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

Anna R Moore,Amy E Ghiretti,Suzanne Paradis,Steven Barnes

doi:10.1371/journal.pone.0074751

Anna R Moore, Amy E Ghiretti + Show 2 more

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https://doi.org/10.1371/journal.pone.0074751

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Journal: PloS one	Publication Date: Aug 26, 2013
Citations: 19	License type: CC BY 4.0

Affiliation: Brandeis University

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.

Highlights

The RGK (Ras, Rem, Rem2, Gem/Kir) protein family is a subclass of small Ras-like GTPases structurally distinct from canonical GTPases
We examined voltage-gated calcium channel (VGCC) current amplitude and find that while we can recapitulate the decrease in calcium current previously observed with Rem2 overexpression, Rem2 knockdown with only one of the two shRNAs used in this study demonstrates the predicted phenotype on VGCC current upon Rem2 knockdown: an increase in calcium current amplitude
We first confirmed that the second hairpin construct could sufficiently knock down Rem2 and that the RNAi-resistant cDNA, which had been engineered to be resistant to both shRNAs, was resistant to RNAi 2 by Western blotting of heterologous cell lysate

Summary

Introduction

The RGK (Ras, Rem, Rem, Gem/Kir) protein family is a subclass of small Ras-like GTPases structurally distinct from canonical GTPases. RGK proteins have been implicated in mediating cytoskeletal rearrangements [2,3,4,5] and inhibition of voltage-gated calcium channel currents [6,7,8,9,10]. Rem is highly expressed in the brain [12] and is upregulated in response to neuronal depolarization [15] Both loss-of-function and overexpression approaches have been used to try to determine the physiological function of Rem. Overexpression studies have shown that Rem can inhibit voltage-gated calcium channel currents in a variety of cell types [8,16,17,18,19], as reported for other RGK proteins [20]. A major caveat of these studies is that they are based on overexpression of Rem protein

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