Cell-type-specific repression reveals MC4R neurons as a site of action for ciliary obesity genes.

Objectives

Understanding how obesity-associated genes function within defined neuronal subpopulations remains a major barrier to translating human genetics into pathophysiological mechanisms. Melanocortin-4 receptor-expressing (MC4R) neurons are key regulators of energy homeostasis and represent a potential site of action for these genes. Here, we investigated whether obesity-associated ciliary genes regulate body weight through MC4R neurons in vivo, using a Cre-dependent CRISPR interference (CRISPRi) strategy that enables temporal, spatial, and cell-type-specific gene repression without permanent DNA modification.

Methods

Gene repression was achieved in MC4R neurons using a Cre-dependent CRISPRi system with AAV-delivered sgRNAs, and physiological outcomes, including body weight and body composition, were assessed following gene downregulation.

Results

CRISPRi-mediated repression of Mc4r in adult PVNMC4R neurons reduced transcript and protein levels and induced progressive obesity, validating the approach. Downregulation of the ciliary adenylyl cyclase Adcy3 specifically in adult PVNMC4R neurons was sufficient to drive weight gain, establishing a cell-type-specific requirement for ADCY3 in body-weight regulation. An in vivo CRISPRi screen of obesity-associated ciliopathy genes (Bbs10, Inpp5e, Ankrd26) demonstrated that their repression in MC4R neurons is sufficient to promote obesity, identifying these neurons as a convergent site linking human ciliopathies to obesity.

Conclusions

These findings define MC4R neurons as a critical neuronal population through which multiple obesity-associated ciliary genes act and establish a direct link between ciliary function in defined neuronal populations and organismal energy balance. This work provides insight into the neural mechanisms underlying obesity and a framework for linking human genetic variations to specific neuronal populations in vivo.