Circadian Rhythms in Neuronal Models of Bipolar Disorder

双相情感障碍(BD)是一种与可遗传的多基因危险因素相关的精神障碍。虽然在临床上 BD的不同的关键临床特征包括日常睡眠和活动周期的中断，表明 在许多患者中，昼夜节律紊乱是这种紊乱的重要组成部分。昼夜节律是 由“时钟基因”组成的分子时钟决定的，时钟基因的表达受环境的调节 光和温度等因素。近年来，我们集团等在发展方面取得了进展 诱导多能干细胞(IPSC)来源的神经前体细胞和BD的细胞模型 神经元。我们已经确定了BD患者的时型(一天中的时间偏好，“早晨”)和 细胞的昼夜节律都可以预测锂维持治疗的临床反应。使用基于IPSC的 方法培养人神经前体细胞和神经元，我们发现BD患者的神经元具有高度兴奋性。 锂可以选择性地逆转锂反应(Li-R)BD供体神经元的表型，但 不在锂无反应者(Li-NR)的电池中。目前，我们建议在这项工作的基础上再接再厉 发展IPSC-神经元模型以进一步研究BD的昼夜节律紊乱。在目标1中，我们将估计 多基因危险因素对兴奋性神经元BD昼夜节律表型的贡献。我们会研究 IPSC来源的鼻咽癌和谷氨酸能神经元的节律来自一个特征良好的大家族 具有患BD的高遗传风险，但与BD诊断不一致。据推测，来自这些基因的细胞 类似的个体将包含跨梯度的BD风险基因，可以使用多基因风险分数进行量化 (RPS)。通过评估PR和节奏参数之间的关系，我们将估计总的 BD的遗传风险对昼夜节律异常表达的贡献。在目标2中，我们建议 评估BD相关基因组对神经元昼夜节律的贡献。全基因组关联 对睡眠和昼夜节律表型的研究表明，情绪存在共同的遗传基础 疾病和时型。在这个目标中，我们将研究24小时内的同步细胞，以有节奏地识别 BD患者和对照组神经前体细胞的基因表达及节律描述 诊断组之间的差异。我们期望发现许多基因改变它们的节律或失去节律 全部在BD神经元中。然后我们将使用一种名为siRNA敲除的分子生物学方法来减少 表达与BD和生物钟相关的候选基因，并评估它们在昼夜节律中的功能丧失 节律分析，在恒定或温度夹带的条件下。在目标3中，我们建议测量昼夜节律 IPSC来源的GABA能中刺神经元样细胞的节律。我们之前对BD的细胞研究 使用兴奋性谷氨酸能神经元。BD动物模型显示时钟的重要贡献 多巴胺基因投射到纹状体中的抑制性、GABA能中棘神经元(MSN)。在这 目的：我们将BD患者的IPSCs(LI-R和LI-NR)分化为抑制性、GABA能的MSN样神经元 使用一种既定的、高效的协议。我们将使用PER2-LUC昼夜节律记录器来测量 首次在活人GABA能神经元中记录节律并评估情绪稳定药物的作用 比如锂和抗精神病药物。在研究结束时，我们将有一个更先进的 了解哪些神经元在BD中失去节律，哪些BD风险基因参与昼夜节律紊乱， 以及昼夜节律紊乱如何影响神经元的功能。这些信息可能有助于更好地识别锂- 反应良好的BD患者，并将他们有效地与有效的治疗联系起来。

Bipolar disorder (BD) is a psychiatric disorder associated with heritable polygenic risk factors. While clinically heterogeneous, key clinical features of BD include disruptions in daily sleep and activity cycles indicating that circadian rhythm disruption is an important part of the disorder in many patients. Circadian rhythms are determined by molecular clocks comprised of “clock genes” whose expression is regulated by environmental factors such as light and temperature. In recent years, our group and others have made progress in developing cellular models of BD using induced pluripotent stem-cell (iPSC) derived neuronal progenitor cells (NPCs) and neurons. We have determined that chronotype (time of day preference, “morningness”) in BD patients and circadian rhythms in cells both predict the clinical response to lithium maintenance therapy. Using iPSC-based methods to grow human NPCs and neurons, we found that neurons from BD patients have a hyperexcitable phenotype that can be reversed by lithium selectively in neurons from lithium-responsive (Li-R) BD donors, but not in cells from lithium non-responders (Li-NR). Presently, we propose to build upon this work and further develop the iPSC-neuron model to further investigate circadian disruption in BD. In Aim 1, we will estimate the contributions of polygenic risk factors to circadian rhythm phenotypes in BD in excitatory neurons. We will study rhythms in iPSC-derived NPC and glutamatergic neurons grown from a well-characterized, extended family who share high genetic risk for BD, but are discordant for the BD diagnosis. Presumably, cells from these genetically similar individuals will contain BD risk genes across a gradient that can be quantified using polygenic risk scores (PRS). By evaluating the relationship between PRS and rhythm parameters, we will estimate the aggregate contributions of genetic risk for BD to the expression of circadian rhythm abnormalities. In Aim 2 we propose to assess the contribution of BD-associated gene sets to circadian rhythms in neurons. Genome-wide association studies (GWAS) of sleep and circadian phenotypes suggest the existence of a shared genetic basis for mood disorders and chronotype. In this aim, we will study synchronized cells over a 24 h period to identify rhythmically expressed genes in neural progenitor cells (NPCs) from BD patients and controls and describe rhythm differences between diagnostic groups. We expect to find that many genes change their rhythm or lose rhythms altogether in BD neurons. We will then use a molecular biology method called siRNA knockdown to reduce the expression of candidate genes linked to BD and the circadian clock, and assess their loss of function in circadian rhythm assays, under constant or temperature-entrained conditions. In Aim 3 we propose to measure circadian rhythms in iPSC-derived GABAergic medium-spiny neuron-like cells. Our previous cell-based studies of BD employed, excitatory glutamatergic neurons. Animal models of BD show important contributions of the clock genes on dopamine projections to inhibitory, GABAergic medium spiny neurons (MSN) in the striatum. In this aim, we will differentiate iPSCs from BD patients (Li-R and Li-NR) into inhibitory, GABAergic MSN-like neurons using an established, high efficiency protocol. We will use the Per2-luc circadian rhythm reporter to measure rhythms in live human GABAergic neurons for the first time and assess the effects of mood stabilizing drugs such as lithium and antipsychotic drugs upon them. At the conclusion of the study, we will have a more advanced understanding of which neurons lose rhythms in BD, which BD-risk genes are involved in the circadian disruption, and how circadian disruption affects the function of neurons. This information may help to better identify lithium- responsive BD patients and link them efficiently to effective treatments.