Ciliary GPCR‐based transcriptome as a key regulator of cilia length control

Abstract

The primary cilium is a plasma membrane-protruding sensory organelle that efficiently conveys signaling cascades in a highly ordered microenvironment. Its signaling is mediated, in part, by a limited set of GPCRs preferentially enriched in the cilium membrane. This includes melanin-concentrating hormone (MCH) receptor 1 (MCHR1), which plays a role in feeding and mood. In addition to its receptor composition, the length of the cilium is a characteristic parameter that is implicated in its function. We previously found that MCH can dynamically shorten cilia length via the Gi/o and Akt pathways in both MCHR1-expressing hTERT-RPE1 cells (hRPE1 cells) and rat hippocampal neurons. However, the detailed mechanisms by which MCH regulates cilia length through ciliary MCHR1 remains unclear. In this study, we aimed to determine the transcriptome changes in MCHR1-expressing hRPE1 cells in response to MCH to identify the target molecules involved in cilia length regulation via MCHR1 activation. RNA-sequencing analysis of ciliated cells subjected to MCH treatment showed upregulation of 424 genes and downregulation of 112 genes compared with static control cells. Validation by quantitative real-time PCR, knocking down, and CRISPR/Cas9-mediated knockout technology identified a molecule, PDZ and LIM domain-containing protein 5 (PDLIM5). Thus, it was considered as the most significant key factor for MCHR1-mediated shortening of cilia length. Additional analyses revealed that the actin-binding protein alpha-actinin 1/4 is a crucial downstream target of the PDLIM5 signaling pathway that exerts an effect on MCHR1-induced cilia shortening. In the endogenous MCHR1-expressing hippocampus, transcriptional upregulation of PDLIM5 and actinin 1/4, following the application of MCH, was detected when the MCHR1-positive cilia were shortened. Together, our transcriptome study based on ciliary MCHR1 function uncovered a novel and important regulatory step underlying cilia length control. These results will potentially serve as a basis for understanding the mechanism underlying the development of obesity and mood disorders.

Physiological Condition Dependent Changes in Ciliary GPCR Localization in the Brain

“…To understand if cilia GPCRs dynamically localize in vivo under physiological contexts associated with receptor activity, we initially chose to assess the known ciliary GPCR, MCHR1. We assessed its ciliary localization alone and in conjunction with the broadly expressed CNS ciliary membrane-associated adenylate cyclase 3 (ACIII) (Bishop, Berbari et al 2007, Berbari, Johnson et al 2008, Hsiao, Munoz-Estrada et al 2021, Kobayashi, Tomoshige et al 2021, Alhassen, Kobayashi et al 2022). We confirmed our MCHR1 antibody immunofluorescence specificity by observing the loss of ciliary staining in a Mchr1 knockout allele mouse brain and colocalization with a Mchr1-mCherry knock-in fusion allele mouse ( Supplemental Figure 1A and B ) (Jasso, Kamba et al 2021).…”

Aging Associates with Cilium Elongation and Dysfunction in Kidney and Pancreas

“…Activation of the melanin-concentrating hormone receptor 1 for instance dynamically shortens primary cilia, which possibly contributes to feeding control. [51] In blood vessels, changes in shear stress alter ciliary length with increased blood flow resulting in shorter and slower blood flow in longer cilia. [52,53] Such altered cilia length likely impacts on cilia function by several means.…”

Neuronal cilia in energy homeostasis

“…For example, under fasted conditions, MCH increases in the lateral hypothalamus ( Segal-Lieberman et al, 2003 ; Simon et al, 2018 ). There is also growing evidence to support the biological importance of ciliary GPCR signaling, such as MCHR1, in regulating cilia length and neuronal function ( Miki et al, 2019 ; Kobayashi et al, 2020 ; Kobayashi et al, 2021 ). Ciliary MCHR1 may regulate different physiological conditions, such as feeding and be a potential target for conditions with impaired cilia function, such as ciliopathies.…”