CRISPR/SaCas9 mutagenesis of stromal interaction molecule 1 in proopiomelanocortin neurons increases glutamatergic excitability and protects against diet-induced obesity.
CRISPR/SaCas9 mutagenesis of stromal interaction molecule 1 in proopiomelanocortin neurons increases glutamatergic excitability and protects against diet-induced obesity.
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DOI：
10.1016/j.molmet.2022.101645
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发表时间：
2022-12
影响因子：
8.1
通讯作者：
Kelly, Martin J.
中科院分区：
文献类型：
--
作者：
Qiu, Jian;Bosch, Martha A.;Stincic, Todd L.;Hunker, Avery C.;Zweifel, Larry S.;Ronnekleiv, Oline K.;Kelly, Martin J.
前阿黑皮素（Proopiomelanocortin，POMC）神经元是下丘脑神经元中整合代谢线索以产生维持能量稳态的适当输出的关键，并表达必要的通道作为一个完美的突触整合器。与代谢激素瘦素和胰岛素类似，谷氨酸也通过I组代谢型谷氨酸受体（mGluR 1和5，mGluR 1/5）兴奋POMC神经元，所述I组代谢型谷氨酸受体激活瞬时受体电位经典（TRPC 5）通道以引起去极化。TRPC 5通道活性的关键调节剂是基质相互作用分子1（STIM 1），其参与TRPC 5通道在钙从内质网耗尽后从受体操作的钙进入到储存操作的钙进入的募集。我们使用含有重组酶依赖性金黄色葡萄球菌Cas9（SaCas）和单一指导RNA（sgRNA）的单一腺相关病毒（AAV）载体来突变雄性小鼠中POMCCre神经元中的Stim 1，通过单一POMC神经元中Stim 1 mRNA表达的qPCR验证。进行全细胞膜片钳实验以验证Stim 1诱变的效果。测量雄性小鼠的体重和食物摄入量，以评估能量平衡的破坏。减少Stim 1的表达增强了mGluR 1/5激动剂3，5-二羟基苯甘氨酸（DHPG）通过Gα q偶联信号通路对POMC神经元的去兴奋作用，Gα q偶联信号通路是兴奋性谷氨酸能输入调节能量稳态的重要部分。TRPC 5通道阻断剂HC 0 70和Pico 14 5可拮抗DHPG的兴奋作用。作为原理的证明，POMC神经元中Stim 1的诱变减少了喂食高脂肪饮食的小鼠的食物摄入，减弱了体重增加，减少了体脂和脂肪垫质量。使用CRISPR技术，我们发现了STIM 1在调节POMC神经元中TRPC 5通道的神经递质激活中的关键作用，这最终对维持能量平衡至关重要。CRISPR/SaCas 9载体选择性地降低成年POMC神经元中的Stim 1表达。减少Stim 1增强mGluR 1/5激动剂DHPG兴奋POMC神经元的功效。选择性TRPC 5通道阻断剂可拮抗DHPG的兴奋作用。Stim 1的突变减少了高脂饮食小鼠的摄食量和体重增加。
Proopiomelanocortin (POMC) neurons are the key anorexigenic hypothalamic neuron for integrating metabolic cues to generate the appropriate output for maintaining energy homeostasis and express the requisite channels as a perfect synaptic integrator in this role. Similar to the metabolic hormones leptin and insulin, glutamate also excites POMC neurons via group I metabotropic glutamate receptors (mGluR1 and 5, mGluR1/5) that activate Transient Receptor Potential Canonical (TRPC 5) Channels to cause depolarization. A key modulator of TRPC 5 channel activity is stromal interaction molecule 1 (STIM1), which is involved in recruitment of TRPC 5 channels from receptor-operated to store-operated calcium entry following depletion of calcium from the endoplasmic reticulum. We used a single adeno-associated viral (AAV) vector containing a recombinase-dependent Staphylococcus aureus Cas9 (SaCas) and a single guide RNA (sgRNA) to mutate Stim1 in POMCCre neurons in male mice, verified by qPCR of Stim1 mRNA expression in single POMC neurons. Whole-cell patch clamp experiments were conducted to validate the effects of Stim1 mutagenesis. Body weight and food intake were measured in male mice to assess disruptions in energy balance. Reduced Stim1 expression augmented the efficacy of the mGluR1/5 agonist 3, 5-Dihydroxyphenylglycine (DHPG) to depolarize POMC neurons via a Gαq-coupled signaling pathway, which is an essential part of excitatory glutamatergic input in regulating energy homeostasis. The TRPC 5 channel blockers HC070 and Pico145 antagonized the excitatory effects of DHPG. As proof of principle, mutagenesis of Stim1 in POMC neurons reduced food intake, attenuated weight gain, reduced body fat and fat pad mass in mice fed a high fat diet. Using CRISPR technology we have uncovered a critical role of STIM1 in modulating glutamatergic activation of TRPC 5 channels in POMC neurons, which ultimately is important for maintaining energy balance. CRISPR/SaCas9 vector selectively reduces Stim1 expression in adult POMC neurons. Reduced Stim1 augments the efficacy of mGluR1/5 agonist DHPG to excite POMC neurons. Selective TRPC 5 channel blockers antagonize the excitatory effects of DHPG. Mutagenesis of Stim1 reduces food intake, weight gain in mice fed a high fat diet.
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影响因子：
29
作者：
Al-Qassab H;Smith MA;Irvine EE;Guillermet-Guibert J;Claret M;Choudhury AI;Selman C;Piipari K;Clements M;Lingard S;Chandarana K;Bell JD;Barsh GS;Smith AJ;Batterham RL;Ashford ML;Vanhaesebroeck B;Withers DJ
通讯作者：
Withers DJ
DOI：
10.1038/oby.2006.314
发表时间：
2006-08-01
期刊：
Obesity (Silver Spring, Md.)
影响因子：
--
作者：
Horvath, Tamas L
通讯作者：
Horvath, Tamas L
DOI：
10.1016/j.cub.2021.07.047
发表时间：
2021-10-11
期刊：
Current biology : CB
影响因子：
--
作者：
Fellinger L;Jo YS;Hunker AC;Soden ME;Elum J;Juarez B;Zweifel LS
通讯作者：
Zweifel LS
影响因子：
11.4
作者：
Falasca, M;Logan, SK;Schlessinger, J
通讯作者：
Schlessinger, J
通过反式编码的小 RNA 和宿主因子 RNase III 进行 CRISPR RNA 成熟。
影响因子：
64.8
作者：
Deltcheva, Elitza;Chylinski, Krzysztof;Sharma, Cynthia M.;Gonzales, Karine;Chao, Yanjie;Pirzada, Zaid A.;Eckert, Maria R.;Vogel, Joerg;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle