An RNAi-Based Approach Identifies Molecules Required for Glutamatergic and GABAergic Synapse Development

Abstract

We report the results of a genetic screen to identify molecules important for synapse formation and/or maintenance. siRNAs were used to decrease the expression of candidate genes in neurons, and synapse development was assessed. We surveyed 22 cadherin family members and demonstrated distinct roles for cadherin-11 and cadherin-13 in synapse development. Our screen also revealed roles for the class 4 Semaphorins Sema4B and Sema4D in the development of glutamatergic and/or GABAergic synapses. We found that Sema4D affects the formation of GABAergic, but not glutamatergic, synapses. Our screen also identified the activity-regulated small GTPase Rem2 as a regulator of synapse development. A known calcium channel modulator, Rem2 may function as part of a homeostatic mechanism that controls synapse number. These experiments establish the feasibility of RNAi screens to characterize the mechanisms that control mammalian neuronal development and to identify components of the genetic program that regulate synapse formation and/or maintenance.

The RGK family of GTP-binding proteins: Regulators of voltage-dependent calcium channels and cytoskeleton remodeling

“…Among the most unique features of RGK proteins is their ability to be transcriptionally regulated [3,5,6,11,[20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] (Table 2). Indeed, Gem/Kir was discovered as a gene upregulated in human T cells after stimulation with mitogens [5] or in BCR-Abl-transformed B cells [11].…”

“…The other RGK subfamily members also demonstrate tissue and molecule-specific transcriptional regulation [3,4,6,20,21,24,25,27,[30][31][32][33][34][35]. Rem is expressed predominantly in cardiac muscle, but also at more modest levels in lung, kidney, and skeletal muscle [3]; Rem2 is highly expressed in the brain and kidney, but also in neuroendocrine tissues [4]; Rad is found in abundance in cardiac and skeletal muscle [6]; while Gem/Kir is found in a diverse set of tissues, including myeloid cells, kidney, liver, and lung [5].…”

“…Similarly, Gem/Kir expression is induced in the same cell line under elevated glucose, or following insulin and KCl treatment [28]. Rem2 is upregulated in developing neurons and siRNA-mediated Rem2 silencing profoundly inhibits the development of glutamatergic and GABAergic synapses [30]. Since Rem2 is the only RGK protein abundantly expressed in neuronal tissues [4], Rem2 signaling appears crucial for synapse development and maturation.…”

Direct inhibition of P/Q-type voltage-gated Ca 2+ channels by Gem does not require a direct Gem/Ca v β interaction

“…For example, in the heart, dominant negative suppression of endogenous Rad increases L-type Ca 2+ -channel currents and action-potential duration in cardiac cells and produces longer QT intervals and arrhythmias (24). Rem2 prevents glucose-stimulated insulin secretion in pancreatic β cells (19) and regulates the development of both excitatory and inhibitory synapses, presumably through a feedback loop that controls Ca 2+ influx (32). Finally, alteration in Gem regulation of Ca v 1.2 L-type Ca 2+ channels may contribute to certain neural phenotypes displayed in Timothy Syndrome, a genetic disorder characterized by cardiac and neurological defects and autism (33).…”

Multifunctional Role of Protein Kinase C in Regulating the Formation and Maturation of Specific Synapses

“…Downstream signaling molecules may include small GTPases (Elia et al, 2006;Paradis et al, 2007) and specific kinases or phosphatases (Kaufmann et al, 2002;Patel et al, 2006;Sytnyk et al, 2006) that affect the organization of the cytoskeleton required for the formation of synapses. One kinase that might regulate synaptic adhesion and assembly is PKC (Loeb et al, 1998;Xia et al, 2003;Schmidt et al, 2004;Kolkova et al, 2005).…”