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.

我们惊讶地发现，TMEM16C功能的损失导致大部分消除 POA神经元，随着前面的前区域的温度升高而增加发射速率下丘脑（POA） - 温敏的神经元，位于温度调节的指挥链之上。到测试TMEM16C参与体温调节和发热性癫痫发作，我们检查了TMEM16C 有条件的敲除（CKO）小鼠，发现带有TMEM16C的突变幼崽从其中央移走神经元无法维持其体温，并且容易发生高温诱导的癫痫发作。到寻找温度敏感POA神经元的分子标记，我们进行了68的单细胞RNASEQ 从这些神经元中记录的脑切片中的POA神经元，受到温度变化，并且验证了温度敏感的POA神经元的分子和遗传标记。通过产生CKO小鼠从温度敏感的POA神经元中除去TMEM16C，我们旨在测试TMEM16C的功能在温敏神经元的规范中，体温调节和高温诱导的癫痫发作。鉴于TMEM16C与高热癫痫发作的GWAS协会，我们发现没有啮齿动物的幼犬没有神经元TMEM16C容易暴露于暴露高温诱导的癫痫发作，TMEM16C CKO小鼠提供动物发热癫痫发作的模型，影响2-8％的幼儿。因为复杂的高温癫痫发作是与海马硬化症作为癫痫病病理学有关，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令人兴奋在来自TMEM16C的小鼠的海马神经元中，通过nestin-cre去除的，这是所有表达的神经元或通过DRD3-CRE靶向约50％的海马锥体神经元。我们还将从 TMEM16C CKO小鼠和兄弟姐妹控制中的温度敏感POA神经元，以确定TMEM16C的方式调节温度敏感的POA神经元活性。我们将进一步检验删除的假设来自中央神经元的TMEM16C会导致这些神经元中钠激活的钾电流的改变。最后，我们将采用最近开发的分裂GFP方法，涉及使用质谱法识别特定神经元类型中与TMEM16C相关的蛋白质。