Using CRISPR tools to uncover the role of CREB-gene regulation in drug abuse

项目总结 物质滥用，包括精神刺激剂和鸦片类药物的滥用，是一个主要的公共健康问题。 这种担忧会带来巨大的财政和社会成本。滥用这些药物仍然顽固不化 条件与有限的FDA批准的治疗。差异神经分子机制的阐明 潜在的精神刺激剂与阿片类药物滥用对于开发新的、有针对性的治疗方法是必要的。 有大量证据表明，表观遗传和转录机制有助于 精神刺激剂和阿片类药物滥用和依赖的发病机制。表达式中的更改 在药物暴露的动物模型中，转录因子的水平和基因组定位已经被确定。 例如，反复接触鸦片类药物或精神刺激剂会升高环磷酸腺苷反应元件 结合蛋白(CREB)介导伏隔核(NAC)的转录，这是大脑的一个主要奖赏区域。 CREB的过度表达降低了精神刺激剂和鸦片类药物的奖励效应，这表明 CREB在该区域的活性可以调节这些药物的成瘾特性。然而，对 这种类型导致数百个基因座的表观遗传和转录变化，限制了对关键机制的洞察力 用于开发有针对性的成瘾疗法。此外，与奖励相关的转录基因调控 机制似乎是细胞类型特有的，对NAC中棘神经元(MSN)有不同的作用。 子类型。因此，为了确定细胞类型特异的、基因位点靶向的CREB的因果贡献 在精神刺激剂和阿片类药物滥用的不同发病机制中的招募，我设计了 CRISPR技术作为CREB有条件的、特定地点招聘的工具。我融合了核酸酶- 死亡的RNA引导的DNA结合蛋白dCas9与活性形式的CREB(S133D)结合，我能够将 靶细胞类型内的CREB结合到所需的基因组区域，以调节体内的转录。在.之下 埃里克·内斯特勒博士和李申博士的主要指导，我将接受体内，细胞靶向CRISPR的培训 给药策略、药物自我给药和生物信息学方法。这次培训将使我能够使用 这些高度具体的操作，以调查其差异贡献的完整视角 精神刺激剂与鸦片类致病意志行为和转录网络。我将验证并 在经过充分研究的FosB位置优化我的技术，允许对我的数据进行有意义的解释，以及 利用CRISPR系统固有的速度来定位一个新的、生物信息预测的基因座Zfp189。在……里面 在我的独立阶段，我计划在我的训练的基础上，将CREB与一种新的药物自身结合 管理部门确定了基因网络。这将确立在生理上使用这些细胞的潜力- 理解复杂事物的相关操作超越传统的单基因操作 神经精神综合症。这次培训将有助于我转变为一名独立的调查员，并取得成果 关于精神刺激剂和阿片类药物滥用的不同致病机制的新见解。

PROJECT SUMMARY Substance abuse, including the abuse of psychostimulants and opiates, represents a major public health concern that exacts tremendous financial and societal costs. Abuse of these drugs remain recalcitrant conditions with limited FDA-approved treatments. Elucidation of the differential neural molecular mechanisms underlying psychostimulant versus opiate abuse is necessary to the development of novel, targeted therapies. There is a substantial body of evidence indicating that epigenetic and transcriptional mechanisms contribute to the pathogenesis of both psychostimulant and opiate abuse and dependence. Changes in the expression levels and the genomic localization of transcription factors have been identified in drug-exposed animal models. For example, repeated exposure to opiates or to psychostimulants elevate cyclic AMP-responsive element binding protein (CREB)-mediated transcription in the nucleus accumbens (NAc), a major brain reward region. Over-expression of CREB decreases the rewarding effects of psychostimulants and opiates, suggesting that CREB activity in this region can regulate the addictive properties of these drugs. However, manipulations of this kind cause epigenetic and transcriptional changes at hundreds of loci, limiting mechanistic insight critical for the development of targeted addiction therapies. Moreover, reward-related transcriptional gene regulatory mechanisms appear to be cell-type specific, with distinct roles for the NAc medium spiny neuron (MSN) subtypes. Therefore, to determine the causal contribution of cell-type specific, gene locus-targeted CREB recruitment in the differential pathogenesis of psychostimulant versus opiate abuse, I have engineered CRISPR technology as a vehicle for the conditional, locus-specific recruitment of CREB. I fused the nuclease- dead, RNA-guided, DNA-binding protein dCas9 to the active form of CREB (S133D), and I am able to direct CREB within targeted cell types to desired genomic regions to modulate transcription in vivo. Under the primary mentorship of Drs. Eric Nestler and Li Shen, I will receive training on in vivo, cell-targeted CRISPR delivery strategies, drug self-administration, and bioinformatic approaches. This training will allow me to use these highly specific manipulations to investigate a complete perspective on their differential contribution to psychostimulant versus opiate pathogenic volitional behaviors and transcriptional networks. I will validate and optimize my technology at the well-studied Fosb locus, allowing for meaningful interpretation of my data, and leverage the speed inherent to the CRISPR system to target a novel, bioinformatically predicted loci, Zfp189. In my independent phase, I plan to build upon my training to multiplex CREB binding to a novel drug self- administration identified gene network. This would establish the potential of using these physiologically- relevant manipulations to move beyond conventional monogenic manipulations in understanding complex neuropsychiatric syndromes. This training will aid in my transition into an independent investigator and yield novel insight on the differential causal pathogenic mechanisms of psychostimulant versus opiate abuse.