Characterisation of primary cilia in human hypothalamic neurons

Primary cilia are sensory organelles that protrude from the plasma membrane of most mammalian cells, including neurons. Among their roles, they are recognised to be involved in the control of feeding and energy expenditure by the hypothalamus. However, how cilia exert their role as signalling hubs in those neurons remains largely unknown. We set out to better understand the role of primary cilia in the control of food intake by hypothalamic neurons. We hypothesised that primary cilia might act as a signal integration centre where different G protein-coupled receptors (GPCRs) are present in close proximity in an isolated cell compartment to then regulate neuronal activity (1). We also speculated that changes in the length of primary cilia would affect ciliary signalling and its effect on cell state (2). We addressed the first hypothesis by characterising the ciliary proteome of human hypothalamic neurons to identify the receptors that localise to these cilia. Gaining better understanding of the signal cilia respond to and the signalling pathways they coordinate, represents a first step in defining the role of cilia in neurons. This was achieved by generating human-induced pluripotent stem cell (hiPSC) lines stably expressing a transmembrane construct that both enables fluorescently labelling of cilia and drives the expression of a peroxidase to allow the streptavidin pull-down and identification of ciliary proteins in human neurons. To address the second hypothesis, we developed an image analysis pipeline for the automated measurement of changes in length of primary cilia in neurons derived from the cilia-targeted hiPSC line. We used this pipeline to screen for annotated compounds with known targets active on the CNS and identify some that produced a change in ciliary length. We hope that these results will improve our understanding of the mechanisms behind regulation of cilia length and will provide a new tool to the field. In summary, we approached our aim by describing both the signalling pathways most enriched in hypothalamic cilia and developing a method to assess cilia length changes. Taken together, these results might represent a first step in developing new therapeutic approaches that target primary cilia in metabolic-related diseases such as obesity.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Macarelli, Viviana

Advisor dc:contributor.advisor

• Merkle, Florian

• primary cilia
• hiPSC
• Hypothalamus
• Obesity

Licence

• https://www.repository.cam.ac.uk/bitstreams/2adb637f-4980-49c2-b38a-218f8dcf4d5e/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.114179

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/377320