Rem2 signaling affects neuronal structure and function in part by regulation of gene expression

Abstract

The central nervous system has the remarkable ability to convert changes in the environment in the form of sensory experience into long-term alterations in synaptic connections and dendritic arborization, in part through changes in gene expression. Surprisingly, the molecular mechanisms that translate neuronal activity into changes in neuronal connectivity and morphology remain elusive. Rem2, a member of the Rad/Rem/Rem2/Gem/Kir (RGK) subfamily of small Ras-like GTPases, is a positive regulator of synapse formation and negative regulator of dendritic arborization. Here we identify that one output of Rem2 signaling is the regulation of gene expression. Specifically, we demonstrate that Rem2 signaling modulates the expression of genes required for a variety of cellular processes from neurite extension to synapse formation and synaptic function. Our results highlight Rem2 as a unique molecule that transduces changes in neuronal activity detected at the cell membrane to morphologically relevant changes in gene expression in the nucleus.

Keywords: Activity-regulated; Excitatory synapse; Glypican-5 (Gpc5); Lrrtm4; Rem2.

Figure 1. Knockout of Rem2 results in changes in gene expression

A) Fold change in Rem2 mRNA by qPCR after 5 days of Cre-mediated Rem2 exon 2 and 3 deletion. mRNA levels were first normalized to Actb and then to the control condition within each replicate then averaged across biological replicates. Data is presented as mean ± SEM. Rem2 KO condition is significantly decreased from control by student’s t-test (P<0.05) B) RNA-seq results: Genes up or downregulated greater than 2-fold with Rem2 knockout. Orange bars indicate genes whose expression change with Rem2 knockout was validated by qPCR while the white bar shows the gene whose expression profile that did not recapitulate the RNA-seq results. The gray bars show genes that were not chosen for qPCR validation. Data is presented as the mean fold change across biological replicates in FPKM (Fragments per kilobase of transcript per million mapped reads) from control ± SEM. C) Validation of gene expression changes (of the genes shown in orange in B) using qPCR. Fold change in mRNA expression for each indicated gene was averaged across 3 biological replicates. First, mRNA levels were normalized to Actb and then to the control condition. Data is presented as mean ± SEM. All Rem2 KO conditions are significantly increased (top row) or decreased (bottom row) from controls by student’s t-test (P<0.05).

Figure 2. Lrrtm4 and Gpc5 are required for proper excitatory synapse formation

A) Immunostaining for PSD-95 (red) and Synapsin I (blue) in the dendrites of transfected neurons. A synapse is defined as the overlap of red and blue puncta (shown in white) on a GFP positive dendrite. Scale bar = 5µm. B) Quantification of PSD-95/Synapsin puncta for neurons transfected with GFP and either: a pSuper empty vector (Control), shRNA targeting Rem2 (Rem2 RNAi), shRNA targeting Gpc5 (Gpc5 RNAi), shRNA targeting Gpc5 and an RNAi resistant Gpc5 cDNA (Gpc5 Rescue), shRNA targeting Lrrtm4 (Lrrtm4 RNAi) or a shRNA targeting Lrrtm4 and an RNAi resistant Lrrtm4 cDNA (Lrrtm4 Rescue). Data is presented as mean of normalized PSD-95/Synapsin puncta density ± SEM. Asterisks indicate p<0.05 compared to control by two-way ANOVA with Tukey post-hoc test. Normalized values are the average of two biological replicates and n ≥ 30 neurons for each condition.

Figure 3. Overexpression of Gpc5 or Lrrtm4 restores the Rem2 RNAi-dependent decrease in synapse density

A) Immunostaining for PSD-95 (red) and Synapsin I (blue) in the dendrites of transfected neurons. A synapse is defined as the overlap of red and blue puncta (shown in white) on a GFP positive dendrite. Scale bar = 5µm. B) Quantification of PSD-95/Synapsin puncta for neurons transfected with GFP and either: (Top) a pSuper empty vector (Control), shRNA targeting Rem2 (Rem2 RNAi), Gpc5 cDNA (Gpc5 OE), or shRNA targeting Rem2 and Gpc5 cDNA (Rem2 RNAi + Gpc5 OE) and (Bottom) a pSuper empty vector (Control), shRNA targeting Rem2 (Rem2 RNAi), Lrrtm4 cDNA (Lrrtm4 OE), or shRNA targeting Rem2 and Lrrtm4 cDNA (Rem2 RNAi + Lrrtm4 OE). Data is presented as mean of normalized PSD-95/Synapsin puncta density ± SEM. * indicates p < 0.005 and # indicates P < 0.085 between conditions indicated by bars using two-way ANOVA with Tukey post-hoc test. In both top and bottom graphs, control versus Rem2 RNAi was different at P < 0.005 but this was not indicated on the graph for clarity. Normalized values are the average of ≥ 2 biological replicates and n ≥ 32 neurons for each condition.

Figure 4. Rem2 knockout causes changes in activity-dependent gene expression

A) Fold change in Rem2 or Fos mRNA expression by qPCR averaged across biological replicates. mRNA levels were first normalized to Actb and then to the control untreated condition within each biological replicate. Data is presented as mean ± SEM. B) Venn diagram showing the number of genes upregulated ≥ 2-fold following KCl stimulation. The 316 genes represented by the yellow circle were upregulated by KCl treatment in control neurons. Of the 316 genes upregulated in control neurons, 94 of those genes were only upregulated ≥ 2-fold in the control condition. The blue circle represents the 345 genes upregulated ≥ 2-fold by KCl stimulation in the Rem2 KO condition. 123 of the 345 genes were upregulated ≥ 2-fold in the Rem2 KO condition only. C) Validation of expression profiles of genes identified in panel B. Fold change in mRNA expression by qPCR for each indicated gene was averaged across biological replicates. Hollow bars represent untreated conditions while yellow bars represent KCl treated conditions for genes promoted by Rem2 signaling and blue bars represent KCl treated conditions for genes repressed by Rem2 signaling. mRNA levels were first normalized to Actb and then to the control untreated (UT) condition within each biological replicate. Data is presented as mean ± SEM. Data shown for the untreated control and Rem2 KO conditions for Rem2 and Slc7a1 are the same data as shown in Figure 1A and 1C respectively. The data is included here again for clarity.

Figure 5. Rem2 signaling promotes the expression of genes encoding membrane targeted proteins and represses the expression of genes encoding cytoplasmic proteins that regulate the cytoskeleton

DAVID Gene Ontology (GO) Cell Component analysis for genes whose expression is promoted by Rem2 signaling (Left, purple) or repressed by Rem2 signaling (Right, blue). Only the GO terms with a p value <0.05 by Fisher’s Exact test were analyzed. The number of genes identified for each GO term are shown by each bar. The P value for each GO term is shown next to the corresponding bar.

Figure 6. Rem2 specifically regulates the activation of activity-dependent transcription factors

Quantification of firefly/renilla luciferase luminescence acquired from cortical neuron lysate from (A) CREB (B) SP1, (C) NFAT, (D) MEF2, (E) NF-κB or (F) RARE reporter transfected cells. Filled bars represent conditions treated with 55mM KCl for 6 hours before harvesting cell lysate while hollow bars represent untreated (UT) conditions. For each reporter, all conditions were normalized to the control UT condition within each biological replicate and averaged between replicates. Data is presented as mean of normalized luciferase activity ± SEM. * indicates p<0.05 by Student’s t-test between KCl treated conditions. # indicates p<0.05 by Student’s t-test between UT conditions. Each bar represents at least 3 biological replicates assayed in triplicate.