The objective of the Section on Transmitter Signaling has been to understand the molecular mechanisms underlying signaling between G-protein coupled receptors and voltage-gated ion channels. On June 16th, 2017 Dr. Stephen R Ikeda, the founder and Chief of the Laboratory of Molecular Physiology, retired from government service. This will therefore be the final report for ZIAAA000430. I will outline the current-status of the three major projects in the section. I will also discuss the disposition of the Sections remaining personnel and resources, towards maximizing the return on the governments investment in these projects and personnel over the past year. Over this past year there has been three Specific Aims of the Section: 1) To examine the role of GPR41(FFA3) in modulating calcium currents in sympathetic neurons. 2) To study neuronal expression and function of Rem2, a member of the RGK family of small GTP-binding proteins. 3) To develop new methodologies for studying signaling between receptors and ion channels.
Specific Aim 1 Over this past year Aim 1 has been pursued primarily by Drs. Colina Prisco and Puhl. LMP and others have previously shown the expression of FFA3 in sympathetic ganglia. To reduce experimental variance, we acquired a transgenic line expressing mRFP in FFA3 expressing cells and have utilized this line to study the FFA3 expression pattern in greater detail and the electrophysiological characteristics of the FFA3 expressing neuron populations. We are also using this reporter line to cross with an FFA3 knockout line to facilitate the study of FFA3 gene ablation on neuron function. Dr. Colina Prisco is funded by NINDS, but has been hosted in LMP. It is our understanding that she will maintain her NINDS funding for the next 3-6 months. She will use this time to finish these experiments and prepare a paper describing her results. She is also actively pursuing positions outside NIH utilizing her training. The promoter region controlling FFA3 gene expression has only been characterized in pancreatic beta cells. In these cells, FFA3 has been shown to be a product of the expression of a bicistronic gene structure contiguous with upstream FFA1 (GPR40, a long chain fatty acid receptor). The FFA3 is proposed to be translated from a message containing FFA1 and FFA3 open reading frames and driven by a single promoter upstream from the FFA1 gene. Surprisingly, we have shown (via qPCR and endpoint RT-PCR) that sympathetic ganglia expressing relatively high levels of FFA3 express little if any FFA1, indicative of the presence of an FFA3 neuron specific promotor. To identify this promoter, we recently isolated a region upstream from the putative transcript end of the mouse FFA3 gene that contains promoter activity in some neuroblastoma cell lines that express FFA3 but not FFA1 mRNA. We are currently characterizing the elements involved in this neuron specific expression.
Specific Aim 2 This past year Specific Aim 2 has been primarily pursued by Drs. Puhl and Liput. Following up on work recently published by LMP showing the expression of Rem2 in the central and peripheral nervous system, Drs Puhl and Liput have begun to characterize the promoter region of this gene. The Rem2 protein is expressed in many neuronal subtypes and the identification of the promoter region could produce a reporter similar to our Scn10a promoter/reporter mouse (Lu VB, Ikeda SR, Puhl HL 3rd A 3.7 kb fragment of the mouse Scn10a gene promoter directs neural crest but not placodal lineage EGFP expression in a transgenic animal. J Neurosci. 2015 May 20;35(20):8021-34.). Such a reporter could facilitate the identification of Rem2 expressing neurons and thereby aid the study of the regional and temporal expression of this gene. Ultimately this reporter could help elucidate the functions of this protein in the basal ganglia, where it is particularly abundant, as well as select locations in the peripheral nervous system such as subpopulations of neurons within the dorsal root and trigeminal ganglia. We have found several neuroblastoma cell lines that endogenously express Rem2 and have used these lines to identify a region upstream of the Rem2 transcription start site containing a CREB responsive region capable of driving reporter expression. This CREB responsive behavior recapitulates that of the endogenous gene as monitored via qPCR. This work will be presented at the 2017 SFN meeting in Washington D.C. in November. The role of the Rem2 gene product in the physiology of the basal ganglia has also been investigated by Dr. Liput. We have shown REM2 is expressed in medium spiny neurons (MSNs) of the striatum and have generated a conditional knockout animal that lacks Rem2 specifically in this population of neurons (MSN-Rem2KO). Electrophysiological characterization of MSN-Rem2KO mice found that Rem2 knockout reduces the frequency of spontaneous EPSCs but not IPSCs in striatal MSNs. This result may be due to an impairment in spinogenesis/synaptogenesis, and current studies are examining this hypothesis by measuring spine morphology and density. Although previous studies have shown that heterologous expression of Rem2 in peripheral neurons inhibits high voltage activated calcium channels, we did not observe an increase in whole cell calcium currents measured in acutely dissociated neurons from MSN-Rem2KO mice, or observe a difference is MSN excitability in an ex vivo slice preparation. Current studies are looking at the effect of Rem2 knockout in various forms of corticostriatal synaptic plasticity. We have also been testing MSN-Rem2KO mice on tasks probing basal ganglia dependent behavioral output. These animals have normal baseline locomotor activity, learn simple motor skills and acquire instrumental learning tasks. However, compared to wildtypes, Rem2 knockout mice show an impairment in a progressive ratio task designed to test an animals motivation to work for an appetitive reinforcer. Current behavioral studies are focused on defining the striatal circuitry responsible for this behavioral impairment. Dr Liput will physically maintain his desk and electrophysiology rig within LMP space, but will be transferring administratively to the Laboratory of Integrative Neuroscience (LIN) on November 16th, 2017 where he will continue his training and REM2 studies under the supervision of Dr. D. M. Lovinger.
Specific Aim 3 Dr. Puhl has been developing luminescent, FRET and BRET based biosensors for the detection of the activation of heterotrimeric G-proteins. We have successfully produced multicomponent biosensors capable of detecting beta/gamma subunit release, upon activation, from the G-protein heterotrimer in heterologous systems. Attempts to produce a more practical unitary construct capable of detecting endogenous activation are still underway. We have focused on BRET based biosensors, due to their inherent low background, but several luminescent complementation and intensity based fluorescent sensors are also being evaluated. Dr. Puhl will remain in LMP but transfer to the Section of Cellular Biophotonics on October 1st, 2017 under the supervision of Dr. Steven S. Vogel.
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