A Loss-Of-Function Analysis Reveals That Endogenous Rem2 Promotes Functional Glutamatergic Synapse Formation and Restricts Dendritic Complexity

Abstract

Rem2 is a member of the RGK family of small Ras-like GTPases whose expression and function is regulated by neuronal activity in the brain. A number of questions still remain as to the endogenous functions of Rem2 in neurons. RNAi-mediated Rem2 knockdown leads to an increase in dendritic complexity and a decrease in functional excitatory synapses, though a recent report challenged the specificity of Rem2-targeted RNAi reagents. In addition, overexpression in a number of cell types has shown that Rem2 can inhibit voltage-gated calcium channel (VGCC) function, while studies employing RNAi-mediated knockdown of Rem2 have failed to observe a corresponding enhancement of VGCC function. To further investigate these discrepancies and determine the endogenous function of Rem2, we took a comprehensive, loss-of-function approach utilizing two independent, validated Rem2-targeted shRNAs to analyze Rem2 function. We sought to investigate the consequence of endogenous Rem2 knockdown by focusing on the three reported functions of Rem2 in neurons: regulation of synapse formation, dendritic morphology, and voltage-gated calcium channels. We conclude that endogenous Rem2 is a positive regulator of functional, excitatory synapse development and a negative regulator of dendritic complexity. In addition, while we are unable to reach a definitive conclusion as to whether the regulation of VGCCs is an endogenous function of Rem2, our study reports important data regarding RNAi reagents for use in future investigation of this issue.

Rem2 Is an Activity-Dependent Negative Regulator of Dendritic ComplexityIn Vivo

“…For control conditions, neurons were also transfected with empty pSuper vector at 33 ng/well or 100 ng/well. For Rem2 RNAi experiments, neurons were also transfected with a pSuper-shRNA plasmid containing an shRNA against Rem2 (Paradis et al, 2007;Ghiretti and Paradis, 2011;Moore et al, 2013) at 33 ng/well. For Rem2 overexpression (OE) experiments, neurons were also transfected with a pCMV-myc-Rem2 cDNA (Ghiretti and Paradis, 2011) at 100 ng/well.…”

“…Flavell and Greenberg, 2008). Given that Rem2 is both upregulated by activity and, functionally, a negative regulator of dendritic complexity (Ghiretti and Paradis, 2011;Ghiretti et al, 2013), we reasoned that a calcium-mediated increase in Rem2 expression might serve to restrict an activity-dependent increase in dendritic arborization. To directly test this hypothesis, we depolarized neurons alone or in combination with RNAi-mediated knockdown of endogenous Rem2 or overexpression (OE) of a Rem2 cDNA in primary cortical cultures and assayed the net effect on dendritic morphology.…”

“…To directly test this hypothesis, we depolarized neurons alone or in combination with RNAi-mediated knockdown of endogenous Rem2 or overexpression (OE) of a Rem2 cDNA in primary cortical cultures and assayed the net effect on dendritic morphology. The shRNA against Rem2 and the Rem2 cDNA have been extensively validated (Ghiretti and Paradis, 2011; Ghiretti et al, 2013; Moore et al, 2013). In addition, recapitulation of the Rem2 RNAi dendritic complexity phenotype by morpholino mediated Rem2 knock-out in X. laevis (Fig.…”

“…Our previous work has shown that the GTPase Rem2 restricts dendritic complexity (a term that describes both the length and degree of branching of the dendritic arbor) in primary cultures of hippocampal neurons (Ghiretti and Paradis, 2011;Ghiretti et al, 2013). Rem2 is a member of the Rad/Rem/Rem2/Gem/Kir (RGK) protein family, a Ras-related subfamily of small GTPases.…”

“…In addition, mRNA expression of Rem2 and other RGK family members is upregulated by extracellular stimuli such as neuronal depolarization, mitogens, and glucose (Maguire et al, 1994;Finlin et al, 2005;Paradis et al, 2007), suggesting that this may be a defining feature of the family. Recently, we demonstrated that Rem2 is a novel substrate for CaMKII, and that phosphorylation of key serine residues by CaMKII is required for Rem2 inhibition of dendritic complexity (Ghiretti et al, 2013). Thus, Rem2 function is regulated by neuronal activity in at least two ways: Rem2 transcription is upregulated in response to neuronal depolarization, and Rem2 is a downstream target of CaMKII signaling.…”