Rem2, a member of the RGK family of small GTPases, is enriched in nuclei of the basal ganglia

Abstract

Rem2 is a member of the RGK subfamily of RAS small GTPases. Rem2 inhibits high voltage activated calcium channels, is involved in synaptogenesis, and regulates dendritic morphology. Rem2 is the primary RGK protein expressed in the nervous system, but to date, the precise expression patterns of this protein are unknown. In this study, we characterized Rem2 expression in the mouse nervous system. In the CNS, Rem2 mRNA was detected in all regions examined, but was enriched in the striatum. An antibody specific for Rem2 was validated using a Rem2 knockout mouse model and used to show abundant expression in striatonigral and striatopallidal medium spiny neurons but not in several interneuron populations. In the PNS, Rem2 was abundant in a subpopulation of neurons in the trigeminal and dorsal root ganglia, but was absent in sympathetic neurons of superior cervical ganglia. Under basal conditions, Rem2 was subject to post-translational phosphorylation, likely at multiple residues. Further, Rem2 mRNA and protein expression peaked at postnatal week two, which corresponds to the period of robust neuronal maturation in rodents. This study will be useful for elucidating the functions of Rem2 in basal ganglia physiology.

Figure 1. Rem2 and Gem mRNA expression in the nervous system.

Relative expression of Rem2 mRNA (A) and Gem mRNA (B) across select regions of the nervous system. mRNA was quantified by qPCR and Rem2 and Gem gene expression was normalized to Actb. Liver samples served as a negative control.

Figure 2. Characterization of Rem2 protein expression in the nervous system.

(A) Rem2 protein expression in select regions of the nervous system. Rem2 was quantified using capillary electrophoresis followed by immunochemistry based detection (see materials and methods for details). (B) Representative electrophoretograms of samples for striatum and cortex showing high and low relative Rem2 signal. Data points in (A) were calculated by integrating the signal peak at approximately 50 kDa, which corresponds to Rem2 protein. (C) Representative images of Rem2 immunofluorescence across various regions of the mouse brain. (D) Validation of the Rem2 antibody by Western blotting and immunohistochemistry. Left, Representative image of Western blot using striatal lysates from wildtype and Rem2 knockout mice with β-actin serving as a loading control. Right, Representative coronal sections from wildtype and Rem2 knockout mice showing Rem2 immunoreactivity in magenta and DAPI counterstain in blue. Scale bars are 500 μm in all images. GP, globus pallidus; IPAC, interstitial nucleus of the posterior limb of the anterior commisure; NAcc; nucleus accumbens; OT, olfactory tubercle; STR, striatum; SNr, substantia nigra reticulata; VP, ventral pallidum.

Figure 3. Rem2 is expressed in striatonigral and striatopallidal medium spiny neurons, but not striatal interneurons.

(A,B) Representative images of Rem2 immunofluorescence on striatal sections from Cre dependent td-tomato reporter mice that express Cre recombinase under the dopamine D1 receptor or adenosine A2a receptor promoter, respectively. Blue arrowheads indicate cells expressing Rem2 and td-tomato, while yellow arrowheads indicate cells expressing Rem2 but not td-tomato. (C–E) Representative images of double-label immunofluorescence for Rem2 and choline acetyltransferase (C), neuropeptide Y (D) or parvalbumin (E), show that Rem2 is not expressed in any striatal interneurons examined. Yellow arrows indicate the cell bodies of interneurons that do not colabel with Rem2. ChAT, choline acetyltransferase; PARV, parvalbumin; NPY, neuropeptide Y. Scale bars are 20 μm and the same in all images.

Figure 4. Rem2-immunoreactivity in dissociated dorsal root ganglion (DRG) neurons.

(A) Representative DRG neurons under phase-contrast (left) and following immunocytochemistry (ICC) staining and fluorescence detection using Rem2 (middle) or Neurofilament 200 (NF200, right) antibodies. White arrowhead indicates a DRG neuron that displays positive-immunoreactivity against Rem2 and NF200 antibodies. Scale bar is 50 μm and is the same for all images. (B) Scatterplot of fluorescence intensity of DRG neurons stained for both NF200 and Rem2 from 1 experiment. Regions of interest were automatically generated around dissociated DRG neurons from the phase-contrast image and fluorescence intensity was measured from subsequent images collected from the Alexa Fluor® 488 or 568 channel. Each dot represents an individual DRG neuron. Dashed lines indicate the boundary between negative and positive-immunoreactivity as determined by staining in negative controls (secondary antibodies alone). (C) Distribution of DRG soma diameter from ICC experiments. White bars indicate the distribution of all DRG neurons (mean = 28.3 ± 0.1 μm, n = 4806, from 4 biological replicates) detected from phase-contrast images. Grey bars indicate the population of DRG neurons positive for NF200-immunoreactivity (mean = 34.5 ± 0.1 μm, n = 1675, from 4 biological replicates). Black bars indicate the population of DRG neurons positive for both NF200 and Rem2-immunoreactivity (mean = 36.5 ± 0.2 μm, n = 501, from 4 biological replicates).

Figure 5. Post-translational modification of Rem2 by phosphorylation.

(A) Representative Rem2 western blot (left) using sample lysates from striatum (STR), hippocampus (HC), and cortex (CTX), and chemiluminescence measurements of each lane (right). Line measurements represent the average signal across the entire width of each lane. (B) Representative Rem2 western blot of striatum lysates used in dephosphorylation experiment (left) and corresponding line graphs of chemiluminescence (right). Striatum lysates were untreated, treated with lambda phosphatase (λ-phos.), treated without lambda phosphatase (no λ-phos.), or treated with lambda phosphatase and phosphatase inhibitors (λ-phos.+PI). (C) Representative Rem2 western blot of hippocampus lysates used in dephosphorylation experiment (left) and corresponding line graphs of chemiluminescence (right). Lane labels are as described in (B). Images are representative of at least 3 independent experiments.

Figure 6. Developmental expression of Rem2 mRNA and protein.

(A) Postnatal expression of Rem2 mRNA in striatum, hippocampus and cortex. Total RNA was isolated from the indicated brain regions at postnatal day (PND) 1 (n = 4), 7 (n = 6), 14 (n = 6), and 21(n = 6), and Rem2 mRNA was quantified by qPCR. Rem2 gene expression was normalized to the geometric mean of the three “housekeeping” genes, Actb, Gapdh, and Tbp. Rem2 gene expression varied significantly across postnatal development in all brain regions examined. *p < 0.05 and **p < 0.01, Kruskal-Wallis test followed by Dunn’s multiple comparisons test. (B) Representative images of Rem2 immunofluorescence in dorsal striatum at PND1, PND3, and PND6. Arrows in PND1 and PND3 images indicated patchy enrichment of Rem2 protein and arrowheads in PND6 images indicate somal rim-like expression. Noteworthy, the bright structures present in the images represent auto-fluorescent blood vessels. Scale bars are 50 μm and the same in all images. cc, corpus callosum; CTX, cortex; STR, striatum.