5-HT 6 receptor blockade regulates primary cilia morphology in striatal neurons

Abstract

The 5-HT6 receptor has been implicated in a variety of cognitive processes including habitual behaviors, learning, and memory. It is found almost exclusively in the brain, is expressed abundantly in striatum, and localizes to neuronal primary cilia. Primary cilia are antenna-like, sensory organelles found on most neurons that receive both chemical and mechanical signals from other cells and the surrounding environment; however, the effect of 5-HT6 receptor function on cellular morphology has not been examined. We confirmed that 5-HT6 receptors were localized to primary cilia in wild-type (WT) but not 5-HT6 knockout (5-HT6KO) in both native mouse brain tissue and primary cultured striatal neurons then used primary neurons cultured from WT or 5-HT6KO mice to study the function of these receptors. Selective 5-HT6 antagonists reduced cilia length in neurons cultured from wild-type mice in a concentration and time-dependent manner without altering dendrites, but had no effect on cilia length in 5-HT6KO cultured neurons. Varying the expression levels of heterologously expressed 5-HT6 receptors affected the fidelity of ciliary localization in both WT and 5-HT6KO neurons; overexpression lead to increasing amounts of 5-HT6 localization outside of the cilia but did not alter cilia morphology. Introducing discrete mutations into the third cytoplasmic loop of the 5-HT6 receptor greatly reduced, but did not entirely eliminate, trafficking of the 5-HT6 receptor to primary cilia. These data suggest that blocking 5-HT6 receptor activity reduces the length of primary cilia and that mechanisms that regulate trafficking of 5-HT6 receptors to cilia are more complex than previously thought.

Restoration of Physiological Expression of 5-HT6 Receptor into the Primary Cilia of Null Mutant Neurons Lengthens Both Primary Cilia and Dendrites

“…Recently, we measured the effect of specific drugs on endogenous 5-HT6R and found that selective antagonists shortened primary cilia, while none of the drug treatments increased dendritic outgrowth (Brodsky et al, 2017). During this study, we coincidentally found that increasing amounts of exogenous 5-HT6R transfection led to drastically increased ectopic expression outside of the primary cilia.…”

“…Published on April 20, 2018as DOI: 10.1124 at ASPET Journals on April 27, 2019 molpharm.aspetjournals.org Downloaded from deleted a potential cilia targeting sequence on the third intracellular loop decreased 5-HT6R trafficking to cilia (Berbari et al, 2008;Brodsky et al, 2017). However, these mutations were unable to prevent cilia targeting entirely; highlighting the robust proclivity for 5-HT6R to traffic into primary cilia.…”

“…Despite mounting evidence for the therapeutic value of this receptor, little is known about the specific mechanisms underlying 5-HT6R signaling. 5-HT6R is expressed nearly exclusively in CNS, in a limited number of brain regions including cortex, hippocampus, and most abundantly in the striatum (East et al, 2002;Hirst et al, 2003;Brodsky et al, 2017); however, localization of 5-HT6R in vivo is notoriously difficult because commercially available antibodies with sufficient specificity have been inconsistently available. Additionally, the subcellular localization of the receptor can be easily overlooked because 5-HT6R is the only serotonin receptor that localizes to the primary neuronal cilium (Brailov et al, 2000;Berbari et al, 2008;Domire and Mykytyn, 2009;Brodsky et al, 2017;Hu et al, 2017).…”

“…5-HT6R is expressed nearly exclusively in CNS, in a limited number of brain regions including cortex, hippocampus, and most abundantly in the striatum (East et al, 2002;Hirst et al, 2003;Brodsky et al, 2017); however, localization of 5-HT6R in vivo is notoriously difficult because commercially available antibodies with sufficient specificity have been inconsistently available. Additionally, the subcellular localization of the receptor can be easily overlooked because 5-HT6R is the only serotonin receptor that localizes to the primary neuronal cilium (Brailov et al, 2000;Berbari et al, 2008;Domire and Mykytyn, 2009;Brodsky et al, 2017;Hu et al, 2017). Primary cilia were described over a century ago and are ubiquitous to almost all non-dividing mammalian cells, including neurons, yet these singular non-motile appendages were frequently misinterpreted as vestigial organelles until recently (Fuchs and Schwark, 2004;Louvi and Grove, 2011).…”

“…Shortly after the cloning and identification of 5-HT6Rs, expression of 5-HT6Rs was reported to be faintly scattered throughout dendrites particularly in striatum, but soon it was recognized that 5-HT6Rs are predominantly found in the primary neuronal cilia (Ruat et al, 1993;Kohen et al, 1996;Hamon et al, 1999;Berbari et al, 2008;Brodsky et al, 2017). As a G-protein coupled receptor, 5-HT6 is positively coupled with G-proteins that stimulate production of cAMP, presumably through adenylyl cyclase III (AC3), the only adenylyl cyclase known to localize only to primary neuronal cilia (Sebben et al, 1994;Kohen et al, 2001;Kang et al, 2005;Bishop et al, 2007;Domire and Mykytyn, 2009).…”