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

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.

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.

CRISPRi-mediated repression of Mc4r in adult PVN^MC4R neurons reduced transcript and protein levels and induced progressive obesity, validating the approach. Downregulation of the ciliary adenylyl cyclase Adcy3 specifically in adult PVN^MC4R 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.

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.