Most monogenic cases of obesity in humans have been linked to mutations in genes encoding members of the leptin–melanocortin pathway. Specifically, mutations in MC4R, the melanocortin-4 receptor gene, account for 3–5% of all severe obesity cases in humans1,2,3. Recently, ADCY3 (adenylyl cyclase 3) gene mutations have been implicated in obesity4,5. ADCY3 localizes to the primary cilia of neurons6, organelles that function as hubs for select signaling pathways. Mutations that disrupt the functions of primary cilia cause ciliopathies, rare recessive pleiotropic diseases in which obesity is a cardinal manifestation7. We demonstrate that MC4R colocalizes with ADCY3 at the primary cilia of a subset of hypothalamic neurons, that obesity-associated MC4R mutations impair ciliary localization and that inhibition of adenylyl cyclase signaling at the primary cilia of these neurons increases body weight. These data suggest that impaired signaling from the primary cilia of MC4R neurons is a common pathway underlying genetic causes of obesity in humans.
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This research was supported by funds from UCSF DRC NIH P30DK063720 and UCSF NORC NIH P30DK098722; an AMC Graduate School PhD Scholarship to J.E.S.; NIH R01AR05439 and NIH R01GM095941, the Burroughs Wellcome Fund and the David & Lucile F. Packard Foundation to J.F.R.; NIH R01DK60540 to C.V.; NIH RO1DK106404 to C.V. and J.F.R.; NIH RO1DA012864 and NIH RO1DA010711 to M.V.Z.; and a New Frontier Research Award through the UCSF Program for Breakthrough Biomedical Research to C.V., J.F.R. and M.V.Z. We thank K. Deisseroth (Stanford University) for providing plasmids.
Integrated supplementary information
a Hypothalamic PVN section of a Tg(Sim1-EGFP) mouse. Sim1 lineage neurons are in green, the neuronal cilia marker Adcy3 is labeled in red, nuclei in pink. b Increased magnification indicating that all Sim1-expressing neurons possess an Adcy3-positive cilium. c Maximal intensity Z projection of a confocal image of a single EGFP-expressing Sim1 neuron in the PVN. Scale bars represent 10 µm.
Mutations found in obese patients are indicated by a black arrow from the mutated amino-acid to the colored circle that indicates the amino-acid in the mutant protein. Red arrows indicate frameshift mutations. Most patients were heterozygous for one mutation. Amino-acid changes causing severe effects on MC4R membrane expression are in red. Such mutations are expected to alter membrane and cilia localization as well as ligand response. Mutations in blue lead to decreased agonist activation (blue) or decreased constitutive activity (purple). Whether these mutations also alter cilia localization has not been tested. Mutations tested in Fig. 3a are in yellow. Mutations leading specifically to decreased ciliary expression are indicated by a green arrow.
a, b Design of DIO AAV-expressing Flag-GPR88* or mCherry, stereotaxically injected in experimental a and control b mice. c Coronal section of the PVN of the hypothalamus of Sim1-Cre mice injected with the AAV DIO Flag-GPR88* + AAV DIO mCherry allows for verification of the accuracy and PVN coverage of the injections. d–e Maximal intensity projections of confocal sections through the PVN region indicated by a red square in c show that all mCherry expressing neurons also express Flag-GPR88* (green). White scale bars represent 10 µm.