding, calmodulin(CaM)-binding, and CaM-dependent protein kinase (CaMK) activity. We validate these findings using biochemical assays that identify interactions with two previously unknown binding partners. We report that RGS14 directly interacts with Ca/CaM and is phosphorylated by CaMKII in vitro. Lastly, we detect that RGS14 associates with CaMKII and CaM in hippocampal CA2 neurons. Taken together, these findings demonstrate that RGS14 is a novel CaM effector and CaMKII phosphorylation substrate thereby providing new insight into mechanisms by which RGS14 controls plasticity in CA2 neurons.
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