Characterization of Lphn3 function using a mutant murine model

描述（由申请人提供）：孤儿GPCR LPHN3虽然相对未知且未被表征，但在全基因组连锁和关联研究中已经独立地与成瘾表型和注意缺陷多动障碍（ADHD）相关。易上瘾和注意力缺陷多动障碍经常是共病，这表明一个共同的病因。此外，初步数据表明，LPHN3基因型可能是预测兴奋剂药物反应的药理学标记。不幸的是，由于在LPHN3中尚未发现与疾病相关的功能多态性，其作用机制完全未知，进一步表征其功能至关重要，因为这一重要基因具有深远的意义。本研究的中心假设是Lphn3信号影响大脑中神经递质释放和单胺浓度，从而影响神经细胞的分化、发育和功能。这些改变最终会对基本行为产生影响，可能会影响多动症和成瘾等表型。我们建议利用新的Lphn3零突变小鼠来研究基因功能。初步数据表明，Lphn3缺失小鼠过度活跃，大脑中DA和5-HT水平发生改变，参与神经元分化和发育的基因以及涉及多巴胺和5-羟色胺能通路（包括多巴胺和5-羟色胺受体）的其他ADHD候选基因的表达水平发生改变。我们利用这些稀少的线索来制定我们的研究计划。我们将从定义大脑中的Lphn3发育表达模式开始，并研究与其他特征明确的神经元标记物的共表达。我们还将利用TaqMan基因表达测定来寻找已知在神经元分化、发育、存活和Lphn3基因型功能中发挥作用的基因表达的变化。测试将在发育时间点和特定的大脑区域进行。接下来，我们将从WT和突变小鼠中分离原代神经元，并基于Lphn3基因型评估其活力、生长和功能。最后，我们设计了一系列行为分析来开始评估Lphn3小鼠的基本行为缺陷。这一系列包括对活动、运动功能、焦虑、抑郁、动机、注意力、冲动、学习和记忆的初步评估。从这个项目中获得的数据将是表征Lphn3基本功能的第一步，这将有助于更好地理解、诊断和治疗多动症和成瘾。

DESCRIPTION (provided by applicant): Though relatively unknown and uncharacterized, the orphan GPCR LPHN3 has already been correlated in genome-wide linkage and association studies to both addictive phenotypes and Attention Deficit Hyperactivity Disorder (ADHD) independently. Vulnerability to addiction and ADHD are frequently comorbid, suggesting a common etiology. Further, initial data indicate that LPHN3 genotype may be a pharmacogenetic marker predicting response to stimulant medication. Unfortunately, as no disease-associated functional polymorphisms have been identified in LPHN3, its mechanism of action is completely unknown, and further characterization of its function is essential as there are far reaching implications for such an important gene. The central hypothesis to be tested in this proposal is that Lphn3 signalling affects neurotransmitter release and concentrations of monoamines in the brain that may affect neural cell differentiation, development, and function. These alterations ultimately play out in an effect on basic behavior that may influence phenotypes such as ADHD and vulnerability to addiction. We propose to utilize the novel Lphn3 null mutant mouse to investigate gene function. Preliminary data indicate that the Lphn3 null mouse is hyperactive, has altered DA and 5-HT levels in the brain, and demonstrates altered expression levels of genes involved in neuronal differentiation and development as well as other ADHD candidate genes involving the dopaminergic and serotonergic pathways including dopamine and serotonin receptors. These scant clues have been used by us to construct our research plan. We will begin with defining the Lphn3 developmental expression pattern in the brain and look at co-expression with other well-characterized neuronal markers. We will also utilize TaqMan Gene Expression Assays to look for changes in expression of genes known to play a role in neuronal differentiation, development, survival, and function as a function of Lphn3 genotype. Assays will be preformed across developmental time points and in specific brain regions. Next we will isolate primary neurons from WT and mutant mice and evaluate their viability, growth and function based on Lphn3 genotype. Finally, we have designed a battery of behavioral assays to begin to evaluate the basic behavioral deficits in the Lphn3 mice. This battery includes preliminary assessment of: activity, motor function, anxiety, depression, motivation, attention, impulsivity, learning and memory. Data derived from this project will be a first step toward characterizing the basic functions of Lphn3, which could enable better understanding, diagnosis, and treatment of ADHD and addiction.