Hedgehog Pathway Activation Alters Ciliary Signaling in Primary Hypothalamic Cultures.

Ruchi Bansal,Patrick J Antonellis,Nicolas F Berbari,Theodore R Cummins,Staci E Engle,Anthony J Baucum,Logan S Whitehouse,Jeremy F Reiter

doi:10.3389/fncel.2019.00266

Abstract

Primary cilia dysfunction has been associated with hyperphagia and obesity in both ciliopathy patients and mouse models of cilia perturbation. Neurons throughout the brain possess these solitary cellular appendages, including in the feeding centers of the hypothalamus. Several cell biology questions associated with primary neuronal cilia signaling are challenging to address in vivo. Here we utilize primary hypothalamic neuronal cultures to study ciliary signaling in relevant cell types. Importantly, these cultures contain neuronal populations critical for appetite and satiety such as pro-opiomelanocortin (POMC) and agouti related peptide (AgRP) expressing neurons and are thus useful for studying signaling involved in feeding behavior. Correspondingly, these cultured neurons also display electrophysiological activity and respond to both local and peripheral signals that act on the hypothalamus to influence feeding behaviors, such as leptin and melanin concentrating hormone (MCH). Interestingly, we found that cilia mediated hedgehog signaling, generally associated with developmental processes, can influence ciliary GPCR signaling (Mchr1) in terminally differentiated neurons. Specifically, pharmacological activation of the hedgehog-signaling pathway using the smoothened agonist, SAG, attenuated the ability of neurons to respond to ligands (MCH) of ciliary GPCRs. Understanding how the hedgehog pathway influences cilia GPCR signaling in terminally differentiated neurons could reveal the molecular mechanisms associated with clinical features of ciliopathies, such as hyperphagia-associated obesity.

Highlights

The hypothalamus is a complex brain region consisting of several nuclei that regulate food and water intake, circadian rhythm, sexual behavior, and body temperature
We confirmed that hypothalamic derived neurons expressed hypothalamic genes such as β-endorphin and that they responded to central (MCH) and peripheral signals with changes in spontaneous firing as occurs in slice recordings
Dysfunction of primary cilia leads to hyperphagia and obesity in patients with genetic disorders such as Bardet-Biedl syndrome (BBS) and Alström syndrome (ALMS) (Vaisse et al, 2017)

Summary

Introduction

The hypothalamus is a complex brain region consisting of several nuclei that regulate food and water intake, circadian rhythm, sexual behavior, and body temperature. The Ciliary Signaling in Hypothalamic Cultures mechanisms underlying genetic disorders associated with hyperphagia and obesity, such as Bardet-Biedl syndrome (BBS) and Alström syndrome (ALMS) remain unclear. Both BBS and ALMS are classified as ciliopathies, as primary cilia dysfunction is thought to be the cellular etiology of the disorders (Vaisse et al, 2017). Hypothalamic neuronal cilia appear critical to normal feeding behavior Both conditional mouse models of BBS and cilia loss become hyperphagic and obese (Mykytyn et al, 2004; Davenport et al, 2007; Berbari et al, 2013; Guo et al, 2016). How primary cilia on these neurons regulate food intake is poorly understood, but several G-protein coupled receptors (GPCRs) have been found to preferentially localize to primary cilia, including melanin concentrating hormone receptor 1 (Mchr1) and melanocortin receptor 4 (Mc4r), both of which are expressed in the hypothalamus along with their ligands, MCH and alpha-melanocyte stimulating hormone (α-MSH), respectively, and have established roles in feeding behavior and obesity (Desy and Pelletier, 1978; Bittencourt et al, 1992; Berbari et al, 2008a; Siljee et al, 2018)

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