Subcellular localization of MC4R with ADCY3 at neuronal primary cilia underlies a common pathway for genetic predisposition to obesity

Abstract

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 humans^1-3. Recently, ADCY3 (adenylyl cyclase 3) gene mutations have been implicated in obesity^4,5. ADCY3 localizes to the primary cilia of neurons ⁶ , 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 manifestation ⁷ . 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.

Figure 1. MC4R localizes to the primary cilia in heterologous cells

A) Representative confocal microscopy images of transiently transfected MEF, RPE and IMCD3 cells, transfected with MC4R-EGFP labeled for the cilia specific protein acetylated Tubulin (Tubulin^Ac, red) and GFP (green), and nuclei with Hoechst 33342 (blue). MC4R-GFP localize to the primary cilium (yellow arrowheads). Scale bars represent 10 μm. (B) Relative ciliary enrichment of melanocortin receptor family members compared to ciliary enrichment of GFP (negative control) and Smoothen (postive control). Data are Mean±sem. Means were compared to GFP (n=12 cells, mean=1.24) and Dunnet’s multiple comparison test was applied. Smo-Myc: n=7 cells, mean= 2.35, p=0,0001; MC1RGFP: n=13 cells, mean=1.29, p=0.9995, MC2RGFGP: n=13 cells, mean=1.23, p=0.9999, MC3RGFP: n=10 cells, mean=1.24, p=0.014; MC4RGFP: n=9 cells, mean=2.68, p=0.0001, MC5RGFP: n=9 cells, mean=1.96, p=0.0008.

Figure 2. MC4R localizes specifically to the primary cilia of a subset of PVN neurons in vivo

A) Strategy used to target the mouse Mc4r locus by Crispr/Cas9. B) Schematic representation of hypothalamic region studied in C–I. C) Coronal section of the PVN of the hypothalamus of a heterozygous MC4R-GFP mouse immunostained for GFP and Adcy3. Note that no immunofluorescence is detectable at this resolution. D–I) Maximal intensity projections of confocal sections through two PVN regions indicated in C reveals MC4R-GFP (green) co-localized with neuronal primary cilia (Adcy3, red) in a subset of PVN neurons. Co-localization of MC4R-GFP with cilia has been observed in the PVN of over 10 mice (male and female) derived from two independent founders. Red scale bars represent 200 μm, White scale bars represent 10 μm.

Figure 3. A subset of human obesity-associated mutations selectively impair ciliary localization of MC4R

A) Relative ciliary enrichment of eight obesity-associated mutant MC4R compared to the wild-type (WT) receptor. Data are Mean±sem. Means were compared to MC4RWT (n=16 cells, mean=2.89) and Dunnet’s multiple comparison test was applied. MC4RR7H: n=10 cells, mean= 2.57, p=0.28; MC4RT150I: n=17 cells, mean=2.57, p=0.21; MC4RP230L: n=12, mean=1.87, p=0.0004, MC4RG231S: n=18, mean=3.10, p=0.39, MC4RR236C: n=22, mean=2.1, p=0.0013, MC4RL250Q: n=10, mean=2.83, p=0.85; MC4RG252S: n=11, mean=2.72, p=0.54; MC4RI301T: n=4, mean=2.59, p=0.47. B) Design of DIO AAV expressing MC4RGFP and MC4RP230LGFP. C) Experimental protocol D) Schematic representation of injection site. E, I) Coronal section of the PVN of the hypothalamus of Sim1 cre mice injected with the MC4RGFP DIO AAV (E) and the MC4RP230LGFP DIOAAV (I) respectively. (F–H and J–L) Maximal intensity confocal projection of sections through the PVN regions indicated in E and I respectively reveals co-localization of MC4RGFP (F–H) but not MC4RP230LGFP (J–L) with neuronal primary cilia in PVN neurons. Expressions of MC4R-GFP (WT vs P230L) have been observed in the PVN of over 6 mice (male and female). Red scale bars represent 200 μm, White scale bars represent 10 μm.

Figure 4. inhibition of Adenylyl cyclase at the primary cilia of Sim1 PNV neurons is sufficient to cause weight gain

A) Experimental protocol (See also supplementary Figure 3). B) Midline stereotaxic injections of AAV DIO Flag-GPR88* + AAV DIO mCherry or AAV DIO mCherry were performed in male Sim1 cre mice. C) Coronal section of the PVN of the hypothalamus of a Sim1 cre mouse injected with the AAV DIO Flag-GPR88* + AAV DIO mCherry. D–F) Maximal intensity projections of confocal sections through the PVN region indicated by a red square in C. Arrows indicate cilia expressing GPR88. Scale bars represent 10 μm. G) Percent weight changes (mean ± SE) of Sim1cre mice following midline PVN injection of AAV DIO GPR88(G283H)+ AAV DIO mCherry (n=12) or AAV DIO mCherry (n=9). Mice were paired at baseline by body weights and litters. Repeated measures of two-way ANOVA followed by Sidak’s multiple comparisons test were performed (treatment F(1, 19) = 8.898, P = 0.0076; time F(5, 95) = 49.07, P < 0.0001; interaction F(5,95) = 8.789, P < 0.0001; p values from Sidak’s multiple comparisons test are shown in the figure). H) Individual weight changes of Sim1cre mice in G) (each line represents one mouse). I) Food intake at baseline and 6 weeks after AAV injections (mean± SD). Repeated measures of two-way ANOVA followed by Sidak’s multiple comparisons test were performed (treatment F(1, 19) = 2.41, P = 0.1370; time F(1, 19) = 9.328, P = 0.0065; interaction F(1, 19) = 9.196, P = 0.0068; p values from Sidak’s multiple comparisons test are shown in the figure). Red scale bars represent 200 μm, White scale bars represent 10 μm.