CaMKII-Dependent Phosphorylation of the GTPase Rem2 Is Required to Restrict Dendritic Complexity

Abstract

The morphogenesis of the dendritic arbor is a critical aspect of neuronal development, ensuring that proper neural networks are formed. However, the molecular mechanisms that underlie this dendritic remodeling remain largely obscure. We previously established the activity-regulated GTPase Rem2 as a negative regulator of dendritic complexity. In this study, we identify a signaling pathway whereby Rem2 regulates dendritic arborization through interactions with Ca2+/calmodulin (CaM)-dependent kinases (CaMKs) in rat hippocampal neurons. Specifically, we demonstrate that Rem2 functions downstream of CaMKII but upstream of CaMKIV in a pathway that restricts dendritic complexity. Further, we show that Rem2 is a novel substrate of CaMKII and that phosphorylation of Rem2 by CaMKII regulates Rem2 function and subcellular localization. Overall, our results describe a unique signal transduction network through which Rem2 and CaMKs function to restrict dendritic complexity.

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

“…However, neither GAPs nor Guanine exchange factors (GEFs) have been identified for Rem2 or the other RGK proteins thus far. These data and other lines of evidence [14] suggest that Rem2 does not transduce signals like a classical GTPase, regulated by its nucleotide binding state, but is instead activated by other means, for example by post-translational modification [20]. In addition, in both cultured rat neurons and in Xenopus optic tectum, Rem2 mRNA expression is upregulated in response to neuronal depolarization [15].…”

“…To perform these experiments, we utilized our previously established assay to quantify excitatory synapse formation [14–18]. Hippocampal neurons were sparsely transfected with GFP and the siRNA Smartpools using the calcium phosphate method such that the vast majority of synaptic connections made onto the transfected neurons are from non-transfected, “wildtype” neurons.…”

“…This approach differs from the high percentage of transduced Rem2 flx/flx cultured neurons achieved with Cre-expressing AAV infection which was necessary for our RNA-seq experiments. As a result, we also included a well-validated RNAi reagent that specifically targets Rem2 [14–18] in our sparse transfection paradigm as a positive control for decreased excitatory synapse density. We have previously shown that the decreased synapse density observed with transfection of this Rem2 shRNA can be rescued by co-transfection of a vector encoding an RNAi-resistant Rem2 cDNA [14–17].…”

“…Rem2 is a positive regulator of excitatory synapse formation in cultured rodent hippocampal neurons and an activity-dependent, negative regulator of dendritic complexity in both cultured rodent neurons and in Xenopus laevis optic tectum [14–18]. Rem2 is a member of the Rad/Rem/Gem/Kir (RGK) family of non-canonical Ras-like GTPases and is primarily expressed in the nervous system [19].…”

Regulation of dendrite morphogenesis by extrinsic cues

“…Upon membrane depolarization, Rem2 and activated CaMKII associate, allowing CaMKII to phosphorylate Rem2. The CaMKII-induced phosphorylation of Rem2 triggers its translocation to the nucleus, where it suppresses CaMKIV signaling and dendrite arborization [96]. …”