Molecular and Genetic Studies of TMEM16C Control of Thermoregulation and Neuronal Excitability

TMEM16C 控制温度调节和神经元兴奋性的分子和遗传学研究

基本信息

批准号：
9885800
负责人：
LILY Y JAN
金额：
$ 35.24万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-15 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9885800
关键词：
Acquaintances Action Potentials Adopted Affect Afferent Neurons Ambystoma Animal Model Anterior Hypothalamus Body Temperature Brain Calcium Child Chloride Channels Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Complex Curiosities Enterobacteria phage P1 Cre recombinase Epidemiologist Epilepsy Excision Exhibits Family Febrile Convulsions Fever Genes Genetic Crosses Genetic Markers Genetic Polymorphism Genetic study Hippocampus (Brain)Induced Hyperthermia Knock-in Mouse Knockout Mice Link LoxP-flanked allele Mass Spectrum Analysis Messenger RNA Modeling Molecular Genetics Mus Neurons Nociception Oocytes Pain Pathology Physiologic Thermoregulation Physiological Potassium Potassium Channel Preoptic Areas Prostaglandin D2 Proteins Publications Rattus Regulation Rest Risk Factors Rodent Seizures Siblings Slice Sodium Synapses Synaptic Potentials System Technology Temperature Terminator Codon Testing Xenopus oocyte anterior hypothalamic nucleus cell type complex febrile seizure conditional knockout early onset expression cloning genome wide association study hippocampal pyramidal neuron hippocampal sclerosis member molecular marker mutant nanobodies nestin protein neuronal excitability pain sensitivity prevent protein distribution pup response single-cell RNA sequencing warm temperature

项目摘要

We made the surprise finding that loss of TMEM16C function led to the elimination of the majority of POA neurons that increase their firing rate with rising temperature of the preoptic area of the anterior hypothalamus (POA) – warm-sensitive neurons that are on top of a command chain for thermoregulation. To test for the involvement of TMEM16C in thermoregulation and febrile seizure, we examined TMEM16C conditional knockout (cKO) mice and found that mutant pups with TMEM16C removed from their central neurons cannot maintain their body temperature and are prone to develop hyperthermia-induced seizure. To look for molecular markers of temperature sensitive POA neurons, we conducted single-cell RNAseq of 68 POA neurons following recording from these neurons in brain slices subjected to temperature changes, and validated a molecular and genetic marker for temperature sensitive POA neurons. By generating cKO mice with TMEM16C removed from temperature sensitive POA neurons, we aim to test the function of TMEM16C in the specification of warm-sensitive neurons, thermoregulation, and hyperthermia-induced seizure. Given the GWAS association of TMEM16C with febrile seizure and our finding that rodent pups without neuronal TMEM16C are prone to exhibit hyperthermia-induced seizures, TMEM16C cKO mice provide animal models for febrile seizure, which affects 2-8% of young children. Because complex febrile seizures are associated with hippocampal sclerosis as the epileptogenic pathology, we will test the hypothesis that TMEM16C regulates hippocampal neuronal excitability by recording action potentials and synaptic potentials in hippocampal neurons from mice with TMEM16C removed either via nestin-Cre that is expressed in all neurons or via Drd3-Cre that targets ~50% of hippocampal pyramidal neurons. We will also record from temperature sensitive POA neurons in TMEM16C cKO mice and sibling controls to determine how TMEM16C modulates temperature sensitive POA neuronal activity. We will further test the hypothesis that removing TMEM16C from central neurons causes alteration of sodium-activated potassium current in these neurons. Finally, we will adopt the recently developed split GFP approach involving the use of mass spectrometry to identify proteins that are associated with TMEM16C in specific neuronal types.

我们惊奇地发现，TMEM16C 功能的丧失导致大多数 POA 神经元随着前视前区温度的升高而增加放电频率下丘脑 (POA) – 位于温度调节命令链顶部的热敏感神经元。为了测试 TMEM16C 参与体温调节和热性惊厥，我们检查了 TMEM16C 条件敲除 (cKO) 小鼠，发现带有 TMEM16C 的突变幼崽的中枢神经系统被移除神经元无法维持体温，并且容易发生高热引起的癫痫发作。寻找温度敏感 POA 神经元的分子标记，我们进行了 68 个单细胞 RNA 测序 POA 神经元在大脑切片中记录这些神经元受到温度变化的影响，以及通过生成 cKO 小鼠，验证了温度敏感 POA 神经元的分子和遗传标记。从温度敏感的 POA 神经元中去除 TMEM16C，我们的目的是测试 TMEM16C 的功能热敏感神经元的规范、温度调节和高热诱发的癫痫发作。鉴于 TMEM16C 与热性惊厥的 GWAS 关联以及我们的发现，啮齿动物幼崽没有神经元 TMEM16C 容易表现出高热诱导的癫痫发作，TMEM16C cKO 小鼠提供动物热性惊厥模型，影响 2-8% 的幼儿，因为复杂性热性惊厥是一种常见疾病。与海马硬化作为致癫痫病理学相关，我们将检验以下假设： TMEM16C 通过记录动作电位和突触电位来调节海马神经元兴奋性在 TMEM16C 小鼠的海马神经元中，TMEM16C 通过在所有细胞中表达的巢蛋白-Cre 被去除。神经元或通过针对约 50% 海马锥体神经元的 Drd3-Cre 我们还将记录。 TMEM16C cKO 小鼠和同胞对照中的温度敏感 POA 神经元，以确定 TMEM16C 如何调节温度敏感的 POA 神经活动我们将进一步测试去除的假设。来自中枢神经元的 TMEM16C 会导致这些神经元中钠激活钾电流的改变。最后，我们将采用最近开发的分离 GFP 方法，涉及使用质谱法来识别特定神经元类型中与 TMEM16C 相关的蛋白质。