腺苷酸环化酶3在主要嗅觉表皮或下丘脑表达对肥胖的作用及其分子机制

The type III adenylyl cyclase (AC3) is an essential component of cAMP signaling. Gene knockout in mice and genome-wide association study in human indicated that AC3 is crucial in body weight control. However, its functional site and molecular mechanisms remains to be determined. AC3 is enriched in neural cilium in the main olfactory epithelium (MOE) and hypothalamus (Hypo). The cilium cAMP signaling is closely related to the body weight control and the effect of autophagy on nutritive molecules sensing. It is presumed that the obesity function of AC3 may be mediated by the coordination of MOE or Hypo cilium with autophagy. This proposal will use the combination method of adeno-associated virus (AAV) packaging of CRISPR/Cas9 and siRNA and stereotaxic injection, specific knockdown of AC3 in the MOE or hypothalamus; restore the expression of AC3 in neural cilium of MOE or hypothalamus in AC3 knockout mice, through transfect constructs with wild type , or sumoylation (small ubiquitin-like modifier) loci (K465) mutant type which will make AC3 not expressed in the cilium; identify the interaction between AC3 and cilium autophagy related genes by co-immunoprecipitation approach, and specific knockdown of the identified genes in the MOE or Hypo by stereotaxic injection. The purpose of this proposal is to elucidate the obesity functional sites of AC3 and its causal relationship with cilia and autophagy. The results of this proposal have important scientific significance and application value for understanding the obesity mechanism of AC3 gene.

腺苷酸环化酶3（AC3）是cAMP信号通路的主要成分。敲除小鼠和人类基因组关联分析均表明AC3对控制体重起关键作用,但其作用部位及分子机制尚待阐明。AC3表达于主要嗅觉表皮组织（MOE）和下丘脑(Hypo)等区域的神经细胞纤毛。纤毛cAMP信号通路与体重和细胞自噬对营养分子的感受密切相关。推测AC3的肥胖功能可能是MOE或Hypo神经纤毛与自噬协调发挥作用。本项目采用腺相关病毒(AAV)包装CRISPR/Cas9、siRNA和脑立体定位注射相结合方法，在MOE或Hypo特异敲低AC3；在AC3敲除小鼠上恢复表达AC3野生型和SUMOylation 位点（K465）突变型，使其表达或不表达于纤毛部位；鉴定AC3与纤毛自噬相关基因的互作关系，并对鉴定出的基因在MOE或Hypo特异敲低。旨在阐明AC3的肥胖功能部位及与纤毛自噬的因果关系。该项目对了解AC3肥胖机理具有重要科学意义和应用价值。

腺苷酸环化酶3(AC3)是cAMP信号通路的主要成分。敲除小鼠和人类基因组关联分析均表.明AC3对控制体重起关键作用, 但其作用部位及分子机制尚待阐明。AC3表达于主要嗅觉表皮组织(MOE)和下丘脑(Hypo)等部位的神经细胞纤毛。纤毛cAMP信号通路与体重和细胞自噬对营养分子的感受密切相关。推测AC3的肥胖功能可能是MOE或Hypo神经纤毛与自噬协调发挥作用。本项目采用腺相关病毒(AAV)包装CRISPR/Cas9和脑立体定位注射相结合方法，在MOE特异敲低AC3表达后发现，与对照小鼠相比，MOE内AC3特异敲低小鼠的体重和摄食量均没有发生改变，说明MOE内的AC3表达并不影响小鼠的体重和摄食量。虽然普通饲料条件下，与NC组小鼠相比较，下丘脑AC3特异敲低小鼠体重无显著改变。但高脂饲料条件下，下丘脑AC3特异敲低小鼠体重显著增加，说明下丘脑组织中AC3调控小鼠肥胖表型。进而在下丘脑组织中特异敲低纤毛上AC3的表达，发现敲低小鼠更加显著肥胖，说明纤毛上AC3表达水平对高脂肪饲料诱导的肥胖更为敏感。细胞、MEF和活体组织，qPCR、WB和电镜观察结果表明，纤毛表达的AC3与GABARAP相互作用。VMH GABARAP KD小鼠AC3+纤毛的数量显著减少，纤毛变短, 呈现更为明显的HFD诱导肥胖表型。总之，小鼠VMH中AC3基因的纤毛表达及其与自噬相关的GABARAP互作，对于维持体重的稳态平衡至关重要。这些结果对了解AC3肥胖机理具有重要科学意义和应用价值。