Hedgehog Pathway Activation Alters Ciliary Signaling in Primary Hypothalamic Cultures

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.

Characterization of primary cilia features reveal cell-type specific variability in in vitro models of osteogenic and chondrogenic differentiation

“…6B and 6C). This is contrary to studies where SAG treatment in neuronal cell-types did not alter cilia length or neuronal activity (Bansal et al, 2019). Modifying cilia length in neural cell-types also did not alter Hh-dependent patterning (Bangs et al, 2015;Bonnafe et al, 2004).…”

Artificial Intelligence Approaches to Assessing Primary Cilia

“…GA3 aids in combining multiple tools in a single workflow to build customized routines for each experiment. We cultured neurons from the hypothalamus and hippocampus from neonatal mice for 10 days and immunolabelled them with the cilia marker adenylate cyclase III (ACIII) 41,21 . After confocal imaging, we measured the lengths of ACIII positive cilia using GA3 after segmenting with segment.ai.…”

“…We next tested the ability of Ai to measure cilia lengths in primary neuronal cultures. We cultured neurons from the hypothalamus and hippocampus of neonatal mice for 10 days and immunolabelled them with the cilia marker adenylate cyclase III (ACIII) 41, 21 . When analyzing neuronal cultures, we found it useful to apply a filter prior to statistically analyzing the lengths.…”

Regulation of Brain Primary Cilia Length by MCH Signaling: Evidence from Pharmacological, Genetic, Optogenetic and Chemogenic Manipulations

“…Thus, the activation of MCH neurons may regulate cilia indirectly through its interaction with serotonin system. Interestingly, MCHR1 and 5-HT6 are among the very few GPCRs that preferentially localize to primary cilia [59], and the inhibition of 5-HT6 receptors is known to shorten cilia length [39, 60].…”